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https://openalex.org/W2172220115
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https://reproductive-health-journal.biomedcentral.com/counter/pdf/10.1186/1742-4755-10-19
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English
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Antenatal care packages with reduced visits and perinatal mortality: a secondary analysis of the WHO Antenatal Care Trial
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Reproductive health
| 2,013
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cc-by
| 5,274
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* Correspondence: vogeljo@who.int
1School of Population Health, Faculty of Medicine, Dentistry and Health
Sciences, University of Western Australia, 35 Stirling Highway, Crawley 6009,
Australia
2UNDP/UNFPA/UNICEF/WHO/World Bank Special Programme of Research,
Development and Research Training in Human Reproduction (HRP),
Department of Reproductive Health and Research, World Health
Organization, Avenue Appia 20, Geneva CH-1211, Switzerland
Full list of author information is available at the end of the article Open Access Open Access Antenatal care packages with reduced visits and
perinatal mortality: a secondary analysis of the
WHO Antenatal Care Trial Joshua P Vogel1,2*, Ndema Abu Habib2, João Paulo Souza2, A Metin Gülmezoglu2, Therese Dowswell3,
Guillermo Carroli4, Hassan S Baaqeel5, Pisake Lumbiganon6, Gilda Piaggio7 and Olufemi T Oladapo8 RESEARCH Antenatal care packages with reduced visits and
perinatal mortality: a secondary analysis of the
WHO Antenatal Care Trial Vogel et al. Reproductive Health 2013, 10:19 Vogel et al. Reproductive Health 2013, 10:19 Vogel et al. Reproductive Health 2013, 10:19
http://www.reproductive-health-journal.com/content/10/1/19 Methods The design of the WHO ANC Trial is described in detail
elsewhere
[10,12]. In
brief,
antenatal
clinics
were
randomized to deliver the new antenatal care package or
the standard antenatal care protocol offered in accord-
ance with local guidelines. The new antenatal package
combined evidence-based screening, therapeutic inter-
ventions and maternal education across four antenatal
visits for low-risk women. A classifying form was used
by the clinics randomized to the new package to identify
any maternal medical and obstetric conditions and these
women received additional antenatal care appropriate to
that condition. The primary fetal/neonatal outcome was
low birth weight, the primary maternal outcome was a
maternal morbidity index defined as the presence of
at least one of the following: pre-eclampsia/eclampsia,
severe postpartum anemia and treated urinary tract in-
fections/pyelonephritis. Maternal and perinatal second-
ary outcomes included among others fetal and neonatal
mortality. The sample size calculations for the trial were
based solely on the primary outcomes, since the mortal-
ity end-points would have required a very large number
of participants [10]. In 2001, Villar and colleagues published the findings
of the WHO Antenatal Care Trial (hereafter referred to
as the WHO ANC Trial), a multi-center, cluster-
randomized trial of an evidence based, reduced visit pack-
age of antenatal care in Thailand, Cuba, Saudi Arabia and
Argentina [10]. The trial found that a reduction in the
number of visits and application of goal oriented, effective
antenatal activities was not inferior to standard antenatal
care packages in the risk of adverse outcomes for mothers
and newborns [10]. The authors also stated that the wide-
spread application of the reduced visit package might, in
some settings, reduce cost. In 2010, the Cochrane systematic review comparing
the effects of reduced visits, goal-oriented antenatal care
programs with the standard model of care for low-risk
women was updated [4]. This review included seven trials
of more than 60,000 women, four in high-income coun-
tries with individual randomization and three in low- and
middle-income countries with cluster randomization,
including the WHO ANC Trial. The four individually
randomized trials defined reduced frequency as six to
eight visits, whereas the three clusters randomized trials
used a reduced schedule of four to six visits. Primary out-
comes were maternal death, pre-eclampsia, perinatal
death, preterm birth and small for gestational age. Abstract Background: In 2001, the WHO Antenatal Care Trial (WHOACT) concluded that an antenatal care package of
evidence-based screening, therapeutic interventions and education across four antenatal visits for low-risk women
was not inferior to standard antenatal care and may reduce cost. However, an updated Cochrane review in 2010
identified an increased risk of perinatal mortality of borderline statistical significance in three cluster-randomized
trials (including the WHOACT) in developing countries. We conducted a secondary analysis of the WHOACT data to
determine the relationship between the reduced visits, goal-oriented antenatal care package and perinatal
mortality. Methods: Exploratory analyses were conducted to assess the effect of baseline risk and timing of perinatal death. Women were stratified by baseline risk to assess differences between intervention and control groups. We used
linear modeling and Poisson regression to determine the relative risk of fetal death, neonatal death and perinatal
mortality by gestational age. Results: 12,568 women attended the 27 intervention clinics and 11,958 women attended the 26 control clinics. 6,160 women were high risk and 18,365 women were low risk. There were 161 fetal deaths (1.4%) in the
intervention group compared to 119 fetal deaths in the control group (1.1%) with an increased overall adjusted
relative risk of fetal death (Adjusted RR 1.27; 95% CI 1.03, 1.58). This was attributable to an increased relative risk of
fetal death between 32 and 36 weeks of gestation (Adjusted RR 2.24; 95% CI 1.42, 3.53) which was statistically
significant for high and low risk groups. Conclusion: It is plausible the increased risk of fetal death between 32 and 36 weeks gestation could be due to
reduced number of visits, however heterogeneity in study populations or differences in quality of care and timing
of visits could also be playing a role. Monitoring maternal, fetal and neonatal outcomes when implementing
antenatal care protocols is essential. Implementing reduced visit antenatal care packages demands careful
monitoring of maternal and perinatal outcomes, especially fetal death. Keywords: Antenatal care, Perinatal mortality, WHO, Developing country © 2013 Vogel et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited. Vogel et al. Reproductive Health 2013, 10:19
http://www.reproductive-health-journal.com/content/10/1/19 Page 2 of 7 Vogel et al. Reproductive Health 2013, 10:19
http://www.reproductive-health-journal.com/content/10/1/19 Introduction or simply a chance effect. The consultation recommended
that researchers from the three trials in question under-
take secondary analyses of their datasets to identify
possible causes. This secondary analysis of the WHO
ANC Trial data explores the relationship between the re-
duced visits antenatal care package and perinatal mortality. Many attempts have been made to rationalize the
content, frequency and implementation of antenatal care
in response to new evidence and technologies [1-4] and
systematic reviews have examined the effectiveness of
individual components of antenatal care programs [5-9]. Evidence-based, validated and safe antenatal care pack-
ages recommending fewer visits for low-risk women
have large implications for developing countries, where
resources are scarce and antenatal care poses both finan-
cial and logistical challenges to patients and health
systems. Results In the WHO ANC Trial, 6,160 women were high risk
and 18,365 were low risk. Table 1 shows selected mater-
nal characteristics stratified by risk and by group. Low-
risk women were less likely to smoke and were younger
and more educated. Robust variance Poisson regression
modeling was used to compare perinatal outcomes be-
tween the new and standard model groups, adjusted for
maternal risk and other significant covariates from uni-
variate analysis. Table 2 shows that adjusted relative risk
of perinatal death is higher in the new model (Adjusted
RR 1.18; 95% CI 1.01, 1.37). This is due to an increased
relative risk of fetal death (Adjusted RR 1.27; 95% CI
1.03, 1.58), which can in turn be attributed to an
increased risk of fetal death at less than 36 weeks Table 3 shows adjusted relative risks for fetal, neonatal
and perinatal mortality, stratified by maternal risk. Perinatal
mortality was not significantly different in high-risk
(Adjusted RR 0.94; 95% CI 0.79, 1.12) or low-risk
women (Adjusted RR 1.24; 95% CI 0.95, 1.63). Within
the high-risk group, the new model was associated with
an increase in the overall risk of fetal death (Adjusted
RR 1.78; 95% CI 1.33, 2.39) and an increased risk of
fetal death at less than 36 weeks (Adjusted RR 1.48;
95% CI 1.17, 1.89). Vogel et al. Reproductive Health 2013, 10:19
http://www.reproductive-health-journal.com/content/10/1/19 Vogel et al. Reproductive Health 2013, 10:19
http://www.reproductive-health-journal.com/content/10/1/19 Page 3 of 7 (Adjusted RR 1.64; 95% CI 1.27, 2.11). Crude and
adjusted relative risks were consistent for all outcomes. Methods The
reviewers found a borderline significant increase in peri-
natal mortality for women randomized to reduced visits
compared with standard care (RR 1.14; 95% CI 1.00, 1.31). On sub-group analysis, the number of perinatal deaths in
the individually randomized trials was relatively low. How-
ever, the three cluster-randomized trials consistently
showed slightly higher perinatal mortality in the reduced
visits group (RR 1.15; 95% CI 1.01, 1.32). A total of 53 clinics in Khon Kaen (Thailand), Havana
(Cuba), Jeddah (Saudi Arabia), and Rosario (Argentina)
were randomized. 12,568 women attended the 27 inter-
vention clinics for a median of five visits. 11,958 women
attended the 26 standard model clinics for a median of
eight visits. Rates of neonatal mortality (0.6% for the
new model vs. 0.7% for the standard model) were rela-
tively similar between groups, but the rates of overall
fetal death (1.4% for the new model vs. 1.1% for the
standard model) were not. For this secondary analysis, only singleton births were
included. Women were stratified by baseline maternal
risk to determine if differences in maternal characteris-
tics between the intervention and control groups con-
tributed to the risk of perinatal mortality. Fetal death
was stratified by gestational age and compared between
intervention and control groups to explore differences in
risks according to timing of fetal death. We used gener-
alized linear modeling and robust variance Poisson
regression
modeling
(adjusted
for
clustering
within
clinics) to determine relative risks for perinatal mortality
and fetal death by gestational age period. All analyses
were conducted using SAS software v9.2 (SAS Institute
Inc., Cary, NC, USA). WHO convened a technical consultation in November
2010 to discuss the implications of these findings and
released a statement [11]. The technical consultation
was unable to identify a specific cause for the increased
risk of perinatal mortality identified in the review. Pos-
sible factors discussed were differences in population,
baseline risk and gaps between visits in the second and
third trimester of pregnancy being too wide for timely
identification of fetal ill-health and appropriate responses, Discussion fetal death was not significantly different (Adjusted RR
1.26; 95% CI 0.91, 1.73). The risk of neonatal mortality
was not significantly different in low-risk women RR
1.35; 95% CI 0.87 – 2.10) but was significantly lower in
high-risk women (Adjusted RR 0.47; 95% CI 0.30, 0.72). We tested baseline maternal risk as an effect modifier,
however this was not significant for any model (data
not shown). fetal death was not significantly different (Adjusted RR
1.26; 95% CI 0.91, 1.73). The risk of neonatal mortality
was not significantly different in low-risk women RR
1.35; 95% CI 0.87 – 2.10) but was significantly lower in
high-risk women (Adjusted RR 0.47; 95% CI 0.30, 0.72). We tested baseline maternal risk as an effect modifier,
however this was not significant for any model (data
not shown). We conducted a secondary analysis of the WHO ANC
Trial dataset following the publication of a Cochrane
review that suggested the risk of perinatal mortality was
slightly higher in reduced-frequency, goal-oriented ante-
natal care packages for low-risk pregnancies in develop-
ing countries [4]. The analysis found that both high- and
low-risk women in the intervention group had an
increased relative risk of fetal death between 32 and 36 -
weeks gestation. While any association between ante-
natal care interventions and fetal death is cause for
concern, these findings are hypothesis-generating rather
than conclusive and must be interpreted with caution. Stratification of fetal deaths by gestational age (22 –
27, 28 – 31, 32 – 36 and >36 weeks) showed a statisti-
cally significant increase in fetal deaths between 32 to
36 weeks of gestation (Adj RR 2.24; 95% CI 1.42, 3.53),
the other periods were not significantly different be-
tween the comparison groups (Table 4). This pattern is
demonstrated in Figure 1, comparing hazard rates for
the reduced visits model and standard model. Results Reproductive Health 2013, 10:19 Vogel et al. Reproductive Health 2013, 10:19
http://www.reproductive-health-journal.com/content/10/1/19 Page 4 of 7 Table 2 Crude and adjusted relative risks of perinatal outcomes
Outcome
New model
Standard model
Crude
relative risk
(95% CI)†
Adjusted
relative risk
(95% CI)‡
N (%)
N (%)
Perinatal mortality
234/11672 (2.0)
190/11121 (1.7)
1.20 (1.04, 1.38)
1.18 (1.01, 1.37)*
Neonatal mortality
73/11511 (0.6)
71/11002 (0.7)
0.93 (0.80, 1.09)
0.95 (0.81, 1.11)
Fetal Death
161/11672 (1.4)
119/11121 (1.1)
1.30 (1.04, 1.62)
1.27 (1.03, 1.58)*
Fetal death <=36 weeks
122/11574 (1.1)
77/11048 (0.7)
1.63 (1.29, 2.08)
1.64 (1.27, 2.11)*
Fetal death >36 weeks
37/11574 (0.3)
42/11048 (0.4)
0.81 (0.52, 1.26)
0.78 (0.51, 1.21)
* RR estimates using robust variance Poisson modeling, taking clustering into account. † Model adjusted for risk strata only. ‡ Multivariate model adjusted for risk strata, smoking, education less than primary, hospital admission in last pregnancy, previous surgery on reproductive tract,
late booking, vaginal bleeding in the first trimester, age, previous low birth weight, parity. Table 2 Crude and adjusted relative risks of perinatal outcomes † Model adjusted for risk strata only. ‡ Multivariate model adjusted for risk strata, smoking, education less than primary, hospital admission in last pregnancy, previous surgery on reproductive tract,
late booking, vaginal bleeding in the first trimester, age, previous low birth weight, parity. Results In the low maternal risk group, the
new model was associated with an increase in the rela-
tive risk of fetal death at less than 36 weeks (Adjusted
RR 1.56; 95% CI 1.05, 2.31), however the overall risk of Table 1 Characteristics of high- and low-risk women stratified by model of antenatal care
Characteristics
High-risk women N = 6,160
Low-risk women N = 18,365
New model
Standard model
New model
Standard model
N = 3,287
N = 2,873
N = 9,281
N = 9,084
Demographics
Married or stable union
2,981 (90.8)
2,544 (89.1)
8,628 (93.0)
8.297 (91.4)
Education less than primary
720 (21.9)
591 (20.6)
1,484 (16.0)
1,291 (14.2)
Smoking during pregnancy
493 (15.0)
460 (16.0)
815 (8.8)
1,035 (11.4)
Substance abuse
64 (2.0)
41 (1.4)
-
-
Ratio persons per room in house (mean, SD)
2.5 (1.4)
2.4 (1.3)
2.4 (1.3)
2.4 (1.2)
Maternal age in years (mean, SD)
26.8 (7.0)
26.7 (7.1)
25.1 (5.5)
24.7 (5.5)
Outcome of previous pregnancy
Abortion
887 (27.0)
716 (24.9)
1,794 (19.3)
1,838 (20.2)
Fetal death
87 (2.7)
68 (2.4)
5 (0.05)
6 (0.07)
Low birth weight (<2500 g)
374 (11.4)
384 (13.4)
-
-
Neonatal death
61 (1.9)
45 (1.6)
5 (0.05)
2 (0.02)
Hospital admission for hypertension or pre-eclampsia/eclampsia
215 (6.5)
132 (4.6)
-
-
Reproductive History
Any previous low birth weight baby
534 (16.3)
537 (18.7)
181 (2.0)
165 (1.8)
Any previous surgery of reproductive tract
227 (6.9)
351 (12.2)
-
-
Any previous abortion
1,443 (50.4)
1,208 (48.9)
2,594 (36.8)
2,601 (38.8)
Any previous fetal death or neonatal loss
563 (19.7)
464 (18.8)
-
-
Present pregnancy
Isoimmunization Rh -ve
143 (4.4)
76 (2.7)
-
-
Vaginal bleeding first trimester
445 (13.5)
333 (11.6)
-
-
Date of LMP unknown
208 (6.3)
153 (5.3)
433 (4.7)
385 (4.2)
Nulliparous
973 (29.8)
903 (31.4)
2,739 (29.5)
2,755 (30.3)
Primigravida
491 (14.9)
463 (16.1)
2,891 (30.4)
2,890 (31.8)
Maternal height in cm
157.2 (6.8)
156.6 (6.6)
156.7 (5.5)
156.4 (6.5)
Maternal weight at first visit in kg
61.4 (13.7)
61.1 (13.2)
58.7 (12.1)
58.3 (11.8)
Duration of gestation at first visit, in weeks
15.6 (8.0)
15.7 (7.8)
16.8 (8.6)
16.1 (8.0)
Late booking for antenatal care (>28 weeks)
368 (11.2)
294 (10.2)
1,285 (13.9)
1.052 (11.6) Characteristics of high- and low-risk women stratified by model of antenatal care Vogel et al. Reproductive Health 2013, 10:19
http://www.reproductive-health-journal.com/content/10/1/19 Vogel et al. Model adjusted for risk strata only.
† Model in high-risk women adjusted for risk strata, married or stable union, hospital admission in last pregnancy, previous surgery on reproductive tract, previous
low birth weight and vaginal bleeding in the first trimester.
‡ Model in low-risk women adjusted for risk strata, married or stable union, smoking, education less than primary, late booking, age, primigravidity and
previous abortion. Discussion between antenatal visits (particularly between the second
and third trimester) or issues with the quality of ante-
natal care delivered. The number of antenatal visits can-
not necessarily act as a proxy for high quality care and
measuring the effective delivery of antenatal interven-
tions is difficult. Reducing the number of antenatal visits
may not necessarily translate into fewer visits of higher
quality for more women in low-resource settings. It may
also be that control clinics were providing other inter-
ventions that were of some benefit. † Model adjusted for risk strata only. trials on numbers of visits, making interpretation difficult
[4]. While the increase in perinatal mortality in the three
cluster-randomized trials was a consistent finding, only
the pooled estimate reached borderline statistical signifi-
cance [4]. This analysis has several limitations. Control clinics in
the WHO ANC Trial used a regimen based on local
protocols; it is therefore likely that visit frequency dif-
fered both within and between control clinics. 47.6% of
women in the new model and 19.6% of women in the
standard model had fewer than five visits, rendering dif-
ferences in visit frequency negligible for this sub-group. Cluster-randomized trials are an efficient study design
for trialing complex interventions in low-resource set-
tings, but it can be difficult to account for all potential
confounders
that
may
differ
between
clusters. For
example, antenatal care providers in the WHO ANC
Trial were known to differ between study sites and
clinics, as well as rates of smoking, education, hospital
admission, previous gynecological surgery and late book-
ing for antenatal care [10]. Complete blinding in trials of
antenatal care is impossible, increasing the chance of a
selection or information bias emerging. However, it is
reassuring that after adjustment for identifiable, poten-
tial risk factors, the crude and adjusted results are simi-
lar. It is also worth noting that in high-risk women the
increased fetal mortality was accompanied by a statisti-
cally significant drop in neonatal mortality (Adjusted RR
0.47; 95% CI 0.30, 0.72). While the reasons for these
findings are unclear, it could be secondary to the higher
number of fetal deaths during pregnancy. Women in these countries differed greatly in baseline
health risks and health resource availability, making com-
parisons problematic. Additionally, the 1996 Zimbabwe
trial and WHO ANC Trial were in mostly urban settings
[10,13], while the 2007 Zimbabwe trial was rural [14]. Discussion The Cochrane review included data from developed and
developing countries and the reviewers acknowledged that
there is considerable variation within and between these Table 3 Crude and adjusted relative risks of fetal and neonatal outcomes stratified by risk and model of antenatal care
High maternal risk group (n = 5,125)
End points
New model
Standard model
Crude relative risk (95% CI)*0
Adjusted relative risk (95% CI)*†
N (%)
N (%)
Perinatal mortality
91/2713 (3.35)
71/2412 (2.94)
1.00 (0.85 – 1.17)
0.94 (0.79 – 1.12)
Fetal death
65/2713 (2.40)
36/2412 (1.49)
2.12 (1.53 - 2.94)
1.78 (1.33 – 2.39)
Fetal death with GA <=36 weeks
51/2713 (1.9)
26/2412 (1.1)
1.77 (1.48 – 2.10)
1.48 (1.17 – 1.89)
Neonatal deaths
26/2648 (1.0)
35/2376 (1.5)
0.48 (0.31 – 0.72)
0.47 (0.30 – 0.72)
Low maternal risk group (n=17,668)
End points
New model
Standard model
Crude relative risk (95% CI)*0
Adjusted relative risk (95% CI)*‡
N (%)
N (%)
Perinatal mortality
143/8959 (1.60)
119/8709 (1.37)
1.14 (0.90, 1.44)
1.24 (0.95, 1.63)
Fetal death
96/8959 (1.07)
83/8709 (0.95)
1.18 (0.91 – 1.52)
1.26 (0.91 – 1.73)
Fetal death with GA <=36 weeks
71/8959 (0.8)
51/8709 (0.6)
1.48 (1.08 – 2.04)
1.56 (1.05 – 2.31)
Neonatal deaths
47/8863 (0.5)
36/8626 (0.4)
1.38 (0.89 – 2.14)
1.35 (0.87 – 2.10)
* Relative risks adjusted for clustering within clinics. 0 Model adjusted for risk strata only. † Model in high-risk women adjusted for risk strata, married or stable union, hospital admission in last pregnancy, previous surgery on reproductive tract, previous
low birth weight and vaginal bleeding in the first trimester. ‡ Model in low-risk women adjusted for risk strata, married or stable union, smoking, education less than primary, late booking, age, primigravidity and
previous abortion. Vogel et al. Reproductive Health 2013, 10:19
http://www.reproductive-health-journal.com/content/10/1/19 Page 5 of 7 Page 5 of 7 Table 4 Relative risk of fetal death by gestational age Table 4 Relative risk of fetal death by gestational age
Gestational
age
New model
Standard
model
Crude relative
risk (95% CI)†
P-value
N (%)
N (%)
22 – 27
52/68 (76.5)
36/54 (66.7)
1.12 (0.98 – 1.29)
0.086
28 – 31
26/77 (33.8)
20/83 (24.1)
1.09 (0.78 – 1.51)
0.62
32 – 36
42/714 (5.9)
20/670 (3.0)
2.24 (1.42 – 3.53)
0.0005
> 36
37/10710 (0.4)
42/10237 (0.4)
0.81 (0.52 – 1.26)
0.35
† Model adjusted for risk strata only. Details of ethics approval 1. Clement S, Candy B, Sikorski J, Wilson J, Smeeton N: Does reducing the
frequency of routine antenatal visits have long term effects? Follow up
of participants in a randomised controlled trial. Brit J Obstet Gynaec 1999,
106:367–370. The WHO Antenatal Care Trial was approved by the
Scientific and Ethical Review Group of the UNDP/
UNFPA/WHO/World Bank Special Programme on Re-
search, Development, and Research Training in Human
Reproduction, the WHO Secretariat Committee for Re-
search into Human Subjects, and the Institutional Re-
view Boards of the individual participating centres and
corresponding health authorities of the regions where
the trial was implemented. 2. Binstock M, Woldetsadik G: Alternative prenatal-care - impact of reduced
visit frequency focused visits and continuity of care. J Reprod Med 1995,
40:507–512. 2. Binstock M, Woldetsadik G: Alternative prenatal-care - impact of reduced
visit frequency focused visits and continuity of care. J Reprod Med 1995,
40:507–512. 3. Mcduffie R, Bischoff K, Beck A: Does reducing the number of prenatal
office visits for low-risk women result in increased use of other medical
services? Obstet Gynecol 1997, 90:68–70. 4. Dowswell T, Carroli G, Duley L, Gates S, Gulmezoglu AM, Khan-Neelofur D,
Piaggio GGP: Alternative versus standard packages of antenatal care for
low-risk pregnancy. Cochrane Db Syst Rev 2010:CD000934. 5. Bergsjo P, Villar J: Scientific basis for the content of routine antenatal
care. 2. Power to eliminate or alleviate adverse newborn outcomes;
some special conditions and examinations. Acta Obstet Gynecol Scand
1997, 76:15–25. Funding
JPV g
JPV is supported by an Australian Postgraduate Award and the A & A Saw
Scholarship. The WHO Antenatal Care Trial was supported by the UNDP/
UNFPA/WHO/World Bank Special Programme of Research, Development and
Research Training in Human Reproduction of WHO. Additional support was
provided by: Municipal Government, City of Rosario, Argentina; Ministry of
Health, Cuba; National Institute of Public Health, Mexico; The Population
Council—Regional Office for Latin America and the Caribbean; Ministry of
Health, Saudi Arabia; Swedish Agency for Research Cooperation with
Developing Countries; Ministry of Public Health and Faculty of Medicine,
Khon Kaen University, Thailand; Department for International Development,
UK; Mother Care—John Snow Inc; National Institute for Child Health and
Human Development, National Institutes of Health, USA; and The World
Bank. For the preparatory phase: University of Western Ontario, Department
of Epidemiology and Biostatistics, Canada; National Institute of Public Health,
Norway; United Nations Development Programme, and the University of
Uppsala, Department of Obstetrics and Gynaecology, Sweden. Acknowledgements
J
N l
(U Jim Neilson (University of Liverpool, UK), Lelia Duley (University of
Nottingham, UK), Franz Majoko (Singleton Hospital, Swansea, UK) for
reviewing the manuscript. Authors’ contributions This analysis was conceived of by several authors (JS, AMG, TD, GC, HB, PL,
GP, OTO) following the WHO technical consultation in November 2010. NH
performed the analysis. The draft manuscript was written by JV. All authors
contributed to and approved interpretation of the findings and the final
manuscript. The views contained herein are those of the named
authors only. Conclusion
h
d This secondary analysis of the WHO ANC Trial data
indicates there is an appreciable increased risk of fetal
death at 32 to 36 weeks gestation for women receiving
the goal-oriented, reduced frequency antenatal care
package. While it is plausible that this was due to re-
duced antenatal visits, differences in settings, popula-
tions or content and quality of care as well as the timing
of visits could also be playing a role; care must be taken
in comparing findings across varied settings, countries
and patient groups. Vogel et al. Reproductive Health 2013, 10:19
http://www.reproductive-health-journal.com/content/10/1/19 Vogel et al. Reproductive Health 2013, 10:19
http://www.reproductive-health-journal.com/content/10/1/19 Page 6 of 7 Page 6 of 7 women receive little or no antenatal care [15], and as
such focusing on the provision of effective antenatal care
at a reduced frequency seems advantageous. On the
other hand, additional antenatal visits may serve to
reinforce maternal education and compliance, or provide
an opportunity for screening and treatment of a condi-
tion that had been missed, omitted or deteriorated since
the previous visit. The reduced visit model has been
adapted and implemented in the north-east region of
Thailand and is currently being scaled up to the whole
country. The model has been modified with an added visit
around 20 weeks for an ultrasound scan. Initial monitor-
ing of perinatal mortality has not revealed any adverse
outcomes in the north-east region. (P Lumbiganon, per-
sonal communication). 2.
Binstock M, Woldetsadik G: Alternative prenatal-care - impact of reduced
visit frequency focused visits and continuity of care. J Reprod Med 1995,
40:507–512. 3.
Mcduffie R, Bischoff K, Beck A: Does reducing the number of prenatal
office visits for low-risk women result in increased use of other medical
services? Obstet Gynecol 1997, 90:68–70. Discussion This
heterogeneity needs to be considered when interpreting the
marginal increase in perinatal mortality on pooled analysis. Furthermore, data from the two Zimbabwean trials
showed that overall, women had similar numbers of
visits
between
intervention
and
controls
groups,
suggesting that any increase in the risk of fetal death in
the intervention groups may be due to factors other
than the number of antenatal visits [13,14]. Secondary
analyses of the two Zimbabwean trials have not yet
been published. In retrospect, the disparities in fetal mortality could
have been more thoroughly investigated and given
greater prominence in the published findings of the
WHO ANC Trial. One possible explanation for these
findings is that lower number of visits or other elements
of the new antenatal care package decrease the identifi-
cation of fetuses at risk of fetal death between 32 and
36 weeks. This may be attributable to prolonged gaps In many low-income countries, where resources are
limited, antenatal care coverage is poor and many Figure 1 Fetal death hazard rate for gestational weeks 22 – 41. Figure 1 Fetal death hazard rate for gestational weeks 22 – 41. Vogel et al. Reproductive Health 2013, 10:19
http://www.reproductive-health-journal.com/content/10/1/19 Author details
1 1School of Population Health, Faculty of Medicine, Dentistry and Health
Sciences, University of Western Australia, 35 Stirling Highway, Crawley 6009,
Australia. 2UNDP/UNFPA/UNICEF/WHO/World Bank Special Programme of
Research, Development and Research Training in Human Reproduction
(HRP), Department of Reproductive Health and Research, World Health
Organization, Avenue Appia 20, Geneva CH-1211, Switzerland. 3Department
of Women’s Health, University of Liverpool, Liverpool Women’s Hospital,
Crown Street, Liverpool 8, UK. 4Centro Rosarino de Estudios Perinatales
(CREP), Moreno 878, Rosario 2000, Argentina. 5King Saud bin Abdulaziz
University for Health Sciences, College of Medicine, Jeddah 21423, Saudi
Arabia. 6Department of Obstetrics and Gynaecology, Faculty of Medicine,
Khon Kaen University, Khon Kaen 40002, Thailand. 7Statistika Consultoria, São
Paulo, Brazil, Divonne-les-Bains, France. 8Department of Obstetrics and
Gynaecology, Olabisi Onabanjo University Teaching Hospital, P.M.B. 2001,
Sagamu, Nigeria. It is critical to monitor maternal, fetal and neonatal
indicators when implementing antenatal care protocols
in any setting. In settings where few women attend ante-
natal care, achieving four antenatal care visits with the
full complement of targeted, evidence-based interven-
tions at each visit is still meaningful. These programs
should be monitored not only in terms of number of
visits but also in terms of actual care delivered and
clinically meaningful maternal and perinatal outcomes. Further research on rates of fetal death in centers using
reduced antenatal care packages would also be of
benefit. Received: 30 January 2013 Accepted: 18 February 2013
Published: 12 April 2013 1.
Clement S, Candy B, Sikorski J, Wilson J, Smeeton N: Does reducing the
frequency of routine antenatal visits have long term effects? Follow up
of participants in a randomised controlled trial. Brit J Obstet Gynaec 1999,
106:367–370. Vogel et al. Reproductive Health 2013, 10:19
http://www.reproductive-health-journal.com/content/10/1/19 Vogel et al. Reproductive Health 2013, 10:19
http://www.reproductive-health-journal.com/content/10/1/19 6. Carroli G, Rooney C, Villar J: How effective is antenatal care in preventing
maternal mortality and serious morbidity? An overview of the evidence. Paediatr Perinat Epidemiol 2000, 15:1–42. 7. de Onis M, Villar J, Gulmezoglu M: Nutritional interventions to prevent
intrauterine growth retardation: evidence from randomized controlled
trials. Eur J Clin Nutr 1998, 52:S83–S93. 8. Gulmezoglu M, de Onis M, Villar J: Effectiveness of interventions to
prevent or treat impaired fetal growth. Obstet Gynecol Surv 1997, 52:139. 9. Villar J, Gülmezoglu A: Nutritional and antimicrobial interventions to
prevent preterm birth: an overview of randomized controlled trials. Obstet Gynecol Surv 1998, 53:575–585. 10. Villar J, Ba'aqeel H, Piaggio G, Lumbiganon P, Miguel Belizan J, Farnot U, Al-
Mazrou Y, Carroli G, Pinol A, Donner A, Langer A, Nigenda G, Mugford M,
Fox-Rushby J, Hutton G, Bergsjo P, Bakketeig L, Berendes H, Garcia J, WHO
Antenatal Care Trial Research Group: WHO antenatal care randomised trial
for the evaluation of a new model of routine antenatal care. Lancet 2001,
357:1551–1564. 10. Villar J, Ba'aqeel H, Piaggio G, Lumbiganon P, Miguel Belizan J, Farnot U, Al-
Mazrou Y, Carroli G, Pinol A, Donner A, Langer A, Nigenda G, Mugford M,
Fox-Rushby J, Hutton G, Bergsjo P, Bakketeig L, Berendes H, Garcia J, WHO
Antenatal Care Trial Research Group: WHO antenatal care randomised trial
for the evaluation of a new model of routine antenatal care. Lancet 2001,
357:1551–1564. 11. World Health Organization: WHO Statement on Antenatal Care. Switzerland:
World Health Organization; 2010. 11. World Health Organization: WHO Statement on Antenatal Care. Switzerland:
World Health Organization; 2010. 12. Villar J, Bakketeig L, Berendes H: 11: The WHO antenatal care randomised
controlled trial: rationale and study design. Paediatr Perinat Epidemiol
2001, 12:27–58. 13. Munjanja S, Lindmark G: Randomised controlled trial of a reduced-visits
programme of antenatal care in Harare, Zimbabwe. Lancet 1996,
348:364–369. 14. Majoko F, Munjanja S, Nyström L, Mason E, Lindmark G: Randomised
controlled trial of two antenatal care models in rural Zimbabwe. BJOG
2007, 114:802–811. 15. United Nations: The Millennium Development Goals Report. 2011 [http://
www.un.org/millenniumgoals/]. 15. United Nations: The Millennium Development Goals Report. 2011 [http://
www.un.org/millenniumgoals/]. doi:10.1186/1742-4755-10-19
Cite this article as: Vogel et al.: Antenatal care packages with reduced
visits and perinatal mortality: a secondary analysis of the WHO
Antenatal Care Trial. Reproductive Health 2013 10:19. Competing interests
Th
h The authors report on competing interest. Several of the authors (GP, GC,
PL, HB) were also co-authors in the original WHO Antenatal Care Trial. Page 7 of 7 Page 7 of 7 doi:10.1186/1742-4755-10-19
Cite this article as: Vogel et al.: Antenatal care packages with reduced
visits and perinatal mortality: a secondary analysis of the WHO
Antenatal Care Trial. Reproductive Health 2013 10:19. Vogel et al. Reproductive Health 2013, 10:19
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Applying Google Trends web-analytic tools in socio-humanitarian and library studies
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Applying Google Trends web-analytic tools in socio-humanitarian and library studies
S. V. Sokolov Institute of Scientific Information on Social Sciences, Moscow, Russia; e-mail: beholder73@gmail.com The article «Application of Google Trends web-analytic tools in socio-humanitarian and library studies» examines the
problems of using web-statistical tools by foreign and domestic experts in the field of library and social sciences. The
article is divided into five parts. The first one sets the objective and main tasks of the work, gives a general description
of both Google services as a whole, and the chosen research method, discusses the history of its creation and imple-
mentation. The second part analyzes the appeal to this method by experts of the International Library Association,
demonstrates this method at the IBA session in Kuala Lumpur in 2018. The third part demonstrates the method use
in modern socio-humanitarian studies presented in the international full-text Springer Databases Link, Oxford University
Press and Sage. The paper considers the works of German and Russian authors actively using this method in library
studies. The fourth part describes the capabilities and limitations of the Google Trends toolkit compared to Yandex-
Wordstat, its Russian counterpart. The fifth part presents the main conclusions and substantiates the significance of
this method for sociological research, sets out prospects for the further use of the Google Trends service to solve cur-
rent library problems. Keywords: web analytics, Google Trends, search query, library marketing, library terminology Keywords: web analytics, Google Trends, search query, library marketing, library terminology
Citation: Sokolov S. V. Applying Google Trends web-analytic tools in socio-humanitarian and library studies. B
sphere. 2018-4. P. 3–9. DOI: 10.20913/1815-3186-2018-4-3-9. Citation: Sokolov S. V. Applying Google Trends web-analytic tools in socio-humanitarian and library studies. Bibl
sphere. 2018-4. P. 3–9. DOI: 10.20913/1815-3186-2018-4-3-9. Р приложение, запущенное в мае 2006 г., показывает
степень популярности ключевых слов среди пользо-
вателей поисковой системы Google по различным
тематикам, на различных языках и в различных ре-
гионах мира. Инструментарий выявления быстро раз-
вивающихся пользовательских запросов адресован
прежде всего маркетологам, журналистам и медий-
ным работникам. Достоинство этого сервиса не только
в выявлении наиболее устойчивых популярных за-
просов в целом, что можно получить и с помощью
большинства традиционных интернет-поисковиков,
но и в получении статистики за короткий промежуток
времени – последние 24 часа или заданный вре-
менной отрезок в прошлом. 5 августа 2008 г. Институт научной информации по общественным наукам РАН,
Москва, Россия; e-mail: beholder73@gmail.com Статья посвящена интеллектуальной поисково-аналитической системе Google Trends, ее использованию как
в рамках научных исследований, проводимых Международной федерацией библиотечных ассоциаций (ИФЛА),
так и в работах зарубежных авторов, статистика по которым была получена при анализе полнотекстовых баз
данных Springer Link, Oxford University Press и SAGE. Рассмотрены различные варианты применения этого инст-
рументария отечественными авторами, дано сравнительное исследование Google Trends и его отечественного
аналога «Яндекс. Вордстат». Ключевые слова: веб-аналитика, Google Trends, поисковый запрос, библиотечный маркетинг, библиотечная тер-
минология Для цитирования: Соколов С. В. Применение веб-аналитического инструментария Google Trends в социогума-
нитарных и библиотековедческих исследованиях // Библиосфера. 2018. № 4. С. 3–9. DOI: 10.20913/1815-3186-
2018-4-3-9. Методология НИР УДК 02:001.891:004.738.5
ББК 78.0+78.6
DOI 10.20913/1815-3186-2018-4-3-9 БИБЛИОСФЕРА, 2018, № 4, с. 3–9 / BIBLIOSPHERE, 2018, no. 4, pp. 3–9 БИБЛИОСФЕРА, 2018, № 4, с. 3–9 / BIBLIOSPHERE, 2018, no. 4, pp. 3–9 Applying Google Trends web-analytic tools in socio-humanitarian and library studies
S. V. Sokolov Google
запустил Google Insights for Search, более сложную
и передовую службу отображения тенденций поиска, оссийские и зарубежные поисковые системы
в настоящее время являются не только агре-
гаторами информации, снабженными продви-
нутым справочно-поисковым аппаратом, но и мощ-
ными статистическими и аналитическими системами. Так, компания Google как автор многих научных, об-
разовательных и культурных проектов предлагает
более 40 служб и инструментов для различных нужд,
которые могут быть использованы специалистами
самых разных направлений, в том числе и в библио-
течно-информационной работе. Р Рассмотрим опыт использования набирающего
популярность сервиса Google Trends, принадлежа-
щего, соответственно, компании Google, в социогу-
манитарных исследованиях и исследованиях, связан-
ных с библиотечной практикой. Это публичное веб- 3 МЕТОДОЛОГИЯ НИР / METODOLOGY OF RESEARCH WORK которая в 2012 г. была объединена с Google Trends. Google Hot Trends является дополнением к Google
Trends, которое отображает 20 «горячих», то есть
с наиболее быстрым ростом, поисковых запросов за
последний час и используется пока лишь в некото-
рых регионах, например, в Соединенных Штатах Аме-
рики. С начала 2016 г. с главной страницы Google
Trends было убрано годовое отображение наиболее
рейтинговых, популярных запросов в интернете и вве-
дено отображение объема поисковых запросов за
последние 24 часа. Этот переход свидетельствует
о большем внимании современных пользователей ин-
тернета к текущим краткосрочным явлениям и про-
цессам. Такое «ускорение времени», как в глобаль-
ных информационных сетях, так и в отдельных биб-
лиотечно-информационных сервисах, фиксируют и ве-
дущие современные исследователи технологий тайм-
менеджмента, например, Мэгги Фарелл из Универси-
тета Клемсона. Ее статья «Управление временем» [19]
в «Журнале библиотечного администрирования» (Jour-
nal of Library Administration) стала в 2018 г. лидером
цитирования среди статей по тематике библиотечно-
информационных исследований в приложении Hot
Articles, размещенном в том числе на центральной
странице электронной библиотеки ГПНТБ России [15]. данным («2.2.2») и по способствованию большему до-
ступу населения к интернет-ресурсам («2.3.1») также
показывают, что существует определенная перспек-
тива развития стандартов по линии открытых дан-
ных в открытых информационных сетях. Как показывают программы конференций ИФЛА
(2010–2017 гг.), проблематика использования серви-
сов Google библиотеками до последнего времени на
уровне отдельных докладов конференций не стави-
лась вообще. В докладе на секции ИФЛА 2016 г. в Коломбо из исследователей Google Trends упоми-
нается только Кай Кахилл (Kay Cahill) [16]. Тем не ме-
нее статистика поисковых запросов в среде Google
к ИФЛА-публикациям применялась, например, в го-
довых отчетах для оценки деятельности организа-
ции. Так, одним из пяти статистических показателей
деятельности ИФЛА в 2017 г. Исследование Google Trends в рамках Международной
федерации библиотечных ассоциаций )
«Взаимодействие в учебном классе – формы
Google и библиотечные руководства» («Engaging the
Classroom – Google Forms and Library Guides»). Эффективность использования инструментов веб-ста-
тистики библиотечными учреждениями и научными
библиотечными институтами показал возрастающий
интерес к ним со стороны Международной библио-
течной ассоциации. Еще в 2006 г., на момент запуска
Google Trends, ИФЛА в публикации Кристины Кунтц
«Использование данных клиентов для улучшения стра-
тегического планирования маркетинга и принятия ре-
шений» [23] показала эффективность использования
информационных баз данных пользователей порта-
лов Amazon.com и Google.com. Длительное время
внедрение нового веб-статистического инструмента-
рия тормозилось в том числе отсутствием необхо-
димых международных библиотечных стандартов на
использование статистики обращений пользователей
в открытом вебе. Так, в руководстве для библиотек
секции по комплектованию и развитию коллекций
ИФЛА августа 2012 г. «Ключевые проблемы развития
коллекций электронных ресурсов: руководство для
библиотек» важнейшим обоснованием необходимости
использования того или иного ресурса было названо
соответствие инструмента статистики принятым стан-
дартам ICOLC (International Coalition of Library Con-
sortia)’s Guidelines for Statistical Measures of Usage of
Web-Based Information Resources и/или SUSHI (Stan-
dard Usage Statistics Harvesting Initiative) [7]. «Отслеживание и прогнозирование роста ме-
дицинской информации с использованием наукомет-
рических методов и Google Trends» («Tracking and
Predicting Growth of Health Information Using Sciento-
metrics Methods and Google Trends»). В последнем докладе [28] авторами из румын-
ского Трансильванского университета была показа-
на возможность коррелированного использования
сетевого ресурса Google Trends вместе с наукомет-
рической базой Web of Science для отслеживания
и прогнозирования информации о медицине для биб-
лиотек в разных странах мира. В последнем докладе [28] авторами из румын-
ского Трансильванского университета была показа-
на возможность коррелированного использования
сетевого ресурса Google Trends вместе с наукомет-
рической базой Web of Science для отслеживания
и прогнозирования информации о медицине для биб-
лиотек в разных странах мира. Applying Google Trends web-analytic tools in socio-humanitarian and library studies
S. V. Sokolov была статистика по
81,246 обращениям к этим публикациям через брау-
зер Google [21]. На последней сессии ИФЛА в Куала-Лумпуре
в 2018 г. виден отчетливый интерес как в целом
к веб-сервисам Google, так и отдельно к сервису
Google Trends. На последнем саммите ИФЛА были
представлены следующие доклады о сервисах Google,
а также спонсируемые Google [17]: «Планы для совместимой архитектуры данных
и протоколов OAIS» («Plans for an OAIS Interoperable
Data Architecture and Protocols»). Google Trends в работах зарубежных авторов
по статистике международных полнотекстовых
баз данных Отдельных монографических исследований, подробно
анализирующих возможности сервиса Google Trends
для решения библиотечных задач, пока не существует. Можно выделить главу исследования Бена Шоуверса
«Библиотечная аналитика и количественные показа-
тели: использование данных для принятия решений
и предоставления услуг» [15]. В этой работе в части
«Внешний инструментарий исследования поведения
посетителей публичного информационно-культурного
учреждения» подробно описывается именно Google
Trends с примерами соотношения популярности по-
нятий «Британский музей» и «Терракотовая армия»,
чтобы сделать вывод о популярности определенной
экспозиции среди всех других выставок музея. В Стратегическом плане ИФЛА на 2016–2021 гг. [20]
зафиксирована необходимость продвижения стандар-
тов для поддержки библиотек, обслуживающих мест-
ное население, а также разработка стандартов для
сбора и сохранения цифрового контента в его разно-
образных формах. Стратегические направления этого
плана по обеспечению большего доступа к открытым 4 С. В. Соколов, 2018, № 4, с. 3–9 / S. V. Sokolov, 2018, no. 4, pp. 3–9 Т а б л и ц а 2
Научные исследования по сервису Google Trends
в коллекции электронных журналов издательства SAGE [29]
T a b l e 2
Research on the Google Trends service in the collection
of electronic journals published by SAGE [29]
Дисциплина
Количество статей
(всего 175)
Исследования средств массовой
информации и коммуникации
18
Социология
9
Политика и международные
отношения
8
Исследования культуры
7
Менеджмент и управление
5
Инженерное дело
и программирование
3
Материаловедение
3
Криминология
2
География
2
Итого
44 Т а б л и ц а 2 Полная картина использования инструментария
Google Trends в современных социогуманитарных
и конкретно в библиотековедческих исследованиях
становится видна при анализе всей современной на-
учной периодики, доступной через международные
полнотекстовые базы данных Oxford University Press,
SAGE и Springer Link. Во всех трех базах доступно чет-
кое отнесение статьи, где хотя бы раз упоминается
искомый термин «Google Trends», к определенной
научной дисциплине или к ряду научных направле-
ний. Чистота подсчета использования этого термина
в научной периодике определяется устойчивостью
самого выражения. При взятии выражения в кавычки
гарантируется наибольшая релевантность поиска. В приведенных ниже трех таблицах представлены
данные по общему количеству упоминаний термина,
присутствию этого термина в наиболее крупных на-
учных рубриках и более подробно – в социально-гу-
манитарных исследованиях. Как видно из приведенной статистики (табл. 1–3),
использование методики Google Trends максимально
задействовано в исследованиях, связанных с меди-
циной, а также в сфере информационных технологий. Google Trends и «Яндекс. Вордстат» На основе сопоставления Google Trends и наиболее
близкого его российского аналога – «Яндекс. Ворд-
стат» – мы составили сравнительную таблицу двух
веб-статистических инструментов (табл. 4). (
)
Сервис Google Trends предполагает проведение
«вертикального поиска» по 24 тематическим рубри-
кам и 10–30 подрубрикам. К сожалению, они пере-
крывают наиболее общие потребительские запросы,
такие как «автотранспортные средства» или «бизнес
и промышленность», и не могут задать область поиска
по узкой научной, социальной и тем более библиоте-
коведческой проблематике. Из рубрики «Наука» сер-
вис выделяет всего 11 подрубрик, связанных только
с естественно-научными дисциплинами, научным обо-
рудованием и научными организациями. Многознач-
ные слова, употребляемые как в библиотековедении,
так и в других дисциплинах, можно искать, обратив-
шись к меткам, всплывающим при наборе слова в по-
исковой строке. Так, слово «библиотека» можно ис-
кать с меткой: Кроме определения популярности ключевых слов
развивается и направление использования сервиса
Google Trends для анализа потребительского (чита-
тельского) спроса. Авторы фундаментального немец-
кого «Справочника библиотечного и информацион-
ного знания», изданного в 2013 г., показали в главе
«Исследование востребованности электронных ре-
сурсов», как с помощью Google Trends можно опреде-
лить стремительный взлет среди немецких интер-
нет-пользователей, а значит, пользователей элек-
тронных ресурсов библиотек, интереса к гибридным
и электронным автомобилям [23]. «поисковый запрос»; «тема»; «тема»; «программирование»; «система публичных библиотек» Список для выбора различных меток задается ав-
томатически, в зависимости от количества тем и руб-
рик, аффилируемых с ключевым словом в поисковой
системе. Для исключения искусственного ограниче-
ния области поиска рекомендуется искать узкие спе-
циализированные ключевые слова сразу по всем те-
матическим рубрикам. МЕТОДОЛОГИЯ НИР / METODOLOGY OF RESEARCH WORK МЕТОДОЛОГИЯ НИР / METODOLOGY OF RESEARCH WORK взаимодополняющих друг друга при решении раз-
личных задач, связанных с исследованиями библио-
тек и открытых баз данных [9]. Необходимо отметить, что именно немецкоязыч-
ная научная периодика образует наибольший регио-
нальный сегмент использования технологии Google
Trends. По мнению известного немецкого исследователя
из Фрейбургского университета современных СМИ,
изложенному в монографии «Цифровой мир и науч-
ная библиотека», в начале своего появления Google
Trends являлся скорее не сервисом, а внутренним
кодом в поисковой машине Google для ранжирова-
ния наиболее популярных ключевых слов [32]. Авторы
исследования «Эволюция информационной инфра-
структуры: библиотеки и наука» [25] также опреде-
ляют поле применимости сервиса Google Trends как
возможность отслеживания популярности научных
неологизмов (например, популярность словосочета-
ний «облачные вычисления», «распределенные вы-
числения» или «большие данные»). Использование Google Trends в трудах российских
исследователей Российские исследователи используют Google Trends
преимущественно для анализа маркетинговой ин-
формации [1, 2], но в последние годы этот анали-
тический инструмент применяется для изучения об-
щественных и политических процессов [4, 6, 13]. Наи-
большее количество исследований сервисов Google
использует их как инструмент веб-статистики, на-
правленный на продвижение библиотечных сайтов
(SEO). Так, Л. Б. Шевченко из ГПНТБ СО РАН рас-
сматривает библиотеку с точки зрения цифрового
маркетинга и предлагает шесть важных требований
к организации библиотечного сайта и пути улучшения
позиционирования библиотеки в интернете [14]. Про-
блемы использования Google Trends в библиотечном
деле активно исследует доктор педагогических наук
Н. С. Редькина [10–12]. Инструментарий Google Trends
применяет в своем исследовании популярности тра-
диционных источников информации в виде печатных
книг доктор филологических наук Челябинского госу-
дарственного университета М. В. Загидуллина [3]. Необходимо отметить, что ряд отечественных иссле-
дователей предпочитает использовать вместо Google
Trends российские аналоги, особенно в области биб-
лиотечной статистики [5], при анализе читательской
аудитории и эффективности работы сайта библио-
теки [8]. Google Trends в работах зарубежных авторов
по статистике международных полнотекстовых
баз данных Доля социально-гуманитарных наук в общем коли-
честве исследований составляет 1/3 в журналах из-
дательства Oxford University Press, 1/4 – в базе SAGE
и всего 1/17 в коллекции электронных журналов из-
дательства Springer Link. Колебания этой величины
связаны с общей структурой той или иной коллекции,
но тем не менее показывают и слабое использо-
вание этой методики науками социального профиля. Из всех исследуемых баз научных статей отдельно Т а б л и ц а 3
Научные исследования по сервису Google Trends
в коллекции электронных журналов издательства
Springer Link [31]
T a b l e 3
Research on the Google Trends service in the collection
of electronic journals published by Springer [31]
Тематика журналов
Количество статей
Информатика Computer Science
297
Бизнес и управление Business
and Management
199
Медицина и здравоохранение
Medicine & Public Health
92
Машиностроение Engineering
66
Социальные науки Social Sciences
56
Library Science
2
Printing and Publishing
2 Т а б л и ц а 1 Научные исследования по сервису Google Trends
в коллекции электронных журналов издательства
Oxford University Press [27] Научные исследования по сервису Google Trends
в коллекции электронных журналов издательства
Oxford University Press [27] T a b l e 1
Research on the Google Trends service in the collection
of electronic journals published by Oxford University Press [27]
Тематика журналов
Количество статей
Медицина и здоровье
93
Право
1
Биология
9
Математика (биоматематика)
5
Публичная политика
1
Проблемы зависимостей
5
Гендерные проблемы
1
Геронтология
1
Здравоохранение
5
Экономика здоровья
5
История
1
Итого
130 Research on the Google Trends service in the collection
of electronic journals published by Oxford University Press [27] проблемы библиотековедения выделены в двух ста-
тьях издательства Springer Link: об исследовании тех-
нологии контент-маркетинга в интернет-продвижении
интереса к художественной литературе [22] и по ис-
пользованию сервиса Google Trends в выявлении по-
пулярности открытых научных данных в странах Аф-
рики южнее Сахары [26]. 5 Заключение Такой многогранный аналитический ресурс, как Google
Trends, только входит в инструментарий специалистов
по общественным наукам. Активное использование
этого инструмента в статьях зарубежных авторов,
в том числе представленных на последней сессии
ИФЛА (Куала-Лумпур), а также в целом ряде иссле-
дований, выполненных в ГПНТБ СО РАН, показывает
растущий интерес к сервисам Google и со стороны
библиотечной науки. Российский аналог этого веб-
инструмента – «Яндекс. Вордстат» – пока не может
конкурировать с Google Trends как по сервисным
опциям, так и по географическому охвату. Возмож-
ности статистических онлайн-сервисов позволяют
представить краткосрочный и долгосрочный анализ
изменения общественного интереса к исследуемой
проблематике и, соответственно, сделать прогноз от-
носительно всех рассматриваемых вопросов. Оценивая перспективы использования статисти-
ческих веб-сервисов, заметим, что сервис Google
Trends позволит исследовать: читательский спрос для составления планов
комплектования и уточнения охвата фонда; читательский спрос для составления планов
комплектования и уточнения охвата фонда; Ряд авторов проводит сравнительные исследо-
вания отечественных и зарубежных инструментов, 6 С. В. Соколов, 2018, № 4, с. 3–9 / S. V. Sokolov, 2018, no. 4, pp. 3–9 Т а б л и ц а 4
Сравнение веб-аналитических инструментов Google Trends и «Яндекс. Вордстат»
T a b l e 5
Comparing web analytic tools of Google Trends and Yandex Wordstat
Google Trends
«Яндекс. Вордстат»
При запросах на английском языке обладает технологией
контекстно-зависимого поиска
Требует дополнительного введения всех возможных ключе-
вых слов для полного охвата семантического поля запроса. Заключение Есть инструмент «Запросы, похожие на...»
Возможность оформления подписки на отслеживаемый
запрос
Обладает возможностью сохранения истории запросов
Не требует регистрации
Требует регистрации
Ищет специальные слова на европейских языках
Ищет преимущественно в русскоязычном сегменте интернета
Адекватно ищет по всем регионам мира
Наиболее эффективен в поиске по России и странам ближнего
зарубежья
Относительный показатель популярности слова (числовое
значение высчитывается относительно пика популярности
самого слова)
Абсолютный показатель популярности слова (числовое зна-
чение равно количеству запросов)
Способен производить поиск по фото- и видеообразам (ви-
деохостингу YouTube и картинкам), а также по персоналиям
Ранжирует поиск по типу устройства, с которого велся поиск
(телефоны, планшеты, стационарные устройства)
Обладает возможностью «вертикального поиска»
Не имеет поискового рубрикатора или классификатора
Обладает расширенными возможностями визуализации
полученных данных:
позволяет поделиться результатами в пяти социальных
сетях;
позволяет встроить результаты в HTML-код своей стра-
ницы на мобильном устройстве или стационарном ком-
пьютере;
позволяет скачать результаты в виде Excel-таблицы
(файл .csv)
Отсутствует дополнительный вывод информации мониторинг региональных научных исследо-
ваний с отслеживанием трендов по популярности
научных запросов в открытом вебе; использован в исследовании, например, такой темы,
как «Библиотека в архитектурно-индустриальном ланд-
шафте региона». Приведенный список вариантов использования
этого метода далеко не полный. Зарубежные иссле-
дования различных сервисов веб-статистики пока-
зывают, что существует значительное количество
направлений их использования, которые могут быть
с успехом применены в постановке, рассмотрении
и решении актуальных социокультурных и библио-
тековедческих задач, стоящих перед российским ис-
следовательским сообществом. популярность библиотек и информационных
сервисов для составления их рейтинга; библиотечные проблемы региона по частотно-
сти запросов по проблемным библиотечным темати-
кам. Это даст нам инструментарий по определению
уровня социально-библиотечной культуры региона. Сервис располагает большим количеством до-
полнительных инструментов, список которых попол-
няется. Так, недавно введенный поиск по фото- и ви-
деообразам «Google Trends – образы» может быть ru/article/v/analiz-marketingovoy-informatsii-na-osnove-inst
rumentariya-publichnogo-web-prilozheniya-google-trends (дата
обращения 04.10.2018).
2. Борочкин А. А. Использование статистики поисковых
запросов в сети интернет для краткосрочного прогно- ru/article/v/analiz-marketingovoy-informatsii-na-osnove-inst
rumentariya-publichnogo-web-prilozheniya-google-trends (дата
обращения 04.10.2018). МЕТОДОЛОГИЯ НИР / METODOLOGY OF RESEARCH WORK МЕТОДОЛОГИЯ НИР / METODOLOGY OF RESEARCH WORK 23. Konrad U., Fühles-Ubach S., Seadle M. (eds.) Handbuch
Methoden der Bibliotheks- und Informationswissenschaft: Bib-
liotheks-, Benutzerforschung, Informationsanalyse. Berlin ;
Boston : Walter de Gruyter, 2013. URL: https://books.Google. ru/books?id=FXV7rK9xTtEC&pg=PA193&dq=bibliothek+Google
+trends&hl=de&sa=X&ved=0ahUKEwjMx877g8DdAhUPt4sKH
Tf2D28Q6AEIRzAH#v=onepage&q=bibliothek%20Google%20
trends&f=false (accessed 04.10.2018). зирования макроэкономических переменных // Деньги
и кредит. 2013. № 8. С. 27-32. р
3. Загидуллина М. В. Книга в медиасреде: исчезнет ли
слово? // Книга: Сибирь – Евразия : тр. I Междунар. науч. конгр. Новосибирск. 2016. Т. 3. С. 4–10. 4. Зиновьева Е. С. Воздействие компаний интернет-индуст-
рии на мировую политику // Вестник МГИМО-Универси-
тета. 2013. № 1. С. 43–47. 24. Koontz C. Using customer data for improved strategic mar-
keting planning and decision making // Management, mar-
keting and promotion of library services based on statistics,
analyses and evaluation. München, 2006. P. 107–115. 5. Канн С. К. «Яндекс-метрика» как инструмент библиотеч-
ной веб статистики // Вопросы образования и науки: тео-
ретический и методический аспекты : сб. науч. тр. по ма-
териалам Междунар. науч.-практ. конф., 2015. С. 60–62. 6. Кириченко Е. В., Сытников Д. А., Петухов А. В., Кацко И. А. Статистика поисковых запросов // Научный журнал
КубГАУ : электрон. журн. 2013. № 9. С. 1–13. URL:
http://ej.kubagro.ru/2013/09/pdf/75.pdf (дата обращения
04.10.2018). 25. Neuroth H., Lossau N., Rapp A. (Hrsg.) Evolution der Informa-
tionsinfrastruktur. Kooperation zwischen Bibliothek und Wis-
senschaft. Glückstadt : Verlag Werner Hülsbusch, 2013. 368 S. 26. Onyancha O. B. Open research data in Sub-Saharan Africa:
a bibliometric study using the Data Citation Index // Publi-
shing Research Quarterly. 2016. Vol. 32. P. 227–246. 7. Ключевые проблемы развития коллекций электронных
ресурсов : рук. для библиотек. Санкт-Петербург, 2012. 38 с. URL: https://www.ifla.org/files/assets/acquisition-collection-
development/publications/Electronic-resource-guide-ru.pdf
(дата обращения 04.10.2018). 27. Oxford University Press. URL: https://academic.oup.com/
journals/search-results?page=1&q=%22google%20trends%22
&SearchSourceType=1 (accessed 04.10.2018). 28. Repanovici A., Landoy A. Tracking and predicting growth of
health information using scientometrics methods and Google
Trends // IFLA Library. URL: http://library.ifla.org/2178/1/
219-repanovici-en.pdf (accessed 04.10.2018). 8. Кулева О. В. Инструменты веб-аналитики в библиотеч-
ной практике // Труды ГПНТБ СО РАН. Новосибирск. 2017. Вып. 12. С. 369–381. 29. SAGE journals. URL: http://journals.sagepub.com (accessed
04.10.2018). 9. Морозов А. М., Кадыков В. А., Пельтихина О. В., Аске-
ром А. М., Морозова А. Д. О возможностях определения
актуальности темы научного исследования // Синергия
наук. 2018. № 19. С. 1018–1024. 30. Showers B. (ed.). Library analytics and metrics: using data to
drive decisions and services facet publishing. London, 2015. 224 p. URL: http://www.facetpublishing.co.uk/title.php?id=
049658#.XAndWOIrnct (accessed 04.10.2018). 10. References 12. Редькина Н. С. Направления развития веб-аналитики //
Научно-техническая информация. Серия 2, Информаци-
онные процессы и системы. 2017. № 5. С. 5–10. 1. Bogdanova S. Yu., Kutusheva N. S., Shaikhumova V. R., Faz-
letdinova Z. I. Analysis of marketing information based on
the Google Trends web app toolkit. Upravlenie ekonomiches-
kimi sistemami, 2017, 4, 1–9. URL: https://cyberleninka.ru/
article/v/analiz-marketingovoy-informatsii-na-osnove-instrumen
tariya-publichnogo-web-prilozheniya-google-trends (accessed
04.10.2018). (In Russ.). 13. Столяренко А. В., Мельничук А. С. Исследование приори-
тетных направлений в туризме республики Крым с по-
мощью Google Trends // Таврический научный обозре-
ватель. 2017. № 7. С. 34–39. 14. Шевченко Л. Б. Шесть трендов цифрового маркетинга
для библиотек // Научные и технические библиотеки. 2015. № 11. С. 12–19. 2. Borochkin A. A. Using statistics of search queries on the
Internet for short-term forecasting of macroeconomic vari-
ables. Den’gi i kredit, 2013, 8, 27–32. (In Russ.). 15. Электронная библиотека ГПНТБ России. URL: http://ellib. gpntb.ru/ (дата обращения: 04.10.2018). 3. Zavgidullina M. V. A book in the media environment: will
a word disappear? Kniga: Sibir’ – Evraziya : tr. I Mezhdunar. nauch. kongr. Novosibirsk, 2016, 3, 4–10. (In Russ.). 16. Cahill K. Google tools on the public reference desk // The
Reference Librarian. 2007. Vol. 48. P. 67–79. 17. Congress programm // IFLA WLIC 2018. URL: https://www. react-profile.org/upload/KIT/system/uploads/17_IFLA%2020
18_Congress%20Programm.pdf (accessed 04.10.2018). 4. Zinov’eva E. S. The impact of Internet industry companies on
world politics. Vestnik MGIMO-Universiteta, 2013, 1, 43–47. (In Russ.). 18. Evelyn L. Curry the reference interview in the Google Age //
IFLA WLIC 2016. URL: http://library.ifla.org/1354/1/125-curry-
en. pdf (accessed 04.10.2018). 5. Kann S. K. «Yandex-metric» as a tool of library web statistics. Voprosy obrazovaniya i nauki: teoreticheskii i metodicheskii
aspekty : sb. tr. po materialam Mezgdunar. nauch.-prakt. konf. Tambov, 2015, 7, 60–62. (In Russ.). 19. Farrell M. Time management // Journal of Library Admini-
stration. 2017. Vol. 57, iss. 2. P. 215–222. 6. Kirichenko E. V., Sytnikov D. A., Petukhov A. V., Kacko I. A. Statistics of search queries. Nauchnyi zhurnal KubGAU, 2013,
9, 1–13. URL: http://ej.kubagro.ru/2013/09/pdf/75.pdf (ac-
cessed 04.10.2018). (In Russ.). 20. IFLA strategic plan 2016-2021. Hague, 2017. 8 p. URL: https://
www.ifla.org/files/assets/hq/gb/strategic-plan/2016-2021.pdf
(accessed 04.10.2018). 21. IFLA annual report 2017. Hague, 2017. 31 p. URL: https://
www.ifla.org/files/assets/hq/annual-reports/2017.pdf (acces-
sed 04.10.2018). 7. Key problems of the development of collections of electronic
resources: a guide for libraries. IFLA. Saint Petersburg, 2012. 38 p. URL: https://www.ifla.org/files/assets/acquisition-col
lection-development/publications/Electronic-resource-guide-
ru.pdf (accessed 04.10.2018). (In Russ.). 22. Pulizzi J. МЕТОДОЛОГИЯ НИР / METODOLOGY OF RESEARCH WORK Редькина Н. С. Google Тrends - динамика интереса к элек-
тронным библиотекам в России и в мире // Научные
основы и практика реализации цифровых проектов
в сфере культуры и образования. Санкт-Петербург, 2016. С. 117–124. 049658#.XAndWOIrnct (accessed 04.10.2018). 31. Springer Link. URL: https://link.springer.com (accessed
04.10.2018). 32. Sühl-Strohmenger W. Digitale Welt und Wissenschaftliche
Bibliothek-Informationspraxis im Wandel. Wiesbaden : Har-
rassowitz Verl., 2008. 294 p. (Bibliotheksarbeit ; Bd 11). 11. Редькина Н. С. Деятельность библиотеки в условиях
формирования информационно технологической пара-
дигмы // Книга: Сибирь – Евразия : тр. I Междунар. науч. конгр. Новосибирск, 2016. Т. 3. С. 216–224. Список источников 1. Богданова С. Ю., Кутушева Н. С., Шайхумова В. Р., Фазлет-
динова З. И. Анализ маркетинговой информации на ос-
нове инструментария публичногоweb-приложения Google
Trends // Управление экономическими системами : элек-
трон. науч. журн. 2017. № 4. С. 1–9. URL: https://cyberleninka. 1. Богданова С. Ю., Кутушева Н. С., Шайхумова В. Р., Фазлет-
динова З. И. Анализ маркетинговой информации на ос-
нове инструментария публичногоweb-приложения Google
Trends // Управление экономическими системами : элек-
трон. науч. журн. 2017. № 4. С. 1–9. URL: https://cyberleninka. 7 7 References The rise of storytelling as the new marketing //
Publishing Research Quarterly. 2012. Vol. 28. P. 116–123. 8 С. В. Соколов, 2018, № 4, с. 3–9 / S. V. Sokolov, 2018, no. 4, pp. 3–9 С. В. Соколов, 2018, № 4, с. 3–9 / S. V. Sokolov, 2018, no. 4, pp. 3–9 8. Kuleva O.V. Web analytics tools in library practice. Trudy
GPNTB SO RAN. Novosibirsk, 2017, 369–381. (In Russ.). 22. Pulizzi J. The rise of storytelling as the new marketing. Pub-
lishing Research Quarterly, 2012, 28, 116–123. 22. Pulizzi J. The rise of storytelling as the new marketing. Pub-
lishing Research Quarterly, 2012, 28, 116–123. g
y
23. Konrad U., Fühles-Ubach S., Seadle M. (eds.) Handbuch Me-
thoden der Bibliotheks- und Informationswissenschaft: Biblio-
theks-, Benutzerforschung, Informationsanalyse. Berlin; Bos-
ton, Walter de Gruyter, 2013. URL: https://books.Google.ru/
books?id=FXV7rK9xTtEC&pg=PA193&dq=bibliothek+Google+
trends&hl=de&sa=X&ved=0ahUKEwjMx877g8DdAhUPt4sKHT
f2D28Q6AEIRzAH#v=onepage&q=bibliothek%20Google%20
trends&f=false (accessed 04.10.2018). 9. Morozov A. M., Kadykov V. A., Peltikhina O. V., Askerom A. M.,
Morozova A. D. On possibilities of determining the relevance
of the research topic. Synergiya nauki, 2018, 19, 1018–1024. (In Russ.). 10. Redkina N. S. Google Trends – the dynamics of interest in
electronic libraries in Russia and in the world. Nauchnye os-
novy i praktika realizatsii tsifrovykh proektov v sfere kul’tury
i obrazovaniya. Saint Petersburg, 2016, 117–124. (In Russ.). 24. Koontz C. Using customer data for improved strategic mar-
keting planning and decision making. Management, marke-
ting and promotion of library services based on statistics,
analyses and evaluation. München, 2006, 107–115. 11. Redkina N. S. Library activity in the conditions of formatting
the information technological paradigm. Kniga: Sibir’ – Evra-
ziya : tr. I Mezhdunar. nauch. kongr. Novosibirsk, 2016, 3,
216–224. (In Russ.). 12. Redkina N. S. Directions for the web analytics development. Nauchno-tekhnicheskaya informatsiya. Seriya 2. Informatsion-
nye protsessy i systemy, 2017, 5, 5–10. (In Russ.). 25. Neuroth H., Lossau N., Rapp A. (eds.) Evolution der Informa-
tionsinfrastruktur. Kooperation zwischen Bibliothek und Wis-
senschaft. Glückstadt, Verlag Werner Hülsbusch, 2013. 368 S. 13. Stolyarenko A. V., Melnichuk A. S. Studying priority areas in
the tourism of the Republic of Crimea using Google Trends. Tavricheskii nauchnyi obozrevatel’, 2017, 7, 34–39. (In Russ.). 26. Onyancha O. B. Open research data in Sub-Saharan Africa:
a bibliometric study using the Data Citation Index. Publishing
Research Quarterly, 2016, 32, 227–246. 14. Shevchenko L. B. Six Trends of Digital Marketing for Lib-
raries. Nauchnye i tekhnicheskie biblioteki, 2015, 11, 12–19. Сведения об авторе: Соколов Сергей Валерьевич – научный сотрудник Фундаментальной библиотеки ИНИОН РАН,
ORCID: 0000-0002-2068-6797 References (In Russ.). 27. Oxford University Press. URL: https://academic.oup.com/
journals/search-results?page=1&q=%22google%20trends%22
&SearchSourceType=1 (accessed 04.10.2018). 15. Elektronnaya biblioteka GPNTB Rossii [Electronic Library of the
Russian National Public Library for Science and Technology]. URL: http://ellib.gpntb.ru/ (accessed 04.10.2018). (In Russ.). 28. Repanovici A., Landoy A. Tracking and predicting growth of
health information using scientometrics methods and Google
Trends. IFLA Library. URL: http://library.ifla.org/2178/1/219-
repanovici-en.pdf (accessed 04.10.2018). 16. Cahill K. Google tools on the public reference desk. The Re-
ference Librarian, 2007, 48, 67–79. 29. SAGE journals. URL: http://journals.sagepub.com (accessed
04.10.2018). 17. Congress program. IFLA WLIC 2018. URL: https://www.react-
profile.org/upload/KIT/system/uploads/17_IFLA%202018_
Congress%20Programm.pdf (accessed 04.10.2018). 30. Showers B. (ed.). Library analytics and metrics: using data to
drive decisions and services facet publishing. London, 2015. 224 p. URL: http://www.facetpublishing.co.uk/title.php?id=04
9658#.XAndWOIrnct (accessed 04.10.2018). 18. Evelyn L. Curry the reference interview in the Google age. IFLA WLIC 2016. URL: http://library.ifla.org/1354/1/125-curry-
en. pdf (accessed 04.10.2018). 31. Springer Link. URL: https://link.springer.com (accessed
04.10.2018). 19. Farrell M. Time management. Journal of Library Administra-
tion, 2017, 57(2), 215–222. 32. Sühl-Strohmenger W. Digitale Welt und Wissenschaftliche Bib-
liothek-Informationspraxis im Wandel. Wiesbaden, Harrasso-
witz Verl., 2008. 294 S. (Bibliotheksarbeit ; Bd 11). 20. IFLA strategic plan 2016-2021. Hague, 2017. 8 p. URL: https://
www.ifla.org/files/assets/hq/gb/strategic-plan/2016-2021.pdf
(accessed 04.10.2018). 21. IFLA annual report 2017. Hague, 2017. 31 p. URL: https://
www.ifla.org/files/assets/hq/annual-reports/2017.pdf (acces-
sed 04.10.2018). Материал поступил в редакцию 19.10.2018 г. Сведения об авторе: Соколов Сергей Валерьевич – научный сотрудник Фундаментальной библиотеки ИНИОН РАН,
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Digital Healthcare
and Expertise Mental Health and
New Knowledge Practices Claudia Egher Claudia Egher HEALTH, TECHNOLOGY AND SOCIETY HEALTH, TECHNOLOGY AND SOCIETY Health, Technology and Society Health, Technology and Society Series Editors
Rebecca Lynch
Wellcome Centre for Cultures and Environments
of Health
University of Exeter
Exeter, UK
Martyn Pickersgill
Usher Institute
University of Edinburgh
Edinburgh, UK Medicine, health care, and the wider social meaning and management of
health are undergoing major changes. In part this reflects developments
in science and technology, which enable new forms of diagnosis, treat
ment and delivery of health care. It also reflects changes in the locus of
care and the social management of health. Locating technical develop
ments in wider socio-economic and political processes, each book in the
series discusses and critiques recent developments in health technologies
in specific areas, drawing on a range of analyses provided by the social
sciences. Some have a more theoretical focus, some a more applied focus
but all draw on recent research by the authors. The series also looks
toward the medium term in anticipating the likely configurations of
health in advanced industrial society and does so comparatively, through
exploring the globalization and internationalization of health. ISSN 2946-3386
ISSN 2946-3378 (electronic)
Health, Technology and Society
ISBN 978-981-16-9177-5 ISBN 978-981-16-9178-2 (eBook)
https://doi.org/10.1007/978-981-16-9178-2 Claudia Egher
Department of Health, Ethics and Society
Maastricht University
Maastricht, The Netherlands Claudia Egher
Department of Health, Ethics and Society
Maastricht University
Maastricht, The Netherlands To Véronique ISSN 2946-3386
ISSN 2946-3378 (electronic)
Health, Technology and Society
ISBN 978-981-16-9177-5 ISBN 978-981-16-9178-2 (eBook)
https://doi.org/10.1007/978-981-16-9178-2 © The Editor(s) (if applicable) and The Author(s) 2023. This book is an open access publication.h © The Editor(s) (if applicable) and The Author(s) 2023. This book is an open access publication. Open Access This book is licensed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing,
adaptation, distribution and reproduction in any medium or format, as long as you give appropriate
credit to the original author(s) and the source, provide a link to the Creative Commons licence and
indicate if changes were made. g
The images or other third party material in this book are included in the book’s Creative Commons
licence, unless indicated otherwise in a credit line to the material. If material is not included in the book’s
Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the
permitted use, you will need to obtain permission directly from the copyright holder. g
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication
does not imply, even in the absence of a specific statement, that such names are exempt from the relevant
protective laws and regulations and therefore free for general use. p
g
g
The publisher, the authors, and the editors are safe to assume that the advice and information in this book
are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or
the editors give a warranty, expressed or implied, with respect to the material contained herein or for any
errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional
claims in published maps and institutional affiliations. This Palgrave Macmillan imprint is published by the registered company Springer Nature Singapore
Pte Ltd.h The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721,
Singapore To Véronique Series Editors’ Preface Series Editors’ Preface Medicine, healthcare, and the wider social meanings and management of
health are continually in the process of change. While the “birth of the
clinic” heralded the process through which health and illness became
increasingly subject to the surveillance of medicine, for example, surveil
lance has become more complex, sophisticated, and targeted—as seen in
the search for “precision medicine” and now “precision public health.”
Both surveillance and health itself emerge as more provisional, uncertain,
and risk-laden as a consequence, and we might also ask what now consti
tutes “the clinic,” how meaningful a concept of a clinic ultimately is, and
where else might we now find (or not find) healthcare spaces and
interventions. Medicine, healthcare, and the wider social meanings and management of
health are continually in the process of change. While the “birth of the
clinic” heralded the process through which health and illness became
increasingly subject to the surveillance of medicine, for example, surveil
lance has become more complex, sophisticated, and targeted—as seen in
the search for “precision medicine” and now “precision public health.” Ongoing developments in science and technology are helping to
enable and propel new forms of diagnosis, treatment, and the delivery of
healthcare. In many contexts, these innovations both reflect and further
contribute to changes in the locus of care and burden of responsibility for
health. Genetics, informatics, and imaging—to name but a few—are
redefining collective and individual understandings of the body, health,
and disease. At the same time, long-established and even ostensibly mun
dane technologies and techniques can generate ripples in local discourse
and practices as ideas about the nature and focus of healthcare shift in
response to global debates about, for instance, One Health and
Planetary Health. vii viii Series Editors’ Preface The very technologies that (re)define health are also the means through
which the individualization of healthcare can occur—through, for
instance, digital health, diagnostic tests, and the commodification of
restorative tissue. This individualization of health is both culturally
derived and state-sponsored, as exemplified by the promotion of “self-
care.” These shifts are simultaneously welcomed and contested by profes
sionals, patients, and the wider public. Hence they at once signal and
instantiate wider societal ambivalences and divisions. This series explores these processes within and beyond the conven
tional domain of “the clinic,” and asks whether they amount to a qualita
tive shift in the social ordering and value of medicine and health. Series Editors’ Preface Locating
technical use and developments in wider socioeconomic and political
processes, each book discusses and critiques the dynamics between health,
technology, and society through a variety of specific cases and draws on a
range of analyses provided by the social sciences. The series has already published more than 20 books that have explored
many of these issues, drawing on novel, critical, and deeply informed
research undertaken by their authors. In doing so, the books have shown
how the boundaries between the three core dimensions that underpin the
whole series—health, technology, and society—are changing in funda
mental ways. In Digital Healthcare and Expertise, Claudia Egher focuses on an area
that has attracted much promise, hope, and investment—but which nev
ertheless remains less well studied than might be expected within the
social studies of medicine: digital mental health. This exciting mono
graph considers compelling questions around the construction and medi
ation of experiences framed often in psychiatry and beyond as “bipolar
disorder” within digital spaces, with particular analytic attention to the
performance and negotiation of expertise. It provides a bold analysis of
the reconfiguring of (the relations between) forms of online (and offline)
epistemic practices, performances of and claims to knowledge and exper
tise, and understandings and experiences of the self. Through Digital
Healthcare and Expertise, Egher illustrates the complexities inherent to
shifting understandings and deployments of expertise relating to bipolar Series Editors’ Preface Series Editors’ Preface ix disorder within online platforms, and presents a novel interpellation of
science and technology studies, medical sociology, and media studies that
will be vital to future analyses of health, technology, and society. London, UK
Rebecca Lynch
Edinburgh, UK
Martyn Pickersgill Rebecca Lynch
Martyn Pickersgill London, UK
Edinburgh, UK Acknowledgments Working on this book has been an incredible adventure, and I am deeply
grateful for all the help and support I have received along the way from
colleagues, family, and friends, from the first stages of my doctoral
research upon which this work is based to the close readings of its penul
timate draft. Having immersed myself in the experiences of people diagnosed with
bipolar disorder for many years now, I have acquired a rich understand
ing of the challenges involved in the management of this condition. Without wanting to be presumptuous, I would therefore like to first and
foremost express my gratitude to the many people diagnosed with bipolar
disorder, who have written about their states, experiments, joys, and dif
ficulties on the digital platforms I studied. In the absence of such endeav
ors, this book would literally not have been possible. I do hope that it
does justice to their tremendous efforts and valuable insights. f
I would particularly like to thank Natasha Tracy and Julie A. Fast, who
kindly allowed me to interview them and use materials from their digital
platforms. I can still vividly remember how impressed I was during the
first year of my PhD, when I discovered their blogs and became aware of
the numerous and varied contributions they were making across different
media. I continue to be impressed by their work, and I would like to
express my appreciation for their dedication and considerable efforts to
help improve the lives of other people diagnosed with bipolar disorder. xi xii Acknowledgments I would also like to thank the representatives of La Haute Autorité de
Santé in France, who gave me permission to reproduce materials from
their website in this book. Two chapters of Digital Healthcare and Expertise are the result of previ
ously published articles that were substantially altered here. An earlier
version of Chap. 4 was published in 2019 under the title “Bipolar Patients
and Creative Online Practices: Sharing Experiences of Controversial
Treatments” in Health 23(4): 458–477. A first version of Chap. 5 was
published in 2020 as “Online Expert Mediators: The Rise of a New
‘Bipolar’ Stakeholder or Going Beyond Interactional Expertise in the
Blogosphere” in Science and Technology Studies 33 (2): 1–22. Both articles
greatly benefitted from the anonymous reviewers and from the editors at
each journal, to whom I am grateful. Acknowledgments I owe many words of thanks to Joshua Pitt and Marion Duval, the edi
tors I worked with on this book, as well as to the series editors, Martyn
Pickersgill and Rebecca Lynch. I am also very grateful to Rubina Infanta
Rani, for her patience, help, and support throughout this process. This
book benefitted tremendously from the very constructive feedback and
suggestions the anonymous reviewers provided on the book proposal and
on the subsequent draft. My heartfelt thanks to them!i My hope is that this book will find its way and will be of relevance to
medical professionals, people diagnosed with mental health conditions,
policy makers, and any other stakeholder groups involved or interested in
the provision of mental healthcare. I am therefore particularly grateful for
the open-access publication grant provided by the Netherlands
Organization for Scientific Research (NWO) and for the financial sup
port obtained from the Maastricht University Library. I would like to
thank Sally Wyatt for all the help she provided me with throughout this
process. I am also very grateful to Olga Zvonareva, Bart Penders, and
Klasien Horstman for their support. The core ideas of this book were developed while I was a PhD candi
date at the Faculty of Arts and Social Sciences at Maastricht University. I
owe a tremendous debt of gratitude to Sally Wyatt and Tamar Sharon for
their guidance, advice, unwavering support, and multiple close readings
of drafts. They have been phenomenal PhD supervisors and they have
inspired me ever since! The tenacity and motivation required to finish Acknowledgments xiii this book during the challenging times of the pandemic have been to a
large extent stimulated by them. I consider myself very lucky for having
made my first steps into STS under the close guidance of Wiebe Bijker,
and I am deeply grateful for his help and for everything that I have
learned from him. Thanks are also due to Ruud Hendriks, who inspired
my first sociological forays into mental health. The shortening and sharp
ening of this book benefitted from the constructive feedback provided by
Tsjalling Swierstra, to whom I am very grateful. Acknowledgments I was also inspired and
invigorated by the presentations and talks I have had over the years with
members of the Maastricht University Science, Technology and Society
Studies (MUSTS) group, of the Centre for Ethics and Politics of Emerging
Technologies (EPET), and of the Netherlands Graduate Research School
of Science, Technology, and Modern Culture (WTMC). This book has also benefitted from the close reading and friendly feed
back provided by very kind and generous friends. Hortense, Valentina,
Lisa, Simone, and Thomas, thank you so much for all your help! h
Finally, I want to express my deep love and gratitude to my family and
friends, to those who have supported me through some pretty hard times
and with whom I have shared countless joyful and amazing moments. Thank you for constantly reminding me that there is a life out there,
while also being understanding and accepting of my need to work. Mamă
şi tată, vă mulțumesc pentru ajutor şi susținere! Many thanks also to
Lucia and Christian for the wonderfully relaxing moments spent together! One of my deep personal regrets is that Pa and my aunt Veronica are no
longer with us to witness this moment. Charlie, I am extremely grateful
that I could rely on your help and support throughout all the years that I
worked on this book and the study preceding it. Krijn and Raquel, thank
you so much for your friendship and for your willingness to read drafts,
double-check references, and help with the editing and any other issue
pertaining to my work for so many years now. Lori, thank you for your
friendship, for your patience, and for the constant and often last-minute
(sorry about that!) efforts to make my graphs and tables look better! Andreea, Oana, PJ, Nico, Julia, Luat, Bart, Laura, Bogdan, Andrei,
Simona, Dina, Kosmas, Marith, Willemine, Dara, and Ricky, thank you
so much for your friendship! Much love and gratitude to Hortense—it is
no exaggeration to say that this book would not have been finalized, had Acknowledgments xiv it not been for your continuous help, encouragement, and support! I am
so grateful that you’re my friend! Véronique, thank you for all the draw
ings and jottings you left on so many of my drafts, and I’m sorry that they
didn’t make it into the actual manuscript. Acknowledgments This book is for you—in the
hope that one day you will understand why mummy played less often
with you than you would have wanted. You are my great love! About This Book About This Book This book explores how expertise about bipolar disorder is performed on
American and French digital platforms by combining insights from STS,
medical sociology, and media studies. It addresses topical questions,
including the following: How do different stakeholders engage with
online technologies to perform expertise about bipolar disorder? How
does the use of the Internet for processes of knowledge evaluation and
production allow people diagnosed with bipolar disorder to re-position
themselves in relation to medical professionals? How do cultural markers
shape the online performance of expertise about bipolar disorder? And
what individualizing or collectivity-generating effects does the Internet
have in relation to the performance of expertise? The book thus consti
tutes a critical and nuanced intervention into dominant discourses which
approach the Internet either as a quick technological fix or as a postmod
ern version of Pandora’s box, sowing distrust among people and threaten
ing unified conceptualizations and organized forms of knowledge. xv —Wiebe E. Bijker, Professor of Technology & Society, Norwegian University of
Science and Technology Praise for Digital Healthcare and Expertise “The digital era sits on an unsteady amalgam of hype and doom scenarios con
cerning the emancipating power of the Internet and the democratization of
expertise. Claudia Egher succeeds in astutely navigating these sweeping claims
to produce a more nuanced and complex picture of technologically mediated
knowledge production. Her relentless search for the how, who, and where of the
enactment of expertise on bipolar disorder online takes the reader on a journey
that shuttles between vast theoretical literatures on expertise and the power of
technology and culture to shaping it, and its actual performance. In the process,
she gives voice to the new experts of bipolar disorder, where user agency is rec
onciled with choice architecture and solidarity persists, as a latent and stubborn
dimension of individualization and personalization.” —Tamar Sharon, Professor of Philosophy, Digitalization and Society, Radboud
University Nijmegen “Adopting a science and technology studies approach, this intriguing book
shows how expertise has different meanings to the diverse actors who are part of
defining and managing bipolar disorder. As Egher’s analysis demonstrates, mate
rial practices and standpoints combine to enact expertise in specific contexts. This book will be of interest to anyone who wants to know more about how
expertise is multiple, dynamic, and complex.” —Deborah Lupton, SHARP Professor in the Centre for Social Research in Health
and Social Policy Research Centre, UNSW Sydney —Deborah Lupton, SHARP Professor in the Centre for Social Research in Health
and Social Policy Research Centre, UNSW Sydney “This book offers a novel approach to expertise as a practical and collective
achievement. With its sharp and effective focus on mental health and digital
communities, it produces wide-ranging and generally applicable conclusions
about expertise and the Internet’s potential to democratize knowledge. The lucid
description of exchanges on digital platforms on bipolar disorder makes this
book a must-read for mental health professionals, STS researchers, and public
health policy makers alike.” —Wiebe E. Bijker, Professor of Technology & Society, Norwegian University of
Science and Technology Contents 1 Studying Expertise Online
1
2 Epistemic Inroads from the Asylum to Digital Psychiatry
37
3 The Drama of Expertise About Bipolar Disorder Online
71
4 Tactical Re-appraisals and Digitally Informed Hypotheses
About the Treatment for Bipolar Disorder
109
5 Online Expert Mediators: Expanding Interactional
Expertise
145
6 Digital Biocommunities: Solidarity and Lay Expertise
About Bipolar Disorder
189
7 Expertise in the Age of Big Data
225
Index
245 1 Studying Expertise Online 1 Studying Expertise Online Index xix List of Acronyms and Abbreviations AA
Alcoholics Anonymous
AI
Artificial intelligence
AIDS
Acquired immunodeficiency syndrome
APSA
L’Association des Psychotiques Stabilisés Autonomes
BPS
British Psychological Society
CESE
Conseil Économique Social et Environnemental
CNSA
Caisse Nationale de Solidarité pour l’Autonomie
DSM-5
Diagnostic and Statistical Manual of Mental Disorders,
5th edition
EBM
Evidence-based medicine
ECT
Electroconvulsive therapy
E-FOIA
Electronic Freedom of Information Act
EIT
Electronic and information technology
EMDR
Eye movement desensitization and reprocessing
EMI
Ecological momentary intervention
ERCIC
Ethical Review Committee of Inter City Faculties, Maastricht
University
GDP
Gross domestic product
GDPR
General Data Protection Regulation
GEM
Groupes d’Entraide Mutuelle
GIA
Groupe d’Information Asile
GP
General practitioner
GPS
Global Positioning System xxi List of Acronyms and Abbreviations HAS
La Haute Autorité de Santé
HONcode
The Health on the Net Foundation Code of Conduct
ICD-10
The International Classification of Disease and Health Related
Problems, 10th edition
ICT
Information and Communication Technology 9
IE
Interactional expertise
ISO
International Organization for Standardization
KTRO
Radio station licensed to transmit in Portland
LFB
Le Forum des Bipotes
NAC
N-acetylcysteine
NIMH
National Institute of Mental Health
PDF
Portable Document Format
PXE
Pseudoxanthoma elasticum
OECD
Organisation for Economic Co-operation and Development
RGAA3
Référentiel Géneral d’Accessibilité pour les Administrations
SAD
Seasonal affective disorder
STS
Science and technology studies
SEE
Studies of Expertise and Experience
UK
The United Kingdom of Great Britain and Northern Ireland
US
The United States of America
VNS
Vagus nerve stimulation
WAI
Web Accessibility Initiation
WCAG
Web Content Accessibility Guidelines
WHO
World Health Organization
W3C
World Wide Web Consortium © The Author(s) 2023
C. Egher, Digital Healthcare and Expertise, Health, Technology and Society,
https://doi.org/10.1007/978-981-16-9178-2_1 List of Figures Fig. 3.1
Images of the upper and lower parts of NIMH’s website
(2016)
90
Fig. 3.2
Images of the upper and lower parts of HAS’ online page on
the diagnostic and treatment of bipolar disorder
95
Fig. 3.3
Images of the upper and lower parts of HAS’ online page
dedicated to bipolar disorder as a chronic condition
96
Fig. 3.4
Image of the affordances—audio and reading ruler—of
a .pdf file provided by HAS
100
Fig. 5.1
Fragment from the first page of Natasha Tracy’s blog,
Bipolar Burble. Retrieved on January 8, 2019
174
Fig. 5.2
Fragment from the blog posts’ overview on Julie A. Fast’s
blog, Bipolar Happens! Retrieved on November 5, 2018
175 Fig. 3.1
Images of the upper and lower parts of NIMH’s website
(2016)
90
Fig. 3.2
Images of the upper and lower parts of HAS’ online page on
the diagnostic and treatment of bipolar disorder
95
Fig. 3.3
Images of the upper and lower parts of HAS’ online page
dedicated to bipolar disorder as a chronic condition
96
Fig. 3.4
Image of the affordances—audio and reading ruler—of
a .pdf file provided by HAS
100
Fig. 5.1
Fragment from the first page of Natasha Tracy’s blog,
Bipolar Burble. Retrieved on January 8, 2019
174
Fig. 5.2
Fragment from the blog posts’ overview on Julie A. Fast’s
blog, Bipolar Happens! Retrieved on November 5, 2018
175 xxiii Studying Expertise Online I have this med down to a science. (…) You also cannot drink any water
after taking it until you wake up. You can take one swig of water here or
there but I try not to. One thing that has worked for me is not eating or
drinking anything from 9:30 until 12:30. This is a trial and error drug and
I have schooled my doctor on what works. (Watson, March 9, 2015, quote
slightly adapted to ensure anonymity) As the online excerpt above suggests, the Internet has facilitated the
“participatory turn” (Prainsack, 2011) in healthcare, with a plethora of
online platforms and mobile health applications claimed to disrupt the
traditional distribution of knowledge and power between medical profes
sionals and people diagnosed (Dedding et al., 2011; Eysenbach et al.,
2004). Yet, the term “participation” has turned out to be rather vague
(Nielsen & Langstrup, 2018; Wyatt et al., 2013), and it is not clear how
active such involvement with one’s health is or should be, nor whether
participation reaches as far as allowing people diagnosed to contribute to
the production of knowledge, and what such contributions consist of. At
the same time, by affording the wider circulation of scientific controver
sies, the Internet has contributed to a growing public awareness about the
disagreements that often exist among experts and about the conditions of 1 C. Egher C. Egher 2 uncertainty under which they make recommendations and decisions. The Internet may have thus served both to weaken the cognitive author
ity of “traditional” experts and to draw attention to the fact that people
lacking official accreditations can also possess substantial knowledge and
experience in certain domains. p
These developments are linked to the rather paradoxical position that
expertise has nowadays come to occupy in Western societies. One may
argue that at no other time in history has expertise been so prevalent, as
it has expanded and now covers areas for which no specific and substan
tial knowledge was previously thought necessary, ranging from child-
rearing and health to interior design, lifestyle choices, and personal
savings. Yet, while this suggests that expertise has become ubiquitous as
well as highly valued, the identity of experts, of those who can rightfully
provide authoritative answers and solutions to complex, “wicked” prob
lems has been challenged. Studying Expertise Online As a consequence, instead of something stable
and well-bounded, expertise has come to mean different things to differ
ent people: it is acquired and manifested in myriad ways across different
locales, it fulfills different functions, and it is importantly shaped by
social, cultural, and economic factors. This raises important questions
about the identity and position of those who can acquire expertise and
about the ways in which they perform it, that is, about the practices,
tools, and standards through which expertise is articulated.h These elements are at the core of this book, as it asks, how is expertise
about bipolar disorder performed on American and French online plat
forms? In so doing, it focuses on how different stakeholders engage with
online technologies to perform expertise about bipolar disorder and
how such activities allow them to re-position themselves in relation to
medical professionals. The book further investigates the individualizing
or collectivity-generating effects that the Internet has in relation to the
online performance of expertise about bipolar disorder, given that it is
still uncertain how it shapes such performances at the level of individu
als or groups, and whether it helps give rise to new configurations. By
focusing on the performance of expertise about bipolar disorder on
French and American digital platforms, it also considers how cultural
factors shape epistemic practices. In addressing these issues, this book
contributes to the fields of science and technology studies (STS), 1 Studying Expertise Online 1 Studying Expertise Online 3 3 medical sociology, and media studies. A brief overview of the public
and theoretical debates that have informed these particular questions
and information about how they are addressed in the chapters of this
book are presented below. The processes through which one can develop expertise are domain-
specific, as they involve the acquisition of particular types of knowledge
and the internalization of relevant practices, norms, and values. This
book focuses on mental health, where the developments sketched above
have had a profound impact, and where expertise has had a particularly
convoluted trajectory. This has been the case partly due to the complex
and elusive nature of mental health conditions and partly to the problem
atic status of psychiatry in relation to medical sciences. The (relative)
success of psychotropic drugs, the development and widespread use of
brain neuroimaging techniques, and advances in genetics have stimulated
in recent years the search for biomarkers for mental health conditions
and have contributed to the proliferation and diversification of profes
sionals working in this field. Nevertheless, mental health expertise con
tinues to be challenged, with some critics denouncing it for pathologizing
variations in average human behavior (Horwitz & Wakefield, 2007), and
others condemning it for medicalizing social and political problems
(Metzl, 2009). Furthermore, people diagnosed have assumed an increas
ingly active role in the production of knowledge about mental health,
and not always in collaboration with medical professionals. Given the
tremendous increase of mental health diagnoses around the world (Rose,
2018), it is more important than ever to understand how expertise about
mental health is currently performed online, by whom, and through
what means. This book addresses these issues by focusing on bipolar disorder, a
mental health condition which has become more prominent over the
last few decades, and which is currently among the top ten causes of dis
ability worldwide (Whiteford et al., 2013). It is a mood disorder charac
terized by the alternation of depressive and manic episodes and marked
by episode-free intervals. While it is thought to be determined by a com
bination of neurological, genetic, and environmental factors, the precise
causes of this condition are currently unknown. It is studied by various
specialists:
psychiatrists,
psychologists,
neuroscientists, C. Egher C. Egher 4 endocrinologists, molecular biologists, epidemiologists, and so on. 1 Studying Expertise Online These
professionals focus on different sites as the origin and location of this
condition, they use different techniques and materials, and so they
understand rather different things by bipolar disorder (Dehue, 2008;
Hacking, 1995; Mol, 2002). Furthermore, the therapeutic approaches
used for its management consist of diverse combinations of chemical
substances, talk and behavioral therapies, as well as technological inter
ventions, such as transcranial magnetic stimulation or vagus nerve stim
ulation. Dominant approaches in personalized medicine have further
enhanced the complexity characterizing this field, as new subgroups
have been distinguished among the people diagnosed with this condi
tion based on whether or not they exhibited specific genetic modifica
tions, and on their responses to certain medications. Furthermore, these
perspectives shape and are shaped by the ways in which people diag
nosed with this condition experience it. Bipolar disorder can therefore
be seen as a “moving target” (Hacking, 1999), since it mobilizes differ
ent types of knowledge, groups of professionals, tools, standards, and
methods in dynamic configurations. This makes the study of this condi
tion relevant for research on expertise, as it can lead to a better under
standing of the ways in which expertise is performed when numerous
factors are involved and when a field is marked by numerous known as
well as unknown unknowns. Expertise about mental health has been traditionally performed in the
hallways of medical institutions, in laboratories and clinics, and on the
pages of compendia and scientific journals. However, since the late
1990s, when the Internet began to be widely adopted, the prominence
of this medium as a new site for the provision of knowledge and the
performance of expertise has grown (Fox et al., 2005; Hardey, 1999; Hu
& Sundar, 2010). This has been particularly the case after the develop
ment of Web 2.0 technologies, such as social media platforms and wikis,
where people could not only consume mental health-related informa
tion, but also actively engage in its production. As medical information
has become accessible to broad audiences and as people diagnosed have
started to play a more active role in the development of knowledge,
some scholars thought that the Internet would contribute to more equal
or balanced relationships between medical professionals and people 1 Studying Expertise Online 1 Studying Expertise Online 5 diagnosed, what some (Kitcher, 2011; Topol, 2015) have referred to as
“democratization.” While more nuanced studies have since been pro
vided (Kivits, 2009; Versteeg & Te Molder, 2018; Ziebland & Wyke,
2012) and while people diagnosed increasingly use digital technologies
for various health-related purposes, it is yet unclear what exactly the lat
ter contribute and how these contributions are used. Nor are there many
results available on the ways in which the Internet has shaped how “tra
ditional” experts perform expertise. This study contributes to these dis
cussions by asking how different stakeholders use the Internet to perform
expertise and how democratizing such practices are. Furthermore, by
combining insights from media studies on different types of online
encounters and their dynamic character with sociological perspectives
on the potential of personalized and precision medicine for the develop
ment of new types of communities (Akrich, 2010; Stommel & Lamerichs,
2014; Tutton & Prainsack, 2011), this book explores the possibility of
new individual-group configurations in the online performance of
expertise about bipolar disorder. p
p
Despite attempts to stabilize expertise about mental health, knowledge
about mental health conditions is not universal, but depends on the
social, cultural, and economic contexts in which it is made manifest
(Kleinman, 1988; Lakoff, 2005). Thus, how bipolar disorder is recog
nized, understood, and intervened upon depends on the ways in which
the provision of mental healthcare is organized in any given country, and
on the interpretative tools used by professionals. It is also informed by the
different ways in which people learn to distinguish and make sense of
problematic experiences from the incessant flow of physical and psycho
logical stimuli and reactions that make up their lives. This raises impor
tant questions about the fate of such local and cultural markers in the
online performance of expertise about bipolar disorder. To cast some
light onto these aspects, this study compares the American and French
perspectives on bipolar disorder and use of online technologies in mental
healthcare. Given the growing popularity of online platforms among
people diagnosed with mental health conditions (Carron-Arthur et al.,
2016; Naslund et al., 2016), such findings are very important, because
cultural and social elements may influence who feels entitled to share
information, what type of information is shared, and how it is C. Egher C. Egher 6 subsequently interpreted and put to use by readers. 1 Studying Expertise Online and contributing to the collective development of lay expertise together
with others with whom they share important similarities (Chap. 6). In the remainder of this chapter, I discuss the details of the research
upon which this book draws and introduce the following chapters. I
begin, however, with an overview of the main sociological approaches to
the study of expertise. 1 Studying Expertise Online In an attempt to
avoid cultural reification, the results of such comparison are presented at
the level of each chapter, thus illustrating how various similarities and
differences developed as an integral part of the specific analyses conducted. f
p
g
p
pi
y
Over the coming chapters we will see that the performance of expertise
about bipolar disorder is not a straightforward process by which offline
practices, tools, and approaches are transferred online, but involves addi
tional skills and complex negotiations, which sometimes lead to unex
pected configurations. The analysis of the empirical materials collected
prompted the realization that the current theoretical perspectives on
expertise do not sufficiently account for the complexity of positions that
relevant stakeholders occupy and for the different types of relations they
need to cultivate to successfully contribute to the development of exper
tise. That is why I put forward a new approach to expertise, wherein
expertise is conceived as a practical achievement, realized through coor
dination and affective labor among stakeholders who occupy multiple
and shifting positions across a complex ecosystem (discussed in more
detail later in this chapter). This new approach is arrived at by engaging
in dialogue with and building upon influential theories on expertise in
STS. In the process, several empirical and theoretical contributions are
made. Building on STS insights on users, this book contributes to medi
cal sociology by showing that through their specific engagement with
the online affordances of blogs and fora, people diagnosed with bipolar
disorder move beyond the performance of lay expertise, and put forward
what I call “digitally enabled hypotheses” about treatment effectiveness
(Chap. 3). By bringing insights from media studies in dialogue with the
recently developed field of Studies of Expertise and Experience (SEE),
the concept of interactional expertise developed by Collins and Evans
(2002) is expanded in this book by taking into consideration the effects
of the medium through which it is performed (Chap. 5). At a time when
the current dominant neoliberal model of governance encourages peo
ple to take up behaviors illustrative of narrowly conceived notions of
individual autonomy and personal responsibility, we will see that some
people diagnosed with bipolar disorder follow up on such encourage
ments and use the Internet to become successful entrepreneurs (Chap. 5), while others develop more nuanced approaches, performing solidarity 7 Theoretical Approaches to Expertise Expertise means different things to different people, it is performed dif
ferently in different contexts, and it leads to different relations between
people who hold expertise and those who do not as well as between those
endowed with different types of expertise. While the Oxford Dictionary
first mentions the term “expertise” in 1869, it was not until the 1970s
that expertise started to gain considerable academic interest, thereby
“reflect[ing] the growth and proliferation of professions with specialized
forms of knowledge, and (…) the increased commodification of knowl
edge production” (McNeil, 1998: 56-57). It has thus far mainly been
studied within the fields of psychology, philosophy, and sociology (Young
& Muller, 2014), and such forays have been characterized by important
disciplinary differences as well as significant distinctions in approach and
conceptualization at the level of each discipline broadly understood
(Williams et al., 1998) (for a discussion of the main approaches to exper
tise in psychology and philosophy, see Annex 1). Realist and constructiv
ist approaches to expertise can be noted in all these fields. According to
realist perspectives, expertise is a real and substantive skill that certain
people possess. Constructivist approaches see expertise as attributional, as
a qualification that experts enjoy based on it having been granted to them
by others who have the prerogative to do so and which is subsequently
socially recognized. Further distinctions can be derived from these two
main perspectives, such as whether expertise is the property of individu
als or groups, and whether it is primarily a form of theoretical or practical
knowledge. In sociology, expertise has initially been studied from the perspective
of the sociology of professions, with early studies seeking to understand C. Egher C. Egher 8 what accounted for the differences between occupations and profes
sions (Kotzee, 2014). The focus has therefore been mainly on expertise
as a property of groups, developed through various processes of accul
turation. Initially, a realist view of expertise seems to have dominated
sociological studies, with various authors showing the differences
between professions and occupations to be substantial and even provid
ing various lists of characteristics for each of them (Evetts et al., 2006). In time, however, as the constructivist approach to expertise has come
to dominate (Koppl, 2010), these authors have been criticized for help
ing maintain the authority of influential professions, such as medicine
and law (Saks, 2012). 1 Studying Expertise Online 1 Studying Expertise Online 9 and Evans (2002, 2007). The second conceives of expertise in terms of its
institutional embedding and is put forward by Jasanoff (2004). The third
understands expertise as a property of discrete networks, and it was devel
oped by Eyal and colleagues in their study on autism (Eyal, 2013; Eyal
et al., 2010; Eyal & Hart, 2010).f Seeking to determine the bases upon which members of different com
munities could be involved in decision-making processes at various levels
depending on the type of knowledge they were endowed with, Collins
and Evans (2007) put forward the Periodic Table of Expertise. In their
view, expertise is characterized by three dimensions: esotericity, or the
degree to which expertise is confined to a particular group; the tacit
knowledge required for it; and the changes in expert performance, which
trace a novice’s trajectory as s/he becomes a member of the expert group. Given the topic of this book, it has been helpful to engage with Collins
and Evans’ insights, as on the one hand they have tried to open up the
concept of expertise by acknowledging that people without official
accreditations could also be experts in a given field, while on the other
they have sought to ensure that expertise continues to designate some
thing “real” (Collins & Evans, 2007:40). Particularly useful has been the
distinction between what they call “contributory” and “interactional
expertise,” which Collins and Evans consider specialist forms of expertise
requiring specialist tacit knowledge. While they conceptualize contribu
tory expertise as the ability to contribute productively to a field, interac
tional expertise refers to the ability to become fluent in the language of
practice of a given domain, thereby being able to engage in substantial
discussions about relevant matters with contributory experts in that field
(I discuss at length this form of expertise in Chap. 5). Thus, Collins and
Evans’ conceptualization focuses on expertise as a matter of one’s knowl
edge and competence.i While this approach is meant to fight relativism, it neglects the strong
relational undertones of expertise, as it is acquired, maintained, and dis
played in complex and often long-lasting exchanges between people with
different levels of knowledge of a field and with different stakes in it. Even
though Collins and Evans’ typology of expertise is fruitfully applied in
this book, it is important to note some of the criticism it has received. Theoretical Approaches to Expertise Such critique is in line with (neo)Marxist per
spectives which conceive of expertise as a social construction, as a means
through which influential groups retain a monopoly over certain ser
vices, with professional training, standards, and evaluations seen as
mechanisms through which outsiders of these groups are denied access
(Illich, 1977; Susskind & Susskind, 2015). Building upon such insights,
feminist and postcolonial scholars have denounced the close relation
between knowledge, authority, and power, and have shown the tenuous
links between expertise, ethnicity, and gender, among others. From this
perspective, rather than something real, based upon substantial ability
in a given field, expertise is seen as an attribute bestowed upon certain
members of society by specific institutions, but which has real and
important consequences in terms of the distribution of power and
privileges. The debate regarding the contribution of STS scholars to the charges
currently brought against expertise in “post-truth” discussions is still
ongoing (Collins et al., 2017; Fuller, 2017; Lynch, 2017; Radder, 2018;
Sismondo, 2017), but there is no doubt that they have been important
proponents and advocates of the constructivist view on expertise. For
instance, influential studies in the field (and in the sociology of scientific
knowledge) have revealed how political and social considerations shape
the production of scientific knowledge (Barnes, 1974; Bijker, 1995;
Bloor, 1976; Shapin & Schaffer, 1985). In STS, currently, three main
approaches can be distinguished in the study of expertise. The first high
lights the substantial character of expertise and challenges the idea that
public engagement means that all views are equal, represented by Collins 1 Studying Expertise Online Jasanoff (2004), for example, views expertise as embedded in practice, 10 C. Egher C. Egher that is, as enacted in specific institutional settings, and she has reproached
Collins and Evans for not having sufficiently taken into account the role
of national and institutional cultures in shaping the development and
content of expertise and the relations between experts and society at large. Jasanoff argues that socio-political elements play an important role in
determining what counts as authoritative knowledge and in ensuring the
obduracy of such understandings (Jasanoff, 2004), be they more or less
well-founded. These elements ascribe authority and credibility, indicating
who the public should trust and defer to in specific matters. Thus, accord
ing to Jasanoff (2003:393), “expertise is not merely something that is in
the heads and hands of skilled persons, constituted through their deep
familiarity with the problem in question, but rather […] it is something
acquired, and deployed, within particular historical, political, and cul
tural contexts.” Whereas Jasanoff conceives of expertise as grounded in institutions,
Eyal has put forward an understanding of expertise as “a network linking
together agents, devices, concepts, and institutional and spatial arrange
ments” (Eyal, 2013: 863). Building upon insights put forward by
Foucault (1972/2010) and Rose (1992), Eyal finds it important to dis
tinguish between expertise and experts, arguing that the study of each
requires different methods and casts light upon different aspects. In this
understanding, expertise is not the attribute of any one individual, but
it is distributed, coming into being through exchanges between “agents”
endowed with different abilities and insights yet committed to solving a
common issue through similar methods. Eyal developed this theory
studying how the parents of autistic children challenged the psychiatric
establishment and succeeded in putting forward a different understand
ing of this condition and in popularizing a new therapeutic approach. These transformations were set into motion, in Eyal’s view, by a checklist
that an army psychiatrist, book author, and parent of an autistic child,
Bernard Rimland, provided on the back of his book for the parents of
autistic children to fill in and send back to him. Thus, the checklist rep
resented an innovative model of knowledge exchange around which the
network was organized, a means which allowed new stakeholders to con
tribute actively to the production of knowledge about autism. His dis
tinction between experts and expertise allows Eyal to conclude that 1 Studying Expertise Online 1 Studying Expertise Online 11 while psychiatrists may have lost in this way some of their territory, as
we will also see in Chap. 2, psychiatric expertise was in fact expanded in
that it became part of a greater network, consisting of more domains and
institutions and touching upon broader areas of life. Expertise as a net
work implies a variable level of flexibility, as it may be more or less easily
rewired depending on the different stakeholder’s resources, skills, and
creativity, on the credibility they enjoy, and on the necessity to develop
new goals. g
This conceptualization of expertise resonates with insights put forward
by scholars working in different fields in response to the highly complex,
dynamic, and interconnected world we live in. Important here are the
insights provided by Edwards (2010), working in the field of professional
learning, in reaction to the realization that people with expertise in a
given field are increasingly required to work outside the boundaries of
their particular institutions, to perform their expertise in collaboration
with specialists from different fields, with different training, methodolo
gies, and perspectives on the issues at hand. She argues that these realities
have led to a “relational turn in expertise” (2010), as they require “an
expertise which includes recognising and responding to the standpoints
of others and is in addition to the specialist knowledge at the core of each
distinct professional practice” (Edwards, 2010:2). Edwards thus seems to
believe that such social skills have now become necessary at a more gen
eral level rather than being required only for some types of expertise
(Kotzee, 2014). While it is indebted to Collins and Evans’ notion of
interactional expertise, this perspective has the merit of seeking to move
toward a more collective and dynamic understanding of expertise. y
g
p
Edwards’ understanding of expertise resonates with a more recent con
tribution from education and communication studies, where Engeström
(2018) has argued in favor of the need to transition to a “collaborative
and transformative expertise.” Expertise derives then from common
activities undertaken by different types of practitioners, who are flexible,
open to new knowledge, and capable of dealing with rapidly changing
environments. Particularly relevant for this book is Engeström’s (2018:1)
argument that “[c]ollaborative and transformative medical expertise is
continuous negotiation and hybridization of the insights of medical pro
fessionals and their patients. Without patients’ insights, accounts, and C. Egher 12 C. 1 Studying Expertise Online 1 Studying Expertise Online 1 Studying Expertise Online Egher actions, medical expertise would at best be merely top-down engineer
ing.” Rather than approaching expertise as an outstanding performance,
Engeström studies it “as everyday work” by focusing on mundane situa
tions when disturbances, breakdowns, and/or rapid transformations
interrupt daily routine. What is particularly interesting about his
approach is that he takes a collective activity as a unit of analysis for
expertise and considers it not only a matter of internalizing authoritative
knowledge, but also as conducive to new ways to produce and manifest
knowledgef Building upon the relational aspect of expertise in a different way,
Kotzee and Smit (2017), philosophers of science, tried to reconcile realist
and constructivist views by putting forward a new conceptualization. Their starting point is the realization that both perspectives conceive of
expertise as relational: in the first case, expertise is seen as consisting of
the relationship between an individual and an ability; in the second, it
consists of the relationship between an individual and others who
acknowledge him/her as an expert in a given domain. Their solution
relies on combining these elements to define expertise as one’s “ability
and/or level of knowledge…that significantly surpasses [that of others]”
(Kotzee & Smit, 2017:647). Given the highly specialized world in which
we live, knowing whose opinion to ask for and whose advice to trust on
a specific issue is highly necessary, and expertise thus understood fulfills
an important public function. Nevertheless, Kotzee and Smit fail to con
sider a third type of relationship, namely that which people with expertise
in a field develop with others who hold expertise in a different field. The
analytical movement between these perspectives on expertise and the
empirical materials collected for the study described in this book high
lighted the fact that this concept could be further refined, especially in
light of the transformations brought about by digitalization. It is to the
new approach to expertise that thus emerged that I now turn. 13 A New Approach to Expertise The engagement with theoretical perspectives on expertise and with the
empirical materials led to a new working definition of expertise, where
expertise is understood as a practical achievement, realized though coor
dination and affective labor among stakeholders who occupy multiple
and shifting positions across a complex ecosystem. Through this defini
tion I position myself among scholars who take a constructivist as well as
practice-oriented approach to expertise in ways which I briefly
describe below.hi This definition is vastly indebted to Mol’s (2002) concept of enact
ment, as the articulation and making manifest of substantial knowledge
and abilities through complex entanglements of people and tools are also
essential elements in this new conceptualization. Thus, the main differ
ence between her perspective and the one developed here may be seen as
a shift in focus dictated by the societal changes and practical transforma
tions that have taken place in recent years. Mol put forward the concept
of enactment because it allowed her to make clear that the distinction
between human subjects and natural objects is blurred: “like (human)
subjects, (natural) objects are framed as part of events that occur and
plays that are staged. If an object is real this is because it is part of a prac
tice. It is a reality enacted” (Mol, 2002:44, emphasis in the original). I
share with Mol the concern to foreground the multiplicity of the object
resulting from such practices, but I add to her perspective the emphasis
on the numerous, fragmented, and dynamic identities of the actors
involved, to use her terminology. These were not sufficiently considered,
since her account focused mainly on the professional identity of the med
ical professionals studied, although some intimations thereof can be
noted in her discussion of the life of “patients” outside the medical set
ting. Enactments or performances thus grant people and objects “fragile
identities” (Mol, 2002), which may shift from one site to the other. Mol’s perspective is combined in this new understanding of expertise
with the insights developed by Engeström (2018), in particular his
emphasis on the collective, dynamic, and adaptable character of exper
tise in current times. According to Engeström (2018), expertise requires 14 C. Egher C. Egher both vertical and horizontal types of movement, as knowledge in a
given area needs not only to be deepened, but has to be enriched with
knowledge from other related areas. 1 Studying Expertise Online interactions between people who may be equally endowed, but in other
domains, and who may have to work together to solve complex problems.h y
g
p
p
This perspective is further indebted to psychological and philosophical
perspectives on expertise, which highlight the importance of affective
reactions in relation to the development of expertise and argue that highly
competent people not only come to know things differently, but also to
feel differently toward them (Dreyfus & Dreyfus, 1986; Selinger &
Crease, 2006). While these perspectives mainly focus on the affective
responses an individual may have in relation to the practices at which s/
he is (becoming) an expert, the online interactions that will be described
in this book highlight the numerous emotions that arise and need to be
managed when different people interact and share insights online. These
emotions played an important role in the development of new knowledge
online, as interactions among people diagnosed could be short or longer-
lasting, superficial, or more substantial, depending on the emotions that
dominated such encounters, which shaped, in turn, the insights that were
shared. Thus, to perform lay expertise online, some people diagnosed had
to overcome their fright or reservations regarding computers and the
Internet, they had to try to make themselves likeable or intriguing enough
for others to interact with them, and they had to care for others, to respect
their views and experiences and to help them develop more positive emo
tions. This means that expertise is not solely a matter of intellectual and
cognitive processes, but that affective labor plays an important part in its
development, as it underlies people’s efforts to coordinate with others.h f
The focus on the multiple and shifting positions that stakeholders can
occupy in relation to the development of expertise is indebted to feminist
theories (Harding, 2004), which have emphasized the different meanings
an issue can acquire depending on the perspective of those who look
upon it, on the identity and position they occupy within a certain social
order. While Richmond (2017) has suggested to approach expertise by
considering the mediation work individuals or groups at the periphery
are forced to undertake in order to (effectively) communicate with those
at the center, her discussion of Lugones’ concept of “mobile positioning”
has been particularly useful here. A New Approach to Expertise By combining this perspective on
the dynamic character of expertise with Jasanoff’s (2004) call to pay
attention to the cultural and institutional elements that shape it, the
understanding of expertise as an achievement across a complex ecosys
tem emerged. This conceptualization prompts the analysts to look
beyond the practices they may be observing, to broaden their focus to
include perspectives on the rights and obligations of the different stake
holders involved, on the prevailing cultural norms and expectations
about their activities. Thus, how expertise is performed constitutes both
an illustration of and a reaction to specific historical developments, to
legal, political, and educational provisions and to the future visions
animating the field at a given moment in time. For instance, new legal
provisions about the acceptability of certain digital practices and the
use of online data may enable and deter people diagnosed with bipolar
disorder to share their insights and seek to engage in epistemic practices
using digital technologies. g
g
g
Edwards’ (2010) views on relational expertise have highlighted the
necessity for different types of professionals or stakeholders to work
together in order to achieve a common goal, but Engeström (2018) sets
the threshold somewhat lower by foregrounding coordination rather
than agreement. This means that the stakeholders involved need not
undergo a substantial transformation and come to share the same under
standing of the various concepts, processes, and tools involved, nor do
they have to use the same standards. What is important is that they agree
to suspend their differences in order to achieve a common goal (a mini
mal form of agreement) under conditions of uncertainty and, often,
within a limited time frame. Thus, one of the advantages of “coordina
tion” in relation to expertise is that it does not emphasize the epistemic
differences between individuals in regard to a specific topic or domain,
which, for instance, Kotzee and Smit’s (2017) conceptualization high
lights. It foregrounds, instead, the development of more similar and equal
(temporary) relations, thereby shifting the focus from people who have
and do not have substantial abilities and knowledge in a field to the 15 1 Studying Expertise Online 1 Studying Expertise Online Thus, by paying attention to the mul
tiple identities one inhabits, one may find ways to escape, obfuscate,
resist, or transform the norms and regulations of the communities one is 16 C. Egher C. Egher part of, and one may develop a more critical perspective on them. This
informed the realization that one and the same stakeholder may occupy
different positions within the ecosystem where expertise is developed and
that these different positionings may need to be both “stirred” and man
aged at different moments throughout this process. For instance, in the
field of mental health, numerous researchers, and medical professionals
are also patients or carers and fulfill executive functions whereby they
contribute to the decision-making regarding the allocation of
research funds.h The new insights that can be acquired by applying this new under
standing of expertise will be illustrated throughout the findings described
in each chapter. Since expertise is a practical achievement in a complex
ecosystem, the important role of historical developments and future
visions in animating the epistemic efforts of various stakeholders will be
brought to the fore in Chap. 2, while the ways in which expertise is
shaped by dominant social values will be discussed in Chap. 6. Chapters
3 and 4 will highlight two dimensions of coordination, both of which
are mediated in different ways by the design and affordances of digital
platforms. Whereas Chap. 3 focuses on the material and epistemic rela
tions that one and the same stakeholder needs to develop and maintain
across different digital spaces to successfully perform expertise, Chap. 4
shows that digital technologies inform coordination in substantial ways
by allowing for the coming together of disparate efforts undertaken by
many, sometimes fleeting, contributors. The multiple and shifting posi
tions that stakeholders can occupy in relation to expertise will be brought
to the fore across Chaps. 4 and 5, as it will be shown how some individu
als diagnosed with bipolar disorder manage to position themselves as
experts by experience, representatives of people diagnosed with bipolar
disorder, patients, advisors, and successful entrepreneurs. As we will see,
it is their successful orchestration of these multiple identities and their
ability to shift the focus from one to the other, depending on the char
acter of their interactions and of their goals, that have enabled them to
become highly influential. 1 Studying Expertise Online While affective practices could be encoun
tered across all the online exchanges studied, they will come to the fore
in Chap. 6, where it will be argued that affective labor shapes the devel
opment of expertise as it contributes to the development of new 17 1 Studying Expertise Online 1 Studying Expertise Online collectives and facilitates the exchange of intensely intimate experiences
about bipolar disorder.i As it must have become clear by now, the concept of performance, first
introduced by Goffman (1959/1990), was used to investigate expertise
about bipolar disorder on digital platforms. According to Goffman, social
interactions represent performances through which individuals seek to
produce desired impressions on their audiences by engaging in various
practices of self-revelation and concealment. Even though some scholars
(Barad, 2003) have understood performance in a very limited way, as
something akin to engaging in an activity, whereas others (Mol, 2002)
have preferred the notion of “enactment” which did not carry within it
the implicit distinction between a real self/“persona” or inner identity
versus a “mask” or external identity, this concept is used here in a broader
sense, which highlights the important coordination required for a par
ticular version of reality (Hafermalz et al., 2016) to be brought into
being. Given the focus on practices, materialities, and events that it allows
for, performance was deemed appropriate in view of the new conceptual
ization of expertise put forward. This way, the impact of the Internet and
its multifaceted character on epistemic practices can be brought to the
fore, as expertise about bipolar disorder can be approached as being dis
tributed across different online platforms, and shaped by the different
technologies available on them. Before moving on to that, however, there
are still a few elements which need to be introduced, namely bipolar dis
order and the role of the Internet in the study of expertise about this
condition, to which I now turn. Bipolar Disorder Mental health conditions are the result of complex interactions between
individuals with a certain biological make-up and their physical and social
environment. Symptoms of what would later be known as bipolar disor
der were first presented in the 1850s to the Académie de Medicine in Paris
by Baillarger, who called it “folie à double forme” (dual form insanity), and
Falret, who referred to it as “folie circulaire” (circular insanity) (Angst &
Sellaro, 2000). Both scientists agreed that this condition had a terrible 18 C. Egher C. Egher prognosis, and Falret postulated that it had a strong genetic basis. In the
1900s, Kraepelin was also pessimistic about the outcome of patients
exhibiting such symptoms, but observed that they also experienced inter
vals when no “abnormal” functioning could be detected. He used the term
“manic-depressive psychosis” to distinguish this mood condition from
“precocious madness,” which later became known as schizophrenia (Angst
& Marneros, 2001). The term “manic-depressive illness” was coined in the
1950s, which roughly coincides with the period when lithium salts started
their successful, still ongoing career as treatment for this condition, fol
lowing a discovery by Australian psychiatrist John Cade (Healy, 2008). In
the 1980s, the name was replaced by “bipolar disorder,” thought to be less
stigmatizing, but this change continues to be debated, as many medical
professionals and people diagnosed consider the former denomination to
convey the character of this condition more appropriately. pp p
Currently, the presumed causes of bipolar disorder represent a mix
ture of neurologic, genetic, and environmental factors, and this condi
tion is managed through a combination of medication, therapy, and
counseling. Because of the similarity in symptoms with major depres
sion, bipolar disorder remains difficult to diagnose correctly, and often
many years (5–12) and numerous encounters with various mental health
professionals are necessary. In the Diagnostic and Statistical Manual of
Mental Disorders (DSM-5, 2013), this condition is coded as bipolar
single manic, bipolar manic, bipolar depressed, bipolar mixed, each cat
egory containing several subtypes. The International Classification of
Disease and Health Related Problems (ICD-10, 2010) groups condi
tions based on their relatedness to each other, so different forms of bipo
lar disorder are spread under the headings of various types of mental
health conditions. An important distinction both in regard to diagnostic
difficulties but also in relation to treatment lies between the types bipo
lar I disorder and bipolar II disorder. Bipolar Disorder These two types differ mainly in
the severity of the manic episodes experienced. Whereas bipolar I disor
der involves severe manic episodes, lasting for several days and at times
requiring hospitalization, those diagnosed with bipolar II disorder expe
rience hypomanic states rather than full-blown manic episodes (Grande
et al., 2016). Even though the enormous increase in people diagnosed is 19 1 The exact numbers may differ depending on the studies consulted and on the forms of bipolar
disorder included. 1 Studying Expertise Online 1 Studying Expertise Online often ascribed to improved diagnostic tools, it may also be due to a posi
tive re-evaluation of this condition. Martin (2009), for example, argues
this is the result of a close connection between the values of capitalism
and some of the traits associated with manic episodes: creativity, passion,
dedication, and intense activity. In contrast, others have explained the
growing number of people diagnosed with this condition by arguing
that the values of capitalism lead to stress, anxiety, and depression
(Hidaka, 2012), while yet others have linked this increase to tendencies
to medicalize social issues (Esposito & Perez, 2014) and to pathologize
variations in human experiences (Horwitz & Wakefield, 2007;
Scott, 2006). Bipolar disorder has also been affected by recent developments in per
sonalized and precision medicine, which provide visions of medical inter
ventions tailored to the specific needs and circumstances of individuals
(Rose, 2018). Doubts about the scientific character of expertise about
mental health have led professionals to embrace perspectives and proce
dures which have deeply anchored this condition in biology, in processes
which could be identified, measured, and acted upon through targeted
approaches. As such, in the aftermath of the Human Genome Project
(1990–2003), numerous research projects (Alda et al., 2005; Cruceanu
et al., 2009; MacQueen et al., 2001) have been undertaken, which have
sought to identity the phenotypes and genetic markers underlying bipo
lar disorder, the predictive factors of response among different (sub)
groups of people diagnosed, and new drug targets. At present, however,
bipolar disorder seems to be characterized by too great genetic and phe
notypic heterogeneity for these insights to be very helpful (Rose, 2018). Furthermore, since treatment response in many of these studies was mea
sured with different instruments, the translation of these new insights
into clinical practice is likely to take some time and to require collabora
tion and intense efforts among a very broad range of professionals,
including not only medical specialists but also engineers and computer
scientists. 20 C. Egher C. Egher
1
2018
2000
2005 21 1 Studying Expertise Online Studying Expertise About Bipolar
Disorder Online While the Internet is intensively used these days for health-related pur
poses, it continues to divide opinions about the ways in which it shapes
(mental) healthcare, and about the benefits and disadvantages of its use
for different stakeholders. Thus, its proponents, many of which are gov
ernment officials, argue that it may help solve the current crisis in mental
healthcare, brought about by a growing number of people diagnosed and
lower budget funds available for this sector, as it may enable the provision
of good quality and cost-effective care. These optimistic views have
acquired renewed impetus with the move toward personalized and preci
sion medicine and with the hopes generated by big data analytics. These
have changed the ways in which health and disease are conceptualized
and have emphasized the need for (self)surveillance and for collecting
highly diverse types of data both from people diagnosed and from those
not (yet) diagnosed (Hogle, 2016; Prainsack, 2018). In this context,
active forms of patienthood have been encouraged not only by public
stakeholders, but also by commercial actors, which have started to become
more involved in healthcare (Sharon, 2016), as we shall see in the next
chapter. p
Critics (Brown & Baker, 2012; Lupton, 2018; Neff, 2013) have
argued, however, that such approaches constitute strategies through
which governments place greater responsibilities upon citizens in a con
text where social provisions are cut and where a market logic is increas
ingly used to guide the provision of mental healthcare. Some
commentators have also criticized the users’ engagement with digital
technologies as a form of free labor (Mitchell & Waldby, 2010; Terranova,
2000; Waldby & Cooper, 2008), where people are encouraged to con
stantly monitor themselves in pervasive and invasive ways, but are
required to give up ownership over their data and any claims over poten
tial profits that can be made from them. Others have also worried about
the different ways in which such data may be used and how they may
affect the individual users of such technologies and the prescription
practices of medical professionals. Such concerns are particularly C. Egher 22 C. Egher well-founded in the US, where Section 2713 of the Affordable Care Act
stipulates the establishment of “guidelines to permit a health insurance
plan to use value-based insurance design” (National Conference of State
Legislatures, 2018). 1 Studying Expertise Online 1 Studying Expertise Online information provided in view of their values. In contrast, smaller stake
holders may need to settle for a platform they can afford or select a design
that will attract many visitors, and attune their message to the (prospec
tive) sponsors' preferences. These choices may affect a platform’s index
score with a search engine, which can have profound consequences, as
studies about people’s online search behavior indicate that users often do
not look beyond the first few results pages (Bar-Ilan et al., 2006;
Höchstötter & Lewandowski, 2009). Thus, the type of platform selected
and its design significantly influence how information is provided, and
the types of knowledge made available. In the early days, the Internet was also seen by some scholars as an
instrument of globalization, as they believed that it would help bring
about cultural homogeneity through the seamless flow of information
among people from all corners of the world and through the subsequent
effacement of local practices in favor of cosmopolitan approaches
(Featherstone et al., 1995). After 2000, however, a growing number of
anthropologists and media scholars have drawn attention to the specific
contexts in which online contributions are made, and have argued that
social and cultural norms importantly shape people’s online behaviors
(Ardichvili et al., 2006; Fox et al., 2005; Miller & Slater, 2000). Yet little
is currently known about the ways in which local and cultural markers
shape online exchanges about mental health, and this is one of the aspects
that this study addresses by comparing how expertise about bipolar dis
order is performed on American and French platforms. In so doing, it
focuses on the ways in which online contributors from these countries
use different digital platforms and the affordances available on them to
determine how local perspectives shape people’s orientations toward
bipolar disorder online. More details about the methodological approaches
used are provided below. Studying Expertise About Bipolar
Disorder Online Thus, while some believe the Internet can be har
nessed to help solve numerous problems in mental healthcare, others
worry about the effects of online practices, about the ways in which
people diagnosed understand themselves and their condition, and about
the ways in which the Internet can shape relations between them and
medical professionals. This book contributes toward a better under
standing of these aspects by engaging with two recurrent ideas about the
Internet’s potential, namely its ability to democratize and to help trans
mit local and cultural norms. In the early days of the Internet, some medical sociologists and media
scholars thought that it would contribute to the democratization of rela
tions between medical professionals and their patients (Hardey, 1999;
Poster, 2001) by allowing people diagnosed to access medical informa
tion previously reserved strictly for medical professionals, by enabling
them to learn about alternative approaches to mental health, and by facil
itating their contributions to epistemic practices. In the meantime, more
nuanced studies (Nettleton & Burrows, 2003; Wyatt et al., 2016) have
been published, which have problematized the Internet’s democratizing
potential, highlighting the multifaceted character of this medium, and
the heterogeneity of people who search for and contribute to health-
related information online. Scholars have also argued that the Internet
leads to new forms of inequality engendered by various algorithms,
including those of search engines, which determine the visibility of digi
tal platforms (Bishop, 2018; Hargittai, 2007; Pasquinelli, 2009). The resources available to people are thus not equally distributed, as
online communication skills, familiarity with various technologies, and
the size and impact of on- and offline (professional) networks can differ
considerably. Furthermore, despite their increasing popularity, interac
tive digital platforms have not replaced non-interactive websites, but co-
exist with them. The choice of an interactive or non-interactive platform
is determined not only by the goals and preferences of users, but also by
their resources and position. Thus, important institutions with a gener
ous budget can invest in their platform, but need to shape the 23 Methods and Sources Methodologically, this book draws upon qualitative empirical material of
two types: data collected from different digital platforms on bipolar dis
order and articles from medical journals. Digital platforms are spaces 24 C. Egher C. Egher which are socially created through interactions and practices between
numerous stakeholders. They can be endowed with different affordances,
and require different levels of skills and resources by their users (Drucker,
2011). Affordances denote mechanisms which are conceptually relational
and which place different opportunities and constraints on both users
and artifacts (Davis & Chouinard, 2016). This means that not all plat
forms and functions embedded on them are equally accessible to all users. Online contributions are therefore informed not only by the availability
or absence of various functions, such as the ability to comment and to
upload texts, graphs, images, and videos, but also by the users’ skills,
preferences, and attitudes toward these technologies as well as by what
they hope to achieve through their sharing practices. The advent of Web
2.0 has heightened the profile of interactive platforms, which are charac
terized by a high media convergence (Herring, 2012), meaning that
information is increasingly provided through a combination of text with
other visual, audio, and video materials. Yet, such platforms exist in an
environment that they share with non-interactive platforms, which are
less dynamic, complex, and open. Non-interactive platforms dedicated to
mental health generally include websites belonging to influential institu
tions, be they governmental bodies, or patient organizations, which
importantly shape the provision of treatment and care for people diag
nosed with bipolar disorder. While the access to the information they
provide is public, the contributors are selected by each particular institu
tion and are generally medical professionals. In order to understand how the Internet shapes the performance of
expertise about bipolar disorder, both interactive and non-interactive
online platforms were selected. The selection was based upon a novel
methodological approach, as I aimed to reproduce the behavior of aver
age Internet users and conducted queries using the index of the search
engine Google as a relevance indicator. A list was thus made of the online
platforms mentioned on the first 30 pages of results. Methods and Sources This list was subse
quently filtered to exclude online platforms in other languages than
English and French, to eliminate multiple pointers to the same item and
websites where the content was not focused on bipolar disorder or which
were not free to access, but required registration or payment. Since lan
guage is not a reliable indicator, the domain of each platform was 25 1 Studying Expertise Online 1 Studying Expertise Online subsequently checked and only the online platforms were retained where
American and French official institutions were mentioned. This was done
to ensure that online data were collected from contributors in these two
countries. Blogs and fora which had been established for less than one
year at the moment when the selection took place (September 2014),
which did not allow the information available on their platforms to be
used for research purposes, and which had few contributors (<10) were
also filtered out. i
The table below gives an overview of the online platforms from which
data were collected: List of selected platforms for data collection List of selected platforms for data collection Platform name and country
Platform type Platform management
National Institute of Mental
Health—US
Non-
interactive
Governmental agency
Bipolar Burble—US
Blog
Person diagnosed
Bipolar Happens!—US
Blog
Person diagnosed
Bp Hope—US
Forum
People diagnosed
La Haute Autorité de Santé
(HAS)—France
Non-
interactive
Governmental agency
Doctissimo—France
Forum
People diagnosed; mediated by
medical professionals
Le Forum des Bipotes—France
Forum
People diagnosed Data were collected at different moments between June 2014 and
September 2018, because online contributors often change their mind
about the online reactions they provide and amend them (multiple times)
or remove them altogether at later moments. By collecting the data from
the same platforms in different periods, it was possible to identify
instances when comments had been edited or removed by the people
who had written them and thus to respect their wishes by removing them
from the data collected. These data were supplemented by the collection
of newspaper and digital articles and communications (Chap. 2), relevant
medical articles (Chap. 4), and online interviews with two highly influ
ential bloggers on bipolar disorder (Chap. 5). To understand how exper
tise about bipolar disorder was performed on these different online
platforms and how meaning and culture were (re)produced online, quali
tative methods which could provide “deep knowledge” of such dynamic 26 C. Egher and situated practices (Markham, 2016) were used. The specific methods
used in each chapter vary, but they include computer-mediated discourse
analysis (Chap. 4), thematic analysis (Chaps. 3, 5, and 6), and conversa
tion analysis (Chap. 6) adapted to online contexts. y
p
p
The approval of the Ethical Review Committee Inner City (ERCIC) of
Maastricht University was sought and obtained on April 6, 2016. 1 Studying Expertise Online It was
not feasible to obtain informed consent from all the online contributors
who posted information on the platforms from which data were. This
was partly due to their sheer number and partly due to the fact that the
contributions collected span roughly 10 years, a period in which many
people who shared their insights may have stopped using these platforms
or may have changed their usernames. Since data were collected from
platforms with a public character, the study upon which this book is
based meets current ethical guidelines for online research. For instance,
according to the British Psychological Society (BPS, 2013:7), “where it is
reasonable to argue that there is likely no perception and/or expectation
of privacy (or where scientific/social value and/or research validity con
siderations are deemed to justify undisclosed observation), use of research
data without gaining valid consent may be justifiable.” To protect the
online contributors from any possible harm, the data were anonymized
by replacing the usernames with pseudonyms, by making slight altera
tions to the dates of the comments directly cited, and by removing the
names of specific items or medications. Even though more substan
tial changes to the content of each of these comments would have further
diminished the chances of re-identification, a decision was made against
this approach. This decision was informed by the consideration that only
the authors of the online contributions are entitled to operate changes to
them. Furthermore, even slight modifications to their content may have
led to shifts in meaning and possible interpretations, which was deemed
particularly undesirable, given that this book’s aim is to show how online
contributors diagnosed with bipolar disorder engage in knowledge prac
tices. All quotes are, therefore, provided verbatim. Given their public
standing, an exception to anonymization was made in the case of the two
bloggers discussed in Chap. 5. Both bloggers were contacted and they are
referred to in this book according to their own indication 27 1 Studying Expertise Online C. Egher Chapter 4 traces how authoritative medical knowledge, such as that
described above, permeates different areas of society, and becomes amena
ble to multiple usages and interpretations. It explores the Internet’s democ
ratizing potential by considering how people diagnosed with bipolar
disorder re-appropriate medical perspectives and combine them with per
sonal insights to contribute to the development of new knowledge through
dynamic and even fleeting online exchanges on blogs and fora. This chapter
is based on two types of sources: articles published by scientists in medical
journals and data collected from blogs and fora, where people diagnosed
shared their treatment experiences. I use de Certeau’s theory (1988) of cre
ative tactics in everyday life to analyze the online data, as it allows to move
beyond domination and resistance as characterizing the main positions
people diagnosed can develop in relation to dominant forms of knowledge,
and to identify more subtle ways through which they can make their agency
manifest. The analysis thus indicates that through their online interactions,
people diagnosed move beyond the performance of lay expertise and col
lectively generate what I call “digitally informed hypotheses” in areas where
the currently available medical knowledge on the effects and side effects of
medications is insufficient. In so doing, the Internet affords individuals
diagnosed a voice, yet one which can have a broad epistemic impact only
when heard and taken seriously by researchers. Chapter 5 shows that the Internet does not always favor the powerful, but
this still does not mean that it has a democratizing effect. It traces the online
activities of two bloggers diagnosed with bipolar disorder using the concept
of interactional expertise developed by Collins and Evans (2002). This chap
ter argues that by combining medical knowledge with their situated experi
ences, and by utilizing the affordances of blogs, these bloggers have become
a new type of stakeholder, what I call “online expert mediators.” This chap
ter makes a theoretical contribution, as the notion of interactional expertise
is extended by taking into consideration the role of the medium through
which interactional expertise is displayed and by showing that its bi-direc
tional character is more substantial than Collins and Evans initially envis
aged. The analysis further indicates that the high standing of online expert
mediators is not the result of a subversive use of the Internet, but of a
dynamic alliance with “traditional” experts and of a strong media presence. Outline of the Remaining Chapters Chapter 2 traces the development of expertise about mental health from
the early days of the asylum to the hopes and anxieties that are currently
generated by the (upcoming) use of digital and AI-based technologies in
the provision of mental healthcare. Based on a review of relevant histori
cal and sociological works, it highlights the trajectory that expertise about
mental health has undergone from the focus on heredity in the nine
teenth century to the more recent embrace of genetics. In so doing, it
engages with the jurisdictional struggles that emerged between psychia
try, psychology, and other disciplines, with the development of self-help
and support groups in the US and France and with the precarious state
that characterizes the provision of mental healthcare in both countries
these days. By building an arch between the past and future of expertise
about mental healthcare, this chapter provides rich contextual informa
tion which is important to better understand the online practices dis
cussed in the remainder of the book and the similarities and differences
among French and American contributors that will be described. g
Chapter 3 describes how expertise about bipolar disorder is performed
by The National Institute of Mental Health (NIMH) in the US and La
Haute Autorité de Santé (HAS) in France. The analysis helps us under
stand how expertise is performed online by influential stakeholders,
which possess substantial resources and have numerous options to choose
from in terms of digital practices. The information NIMH and HAS put
forward online about bipolar disorder was analyzed by combining insights
from Latour (1987) and media studies with a dramaturgical perspective
(Goffman, 1959/1990). This approach allowed for a better understand
ing of the material and epistemic relations that these institutions had to
develop and manage online to successfully perform expertise. I argue that
both stakeholders are rather reluctant Internet users, who perform exper
tise in a highly conservative fashion, which in turn allows them to articu
late the knowledge currently available on this condition as stable and
precise. While both institutions use similar performative techniques, they
adapt them to subtly redefine bipolar disorder in ways which seem better
aligned to the priorities characterizing their national health system and
their institutional prerogatives and goals. 28 C. Egher C. Egher g
Chapter 6 builds upon recent calls made by medical sociologists and
STS scholars to focus on the relational character of illness, thereby 1 Studying Expertise Online 1 Studying Expertise Online 29 exploring the Internet’s potential for solidarity. It shows that mental
health-related online exchanges enable people diagnosed with bipolar
disorder to perform solidarity. This has important epistemic conse
quences, because online solidaristic practices allow individuals both to
perform lay expertise and to contribute to its collective development, as
new knowledge is distilled from the personal experiences and insights
that are brought together. Such activities are underpinned by affective
labor, which facilitates the emergence of digital biocommunities and
the development of lay expertise. Based on Prainsack and Buyx (2017)’s
concept of solidarity and Gershon’s (2010) notion of idioms of prac
tice, the notion of digital biocommunities denotes a new type of sub
group, developed not only upon a common diagnosis, life circumstances,
experiences, perspectives, and values, but also on similar engagements
with the technologies of fora. By putting forward this concept, I high
light that despite an increased focus on individualization in mental
healthcare, people diagnosed experience their condition in relational
terms, even in regard to lived, embodied experiences.i Chapter 7 brings together the main findings and conclusions that have
emerged from the study of the online performance of expertise about
bipolar disorder described in this book. By building upon the theoretical
perspectives discussed in this introductory chapter and by combining
them with insights acquired from the empirical chapters, a new perspec
tive on expertise was put forward. This new approach conceives of exper
tise as a practical and collective achievement realized through coordination
and affective labor among stakeholders who occupy multiple and shifting
positions within a complex ecosystem. This approach seeks to do justice
to the important ways in which cultural and institutional factors shape
expertise, while acknowledging the agency and complex identities of rel
evant stakeholders, who can be in turn or at the same time individuals
diagnosed with a condition, professionals, scientific contributors, and
information mediators. I discuss the significance of the main findings by
considering them within the context of broader transformations that
digital technologies have contributed to in processes of knowledge pro
duction, circulation, and evaluation, and which were already touched
upon in Chap. 2. I argue that we need to move beyond rather simplistic
approaches which see the Internet either as a quick technological fix or a
postmodern version of Pandora’s box. 30 C. Egher 1 Studying Expertise Online 31 Collins, H., & Evans, R. (2007). Re-thinking Expertise. The University of
Chicago Press. Collins, H., & Evans, R. (2007). Re-thinking Expertise. The University of
Chicago Press. g
ins, H., Evans, R., & Weinel, M. (2017). Interactional Expertise. In U. Felt Collins, H., Evans, R., & Weinel, M. (2017). Interactional Expertise. In U. Felt,
R. Fouché, C. Miller, & L. Smith-Doerr (Eds.), The Handbook of Science and
Technology Studies (4th ed., pp. 765–792). The MIT Press. h
Cruceanu, C., Alda, M., & Turecki, G. (2009). Lithium: A Key to the Genetics
of Bipolar Disorder. Genome Medicine, 1(8), 79.hf Davis, J., & Chouinard, J. (2016). Theorizing Affordances: From Request to
Refuse. Bulletin of Science, Technology & Society, 36(4), 241–248. d C
M (1988) Th P
i
fE
d
Lif U i
i
fC lif
i P h
f
y
y
f
y
Dedding, C., van Doorn, R., Winker, L., & Reis, R. (2011). How Will e-health
Affect Patient Participation in the Clinic? A review of e-health Studies and
the Current Evidence for Changes in the Relationship between Medical
Professionals and Patients. Social Science & Medicine, 72(1), 49–53. Dehue, T. (2008). De Depressie-Epidemie. Augustus. Dehue, T. (2008). De Depressie-Epidemie. Augustus. Dreyfus, H., & Dreyfus, S. (1986). Mind Over Machine: The Power of Human
Intuition and Expertise in the Era of the Computer. Free Press.h Drucker, J. (2011). Humanities Approaches to Interface Theory. Culture
Machine, 12. Available at: http://www.culturemachine.net/index.php/cm/
article/view/434/462 Edwards, A. (2010). Being an Expert Professional Practitioner. The Relational Turn
in Expertise. Springer. Engeström, Y. (2018). Expertise in Transition. Expansive Learning in Medical
Work. Cambridge University Press.i Esposito, L., & Perez, F. (2014). Neoliberalism and the Commodification of
Mental Health. Humanity & Society, 38(4), 414–442.i Evetts, J., Mieg, H., & Felt, U. (2006). Professionalization, Scientific Expertise
and Elitism: A Sociological Perspective. In K. Ericsson, N. Charness,
P. Feltovich, & R. Hoffman (Eds.), The Cambridge Handbook of Expertise and
Expert Performance (pp. 105–123). Cambridge University Press. Eyal, G. (2013). For a Sociology of Expertise: The Social Origins of the Autism
Epidemic. American Journal of Sociology, 118(4), 863–907. Eyal, G., & Hart, B. (2010). How Parents of Autistic Children Became “Experts
on Their Own Children”: Notes Towards a Sociology of Expertise. Annual
Conference of the Berkeley Journal of Sociology, 54, 1–38. Available at https://
works.bepress.com/gil_eyal/1/ Eyal, G., Hart, B., Onculer, E., Oren, N., & Rossi, N. (2010). References Akrich, M. (2010). From Communities of Practice to Epistemic Communities:
Health Mobilizations on the Internet. Sociological Research Online, 15(2), 10. Available at: http://www.socresonline.org.uk/15/2/10.html Alda, M., Grof, P., Rouleau, G., Turecki, G., & Young, T. (2005). Investigating
Responders to Lithium Prophylaxis as a Strategy for Mapping Susceptibility
Genes for Bipolar Disorder. Progress in Neuro-Psychopharmacology and
Biological Psychiatry, 29(6), 1038–1045. American Psychiatric Association. (2013). Diagnostic and Statistical Manual of
Mental Disorders, fifth edition (DSM-5). i
Angst, J., & Marneros, A. (2001). Bipolarity from Ancient to Modern Times:
Conception, Birth and Rebirth. Journal of Affective Disorders, 67, 3–19. Angst, J., & Sellaro, R. (2000). Historical Perspectives and Natural History of
Bipolar Disorder. Biological Psychiatry, 48, 445–457. Ardichvili, A., Maurer, M., Wei, L., Wentling, T., & Stuedemann, R. (2006). Cultural Influences on Knowledge Sharing Through Online Communities of
Practice. Journal of Knowledge Management, 10(1), 94–107. Barad, K. (2003). Posthumanist Performativity: Toward an Understanding of
How Matter Comes to Matter. Signs, 28(3), 801–831. Bar-Ilan, J., Mat-Hassan, M., & Levene, M. (2006). Methods for Comparing
Rankings of Search Engine Results. Computer Networks, 50, 1448–1463. Barnes, B. (1974). Scientific Knowledge and Sociological Theory. Routledge &
Kegan Paul.h Bijker, W. (1995). Of Bicycles, Bakelites, and Bulbs: Toward a Theory of the
Sociotechnical Change. The MIT Press. gh
Bishop, S. (2018). Anxiety, Panic and Self-Optimization: Inequalities and the
YouTube Algorithm. Convergence, 24(1), 69–84. g
g
Bloor, D. (1976). Knowledge and Social Imagery. Routledge & Kegan Paul. British Psychological Society (BPS). (2013). Ethics Guidelines for Internet-
Mediated Research. British Psychological Society. Brown, B., & Baker, S. (2012). Responsible Citizens. Individuals, Health and
Policy under Neoliberalism. Anthem Press. Carron-Arthur, B., Reynolds, J., Bennett, K., Bennett, A., Cunningham, J., &
Griffiths, K. (2016). Community Structure of a Mental Health Internet
Support Group: Modularity in User Thread Participation. JMIR Mental
Health, 3(2), e20. Collins, H., & Evans, R. (2002). The Third Wave of Science Studies. Studies of
Expertise and Experience. Social Studies of Science, 32(2), 235–296. 1 Studying Expertise Online 1 Studying Expertise Online The Autism
Matrix: The Social Origins of the Autism Epidemic. Polity Press. h
g
f
p
Eysenbach, G., Powell, J., Englesakis, M., Rizo, C., & Stern, A. (2004). Health
Related Virtual Communities and Electronic Support Groups: Systematic 32 C. Egher Review of The Effects of Online Peer to Peer Interactions. British Medical
Journal, 328(7449), 1166–1170. Review of The Effects of Online Peer to Peer Interactions. British Medical Journal, 328(7449), 1166–1170. Featherstone, M., Lash, S., & Robertson, R. (Eds.). (1995). Global Modernities. Sage Publications. Featherstone, M., Lash, S., & Robertson, R. (Eds.). (1995). Global Modernities. Sage Publications. Foucault, M. (1972/2010). History of Madness. Routledge. Translated by
Jonathan Murphy and Jean Khalfa.h Fox, N., Ward, K., & O’Rourke, A. (2005). The “Expert Patient”: Empowerment
or Medical Dominance? The Case of Weight Loss, Pharmaceutical Drugs and
the Internet. Social Science & Medicine, 69, 1299–1309. Fuller, S. (2017). The Post-Truth about Philosophy and Rhetoric. Philosophy &
Rhetoric, 50(4), 473–482. Gershon, I. (2010). The Breakup 2.0. Disconnecting over New Media. Cornell
University Press. E-book. Goffman, E. (1959/1990). The Presentation of Self in Everyday Life (8th ed.). Penguin.Mol.h Grande, I., Berk, M., Birmaher, B., & Vieta, E. (2016). Bipolar Disorder. The
Lancet, 387(10027), 1561–1572. Hacking, I. (1995). Rewriting the Soul: Multiple Personality and the Sciences of
Memory. Princeton University Press.h y
y
Hacking, I. (1999). The Social Construction of What? Harvard University Press. Hafermalz, E., Riemer, K., & Boell, S. (2016). Enactment of Performance? A
Non-dualist Reading of Goffman. In L. Introna, D. Kavanagh, S. Kelly,
W. Orlikowski, & S. Scott (Eds.), Beyond Interpretivism? New Encounters with
Technology and Organization (pp. 167–181). Springer.h Hacking, I. (1999). The Social Construction of What? Harvard University Press. Hafermalz, E., Riemer, K., & Boell, S. (2016). Enactment of Performance? A f Hardey, M. (1999). Doctor in the House: The Internet as A Source of Lay
Health Knowledge and the Challenge to Expertise. Sociology of Health &
Illness, 21, 820–835. Harding, S. (Ed.). (2004). The Feminist Standpoint Theory Reader. Intellectual
and Political Controversies. Routledge.h Hargittai, E. (2007). The Social, Political, Economic and Cultural Dimensions
of Search Engines: An Introduction. Journal of Computer-Mediated
Communication, 12(3), 769–777. Healy, D. (2008). Mania: A Short History of Bipolar Disorder. John Hopkins
University Press. Herring, S. (2012). Discourse in Web 2.0.: Familiar, Reconfigured, and
Emergent. In D. Tannen & A. 1 Studying Expertise Online Tester (Eds.), Georgetown University Round
Table on Languages and Linguistics 2011: Discourse 2.0.: Language and new
media (pp. 1–29). Georgetown University Press. pp
g
Hidaka, B. (2012). Depression as a Disease of Modernity: Explanations for
Increasing Prevalence. Journal of Affective Disorders, 140, 205–214. 1 Studying Expertise Online 1 Studying Expertise Online 33 Höchstötter, N., & Lewandowski, D. (2009). What Users See — Structures in
Search Engine Results Pages. Information Sciences, 179, 1796–1812. Hogle, L. (2016). Data-intensive Resourcing in Healthcare. BioSocieties,
11(3), 372–393.ih Horwitz, A., & Wakefield, J. (2007). The Loss of Sadness. How Psychiatry
Transformed Normal Sorrow into Depressive Disorder. Oxford University Press. Hu, Y., & Sundar, S. (2010). Effects of Online Health Sources on Credibility
and Behavioral Intentions. Communication Research, 31(1), 105–132. Hu, Y., & Sundar, S. (2010). Effects of Online Health Sources on Credibility
and Behavioral Intentions. Communication Research, 31(1), 105–132. Illich, I. (1977). Disabling Professions. Marion Boyars. Illich, I. (1977). Disabling Professions. Marion Boyars.f Jasanoff, S. (2003). Breaking the Waves in Science Studies: Comment on
H.M. Collins and Robert Evans, ‘The Third Wave of Science Studies’. Social
Studies of Science, 33(3), 389–400. f
Jasanoff, S. (Ed.). (2004). States of Knowledge. The Co-Production of Science and
Social Order. Routledge. g
Kitcher, P. (2011). Science in a Democratic Society. Prometheus Books. Kivits, J. (2009). Everyday Health and the Internet: A Mediated Health
Perspective on Health Information Seeking. Sociology of Health and Illness,
31(5), 673–687. Kleinman, A. (1988). Rethinking Psychiatry from Cultural Category to Personal
Experience. The Free Press. Koppl, R. (2010). The Social Construction of Expertise. Society, 47(3), 220–226.f Kleinman, A. (1988). Rethinking Psychiatry from Cultural Category to Personal
Experience. The Free Press.h Kotzee, B. (2014). Differentiating Forms of Professional Expertise. In M. Young
& J. Muller (Eds.), Knowledge, Expertise and the Professions (pp. 61–76). Routledge. Kotzee, B., & Smit, J. (2017). Two Social Dimensions of Expertise. Journal of
Philosophy of Education, 51(3), 640–654.f Lakoff, A. (2005). Pharmaceutical Reason: Knowledge and Value in Global
Psychiatry. Cambridge University Press. Latour, B. (1987). Science in Action. How to Follow Scientists and Engineers
Through Society. Harvard University Press. h
Lupton, D. (2018). Digital Health. Routledge. Lynch, D. (2017). STS, Symmetry and Post-Truth. Social Studies of Science,
47(4), 593–599. MacQueen, G., Young, L., & Joffe, R. (2001). A Review of the Psychosocial
Outcomes in Patients with Bipolar Disorder. Acta Psychiatrica Scandinavica,
103(3), 163–170. Markham, A. (2016). Method as Ethic, Ethic as Method. Journal of Information
Ethics, 15(2), 37–55. Martin, E. (2009). Bipolar Expeditions. Mania and Depression in American
Culture (2nd ed.). Princeton University Press. 34 C. Egher McNeil, M. (1998). Gender, Expertise and Feminism. In R. Williams,
W. Faulkner, & J. Fleck (Eds.), Exploring Expertise: Issues and Perspectives
(pp. 55–79). Macmillan Press. Metzl, J. 1 Studying Expertise Online 35 Radder, H. (2018, April 16). Post-Truth en de Vraag Wat Goede Wetenschap Is. Science Guide. Retrieved April 20, 2018, from https://www.scienceguide. nl/2018/04/de-vraag-wat-is-goede-wetenschap-blijft-relevant/ Richmond, K. (2017, August 29). Towards a Feminist Theory of Expertise. The
Postgraduate Gender Research Network of Scotland. Retrieved May 21, 2018,
from https://pgrnscotland.wordpress.com/2017/08/29/towards-a-feminist-
theory-of-expertise/ Rose, N. (1992). Engineering the Human Soul: Analyzing Psychological
Expertise. Science in Context, 5(2), 351–369. Rose, N. (2018). Our Psychiatric Futures. Polity.i Saks, M. (2012). Defining a Profession. Professions and Professionalism, 2(11), 1–10.hi Scott, S. (2006). The Medicalisation of Shyness: From Social Misfits to Social
Fitness. Sociology of Health & Illness, 28(2), 133–153.h Selinger, E., & Crease, R. (2006). Dreyfus on Expertise. The Limits of
Phenomenological Analysis. In E. Selinger & R. Crease (Eds.), The Philosophy
of Expertise. Columbia University Press.f Shapin S, Schaffer S (1985/2011). Leviathan and the Air-Pump. Hobbes, Boyle,
and the Experimental Life. 2nd ed. Princeton: Princeton University Press.h Sharon, T. (2016). The Googlization of Health Research: From Disruptive
Innovation to Disruptive Ethics. Personalized Medicine, 13(6), 563–574. Sismondo, S. (2017). Post-Truth? Social Studies of Science, 47(1), 3–6. Stommel, W., & Lamerichs, J. (2014). Interaction in Online Support Groups. Advice and Beyond. In H. Hamilton & W. Chou (Eds.), The Routledge
Handbook of Language and Health Communication (pp. 198–211). Routledge. Susskind, R., & Susskind, D. (2015). The Future of the Professions. How Technology
Will Transform the Work of Human Experts. Oxford University Press. Stommel, W., & Lamerichs, J. (2014). Interaction in Online Support Groups. Advice and Beyond. In H. Hamilton & W. Chou (Eds.), The Routledge
Handbook of Language and Health Communication (pp. 198–211). Routledge. S
ki d R & S
ki d D (2015) Th F
f h P f
i
H
T h
l Susskind, R., & Susskind, D. (2015). The Future of the Professions. How Technology
Will Transform the Work of Human Experts. Oxford University Press. Terranova, T. (2000). Free Labor: Producing Culture for the Digital Economy. Social Text 63, 18(2), 33–58.hh Topol, E. (2015). The Patient Will See You Now. The Future of Medicine Is in Your
Hands. Basic Books. Tutton, R., & Prainsack, B. (2011). Enterprising or Altruistic Selves? Making
Up Research Subjects in Genetics Research. Sociology of Health & Illness,
33(7), 1081–1095. Vaugrente, C. (2018, March 29). Troubles Bipolaires: Les Différences Femmes/
Hommes. Retrieved from https://www.e-sante.fr/troubles-bipolaires-les-
differences-femmeshommes/actualite/615617. Accessed on April 15, 2018. Versteeg, W., & Te Molder, H. (2018). 1 Studying Expertise Online (2009). The Protest Psychosis. How Schizophrenia Became a Black Disease. Beacon Press. Miller, D., & Slater, D. (2000). The Internet. An Ethnographic Approach. Berg. Mitchell, R., & Waldby, C. (2010). National Biobanks: Clinical Labor, Risk
Production, and the Creation of Biovalue. Science, Technology & Human
Values, 35(3), 330–355. Mol, A. (2002). The Body Multiple: Ontology in Medical Practice. Duke
University Press. Naslund, J., Aschbrenner, K., Marsch, L., & Bartels, S. (2016). The Future of
Mental Health Care: Peer-To-Peer Support and Social Media. Epidemiology
and Psychiatric Sciences, 25(2), 113–122. National Conference of State Legislatures. (2018, February 20). Value-Based
Insurance Design. Retrieved from http://www.ncsl.org/research/health/
value-based-insurance-design.aspx. Accessed on December 30, 2018.f Neff, G. (2013). Why Big Data Won’t Cure Us. Big Data, 1, 117–123. PMID:25161827. Nettleton, S., & Burrows, R. (2003). E-Scaped Medicine? Information,
Reflexivity, and Health. Critical Social Policy, 23(2), 165–185. Nielsen, K., & Langstrup, H. (2018). Tactics of Material Participation: How
Patients Shape Their Engagement through E-health. Social Studies of Science,
48(2), 259–282. Orgad, S. (2005). The Transformative Potential of Online Communication. Feminist Media Studies 5(2), 141–161. Pasquinelli, M. (2009). Google’s PageRank Algorithm: A Diagram of the
Cognitive Capitalism and the Rentier of the Common Intellect. In K. Becker
& F. Stalder (Eds.), Deep Search: The Politics of Search Beyond Google
(pp. 152–162). Transaction Publishers. Poster, M. (2001). What’s The Matter with The Internet? University of
Minnesota Press.h Prainsack, B. (2011). Voting with Their Mice: Personal Genome Testing and the
“Participatory Turn” in Disease Research. Accountability in Research,
18(3), 132–147. Prainsack, B. (2018). The “We” in the “Me”: Solidarity and Health Care in the Era
of Personalized Medicine. Science, Technology, & Human Values, 43(1), 21–44. Prainsack, B., & Buyx, A. (2017). Solidarity in Biomedicine and Beyond. Cambridge University Press. 1 Studying Expertise Online 1 Studying Expertise Online “You Must Know What You Mean When
You Say That”: The Morality of Knowledge Claims about ADHD in Radio
Phone-Ins. Sociology of Health & Illness, 40(4), 1–17. C. Egher 36 Waldby, C., & Cooper, M. (2008). The Biopolitics of Reproduction. Australian
Feminist Studies, 23(55), 57–73. Whiteford, A., Degenhardt, L., Rehm, J., Baxter, A., Ferrari, A., Erskine, H.,
Ch rl
n F N rm n R Fl
m n A J hn
N B r t in R M rr
C Whiteford, A., Degenhardt, L., Rehm, J., Baxter, A., Ferrari, A., Erskine, H.,
Charlson, F., Norman, R., Flaxman, A., Johns, N., Burstein, R., Murray, C.,
& Vos, T. (2013). Global Burden of Disease Attributable to Mental and
Substance Use Disorders: Findings from the Global Burden of Disease Study
2010. The Lancet, 382(9904), 1575–1586. h
Williams, R., Faulkner, W., & Fleck, J. (Eds.). (1998). Exploring Expertise. Issues
and Perspectives. Macmillan Press. World Health Organization. (WHO). (2010). International Statistical
Classification of Diseases and Related Health Problems, 10th Revision
(ICD-10). World Health Organization. Wyatt, S., Harris, A., Adams, S., & Kelly, S. (2013). Illness Online: Self-
Reported Data and Questions of Trust in Medical and Social Research. Theory, Culture & Society, 30(4), 131–150. Wyatt, S., Harris, A., & Kelly, S. (2016). Controversy Goes Online: Schizophrenia
Genetics on Wikipedia. Science & Technology Studies, 29(1), 13–29. Young, M., & Muller, J. (Eds.). (2014). Knowledge, Expertise and the Professions. Routledge. Ziebland, S., & Wyke, S. (2012). Health and Illness in a Connected World:
How Might Sharing Experiences on the Internet Affect People’s Health? The
Milbank Quarterly, 90(2), 219–249. Open Access This chapter is licensed under the terms of the Creative Commons
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C. Egher, Digital Healthcare and Expertise, Health, Technology and Society,
https://doi.org/10.1007/978-981-16-9178-2_2 Epistemic Inroads from the Asylum
to Digital Psychiatry Expertise about mental health has been marked since its early days by an
important set of challenges, which it has not yet managed to fully over
come. Psychiatry’s recognition as a medical specialty in its own right, the
scientific character of its methods, the effectiveness of its therapeutic
interventions, its political functions, and the struggle between care and
cure have marked its history (Rose, 2018). Important have also been the
various jurisdictional struggles in which psychiatry has been embroiled,
as authority over various areas of mental health has been claimed by dif
ferent disciplines, which have developed or become more influential over
the years due to the availability of new types of tools and knowledge. These aspects are important in view of the new conceptualization of
expertise that I put forward, where epistemic practices are shaped by the
ecosystem within which they develop, which frames their conditions of
possibility. Based on this understanding, expertise about bipolar disorder
online emerges at the confluence of specific historical trajectories that
have shaped how and what has been studied in relation to mental health
conditions, of current needs and circumstances in this healthcare sector,
and of expectations about the future. To better understand the online
practices that this book focuses on, this chapter draws an arch, stretching 37 © The Author(s) 2023
C. Egher, Digital Healthcare and Expertise, Health, Technology and Society,
https://doi.org/10.1007/978-981-16-9178-2_2 38 C. Egher C. Egher from the establishment of asylums at the beginning of the nineteenth
century to the current provision of mental healthcare in the US and
France and the future visions animating it. Such a broad longitudinal
perspective means that while important elements will be highlighted,
many complex debates will be simplified and a series of aspects that are
not directly relevant for the argument made in this book will be over
looked. These shortcomings are mitigated, however, by the fact that the
understanding of the online practices discussed in the following chapters
will be enriched through the historical insights and future hopes and fears
about the digitalization of mental healthcare (Pickersgill, 2019) described
here. This will allow us to better appreciate the novelty but also the con
tinuity that underscores them. Historical Overview of the Development
of Expertise About Mental Health Significant for the development of expertise about mental health are the
changes that took place at the end of the eighteenth century, when the
realization that community care for the “insane” often involved abusive
approaches prompted many to advocate for the necessity of “moral treat
ments” and the establishment of asylums as the means to achieve this. Since it was largely thought that “madness” was triggered and/or aggra
vated by the circumstances one found oneself in, asylums were envisioned
as tranquil, orderly places, where one could recover from the humdrum
of modernity and industrialization. Thus, in the early days, at least, the
establishment of asylums was animated by humanistic tendencies, by the
desire to cure those afflicted by “madness” and to provide them and their
families with support and solace. From this point of view, the asylum
system could be understood as a precursor and important influence on
the development of the welfare state, as Porter (2018) convincingly
argued. Its spread was encouraged in France by the 1838 law which
required mental health facilities to be established in each département,
and similar legislation was soon passed also in the US. Throughout the
nineteenth century and beyond, mental healthcare continued to be 2 Epistemic Inroads from the Asylum to Digital Psychiatry 39 provided in various ways, within the community and across other institu
tions, such as university hospitals and private clinics (Rose, 2018). Where
one received mental healthcare depended not only on the facilities that
were available in one’s region, but also on one’s socioeconomic status. In
France, asylums were funded by the state, which helped inform a greater
degree of centralization and standardization of practices, even though
important differences were recorded between departments depending on
the availability of such facilities. In the US, their funding depended on
legislation and the preferred policies and approaches at the level of the
individual states, which led to greater variability and disparities. As we
shall see in the next chapter, such differences made a durable mark upon
the organization of the mental healthcare systems in these two countries
and can also be noted these days. The establishment of asylums played an important role in the develop
ment of “mental medicine” as it made it possible for alienists, the doctors
treating the “mad,” to study the behaviors of a great number of patients
and to engage in various experiments. Historical Overview of the Development
of Expertise About Mental Health At the time, mental healthcare was
provided based on the symptoms patients experienced and consisted of a
combination of scientific and behavioral measures, which varied in dura
tion, harshness, and intensity. Mental health conditions were distin
guished based on groups of symptoms and they were thought to be
brought about by physical, moral causes, or a combination of both. Distinctions were made between predisposing and effective causes, as it
was thought that whereas one may have been susceptible to develop men
tal health issues due to bodily factors, a triggering event was needed to set
such processes into motion. Such events were often of a moral nature, as
can be noted by the numerous causes for mental illness that were circu
lated at the time, ranging from revolutionary excess and participation in
political events, to sedentary occupations, and a low level of instruction
(Porter, 2018). This understanding of causes greatly shaped how mental health condi
tions were studied as well as how they were intervened upon. Alienists
initially combined clinical and laboratory expertise, as they sought to
locate these conditions within the body, and apart from the various
examinations on asylum residents, they also engaged in postmortem
investigations, focusing primarily on the brain. Yet, by the second decade 40 C. Egher C. Egher of the nineteenth century, the lack of any reliable indication that markers
of “madness” could be indisputably identified within pathological auton
omy “were drawing the profession into crisis” (Arribas-Ayllon et al.,
2019: 28). This prompted the alienists to focus on heredity, for which
they had shown little interest prior to 1812, as a fundamental cause
(Arribas-Ayllon et al., 2019; Foucault, 1972/2010). They understood
heredity as a predisposing cause, which could trigger mental health con
ditions in combination with what were thought at the time as morally
reprehensible behaviors, such as the consumption of alcohol, masturba
tion, and overwork. The study of heredity was accompanied by the devel
opment of new approaches and techniques, as it broadened the focus
from the individuals afflicted by mental health issues to their families and
made new types of data necessary, which could be acquired through
detailed questioning, family history searches, and the development of
family pedigrees. Even though the alienists enthusiastically engaged in
the collection of vast amounts of data, the latter were not equally avail
able across institutions, nor were they systematically collected from the
very beginning. Historical Overview of the Development
of Expertise About Mental Health Substantial efforts were therefore dedicated to improve
the quality of the data collected and to standardize the data collection
methods, so as to facilitate comparisons and to enhance the scientific
character of the insights acquired. An important landmark in this sense
was Esquirol’s use of the statistic table as a means to organize mental
health cases in France, practice which became popular among many
alienists, who soon improved on this technology in order to better deter
mine correlations (Porter, 2018). Psychiatric expertise thus came to rely on a combination of clinical and
statistical knowledge, and the latter informed its development as a par
tially international enterprise. Knowledge was intensely exchanged
among alienists through professional tours in the US and Europe, at
international meetings organized by the numerous professional associa
tions that were being established, and through the eager publication of
their statistics in the specialty journals that were founded in considerable
number from the 1840s onward (Porter, 2018). There were, however,
also important differences among countries concerning the role ascribed
to statistical knowledge in relation to psychiatric expertise and to what
were considered the best means to study the impact of heredity on mental 2 Epistemic Inroads from the Asylum to Digital Psychiatry 41 health. Thus, whether or not the deployment of statistical methods was
an indication of scientific rather than merely administrative or bureau
cratic expertise was the object of heated debates in France, where many
mental healthcare professionals reproached their statistically bent col
leagues for having a simplistic understanding of heredity and mental ill
ness. Under their influence, the dominant understanding of “insane
heredity” in France became that of a process of physical and mental decay
where the environment played a complex role, and “insane heredity” con
tinued to be studied through cases. This marks an important difference
between the US and other European countries, such as Germany, where
expertise about the heredity of mental health conditions was successfully
claimed by statisticians and geneticists (Porter, 2018). g
Despite the more standardized data collection and statistical methods
used, the mechanisms through which heredity affected mental and bodily
processes continued to remain unclear. The alienists managed, however,
to successfully mobilize this uncertainty to position mental health as an
important social issue, which required not only treatment, but also urgent
social reforms focusing on prevention at the national level. Historical Overview of the Development
of Expertise About Mental Health In France, this
process was facilitated by political developments, as medical practitioners
came to play an important role in public health due to the Napoleonic
reforms. As heredity’s influence on the development of mental illness was
thought to be rather grim, the alienists warned that it led to degeneration
through its cumulative effects across multiple generations. Hereditary
mental defects thus became a national concern, as they could impede a
country’s progress and competitiveness, and their management required
a combination of scientific and moral approaches. The same ethos was
exuded in the US by many asylum supporters, who argued in favor of a
greater provision of funds for these institutions and for important social
measures as an adequate response. In this context, the alienists success
fully positioned the moral expertise they claimed to be endowed with as
highly relevant, and came to “moralize the masses” (Arribas-Ayllon et al.,
2019:30) by directing nation-wide efforts to eliminate the moral behav
iors they found problematic. Through their work, from the 1840s
onward, both in France and in the US, the population censuses started to
collect data through which the spread of mental health conditions and
the role of heredity in such processes were hoped to be determined at the
level of the nation. 42 C. Egher C. Egher C. Egher Thus, in Meyer’s view, mental health conditions were
not so much the result of the cumulated effects of faulty genes but rather 2 Epistemic Inroads from the Asylum to Digital Psychiatry 43 inadequate reactions to life circumstances that could be made sense of
within the context of a patient’s life (Healy, 2008; Hochmann, 2017;
Rose, 2018) and that could be partially addressed and prevented through
an adequate mental hygiene. q
yg
The search for diagnosis criteria based on etiology or prognosis marked
a durable distinction among mental healthcare professionals of a different
bent and was also reflected in their understanding of the role of genes in
the development of mental health conditions. As the statistical data of
populations came to be seen as a form of scientific capital at the begin
ning of the twentieth century (Arribas-Ayllon et al., 2019), it galvanized
collaborations among alienists, statisticians, biologists, and so on and
thereby challenged the separation between mental health expertise and
“ordinary medicine” which had strongly persisted until then (Porter,
2018). Hopes of establishing mental health diagnoses based on etiology
were revitalized by such collaborations through a renewed focus on the
brain, on the one hand, and on the influence of genes, on the other. Thus,
neurological and experimental approaches regained popularity among
some mental healthcare professionals in the US, who thought the psy
chiatry of the asylums with its focus on clinical observations was out
dated. For instance, the New York asylums purchased freezing microtomes
for slicing brain samples, which they used for various investigations and
preserved along with cards describing the behavioral profile of the person
they were coming from, as even after the 1930s, some hoped to correlate
characteristics of the preserved brain with the actions and behaviors of
“insane” criminals. Also in France, a renewed focus on the neural and
molecular mechanisms underlying specific symptoms could be noted,
with neuropsychiatrists such as Clérambault arguing that delirious and
hallucinatory states were the result of the irritation of nerve centers that
could gradually engulf the entire cortex (Hochmann, 2017).h The role of genes in the development of mental health conditions
acquired renewed attention, as important breakthroughs in the realm of
statistics, such as Galton’s probabilistic laws, allowed for mathematical
relationships to be determined among generations in terms of hereditary
transmission (Arribas-Ayllon et al., 2019). C. Egher Such data collection processes went hand in hand with attempts to
standardize diagnoses, yet mental health conditions proved difficult to
classify. Initially, alienists such as Pinel sought to distinguish between
mental health conditions based on their etiology, that is, on their causes
and origins, but the failure to identify specific physical causes brought
such an approach under strain. As asylums made possible the observation
of the pattern of symptoms experienced by an individual over a period of
time, at the end of the nineteenth century, the German psychiatrist Emil
Kraepelin advanced the idea of establishing diagnoses based on prognosis
rather than etiology through the collection of detailed histories of the
course of illness. Kraepelin put forward a new nosology, where he identi
fied 13 major groups of mental health conditions. Relevant here is the
division of psychotic illnesses into “manic-depressive psychosis” and
“dementia praecox,” known these days as schizophrenia, which he intro
duced based on the presence or absence of mood changes and by focusing
on their outcome. Whereas the latter was understood to lead to cognitive
and clinical decline, the former allowed for a less pessimistic perspective
(Healy, 2008), although the overall outlook remained grim. Kraepelin’s
approach was met with reserve in France, partly due to recent memories
of the war between this country and Germany and to persistent political
animosities. But it also stemmed from the fact that French psychiatrists
did not share his negative perspective on the outcome of these condi
tions, with many of them arguing that Kraepelin’s views had been skewed
by his observations of asylum patients, who presented more aggravated
forms of mental health conditions than those who could be seen by city
doctors, for instance, in other medical institutions (Hochmann, 2017). In contrast, Kraepelin’s focus on prognosis was initially enthusiastically
received by Adolf Meyer, director of the New York State Psychiatric
Institute and, through his influence, by many other American psychia
trists, who appreciated the return to a clinical focus in psychiatry. While
Kraepelin’s perspectives remained generally popular in the US, in the
1920s Meyer himself changed course, as he reproached the German psy
chiatrist for a too strong neurological focus, and he highlighted, instead,
the role of the environment in the development and outcome of mental
health conditions. C. Egher Further impetus was provided
by the (re)discovery of the Mendelian ratios and the attempts to apply
them in the study of the development of mental health conditions. 44 C. Egher C. Egher Noteworthy in this sense are the activities of the American researchers
Davenport, Goddard, and Rosanoff, who tried to persuade their European
counterparts that insanity could be explained as single-factor Mendelian
traits, that is, that discrete genetic units could be linked with specific
mental health conditions, such as manic-depressive illness, as bipolar dis
order was largely known at the time. Heated debates emerged about the
role of genes between such Mendelian supporters, many of whom were
neuropsychiatrists, who considered different mental health conditions to
be determined by specific groups of rare genes, and biometricians and
clinical psychiatrists who argued that mental health conditions were trig
gered through the concerted influence of rare variants and multiple genes
of small effects. The biometricians and statisticians found issue with the
quality of the data and of the calculations upon which those in favor of
Mendelism based their claims, whereas the psychiatrists found a clinical
approach based on the long-term observation of the patient and the
development of the disease more reliable and useful (Arribas-Ayllon
et al., 2019; Porter, 2018). By the end of the nineteenth century, the failure of the asylums had
become obvious in both the US and France, as the number of people
diagnosed with mental health conditions was on the rise, while the effec
tiveness of the treatments used remained limited. Different types of men
tal healthcare professionals embraced therefore hereditarianism. Some
did this because the broader correlations heredity allowed for between
physical and moral causes enabled them to claim expertise on various
social matters (Arribas-Ayllon et al., 2019; Porter, 2018). Others became
involved in social reforms and other political decisions pertaining to the
management of populations, as it provided them with access to the
resources and influence needed to continue their neurological and genetic
studies (Porter, 2018). Heredity thus became entwined with themes of
family and racial hygiene, as the research activities pursued by Davenport
and his supporters in the US illustrate. Furthermore, next to warning
against certain behaviors, the alienists came to give advice on reproduc
tive practices. C. Egher Psychiatry was thus from its early days a political science
(Rose, 2018), which not only exerted negative power through prohibi
tions and forced commitment into asylums, but also positive power, as it
encouraged the “healthy” population to “breed.” Nevertheless, it is 2 Epistemic Inroads from the Asylum to Digital Psychiatry 45 important to bear in mind that, for at least the first half of the twentieth
century, the trajectories of psychiatric genetics and neuropsychiatry were
marked by the suffering, exclusion, and extermination brought about by
mental healthcare professionals involved in the eugenics movement or
collaborating with authoritarian political regimes. This, however, did not
put an end to the claims of mental healthcare professionals to moral
expertise, as they remain important to our present time, as we will now
see, as we turn our focus to Freud and psychoanalysis. While psychiatrists sought to determine the causes of mental health
conditions by focusing on genes or on the brain, their jurisdiction over
the field of mental health came to be disputed at the end of the nine
teenth century by dynamic psychologists, who introduced a different per
spective by focusing on trauma and its impact on individual development. Having studied in France, Freud was critical of the wide role ascribed
there to heredity and degeneration in relation to mental health condi
tions (Hochmann, 2017). He argued, instead, in favor of a focus on the
individual and its development, which Freud thought to be importantly
shaped by circumstances unfolding in the private sphere (Hochmann,
2017; Illlouz, 2008). In France, the uptake of psychoanalysis was very
slow and Freud’s views remained largely unpopular until the 1960s
(Turkle, 1981). The first French psychoanalytic association was founded
only in 1926, more than a decade after its American counterpart, and this
new approach to mental healthcare was generally disregarded by reputed
French mental healthcare professionals as well as by vast numbers of the
population, who considered it a new form of bourgeois self-indulgence
(Turkle, 1981). C. Egher In the US, however, psychoanalysis enjoyed tremendous
popularity from its very beginning, at the confluence of three main fac
tors: the ongoing jurisdictional struggles among medical professionals
and representatives of the clergy about the provision of mental health
care; the popularity of spiritual approaches and interventions focusing on
the mind; and the ambivalence of Freud’s theories which allowed various
stakeholders to adopt them in the pursuit of distinct goals (Illlouz, 2008).h The support of the American medical elites for psychoanalysis ensured
its development as a medical specialty in its own right and its embedding
within influential institutions. Already in the second decade of the twen
tieth century, numerous professional psychoanalytic societies were C. Egher 46 C. Egher formed, the first American psychoanalytic journals, The Psychoanalytic
Review and The Psychoanalytic Quarterly, were published, and uniform
professional criteria were developed in a bid to heighten its authority and
legitimacy. Furthermore, in 1927, the American Psychoanalytic Society
decided that only medical doctors were allowed to train and practice as
psychoanalysts (Illlouz, 2008), which enhanced its “domestication”
(Turkle, 1981). The treatment of bipolar disorder in the US, over which
psychiatrists had dominated, thus also came under the purview of psy
choanalysts, who were importantly influenced by the works of Abraham,
one of Freud’s supporters and collaborators on On Murder, Mourning and
Melancholia (1917/2005). Abraham focused on the study of psychoses
and considered bipolar disorder to be the result of libidinal fixations that
ensued as an infant’s sexual development was frustrated. In his view,
bipolar disorder thus constituted a reenactment of past conflicts informed
by the ambivalence of the loved object. Abraham developed various
methods through which psychoanalytic approaches could be employed as
therapeutic practices for bipolar disorder and exerted a strong influence
on Lewin’s thinking, an important member of the New York Psychoanalytic
Society. Under the influence of Erikson and Maslow, the ultimate goal of
therapy came to be self-realization (Illlouz, 2008), which enabled psy
chologists to claim expertise over broad areas of social and private life,
thus continuing the moral careers initiated by nineteenth-century
alienists. The publication of the first Diagnostic and Statistical Manual (DSM-
I) in 1952 indicated that expertise about mental healthcare in the US was
largely shaped at the time by psychoanalytic perspectives and by Meyer’s
psychobiological style of thought (Illlouz, 2008; Rose, 2018). C. Egher The man
ual distinguished between mental health conditions that developed as a
result of impaired brain functioning and psychiatric conditions, such as
depression of phobia, which were thought to arise as the result of mal
adaptive behaviors. This latter group of conditions was seen as reactions
to environmental circumstances rather than as full-fledged disease enti
ties (Rose, 2018). This meant that an important aspect of therapeutic
practice consisted of mental healthcare professionals’ activities of inter
pretation of the symptoms exhibited by a person, which had to be made
sense within the life story of the person diagnosed. This perspective 2 Epistemic Inroads from the Asylum to Digital Psychiatry 47 triggered, however, intense reactions from mental healthcare profession
als who considered this a threat to the scientific character of psychiatric
diagnoses, leading to the removal of the term “reactions” from DSM-II
(1968) and to the pronounced biological and clinical focus at the heart of
DSM-III (Healy, 2008; Rose, 2018). Under the strong influence of a
group of psychiatrists at the Department of Psychiatry of the University
of Washington, the 1970s were marked by a “major epistemological and
ontological shift” (Rose, 2018:79) with the return to etiology in diagno
sis, and the search for biological causes as underlying, even when partial,
causes of disease. Expertise about mental healthcare was thus hoped to
become more objective, as diagnoses were based upon observable symp
toms, and, where applicable, upon laboratory tests, which were expected
to be understood in the same way by any mental healthcare professional
assessing them. g
The 1980s were therefore marked by an acceleration of studies con
ducted to discover the neurological processes or the genetic factors under
lying bipolar disorder and other related conditions. In psychiatric
genetics, family and twin studies were taken up again, whereas techno
logical developments, such as recombinant DNA and advances in chro
mosomal mapping, allowed for the construction of genetic linkage maps,
thereby briefly bringing back to life and popularity the idea of single
dominant genes the Mendelians had been so fond of (Arribas-Ayllon
et al., 2019). Several claims about the identification of genes for bipolar
disorder were made in the second part of the 1980s (Baron et al., 1987;
Egeland et al., 1987), but they failed to be replicated. C. Egher Even though the
more advanced technologies developed in the aftermath of the Human
Genome Project in early 2000 resurrected hopes about a more thorough
understanding of the mechanisms through which genes were involved in
the development of mental health conditions such as bipolar disorder, the
results remained rather disappointing. Thus, at the entry into the new
millennium bipolar disorder was considered to be the result of complex
genetic traits, which “provide[d] theoretical coherence and respectability
to an otherwise ambivalent relationship between genetic and non-genetic
factors” (Arribas-Ayllon et al., 2019:75). Even though no biomarkers
could be used as reliable indicators of diagnoses even by the time the lat
est DSM-5 (2013) was published, the widespread use of digital 48 C. Egher C. Egher technologies and advances in AI have nourished hopes that before long a
precision medicine psychiatry will be within reach. The efforts to develop
medical knowledge and therapeutic approaches for mental health condi
tions sketched thus far were also accompanied by important activities
focusing on the provision of care, whereby support and self-help groups
played an important role, as I briefly highlight below. Self-help and Support Groups The history of psychiatry has been profoundly marked by its confronta
tion with a double “temptation”—the provision of therapeutic care and
that of social assistance (Swain, 1988). Given this book’s empirical focus
on the activities of people diagnosed with bipolar disorder and since “the
emergence of support groups should be understood as the other side of
the cultural coin of institutionalized therapeutic language” (Illlouz,
2008:186), it is important to briefly consider the role self-help and social
support groups have historically played. In France, such groups have
started to develop in the aftermath of the Second World War. A notewor
thy landmark in the development of support groups, which consisted of
both medical professionals and people diagnosed, was the founding of
the Croix Marine movement (Fédération d’Aide à la Santé Mentale Croix
Marine). It was initiated by three psychiatrists—Pierre Doussinet, Alice
Delaunay, and Elizabeth Jacob—in 1952, with the aim of providing pro
tection and mutual psychological and social help to people diagnosed
with mental health conditions. While over the following two decades the
provision of ambulatory care launched this way developed further,
changes in French legislation in the 1970s regarding the status and pre
rogatives of social and medico-social institutions led to a strict separation
between the provision of medical care and social action. Inspired by the 1968 protests, a number of psychiatrists together with
people diagnosed with mental health conditions founded the Groupe
d’Information Asile (GIA) in the early 1970s (Bernadet et al., 2002), to
fight against repressive practices in psychiatry. The group has since devel
oped a strong juridical orientation and claims to have played a major
contribution in the 2010 decision of the Constitutional Court, by which 2 Epistemic Inroads from the Asylum to Digital Psychiatry 49 all methods of involuntary commitment previewed under French law
were declared unconstitutional (Troisoeufs & Eyraud, 2015). In the
1980s, the first association of people diagnosed with mental health con
ditions focusing on defending the rights of patients, L’Association des
Psychotiques Stabilisés Autonomes (APSA), was founded with the support
of psychiatrists. The first patient group with a specific focus on advocacy,
Advocacy France, only came into being in the 1990s, drawing inspiration
from advocacy groups in the US (Laval, 2015). Nevertheless, most French
self-help and support groups still focus primarily on the provision of sup
port and education for people diagnosed and their families (Troisoeufs &
Eyraud, 2015). Self-help and Support Groups Such groups became all the more popular after the law
stipulating the creation of mutual help groups—Groupes d’Entraide
Mutuelle (GEMs)—was adopted in 2005. By 2008, 300 GEMs had
developed (Girard, 2008), and in 2016, 430 GEMs were counted
throughout the French territories (CNSA, 2017). Nevertheless, most of
them continue to function locally, they do not reach the broad public,
and have little political influence. l
In the US, the peer support movement has its origins in the practices
of hiring people diagnosed with mental health conditions in asylums and
other psychiatric institutions in the 1920s (McCosker, 2018). The focus
on self-help acquired impetus due to the popularity of psychoanalytic
approaches in this country and its espousal of dominant American values,
such as individual self-determination and entrepreneurialism. Under its
influence, the self came to be perceived as an ongoing project, as mallea
ble, adjustable, and improvable. And a lot of attention started to be paid
to the development of behavioral approaches in mental healthcare, meant
to help people diagnosed with mental health conditions better “cope”
and “adjust” (Illlouz, 2008). Self-help and support groups in this country
have their origin in two different types of organizations. Thus, self-help
groups are linked to the funding of Alcoholics Anonymous (AA) in 1935,
from which self-help groups have borrowed important organizational as
well as ideological elements. In 1948, the first Fountain House, a social
club for people diagnosed with mental health conditions, was founded in
New York by We Are Not Alone, a group of ex-patients from the State
Hospital. In the 1950s, the Fountain House came under the leadership of
a social worker and broadened its focus to include, next to socialization, 50 C. Egher C. Egher employment training, so that its members could gain and retain jobs
(Dincin, 1975). This model has spread and thrived over the years, now
counting clubhouses across the US, the UK, and Scandinavia. One of the
first instances of support groups in the US is Recovery Inc., which was
founded by neuropsychiatrist Abraham Low in 1937 in Chicago, to care
for people with mental health conditions after their discharge from hos
pital. While soon thereafter Recovery Inc. also turned into a self-help
group, its success has been more modest compared to AA and other simi
lar groups. Self-help and Support Groups These developments took place in a context marked by calls to reform
mental health hospitals and turn them from places of confinement into
spaces of care. They were also informed by the growing realization that
the availability and accessibility of community care provisions were insuf
ficient due to the rapid pace of de-institutionalization, ensuing organiza
tional loopholes, and insufficient funding (Brown, 1988; Estroff,
1985/2001). Support groups and self-help groups thus became popular
at a time when different expectations were being formulated about the
relations between medical professionals and people diagnosed, and when
new types of professionals and more social actors were becoming engaged
in the provision of mental healthcare services (Norman, 2006). The
1980s inaugurated a lasting period of proliferation and diversification for
self-help and (mutual) support groups in the US. For instance, a national
survey conducted in 2002 revealed that there were 7467 organizations
led by and for consumers of mental health services and their families, a
substantial number compared to the 4546 traditional, professional-led
mental health organizations (Goldstrom et al., 2006). In recent years,
many self-help and support groups have also proliferated and diversified
online (Kaufman & Whitehead, 2016) and the active role of “informed
supporters” (Barak et al., 2009), that is, people diagnosed with the same
mental health condition, in helping others with the same diagnosis by
providing them with “more tailored feedback” (Barak et al., 2009:8) has
become widely acknowledged. These activities have been shaped by the
ways in which the provision of mental healthcare has been organized in
the US and France, and by the challenges experienced by the mental
healthcare systems in both countries, which I now briefly describe. 2 Epistemic Inroads from the Asylum to Digital Psychiatry 51 Mental Healthcare in the US and France The development of different types of mental healthcare expertise and the
dominant approaches to the study of mental health conditions also rever
berated in the ways in which mental healthcare was provided. While asy
lums and psychiatric hospitals continued to be the main institutions
focusing on the diagnosis and treatment of people diagnosed with mental
health conditions throughout the first half of the twentieth century,
important changes started to take place in the 1960s. In France, psychi
atric expertise came under heavy criticism and mental healthcare came to
be organized in sectors, with each sector providing care to roughly 70,000
adult inhabitants (Verdoux, 2003; Verdoux & Tignol, 2003). Developed
largely in response to calls for reform made by the antipsychiatry move
ment (Castel, 1981), the sector was designed as a means through which
mental healthcare could be provided by a multidisciplinary team of pro
fessionals headed by a psychiatrist. This team was expected to be familiar
with the community and to be able to guide and assist the patient’s rein
tegration, thereby importantly ensuring continuity of care (Coldefy,
2007; Petitjean, 2009). The events of May 1968 also marked a turning
point for the standing of psychoanalysis in France, as it came to be widely
adopted, both as a therapeutic approach and as a cultural phenomenon
(Turkle, 1981). In contrast, in the US, psychoanalysis was the object of criticism by the
antipsychiatry movement along with psychiatry (Turkle, 1981) due to its
medicalization and close integration in dominant institutions (Illlouz,
2008). In 1963 the Community Mental Health Act was signed in the
US, which significantly changed the provision of mental healthcare
through the establishment of community mental health centers through
out the country. As people diagnosed who had been previously treated in
asylums and hospitals could receive mental healthcare services within
their communities, this set into motion the process of de-institutionaliza
tion. Economic considerations played an important part in these devel
opments, as the availability of new medications and therapeutic
approaches rendered mental healthcare in the community more cost-
effective. The process of deinstitutionalization unfolded at a higher rate 52 C. Egher C. Egher in the last few decades of the twentieth century, which led to new chal
lenges for people diagnosed with health conditions and mental health
professionals. Mental Healthcare in the US and France p
Even though the process of de-institutionalization was meant to
improve the provision of mental healthcare and render it economical,
over the last few decades, the French and the American healthcare sys
tems have found themselves in a precarious state. In France, most citizens
are insured and have free access to mental healthcare in the public sector. Although at the beginning of the twenty-first century, the French health
care system was voted the best out of 191 nations (WHO, 2000), such an
extraordinary ranking came at a very high cost. In 2013, for instance,
roughly 10.9% of the country’s GDP was allocated to the health sector
(OECD Health Statistics, 2015). In the field of mental healthcare more
specifically, the French government has been confronted with multiple
challenges, leading researchers and journalists to note at various moments
in time that French psychiatry was in crisis (Castel, 1981; Coffin, 2009;
Pignarre, 2006) or that it was experiencing a “severe depression” (Le
Monde, 2018). In 2008, mental health conditions represented about
32% of the country’s overall disease burden (WHO, 2008) and their
incidence has been steadily increasing (OECD, 2016), thereby placing
tremendous pressure on the mental healthcare system. For instance, in
2016, 2.1 million patients were admitted either to one of the 3900 cen
ters of medico-psychological expertise spread throughout France or to
psychiatric hospitals (about 25%). Although the process of deinstitutionalization has occurred at a much
slower rate in France than in other countries (Petitjean, 2009), over the
last three decades the number of hospital beds available for people diag
nosed with mental health conditions has been reduced by 70%. Most of
the state’s budget for mental health continues, however, to be allocated to
in-hospital forms of treatment (OECD, 2016; Petitjean, 2009), while
outpatient alternatives are insufficient. Furthermore, the centers of
medico-psychological expertise, which were developed along with the
sector as a link between general practitioners and psychiatric hospitals,
have been the victims of several restructuring measures brought about by
reforms that will be discussed in more detail in the following chapter. The
lack of personnel and other resources has thus led to considerable delays 2 Epistemic Inroads from the Asylum to Digital Psychiatry 53 in the provision of mental healthcare, as individuals experiencing symp
toms may wait up to one year before having a first appointment, and has
challenged the monitoring and timely modifications of treatment for the
already registered patients. Mental Healthcare in the US and France Furthermore, since the psychoanalytical
model remained the dominant approach to mental health until the
2000s, some claim that the French mental healthcare system is character
ized by “underdevelopment in community psychiatry, accessibility of
mental health professionals trained in cognitive-behavioral psychother
apy, and psychiatric research” (Verdoux, 2003:85). Yet others criticize the
“scientism” currently characterizing dominant approaches in the provi
sion of mental healthcare in France and deplore the decline of psycho
analysis as the demise of one of the last “humanistic” approaches focusing
on the individual within the full context of his/her own life
(Roudinesco, 2019). The discrepancies noted in terms of the availability of asylums at the
beginning of this chapter resonate nowadays with significant differences
between regions regarding the resources at their disposal, the distribution
of outpatient clinics, and the number of existing mental hospitals
(Coldefy et al., 2009; Coldefy, 2007; Provost & Bauer, 2001; Verdoux,
2003). The distribution of medical professionals is also skewed, with
rural areas (Coldefy, 2007) and regions in Northern France (Petitjean,
2009; Verdoux, 2003) struggling due to a low number of specialists. Furthermore, while France was once the country with one of the highest
number of psychiatrists in the world, their amount has been decreasing
steadily, not only as the result of measures meant to render the provision
of mental healthcare more efficient, but also because of the unpopularity
of psychiatry as a specialization among young doctors. Thus, since 2012,
the internship positions available in psychiatry have no longer been filled,
with a turn for the worse signaled in 2019, when 17% of positions
remained vacant, and only a minor improvement booked in 2020, when
11% of positions remained unoccupied (Raybaud, 2021). This is bound
to affect people diagnosed with bipolar disorder or in need of such a diag
nosis, since in France the diagnosis, treatment, and evaluation of the con
dition’s evolution are determined by psychiatrists. General practitioners
(GPs) play a different role, as they function as first points of contact and 54 C. Egher C. Egher subsequently as the ones who administer the treatment and who are fre
quently in touch with the patients.h q
y
p
The American mental healthcare system has also been confronted with
important challenges (Boyle & Callahan, 1995). Unlike in France, dein
stitutionalization occurred in the US at a very high rate. Mental Healthcare in the US and France Yet, few solu
tions were put in place to enable people diagnosed to receive the care they
needed within the community (Estroff, 1985/2001), and not many peo
ple knew about them, when such solutions existed (Grob, 2005). The
situation worsened toward the turn of the century, prompting the chair
of the President’s New Freedom Commission on Mental Health to state
in 2002 that “the system needs dramatic reform because it is incapable of
efficiently delivering and financing effective treatments—such as medica
tions, psychotherapies, and other services—that have taken decades to
develop. Responsibility for these services is scattered among agencies,
programs, and levels of government” (Hogan, in Gijswijt-Hofstra,
2002:156f). The delivery of cost-effective mental healthcare services con
tinues to remain a problem, as spending has been increasing at alarming
rates. In 2019, $225 billion was dedicated to this sector, marking an
increase of 52% compared to the expenditure in 2009 (Open Minds
Market Intelligence Report, 2019). At the same time, the delivery of
mental healthcare continues to remain highly fragmented and insurance
companies often distinguish between medical and behavioral types of
interventions, prompting differences in facilities, data collection systems,
and reimbursement requirements (Mou & Insel, 2021). While some American people diagnosed with mental health condi
tions nowadays receive better quality care than they would have a few
decades earlier, the system continues to be marked by important inequal
ities. Even though the passage of the Affordable Care Act has enabled
more individuals to have access to healthcare, over 10% of the people
diagnosed with mental health conditions continue to be uninsured
(Mental Health America, 2022). Furthermore, the quality of care varies
depending on one’s type of insurance. For instance, the coverage pro
vided by Medicare is limited, as it only enables access to about 25% of
the mental healthcare professionals registered in the US. Moreover, it
allows for a total of 190 days of in-hospital psychiatric care, even for
people diagnosed with severe mental health conditions, such as bipolar 2 Epistemic Inroads from the Asylum to Digital Psychiatry 55 disorder. At the same time, only 56% of psychiatrists accept commercial
insurances (Leonhardt, 2021). Mental Healthcare in the US and France As a consequence, not all insured people
diagnosed with mental health conditions have access to the same type of
treatment (Hogan, 2003), with evidence indicating that the mental
healthcare received by members of ethnic minorities continues to be lim
ited and of lower quality (National Institute on Minority Health and
Disparities, 2019; Kataoka et al., 2002). p
There are also significant geographic differences in terms of access to
mental healthcare, which echoes somewhat the situation in France. Thus,
in different states, mental healthcare services are reimbursed to varying
degrees, and managed care controls limit access to costly services while
seeking to promote cheaper options more widely (Scheid, 2000). Not only
does the availability of community services differ, but the system is also
marked by important discrepancies regarding the number and type of
medical professionals available (Mental Health Care Professional Shortage
Areas (HPSAs), 2020), with more than 112 million Americans living now
adays in areas with few mental healthcare providers (Leonhardt, 2021). Thus, while in New York, Massachusetts, and Vermont there are more
than 15 psychiatrists per 100,000 people, in Texas and Idaho there are
fewer than 6 (Simon, 2015). Furthermore, there appear to be over 4000
areas across the US with only one psychiatrist for 30,000 people (Simon,
2015). These issues are further complicated by the fact that the overall
number of psychiatrists available is bound to decrease over the coming
years. This is due to a diminished interest among young medical doctors to
specialize in psychiatry and to the upcoming retirement of a large number
of psychiatrists, as 59% of them are 55 or older (National Council for
Mental Wellbeing, 2017; Simon, 2015). In this context, matters are not
made any easier by differences in legislation among states, which prevent
mental healthcare practitioners from practicing everywhere in the US. It is against this background that the Internet and, more recently, the
development and spread of digital and AI-based technologies have led to
great hopes that they may enhance the accessibility and quality of mental
healthcare, both in the US and in France. At the same time, these tech
nologies have also given rise to grave concerns about their potential to
further inequalities and fragmentation. It is to these utopian and dysto
pian expectations that I now turn. 56 C. Egher 1 In the US, the Department of Veterans Affairs and Department of Defense has developed mental
health apps (lagan et al., 2020). 2 Digital phenotyping is used by clinicians with the aim of creating objective parameters that cor
relate with diagnostic criteria by using extensive data about a person to refine diagnosis and predict
behavior. It is a form of population monitoring/surveillance. Digital and AI-Based Technologies
in Mental Healthcare
2017
2019
2021
2019
2021
2019
1
2019
h The search for objective criteria to establish mental health diagnoses,
which started in the early days of the asylum, continues nowadays through
the use of AI-based technologies. Thus, machine-learning algorithms are
currently being trained in collaboration with psychiatrists to identify
people with various mental health diagnoses at a prodromal stage, when
symptoms have not yet manifested themselves clearly (Bauer et al., 2019;
Miller, 2019; Rudin & Ustun, 2018; Shatte et al., 2019). Promising in
this sense are various natural language processing algorithms, which are
meant to analyze linguistic as well as paralinguistic aspects to help in
diagnosis as well as in the prediction of an upcoming mental health 2 Epistemic Inroads from the Asylum to Digital Psychiatry 57 episode. It is thus hoped that patterns of speech, one’s vocabulary, as well
as acoustic elements can be rendered legible as mental health markers and
indicators. As we have seen in the first section of this chapter, a lot of
attention has traditionally been paid to these elements in psychiatric
expertise. What is new here, however, is the breadth and level of precision
and granularity with which these elements are monitored, recorded, and
assessed. This is often invoked as a cause for celebration by the techno
logical companies involved in such developments, as the following quote
illustrates: “With AI, our words will be a window into our mental health”
(IBM, 2017).
2
2018h The use of digital technologies in the provision of existing therapeutic
approaches has been accompanied by visions whereby significant changes
are operated in the frequency, length, and content of such therapies. For
instance, proponents of Ecological Momentary Interventions (EMIs)
have advocated for the provision of multiple psychological interventions
or behavioral prompts spread throughout the day and informed by sen
sory data acquired through digital technologies (D’Alfonso, 2020). This
way, the delivery of personalized therapy is understood as not only being
attuned to a specific person, but also as adjustable to the particular con
text in which that person may find him/herself in and to the best timing 58 C. Egher C. Egher when it should be provided or ingested. There are also technologies which
aim for the personalization of therapeutic interventions by facilitating
common decision-making. Digital and AI-Based Technologies
in Mental Healthcare Such technologies elicit information about
each person’s preferences, needs, and values and subsequently make it
available to medical professionals, so that they can decide about the best
medical treatment based, ideally, on a richer understanding of the per
son’s symptoms, challenges, life circumstances, and goals. An example of
such a digital decision-making aid is common ground, which was shaped
by the experiential knowledge of its developer, who is allegedly diagnosed
with a mental health condition. Importantly, many also hope that AI-
based technologies will enable the development of novel therapeutic
interventions based on an individual’s genes, lifestyle, and other relevant
environmental markers (Fernandes et al., 2017), thereby turning preci
sion psychiatric medicine into a reality. Digital and AI-based technologies have also contributed to changes in
how already available therapeutic approaches are provided and by whom. Thus, the use of computers for the provision of various mental health
services ranging from online chats to text messaging between people
diagnosed with mental health conditions and medical professionals or
other people diagnosed is widespread. Videoconferencing tools are being
increasingly used to arrange mental healthcare appointments both in the
US and in France, as many technological companies have started to
develop technologies to address the geographic disparities in the provi
sion of mental healthcare discussed above and to help save time for both
mental healthcare practitioners and the people diagnosed. For instance,
Doctolib, which is the current leader of digital healthcare services in
France, as it comprises 80% of this market, launched a smartphone appli
cation in January 2019. The application allows people diagnosed with
mental health conditions or experiencing symptoms to book video con
sultations with mental healthcare professionals of their choice, regardless
of where they find themselves (Blaquière, 2019). The intention is to
enhance convenience for both parties, as the times and dates at which
different mental healthcare professionals are available are clearly indi
cated. While only medical professionals have to pay for the use of
Doctolib and its application, in the US people diagnosed are required to 2 Epistemic Inroads from the Asylum to Digital Psychiatry 59 pay for online counseling services, but many of them, such as BetterHelp
and Talkspace, are currently reimbursed by some insurance companies. p
y
y
p
Chatbots are also becoming increasingly popular not only for mood
tracking, but also for the provision of cognitive-behavioral therapy, mind
fulness, and behavioral reinforcement. Digital and AI-Based Technologies
in Mental Healthcare Of notoriety in France is the
application My Sherpa, developed by Doctorpsy, which is claimed to
have been downloaded by over 220,000 people. It allows people experi
encing various mental health issues to access psychotherapy and to inter
act with a chatbot about their mental states. In the US and many other
countries, numerous services are provided by three of the most promi
nent chatbots in mental healthcare—Tess, Wysa, and Woebot. Chatbots
are seen as viable solutions for people diagnosed with mental health con
ditions who may have difficulties accessing conventional therapeutic ser
vices, and who may appreciate the constant availability of such “therapist
robots” and their supposedly neutral, non-judgmental character. Furthermore, “machine counselors” have also been used rather success
fully in suicide prevention services, and great hopes are attached to virtual
therapeutic agents using avatar representations, such as ELLIE, which are
meant to move beyond language processing and to engage in the analysis
of nonverbal signals. Digital and AI-based technologies are not only expected to contribute
to better diagnosis and treatment, but also expected to bring about
important changes in the work of medical professionals and in their rela
tions to people diagnosed. For instance, many hope AI will help improve
the quality of care by reducing clinicians’ paperwork-related workload
and by summarizing important information from a person’s patient
record. Whereas monitoring devices are often discussed in relation to
acquiring insights into people’s physical and mood states, some have also
been implemented to keep an eye on the mental healthcare provided by
professionals. For instance, in the US “Electronic Visit Verification” is
used to log in the precise duration of home visits by mental health service
providers (Olowu, 2015). Importantly, the widespread use of digital
technologies in mental health may be accompanied by the blurring of
numerous categories, given their more malleable character. For instance,
depending on the context of use, a digital pill may be a digital treatment,
but it may also be a form of surveillance or control (Cosgrove et al., 60 C. Egher 2020). Such blurring of boundaries may lead to challenges for the ways
in which expertise is performed, and may require new sets of skills to
navigate the changes they may bring to the relations between people
diagnosed and mental healthcare professionals. Digital and AI-Based Technologies
in Mental Healthcare It may also require both
groups to acquire greater insights into the available legislation, in order to
better understand how such technologies and the data acquired through
them can be used and shared. Whereas proponents of AI and digital technologies in mental healthcare
are enthusiastic about the transformations their implementation could
lead to, critics have drawn attention to some problematic aspects regarding
their (future) use (Fiske et al., 2019). Since many of these concerns are also
valid in relation to the data currently collected from online platforms such
as those that this book focuses on, they will be discussed in more detail, as
they are important to consider when engaging with the insights provided
in the next chapters. The main types of criticism identified focus on the
feasibility and efficiency of the collaborations required, the quality of the
data, and the epistemic character of the insights that can be derived
through the use of digital technologies. Thus, some commentators (Carr,
2020; Pasquale, 2020) have emphasized that the responsible and reliable
development, assessment, and implementation of such technologies
require the collaboration of a diverse community of experts, including
researchers, clinicians, regulators, and people diagnosed. This is bound to
be an arduous process, as the development of a common understanding,
familiarity with core approaches in each discipline, new research methods,
and novel ways to redistribute responsibility will likely be required. Other scholars have raised concerns about the type of data that can be
obtained and from whom and the consequences this may lead to. Thus,
the data that are currently collected through monitoring devices and used
to train algorithms that are supposed to help in decision-making do not
(sufficiently) capture personal, social, cultural, and economic factors, yet
these importantly shape one’s mental state (Birk & Samuel, 2020;
Bradstreet et al., 2019). This situation is partly due to the quantitative
logic underlying these technologies, as they mainly record aspects that can
be measured and analyzed through statistical methods. At the same time,
it is also informed by the biological language surrounding digital pheno
typing, which orients attention in particular directions and may thus lead 2 Epistemic Inroads from the Asylum to Digital Psychiatry 61 to the reification of mental health conditions as biological (Birk & Samuel,
2020). From this point of view, Bemme et al. Digital and AI-Based Technologies
in Mental Healthcare (2020, not paginated) con
vincingly warned that “[t]he quest for holism through big data may thus
lead to a re-emergence of the tyranny of reductionism.” Apart from the
decontextualization and reductionism that might be operated through
digital data collection practices, scholars have also warned about impor
tant inequalities among people diagnosed with mental health conditions
in terms of access and representation. While in the days of the asylum,
mostly the poor and the destitute were overrepresented in the data col
lected (Porter, 2018), nowadays socioeconomic status and location inform
the availability of data, as almost half of the world’s population still does
not have access to the Internet and digital technologies. Another prob
lematic aspect is that thus far people diagnosed with mental health condi
tions and their carers have not been involved in the development of
AI-based interventions (Bradstreet et al., 2019). Bradstreet et al. (2019:128) warned in this sense that “[t]here are risks of replicating exist
ing and even creating new inequalities in health and mental health as well
as risks that new forms of coercion or compulsory treatment could emerge. Scrutiny, transparency and algorithmic accountability are essential.” Noteworthy concerns have also been raised about the epistemic char
acter of the insights acquired from such data and the validity of the deci
sions based on them. For instance, critics have highlighted that algorithms
are trained on insights acquired through the subjective and selective work
of human professionals. From this point of view, algorithms are not
objective, as they reflect current hierarchies of knowledge and patterns of
exploitation in their functioning (Bemme et al., 2020). Another relevant
perspective is provided by Coghlan and D’Alfonso (2021), who put for
ward four types of possible relations between the information generated
using digital devices and mental health phenomena: two types of causal
relations, a correlative and a constitutive relation. Through these four
scenarios, Coghlan and D’Alfonso (2021) show that the availability of
data collected through digital technologies does not automatically lead to
reliable insights about people’s mental health. To arrive at the latter, infer
ences need to be made and their quality depends on the availability of
accurate and precise definitions, adequate measurement tools, the possi
bility to correctly identify distorting effects, and the opportunity to draw C. Egher 62 C. Egher upon additional types of data. Digital and AI-Based Technologies
in Mental Healthcare Thus, while digital phenotyping may con
tribute to new and more reliable knowledge about mental health, caution
is needed not to misinterpret and misrepresent the epistemic character of
the data collected through digital technologies. This is particularly impor
tant, given that algorithms have thus far had a hard time distinguishing
between different disease categories from the same data, while people
diagnosed with mental health conditions present a high level of comor
bidity (Birk & Samuel, 2020). Criticism regarding the use of AI and digital technologies in mental
healthcare has also focused on the changes they have prompted to the
ways in which the psychiatric subject can be constituted and studied. In
this sense, scholars have highlighted the blurring of boundaries between
those who make and who are made by the data collected through such
tools. They have also argued that the real-time collection of different
types of psychological data and the countless possibilities to aggregate
them contribute to the development of an “aggregate human” that defies
stable categories as well as micro and macro distinctions (Bemme et al.,
2020). This raises questions about the types of mental healthcare that
would be appropriate for such a human and about the methods through
which s/he can best be studied. Other scholars have noted the relatively narrow domain of application
of these digital technologies, as most technological companies have
focused their investments on tools meant to alleviate mild to moderate
symptoms and have manifested less interest for the development of
instruments able to address the more severe symptoms of conditions such
as bipolar disorder or schizophrenia. This way, those who most need
mental healthcare services might be further disadvantaged and the wide
spread use of digital tools will most likely fail to contribute to curbing
current mental healthcare costs (Mou & Insel, 2021). Furthermore, con
siderable doubts have also been expressed about the quality of the thera
peutic approaches enabled through these technologies, as several reviews
have indicated that many of the technologies and applications that people
diagnosed with mental health conditions can access freely or at a low cost
have not been scientifically tested or have only been assessed through
short, small-scale studies. 2 Epistemic Inroads from the Asylum to Digital Psychiatry 2 Epistemic Inroads from the Asylum to Digital Psychiatry 63 Whereas numerous mobile phone applications are available for use for
people diagnosed with various mental health conditions (Faurholt-Jepsen
et al., 2018; Faurholt-Jepsen et al., 2019) and are downloaded millions of
times per month (Marathe & Ravi, 2020; Nicholas et al., 2015), their
quality can vary widely and they are much less regulated than medicine-
based treatments. Furthermore, since most information available thus far
consists of engagement metrics, there is limited understanding about the
ways in which these technologies shape the quality of mental healthcare
care. While some people diagnosed may feel empowered to use digital
technologies to better understand and manage their conditions, others
may feel overwhelmed. Important questions have also been raised about
the long-term impact of such technologies on people’s abilities to manage
their mental health, with some critics worrying that intensive engage
ment with digital technologies may lead to “de-skilling,” as individuals
would come to rely more on these tools and spend less time and effort
actively managing their condition. Thus, even in the case of applications
and digital technologies of proven quality, it is unclear how to optimally
deploy them in practice, and how the preferences of individuals diag
nosed with mental health conditions and the specificity of their daily lives
could best be considered in this sense. Numerous critics have also raised concerns about various legal and
regulatory aspects in regard to the use of digital technologies. Thus, many
commentators have highlighted the highly intrusive character of these
devices (Carr, 2020), as they imply continuous video and audio monitor
ing, which makes their acceptability questionable. In this sense, Guta
et al. (2018) argued that such technologies should be seen as part of a
“larger integrated surveillance apparatus” or of a “digital medicine panop
ticon,” which focuses on already marginalized communities. People diag
nosed with mental health conditions enjoy different degrees of legal
protection in this sense, depending on the country they live in. For
instance, in France and other countries of the European Union, the
General Data Protection Regulation (GDPR) should afford them greater
protection, whereas in the US the legal provisions available remain lim
ited and differ among states. Other critics (Carr, 2020) have raised con
cerns about the degree to which people diagnosed with mental health
conditions, whose state can fluctuate over time, can give informed 64 C. Egher C. 2 Epistemic Inroads from the Asylum to Digital Psychiatry Egher consent and about the time frame within which such consent could be
considered valid. Another important concern stems from the fact that
data collected through such technologies could become available to third
parties who may use it in a discriminatory fashion or in other ways disad
vantageous to the individuals from which they have been collected. Such
data are already collected and used in the judiciary, as some people with
mental health conditions who would otherwise be hospitalized or incar
cerated are allowed to stay home under GPS monitoring (Boone et al.,
2017). While at first glance such digital approaches may seem more
humane and affording better care, there are also concerns that they may
entail new types of coercive measures, including the mandatory sharing
of mental healthcare information, such as the number of hospitalizations
or suicidal behavior, across institutions. Some commentators therefore
expect digital technologies to be intensively used in coercive psychiatric
interventions (Gooding, 2019). The developments described here sketch the conditions of possibility
for the epistemic practices that this book focuses on. The online perfor
mances of expertise about bipolar disorder to which we will now turn our
attention carry therefore vestiges of the different theoretical approaches
that shaped the development of knowledge about mental health, of the
various tools and instruments used for the collection of data that have
been discussed in the first part of this chapter. They are also shaped by the
new practices and forms of knowledge that digital technologies currently
allow for and by the hopes and fears that AI-based technologies have
generated among different stakeholders. How expertise about bipolar dis
order is performed in this context by official bodies in the US and France
is discussed at length in the following chapter. American Psychiatric Association. (2013). Diagnostic and Statistical Manual of
Mental Disorders, fifth edition (DSM-5).
American Psychiatric Association. (1968). Diagnostic and Statistical Manual of
Mental Disorders, second edition (DSM-II).
Arribas-Ayllon, M., Bartlett, A., & Lewis, J. (2019). Psychiatric Genetics. From
Hereditary Madness to Big Biology. Routledge. References American Psychiatric Association. (2013). Diagnostic and Statistical Manual of
Mental Disorders, fifth edition (DSM-5). American Psychiatric Association. (1968). Diagnostic and Statistical Manual of
Mental Disorders, second edition (DSM-II). Arribas-Ayllon, M., Bartlett, A., & Lewis, J. (2019). Psychiatric Genetics. From
Hereditary Madness to Big Biology. Routledge. 2 Epistemic Inroads from the Asylum to Digital Psychiatry 2 Epistemic Inroads from the Asylum to Digital Psychiatry La Documentation Française. Coldefy, M., Le Fur, P., Lucas-Gabrielli, V., & Mousquès, J. (2009). Une Mise
en Perspective de l’Offre de Soins des Secteurs de Psychiatrie Générale et du
Recours à la Medicine Générale. Pratiques et Organization des Soins,
40(3), 197–206. Cosgrove, L., Karter, J., McGinley, M., & Morrill, Z. (2020). Digital
Phenotyping and Digital Psychotropic Drugs: Mental Health Surveillance
Tools That Threaten Human Rights. Health and Human Rights, 22(2), 33–39. Tools That Threaten Human Rights. Health and Human Rights, 22(2), 33–39. D’Alfonso, S. (2020). AI and Mental Health. Current Opinion in Psychology,
36, 112–117. hh
D’Alfonso, S. (2020). AI and Mental Health. Current Opinion in Psychology,
36, 112–117. Dincin, J. (1975). Psychiatric Rehabilitation. Schizophrenia Bulletin, 13, 131–147.f Dincin, J. (1975). Psychiatric Rehabilitation. Schizophrenia Bulletin, 13, 131–147. Egeland J A
Gerhard D S
Pauls D L
et al (1987) Bipolar Affective Dincin, J. (1975). Psychiatric Rehabilitation. Schizophrenia Bulletin, 13, 131–147. Egeland, J. A., Gerhard, D. S., Pauls, D. L., et al. (1987). Bipolar Affective
D
d
L k d
DNAM k
Ch
11 N
325 783 787 Dincin, J. (1975). Psychiatric Rehabilitation. Schizophrenia Bulletin, 13, 131–147. Egeland, J. A., Gerhard, D. S., Pauls, D. L., et al. (1987). Bipolar Affective
Disorder Linked to DNA Markers on Chromosome 11. Nature, 325, 783–787. Estroff S (1985/2001) Making it Crazy. An Ethnography of Psychiatric Clients
i
A
i
C
i
2 d d
U i
i
f C lif
i P Dincin, J. (1975). Psychiatric Rehabilitation. Schizophrenia Bulletin, 13, 131–147. Egeland, J. A., Gerhard, D. S., Pauls, D. L., et al. (1987). Bipolar Affective
Disorder Linked to DNA Markers on Chromosome 11. Nature, 325, 783–787.f Egeland, J. A., Gerhard, D. S., Pauls, D. L., et al. (1987). Bipolar Affective
Disorder Linked to DNA Markers on Chromosome 11. Nature, 325, 783–787.f Estroff S (1985/2001) Making it Crazy. An Ethnography of Psychiatric Clients
in an American Community 2nd ed. : University of California Press. Faurholt-Jepsen, M., Bauer, M., & Kessing, L. (2018). Smartphone-Based
Objective Monitoring in Bipolar Disorder: Status and Considerations. International Journal of Bipolar Disorders, 6, 6. Faurholt-Jepsen, M., Torri, E., Cobo, J., et al. (2019). Smart-phone based Self-
monitoring in Bipolar Disorder: Evaluation of Usability and Feasibility of
Two Systems. Journal of Bipolar Disorders, 7(1), 1–11. Fernandes, B. S., Williams, L. M., Steiner, J., Leboyer, M., Carvalho, A. F., &
Berk, M. (2017). The New Field of ‘Precision Psychiatry. BMC Medicine,
15(1), 80. 2 Epistemic Inroads from the Asylum to Digital Psychiatry 65 Barak, A., Klein, B., & Proudfoot, J. (2009). Defining Internet-Supported
Therapeutic Interventions. Annals of Behavioral Medicine, 38(1), 4–17. h
Baron, M., Risch, N., Hamburger, R., et al. (1987). Genetic Linkage between
X- chromosome Markers And Bipolar Affective Illness. Nature, 326, 289–292. f
Bauer, M., Monteith, S., Geddes, J., Gitlin, M. J., Grof, P., Whybrow, P. C., &
Glenn, T. (2019). Automation to Optimise Physician Treatment of Individual
Patients: Examples in Psychiatry. The Lancet Psychiatry, 6(4), 338–349. h
Bemme, D., Brenman, N., & Semel, B. (2020, October 13). The Subjects of
Digital Psychiatry’. Somatosphere. Available at http://somatosphere.net/2020/
subjects-of-digital-psychiatry.html/. Bernadet, P., Douraki, T., & Vaillant, C. (2002). Psychiatrie, Droits de l’Homme
et Défense des Usagers en Europe. Écrès. Bhugra, D., Tasman, A., Pathare, S., Priebe, S., Smith, S., Torous, J., et al. (2017). The WPA-Lancet Psychiatry Commission on the Future of Psychiatry. The Lancet Psychiatry, 4(10), 775–818. h
Birk, R., & Samuel, G. (2020). Can Digital Data Diagnose Mental Health
Problems? A Sociological Exploration of ‘Digital Phenotyping’. Sociology of
Health & Illness, 42(8), 1873–1887. Blaquière, J. (2019, January 14). Doctolib se lance dans la téléconsultation. Le
Figaro. Available at https://www.lefigaro.fr/societes/2019/01/14/20005-
20190114ARTFIG 00128-doctolib-se-lance-dans-la-teleconsultation.phph Boone, M., van der Kooij, M., & Rap, S. (2017). The Highly Reintegrative
Approach of Electronic Monitoring in the Netherlands. European Journal of
Probation, 9(1), 46–61.h Boyle, P., & Callahan, D. (1995). Managed Care in Mental Health: The Ethical
Issues. Health Affairs, 14, 7–23. f
Bradstreet, S., Allan, S., & Gumley, A. (2019). Adverse Event Monitoring in
mHealth for Psychosis Interventions Provides an Important Opportunity for
Learning. Journal of Mental Health, 28(5), 461–466.h Brown, P. (1988). The Transfer of Care: Psychiatric Deinstitutionalization and Its
Aftermath. Routledge Kegan & Paul. Caisse Nationale de Solidarité pour l’Autonomie (CNSA) (2017, Mai). Les
GEM. Groupes d’entraide mutuelle. Les cahiers pédagogiques de la CNSA. Carr, S. (2020). ‘AI gone mental’: Engagement and Ethics in Data-driven
Technology for Mental Health. Journal of Mental Health, 29(2), 125–130. Castel, R. (1981). La Gestion des Risques. De l’Anti-Psychiatrie à l’Après-
Psychanalyse. Minuit. Coffin, J.-C. (2009). La Psychiatrie par Temps de Crise. Études, 6(410), 751–761. 66 C. Egher Coghlan, S., & D’Alfonso, S. (2021). Digital Phenotyping: An Epistemic and
Methodological Analysis. Philosophy & Technology, 34(4), 1905–1928. Coghlan, S., & D’Alfonso, S. (2021). Digital Phenotyping: An Epistemic and
Methodological Analysis. Philosophy & Technology, 34(4), 1905–1928. Coldefy, M. (Ed.). (2007). La Prise en Charge de la Santé Mentale. Recueil
d’Études Statistiques. 2 Epistemic Inroads from the Asylum to Digital Psychiatry Fiske, A., Henningsen, P., & Buyx, A. (2019). Your Robot Therapist Will See
You Now: Ethical Implications of Embodied Artificial Intelligence in
Psychiatry, Psychology, and Psychotherapy. Journal of Medical Internet
Research, 21(5), 1–12. Flore, J. (2021). Ingestible Sensors, Data, and Pharmaceuticals: Subjectivity in
the Era of Digital Mental Health. New Media & Society, 1–18. Foucault M (1972/2010). History of Madness. : Routledge. Translated by
Jonathan Murphy and Jean Khalfa. Freud S (1917/2005) On Murder, Mourning and Melancholia. : Penguin Books. Trans. by Shaun Whiteside Girard, V. (2008). Dossier n° 13- Auto Support en Santé Mentale en France. Bulletin Amades, 75, 1–9. 2 Epistemic Inroads from the Asylum to Digital Psychiatry 2 Epistemic Inroads from the Asylum to Digital Psychiatry 67 m, I., Campbell, J., Rogers, J., Lambert, D., Blacklow, B., Henderseon, M., & Manderscheid, R. (2006). National Estimates for Mental Health
Mutual Support Groups, Self-Help Organizations, and Consumer-Operated
Services. Administration and Policy in Mental Health and Mental Health
Services Research, 33(1), 92–103. Gooding, P. (2019). Mapping the Rise of Digital Mental Health Technologies:
Emerging Issues for Law and Society. International Journal of Law and
Psychiatry, 67, 1–11. Grob, G. (2005). The Transformation of Mental Health Policy in Twentieth
Century America. In M. Gijswijt-Hofstra, H. Oosterhuis, J. Vijselaar, &
H. Freeman (Eds.), Psychiatry and Mental Health Care in the Twentieth-Century:
Comparisons and Approaches (pp. 141–161). Amsterdam University Press. Comparisons and Approaches (pp. 141–161). Amsterdam University Press. Guta, A., Voronka, J., & Gagnon, M. (2018). Resisting the Digital Medicine
Panopticon: Toward a Bioethics of the Oppressed. The American Journal of
Bioethics, 18(9), 62–64. Healy, D. (2008). Mania: A Short History of Bipolar Disorder. John Hopkins
University Press. Hochmann, J. (2017). Histoire de la Psychiatrie. Paris: Presses Universitaires de
France (4th ed.). Hogan, M. (2003). The President’s New Freedom Commission: Recommen
dations to Transform Mental Health Care in America. Psychiatric Services,
54(11), 1467–1474. Hogan, M., & to President George W Bush. (2002). Interim Report of the
President’s New Freedom Commission on Mental Health. In M. Gijswijt-
Hofstra, H. Oosterhuis, J. Vijselaar, & H. Freeman (Eds.), Psychiatric Cultures
Compared. Psychiatry and Mental Health Care (p. 156). Amsterdam
University Press. IBM Research Editorial Staff (January 5, 2017). ‘IBM 5 in 5: With AI, our words
will be a window into our mental health.’ Retrieved from: https://www.ibm. com/blogs/research/2017/01/ibm-5-in-5-our-words-will-be-the-windows-to-
our-mental-health/. Accessed on 23.09.2021 Illlouz, E. (2008). Saving the Modern Soul. Therapy, Emotions, and the Culture of
Self-Help. University of California Press. Kataoka, S., Zhang, L., & Wells, K. (2002). Unmet Need for Mental Health
Care Among U.S. Children: Variation by Ethnicity and Insurance Status. American Journal of Psychiatry, 159, 1548–1555. Kaufman, S., & Whitehead, K. (2016). Producing, Ratifying, and Resisting
Support in an Online Support Forum. Health, 1–17. Advance online publi
cation. https://doi.org/10.1177/1363459315628043 68 C. Egher Lagan, S., Ramakrishnan, A., Lamont, E. et al. (2020). Digital Health
Developments and Drawbacks: A Review and Analysis of Top-Returned
Apps for Bipolar Disorder. Int J Bipolar Disord, 8, 39. https://doi.org/10.1186/
s40345-020-00202-4 Laval, C. (2015). Edito. Rhizome, 58(4), 1–2. Le Monde. (2018, August 18). La Psychiatrie, un secteur en état d’urgence. Available at https://www.lemonde.fr/idees/article/2018/08/18/la-psychiatrie-
un-secteur-en-etat-d-urgence_5343765_3232.html Leonhardt, M. 2 Epistemic Inroads from the Asylum to Digital Psychiatry (2021, May 10). What You Need to Know About the Cost and
Accessibility of Mental Health Care in America. Available at https://www.cnbc. com/2021/05/10/cost-and-accessibility-of-mental-health-care-in-
america.htmlh Marathe, P., & Ravi, S. (2020, December 8). The Mental Health Dilemma:
If Technology is the Problem, Can it also be the Solution?. STAT News. Available at https://www.statnews.com/2020/12/08/mental-health-dilemma-
technology-cause-and-solution/ Martinez-Martin, N., Insel, T., Dagum, P., Greely, H., & Cho, M. (2018). Data
Mining for Health: Staking Out the Ethical Territory of Digital Phenotyping. Npj Digital Medicine, 1(1), Article 68. McCosker, A. (2018). Engaging Mental Health Online: Insights from beyond
blue’s forum influencers. New Media & Society, 20(12), 4748–4764.h l
Mental Health America. (2022). The State of Mental Health in America. Available
at https://mhanational.org/issues/state-mental-health-america Mental Health Care Health Professional Shortage Areas (HPSAs). (2020). Available at Mental Health Care Health Professional Shortage Areas
(HPSAs) | KFF Miller, K. (2019). A Matter of Perspective: Discrimination, bias and in- equality
in AI. In T. Walsh (Ed.), Closer to the Machine. Technical, Social, and Legal
Aspects of AI. Office of the Victorian Information Commissioner. Available at
https://ovic.vic.gov.au/wp-content/uploads/2019/08/closer-to-the-
machine-web.pdf Mou, D., & Insel, R. (2021, January 19). Startups Should Focus on Innovations
that Truly Improve Mental Health. Available at https://www.statnews. com/2021/01/19/startups-innovations-truly-improve-mental-health/h National Council for Mental Wellbeing. (2017). The Psychiatric Shortage. Causes
and Solutions. Available at https://www.thenationalcouncil.org/wp-content/
uploads/2017/03/Psychiatric-Shortage_National-Council-.pdf?daf=375
ateTbd56 2 Epistemic Inroads from the Asylum to Digital Psychiatry 2 Epistemic Inroads from the Asylum to Digital Psychiatry 69 Nicholas, J., Larsen, M., Proudfoot, J., & Christensen, H. (2015). Mobile Apps
for Bipolar Disorder: A Systematic Review of Features and Content Quality. Journal of Medical Internet Research, 17(8), e198. Norman, C. (2006). The Fountain House Movement, an Alternative
Rehabilitation Model for People with Mental Health Problems, Members’
Descriptions of What Works. Scandinavian Journal of Caring Science,
20, 184–192. OECD. (2015). Country Note: How does health spending in FRANCE compare? OECD Health Statistics. OECD (2016) H l h P li
i F
OECD H l h P li
O
i
OECD D. (2016). Health Policy in France. OECD Health Policy Overview. OECD. Olowu, A. (2015). Delivering Proof of Care at the Point of Care. How Electronic
Visit Verification Can Benefit Clinicians, Home Health Workers and Patients. Health Management Technology, 36(4), 16.h Open Minds. (2019). The U.S. Mental Health Market: $225.1 Billion In
Spending. Market Intelligence Report. Pasquale, F. (2020). New Laws of Robotics. Defending Human Expertise in the Age
of AI. The Belknap Press of Harvard University Press.h h
Petitjean, F. (2009). The Sectorization System in France. International Journal of
Mental Health, 38(4), 25–38. Pickersgill, M. (2019). Digitising Psychiatry? Sociotechnical Expectations,
Performative Nominalism and Biomedical Virtue in (Digital) Psychiatric
Praxis. Sociology of Health & Illness, 41(1), 16–30. Pignarre, P. (2006). Les Malheurs des Psys. Psychotropes et Médicalisation du Social. La Découverte.h Porter, T. (2018). Genetics in the Madhouse. The Unknown History of Human
Heredity. Princeton University Press. Provost, D., & Bauer, A. (2001). Trends and Developments in Public Psychiatry
in France since 1975. Acta Psychiatrica Scandinavica, 104(410), 63–68. Raybaud, A. (2021, June 16). Chez les étudiants en medicine, la psychiatrie plus
délaissée que jamais. Le Monde. Available at https://www.lemonde.fr/cam
pus/article/2021/06/16/chez-les-etudiants-en-medecine-la-psychiatrie-plus-
delaissee-que-jamais_6084290_4401467.html Rose, N. (2018). Our Psychiatric Futures. Polity Press. Roudinesco, E. (2019, February 8). Les psychanalystes ont contribué à leur pro
pre déclin. Le Monde. Rudin, C., & Ustun, B. (2018). Optimized Scoring Systems: Toward Trust in
Machine Learning for Healthcare and Criminal Justice. Interfaces,
48(5), 449–466. 70 C. Egher Scheid, T. (2000). Rethinking Professional Prerogative: Managed Mental Health
Care Providers. Sociology of Health & Illness, 22(5), 700–719. Shatte, A., Hutchinson, D., & Teague, S. (2019). Machine Learning in Mental
Health: A Scoping Review of Methods and Applications. Psychological
Medicine, 49, 1426–1448. Simon, C. (2015, September 9). US Faces Severe Shortage of Psychiatrists as
Demand Grows-Report. RT News. © The Author(s) 2023
C. Egher, Digital Healthcare and Expertise, Health, Technology and Society,
https://doi.org/10.1007/978-981-16-9178-2_3 2 Epistemic Inroads from the Asylum to Digital Psychiatry Accessed August 12, 2017, from https://
/
/314777
h
hi
i
d
d
/ (
p
)
g
y
Demand Grows-Report. RT News. Accessed August 12, 2017, from http
www.rt.com/usa/314777-us-shortage-psychiatrists-demand-grows/ p
g
,
,
www.rt.com/usa/314777-us-shortage-psychiatrists-demand-grows/ Swain, G. (1988). Le Sujet de la Folie. Naissance de la Psychiatrie. Privat. Troisoeufs, A., & Eyraud, B. (2015). Psychiatrisés en Lutte, Usagers, Gemeurs…:
Une Cartographie des Différentes Formes de Participation. Rhizome,
58(4), 3–4. Turkle, S. (1981). Psychoanalytic Politics. Freud’s French Revolution. The
MIT Press. Verdoux, H. (2003). Psychiatry in France. International Journal of Social
Psychiatry, 49(2), 83–86. y
y
Verdoux, H., & Tignol, J. (2003). Focus on Psychiatry in France. British Journal
of Psychiatry, 183, 466–471. WHO. (2000). Health Systems: Improving Performance. World Health Report
2000. World Health Organization. 2000. World Health Organization.h WHO. (2008). The Global Burden of Disease: 2004 update. World Health
Organization. Open Access This chapter is licensed under the terms of the Creative Commons
Attribution 4.0 International License (http://creativecommons.org/licenses/
by/4.0/), which permits use, sharing, adaptation, distribution and reproduction
in any medium or format, as long as you give appropriate credit to the original
author(s) and the source, provide a link to the Creative Commons licence and
indicate if changes were made.h The images or other third party material in this chapter are included in the
chapter’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the chapter’s Creative Commons
licence and your intended use is not permitted by statutory regulation or exceeds
the permitted use, you will need to obtain permission directly from the copy
right holder. The Drama of Expertise About Bipolar
Disorder Online The Internet has been increasingly used by governments around the
world as a cost-effective way to provide health-related information to
various audiences (Barak, 1999; Bennett & Glasgow, 2009; Christensen
et al., 2004; Griffiths et al., 2006; Levy & Strombeck, 2002). Whereas
the US was an early enthusiast and France a relative latecomer, for almost
two decades now, important governmental agencies and mental health
care providers in both countries have been sharing insights about bipolar
disorder online. In so doing, they have been confronted with two major
challenges. On the one hand, they need to conform with legislation
requiring governmental agencies that have online platforms to make sure
that the information they share online is accessible to people with dis
abilities. On the other hand, they are required to make their views public
in a context where many people, including mental health professionals,
are critical of psychiatry (Morrison, 2013), and where important strug
gles take place between the different types of professionals involved. This
means that the official character of an institution is no longer a sufficient
guarantee that the psychiatric insights it provides are accepted as knowl
edge, so when sharing information online, official bodies need to make
proof of their expertise. Because of these challenges, “science 71 © The Author(s) 2023
C. Egher, Digital Healthcare and Expertise, Health, Technology and Society,
https://doi.org/10.1007/978-981-16-9178-2_3 C. Egher 72 C. Egher communication represents a crucial activity” (Horst et al., 2017:881)
nowadays and the “investigation of the Internet’s applicability as a tool
for public mental health interventions is important” (Ybarra & Eaton,
2005:75).h This chapter therefore focuses on the performative techniques that
highly authoritative governmental agencies—The National Institute of
Mental Health (NIMH) in the US and La Haute Autorité de Santé (HAS)
in France—deploy to convincingly perform expertise about bipolar dis
order on their online platforms. Coordination plays an important role in
the development of expertise, based on the new conceptualization I put
forward. Coordination can refer to how different stakeholders come to
work together toward a common goal, as we shall see in the next chapter,
but it also denotes the fine-tuning activities one and the same stakeholder
needs to engage in across different settings, for the knowledge displayed
to be authoritative. The Drama of Expertise About Bipolar
Disorder Online While such activities are always necessary, online they
become all the more important given the multiple presences that one
may have, the haphazard way in which the audience might arrive at infor
mation, and the manifold ways in which disparate elements can be linked
together, thereby acquiring new meaning. This chapter focuses on this
dimension of coordination and discusses the activities NIMH and HAS
undertook as they had to create and manage relations across different
online stages and materials (Drucker, 2011) to convincingly perform
expertise about bipolar disorder online. To set the stage, I briefly discuss
the context in which the American and the French governments have
started to promote the use of the Internet as a cost-effective way to pro
vide mental health-related information. 1 The other participating countries are the Netherlands (program leader), Belgium, Germany,
Ireland, and the UK. 2 The US Access Board is a federal agency that aims to enhance the access of people with disabilities
by providing guidelines and standards on various aspects, such as information technology, trans
portation, and medical equipment. The Internet in Mental Healthcare in France
and the US
3 The Drama of Expertise About Bipolar Disorder Online 73
2017
2016
2016
1
2017 Similarly to France, also in the US the authorities started to look for
online solutions in the provision of mental healthcare out of financial
considerations and because of a dramatic expected decrease in the num
ber of psychiatrists in the near future, due to retirement and low numbers
of student applications in relevant fields. It is in this context that the
Internet came to be seen as an effective and relatively cheap medium that
could be efficiently used (1) to educate people about mental health in an
attempt to prevent and to timely diagnose; (2) to facilitate, expedite, and
enhance communication between people diagnosed and medical profes
sionals; (3) to enable access to care for people living in remote areas
(Farrell & McKinnon, 2003); and (4) to provide treatment in the form
of various online therapies (Barak & Grohol, 2011; Ybarra & Eaton,
2005). Like in France, such tendencies were encouraged by the C. Egher C. Egher 74 development of various strategies and pieces of legislation. For instance,
already in 1996, the Electronic Freedom of Information Act (E-FOIA)
Amendments mandated that governmental agencies provide information
and make their records available in an electronic format (BIS, 2016;
Department of Justice, 2014). Aware of people’s increasing tendencies to
look for information online, in May 2012, the White House launched
the Digital Government Strategy, which aimed to further encourage
agencies to use information and communication technologies (ICTs). It
also provided guidance meant to assist them “to improve digital services
and use emerging technologies to serve the public as effectively as possi
ble” (OMB Memo 17-06, 2016). As a follow-up on this strategy, the
White House released the U.S. Digital Service Playbook in 2014, which
offered 13 main recommendations drawn from successful practices devel
oped in the public as well as private sector (ibid.; The U.S. Digital Service,
2018). Despite such encouragement, agencies in both countries also
encountered considerable challenges, as I will now discuss. 3 The International Organization for Standardization (ISO) is an international standard-setting
body composed of representatives from various national standards organizations, which develops
voluntary standards. In March 2017 ISO was working in 162 countries. g
4 The resolution is registered as COM (2001) 529–C5-0074/2002–2002/2032 (COS). 3 The International Organization for Standardization (ISO) is an international standard-setting
body composed of representatives from various national standards organizations, which develops
voluntary standards. In March 2017 ISO was working in 162 countries.
4 The resolution is registered as COM (2001) 529–C5-0074/2002–2002/2032 (COS). Technical Challenges: Accessibility Regulations
for Online Platforms
2017
2 3 The Drama of Expertise About Bipolar Disorder Online 75 2017
3
4
According to WCAG 2.0, websites should be perceivable, operable,
understandable, and robust. This means that the content put forward
should be easy to see and hear and that any non-text content should be
accompanied by text options, which can be more easily accessed using
braille or speech, among others. At the same time, websites should be
designed so that users can easily find their way around them, the infor
mation provided on them should be understandable, and the functions
they contain should all be accessible using a keyboard. Furthermore, the
content provided on websites should not cause seizures, and the compat
ibility of online platforms with “future user agents, including assistive
technologies” should be enhanced (WCAG2.0). Governmental agencies
and mental healthcare providers need therefore to make sure that the C. Egher 76 C. Egher information they provide on their online platforms is accessible to people
with different types of disabilities, to people with different levels of edu
cation, and to people whose modest income may mean that they cannot
afford a computer, but can only look up such information using cheaper,
less developed, or outdated technologies. While such requirements are
necessary and laudable, it is important to note that they place significant
constraints on these stakeholders regarding the ways in which they can
use the Internet and the type of affordances they select for their websites. 3 The Drama of Expertise About Bipolar Disorder Online 3 The Drama of Expertise About Bipolar Disorder Online The degree to which such critics have opposed and continue to chal
lenge psychiatry has varied as has their identity. Sometimes, criticism has
been radical and has included, next to intellectuals, mental health profes
sionals, with psychiatrists such as Szasz arguing that mental illness was a
myth, a labeling mechanism through which the social and economic cir
cumstances that dramatically affected people’s lives were occluded from
view (Szasz, 1961). Other mental health professionals such as Laing
sought for a middle ground, founding residential homes and striving to
develop more equal therapeutic approaches (Fussinger, 2011; Roberts &
Itten, 2006). Similar variety has characterized the responses of people
diagnosed, with some wholeheartedly embracing the medical model,
with others arguing against specific medical interventions, such as forced
containment and electroconvulsive therapy (ECT), and with yet others,
ex-patients or self-entitled “survivors” of the mental health system (espe
cially in the US) rejecting the medical model altogether (McLean, 2000). Some authors (McLean, 2003; Rissmiller & Rissmiler, 2006) suggest
that such antipsychiatric tendencies have been transformed and even
integrated within the mental healthcare system they were once so critical
of, in part due to psychiatry’s reaction to the criticism received. Thus,
psychiatry embraced a biomedical approach in efforts to render itself
more scientific, a growing number of medical professionals started to
value the insights of their patients, and the rights of the latter came to be
codified in patient charters (Hopton, 2006). Furthermore, antipsychiatry
supporters are claimed to have morphed in time into members of the
broad consumer movement (McLean, 2003; Rissmiller & Rissmiler,
2006), which argues for the inclusion of people diagnosed in decision-
making at all levels but which accepts the medical model of mental ill
ness. Such stakeholders are satisfied with the fact that (in principle, at
least) people diagnosed have the opportunity to choose the medical pro
fessionals they see and also have a say in the treatment they receive. According to proponents of such views, while more radical ex-
patients/“survivors” still exist, a new type of consumer has come into
being, who no longer shares the feelings of hopelessness of the ex-patients
from the 1970s, nor the latter’s strong criticism and suspicion toward
mental healthcare professionals The degree to which such critics have opposed and continue to chal
lenge psychiatry has varied as has their identity. Epistemic and Social Challenges: Critique
of Psychiatry and Divergent Interests While the accessibility requirement set upon governmental agencies and
mental healthcare providers has led to challenges of a more technical
nature, the public character of the Internet has contributed to some epis
temic and social difficulties. This second set of challenges refers to the
current context in which NIMH and HAS provide insights about bipolar
disorder online, where the authority of such bodies is no longer readily
accepted and their recommendations are not taken up without critical
consideration. According to Rose, (2018:20), “[psychiatry’s] very foun
dations came under attack from all sides” in the 1960s–1970s, and ever
since, the expertise and authority of governmental agencies and mental
healthcare providers has been challenged in various ways. Antipsychiatry
emerged in that period as a movement which challenged the validity of
psychiatric diagnostic and therapeutic practices, considering psychiatry
to be an instrument of social oppression and control (Castel, 1976; Rose,
2018). Supporters of the movement further criticized the power imbal
ance at the heart of all forms of psychiatric treatments and the alienation
of medical professionals from their patients. At the same time, many
questioned the validity of psychiatric diagnoses, which they saw as arbi
trary (McPherson & Armstrong, 2006; Wright & Cummings, 2005) and
over-pathologizing (Horwitz & Wakefield, 2007; Scott, 2006), while
others denounced the inhumane treatment of people placed in mental
hospitals (Gostin, 2008; Morrison, 2013). 77 3 The Drama of Expertise About Bipolar Disorder Online Sometimes, criticism has
been radical and has included, next to intellectuals, mental health profes
sionals, with psychiatrists such as Szasz arguing that mental illness was a
myth, a labeling mechanism through which the social and economic cir
cumstances that dramatically affected people’s lives were occluded from
view (Szasz, 1961). Other mental health professionals such as Laing
sought for a middle ground, founding residential homes and striving to
develop more equal therapeutic approaches (Fussinger, 2011; Roberts &
Itten, 2006). Similar variety has characterized the responses of people
diagnosed, with some wholeheartedly embracing the medical model,
with others arguing against specific medical interventions, such as forced
containment and electroconvulsive therapy (ECT), and with yet others,
ex-patients or self-entitled “survivors” of the mental health system (espe
cially in the US) rejecting the medical model altogether (McLean, 2000). Some authors (McLean, 2003; Rissmiller & Rissmiler, 2006) suggest
that such antipsychiatric tendencies have been transformed and even
integrated within the mental healthcare system they were once so critical
of, in part due to psychiatry’s reaction to the criticism received. Thus,
psychiatry embraced a biomedical approach in efforts to render itself
more scientific, a growing number of medical professionals started to
value the insights of their patients, and the rights of the latter came to be
codified in patient charters (Hopton, 2006). Furthermore, antipsychiatry
supporters are claimed to have morphed in time into members of the
broad consumer movement (McLean, 2003; Rissmiller & Rissmiler,
2006), which argues for the inclusion of people diagnosed in decision-
making at all levels but which accepts the medical model of mental ill
ness. Such stakeholders are satisfied with the fact that (in principle, at
least) people diagnosed have the opportunity to choose the medical pro
fessionals they see and also have a say in the treatment they receive. According to proponents of such views, while more radical ex-
patients/“survivors” still exist, a new type of consumer has come into
being, who no longer shares the feelings of hopelessness of the ex-patients
from the 1970s, nor the latter’s strong criticism and suspicion toward
mental healthcare professionals. 78 C. Egher C. Egher C. Egher An overview of books and articles published in the last two decades
suggests, however, that such claims about the successful rapprochement
between former antipsychiatry supporters and medical institutions
underestimate the critical atmosphere which continues to surround psy
chiatry. Numerous psychiatrists remain critical of their specialty and have
come together in various organizations, such as The International Critical
Psychiatry Network, to exchange views and to seek to develop alternatives
to the current dominant approach. At the same time, they call for drastic
reform of the mental healthcare system, arguing that accessibility and
quality of care remain importantly dependent on markers of identity,
such as class, race, and gender (Hopton, 2006; Metzl, 2009). Another
group of critics accuses current psychiatry of medicalization or imperial
istic tendencies, as normal aspects of life and behavior, such as mourning,
have become pathologized (Lane, 2009). While some commentators
consider psychiatry as a political science since its very inception (Rose,
2018), others see an augmentation of its politicization and argue that it
puts forward views that have little scientific backing in order to serve
particular interests and to uphold certain social values (Wright &
Cummings, 2005). Yet others decry the medicalization of mental health
conditions in that it focuses solely on medications and neglects social
provisions, which are highly necessary for the recovery and social reinte
gration of people diagnosed (Kinderman, 2014). Psychiatrists and journalists alike have criticized the close relation
between psychiatrists and pharmaceutical companies (Carlat, 2010;
Kirsch, 2010; Whitaker, 2010). From this point of view, some deplore
the fact that most research on the effectiveness of specific medications is
conducted by the pharmaceutical companies themselves, which suggests
the results may be biased (Whitaker, 2010). Others downright challenge
the effectiveness of medical treatments, arguing, for instance, that there is
no significant difference between the effects of antidepressants and those
of placebo (Kirsch & Sapirstein, 1998). There are also voices who warn
that the promotion of self-determination and empowerment of people
diagnosed with mental health conditions may be superficial and repre
sent a political move rather than genuine interest and appreciation for
their insights (Bernstein, 2006; Hopton, 2006). A staunch opponent of
psychiatry remains the Church of Scientology, which funds the Citizens 79 3 The Drama of Expertise About Bipolar Disorder Online 3 The Drama of Expertise About Bipolar Disorder Online Commission on Human Rights, the museum Psychiatry: Industry of Death,
and which disseminates various materials harshly criticizing the effects of
psychotropic drugs as well as the motives and intentions of this profession. Apart from medical professionals, sociologists, and journalists, critical
psychiatric tendencies continue to be put forward by people diagnosed. From this point of view, the Internet has enabled many opponents to
come together. According to Whitley (2012:1040), “[t]he Internet has
given a means for current and former psychiatric patients, who some
times refer to themselves as ‘survivors’, to widely disseminate often nega
tive attitudes, beliefs, experiences, and opinions vis-a-vis psychiatry.” An
example is The Antipsychiatry Coalition, an organization which aims “to
warn you of the harm routinely inflicted on those who receive psychiatric
‘treatment’ and to promote the democratic ideal of liberty for all law-
abiding people” at an international level. They challenge the medical
understanding of mental health conditions and the scientific bases for the
medical treatment prescribed, accusing it to be “quackery,” and organize
various actions to raise awareness, such as the Electroshock Protest, which
took place on May 16, 2015, in the US. Highly influential in this sense
is also Monica Cassani’s blog, Beyond Meds. An ex-patient and mental
health professional, Cassani claims that this dual position enables her to
share “some interesting and sometimes uncomfortable insights into the
mental health system in the United States” (Cassani, 2017). Other ex-
patients continue to refuse the medical model of mental illness, arguing
instead that their experiences represent different ways of being in the
world, and such views are promoted by groups such as the Hearing Voices
Network (Hopton, 2006; Romme & Escher, 1993).h p
The various types of critique enumerated above indicate that there
continue to be important differences even among mental health profes
sionals regarding their understanding and approach to mental health. Such differences of opinion are augmented by the various reforms which
have been brought to the mental healthcare system in the US and France
(Hochmann, 2017), as by limiting insurance coverage and the number of
(prospective) specialists, these reforms have led to the marginalization of
previously successful professionals, such as psychoanalysts in France
(Pignarre, 2006). At the same time, different types of mental healthcare
professionals often find themselves in competition for limited resources 80 C. Egher C. 3 The Drama of Expertise About Bipolar Disorder Online Egher or have to take over functions and tasks previously fulfilled by other spe
cialists (Desmettre, 2009; Gill et al., 2014). Furthermore, this also seems
to prompt some of them to embrace divergent interests and to advocate
different approaches. Psychologists often reduce psychiatry to the mere
provision of medical treatment and accuse it of neglecting the full person
of the person diagnosed. Furthermore, by focusing too much on genetic
and neurological factors, psychologists and social therapists argue that
important environmental factors are neglected. To the accusation that
they merely prescribe psychotropic drugs, psychiatrists reply by pointing
to general practitioners as the professionals who often prescribe higher
dosages and more medicines than they recommend. As a reaction to
extreme biomedicalization, psychoanalysts seem to be making a come
back in the US, even though access to them is heavily restricted by insur
ance policies (Chessick, 2006; Maness, 2017; O’Sullivan, 2016). In
France, the conflict between psychoanalysts and psychiatrists is still fresh
(Pignarre, 2006). For instance, a report from 2009 for the Minister of
Health and Sports, Roselyne Bachelot, where three approaches to mental
health were evaluated, caused a lot of uproar. At a more general level,
mental healthcare providers decry the influence of managed care controls
and the cost containment policies which have been taken up over the last
few decades, and which severely reduce their autonomy and ability to
make treatment decisions freely (Scheid, 2000). It is in this context,
fraught by different types of challenges, that the online provision of infor
mation about bipolar disorder by governmental agencies and mental
healthcare providers takes place. Succeeding to perform expertise about
bipolar disorder online in such circumstances becomes a rather remark
able feat, which needs to be carefully studied. Rhetoric and the Performance
of Expertise Online In the field of STS, there is a rich tradition of studies on the construction
of scientific knowledge (Bijker et al., 2009; Knorr Cetina, 1999;
MacKenzie, 1990; Shapin & Schaffer, 1985/2011), whereby the 3 The Drama of Expertise About Bipolar Disorder Online 81 importance of social, political, and economic factors in processes which
for a long time have been claimed to be neutral has been highlighted. In
this sense, Felt remarked that “[m]aking knowledge is …never an ‘inno
cent’ activity; nothing can be regarded as ‘natural’ or ‘simply given’” (Felt,
2017:253). Important to understand the work that goes into the con
struction of scientific facts is the work of Latour (1987), who shows that
“science in the making” is messy, subject to heated debates and controver
sies, which are often solved by making strategic alliances or by using one’s
social capital (Bourdieu, 1975). In what follows, I will discuss how gov
ernmental agencies perform expertise about bipolar disorder online,
which means that I will not focus on the construction of scientific facts,
but will trace, instead, the manners in which they are made available
online, their unfolding destiny on these platforms. For this purpose, I
adjust the notion of performance put forward by Goffman (1959/1990)
building upon insights acquired from its application in the study of sci
entific authority (Hilgartner, 2000) and the suggestion that this concept
may be amenable to the investigation of phenomena involving digital
technologies (Hafermalz et al., 2016). Performance thus (re)conceived
was combined with insights from Latour on the rhetorical techniques
through which scientific facts are constructed and with perspectives from
media studies (Drucker, 2011) on the role of various web interface ele
ments on users’ experiences. This innovative approach allowed for various
digital objects and technologies to be approached as actors fulfilling dif
ferent roles and functions in the performance of the two institutions
studied here, and focused the analysis on the ways in which seemingly
disparate elements—aesthetic, functional, content-related—were com
bined to put forward specific perspectives on bipolar disorder for particu
lar audiences. Rhetoric plays an important role in the complex trajectory statements
that follow from mere hypotheses or “hunches” to scientific facts (Latour,
1987; Latour & Woolgar, 1979). Rhetoric and the Performance
of Expertise Online It also lends itself to the analysis of the
relations between the different types of knowledge used in mental health
care, as positive or negative modalities, which imbue statements with
lower or greater degrees of certainty, can convey the dominance of par
ticular ways of understanding while subtly discarding or diminishing
others. Such rhetorical techniques are integral part of a performance, but 82 C. Egher C. Egher their effects are shaped by other important elements: the team and team
mates, who put up the performance, and who can take up different roles,
that is, director, actors; the setting, that is the environment where the
performance takes place; sign equipment consisting of various props that
help foster the impression intended by the performance; and the audi
ence, consisting of those for whom the performance is put up (Goffman,
1990/1959). Whereas the sign equipment and stage Goffman had in
mind were of a different material character, the choice of web design ele
ments, the visual cues provided contribute to the production of meaning
on online platforms (Drucker, 2013). I therefore suggest that the online
technologies these institutions use and the online practices they engage in
help foreground particular insights about bipolar disorder while down
playing others. This has implications not only for how this condition is
understood by online readers, but also for the credibility and standing of
NIMH and HAS. Important for the success of a performance is the division of the stage
into two regions—the front and backstage—which can be accessed by
different people and where different behaviors can be taken up. Whereas
the frontstage refers to the totality of actions and props that the actors
engage with and use in their performance that are visible to the audience,
the backstage refers to the elements that one needs to occlude from view
in order to guarantee a successful performance, information to which the
audience’s access is purposefully impeded. The comparison of the infor
mation on bipolar disorder made available online by NIMH and HAS at
different moments in time allowed for the identification of novel ele
ments entering the frontstage as well as of insights and perspectives which
were downplayed or given up upon. Rhetoric and the Performance
of Expertise Online I therefore suggest that in the case of
online performances, there may be two types of backstage worth consid
ering: the “conventional” one that Goffman (1959/1990) described, con
taining interactions and negotiations among the online platform
developers, debates among scientists, drafts of the information intended
to be made available, and the tools and technologies used for these activi
ties; and a “digital” backstage, containing older versions of the perfor
mance and revealing the affordances previously available on these
platforms. By comparing them with the current performance, elements
which NIMH and HAS may seek to conceal are unearthed. Whereas 3 The Drama of Expertise About Bipolar Disorder Online 83 access to the first type of backstage was not possible for the study described
here, the “digital” backstage could be visited by collecting and comparing
data from the online platforms at three different moments in time. Data
used in this analysis consist therefore of the online pages dedicated to
bipolar disorder on the website of NIMH and HAS, and they were col
lected in 2014, 2015, and 2016. The success of a performance does not only depend on the talent of the
actors and the quality of their parts, but it is also importantly shaped by
the stage decorum. Insights from media studies reveal that elements of
visual design importantly shape the meaning of the information made
available on an online platform. Thus, the quantity of information pro
vided on a particular aspect, where the information is placed on a web
site, the font size and type, how the information is visually framed by
banners and advertisements, a dynamic or static environment, and the
type and position of the menu as well as the writing style used guide read
ers toward particular bits of information, and help them distinguish
important insights from less relevant ones (Moshagen & Thielsch, 2010). The use of color is also very important, as color patterns help readers
recognize how information is structured and organized; the contrast
between foreground and background importantly affects a website’s read
ability, while the number and kind of colors used and their distribution
on the website affect readers’ ability to concentrate and may imbue the
information with particular connotations (Cyr & Trevor-Smith, 2004;
Flemming, 1998). Next to these elements, the navigability of an online
platform and the affordances available to their users importantly shape
users’ attitude toward the insights provided. Rhetoric and the Performance
of Expertise Online Given the important role
they play in the production of meaning and the great variety of ways in
which they can be combined online, these elements were also considered
when analyzing how NIMH and HAS perform expertise about bipolar
disorder online. The data collected and analyzed consist therefore of texts,
videos, images, and hyperlinks. In the following, I describe the main per
formative techniques identified and present images and extracts to illus
trate the approaches and online elements that helped put forward
particular understandings of bipolar disorder. 84 C. Egher NIMH and the Quest for the Redefinition
of Bipolar Disorder NIMH is the main agency of the American government responsible for
biomedical and health-related research. It is the largest research organiza
tion in the world focusing on mental health. With a budget of about $1.5
billion, NIMH conducts its own research, but also largely determines the
national research agenda by providing grants to other institutes and orga
nizations throughout the US. In what may be seen as an attempt to coun
ter anti-psychiatric tendencies, since the 1980s, NIMH has also started
to pay more attention to the perspectives and insights of people diag
nosed with mental health conditions. For instance, it has funded self-help
agencies managed by former patients or self-titled “consumers,” which
nowadays together constitute The Center for Mental Health Services. At
the same time, NIMH has launched two research centers with the task to
study the activity of self-help groups and the (therapeutic) effectiveness of
such initiatives among people diagnosed with severe mental health condi
tions (Borkman, 1997). Important to understand the highly influential
position NIMH occupies is the distinction between being “in authority”
and being “an authority” put forward by Jongen (2017). Being in author
ity refers to the mandate certain governmental bodies receive to develop
rules and regulations and even to make decisions for others. Being an
authority is linked to the epistemic authority of certain institutions or
people, and highlights the relation between the bearers of such authority
and those who grant it. NIMH is therefore both in authority, by actively
shaping the activities of numerous institutions and self-help groups, and
an authority because of the prestige it enjoys. Furthermore, it is endowed
with sufficient resources to shape its online presence as its representatives
best see it fit. i
By analyzing the online materials described above, I found that NIMH
performed expertise about bipolar disorder online as stable and authori
tative and achieved this by prioritizing on its platform understandings 3 The Drama of Expertise About Bipolar Disorder Online 85 and approaches that could lend themselves more easily to observable and
quantifiable investigations. Important in this sense was the understand
ing of bipolar disorder that NIMH put forward. Whereas this condition
is generally conceived as a “mood disorder” and the role of the brain in its
onset and development is still debated, NIMH referred to the brain being
its main causal factor as an unproblematic fact. NIMH and the Quest for the Redefinition
of Bipolar Disorder According to the institute
(2014, 2015, 2016), “[b]ipolar disorder, also known as manic-depressive
illness, is a brain disorder that causes unusual shifts in mood, energy,
activity levels, and the ability to carry out day-to-day tasks.”hi y
y
y
y
y
This seemingly stable and unproblematic definition of bipolar disorder
was maintained through the deployment of three sets of omissions and by
operating an important modification to a related concept. Thus, while
bipolar disorder was defined rather unproblematically as a brain condi
tion throughout the time period studied, the definition of a mood epi
sode was broadened to include next to emotional states (2014) also
different levels of energy and types of behavior (2016). Thus, NIMH
modified the weight ascribed to the various markers of this condition
and, in so doing, put forward a more complex image of bipolar disorder. This challenges the views of commentators who have expressed concern
that the rise of “the brain” in mental health research would oversimplify
how mental health conditions are understood and approached. The fact
that moods were brought by NMH on a par with other aspects, such as
one’s ability to engage in various acts, does foreground, however, more
quantifiable approaches to how bipolar disorder is diagnosed, as medical
professionals need to assess a person’s behaviors along a growing number
of dimensions and at a more granular level. Thus, whereas the definition
of bipolar disorder seemingly stayed the same, the means by which this
condition can be known were transformed. Bipolar disorder thus gradu
ally became a condition which can be more easily recognized and moni
tored from the outside, even in the absence of highly deviant behaviors,
and, importantly given the various digital and AI-based technologies dis
cussed in the first chapter, along quantifiable markers. ii
Despite this important change, NIMH succeeded to perform expertise
about bipolar disorder as stable and unproblematic by operating three
sets of omissions: (1) keeping backstage insights about its changing per
spective on bipolar disorder; (2) not providing information on how C. Egher C. Egher 86 scientific, social, and technological factors inform this perspective; and
(3) not engaging with its societal significance. These omissions were suc
cessfully achieved through a shrewd combination of rhetoric and the use
of specific sign equipment, that is, of specific digital affordances. NIMH and the Quest for the Redefinition
of Bipolar Disorder Thus,
the impression of epistemic stability was supported by NIMH’s decision
to dress its main character in the modest costume of digital text, which
gives the illusion of permanence and immutability. No further details
were given regarding the designer(s) of this costume, when it was pro
duced and how. Building upon Latour’s (1987) insights, this choice may
have been informed by NIMH’s desire to highlight the scientific charac
ter of the information it provided, by rendering it “devoid of any trace of
ownership, construction, time and place” (Latour, 1987:23). Yet, a look
into the backstage of NIMH’s platform, that is, at records of the informa
tion previously available on its main page dedicated to bipolar disorder,
revealed that in 2014 the institute was more optimistic about the genetic
causes of this condition, both in terms of content and the amount of
space dedicated to them: Bipolar disorder tends to run in families. Some research has suggested that
people with certain genes are more likely to develop bipolar disorder than
others. Children with a parent or sibling who has bipolar disorder are
much more likely to develop the illness, compared with children who do
not have a family history of bipolar disorder. However, most children with
a family history of bipolar disorder will not develop the illness. (…) But
genes are not the only risk factor for bipolar disorder. Studies of identical
twins have shown that the twin of a person with bipolar illness does not
always develop the disorder, despite the fact that identical twins share all of
the same genes. (NIMH, 2014) Whereas in 2014, NIMH did not hesitate to suggest bipolar disorder
may be a condition occurring in families, by 2016 the language it used
about genetic causes had become more tentative: Some research suggests that people with certain genes are more likely to
develop bipolar disorder than others. But genes are not the only risk factor
for bipolar disorder. Studies of identical twins have shown that even if one
twin develops bipolar disorder, the other twin does not always develop the 3 The Drama of Expertise About Bipolar Disorder Online 3 The Drama of Expertise About Bipolar Disorder Online 87 disorder, despite the fact that identical twins share all of the same genes. (NIMH, 2016) disorder, despite the fact that identical twins share all of the same genes. (NIMH, 2016) The 2016 text may be seen as an attempt to preempt alarm among the
relatives of people diagnosed with bipolar disorder, and its brevity sug
gests that NIMH may have assumed this information not to be particu
larly interesting for its audience. The use of indefinite adverbs and
adjectives, such as “some” and “certain,” and the omission of any refer
ences does not encourage the audience to look further into these matters. The audience is therefore expected to believe these statements simply
because they have been uttered by a stakeholder already recognized as an
authority. While it makes sense to retain on the website the most up-to-
date insights about bipolar disorder, this comparison highlights the per
formative effects of the “presentism” that digital platforms afford,
particularly when the focus can be maintained on the frontstage. Thus,
the possibility to avoid any discussion of the processes and evaluations
whereby content is modified helped promote an image of NIMH’s exper
tise about bipolar disorder as stable and unquestionable. p
q
This impression of stability was also reinforced by NIMH’s omitting of
any insights about the factors that influence its orientation as to the most
fruitful areas of research into the causes of bipolar disorder. Whereas
describing bipolar disorder as a brain condition favors a neuroscientific
approach to it, NIMH did not share any details regarding the type and
amount of scientific evidence which led it to support this conceptualiza
tion. Nor did it indicate to what extent advances in neuroimaging tech
nologies and techniques prompted it to consider that “the brains of
people with bipolar disorder may differ from the brains of healthy people
or people with other mental disorders” (NIMH, 2017). In so doing,
NIMH occluded from the audience’s view the role factors such as the
dynamic assessments of scientific feasibility, the changing popularity and
influence of specific scientific fields, charismatic scientists, available tech
nologies, financial costs, and administrative and organizational obstacles
play in such processes of knowledge production.i NIMH further omitted to publicly consider the societal significance of
its conceptualization, to ponder on how the understanding of bipolar
disorder as a “brain condition” that can be measured and monitored 88 C. Egher C. 3 The Drama of Expertise About Bipolar Disorder Online Egher along numerous parameters may impact on the types of mental health
professionals involved and on the work they do, on the experiences of
people diagnosed with this condition, and on the social provisions they
are entitled to. Through such omissions, the institute positioned this
knowledge as natural, obvious, unproblematic. In so doing, I argue that
NIMH performed yet another type of omission, as it appeared rather
oblivious to the distrust some people experience in regard to psychiatry
and governmental institutions, discussed in the earlier sections of this
chapter. All these are issues that NIMH kept backstage, successfully
occluding them from view in its performance. 5 For more information on some sign equipment elements and an overview of the specific roles they
play in NIMH’s performance, see Table 2.2 in the Appendix. The red numbers in these images were
added by the author for analytic purposes. The Role of Sign Equipment in NIMH’s Performance
on Bipolar Disorder As already mentioned in the previous chapter, the type of platform a par
ticular stakeholder chooses depends on its status, on the resources it has
available, and on its goals. From a financial and technical point of view,
non-interactive online platforms are less challenging, as they represent
variations upon options which have been available since the early days of
the Internet. Considering the relatively simple technologies and pro
gramming functions required for their development as well as their lim
ited interactive potential, non-interactive online platforms may be seen as
rather conservative options in the current digital environment. They are
hardly ideal for governmental agencies which aim to educate their audi
ences and encourage them to use (certified) online resources. Furthermore,
in a context where such official bodies find themselves under the obliga
tion to have an online presence, opting for a non-interactive online plat
form does not help them position themselves as open and transparent. Nevertheless, such platforms continue to be the preferred choice for
many official institutions, which seem to be more interested in making
information available to the public rather than also acquiring direct
insights into the public’s views and experiences. 3 The Drama of Expertise About Bipolar Disorder Online 89
3.1
2007 3.1 5
While the visual design of the site is how NIMH chose to decorate the
stage for its performance, I consider the platform’s rubrics to be stage
props, which contributed to the institution’s self-presentation and revealed
the targeted audience and the type of relation NIMH envisaged with it. Thus, the information it made available about bipolar disorder was placed
under the more general rubric entitled “Health & Education,” which
indicated that the insights provided were meant for the general popula
tion and not for mental healthcare professionals. In so doing, NIMH
seemed to be positively responding to the various measures taken by US 90 C. Egher 3 The Drama of Expertise About Bipolar Disorder Online 91 authorities to encourage the use of the Internet for mental health-related
education. The horizontal rubrics at the top—mental health informa
tion; statistics; consumer health publications; help for mental illnesses;
clinical trials—brought together different types of knowledge and posi
tioned NIMH as an institution devoted to furthering scientific knowl
edge while being appreciative of the insights shared by people diagnosed
with mental health conditions. At the same time, these rubrics helped
remind the audience that the institute is dedicated not only to research
but also to improving the lives of people diagnosed. Whereas the rubrics at the top of the website served self-presentation
purposes and focused on the institute’s missions and prerogatives, those
in the lower part of the page revealed how it used its online platform. Thus, they contained brief clarifications regarding NIMH’s position on
FOIA, accessibility, privacy, its policies, and the ways in which it could be
contacted. Regarding accessibility, NIMH assured its readers that it “is
making every effort to ensure that the information available on our web
site is accessible to all. To meet this commitment, we have designed our
site to comply with Section 508 of the Rehabilitation Act.” Nevertheless,
NIMH reserved for itself the right to decide how to fulfill this commit
ment. Thus, while the static character of its website facilitated its acces
sibility, it did not provide text alternatives for some of the videos put up. From this point of view, it would appear that NIMH used the accessibil
ity guidelines in ways which allowed it to opt for the platform design and
online affordances that it preferred. f
Important insights regarding the goals of the performance NIMH put
up and about its intended audience were provided under the rubric
“Policies,” where it became apparent that the institute mainly focused on
the general public rather than medical professionals. In so doing, NIMH
was wary not to jeopardize or diminish the authority and prerogatives of
medical professionals, as it does not intend to provide specific medical advice on our Web sites, but
rather to help visitors better understand mental health and disorders. NIMH will not provide specific medical advice and urges you to consult
with a qualified mental health or health care provider for diagnosis and for
answers to your personal questions. 92 C. Egher C. Egher C. Egher Thus, whereas the institute was
rather conservative in how it performed expertise, it did use the latest
approaches to online technologies when they served its purposes.fi Goffman’s dramaturgical approach helped highlight some specific
ways in which the (visual) design of NIMH’s online platform and the 3 The Drama of Expertise About Bipolar Disorder Online 93 online affordances available on it participated in shaping the meaning of
bipolar disorder and the institute’s public image. Sharing information
online requires important coordination work between distinct elements
and the choice of these elements is often indicative of the type of audi
ence targeted and how it is expected to engage with the information pro
vided, as the discussion of HAS’ performative techniques will indicate. C. Egher In line with the institute’s penchant for conservative choices regarding
its online platform, the role of the audience to the performance was lim
ited. It could not engage in processes of knowledge production and eval
uation through direct reactions, nor could it publicly challenge in this
online space the vision of bipolar disorder NIMH put up. Furthermore,
the peripheral position ascribed to the option “Contact Us” suggested
that dealing with public inquiries was not a function NIMH prioritized. While localized engagement or resistance was this way discouraged, the
audience was allowed, however, to play an active role in ensuring the
circulation of NIMH’s insights by printing, e-mailing, or sharing them
on social media. Even though these online affordances are common ele
ments on most websites nowadays, I argue that they fulfilled a performa
tive function nonetheless, as they suggested that the knowledge NIMH
made available was relevant and interesting enough for people to want to
keep it or to inform others about. p
The simplicity and clarity of the platform was aligned with the ways in
which the part of the main actor was structured and worded. Thus,
NIMH’s perspective on bipolar disorder was organized along the follow
ing rubrics: definition; signs and symptoms; risk factors; treatments and
therapies; join a study; and learn more. As these rubrics already suggest,
the information provided contributed to a performance whereby bipolar
disorder was presented as a complex but manageable condition. A hope
ful, optimistic tone was maintained by mentioning ongoing studies
meant to shed further light on its causes and reveal fruitful new forms of
treatment. The vertically organized rubrics on the right played an impor
tant role in supporting this perspective, as they helped convey a dynamic,
productive view on bipolar disorder research, with new insights being
frequently put forward and new studies developed for people to join. The
mobile character of these videos and hyperlinks had a double function, as
they helped highlight the stability and importance of the main actor, and
also reminded the audience of NIMH’s prerogative and dedication to
tackle the complexity of bipolar disorder. HAS’ Performative Techniques to Redefine
Bipolar Disorder HAS is an independent public institution with a scientific character, cre
ated in 2004. Its board consists of eight members appointed for six years
(with the possibility of renewal every three years) by the President of
France (two members can be proposed by the President, two by the
President of the Senate, two by the President of the National Assembly,
and two by the President of the Economic, Social, and Environmental
Council (CESE)). HAS fulfills three main functions: (i) to evaluate from
a medical and economic point of view health products, technologies, and
practices in view of their admission for reimbursement (a French version
of Health Technology Assessment); (ii) to provide recommendations on
healthcare practices and public health; to create guide books on treat
ment for patients and medical professionals; to develop medico-economic
studies; to advise public institutions in their decisions regarding public
health, and to define the trajectory of personalized care to which one is
entitled; and (iii) to certify healthcare establishments and to provide
accreditations for medical professionals. Very important for this study is
that HAS also certifies health-related online platforms. Its current annual
budget is €60 million, and its revenue comes from taxes on promotional
spending by drug companies, National Health Insurance, state funding,
HONcode accreditation fees, payment for assessing applications for
inclusion on reimbursement lists, and so on. 94 C. Egher C. Egher
3.3
3.2
3.3
3.3
3.3
3.2
3.3
3.3
3.2
95
3 The Drama of Expertise About Bipolar Disorder Online 3 The Drama of Expertise About Bipolar Disorder Online 95 95
3 The Drama of Expertise About Bipolar Disorder Online 96
C. Egher C. Egher 96 To this aim, HAS further operated another and, arguably, most dra
matic twist in its performance of expertise about bipolar disorder, as it
depicted suicidal attempts as symptoms of this condition and described
bipolar disorder as “a highly suicidogenic pathology” (2016). Since 3 The Drama of Expertise About Bipolar Disorder Online 97 statistics confer an aura of objectivity and credibility to the information
provided, making it seem more factual and more urgent (Potter, 1996),
the main actor called upon them, to legitimate this understanding of
bipolar disorder. HAS’ Performative Techniques to Redefine
Bipolar Disorder Thus, it warned the audience that one out of two people
diagnosed with this condition would make at least one suicide attempt
and that at least one out of ten untreated patients would commit suicide,
which accounted for 15% of the population of people diagnosed with
bipolar disorder. The performance HAS put up on this frontstage was
aligned with its institutional role, as through it, the agency guided medi
cal professionals in the assessment of the suicide risk their patients posed. It did so by developing indicators specific to bipolar disorder: an early
onset of the condition; the presence of mixed characteristics; rapid cycles;
the presence of psychotic symptoms; alcohol addiction; and addiction to
illicit substances or to other psychoactive substances. Furthermore, HAS
engaged in the construction of new categories, as it put forward a specific
assessment of a suicidal crisis—low, medium, high—and positioned
social isolation as an indicator of medium or high level. In so doing, the
agency framed the ability to assess the risk of suicide as one of the com
petencies that medical professionals involved in the management of this
condition needed to master. The attention to the new skills and abilities
required marks an important difference between HAS and NIMH, as the
latter omitted to include in its performance of expertise considerations
about the ways in which changes in its understanding and approach to
bipolar disorder would shape the work of medical professionals. The presence of the report on the elaboration of the memo card as another
main actor of this frontstage illustrates HAS’ commitment to transparency
and public accountability. This document indicated the credentials of the
team of experts involved, testified to the depth and breadth of the literature
reviewed, and revealed the numerous actors that were consulted before the
final product—the memo card—was publicly made available: different
types of medical professionals, several patient organizations, non-govern
mental organizations, etc. It thus rendered transparent the processes by
which the memo card came into being and transported the audience onto
the backstage. Whereas such information was absent from the online plat
form of NIMH, possibly in order to avoid any liability and potential causes
for litigation, the audience of HAS had thus the opportunity to understand 98 C. Egher C. Egher the tremendous scientific and diplomatic effort involved in such an under
taking, and was invited to appreciate this institution’s inclusive character. HAS’ Performative Techniques to Redefine
Bipolar Disorder By
placing on the frontstage a document which one generally expects to find
relegated to the backstage, HAS sought to legitimate and heighten the
acceptability and authority of the memo card, while also positioning itself as
a reliable, transparent, and democratic institution. The Role of Sign Equipment in HAS’ Performance
of Expertise The structure of the performance HAS put up and its choice of stage
equipment were by no means accidental, but were aligned with the
intended audience and educational purposes. HAS’ performance was
organized as a series of monologues, with each individual character tak
ing its turn to play its part. The main characters were dressed in the
simple and conservative outfit of portable document format (pdf) files,
whereas some of the side actors were wrapped in the equally conservative
suit of digital text. These choices enabled the audience to focus upon
what each character had to say without any interruptions, contradictions,
or divagations. At the same time, by wearing such hard and impenetrable
costumes, each part the characters had to say, every insight and practical
advice about bipolar disorder that they provided acquired stability and
authority, which befit scientific facts. Furthermore, pdf files allow for rel
evant information to be highlighted and easily identified, they enable
users to add their own thoughts and ideas in the form of comments and
notes, and they can be accessed from a variety of devices. Thus, not only
did such attires allow HAS to fulfill the WCAG 2.0 accessibility recom
mendations, but they could also be more readily consumed by careful
and goal-oriented readers, such as treating doctors, who need to act
quickly and who require stability and coherence in their practices. 99 3 The Drama of Expertise About Bipolar Disorder Online 3.4 As the distribution of roles and the choice of decorum indicate, HAS
staged its performance on bipolar disorder as a rather idyllic world, where
the best results could be achieved when treating doctors corroborate their
treatment decisions with legal provisions regarding the insurance and
reimbursement of medical care and when they provide their patients with
documentation meant “to support the dialogue” between them. Given
the currently fraught relations between different types of mental health
professionals in France, this approach may have been taken in order to
provide an example for its medical audiences to follow, as an attempt to
achieve not only informational but also behavioral changes. These con
siderations may have therefore informed HAS’ choice of costume for its
characters as well as the way in which it organized the reverberations of
several plot twists across different regions of its three stages. 100 C. Egher Discussion 101 3 The Drama of Expertise About Bipolar Disorder Online Similarly, NIMH’s definition of bipolar disorder as a neurological condi
tion is understandable in the American context, where the biomedical
model remains dominant and where Congress has been increasingly
approving the allocation of funds in areas of research which can provide
hard evidence and lead to findings that are easier to render profitable. Nonetheless, while politics play an important role in the functioning and
orientation of both agencies, they both successfully managed to relegate
it to the backstage, presenting an image of governmental bodies moved
by the sole purpose of furthering science and improving the treatment
and provision of care to people diagnosed with bipolar disorder.hi Similarly, NIMH’s definition of bipolar disorder as a neurological condi
tion is understandable in the American context, where the biomedical
model remains dominant and where Congress has been increasingly
approving the allocation of funds in areas of research which can provide
hard evidence and lead to findings that are easier to render profitable. Nonetheless, while politics play an important role in the functioning and
orientation of both agencies, they both successfully managed to relegate
it to the backstage, presenting an image of governmental bodies moved
by the sole purpose of furthering science and improving the treatment
and provision of care to people diagnosed with bipolar disorder.hi p
p
p
g
p
The analysis discussed here was colored by a specific normative per
spective, which sees non-interactive online platforms as rather conserva
tive, whereas interactive online platforms are perceived as more dynamic
and amenable to varied purposes. Choosing for an interactive online plat
form can be problematic for official institutions, because of the inher
ently generative character of such platforms, where users can engage with
the content made available in ways that may lead to interpretations and
evaluations of varying quality, and may put forward suggestions that may
be detrimental to others. It is regrettable, however, that neither NIMH
nor HAS tried to reach a balance between control over the platform and
more distributed forms of agency, where users would have had some pos
sibility to contribute, if not to the production, then, at least, to the evalu
ation of the insights these institutions shared. Discussion This chapter discussed the performative techniques through which two
highly influential institutions performed expertise about bipolar disorder
online, in a context in which official bodies are required to make their
insights available via the Internet, but face important challenges to their
authority, and need to respect specific technical provisions. It showed
that NIMH and HAS performed expertise about bipolar disorder in dif
ferent ways, as NIMH used various strategies to depict its perspective on
this condition as stable and precise, whereas HAS actively and transpar
ently engaged in its redefinition to assist in the reform of the French
mental healthcare. The type of information shared depended on the
intended audience, and the visual design and most of the affordances
available on the online platforms of these institutions helped the audi
ence better find their way while reiterating the authoritative position of
NIMH and HAS. As we have seen, these institutions were not enthusias
tic users of the Internet, exploring its full potential and experimenting
with the latest online technologies. Instead, they opted for non-interactive
online platforms, reminiscent of the Web 1.0 era, with static content and
limited interactivity. Combined with various rhetorical strategies, this
conservative choice enabled, however, these institutions to put forward
their knowledge on bipolar disorder as reliable and authoritative. This
means that the accessibility commitment need not only pose difficulties
or bring additional challenges to public institutions. Instead, it might
allow such bodies to engage with preferred digital technologies to put
forward a desired public image and to bestow their insights in ways that
highlight their legitimacy. Even though both NIMH and HAS used largely similar performative
techniques, they approached bipolar disorder in different ways, which
seems to confirm yet again that national characteristics and priorities spe
cific to any given healthcare system shape the content public bodies share
online. For instance, the higher suicide rate in France (14.3%) as com
pared to the US (13.1%) (OECD, 2015) may be the reason why HAS
chose to perform expertise about bipolar disorder by developing catego
ries of suicide risk and by defining this condition as highly suicidogenic. Discussion The need to educate people
of different socioeconomic levels and to render insights available to those
with disabilities is not a sufficient explanation for their choice of platform
type, since online technologies with more open and flexible affordances
exist, which can be accessed also from affordable devices. From this point
of view, NIMH and HAS’ approach is in stark contrast to initiatives
developed in recent years in personalized health and e-health (Sardi et al.,
2017), where diverse, interactive, gamified elements are integrated to
digital technologies and applications to encourage users to engage with
the information provided in specific ways. While important reservations
exist in regard to such approaches (Lupton, 2020; Prainsack, 2017;
Sharon, 2016; Swierstra, 2016; van Dijk et al., 2018) and they need to be
carefully considered on a case-by-case basis, the use of interactive online 102 C. Egher C. Egher platforms endowed with a diverse array of affordances may help people
acquire a better and longer-lasting understanding of complex issues and
may further public engagement. g g
Both NIMH and HAS took up the incentive to share knowledge about
bipolar disorder online, but they did so in ways which suggest that they
were either oblivious or unwilling to substantially engage with the fact
that their authority is questionable and questioned and that they need to
convince as experts if they want to succeed in educating their audience. hf Both NIMH and HAS took up the incentive to share knowledge about
bipolar disorder online, but they did so in ways which suggest that they
were either oblivious or unwilling to substantially engage with the fact
that their authority is questionable and questioned and that they need to
convince as experts if they want to succeed in educating their audience. The analysis indicated that whereas HAS had made some efforts toward
acknowledging its public accountability, NIMH largely continued to
issue pronouncements in a technocratic fashion. While this approach
might be a defense mechanism, a way to steer off the public critical cli
mate, it renders these agencies less effective in the convincing provision
of insights. Through such practices, they also do not live up to the image
they seek to perform online as supporters of public engagement. Discussion Whereas
this chapter has described how influential governmental agencies per
formed expertise about bipolar disorder online, in the next chapter I will
argue that a particular type of interactive online platform—blogs—allows
people diagnosed with bipolar disorder to engage in more substantial
ways in processes of knowledge production and evaluation of treatment. References Barak, A. (1999). Psychological Applications on the Internet: A Discipline on
the Threshold of a New Millennium. Applied & Preventive Psychology,
8, 231–245. Barak, A., & Grohol, J. (2011). Current and Future Trends in Internet-
Supported Mental Health Interventions. Journal of Technology in Human
Services, 29(3), 155–196. Bennett, G., & Glasgow, R. (2009). The Delivery of Public Health Interventions
Via the Internet: Actualizing Their Potential. Annual Review of Public Health,
30, 273–292. Bernstein, R. (2006). A Seat at the Table: Trend or Illusion? Health Affairs,
25(3), 730–733.hi Bijker, W., Bal, R., & Hendriks, R. (2009). The Paradox of Scientific Authority. The Role of Scientific Advice in Democracies. The MIT Press. 3 The Drama of Expertise About Bipolar Disorder Online 103 Borkman, T. (1997). A Selective Look at Self-Help Groups in the United States. Health and Social Care in the Community, 5, 357–364. Bourdieu, P. (1975). The Specificity of the Scientific Field and the Social
Conditions of the Progress of Reason. Social Science Information, 14(6), 19–47. Bureau of Industry and Security (BIS). (2016). Electronic FOIA. Accessed
August 12, 2017, from https://efoia.bis.doc.gov/ g
f
( ),
Bureau of Industry and Security (BIS). (2016). Electronic FOIA. Accessed
August 12, 2017, from https://efoia.bis.doc.gov/h Carlat, D. (2010). Unhinged: The Trouble with Psychiatry- A Doctor’s Revelations
About a Profession in Crisis. Free Press. Carlat, D. (2010). Unhinged: The Trouble with Psychiatry- A Doctor s Revelations
About a Profession in Crisis. Free Press. Carlat, D. (2010). Unhinged: The Trouble with Psychiatry A Doctor s Revelations
About a Profession in Crisis. Free Press. Cassani M (2017) Everything Matters Beyond Meds About Accessed August About a Profession in Crisis. Free Press. Cassani, M. (2017). Everything Matters. Beyond Meds. About. Accessed August f
Cassani, M. (2017). Everything Matters. Beyond Meds. About. Accessed August
12, 2017, from https://beyondmeds.com/about/ 12, 2017, from https://beyondmeds.com/about/
Castel, R. (1976). L’Ordre Psychiatrique. L’Âge d’Or de l’Aliénisme. Minuit. Chessick R (2006) The Future of Psychoanalysis SUNY Press 12, 2017, from https://beyondmeds.com/about/
Castel, R. (1976). L’Ordre Psychiatrique. L’Âge d’Or de l’Aliénisme. Minuit. Chessick, R. (2006). The Future of Psychoanalysis. SUNY Press. Christensen H Griffiths K & Jorm A (2004) Delivering Interventions fo Christensen, H., Griffiths, K., & Jorm, A. (2004). Delivering Interventions for
Depression by Using the Internet: Randomized Controlled Trial. BMJ,
328, 265–269. Cyr, D., & Trevor-Smith, H. (2004). Localization of Web Design: An Empirical
Comparison of German, Japanese, and United States Web Site Characteristics. References Journal of the American Society for Information, Science and Technology, 55(13),
1199–1208. Department of Justice. (2014). Department of Guide to the Freedom of
Information Act. Proactive Disclosures. Accessed on August 12, 2017. Desmettre, S. (2009). La Prise en Charge des Troubles Psychiatriques. Regards
Croisés Sur L’Économie, 1(5), 168–170. Djonov, E. (2007). Website Hierarchy and the Interaction Between Content
Organization, Webpage and Navigation Design: A Systemic Functional
Hypermedia Discourse Analysis Perspective. Information Design Journal,
15(2), 144–162. Drucker, J. (2011). Humanities Approaches to Interface Theory. Culture
Machine, 12. Available at http://www.culturemachine.net/index.php/cm/
article/view/434/462 Drucker, J. (2013). Reading Interface. PMLA, 128(1), 213–220. eMEN. (2017). NEWS Summer 2017. Interreg North-West Europe. E-Mental
Health Innovation and Transnational Implementation Platform North West
Europe: Lille. Farrell, S., & McKinnon, C. (2003). Technology and Rural Mental Health. Archives in Psychiatric Nursing, 17, 20–26. Felt, U. (2017). Making Knowledge, People, and Societies. In U. Felt, R. Fouché,
C. Miller, & L. Smith-Doerr (Eds.), The Handbook of Science and Technology
Studies (4th ed., pp. 253–257). The MIT Press. 104 C. Egher Flemming, J. (1998). Web Navigation: Designing the User Experience. O’Reilly Media. Flemming, J. (1998). Web Navigation: Designing the User Experience. O’Reilly Media.h Fussinger, C. (2011). “Therapeutic Community”, Psychiatry’s Reformers and
Antipsychiatrists: Reconsidering Changes in the Field of Psychiatry after
World War II. History of Psychiatry, 22(2), 146–163. Gill, K., Kauser, S., Khattack, K., & Hynes, F. (2014). Physician Associate: New
Role within Mental Health Teams. The Journal of Mental Health Training,
Education and Practice, 9(2), 79–88.fh Goffman E (1959/1990). The Presentation of Self in Everyday Life 8th ed.:
Penguin. Gostin, L. (2008). “Old” and “New” Institutions for Persons with Mental
Illness: Treatment, Punishment or Preventive Confinement? Public Health,
122(9), 906–913. Griffiths, F., Lindenmeyer, A., Powell, J., Lowe, P., & Thorogood, M. (2006). Why Are Health Care Interventions Delivered Over the Internet? A
Systematic Review of the Published Literature. Journal of Medical Internet
Research, 8(2), e10. https://doi.org/10.2196/jmir.8.2.e10 Hafermalz, E., Riemer, K., & Boell, S. (2016). Enactment of Performance? A
Non-dualist Reading of Goffman. In L. Introna, D. Kavanagh, S. Kelly,
W. Orlikowski, & S. Scott (Eds.), Beyond Interpretivism? New Encounters with
Technology and Organization (pp. 167–181). Springer. Hilgartner, S. (2000). Science on Stage: Expert Advice as Public Drama. Stanford
University Press. Hochmann, J. (2017). Histoire de la Psychiatrie (4th ed.). Presses Universitaires
de France. Hopton, J. (2006). The Future of Critical Psychiatry. Critical Social Policy,
26(1), 57–73. References Horst, M., Davies, S., & Irwin, A. (2017). Reframing Science Communication. In U. Felt, R. Fouché, C. Miller, & L. Smith-Doerr (Eds.), The Handbook of
Science and Technology Studies (4th ed., pp. 881–907). The MIT Press.ih h
Horwitz, A., & Wakefield, J. (2007). The Loss of Sadness. How Psychiatry
Transformed Normal Sorrow into Depressive Disorder. Oxford University Press. Horwitz, A., & Wakefield, J. (2007). The Loss of Sadness. How Psychiatry
Transformed Normal Sorrow into Depressive Disorder. Oxford University Press. Jongen, H. (2017). Combating Corruption the Soft Way: The Authority of Peer
Reviews in the Global Fight Against Graft. Datawyse. Jongen, H. (2017). Combating Corruption the Soft Way: The Authority of Peer
Reviews in the Global Fight Against Graft. Datawyse. g
g
f
y
Kinderman, P. (2014). A Prescription for Psychiatry. Why We Need a Whole New
Approach to Mental Health and Wellbeing. Palgrave Macmillan. Kirsch, I. (2010). The Emperor’s New Drugs: Exploding the Antidepressant Myth. Basic Books. 3 The Drama of Expertise About Bipolar Disorder Online 105 Kirsch, I., & Sapirstein, G. (1998). Listening to Prozac but Hearing Placebo: A
Meta-Analysis of Antidepressant Medication. Prevention & Treatment, 1(2),
0002a. Accessed August 20, 2017, from http://journals.apa.org/prevention/
volume1/pre0010002a.html p
Knorr Cetina, K. (1999). Epistemic Cultures. How the Sciences Make Knowledge. Harvard University Press. Lane, C. (2009). Comment la Psychiatrie et L’Industrie Pharmaceutique Ont
Médicalisé Nos Émotions. Flammarion. Latour, B. (1987). Science in Action. How to Follow Scientists and Engineers
Through Society. Harvard University Press.h h
Latour, B., & Woolgar, S. (1979). Laboratory Life: The Social Construction of
Scientific Facts. Sage. i
Levy, J., & Strombeck, R. (2002). Health Benefits and Risks of the Internet. Journal of Medical Systems, 26, 495–510. Loi no. 2005-102 du 11 février 2005 pour l’égalité des droits et des chances, la
participation et la citoyenneté des personnes handicapées (1). Accessed
August 12, 2017, from https://www.legifrance.gouv.fr/affichTexte.do?cidTex
T
T
64 te=JORFTEXT000000809647 te=JORFTEXT000000809647 Lupton, D. (2020). Data Selves. Polity Press. MacKenzie, D. (1990). Inventing Accuracy. A Historical Sociology of Nuclear
Missile Guidance. The MIT Press.h Maness, C. (2017). Saving Sigmund: Psychoanalysts Fight to Make Their
Profession Relevant. STAT. Accessed August 12, 2017, from https://www. statnews.com/2017/03/15/psychoanalyst-freud-relevant/ McLean, A. (2000). From Ex-Patient Alternatives to Consumer Options:
Consequences of Consumerism for Psychiatric Consumers and the Ex-Patient
Movement. International Journal of Health Services, 30(4), 821–847. McLean, A. (2003). Recovering Consumers and a Broken Mental Health System
in the United States: Ongoing Challenges for Consumers/Survivors and the
New Freedom Commission on Mental Health. International Journal of
Psychosocial Rehabilitation, 8, 58–70. McPherson, S., & Armstrong, D. (2006). Social Determinants of Diagnostic
Labels in Depression. Social Science & Medicine, 62(1), 50–58. Metzl, J. (2009). The Protest Psychosis. How Schizophrenia Became a Black Disease. Beacon Press. Ministère Des Affaires Sociales Et De La Santé. (2016, July 4). Stratégie natio
nale e-santé 2020. Paris. 106 C. Egher Morrison, L. (2013). Talking Back to Psychiatry. The Psychiatric Consumer/
Survivor/Ex-Patient Movement. Routledge.h Moshagen, M., & Thielsch, M. (2010). Facets of Visual Aesthetics. International
Journal of Human-Computer Studies, 68, 689–709. O’Sullivan, S. (2016). It’s All in Your Head: True Stories of Imaginary Illness. Vintage Books. g
OECD. (2015). Country Note: How Does Health Spending in FRANCE
Compare? OECD Health Statistics. Compare? OECD Health Statistics. Office of Management and Budget (OMB). (2016). Memorandum for the Heads
of Executive Departments and Agencies M-17-06 Office of Management and Budget (OMB). (2016). Memorandum for the Heads
of Executive Departments and Agencies. M-17-06. f
p
g
Pignarre, P. (2006). Les Malheurs des Psys. Psychotropes et Médicalisation du Social. La Découverte. Potter, J. (1996). Representing Reality. Discourse, Rhetoric, and Social Construction. Sage Publications. g
Prainsack, B. (2017). Personalized Medicine. Empowered Patients in the 21st
Century? New York University Press. y
Référentiel Général D’Accessibilité Pour Les Administrations (RGAA) Version 3
2017. Accessed August 12, 2017, from https://references.modernisation. f /
ibili / Référentiel Général D’Accessibilité Pour Les Administrations (RGAA) Version 3
2017 A
d A
12 2017 f
h
// f
d
i
i (
)
2017. Accessed August 12, 2017, from https://references.modernisation. gouv.fr/rgaa-accessibilite/ Rissmiller, D., & Rissmiler, D. (2006). Open Forum: Evolution of the
Antipsychiatry Movement into Mental Health Consumerism. Psychiatric
Services, 57(6), 863–866. Roberts, R., & Itten, T. (2006). Laing and Szasz: Anti-Psychiatry, Capitalism,
and Therapy. Psychoanalytic Review, 93(5), 781–799. h
y
y
Romme, M., & Escher, S. (Eds.). (1993). te=JORFTEXT000000809647 Accepting Voices. MIND. Rose, N. (2018). Our Psychiatric Futures. Polity Press. ., Idri, A., & Fernández-Alemán, J. (2017). A Systematic Review of
fi
i
i E H l h J
l f Bi
di l I f
i
71 31 48 Sardi, L., Idri, A., & Fernández-Alemán, J. (2017). A Systematic Review of
Gamification in E-Health Journal of Biomedical Informatics 71 31–48 Sardi, L., Idri, A., & Fernández-Alemán, J. (2017). A Systematic Rev Sardi, L., Idri, A., & Fernández-Alemán, J. (2017). A Systematic Review of
Gamification in E-Health. Journal of Biomedical Informatics, 71, 31–48. S h d T (
) R h k
P f
l P
M
d M
l H l h Gamification in E-Health. Journal of Biomedical Informatics, 71, 31 i
Scheid, T. (2000). Rethinking Professional Prerogative: Managed Mental Health
Care Providers. Sociology of Health & Illness, 22(5), 700–719.hi gy f
Scott, S. (2006). The Medicalisation of Shyness: From Social Misfits to Social
Fitness. Sociology of Health & Illness, 28(2), 133–153. gy f
Section 508.gov. It Accessibility Law and Policies. Accessed August 12, 2017.,
f
h
//
i
508
/
/l
d
li i Section 508.gov. It Accessibility Law and Policies. Accessed August 12, 2017.,
from https://section508.gov/manage/laws-and-policiesf g
y
from https://section508.gov/manage/laws-and-policiesf Shapin S, Schaffer S (1985/2011) Leviathan and the Air-Pump. Hobbes, Boyle,
and the Experimental Life 2nd ed. : Princeton University Press.h Sharon, T. (2016). The Googlization of Health Research: From Disruptive
Innovation to Disruptive Ethics. Personalized Medicine, 13(6), 563–574. 3 The Drama of Expertise About Bipolar Disorder Online 3 The Drama of Expertise About Bipolar Disorder Online 107 Swierstra, T. (2016). Introduction to the Ethics of New and Emerging Science
and Technology. In R. Nakatsu, M. Rauterberg, & P. Ciancarini (Eds.),
Handbook
of
Digital
Games
and
Entertainment
Technologies
(pp. 1271–1295). Springer.h Szasz, T. (1961). The Use of Naming and the Origin of the Myth of Mental
Illness. American Psychologist, 16, 59–65.h The U.S. Digital Service. (2018). Digital Services Playbook. Accessed August 12,
2017, from https://playbook.cio.gov/ Van Dijk, J., Poell, T., & de Waal, M. (2018). The Platform Society. Oxford
University Press. VPH Institute. (2016). France National E-Health Strategy 2020. Accessed August
14, 2017, from http://www.vph-institute.org/news/france-national-e-health-
strategy-2020.html Whitaker, R. (2010). Anatomy of An Epidemic: Magic Bullets, Psychiatric Drugs,
and the Astonishing Rise of Mental Illness. Crown Publishing Group.h Whitley, R. (2012). The Antipsychiatry Movement: Dead, Diminishing, or
Developing? Psychiatric Services, 63(10), 1039–1041. World Wide Web Consortium (W3C). (2017). Web Content Accessibility
Guidelines (WCAG). 2.0. Accessed August 12, 2017, from https://www. w3.org/TR/WCAG20/ w3.org/TR/WCAG20/ g
Wright, R., & Cummings, A. (2005). Destructive Trends in Mental Health. The
Well-Intentioned Path to Harm. Routledge. Ybarra, M., & Eaton, W. (2005). Internet-Based Mental Health Interventions. Mental Health Services Research, 7(2), 75–87. 108 C. Egher Open Access This chapter is licensed under the terms of the Creative Commons
Attribution 4.0 International License (http://creativecommons.org/licenses/
by/4.0/), which permits use, sharing, adaptation, distribution and reproduction
in any medium or format, as long as you give appropriate credit to the original
author(s) and the source, provide a link to the Creative Commons licence and
indicate if changes were made. Open Access This chapter is licensed under the terms of the Creative Commons
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by/4.0/), which permits use, sharing, adaptation, distribution and reproduction
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indicate if changes were made. The images or other third party material in this chapter are included in the
chapter’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the chapter’s Creative Commons
licence and your intended use is not permitted by statutory regulation or exceeds
the permitted use, you will need to obtain permission directly from the copy
right holder. The materials used in this chapter from the blog Bipolar Burble have been included in an article
titled “Bipolar Patients and Creative Online Practices: Sharing Experiences of Controversial
Treatments,” which was published in the journal Health, https://doi.
org/10.1177/1363459319838315. © The Author(s) 2023
C. Egher, Digital Healthcare and Expertise, Health, Technology and Society,
https://doi.org/10.1007/978-981-16-9178-2_4 Tactical Re-appraisals and Digitally
Informed Hypotheses About
the Treatment for Bipolar Disorder While patient engagement in mental healthcare has a long tradition, as
already mentioned in the first chapter, since the late 1980s a series of fac
tors has led to a growing responsibilization of people in regard to their
health (Petersen & Lupton, 1996) and has stimulated them to contribute
to the production of knowledge. The Internet has played an important
role in such developments (Wyatt et al., 2013), as it has allowed people
diagnosed to enroll in medical studies more easily, to engage in practices
of self-monitoring and -experimentation, and to exchange information
with more people with the same diagnosis than was previously possible. Yet, while patient engagement is welcomed and encouraged, what exactly
is meant by it, what patients are expected to contribute, and what the
limits to such contributions are or should be remain debatable (Adams,
2011; Lupton, 2018). These issues are exacerbated online by the variety
and specificity characterizing both mental health conditions and online
platforms, which shape the content and character of interactions. More 109 110 C. Egher C. Egher research is needed to map out what patients contribute online, and to
understand how such contributions are contextually shaped. y
p
1988
1
1 I was inspired to use the term “hypothesis” by some French online contributors, who used this
notion to describe a suggestion regarding the effects of certain environmental factors on treatment
effectiveness put forward by other people diagnosed with bipolar disorder on the forum Le Forum
des Bipotes. Problematizing Patient Engagement People diagnosed with mental health conditions have assumed a growing
role in the production of knowledge, as the provision of treatments and
caring practices has shifted in the context of deinstitutionalization from
medical environments to more private and non-clinical settings, such as
homes and community centers. As such, patient engagement in mental
healthcare has developed under various forms, ranging from clubhouses
and self-help and support groups (McLean, 2003), focusing on the 4 Tactical Re-appraisals and Digitally Informed Hypotheses… 111 societal reintegration of people diagnosed with mental health conditions,
to social movements, such as the psychiatric consumer/survivor/ex-patient
movement, whose members “attempt to shape treatment to respond to
their own needs” (Morrison, 2013: ix). Since the early 1990s, people diag
nosed have also participated in the provision of psychiatric care, as paid or
voluntary case managers, facilitators, or peer support providers (Davidson,
2005). As such, they have been involved in numerous ways not only in
care but also in the production of knowledge about treatment. More recently, patient engagement has been encouraged through top-
down and grassroots initiatives meant to improve the provision of mental
healthcare and to render it more cost-efficient. Social media and digital
technologies have played an important role in these developments, as
they have provided new avenues for patient engagement, which have
been both celebrated and critiqued. In the early days of the public
Internet, some commentators expected this medium to empower patients,
contributing to the re-appreciation of lay expertise (Hardey, 1999). Others have criticized digital technologies as a means for creating free
labor, as a neoliberal practice of outsourcing tasks and responsibilities
onto individuals while decreasing social provisions (Lupton, 2020; Rose,
2018; Thomas, 2016). Most medical sociologists and media scholars
agree, however, that digital technologies have contributed to more active
conceptualizations of the role of patients (Felt, 2015). The personal expe
riences of (pre)patients have become all the more important (Prainsack,
2017), as the adoption of big data analytics in healthcare and the drive
toward precision medicine make highly diverse data necessary (Lupton,
2020), including next to traditionally “medical” markers, information
such as credit card purchases, and social media interactions (Weber et al.,
2014). This has contributed to new perspectives on what counts as evi
dence (Hogle, 2016) and to intensified calls for individuals to engage in
self-monitoring practices and to contribute information. Problematizing Patient Engagement As the emergence and popularity of movements such as “The
Quantified Self” (Lupton, 2016) indicate, many people have responded
enthusiastically to such calls and have engaged with numerous digital
technologies, such as self-tracking devices, wearables, implantables, and
external sensors, to track diverse bodily states, patterns of activities, and
moods, thereby contributing to the generation of new types of data. 112 C. Egher C. Egher Most such data have been numerical, thus allowing the users of these
technologies to identify patterns in the data, to combine different data
sets to arrive at novel insights, and to engage in various comparisons. Depending on their preferences and the available data policies, indi
viduals have thus been able to contribute to the development of new
medical knowledge and products in more and less direct ways. Thus,
they could share their insights at collective, “show-and-tell” events,
such as those organized by supporters of The Quantified Self move
ment, and they could carefully monitor and provide relevant health
data at specific intervals on online platforms such as PatientsLikeMe or
CureTogether or participate in various one-person trials (Schork,
2015). But they could also be among the (knowing or unknowing)
providers of health data aggregated by initiatives such as IBM Watson
Health, the Apple ResearchKit, or Genomera. While researchers and
medical professionals have been mainly interested in data generated
through digital self-tracking devices, patients and their families have
also provided valuable qualitative information, in the form of accounts
shared on interactive digital platforms, such as blogs and fora, where
they could share their experiences and enrich their knowledge through
the narratives provided by other contributors. Attempts to determine the knowledge of patients have a long history
(Segall & Roberts, 1980), and more recently patients’ use of online plat
forms has served to further problematize their relations with medical pro
fessionals and to nourish ongoing discussions about the type and relevance
of the knowledge each party contributes (Versteeg et al., 2018). While
medical knowledge is generally seen as relying upon scientific and clinical
insights, patients are often ascribed experiential knowledge, that is, “truth
learned from personal experience with a phenomenon rather than truth
acquired through discursive reasoning, observations, or reflection on
information provided by others” (Borkman, 1976:446). Two main strands can be distinguished in the debate about the mean
ing and relevance of patient engagement. Problematizing Patient Engagement Building upon Borkman
(1976), one group (Collins & Evans, 2002; 2007; Prior, 2003) focuses on
the experiential knowledge of people diagnosed, which they see as stem
ming from their direct experiences with a condition and argue that such
knowledge is different from that acquired formally, through specialized 4 Tactical Re-appraisals and Digitally Informed Hypotheses… 113 training. Proponents of the other perspective (Arksey, 1994; Epstein,
1995) reject such a strict distinction and highlight the substantial role
people diagnosed can play in the development of medical knowledge. Arksey (1994) put forward the notion of “lay expertise” to acknowledge
the substantial technical knowledge people could acquire informally. Epstein further developed these insights, showing how some AIDS activ
ists had “learned the language and culture of medical science” (1995:17),
and were thereby able to engage in and change medical research and clin
ical practices. Both positions have shortcomings: while the first perspec
tive conceives of expertise and experience as opposite poles on a
continuum, the second runs the risk of expertise being depleted of its
substantial character, if applied too generously. Wilcox (2010) provides a helpful perspective for the analysis in this
chapter. She argues that lay expertise should be understood as “collective
knowledge that may be widely available yet is still unevenly socially dis
tributed” (2010:45). This is reinforced by studies of health-related online
behaviors that have shown that “[s]ocial media platforms facilitate the
sharing of health information between users and the co-production of
new knowledge that is shaped by personal experience” (Sosnowy,
2014:316). While previously patient associations collected and processed
the experiences of numerous individuals diagnosed to transform them
into collective knowledge, I argue that such practices are nowadays facili
tated by blogs and fora. Authored by one or several individuals and
updated regularly, blogs comprise posts on a variety of topics, listed typi
cally in reverse chronological order. Fora are online platforms where indi
viduals can post messages and read those of others along “threads” where
insights on a specific topic accumulate, turning fora into rich discussion
databases. While on blogs, people diagnosed mainly contribute as reac
tions to the posts shared by the blog author(s), on fora, even though
threads can also be initiated by the forum owner/administrator, they are
largely developed by individual users, who want to receive advice on a
particular issue. Problematizing Patient Engagement Importantly, both blogs and fora may enable collective
processes of knowledge production by bringing together people with the
same diagnosis but endowed with different types of knowledge, skills,
and resources, by facilitating their dialogue, and by preserving their
exchanges. These online platforms are worthy of scholarly attention, since 114 C. Egher “[h]ealth issues are today often negotiated in parallel with professionals in
institutional settings like hospitals and among peers in activities taking
place online” (Bellander & Landqvist, 2018:1). Lay Expertise and Bipolar Disorder Lay expertise is often articulated in situations when scientific knowledge
is lacking, when it has not yet stabilized or when issues are too complex
to be solved using only one type of knowledge (Baillergeau & Duyvendak,
2016). This makes its study in relation to bipolar disorder relevant. As
already mentioned in Chap. 1, while its causes are not precisely known
(Frey et al., 2013), bipolar disorder is thought to be determined by a
combination of genetic, neurological, and environmental factors. Treatment is prescribed in a rather formulaic fashion, and there is still
limited understanding of how the prescribed medicines work. Finding an
effective treatment regimen for any individual patient may take several
months or years. In the case of bipolar disorder, therefore, there is a sig
nificant amount of space for people diagnosed to contribute to knowl
edge, making it an interesting site to study patient engagement and the
performance of expertise. p
p
According to Britten and Maguire (2016), while medical professionals
appreciate patient engagement in various aspects of clinical practice and
research, patients’ experiences about treatment have not been sufficiently
acknowledged. Furthermore, whereas new drugs prescribed for mental
health conditions are assessed based on short clinical trials (involving
typically six weeks of exposure), many of them are used as maintenance
treatment. This means that “the effects of these drugs as used in practice
are not known” (Frank et al., 2005: 292) and “decisions about payment,
inclusion, placement in formularies and clinical management are usually
not informed by data on long-term clinical or economic consequences”
(ibid.). What further complicates matters in the field of mental health is
that treatment compliance and adherence remain problematic, leading to
important tensions between medical professionals and people diagnosed. Nonetheless, in a highly influential study, Martin (2009) has described
the complex attitudes people diagnosed with bipolar disorder develop 4 Tactical Re-appraisals and Digitally Informed Hypotheses… 115 toward their medication and has highlighted the substantial knowledge
they have about their own symptoms and reactions to treatment. These
are therefore aspects about which people diagnosed with bipolar disorder
may be very insightful, and the Internet could provide a welcoming space
for those among them who want to share their treatment experiences and
contribute to the development of new knowledge. Studying Tactics Online Over the last few decades, official institutions have increasingly empha
sized the need to inform and consult the public about scientific findings
and research agendas. This has contributed to the distribution of scien
tific information in numerous shapes and across different media. According to Epstein (1996:177), “debates about the safety and efficacy
of treatments travel with particular ease between the pages of scientific
publications, the mass media…” due to their highly politicized character
and the different types of stakeholders involved. Since finding effective
treatment is a priority for people diagnosed (Thompson et al., 2012),
such debates no doubt reach them, and they may subsequently adapt the
information conveyed for their own purposes (Sharon, 2015). The con
cept of “tactics” developed by de Certeau is therefore useful, as it allows
me to analyze the creative ways in which people diagnosed with bipolar
disorder take up elements of the dominant discourse regarding the effec
tiveness of medications, and transform them in their attempts to adjust
treatment to their personal needs and preferences. De Certeau (1988:
xix) defines tactics as ingenious actions through which individuals seek to
re-appropriate dominant representations by adapting them to their own
needs, rules, and goals. Tactics “must constantly manipulate events in
order to turn them into ‘opportunities’” (De Certeau, 1988: xix). The concept of tactics is pertinent because the online contributions of
people diagnosed with bipolar disorder, which I argue that collectively
contribute to the development of digitally informed hypotheses, are not
always organized and institutionalized, nor do they occur outside the
confines of the dominant medical discourse. They rather represent cre
ative practices undertaken from within the dominant order, whereby 116 C. Egher C. Egher people diagnosed seek to render the space of medical knowledge about
the treatment of bipolar disorder more “habitable.” Thus, in the analysis
I focus on the perspectives of online contributors, on how current medi
cal views on the treatment of bipolar disorder are understood, taken up,
and adapted by them. Tactics are time-bound and therefore appropriate
for studying blogs and fora, where people use their creativity and differ
ent types of knowledge to interact with others, where the meaning of
each individual contribution is co-determined by the content put for
ward by others, and where it is positioned, in time as well as in space, in
relation to it. Studying Tactics Online This concept is also useful because online contributors need
to take into account the restrictions imposed on them by platform own
ers or administrators. Tactics emphasize the malleable, equivocal nature
of lay expertise, as the study of blogs and fora makes us more aware of
how the engagement with medical insights on bipolar disorder changes
depending on the readers’ interpretations and needs, and on how the
experiences of the contributors putting them forward develop. Since in de Certeau’s framework, tactics represent creative adaptations
of dominant representations, I first sought to delineate the main charac
teristics of current medical knowledge on the treatment of bipolar disor
der by engaging with the literature. This involved an initial consultation
of relevant sociological studies (Collin, 2015; Healy, 2008; Lakoff, 2005),
followed by the review of 30 medical articles published between 2000
and 2016. The characteristics arrived at this way were further refined by
reading the abstracts of 15 medical articles published between 2010 and
2018. Three main characteristics were identified and they guided the
analysis of the online data. Online data were gathered from one French and one American inter
active platform. Le Forum des Bipotes (LFB) is a forum developed by a
person diagnosed with bipolar disorder. LFB was founded in 2007 and
functioned until 2014. While it is no longer active at the moment, it
enjoyed great popularity and it is still maintained online as a source of
information. Bipolar Burble is the personal blog of Natasha Tracy, who is
diagnosed with bipolar disorder. She enjoys celebrity status in this world,
as the blogs she has authored have repeatedly been listed among the top
best blogs about bipolar disorder, and appear on the first page of results
by search engines such as Google. Blogs and fora have the advantage of 4 Tactical Re-appraisals and Digitally Informed Hypotheses… 117 containing contributions by different stakeholders in relation to bipolar
disorder, allowing for a comparison of the ways in which medical infor
mation and other types of knowledge are dealt with online by people in
different positions of authority and endowed with different resources. f
pf
In choosing these online platforms, I aimed to mimic the approach of
regular users, and, using the Google index as an indicator of relevance, I
limited the selection to the results provided on the first 30 pages. Studying Tactics Online The selec
tion was further refined by excluding multiple pointers to the same item,
and by filtering out blogs which were not in English, which had been estab
lished for less than one year at the moment when the selection took place
(September 2014), and which had few contributors (<10). From these
online platforms, I selected 10 entries/threads which covered information
on the treatment of bipolar disorder, broadly understood, and which had
more than 30 comments each. The data were analyzed using computer-
mediated analysis (Herring, 2012) and building upon insights from socio
linguistics (Blommaert, 2005), which highlight the action-oriented and
power-laden character of language. An asset in itself, language is also a
means to acquire other resources and to achieve specific goals, such as
claiming a particular identity, displaying a certain type of expertise, and
distinguishing between different claims. For the sake of accuracy, excerpts
are reproduced as they appeared online, with no spelling or grammar cor
rections, and French quotes were translated by the author. As already men
tioned in Chap. 1, to respect the privacy of the online contributors and to
avoid their identification, their usernames were replaced by pseudonyms
and the dates were slightly modified. I will first describe the three charac
teristics of medical knowledge on the treatment of bipolar disorder identi
fied and will then discuss their re-appropriation on blogs and fora. Three Characteristics of Medical Knowledge
on the Treatment for Bipolar Disorder Treatment for bipolar disorder focuses on mood stabilization and main
tenance and combines medications and psychotherapy. Its prescription
occurs in a context where the production and interpretation of clinical
evidence remains problematic in mental health, as 118 C. Egher [p]sychiatry has a long history of trying to identify predictors of differential
pharmacological response, but these attempts began before the evidence-
based medicine (EBM) approach was born, were not deployed in the con
text of RCTs, and were not tested in RCTs. These attempts came from a
different tradition, the mechanistic tradition.… (de Leon, 2012: 156) In the mechanistic tradition, the effectiveness of psychiatric drugs is
due to their action upon specific mechanisms in the brain. In the after
math of the Human Genome Project, the rise of personalized and preci
sion medicine has contributed to numerous attempts to identify genetic
markers for bipolar disorder and the biomarkers that render some of the
people diagnosed with it responsive to specific treatments. As “the rapid
progress in the ‘-omics’ fields makes the notion of evidence a moving
target” (Khoury et al., 2008: 1606), nowadays different types of evidence
can be produced in multiple, innovative ways, in different settings and
involving different stakeholders (Collins & Varmus, 2015). Nevertheless,
the studies undertaken thus far have only been mildly successful in pro
viding clear insights on the effectiveness of treatments for bipolar disor
der, whereas attempts to identify biomarkers to explain the heterogeneity
of drug responses among patients have generally been unsuccessful. Based
on the literature review I undertook, I propose that current medical
knowledge about the treatment of bipolar disorder is characterized by
uncertainty, complexity, and individualization. y
p
y
Uncertainty is “characterized by self-awareness of incomplete knowl
edge about some aspect of the world” (Han, 2013:16). In the medical
field, “[t]he evidence in which different uncertainties are manifest ranges
from anecdotal clinical observations to data from randomized clinical
trials” (ibid.). Uncertainty about the treatment of bipolar disorder is
informed by methodological issues derived from important characteris
tics of this condition, such as the considerable heterogeneity in the defi
nition and assessment of a mood episode, relapse (Young & Newham,
2006), and therapeutic response. Three Characteristics of Medical Knowledge
on the Treatment for Bipolar Disorder While EBM has led to “an ever-
increasing demand for standardization and improved quality in psychiat
ric treatment” (Geddes & Goodwin, 2001:191), there is still a lot of
uncertainty regarding the mechanism of action of various drugs used for
the treatment of bipolar disorder. For example, anticonvulsants were 4 Tactical Re-appraisals and Digitally Informed Hypotheses… 119 introduced in the treatment of bipolar disorder because of certain simi
larities between this condition and epilepsy, but the current understand
ing of their action mechanism remains superficial. While the use of any
antidepressants in the treatment of bipolar disorder is controversial, stud
ies have reported important variations in their efficacy and tolerability. Yet, there is limited understanding as to the causes of such heterogeneity. Uncertainty also exists in relation to side effects, and this is the case even
for substances which have been long prescribed in the treatment of bipo
lar disorder. For instance, reports on the degree to which long-term lith
ium use may lead to renal failure or to congenital malformations, when
taken during pregnancy, are ambiguous. g p g
y
g
Uncertainty is sometimes due to a lack of clarity, but there are also
situations when it is due to a gap in knowledge. Various treatment com
binations are often prescribed in clinical practice in response to patients’
needs, side effects, or other medications they take, while there are no
study results available to confirm or discourage such practices. Another
type of uncertainty is linked to patient behavior, particularly treatment
adherence. For instance, even though lithium is frequently prescribed
and is considered to be highly effective for mood stabilization, it may
prompt more frequent episodes if it is abruptly interrupted. This is
another aspect that renders treatment effectiveness more difficult to
define and assess. At the same time, it indicates that treatment decisions
need to be based not only on the best available evidence regarding the
effectiveness of particular medications, but have to consider treatment
adherence and the elements which mostly influence it (Levin et al., 2016). l
Complexity denotes the multiple factors which may play a role in the
development of a disease and/ or in an organism’s reactions to treatment
and the awareness that changes in any of these factors may affect others in
unpredictable ways, while sometimes remaining themselves hard to fore
see (Plsek & Greenhalgh, 2001). Three Characteristics of Medical Knowledge
on the Treatment for Bipolar Disorder In the case of bipolar disorder, complex
ity is derived from the diverse causes of this condition and the multiplicity
of factors involved in its therapeutic approach, which make it difficult to
assess the effects of specific elements and interactions and to make
informed decisions about treatment. Numerous findings show that the
effects of various medications used in the treatment of bipolar disorder not
only are influenced by the level of specific hormones and other bodily 120 C. Egher C. Egher values but are also importantly shaped by one’s genetics (Craddock &
Sklar, 2013) and hereditary make-up. For instance, only 30% of people
diagnosed with bipolar disorder are responsive to lithium and researchers
have been able to develop a general molecular and functional profile of
this group. While such responsiveness was thought to indicate a subtype
of bipolar disorder, more recent insights suggest that lithium responsive
ness is linked with certain symptoms and is heritable (Tighe et al., 2011). Genetic insights indicating that bipolar disorder is not a discrete entity
have further contributed to the complexity characterizing the search for
treatment, guiding such endeavors across traditional diagnostic boundar
ies (Harrison et al., 2016). The environment in which one finds oneself
provides another complicating dimension (Harrison et al., 2016). Factors
such as climate, family situation, workplace stress, and especially shift
work, which disrupts night and day rhythms, also influence treatment
effectiveness. Comorbidity further complicates current understandings on
treatment effectiveness, as medicines prescribed for other conditions may
interact with the bipolar disorder treatment, leading either to different
effects altogether or to weaker or stronger effects than expected. The tim
ing when particular interventions are used also seems to importantly
determine treatment selection and effectiveness. Thus, different medical
combinations are considered depending on the condition’s developmental
stage (Sachs, 2004) and on the age of the people diagnosed. For instance,
studies suggest that the use of psychoeducation to prevent relapses is most
effective during the first years after diagnosis, with much more modest
effects when taken up later (Miziou et al., 2015). Furthermore, the effects
of particular medications only become fully manifest after being taken for
a long period of time without interruptions. Individualization understood as individual variations in treatment
response has recently come more and more to the attention of researchers
(Bates, 2010), and constitutes a move away from “standard” approaches,
where reactions to medications are studied among relatively large groups. Three Characteristics of Medical Knowledge
on the Treatment for Bipolar Disorder While the hope is that at some point treatment response will be studied
at the level of each person of interest, individualization currently denotes 4 Tactical Re-appraisals and Digitally Informed Hypotheses… 121 practices which focus on subgroups of increasingly smaller sizes, as dis
tinctions are made at greater levels of specificity. From this perspective,
attempts at determining treatment effectiveness in the field of mental
health have also been strongly influenced by developments in the field of
pharmacogenetics, as various studies have shown that determining a
patient’s genotype can help when deciding upon the prescription of spe
cific antipsychotic drugs (Tanaka & Hisawa, 1999). At the same time,
various studies have focused on how and why particular subgroups diag
nosed with bipolar disorder react differently to specific substances, requir
ing higher or lower dosages for the intended effects. Insights from
personalized medicine have led to a growing awareness that evidence
about treatment effectiveness requires taking into account parameters
such as dosage, form, and frequency and that genetic, hereditary, and
environmental factors may trigger different reactions in different indi
viduals (Hedgecoe & Martin, 2003). Developments in genetics have
prompted medical researchers to hope that genetic loci playing a role in
the development of bipolar disorder will be found, leading to the identi
fication of biomarkers and to the development of more effective treat
ment pathways and targets (Squassina & Pisanu, 2013). There has also
been a growing recognition that “an individual’s unique life circum
stances… influence disease susceptibility, phenotype, and response to
treatment” (Ziegelstein, 2015: 888). Next to genetic or genomic markers,
various personal categories, many of which are dynamic and change
numerous times throughout the life of a particular individual (Naylor &
Chen, 2010), have thus come to play a role in the development of knowl
edge about the treatment of bipolar disorder. This way, the evidence
about treatment effectiveness has been expanded to include “psychologi
cal, social, cultural, behavioral, and economic factors of each person”
(Ziegelstein, 2015:888). practices which focus on subgroups of increasingly smaller sizes, as dis
tinctions are made at greater levels of specificity. From this perspective,
attempts at determining treatment effectiveness in the field of mental
health have also been strongly influenced by developments in the field of
pharmacogenetics, as various studies have shown that determining a
patient’s genotype can help when deciding upon the prescription of spe
cific antipsychotic drugs (Tanaka & Hisawa, 1999). Three Characteristics of Medical Knowledge
on the Treatment for Bipolar Disorder At the same time,
various studies have focused on how and why particular subgroups diag
nosed with bipolar disorder react differently to specific substances, requir
ing higher or lower dosages for the intended effects. Insights from
personalized medicine have led to a growing awareness that evidence
about treatment effectiveness requires taking into account parameters
such as dosage, form, and frequency and that genetic, hereditary, and
environmental factors may trigger different reactions in different indi
viduals (Hedgecoe & Martin, 2003). Developments in genetics have
prompted medical researchers to hope that genetic loci playing a role in
the development of bipolar disorder will be found, leading to the identi
fication of biomarkers and to the development of more effective treat
ment pathways and targets (Squassina & Pisanu, 2013). There has also
been a growing recognition that “an individual’s unique life circum
stances… influence disease susceptibility, phenotype, and response to
treatment” (Ziegelstein, 2015: 888). Next to genetic or genomic markers,
various personal categories, many of which are dynamic and change
numerous times throughout the life of a particular individual (Naylor &
Chen, 2010), have thus come to play a role in the development of knowl
edge about the treatment of bipolar disorder. This way, the evidence
about treatment effectiveness has been expanded to include “psychologi
cal, social, cultural, behavioral, and economic factors of each person”
(Ziegelstein, 2015:888). In the remainder of this chapter, I will show that such realizations are
not restricted to the pages of academic publications, but reach people
diagnosed with bipolar disorder, who engage with them both to achieve
practical goals and to contribute to the development of new knowledge. 122 C. Egher Engaging with Medical Knowledge About
the Treatment of Bipolar Disorder Online The analysis of the online data revealed that people diagnosed were aware
of current medical knowledge on the treatment of bipolar disorder, as the
following three tactics were identified: the mobilization of the notions of
uncertainty, complexity, and individualization. Rather than being merely
neutral spaces where these exchanges unfolded, I argue that blogs and
fora allowed for individual hunches or suppositions to thicken into what
I call “digitally informed” hypotheses about new factors that may influ
ence the effectiveness of treatment. As we will see below, the narrative
format specific to contributions on such platforms enabled people diag
nosed with bipolar disorder to describe in great detail their various states,
to select the aspects each of them found important and meaningful to
share in the absence of pre-set rubrics and criteria, and to convey contex
tual information and aspects of the qualities of their experiences that
generally resist quantification. This way, online contributors could pro
vide more clues about the applicability and relevance of their insights,
they engaged dialogically in the assessment of the views and experiences
shared by others, and they used different, additional standards to evaluate
the credibility of various statements. y
The design and affordances of blogs and fora played an important role
in the development of “digitally informed hypotheses.” Thus, they
enabled online contributors to engage in various interactive practices
whereby personal views or assumptions could solidify and acquire a more
general character: by liking specific contributions, by directly reacting to
them through comments and expressing similarities of experience, and
by providing hyperlinks to studies and other resources in their support. Furthermore, blogs and fora allowed for the longitudinal accumulation
in the same space of similar experiences, even when conveyed through
fleeting exchanges by occasional contributors. This made possible the
gradual emergence of a limited body of evidence, pointing to fruitful
areas for further inquiry or, as was mostly the case here, self-
experimentation. Whether or not certain personal insights turned into
collectively generated hypotheses was often a question of repetition, 4 Tactical Re-appraisals and Digitally Informed Hypotheses… 123 accumulation, and visibility. This process was thus not only informed by
the urgency of the aspects under discussion, but also informed by how
skillfully various online contributors could mobilize online affordances
and by the flexibility allowed by the platform owners/administrators. Engaging with Medical Knowledge About
the Treatment of Bipolar Disorder Online The
latter were influential through the various rules they set regarding the
type of content that could be made available, the word limit for each
contribution, and the modality in which insights could be shared. Worth
to be mentioned here are, for instance, Tracy’s decisions to provide links
on her blog’s homepage to the blog entries with the highest number of
comments and to those with the most recent comments, which kept
them in the attention of her audience and increased the likelihood that
they would accumulate more contributions. The curatorial work the
forum administrator engaged in also shaped the visibility of some insights,
as he moved comments made on the one thread onto another which he
considered more suitable, and re-positioned certain threads on the first
page of the forum when major social and cultural events suggested they
would be of interest. Thus, “digitally informed hypotheses” about the
effects and side effects of medications emerged as online contributors re-
appropriated the notions of uncertainty, complexity, and individualiza
tion through interactions mediated by the technologies of blogs and fora. C. Egher I started taking it about 6 months ago after reading your blog about it. I
have had no side effects and have had no depressive episodes either. I have
had a mixed episode but the depressive symptoms were much less than they
would normally be. I’m still cautious about saying it has helped and still
monitoring but so far so good. Thank you for mentioning it in the first
place. We are all different and some people may have negative effects, that’s
the same with anything. I would say give it a go. (Jane, November 7, 2014) I’ve been taking [substance name] for about 18 months now, I have had
no side effects, the depressions have not been as bad and I think possibly
the highs are less too. I do get psychosis and I haven’t noticed any effect on
this. Although my doctor is sceptical I will continue to take it. Hopefully
if the trials are successful doctors will be more likely to suggest this treat
ment. This same doctor recommended glucosamine for my arthritis so it’s
not that he is against supplements. (Jane, November 19, 2015) Something akin to a hierarchy or an attempt at a systematic assessment
becomes apparent in both comments, as Jane focuses first on the presence
or absence of side effects, then on this substance’s effects on depression,
for which it is intended, and only later on its impact on other symptoms. While the first quote testifies to the influence online bloggers have upon
their readers’ treatment, both excerpts suggest that people diagnosed
make sense of their experiences with medications in a relational way. In
her first contribution, Jane solves the dissonance between her findings
and those of other people diagnosed by invoking the uniqueness of each
person and echoes Tracy in recommending it to others. In her second
comment, experiential and medical knowledge are described as being at
odds with each other, as Jane’s tentatively positive findings and intention
to continue taking the pills are set against her doctor’s doubts. Given
Jane’s awareness about the limited amount of clinical evidence available,
her sharing activity and encouragement for others to try this sub
stance may be seen as an attempt to help fill these gaps in medical knowl
edge. The mild improvements she describes further suggest that she may
make treatment decisions using lower effectiveness standards than medi
cal professionals. Uncertainty People diagnosed sought to address medical uncertainty about the effects
of certain substances by engaging in self-experiments. In so doing, they
mobilized uncertainty through their ability to locate and manipulate
important gaps in relevant medical knowledge, both at the scientific and
clinical level, thereby identifying a space which could mainly be furbished
through the insights they provided. For instance, in a post from October
2011, Tracy argued that a certain chemical substance might be a new
cheap and effective supplement in the treatment of bipolar depression. She cautioned, however, that, while promising, the evidence was limited. In the aftermath, her readers tried this substance, kept careful track of
their reactions to it, and shared their experiences at different moments
in time: 124 C. Egher C. Egher Medical uncertainty may therefore be a cause for hope
in certain instances and may help to keep people diagnosed motivated
and actively engaged with their treatment. 4 Tactical Re-appraisals and Digitally Informed Hypotheses… 125 French online contributors re-conceptualized uncertainty to test medi
cal claims about the benefits some of the medications they took for bipo
lar disorder could have upon other bodily processes. Thus, various online
contributors shared insights which they had acquired from their doctors
about the neuroprotective effects of lithium, as well as their own opinions
and experiences in this respect: C. Egher C. Egher The highest level of credibility ascribed to this view was provided by
Bipote_Admin, who referred to lithium’s neuroprotective properties
as a fact: Apart from that, as Annemarie says, the neuroprotective and even trophic
effect of lithium is worth mentioning because it opens up new therapeutic
perspectives. An increase in the volume of gray matter, especially in the
frontal lobe, has been observed in patients undergoing lithologic therapy. A thymic episode is neurotoxic and its repetition can cause neurobiological
damage. (Bipote_Admin, September 8, 2012) The use of medical terminology and the passive voice serve to render
this claim more credible and neutral. The last sentence echoes the views
NIMH put forward that we discussed in the previous chapter, and it
indicates that Bipote_Admin’s insights were informed by an understand
ing of bipolar disorder as a brain condition. Since many of the clinical
studies which had confirmed this hypothesis were based on neuroimag
ing techniques (Bearden et al., 2007; Machado-Vieira et al., 2009), this
can be seen as an indication of the degree to which this online contribu
tor had internalized medical knowledge. The excerpts above show that reframing uncertainty in terms of tactics
is helpful to understand how people diagnosed with bipolar disorder
negotiate medical knowledge to turn their personal experiences into valu
able contributions. Other complex factors that can influence treatment
effectiveness are discussed below. This is what my shrink says: Lithium protects against Alzheimer’s. For my mother this seems to be
true thus far…. (….)
Sometimes I don’t know who or what to believe… (Oliane,
September 7, 2012) (
)
Sometimes I don’t know who or what to believe… (Oliane,
September 7, 2012) At the time when contributions such as this were made, the mecha
nisms through which lithium achieved its neuroprotective effects
remained unclear (Forlenza et al., 2014). The available evidence about
these effects was largely derived from preclinical trials and from retro
spective registry studies conducted on people diagnosed with bipolar dis
order. Whereas Oliane invoked the psychiatrist as well as the experiences
of her mother to legitimate the claim about lithium’s neuroprotective
effects, it gradually acquired more credibility, as more contributors con
firmed having heard about these effects, and having taken the claim seri
ously enough to base treatment decisions on them: I’ve also heard this about Alzheimer’s, and also for multiple sclerosis. (Kat,
September 8, 2012)
* I’ve also heard this about Alzheimer’s, and also for multiple sclerosis. (Kat,
September 8, 2012)
* My psychiatrist at the expert center in Marseille says that lithium recon
stitutes the neural connections that explode under the effect of bipolarity. It also protects from Alzheimer’s disease.h These arguments have tipped the scales even for me, who am a rebel
when it comes to taking drugs. I agreed to resume a lithium treatment. I’m
starting tonight. He also prescribed Xéroquel. But to that one I say no! I’m
still fighting it. (Annemarie, September 8, 2012) 126 C. Egher Complexity People diagnosed with bipolar disorder mobilized the notion of complex
ity online, as they sought to put forward new factors informing variations
in the effectiveness of medications. Not content with merely describing
the particular effects they experienced, online contributors actively sought
confirmation or additional information from others regarding these
experiences, often so that they could use such insights as resources to bet
ter negotiate with medical professionals in favor or against the prescrip
tion of specific medications. Generics were often mentioned in such 4 Tactical Re-appraisals and Digitally Informed Hypotheses… 127 contexts by American online contributors. For instance, they invoked the
complexity of interactions between the various substances contained in
this type of medicines and the role variation in their different dosages
may have to put forward the hypothesis that their effectiveness varied: Ugh! This weekend I picked up a refill (…) At home, I realized the pills
looked different. I took them but experienced NO relief whatsoever. I took
the bottle down to the pharmacy and insisted they were not what I had been
receiving only to be told they WERE. I know what I take and what my pills
look like after all this time. A second visit with another pharmacist at the
same pharm told me that indeed they had switched generics on me. Did you
know that the FDA allows a 20–30% variable amount of the active ingredi
ent in generics. I did not until LOTS of research. You have to be your own
doctor AND pharmacist, apparently… (Marina, October 6, 2014) Marina’s comment depicts this hypothesis as the outcome of a discov
ery journey and traces this contributor’s development from the classical
“good patient” described by Freidson (1970), who was willing to comply
with medical advice even when the pills looked differently, to more recent
understandings of patienthood, which conceive of people diagnosed as
interested in educating themselves about their condition and assuming
an active role in its management. The unfolding of the events described
in this quote further serves to reinforce the unbiased character of Marina’s
claims, as she only became distrustful when confronted with an embod
ied experience—the pills’ lack of effect—and set out to find out more
information about generics, their compositions, and pharmaceutical reg
ulations once this experience was legitimated by a “traditional” expert. Complexity That people diagnosed with bipolar disorder take up the role of investiga
tors to make sense of their personal experiences about medication is illus
trated by one of the reactions to Marina’s contribution: My doc told me it’s a 40 percent swing… Issue is the filler… Different
manufacturers use different fillers which can effect how the med is used
by your system. Some can come on strong while others are weak… Many
braded pills have 10 manufacturers or more and they are mostly over
seas. The FDA could give a hoot. I find that most pharmacists know
very little also… Probably because they have so many different meds 128 C. Egher to deal with… And, if you notice many generics have gone up in price
tremendously since the branded aren’t available…Money. Money, Money. (Jack, October 6, 2014) to deal with… And, if you notice many generics have gone up in price
tremendously since the branded aren’t available…Money. Money, Money. (Jack, October 6, 2014) This exchange illustrates how people diagnosed take up a potential fac
tor an online contributor suggested to influence the effectiveness of med
ication and try to make sense of it building upon their own experiences
and insights. In this case, the varying effectiveness of generics is con
firmed and further explanation for it is sought not only by considering
the different action of the chemical compounds used, but also by relating
it to their manufacturers and to the more or less strict legislation existing
in the countries where they are based. Importantly, these comments indi
cate that online contributors ascribe the limited or incorrect information
they receive from different sources to different causes: some unintentional
and due to systemic issues, such as the pharmacists having a hard time to
keep up with all the new types of medications available, while others
intentional and due to corruption, such as official bodies failing to inter
vene due to their close ties with the pharmaceutical industry. Since the
latter are depicted as mainly motivated by commercial interests, the
hypotheses people diagnosed develop based on their personal experiences
about treatment acquire more credibility among online contributors. The
failure of certain governmental agencies to involve and inform the public
in more effective ways about their regulatory procedures may thus have a
negative impact on their public image and contribute to shifts in the
tasks and cognitive authority of different stakeholders. Complexity In this context,
people diagnosed with bipolar disorder may become more influential and
succeed in re-positioning themselves in relation to medical professionals
and even researchers through their more active engagement in the pro
duction of new knowledge and through the existence of an audience will
ing to take their insights into account. People diagnosed with bipolar disorder also re-appropriated the notion
of complexity to refine medical insights about some of the factors influ
encing treatment effectiveness. This was, for instance, the case for a, cer
tain atypical antipsychotic which is recommended to be taken with a
meal. Whereas in her blog post Tracy described recent study results which
specified the number of calories required per meal for this medicine to be 4 Tactical Re-appraisals and Digitally Informed Hypotheses… 129 effective, online contributors further specified these insights by arguing
that not only the number of calories influenced treatment effectiveness,
but also the types of proteins consumed, as the following quote illustrates: I don’t know the correct spelling [of medication] (…) I have this med
down to a science. I eat a homemade hamburger … at 12:30 p.m. and then
I drink a small chug of milk at 1:15 p.m. and then finally another ham
burger and glass of skim milk at 1:45 p.m. If you eat the second hamburger
and glass of milk any earlier than 1:45 p.m. it will not work and you will
be sick for the next 12 hours. Then you must repeat the process at 12:30
a.m. You also cannot drink any water after taking [name of medicine] until
you wake up. You can take one swig of water here or there but I try not to. One thing that has worked for me is not eating or drinking anything from
9:30 until 12:30. [name of medicine] is a trial and error drug and I have
schooled my doctor on what works. (Watson, March 9, 2015, quote
slightly adapted to ensure anonymity) Striking here is the level of detail and precision provided by Watson,
whose claim to expertise is based on the substantial knowledge he acquired
through the varied tinkering practices that he engaged in to fine-tune
what he considers to be the most effective approach to the intake of this
medicine. Complexity Even though Watson spells the name of this medication
wrongly, his recommendation is bracketed at the beginning and at the
end by his claim to scientific authority, which is further emphasized by
the description of a reversal in positions between himself and his doctor. Such statements serve to increase the legitimacy of the insights Watson
provides, whereas the dissonance between his incorrect spelling and the
authoritative character of his statement suggests that he may consider
practical knowledge, with which he believes to be endowed, more impor
tant than abstract, theoretical insights. Other online contributors re-appropriated the complexity of symp
toms of bipolar disorder to advocate for an expansion of therapeutic
interventions, so as to include, next to various combinations of medical
treatments and “talk” therapies, elements of interior design, atmospheric
aspects, such as air pressure and humidity, and the use of specific objects: Have you done EMDR [Eye Movement Desensitization and Reprocessing]? What about a Sun Lamp? There is 1 on Amazon by Sphere Technologies 130 C. Egher that is $69. It has the highest reviews on Amazon. My friend lent me one
and I have been using it for a 8 days. You want to get one that is 10,000 lux
and I have seen them as low as $49. (…) I think the sun lamp is worth a
shot. It’s primarily made for people with SAD [seasonal affective disorder]. I am also going to start volunteering at the animal shelter as a “Cat
Socializer”. You just go and play with the cats and it makes you feel better,
and of course the cats too, and makes them more adoptable. (Roger,
April 11, 2015) This quote suggests that for this particular contributor personal experi
ences as well as the evaluations provided by others on online platforms such
as Amazon constitute reliable evidence in favor of taking up potentially
therapeutic procedures. At the same time, Roger is dedicated to providing
other contributors with accurate insights, as he carefully situates his claims
by mentioning for how long he had been using the sun lamp and by clearly
stating that it was primarily developed for another condition. Complexity The details
about the price of this technology and how he came to use it are illustrative
of the financial considerations that people diagnosed with bipolar disorder
who live in the US need to take into account when evaluating their treat
ment options, and were echoed by many other contributors.h The comparison between the French forum and the American blog
revealed that these considerations were importantly shaped by social and
cultural factors. While the effectiveness of generics or the cost of objects
ascribed therapeutic value were important topics among online contribu
tors in the US, they were not mentioned by French contributors, whose
insurance coverage spared them such worries. Furthermore, the analysis of
the online data suggested that the blog studied here was at times an impor
tant venue through which American contributors, who were no longer
insured, could benefit from up-to-date insights on available treatments, as
they gained access to medical information. Online exchanges thus came to
replace, to a certain extent, medical encounters, as some uninsured online
contributors, who had to pay out of pocket for medication, used the treat
ment experiences and information shared by others to determine what
medication would be most effective for them. In contrast, American and
French contributors who were insured claimed to have other motivations
for engaging in online exchanges. Thus, some wanted to share the insights 4 Tactical Re-appraisals and Digitally Informed Hypotheses… 131 they acquired on the factors affecting the effectiveness of treatment with
their doctors, whereas others wanted to determine whether or not to con
tact other medical professionals when their own doctors were away or, in
some cases, to figure out whether their experiences were serious enough to
warrant disturbing their doctor while on holiday. The tactics through which American and French online contributors re-
appropriated complexity were also influenced by national institutional per
spectives on mental health. It was thus obvious that many online
contributors on the French forum supported a biopsychosocial model of
disease, which, as I mentioned in the previous chapter, was the dominant
approach to mental healthcare in France up until the early 2000s, as they
emphasized the need to tackle bipolar disorder by addressing it simultane
ously as a biological, psychological, and social condition. Complexity In general, French
contributors advocated an understanding of treatment effectiveness as not
solely the result of the actions of the various chemical substances they took,
but also of the various types of therapies and social support available. Furthermore, many of the additional therapies they suggested had a dia
logical or interactional character, such as psychoanalytic approaches and
eye movement desensitization and reprocessing therapy. While “talk” ther
apy or Alcoholics Anonymous (AA) meetings were often mentioned by
American contributors, approaches focusing on dietary changes and on
technological interventions, such as vagus nerve stimulation (VNS) and
even electroconvulsive therapy (ECT), were by far more popular. At the
same time, a pronounced overreliance on medications could be noted,
which some of the blog contributors were aware and critical of: You miss 1 day of your Seroquel, or your Cymbalta, or your Depakote…
seriously, it will be okay… if not, use your psychotherapy techniques. Oh,
that’s right… not too many actually do psychotherapy… it’s all the meds
baby. (Mandy, May 12, 2011) Overall, both American and French online contributors argued in
favor of acknowledging a more diverse array of chemical interactions and
practices as influencing treatment effectiveness. Personal preferences also
informed the choice of therapeutic intervention, and they are dis
cussed below. 132 C. Egher C. Egher Individualization People diagnosed with bipolar disorder creatively engaged with medical
insights about individualization in treatment response to argue for the
recognition of diverse personal preferences and leisurely pursuits as
important influences on treatment effectiveness. For researchers, such
variations refer to the identification of specific, small(er) groups sharing
common molecular, environmental, and personal attributes. In contrast,
online contributors often interpreted individualization so that each per
son’s health and illness trajectory became unique, which sometimes
prompted them to argue against evidence derived from RCTs. In such
instances, they advised others not to focus too much on statistics, but to
bear in mind that the challenges they were facing were deeply personal
and unlike those of anyone else. In general, online contributors re-interpreted individualization to
argue that lifestyle choices or leisurely activities which fell outside of the
medical domain had therapeutic value, and often engaged in compari
sons between their effects and those of medications, as the quote below
illustrates: Recently (I did something very random though less so for my brain), as I
was coming out of a big depression, I went horse-riding, a great passion …
well this session was the equivalent of an antidepressant and an anxiolytic,
I was in the seventh heaven … Zen, feeling well … and this word ‘well’, we
often look for it in our illness.hfi The therapeutic effect: it stimulates, it “zenifies”, it has an anti-depressant
effect without a change of mood! You and other bipolars shook me up, my shrink as well … but very
frankly I didn’t want to listen… I had to find the “drive”, to set the machine
in motion again… For the moment I have found it again… and I am much better. (Sunset,
Xeroquel, August 7, 2011) This contributor seems to suggest that the pursuit of a hobby might be
better than the categories of medications invoked, since its anti-depressive
effects are not accompanied by the risk of mania or hypomania. The 4 Tactical Re-appraisals and Digitally Informed Hypotheses… 133 suggestion that leisurely pursuits have therapeutic effects is strengthened
in the second part of the quote, where the state of well-being thus acquired
is reported to be lasting. Individualization Other people diagnosed with bipolar disorder
took up this suggestion and contributed to its development into a hypoth
esis, by reporting similar positive effects they were experiencing when
engaging in their favorite activities, be they reading, cooking, or contrib
uting on online platforms: this blog and your collective experiences have been better for me than any
medications as they usually have side-effects that are not welcomed. (Damian, February 25, 2014) While these comments may indicate that the borders between the
treatment and (self)management of a condition are rather porous for the
people diagnosed with bipolar disorder studied here, only some of these
digitally informed hypotheses have thus far coincided with scholarly
interest. The last two quotes refer to practices that have been more suc
cessful from this point of view. For instance, whereas a focus on horses
has been missing thus far in therapeutic approaches to bipolar disorder,
this topic has received more attention over the last few years in relation to
autism (Malcolm et al., 2018). Similarly, the idea that participation in
online support communities would have positive effects on the well-
being of a person diagnosed is the object of ongoing research. Whereas
some of the results produced thus far have been inconclusive (Lagan
et al., 2020; Naslund et al., 2016), others give some cause for hope that
digital interactions with certified peer specialists can improve treatment
outcomes in mental healthcare (Fortuna et al., 2019). Unlike the contributors discussed above, another group of people
diagnosed with bipolar disorder also re-interpreted individualization in
treatment response by using the degree to which they could engage in
activities that were meaningful to them as new and, arguably, more rele
vant standards to assess treatment effectiveness. In such instances, they
went beyond considerations as to whether or not a certain medication
stabilized their mood, and focused, instead, on the extent to which it
allowed them to perform social roles, professional duties, or activities
they enjoyed, be that the joy of interacting with their children, to fulfill 134 C. Egher C. Egher their athletic aspirations, or to have a body image more aligned to their
personal aesthetic ideals. Such accounts succeeded in rendering the effects
of specific treatments “thick,” meaningful, understandable to people
diagnosed as well as undiagnosed. Individualization In so doing, they helped others decide
whether a certain medicine or therapeutic approach might be worth a try,
based on similarities in life circumstances, hobbies, personal values, and
preferences. p
The identification of successful treatments according to such individu
alized standards was often accompanied by effusive displays of gratitude
and appreciation: I bless every day the medical team who discovered the effects of lithium,
even though I’m suffering from its toxicity to the kidneys today, and I don’t
regret having taken this treatment over a long period of time. I owe it the
best years of life, calmer and more serene than I could have imagined. (hon
eysuckle, September 19, 2012) As this quote indicates, such enthusiasm was not only reserved for
medications with minimal side effects or where the side effects had not
yet become apparent, but emerged as the result of a careful retrospective
analysis. The accumulation of superlatives attached to the positively qual
ifying adjectives in the second sentence is illustrative of the ways in which
affective markers functioned as indicators of the degree of confidence
online contributors had in the effectiveness of medications. Unlike scien
tific accounts of treatment assessment which steer away from sentimen
tality and subjectivity, people diagnosed with bipolar disorder used
sentimental effusions judiciously, but they did mobilize them for specific
medications, to lend additional persuasive strength to their accounts
about treatment effectiveness. Given that individual perceptions were
accepted as reliable and authoritative by the other contributors, this
reframing of individualization might contribute to heighten the epis
temic relevance of emotional and affective personal markers in a field
where authoritative knowledge has traditionally been acquired based on
groups and the calculation of averages. Other online contributors re-appropriated individualization to suggest
that personality traits, one’s attitude toward treatment, and the level of 4 Tactical Re-appraisals and Digitally Informed Hypotheses… 135 personal commitment could impact on the effectiveness of medical treat
ment. Thus, in numerous comments the role of personal characteristics,
such as risk aversion, tolerance, patience, and curiosity, on treatment
response was seriously considered. This is, for instance, how a contributor
made sense of the absence of positive effects the first time she took
lithium: I think the first time I wasn’t patient enough. I was expecting too much,
too quickly. I don’t really know what it means to be balanced and I was
expecting an influx of positive emotions. C. Egher Overall, online contributors mobilized individualization to provide
insights meant to enable other people diagnosed with bipolar disorder to
decide upon treatment, depending on their lifestyle preferences, on what
they appreciated most about their existence and wanted to maintain,
restore, or improve. They thus rendered the space of medical knowledge
about treatment meaningful to them by inscribing in it elements and
experience they found fulfilling. The implications of these findings are
discussed below. Individualization Until then I had only seen life in
black, in gray … so I wanted to see it in pink, at least from time to time. Whereas normal life is not like that at all! And it’s true that I’m used to
being constantly overwhelmed by emotions. I thrive on adrenaline. I think
I have never known anything else, because the disease appeared too early. I
don’t even know what it’s like to live normally … to live without having to
struggle, without making any excesses. That’s why I didn’t think that I was
that sick! By force of habit! (Annemarie, September 8, 2012) This excerpt suggests that the performative effect of expectations
(Engel & Van Lente, 2014) is also applicable when it comes to embodied
experiences, as in Annemarie’s view, individuals need to be ready for cer
tain medications and experienced enough to recognize when they are
effective. Making the right decisions about treatment is thus not only
understood as a matter of one’s mental and physical state, but also
depends on the time of onset of the disease and the degree of self-
knowledge that one had by then acquired, a point to which we will return
in Chap. 5. Annemarie further points to an important element which
informs treatment non-adherence among people diagnosed with bipolar
disorder, as some of them enjoy their (hypo)manic states and have a hard
time appreciating (clinical) stability, which they experience as a flattening
of affect. It thus illustrates that lay expertise is not only a matter of acquir
ing vast experiential knowledge combined with medical insights, but that
it also entails the capacity to manage one’s expectations, to acquire a bet
ter understanding of what living with bipolar disorder when receiving
effective treatment can feel and look like. 136 C. Egher Discussion People diagnosed with mental health conditions have been actively
engaged in their health for a long time. In a context where medical
knowledge has permeated different areas of society, and has, thus, become
amenable to multiple usages and interpretations, the Internet provides
new avenues for them to exchange insights and to contribute to the pro
duction of knowledge. Using de Certeau’s (1988) theory of creative tac
tics it has been possible to show that people diagnosed with bipolar
disorder develop more nuanced positions than challenging or accepting
medical perspectives online, to take their insights and suggestions about
treatment effectiveness seriously, and to approach them as productive
exchanges which may lead to new knowledge. Thus, by mobilizing the
notions of uncertainty, complexity, and individualization, online con
tributors sought to state the importance of individual experiences as epis
temic resources, to put forward new factors influencing treatment
effectiveness, and to advocate for an expansion of therapeutic interven
tions. In so doing, I have argued that they went beyond the performance
of lay expertise and collectively developed “digitally informed hypothe
ses” about treatment effectiveness in an attempt to render the space of
their interactions with medical professionals and of daily life with bipolar
disorder more comfortable. The findings discussed in this chapter thus
contribute to a substantial body of literature which has highlighted the
value of blogs in providing people diagnosed with more tailored resources
to navigate daily life (Adams, 2010) and the Internet’s ability to facilitate 4 Tactical Re-appraisals and Digitally Informed Hypotheses… 137 collective learning and the development of epistemic communities
(Akrich, 2010). collective learning and the development of epistemic communities
(Akrich, 2010). The epistemic practices described here took place in a context marked
by a broadening of the conceptualization of health-relevant data, as the
growing number of wearable technologies people use and the digital
traces they leave behind has made available tremendous amounts of
information. Yet, unlike individuals who engage with digital self-tracking
tools as “interpreters of the body” (Lupton, 2013), online contributors
on the blog and forum studied relied upon other people diagnosed with
bipolar disorder as their sounding board, as they made sense of the vari
ous states, moods, and other insights they collected dialogically. As we
will discuss in more detail in Chap. Discussion 5, these activities support an under
standing of such interactive online platforms as spaces for biosociality
(Kingod, 2018), where contributors share their experiences and the cre
ative practices they develop to better manage their conditions (Pols,
2014). The design and affordances of blogs and fora importantly shaped
the development of “digitally informed hypotheses,” as the coordination
required for such epistemic practices was facilitated through the repeti
tion, accumulation, and visibility of particular ideas over extended peri
ods of time that they allowed for. The fact that information from different
years can be located in the same place has, however, potential drawbacks,
as proximity on the blog or forum might obliterate important contextual
factors, and unreflectively equate experiences shaped by specific temporal
and social coordinates. This may have negative consequences for the reli
ability of the inferences made based on such insights, as they may lack
internal consistency, but also on their validity, since elements that are
important to correctly interpret the data used are missing or not taken
into consideration. While the de-contextualized use of data is already
common in data analytics, many scholars have warned against the conse
quences such practices may have upon the quality of the scientific claims
inferred from them and about the societal transformations they may lead
to (Gregory et al., 2019; Prainsack, 2017; Wyatt et al., 2013). Through their comments, contributors showed that the effects of med Through their comments, contributors showed that the effects of med
ications do not manifest themselves in pristine laboratory conditions but
occur in the messy context of daily life of people with the same diagnosis,
but perhaps with different symptoms, bodily reactions, needs, and 138 C. Egher preferences. They also suggested that the effects and side effects of medi
cations are shaped by the specific circumstances of the lives they act upon,
and such knowledge is still insufficient at the medical level. While the
recommendations of medical professionals are based on evidence obtained
in conditions where high levels of validity and reliability can be guaran
teed, the online interactions between people diagnosed with bipolar dis
order indicate that they often value insights acquired through the
accumulation of personal accounts, whose reliability is indicated through
detailed descriptions and the presence of affective and emotional markers
next to medical information. Discussion These perspectives are in line with findings
which suggest that medical professionals and people diagnosed have con
trasting views on knowledge and validity (Bellander & Landqvist, 2018),
and which argue that “[t]he epistemic authority of the patient’s experi
ence as a source of knowledge emerges not in spite, but precisely because,
of its highly emotive and embodied dimensions” (Mazanderani,
2014:141). The comparison of the tactics through which American and French
online contributors re-appropriated medical uncertainty, complexity, and
individualization as treatment response revealed important similarities
and differences. Both French and American online contributors engaged
in online exchanges to achieve specific pragmatic goals, as they tried to
identify more suitable treatments for themselves depending on their life
style and personal preferences, to expand the meaning of treatment to
include various practices, or to consider its effects in interaction with a
more complex array of substances and activities. They also tried to acquire
more agency in their interactions with medical professionals by having
their experiences confirmed by many others. The analysis further indi
cated that cultural, social, and institutional differences importantly shape
online contributions, leading to noteworthy distinctions. More cross-
cultural studies on the treatment experiences of people diagnosed with
bipolar would be highly valuable, as they would cast light upon impor
tant similarities and differences in reactions hitherto considered as mainly
biological, and may reveal what factors inform them. While this chapter discussed how people diagnosed with bipolar disor
der contributed collectively to the development of new insights about
treatment effectiveness, the next one will show that the ideal of active 4 Tactical Re-appraisals and Digitally Informed Hypotheses… 139 patienthood combined with the skillful use of the Internet and an entre
preneurial spirit can render some individuals diagnosed with bipolar dis
order highly influential. One of these individuals is Natasha Tracy, who
in this chapter received limited attention as the owner of the blog from
which data were collected, but who will come into the limelight due to
another position she occupies and a more substantial engagement in
expertise about bipolar disorder. Borkman, T. (1976). Experiential Knowledge: A New Concept for the Analysis
of Self-Help Groups. Social Service Review, 50(3), 445–456. References Studies of h
p
g
( )
Collins, H., & Evans, R. (2002). The Third Wave of Science Studies. Studies of
Expertise and Experience. Social Studies of Science, 32(2), 235–296.h Collins, H., & Evans, R. (2002). The Third Wave of Science Studies. Studies of
Expertise and Experience. Social Studies of Science, 32(2), 235–296.h hh
Expertise and Experience. Social Studies of Science, 32(2), 235–296.h Collins, H., & Evans, R. (2007). Re-thinking Expertise. Chicago: The University
of Chicago Press. Collins, F., & Varmus, H. (2015). A New Initiative on Precision Medicine. New
England Journal of Medicine, 372(9), 793–795.h Craddock, N., & Sklar, P. (2013). Genetics of Bipolar Disorder. The Lancet,
381, 1654–1662. Davidson, L. (2005). Recovery, Self Management and the Expert Patient-
Changing the Culture of Mental Health from a UK Perspective. Journal of
Mental Health, 14(1), 25–35. de Certeau, M. (1988). The Practice of Everyday Life. University of California Press. de Certeau, M. (1988). The Practice of Everyday Life. University of California Press. De Leon, J. (2012). Evidence-based Medicine Versus Personalized Medicine:
Are They Enemies? Journal of Clinical Psychopharmacology, 32, 153–164. De Leon, J. (2012). Evidence-based Medicine Versus Personalized Medicine:
Are They Enemies? Journal of Clinical Psychopharmacology, 32, 153–164. Engel, N., & Van Lente, H. (2014). Organizing Innovation and Control
Practices: The Case of Public-Private Mix in Tuberculosis Control in India. Sociology of Health and Illness, 36(6), 917–931.h Epstein, S. (1995). The Construction of Lay Expertise: AIDS Activism and the
Forging of Credibility in the Reform of Clinical Trials. Science, Technology &
Human Values, 20(4), 408–437. Epstein, S. (1996). Impure Science: AIDS, Activism, and the Politics of Knowledge. University of California Press. Felt, U. (2015). Sociotechnical Imaginaries of “the Internet”, Digital Health
Information and the Making of Citizen-Patients. In S. Hilgartner, C. Miller,
& R. Hagendijk (Eds.), Science and Democracy: Making Knowledge and
Making Power in the Biosciences and Beyond (pp. 176–197). Routledge. Forlenza, O., De-Paula, V., & Diniz, B. (2014). Neuroprotective Effects of
Lithium: Implications for the Treatment of Alzheimer’s Disease and Related
Neurodegenerative Disorders. ACS Chemical Neuroscience, 5(6), 443–450. Fortuna, K., Brook, J., Umucu, E., Walker, R., & Chow, P. (2019). Peer Support:
A Human Factor to Enhance Engagement in Digital Health Behavior Change
Interventions. Journal of Technology in Behavioral Science, 4, 152–161. Frank, R., Conti, R., & Goldman, H. (2005). Frank, R., Conti, R., & Goldman, H. (2005). Mental Health Policy and
Psychotropic Drugs. The Millbank Quarterly, 83(2), 271–298. References Adams, S. (2010). Blog-based Applications and Health Information: Two Case
Studies That Illustrate Important Questions for Consumer Health Informatics
(CHI) Research. International Journal of Medical Informatics, 79, 89–96.h Adams, S. (2011). Sourcing the Crowd for Health Services Improvement: The
Reflexive Patient and “Share-Your-Experience” Websites. Social Science &
Medicine, 72(7), 1069–1076. Akrich, M. (2010). From Communities of Practice to Epistemic Communities:
Health Mobilizations on the Internet. Sociological Research Online, 15(2), 10. Available at http://www.socresonline.org.uk/15/2/10.html Arksey, H. (1994). Expert and Lay Participation in the Construction of Medical
Knowledge. Sociology of Health & Illness, 16(4), 448–468. Baillergeau, E., & Duyvendak, J. W. (2016). Experiential Knowledge as a
Resource for Coping with Uncertainty: Evidence and Examples from the
Netherlands. Health, Risk & Society, 18(7–8), 407–426. Bates, S. (2010). Progress Towards Personalized Medicine. Drug Discovery Today,
15(3), 115–120. Bearden, C., Thompson, P., Dalwani, M., Hayashi, K., Lee, A., Nicoletti, M., Bearden, C., Thompson, P., Dalwani, M., Hayashi, K., Lee, A., Nicoletti, M.,
& …Soares, J. (2007). Greater Cortical Gray Matter Density in Lithium-
Treated Patients with Bipolar Disorder. Biological Psychiatry, 62(1), 7–16. Bellander, T., & Landqvist, M. (2018). Becoming the Expert Constructing
Health Knowledge in Epistemic Communities Online. Information,
Communication & Society, 23(4), 507–522. https://doi.org/10.108
0/1369118X.2018.1518474 Blommaert, J. (2005). Discourse: A Critical Introduction. Cambridge
University Press. Borkman, T. (1976). Experiential Knowledge: A New Concept for the Analysis
of Self-Help Groups. Social Service Review, 50(3), 445–456. 140 C. Egher Britten, N., & Maguire, K. (2016). Lay Knowledge, Social Movements and The
U
f M d
P
l R fl
H l h
( ) Britten, N., & Maguire, K. (2016). Lay Knowledge, Social Movements and The
Use of Medicines: Personal Reflections. Health, 20(2), 77–93. l
( )
Collin, J. (2015). Universal Cures for Idiosyncratic Illnesses: A Genealogy of l
Collin, J. (2015). Universal Cures for Idiosyncratic Illnesses: A Genealogy of
Therapeutic Reasoning in the Mental Health Field Health 19(3) 245 262 l
Collin, J. (2015). Universal Cures for Idiosyncratic Illnesses: A Genealogy of
Therapeutic Reasoning in the Mental Health Field. Health, 19(3), 245–262.hh , J (
5)
y
gy
Therapeutic Reasoning in the Mental Health Field. Health, 19(3), 245–262. Collins, H., & Evans, R. (2002). The Third Wave of Science Studies. Studies of
Expertise and Experience. Social Studies of Science, 32(2), 235–296. Therapeutic Reasoning in the Mental Health Field. Health, 19(3), 245–262. Collins, H., & Evans, R. (2002). The Third Wave of Science Studies. References Mental Health Policy and
P
h
D
Th M llb
k Q
l 83(2) 2 1 298 Fortuna, K., Brook, J., Umucu, E., Walker, R., & Chow, P. (2019). Peer Support:
A Human Factor to Enhance Engagement in Digital Health Behavior Change
Interventions. Journal of Technology in Behavioral Science, 4, 152–161. Frank, R., Conti, R., & Goldman, H. (2005). Mental Health Policy and
Psychotropic Drugs. The Millbank Quarterly, 83(2), 271–298. 4 Tactical Re-appraisals and Digitally Informed Hypotheses… 141 Freidson, E. (1970). The Profession of Medicine. Harper and Row. Frey, B., Andreazza, A., Houenou, J., Jamain, S., Goldstein, B., Frye, M., et al. (2013). Biomarkers in Bipolar Disorder: A Positional Paper from the
International Society for Bipolar Disorders Biomarkers Task Force. Australian
& New Zealand Journal of Psychiatry, 47(4), 321–332. Geddes, J., & Goodwin, G. (2001). Bipolar Disorder: Clinical Uncertainty,
Evidence-Based Medicine and Large-Scale Randomised Trials. The British
Journal of Psychiatry, 178(S41), 191–194. Gregory, K., Cousijn, H., Groth, P., Scharnhorst, A., & Wyatt, S. (2019). Understanding Data Search as a Socio-Technical Practice. Journal of
Information Science, 46, 1–17. https://doi.org/10.1177/0165551519837182 Han, P. (2013). Conceptual, Methodological, and Ethical Problems in
Communicating Uncertainty in Clinical Evidence. Medical Care Research
and Review, 70(1), 14–36.h Hardey, M. (1999). Doctor in the House: The Internet as A Source of Lay
Health Knowledge and the Challenge to Expertise. Sociology of Healthy &
Illness, 21, 820–835. Harrison, P., Cipriani, A., Harmer, C., Nobre, A., Saunders, K., Goodwin, G.,
& Geddes, J. (2016). Innovative Approaches to Bipolar Disorder and Its
Treatment. Annals of the New York Academy of Sciences, 1366(1), 76–89. f
y f
Healy, D. (2008). Mania: A Short History of Bipolar Disorder. John Hopkins
University Press. Hedgecoe, A., & Martin, P. (2003). The Drugs Don’t Work. Social Studies of
Science, 36, 723–752. Herring, S. (2012). Discourse in Web 2.0.: Familiar, Reconfigured, and
Emergent. In D. Tannen & A. Tester (Eds.), Georgetown University Round
Table on Languages and Linguistics 2011: Discourse 2.0.: Language and new
media (pp. 1–29). Georgetown University Press. Hogle, L. (2016). Data-intensive Resourcing in Healthcare. BioSocieties,
11(3), 372–393. Khoury, M., Berg, A., Coates, R., Evans, J., Teutsch, S., & Bradley, L. (2008). The Evidence Dilemma in Genomic Medicine. Health Affairs, 27(6),
1600–1611. Kingod, N. (2018). The Tinkering M-Patient: Co-Constructing Knowledge on
How to Live with Type I Diabetes Through Facebook Searching and Sharing
and Offline Tinkering with Self-Care. Health, 1–17. Lagan, S., Ramakrishnan, A., Lamont, E., Ramakrishnan, A., Frye, M., &
Torous, J. (2020). Digital Health Developments and Drawbacks: A Review 142 C. Egher and Analysis of Top-Returned Apps for Bipolar Disorder. International
Journal of Bipolar Disorders, 8(39), 1–8.f Lakoff, A. (2005). Pharmaceutical Reason. Knowledge and Value in Global
Psychiatry. Cambridge University Press. Levin, J., Krivenko, A., Howland, M., Schlachet, R., & Sajatovic, M. (2016). Medical Adherence in Patients with Bipolar Disorder: A Comprehensive
Review. CNS Drugs, 30(9), 819–835. Lupton, D. (2013). 4 Tactical Re-appraisals and Digitally Informed Hypotheses… Quantifying the Body: Monitoring and Measuring Health
in the Age of mHealth Technologies. Critical Public Health, 23(4), 393–403.hi Lupton, D. (2016). The Quantified Self. A Sociology of Self-Tracking. Polity hi
Lupton, D. (2018). Digital Health. Routledge. Lupton, D. (2020). Data Selves. Polity Press. Machado-Vieira, R., Manji, H., & Zarate, C. (2009). The Role of Lithium in
the Treatment of Bipolar Disorder: Convergent Evidence for Neurotrophic
Effects as a Unifying Hypothesis. Bipolar Disorder Suppl., 2, 92–109. Malcolm, R., Ecks, S., & Pickersgill, M. (2018). ‘It just opens up their world’:
Autism, Empathy, and the Therapeutic Effects of Equine Interactions. Anthropology & Medicine, 25(2), 220–234. Martin, E. (2009). Bipolar Expeditions. Mania and Depression in American
Culture (2nd ed.). Princeton University Press.h Mazanderani, F. (2014). The Patient’s View: Perspectives from Neurology and
the ‘New’ Genetics. Science as Culture, 23(1), 135–144. McLean, A. (2003). Recovering Consumers and a Broken Mental Health System
in the United States: Ongoing Challenges for Consumers/Survivors and the
New Freedom Commission on Mental Health. International Journal of
Psychosocial Rehabilitation, 8, 58–70. Miziou, S., Tsitsipa, E., Moysidou, S., et al. (2015). Psychosocial Treatment and
Interventions for Bipolar Disorder: A Systematic Review. Annals of General
Psychiatry, 14(19), 1–11. Morrison, L. (2013). Talking Back to Psychiatry. The Psychiatric Consumer/
Survivor/Ex-Patient Movement. Routledge.h Naslund, J., Aschbrenner, K., Marsch, L., & Bartels, S. (2016). The Future of
Mental Health Care: Peer-To-Peer Support and Social Media. Epidemiology
and Psychiatric Sciences, 25(2), 113–122. Naylor, S., & Chen, J. (2010). Unraveling Human Complexity and Disease
with Systems Biology and Personalized Medicine. Personalized Medicine,
7(3), 275–289. 4 Tactical Re-appraisals and Digitally Informed Hypotheses… 143 Petersen, A., & Lupton, D. (1996). The New Public Health. Health and Self in
the Age of Risk. Sage.h Plsek, E., & Greenhalgh, T. (2001). Complexity Science: The Challenge of
Complexity in Health Care. BMJ, 323, 625–628. Complexity in Health Care. BMJ, 323, 625–628. Pols, J. (2014). Knowing Patients: Turning Patient Knowledge into Science. Science Technology & Human Values 39(1) 73–97 Pols, J. (2014). Knowing Patients: Turning Patient Knowledge into Science. Science, Technology & Human Values, 39(1), 73–97. Science, Technology & Human Values, 39(1), 73–97.h Prainsack, B. (2017). The “We” in the “Me”: Solidarity and Health Care in the
Era of Personalized Medicine. Science, Technology, & Human Values,
43(1), 21–44. Prior, L. (2003). Belief, Knowledge and Expertise: The Emergence of the Lay
Expert in Medical Sociology. Sociology of Health & Illness, 25, 41–57. Rose, N. (2018). Our Psychiatric Futures. Polity Press. Sachs, G. (2004). Strategies for Improving Treatment of Bipolar Disorder:
Integration of Measurement and Management. Acta Psychiatrica Scandinavica,
110(S422), 7–17. Schork, N. (2015). Personalized Medicine: Time for One-Person Trials. Nature,
520, 609–611. Segall, A., & Roberts, L. (1980). A Comparative Analysis of Physician Estimates
and Levels of Medical Knowledge Among Patients. Sociology of Health &
Illness, 2(3), 317–334. Sharon, T. (2015). Healthy Citizenship Beyond Autonomy and Discipline:
Tactical Engagements with Genetic Testing. BioSocieties, 10(3), 295–316. Sosnowy, C. (2014). Practicing Patienthood Online: Social Media, Chronic
Illness, and Lay Expertise. Societies, 4, 316–329. Squassina, A., & Pisanu, C. (2013). Personalized Medicine in Bipolar Disorder:
How Can We Overcome the Barriers to Clinical Translation? Personalized
Medicine, 10(8), 765–768. Tanaka, E., & Hisawa, S. (1999). Clinically Significant Pharmacokinetic Drug
Interactions with Psychoactive Drugs: Antidepressants and Antipsychotics
and the Cytochrome P450 System. Journal of Pharmacy and Therapeutics,
24(1), 7–16.h Thomas, P. (2016). Psycho Politics, Neoliberal Governmentality and Austerity. Self & Society, 44(4), 382–393. Thompson, J., Bissell, C., Cooper, C., Armitage, C., & Barber, R. (2012). Credibility and the “Professionalized” Lay Expert: Reflections on the
Dilemmas and Opportunities of Public Involvement in Health Research. Health, 16(6), 602–618. 144 C. Egher Tighe, S., Mahon, P., & Potash, J. (2011). Predictors of Lithium Response in
Bipolar Disorder. Therapeutic Advances in Chronic Disease, 2, 209–226. Versteeg, W., Te Molder, H., & Sneijder, P. (2018). “Listen to Your Body”:
Participants’ Alternative to Science in Online Health Discussions. Health,
22(5), 432–450. Weber, G., Mandl, K., & Kohane, I. (2014). Finding the Missing Link for Big
Biomedical Data. A modified version of this chapter will be published in the special issue “Expertise and Its
Tensions” in Science and Technology Studies. Whereas the chapter focuses on two bloggers—
Natasha Tracy and Julie A. Fast—in the article, the online and offline activities of another
blogger—Charlotte Walker—are also discussed. 4 Tactical Re-appraisals and Digitally Informed Hypotheses… JAMA, 311(24), 2479–2480. Wilcox, S. (2010). Lay Knowledge: The Missing Middle of the Expertise
Debates. In R. Harris, N. Wathen, & S. Wyatt (Eds.), Configuring Health
Consumers. Health Work and the Imperative of Personal Responsibility
(pp. 45–64). Palgrave Macmillan. Wyatt, S., Harris, A., Adams, S., & Kelly, S. (2013). Illness Online: Self-
Reported Data and Questions of Trust in Medical and Social Research. Theory, Culture & Society, 30(4), 131–150.h h
Young, A., & Newham, J. (2006). Lithium in Maintenance Therapy for Bipolar
disorder. Journal of Psychopharmacology, 20(2), 17–22.h Ziegelstein, R. (2015). Personomics. The Journal of the American Medical
Association Internal Medicine, 175(6), 888–889. Open Access This chapter is licensed under the terms of the Creative Commons
Attribution 4.0 International License (http://creativecommons.org/licenses/
by/4.0/), which permits use, sharing, adaptation, distribution and reproduction
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author(s) and the source, provide a link to the Creative Commons licence and
indicate if changes were made.h The images or other third party material in this chapter are included in the
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right holder. © The Author(s) 2023
C. Egher, Digital Healthcare and Expertise, Health, Technology and Society,
https://doi.org/10.1007/978-981-16-9178-2_5 Online Expert Mediators: Expanding
Interactional Expertise Blogs are interesting. They show that humans want to communicate. They
show that we want to share our stories. They also became money making
opportunities and vanity projects that sometimes make me question my
own motives. I am VERY careful about what I share. I am personal without
over-sharing. I’m careful of my brand. I protect it every day. I know what I
write and I know the effect it has on my audience. (Fast, May 8, 2017,
e-mail interview) This is how Julie A. Fast, one of the best-known bloggers on bipolar
disorder and a person who, based on her own admission, has helped
shape this genre, describes this type of interactive online platform and her
engagement with it. This quote is impressive in its honesty and it also
highlights specific opportunities the Internet has contributed to as well as
the need for a particular type of expertise to be able to take advantage of
them. This chapter is dedicated to the study of interactional expertise, 145 © The Author(s) 2023
C. Egher, Digital Healthcare and Expertise, Health, Technology and Society,
https://doi.org/10.1007/978-981-16-9178-2_5 146 C. Egher C. Egher focusing on the activities of two highly successful bloggers diagnosed
with bipolar disorder. Whereas in the previous chapter one of them fig
ured in her function of platform owner, shaping the conditions of possi
bility for other people diagnosed with bipolar disorder to contribute
online, here their personal substantial engagement in the development of
new knowledge will be focused on. In so doing, the relevance of the mul
tiple and shifting positions that stakeholders can occupy in relation to the
development of expertise, which is foregrounded in the new conceptual
ization thereof that I put forward, will be highlighted.i p
g
g
Relations between important stakeholders in the field of mental health
have been significantly transformed by the Internet (Barak & Grohol,
2011). This medium has affected the identity and the type of interactions
between knowledge producers and users (Wyatt et al., 2013), contribut
ing to the diversification of sources of medical knowledge away from
clinical environments (Nettleton, 2004), closer to the everyday settings
of people diagnosed (Lucivero & Prainsack, 2015), and leading to the
re-appreciation of other types of knowledge (Schaffer et al., 2008). Online Expert Mediators: Expanding
Interactional Expertise Such
changes have taken place in a context where pronounced neoliberal ten
dencies have introduced a market logic in the provision of healthcare and
have encouraged individuals to assume responsibility for their health
(Novas, 2006; Rose, 2007). Web 2.0 technologies enable users not only
to consume information but also to engage in its production (Lupton,
2014). Thus, the current dominant imperatives to stay or become healthy
by seeking and sharing health-related information have contributed to
the development of a space where new forms of agency can develop
(Kivits, 2013). Whereas the previous chapter has shown how people can
perform lay expertise and collectively contribute new insights about
bipolar disorder, the focus shifts here to the new entrepreneurial subjec
tivities (Tutton & Prainsack, 2011) that these technologies have contrib
uted to. By studying the activities of two bloggers using Collins and
Evans’ (2002) concept of interactional expertise, I show that through
their skillful use of the Internet, some individual patients have become
highly influential, and argue that this medium has thus helped facilitate
the emergence of a new type of stakeholder—the online expert mediator. In so doing, I expand the notion of interactional expertise by arguing that
it has more of a bi-directional nature than Collins and Evans (2002) and 5 Online Expert Mediators: Expanding Interactional Expertise 147 Collins et al. (2017) assume and that it is important to consider the
effects of the medium through which it is performed. But first, let us
consider how the role of patients in mental health has changed over the
last few decades, focusing on the Internet’s influence in these
transformations. Greater Mental Health Patient Engagement
and the Internet As many medical sociologists have indicated, since the last few decades of
the twentieth century patient engagement has been promoted in differ
ent areas and for different goals (Barello et al., 2014; Turner, 1995),
through top-down processes (Godfrey et al., 2003; Hogg, 2009) or as a
result of grassroots activities (Barbot & Dodier, 2002; Kushner, 2004;
Landzelius, 2006; Novas, 2006; Rabeharisoa et al., 2013; Taussig et al.,
2003). The meaning and consequences of patient engagement vary
(Hickey & Kipping, 1998; Rowland et al., 2017), yet, as the findings in
the previous chapter show, people diagnosed have also come to grasp the
conditions of complexity and uncertainty under which medical profes
sionals operate, leading to a growing awareness of the limits of medical
expertise. These realizations have had a profound resonance in mental
health, where the authority of medical professionals has been challenged
since the late 1960s (Pickersgill, 2012), in manners which were discussed
in more detail in the introductory chapter. Combined with official
restructuring initiatives and considerable openness among people diag
nosed toward new approaches and types of knowledge, such challenges
have contributed to the proliferation and diversification of mental health
professionals (Brown, 1988; Grob, 2005). The relations between existing
stakeholders have thus been modified, and the role of patients has
changed from passive recipients of care (Barnes & Shardlow, 1997) to
consumers who feel entitled to choose the type of care they receive
(McLean, 2000). While some patients consider themselves survivors and
actively militate against medical conceptualizations and interventions
(Crossley & Crossley, 2001; Speed, 2006; Whitley, 2012), many others 148 C. Egher C. Egher have engaged in processes of knowledge production (Gillard et al., 2012;
Kemp, 2010), evaluation (Director, 2005), and implementation
(Davidson, 2005), thereby acquiring a greater role in mental health.f y
q
g
g
People diagnosed have used the Internet for different types of epis
temic engagements. Some patients have used the knowledge thus acquired
to question and/or challenge the expertise of medical professionals in
several ways (Fox et al., 2005; Gowen et al., 2012; Mulveen & Hepworth,
2006; Orsini & Smith, 2010). Others have engaged in various scientific
activities, ranging from monitoring themselves using self-tracking devices
and sharing their data with others to using collaborative platforms, such
as PatientsLikeMe, to test medical hypotheses (Kallinikos & Tempini,
2014). Greater Mental Health Patient Engagement
and the Internet Through their use of the Internet, such “citizen scientists” or
“health hackers” have gone beyond the mere provision and exchange of
medically interesting information, connecting with other people with the
same diagnosis to “conduct clinical trials on their own diseases” (Bottles,
2013:88), thereby enacting particular values and ideals of patienthood
(Sharon, 2017). Such online opportunities have been all the more impor
tant in the field of mental health, where study participation has tradition
ally been difficult, as the symptoms of people diagnosed often rendered
their adherence to specific interventions problematic, while the desire to
avoid stigmatization made them reluctant to attend face-to-face meetings
(Naslund et al., 2015). Used in mental health since its early days, the Internet has importantly
shaped the participation of people diagnosed in knowledge production. Already in 1999, Barak (1999: 231) noted that “the rapid developments
in computers and information technology over the past decade have had
an impact on psychology, which has moved (…) from local computer
applications to network applications that take advantage of the Internet.”
By now, numerous studies have indicated the potential (Barak et al.,
2008; Carlbring & Andersson, 2006; Proudfoot, 2004; Smith et al.,
2011) and variety of online interventions for mental health (Barak &
Grohol, 2011; Kraus et al., 2010; Marks et al., 2007; Ybarra & Eaton,
2005). Bipolar disorder is among the mental health conditions affected
by such approaches, as various online therapies and different types of
mobile phone applications have been developed (Nicholas et al., 2015). 149 5 Online Expert Mediators: Expanding Interactional Expertise There are important differences in approach, motivation, and goals
among patient organizations focusing on the same condition (Barbot,
2006) and even among members of the same group (Epstein, 1996). The
Internet has helped render more visible the heterogeneity of bipolar
patients, as various online platforms testify to their different needs and
preferences. It has also contributed to the emergence of new types of
involvement for people diagnosed with bipolar disorder, by diversifying
the range of stances at their disposal. By using the Internet, they have
been able to develop new skills and to acquire various resources. This has
not only rendered bipolar patients more salient stakeholders, but it has
also contributed to a diversification of the type of stakeholdership they
could take up. Since the emergence of surveillance medicine in the twentieth century
(Armstrong, 1995), and particularly after the adoption of a consumerist
culture in healthcare (Lupton, 1995), individuals have been encouraged
to engage in self-surveillance practices and to actively manage their health
by staying informed. The development of digital technologies has con
tributed to the diversification and intensification of these tendencies
(Kopelson, 2009), but has also “promoted the individual expression of a
personal experience of health” (Kivits, 2013:222), as people have been
increasingly exhorted not only to seek information but also to share per
sonal insights. Thus, the Internet has enabled not only patient groups,
but also individuals to become influential by achieving high levels of vis
ibility and by acquiring numerous readers. While most researchers have
studied the changing identity and growing influence of patients as a result
of collective actions, several academic works have highlighted the impor
tance of particular individuals in shaping the character of patient organi
zations and of their interactions with medical professionals (Klawiter,
1999; Lerner, 2001). This chapter makes a contribution in this sense, by
showing that some individual patients have become highly influential in
mental health by taking advantage of some of the opportunities gener
ated by the development of Web 2.0 platforms in the context of growing
tendencies to responsibilize individuals for their health (Nettleton, 2004). Among the multiple forms of self-expression the Internet has enabled,
illness blogs represent a highly popular genre (De Boer & Slatman, 2014). Among the multiple forms of self-expression the Internet has enabled,
illness blogs represent a highly popular genre (De Boer & Slatman, 2014). Given their popularity, malleable architecture, and primarily individual 150 C. 5 Online Expert Mediators: Expanding Interactional Expertise Egher character, blogs represent an excellent site to study the activities, knowl
edge practices, and alliances through which individuals achieve an influ
ential position. Illness blogs are a specific type, as they “are used to express
the experience of illness and to connect with readers via the internet”
(Heilferty, 2009:1542). They differ based on their design, accessibility,
and interactive character, and it is the more or less skillful combination of
affordances related to these aspects that largely determines a blog’s
standing. Two Bloggers on Bipolar Disorder On December 3, 2016, an online search using the keywords “bipolar
blog” generated 12,600,000 results on Google and 6,870,000 on Yahoo. Regardless of the search engine used, the blog of Natasha Tracy, which
was discussed from a different perspective in the previous chapter, and of
Julie A. Fast came up on the first page of results, either directly or men
tioned under rubrics such as “the best bipolar blogs of the year” on several
health platforms. They are thus likely to come to the attention of many
Internet users, especially since both of them can be accessed freely by
readers. Each of these bloggers has been diagnosed with bipolar disorder for
about two decades. Tracy is an award-winning mental health speaker and
writer on topics such as bipolar disorder, depression, pharmacology, and
other mental health–related issues. She has authored three blogs—Break
ing Bipolar, Bipolar Burble, and Bipolar Bites—and has been a contributor
to the Huffington Post. Her blogs attract large numbers of visitors, and
many of her posts receive hundreds of comments. Fast is “a world leading
mental health expert on the topics of bipolar disorder, depression, sea
sonal affective disorder, personality disorders and mood management.”
She states that her site and blog together have been visited by one million
visitors. Unlike Tracy, her personal blog, Bipolar Happens!, only gathers a
very modest number of comments (< 10), but there is significantly more
interaction on her blog on the bipolar disorder “Hope” magazine web
site, Fast Talk. Fast also works as a “bipolar disorder management special
ist” at Share.com, the website created by Oprah and Dr. Oz. To reach Each of these bloggers has been diagnosed with bipolar disorder for
about two decades. Tracy is an award-winning mental health speaker and
writer on topics such as bipolar disorder, depression, pharmacology, and
other mental health–related issues. She has authored three blogs—Break
ing Bipolar, Bipolar Burble, and Bipolar Bites—and has been a contributor
to the Huffington Post. Her blogs attract large numbers of visitors, and
many of her posts receive hundreds of comments. Fast is “a world leading
mental health expert on the topics of bipolar disorder, depression, sea
sonal affective disorder, personality disorders and mood management.” She states that her site and blog together have been visited by one million
visitors. Two Bloggers on Bipolar Disorder Unlike Tracy, her personal blog, Bipolar Happens!, only gathers a
very modest number of comments (< 10), but there is significantly more
interaction on her blog on the bipolar disorder “Hope” magazine web
site, Fast Talk. Fast also works as a “bipolar disorder management special
ist” at Share.com, the website created by Oprah and Dr. Oz. To reach 5 Online Expert Mediators: Expanding Interactional Expertise 151 audiences of different ages, in recent years, Fast has also actively engaged
in the provision of information about bipolar disorder on Instagram and
Facebook. While Internet users have been studied as health-related information
seekers and/or producers, less attention has been paid to their potential as
information mediators. Illness blogs are important mediation sites, as
experiential knowledge is combined with medical, pharmaceutic, and
socioeconomic information. These bloggers function as mediators, as
they “transform, translate, distort, and modify” (Latour, 2005:39) the
information they share in order to adapt it to the opportunities and limi
tations of the medium and to the requirements of different audiences
(Wathen et al., 2008). Importantly, the development of this new stake
holder category occurs in a context where patient experiences have come
to be valued, elicited in various ways online, and, subsequently, com
modified (Adams, 2013; Lupton, 2014; Mazanderani et al., 2012). I
argue that through their practices and collaborations with different stake
holders, these two bloggers move beyond the role bipolar patients gener
ally have in the field of mental health, and turn themselves into a new
type of stakeholder—the online expert mediator. Theoretical Approaches
to Interactional Expertise The online activities of these bloggers are analyzed instead using the con
cept of interactional expertise (Collins & Evans, 2002), which bridges
the divide between practical, experiential, and scientific knowledge. This
notion is particularly useful, because it allows me to identify people
endowed with substantial knowledge but missing official credentials, and
provides an appropriate explanatory framework when studying phenom
ena “involving different expert communities” (Collins et al., 2017: 782). While contributory expertise denotes one’s ability to contribute produc
tively to a field (Collins & Evans, 2007), interactional expertise “is exper
tise in the language of a specialism in the absence of expertise in its
practice” (emphasis in the original) (Collins & Evans, 2007: Loc. 520). 152 C. Egher C. Egher This means that people endowed with interactional expertise have spe
cialist tacit knowledge and they understand the language of a domain of
practice and can engage in interesting conversations with those with con
tributory expertise in that domain, but they cannot make practical con
tributions to it. Thus, people may acquire interactional expertise through
immersion in a field while following a different trajectory than contribu
tory experts (Collins et al., 2006). Interactional expertise is also highly
specific: just like contributory experts in a field can contribute success
fully only in some areas, interactional experts can be more competent
about particular subdomains of a field. Furthermore, the acquisition of
interactional expertise enables people to function as mediators between
contributory experts in a field and the group(s) they represent. i
Collins and Evans (2015, 2017) have studied interactional expertise
using the Imitation Game, which is an adaptation of the test Alan Turing
developed to assess the intelligence of computers. In so doing, they resist
calls to expand the initial definition of interactional expertise in ways
which they believe would diminish its “real” character. Nevertheless, in
this chapter I follow the lead of scholars who have argued for a broaden
ing of the way in which this concept is understood (Goddiksen, 2014). I
thus take up Plaisance and Kennedy’s (2014) recommendation to study
interactional expertise by considering the “fruitful” contributions people
endowed with it can bring to a field due to “the various profiles that inter
actional experts can have as a result of who they are, why they’ve sought
to acquire IE [interactional expertise], and how they make use of it”
(Plaisance & Kennedy, 2014:65). Theoretical Approaches
to Interactional Expertise In so doing, I extend interactional
expertise by considering the effects of taking seriously the medium
through which it is displayed and I show that it has more of a bi-directional
character than Collins and Evans had envisaged.f I argue that there are important differences between the activities peo
ple can engage in and the approaches that they can choose from to dis
play interactional expertise, depending on the medium they use. While
some people may be able to understand their interlocutors better and
express themselves more eloquently during face-to-face encounters, they
may have a more difficult time displaying their interactional expertise 5 Online Expert Mediators: Expanding Interactional Expertise 153 convincingly via the telephone, in writing, or online. Furthermore, each
medium might bring them in touch with different audiences, with differ
ent criteria for assessing credibility, different expectations, and informa
tional needs. By studying the activities of Tracy and Fast on different
online platforms, I identify various ways in which these bloggers make
use of online affordances in order to successfully deploy interactional
expertise and thereby establish themselves as authoritative figures in
the field. i
These two bloggers were identified using the Google index as a rele
vance indicator, as I aimed to mimic the approach of regular users. Data
were collected between July 2014 and September 2018 and initially con
sisted of bloggers’ posts about the treatment of bipolar disorder and
information provided under the “about” rubric of every blog. To acquire
a better understanding of the bloggers’ standing and other public activi
ties, additional online queries were subsequently conducted, using the
bloggers’ names as search terms in the search engine Google. The search
“Natasha Tracy” generated 19,600,000 results, while “Julie A. Fast” 349
million. The biographical and social data were collected from the first ten
pages of results. I also conducted an e-mail interview with Julie A. Fast
and an online interview with Natasha Tracy using the Skype telecommu
nications application. I performed thematic analysis of all the texts col
lected, including hyperlinks and images, by identifying important themes
through repeated readings (Lupton, 1995). I operationalized interac
tional expertise based on Collins and colleagues’ approach (2006) into
three main dimensions: linguistic fluency in the field of medical knowl
edge about bipolar disorder; ability to evaluate and distinguish between
medical professionals; and ability to provide practical advice about rele
vant matters in the field. Theoretical Approaches
to Interactional Expertise Given the aim of expanding the notion of inter
actional expertise, the following aspects were additionally focused on
how and when bloggers invoked and displayed medical knowledge; the
bloggers’ relations with medical professionals; the alliances they forged;
elements conveying the bloggers’ standing; and the bloggers’ use of online
affordances. In what follows, I discuss how these bloggers have turned
themselves into online expert mediators. 154 C. Egher Tracing the Development of a New
Stakeholder Category Technical Prowess 5 Online Expert Mediators: Expanding Interactional Expertise I say it was a perfect storm because on the exact month that I started my
webpage, Google started something new called ADWORDS. I was one of
their first customers. I created an ADWORDS account and started to
advertise my treatment plan from the first week it was online. This was
perfect timing. Believe me, so much of what happens online is LUCK. Yes,
I was prepared and I had a truly great product, but the timing was perfect. Often, you have to be in the right place in order to adapt new technology. (Fast, May 8, 2017, e-mail interview) In this blogger’s view, therefore, her considerable influence is partly
due to her innovative approach and the active role she played in the
development of the field of self-help e-books from its inception.i i
Both bloggers credit their success to the specific character of online
communication and digital technologies, which allowed them for advance
planning and for the content they created to be made available to their
audiences regardless of their health state at any given moment in time. Fast mentioned, “I was sick a lot- so having an internet business was a
miracle for me. I could be sick and still sell my books” (Fast, May 8,
2017, personal communication), and argued that “[a]n internet business
is the ONLY business I can do considering my brain limitations” (Fast,
May 8, 2017, e-mail interview). At the same time, the bloggers empha
sized that different online platforms afford varying degrees of control and
power and that appropriate skills have to be acquired to use each type of
platform to one’s benefit: THEN, Facebook happened. I can’t tell you enough how this changed
everything. MySpace simply couldn’t do what Facebook did. Facebook
made talking about yourself very easy. I had a love/hate relationship with
Facebook for many years. I was bullied a lot and didn’t know how to con
trol the flow of information. Webpages and blogs are safe spaces- the author
controls who says what. Facebook was a free for all. It was amazing and
destructive at the same time. I now know exactly how to use it, but it’s an
art. I can say the same for Twitter. Technical Prowess A first characteristic of online expert mediators is their endowment with
or access to substantial technological skills. Fast and Tracy managed to
become online expert mediators because they were among the first to
realize the Internet’s potential and to understand how much people diag
nosed with bipolar disorder needed their insights. While Tracy has a
degree in computer science and used to work for Microsoft, Fast’s long-
term partner at the time when she started sharing her perspectives on
bipolar disorder online was a gifted programmer. She also mentioned
that both of them were technology enthusiasts. In the e-mail interview I
conducted with her, Fast stated that her “internet career was a perfect
storm of events” (Fast, May 8, 2017, e-mail interview) and noted the role
that various technologies and her partner’s abilities to make use of them
played in her career: In that EXACT moment in the spring of 2002, I had the idea that I could
take my first two books and sell them as download books. No one was
doing this except a few guys who were selling sales tools and real estate
guides. I found NO books on the internet about any psychology topic or
even any self help topics. I knew I had a good idea. (…) We spent the next
month building a website to sell my two books. I turned my manuscripts
into PDF files and because he was a programmer and a computer genius-
we were able to build something that hardly existed at the time. An ebook
website! The books were Bipolar Happens! and my Health Cards Treatment
System for Bipolar Disorder. I wrote a home page- Ivan created links for
people to buy the books through something called a SHOPPING CART
and the business was born. It wasn’t that long ago, but can you believe that
the words Ebooks and shopping cart were so new, we were not even sure
what they meant. My business helped define the process. I was the first
person in the world to sell a psychology or self help ebook online. (my emphasis) 155 5 Online Expert Mediators: Expanding Interactional Expertise 5 Online Expert Mediators: Expanding Interactional Expertise (Fast, May 8, 2017, e-mail interview) To render their online undertakings successful, the bloggers also had to
call upon different types of knowledge and make wise decisions about the
involvement of other professionals. Just as the Internet is multiple, so are 156 C. Egher C. Egher the skills necessary to use it in one’s favor, and developing such insights
requires time and numerous other resources that few people may have at
their disposal. Reflecting on the important role of website statistics in her
online activities, Fast stated: It’s important to know that the internet is incredibly ALIVE- we do things
that you can’t do in a regular business…So many internet business owners
make decisions that are creative instead of business oriented and creative. You need both. Creative people who don’t really like looking at stats tend
to just post and hope for the best. In reality, a successful internet entrepre
neur always has the business in mind and must look at stats from every
source possible. It’s an ever changing environment, but stats are always the
friend of an online business. You can hire someone to do this, but you need
to understand it yourself first. (Fast, March 20, 2021, personal
communication) Next to statistics, timing and the early development of relevant rela
tionships seem to have been of great importance for the online careers of
these bloggers. Fast’s early start using one particular online technology
enabled her to be among the first to embrace many others, which allowed
her to increase her online visibility: “I also feel… these three sites [her sales
website, her PR page, and her blog] have helped with Google rankings” (Fast,
May 23, 2017, personal communication). From this other point of view,
these online technologies also contributed to important inequalities, as
her status as a successful early adaptor provided her with more authority
and influence than people who started using them later, thereby enabling
her, based on her own admissions, also to shape the genre of illness blogs. Interactional Expertise Next to technical skills, Fast and Tracy also needed to develop and per
form interactional expertise about medical knowledge on bipolar disor
der to successfully function as online expert mediators. The display of
linguistic fluency in a field is the main mark of people endowed with
interactional expertise (Collins & Evans, 2002). While Tracy and Fast are
not medical professionals, nor did they study medicine, the many years 5 Online Expert Mediators: Expanding Interactional Expertise 157 since they have been diagnosed with bipolar disorder, the multitude of
treatments they have tried, and the great variety of professionals they
have consulted have provided them with ample opportunities to observe
the practices of the medical community. Furthermore, their own proac
tive attitudes have enabled them to deepen their medical knowledge
about bipolar disorder. These bloggers display their linguistic prowess
throughout their posts and interactions with commentators, as they
explain medical phenomena using a more accessible vocabulary and pro
viding examples, they give advice about the most appropriate therapeutic
approaches depending on one’s symptoms and/or life circumstances, and
they are aware of the latest developments in the field. The excerpt below
is illustrative of such activities: Drug tolerance is also known to occur upon drug-discontinuation. In
other words, someone who has previously responded well to lithium dis
continues the drug, symptoms reemerge, the person goes back on lithium
but does not find it effective. Again, we don’t know why this occurs but it
does appear to in a small percentage of patients. In one study, it occurred
in 13.6 percent of people taking lithium. Warning, this is a preclinical study and as such the implications from it
may not be fully understood. Please make sure to make any medication
changes only with doctor oversight. For more information please see the
study Tolerance to the Prophylactic Effects of Carbamazepine and Related
Mood Stabilizers in the Treatment of Bipolar Disorders [hyperlink pro
vided]. (Tracy, Bipolar Bites, May 30, 2012) This quote indicates Tracy’s position as mediator between medical pro
fessionals and bipolar patients, a position which I argue is characteristic
for this new type of stakeholder. While it may be that it refers to the level
of knowledge available to the whole of humanity, the use of “we” in a
context where study results are discussed suggests that Tracy sees herself
more as a member of the medical community. Interactional Expertise At the end of the post,
however, she reclaims her subordinate position to medical professionals,
while by sharing the source she used, Tracy reveals her awareness of the
need to legitimize her claims. 158 C. Egher C. Egher Mediators importantly transform the meaning of the information they
transmit, and this is obvious in the posts authored by both bloggers. While they convincingly use medical vocabulary, they do so in particular
ways. For instance, in a manner which reiterates the tactic of individual
ization discussed in the previous chapter, Tracy puts forward her own
reading of personalized medicine, as on numerous occasions she seems to
believe that each person displays an individual mix of symptoms and
reacts differently to treatment, as the quote below illustrates: And if 99 people say the med is bad, but 1 says it’s good, what benefit is
that? Should the patient not try it? Should the patient assume the med
won’t work or will have too many side effects? The 99:1 ratio essentially
means nothing because we’re all different. (Tracy, Breaking Bipolar,
June 30, 2011) Furthermore, Tracy often uses statistics and results obtained through
randomized controlled trials to support her claims. This shows that she
makes strategic choices about the ways in which she refers to medical
information, an approach previously identified among patient organiza
tions (Treichler, 1999). This rather complicated balancing act is necessary
as it allows her not to alienate readers with experiences different from the
ones she describes while maintaining her authority. At the same time, it
enables her not to stray too far from the prevailing medical consensus,
thereby retaining her ties with the medical community.h The bloggers display their linguistic prowess also by distinguishing
between different medical professionals in the field of bipolar disorder,
and they often criticize the prescription habits of general practitioners, as
the quote below illustrates: Interestingly, many fewer people being treated by bipolar disorder experts
are on antidepressants: Interestingly, many fewer people being treated by bipolar disorder experts are on antidepressants: are on antidepressants: • Treated by community psychiatrists—80 percent of patients are on
antidepressants • Treated by mood disorder clinics—50 percent of patients are on
antidepressants • Treated by mood disorder clinics—50 percent of patients are on
antidepressants 1 https://natashatracy.com/bipolar-and-mental-health-resources/. Accessed on May 13, 2016. 5 Online Expert Mediators: Expanding Interactional Expertise 159 5 Online Expert Mediators: Expanding Interactional Expertise • Treated by specialty bipolar clinics—20 percent of patients are on
antidepressants • Treated by specialty bipolar clinics—20 percent of patients are on
antidepressants So it would seem that the more specialized the care, the more profession
als recognize the concerns over antidepressants. (Tracy, Breaking Bipolar,
July 10, 2013) So it would seem that the more specialized the care, the more profession
als recognize the concerns over antidepressants. (Tracy, Breaking Bipolar,
July 10, 2013) As such views are expressed in posts where they provide the latest
insights into a particular treatment, it would appear that these bloggers
position themselves as more up to date than some medical professionals. Collins and Evans’ (2002) conceptualization of expertise is based upon
the idea that no contributory expert is equally competent in all areas
pertaining to a particular domain. From this point of view, it remains
open for debate whether these online contributions are attempts to fill
relevant epistemic gaps or whether they represent interventions through
which the bloggers challenge the authority and standing of medical pro
fessionals who are lower positioned than specialists and scientists, for
instance. This ambiguity is further exacerbated by the fact that such online com
ments are balanced by entries where Tracy and Fast warn readers about
their lack of medical credentials and take up a complementary function
to medical professionals. They try, for instance, to prevent people from
quitting their medication when scandals related to pharmaceutical com
panies emerge. Fast even depicts herself (and people diagnosed) as useful
allies, helping doctors identify dishonest claims made by pharmaceutical
companies through their experiential knowledge of the effects and side
effects of medications (Fast, Bipolar Happens!, October 16, 2016). Furthermore, multiple entries (Tracy, Breaking Bipolar, July 5, 2012)
show that through their immersion in the community of medical profes
sionals, these bloggers have also become familiar with the political econ
omy of the pharmaceutical industry.l Another way in which they display their fluency in medical knowledge
is by evaluating the merits of various studies and by distinguishing
between medical information based on its source. In so doing, Tracy and
Fast often clarify the status of the knowledge on bipolar disorder cur
rently available and the inferences that can be made on it, as the follow
ing excerpt indicates: 160 C. Egher 5 Online Expert Mediators: Expanding Interactional Expertise 161 5 Online Expert Mediators: Expanding Interactional Expertise C. Egher It’s time to get clear on what we really know about brain scans and #bipo
lar. It’s so frustrating to read articles and studies about bipolar and brain
imaging. At this time, there is NO brain image scan for the diagnosis of
bipolar. Please do not pay someone who tells you that they can determine
bipolar from an MRI or PET scan. It simply isn’t true. This is nascent science. One study shows some grey matter thinning in
3000 patients, another shows ‘abnormal’ activity in the amygdala and fron
tal lobes. There is nothing definitive and even if someone did find a change
in the brain, without having a management plan that works, the informa
tion is just that… information. (Fast, Bipolar Happens!, July 18, 2018) Fast performs interactional expertise by showing her familiarity with
medical technology and terminology, and by being able to distinguish
hopes and visions from the current relevance of brain scans in the diag
nosis of bipolar disorder. Concerned about the quality of information
that people diagnosed may acquire even from medical sources, Fast posi
tions herself as a mediator, by using her own knowledge in order to cor
rect erroneous assumptions and expectations. The bloggers also perform
interactional expertise through their careful selection of the sources of
information they use in their posts, as the excerpt below shows: 1 Through their online posts, Tracy and Fast thus show that they have
become fluent in the language of medical professionals and have there
fore successfully developed interactional expertise. A Strong Media Presence While important, having (access to) considerable technical skills and
developing interactional expertise are not sufficient for these bloggers to
become online expert mediators. To function as successful mediators
between medical professionals and people diagnosed, Fast and Tracy not
only require ample opportunities to perform interactional expertise, but
they also need a strong media presence, to increase their public visibility
and standing. The two bloggers have achieved this by developing close
relations with mass media outlets. Tracy has often been interviewed and
has participated in documentaries about bipolar disorder. In 2008, Fast
hosted a weekly radio program, The Julie Fast Show, on KTRO in
Portland, during which she had a number of “special guests,” medical
professionals or people diagnosed with various mental health conditions,
who often wrote about their experiences and participated in advocacy
actions. She is regularly interviewed on diverse mental health issues, such
as pop artist Britney Spears’ nervous breakdown and actress Carrie Fisher’s
death, and writes on mental health in magazines such as People and US
Weekly. Fast was also the original consultant for the character played by
Claire Danes, the main protagonist who suffered from bipolar disorder in
the popular drama series Homeland. Fast and Tracy have also published
books about their experiences with bipolar disorder, thereby further
extending their reach. In 2016, Tracy wrote Lost Marbles: Insights into My
Life with Depression and Bipolar. Fast is the author of five books, which
have sold over 250,000 copies, four of which are “on the Amazon.com
mood disorder bestselling book list” (Fast, Bipolar Happens!, 2016).h Through such activities, the bloggers also reach broader audiences than
bipolar patients and their families, thereby contributing to how bipolar
disorder and other related conditions are understood by the general pub
lic. As they become more familiar with other media, these bloggers can
use their skills for more political purposes, as they may generate public
sympathy, emphasize the urgency of particular pieces of legislation or
treatment provisions, or put forward more complex images of life with
bipolar disorder. In so doing, Tracy and Fast expand their mediation
work beyond the more immediately responsive online medium, 162 C. Egher C. Egher translating, synthesizing, bringing together, and refining different types
of knowledge about bipolar disorder in formats in which interaction is
more difficult, takes more time, and occurs more frequently away from
the public. A Strong Media Presence Yet, it is precisely through their ability to use different media
and to retain a coherent image across them that these bloggers further
increase their influence and standing.h l
That their standing goes beyond that of the average blogger is indi
cated by the numerous awards Tracy and Fast have received. Tracy received
the Beatrice Stern Media Award and the #ErasingtheStigma Leadership
Award and has been listed as the fourth Health Maker in the top ten
online influencers in the area of mental health by Sharecare.com. She was
also a speaker at the National Council on Mental Health and Addictions
Conference and is hailed as one of the “heroic” figures of people diag
nosed with bipolar disorder. Another indicator that her reach goes beyond
the small circle of family and friends of regular illness bloggers is the fact
that she has been a contributor on health platforms and a subject matter
expert on bipolar disorder at Answers.com, all of which have millions of
visitors. Fast received the Mental Health American Journalism award for
the Best Mental Health Column in the US. Furthermore, the blogs Tracy
and Fast have authored have been voted many times among the best
bipolar blogs. Whereas Fast and Tracy’s activities across multiple media help them
acquire greater audiences, the distinctions they received function as refer
ences or recommendations. Consequently, they can use their public visi
bility and standing as important resources to facilitate the development
of more varied and substantial collaborations, not only with people diag
nosed, but also with medical professionals. While this is an important
characteristic of online expert mediators, in the next part of the analysis I
use these insights to make a theoretical contribution, by expanding the
notion of interactional expertise. I do so by arguing that interactional
expertise has a stronger bi-directional nature than Collins and Evans
assume and that the effects of the medium through which interactional
expertise is performed need to be taken seriously. 163 5 Online Expert Mediators: Expanding Interactional Expertise Substantial Interactions and Bi-directionality Bi-directionality refers to the ability of people endowed with interac
tional expertise to function as mediators between others with the same
kind of contributory expertise as they and with individuals who have
contributory expertise in the field where they hold interactional exper
tise. Whereas bi-directionality is an important aspect of interactional
expertise, Collins and Evans do not sufficiently theorize it. For Collins
and Evans (2002), interactional experts translate the scientific practices
of contributory experts in one field for people with contributory exper
tise in another field, and shape the knowledge contributory experts pro
duce by questioning some of their practices or by making them aware of
other perspectives on an issue of interest. Thus, Collins and Evans see
interactional experts as providing contributory experts with sources of
inspiration. Whereas they see such exchanges as taking place in both con
ditions of symmetry and asymmetry, in the latter case, they only seem to
conceive of one direction for the acquisition of interactional expertise, as
I explain below. Collins and Evans (2002) do not provide much information about the
acquisition of interactional expertise in conditions of symmetry, but they
suggest that it occurs between experts who may find themselves equally
well positioned in order to productively contribute to the solution of a
certain problem. In such conditions, Collins and Evans (2002) state that
any of the two groups may absorb the expertise of the other one by devel
oping interactional expertise. One may imagine such a situation occur
ring, for instance, as two different types of medical professionals are
consulted for the treatment of a difficult case. Which one of the two
specialists takes charge and oversees the patient’s treatment is “arbitrary”
from Collins and Evans’ point of view, as long as one of the doctors has
or develops interactional expertise into the other medical field, in order
to be able to make informed decisions about the therapeutic approaches
based on relevant insights from both medical fields. While in this 164 C. Egher C. Egher example, the two contributory experts need to solve a common problem,
Collins and Evans (2002) also give examples of situations where interac
tional expertise is developed by experts to address their own specific goals. For instance, they often invoke sociologists, who need to become fluent
in the practice language of the scientific group they study, to be able to
successfully conduct a sociological analysis. Substantial Interactions and Bi-directionality In such cases, however,
Collins and Evans (2002, 2017) conceive of the development of interac
tional expertise as the responsibility of the group doing the study or need
ing to solve a specific problem. i
Collins and Evans (2002) tie the development of interactional exper
tise under conditions of asymmetry to instances when this type of exper
tise is needed to facilitate the integration of a certain (sub)type of
contributory expertise into another, broader, form of contributory exper
tise, with which the first is continuous, for the satisfactory resolution of a
complex problem. They give examples both of situations when third par
ties are involved and of instances when the integration of one type of
expertise into the other occurs without external involvement. In the first
case, Collins and Evans (2002) mention that such absorption may be
mediated by people who are not contributory experts in any of the two
fields, but who hold interactional expertise in both. For instance, a soci
ologist may translate the knowledge and perspectives of a (smaller) group
of unaccredited and less influential experts into a language that the
accredited experts can understand, in order to appreciate and be able to
use the insights of the unaccredited experts to solve a common problem. When discussing the development of interactional expertise under
conditions of asymmetry, Collins and Evans (2002) tend, however, to
ascribe the task or capability of acquiring interactional expertise to the
group of experts which are better positioned to solve a certain problem
because of the epistemic authority, legitimacy, and other resources they
already enjoy. Thus, they provide a new reading of Wynne’s (1992) study
on the relationship between scientists and the Cumbrian farmers in the
aftermath of the Chernobyl disaster, where the scientists failed to recog
nize the contributory expertise of the farmers, who lacked official accredi
tation. Collins and Evans (2002:255) state that for the farmers’ insights
to be taken seriously, the latter “would not have had to engage in a sym
metrical conversation” (emphasis in the original), but the scientists would 5 Online Expert Mediators: Expanding Interactional Expertise 165 have had to be willing to incorporate the former’s insights by developing
the relevant interactional expertise. Another example testifying to the
same perspective is that of medical professionals becoming interactional
experts on the lived experiences of their patients, which Collins et al. (2017) more recently invoked. Substantial Interactions and Bi-directionality Importantly, in Collins and Evans’
(2002:256) view, “only the party with interactional expertise can take
responsibility for combining the [contributory] expertises.”h p
y
g
y
p
This argument suggests that under conditions of asymmetry, Collins
and Evans believe the more influential party has the necessary resources
to develop interactional expertise. I argue, however, that interactional
expertise can not only develop in a bottom-up direction, whereby stake
holders who already enjoy epistemic authority in a given field expand
their expertise by absorbing knowledge from unofficially recognized con
tributory experts in a (sub)field of interest, but it can also be acquired in
what may seem like a top-down manner. Thus, people who have con
tributory expertise in a field but are not officially accredited can become
fluent in the practice language of relevant epistemic groups, if they are
endowed with other necessary resources. The acquisition of interactional
expertise might this way contribute toward the development of more
symmetric relationships between people who enjoy different standing
due to the status of the field in which they have contributory expertise. As the description below will show, even under conditions of asymmetry,
people endowed with interactional expertise can engage in exchanges that
are more substantial than having interesting conversations with contribu
tory experts, as Collins and Evans (2002) believe. Furthermore, whereas
in determining the (a)symmetric character of an interaction, Collins and
Evans seem to focus mainly on epistemic standing and authority as the
determining factors, I argue that the availability of other resources may
help balance such exchanges. More attention should therefore be paid to
the type and quality of the interactions between interactional and con
tributory experts in different fields and to how different conditions or
types of asymmetry may affect them. The exchanges of the bloggers stud
ied here are revelatory in this sense. One of the challenges encountered by researchers interested in collabo
rating with patients is to enable their contributions (Hewlett et al., 2006). This is an area where online expert mediators engage in mediation work, 166 C. Egher C. Egher as they succeed to develop a space where their readers can articulate their
experiences and negotiate how they position themselves in relation to
their condition and the medical community. The bloggers educate people
diagnosed about medical terminology and perspectives, so that the latter
are better able to engage in collaborative projects with researchers. Substantial Interactions and Bi-directionality This is
important, because not all bipolar patients may have the time and be in
the physical and mental state necessary to grapple with medical terminol
ogy and research methodology. Moreover, Tracy and Fast may provide
people diagnosed with the confidence that the insights they have are rel
evant and valuable, thus enabling them to interact with medical profes
sionals with the assurance and determination necessary to move toward
more equal exchanges. They may also help those interested in research
participation to develop the patience and distance necessary to accept
results, which may contradict their personal views. Next to bipolar patients, the bloggers have constituted themselves as
valuable allies for medical professionals who lack but need their insights
derived from the lived experience with bipolar disorder for various aims. Thus, online expert mediators can assist medical professionals to acquire
interactional expertise regarding the embodied experience of bipolar dis
order, and thus help them develop a broader perspective about this con
dition and novel research ideas. By positioning themselves as representatives
of their bipolar readers, the bloggers provide medical professionals with
important information regarding the research directions people diag
nosed with bipolar disorder would find relevant. In a context where med
ical expertise continues to be challenged, online expert mediators further
serve the interests of the medical community by bestowing additional
credibility upon the scientific approaches they champion.hfi i
The bloggers have also acquired sufficient medical knowledge and
other relevant resources for medical professionals to agree to collaborate
with them. Tracy noted that in my role I’ve been lucky enough to meet some really great professionals,
some really great psychiatrists and researchers in their own right. And
because I’ve gotten to know them through various acquaintances and
through various things that I’ve done, if I want to do something that is
scientific in nature, which I have done, then I can actually approach them in my role I’ve been lucky enough to meet some really great professionals,
some really great psychiatrists and researchers in their own right. And
because I’ve gotten to know them through various acquaintances and
through various things that I’ve done, if I want to do something that is
scientific in nature, which I have done, then I can actually approach them 5 Online Expert Mediators: Expanding Interactional Expertise 167 and say: “Look, I really have an interest in doing this. Substantial Interactions and Bi-directionality Do you have an
interest in supporting this?” And we can work together to make it happen. (Tracy, March 29, 2021, online interview) and say: “Look, I really have an interest in doing this. Do you have an
interest in supporting this?” And we can work together to make it happen. (Tracy, March 29, 2021, online interview) For instance, together with Prakash Masand, M.D., Tracy wrote an
article published in 2014 in the medical journal The Primary Care
Companion for CNS Disorders. Furthermore, in July 2016, she initiated a
survey about patients’ experiences concerning electroconvulsive therapy
(ECT) on her personal blog: My name is Natasha Tracy and this ECT survey was my idea. I am running
this survey with Dr. Prakash Masand [hyperlink provided], the psychiatrist
behind the site Global Medical Education [hyperlink provided] which
aims to educate others, particularly doctors, about medical issues such as
those surrounding mental illness. For my part, I have bipolar disorder and have had ECT for bipolar depres
sion. This has made me passionate about the subject as I see the extreme
debate that goes on about this treatment online. (Tracy, Bipolar Burble,
July 3, 2016) While she is knowledgeable enough to come up with this idea and for
an authoritative medical figure to collaborate with her, Tracy needs this
partnership to legitimate her endeavor, since she lacks the apparently still
necessary official accreditations. fi
Like Tracy, Fast has also used her blog to encourage people to partici
pate in studies she champions: Can We Diagnose Bipolar Disorder Using Eye Images? (…)h This is the question a new study from Souther [sic] Methodist University
poses based off of my work on recognizing signs of mania in the eyes. Please visit the website and read more about this potently life changing
study. What if we could see that we are manic through a physical sign even
when our brain is telling us we are just fine? Think of the possibilities. ih
Click here to read more about the SMU Mania in the Eyes Research
Study. [hyperlink provided] 168 C. Egher C. Egher In her turn, Fast has co-authored the books Take Charge of Bipolar
Disorder: A 4-Step Plan for You and Your Loved Ones to Manage the Illness C. Egher In her turn, Fast has co-authored the books Take Charge of Bipolar
Disorder: A 4-Step Plan for You and Your Loved Ones to Manage the Illness 5 Online Expert Mediators: Expanding Interactional Expertise 169 and Create Lasting Stability (2004), Loving Someone with Bipolar Disorder
(2004), and Get It Done When You’re Depressed (2008) together with Dr. John Preston. He is now professor emeritus with Alliant International
University in Sacramento, the author of 21 books, and the recipient of
the “President’s Award” from the Mental Health Association and of
“Distinguished Contributions to Psychology Award” from the California
Psychological Association. Fast is also claimed to “train pharmacists, psy
chiatric residents, social workers, alternative health care practitioners,
general physicians, nurse practitioners, therapists and many more health
care professionals on the topics of depression and bipolar disorder man
agement” (Amazon, 2016). Reflecting on The Health Cards Treatment
System for Bipolar Disorder, which she developed for people diagnosed
with bipolar disorder and their family members, and which “works with
or without medications,” as she claims (Bipolar Happens!, 2016), Fast
states: “I know that tens of thousands of my readers use the Health Cards
daily… (…) Even my health care professionals use them!” (Fast, Bipolar
Happens!, May 6, 2010). While using Fast’s cards attests to an awareness
by medical professionals that bipolar patients and their families may have
needs which traditional medical approaches insufficiently address, it may
also be a means for them to retain monopoly over medical knowledge at
a time when other professionals challenge it. For the time being, however,
both Fast and Tracy as well as the medical professionals they work with
profit from forging alliances, and such substantial exchanges are charac
teristic for the activities of online expert mediators. These bloggers are thus more than interesting and inspiring conversa
tion partners for medical professionals. They are stakeholders that
researchers want to collaborate with substantially, as they can facilitate
the enrolment of a high number of study participants, and they can pro
vide experiential knowledge and important insights into relevant areas for
future research. The way for such partnerships has already been paved by
patient organizations, but there have also been several substantial collab
orations between researchers and particular individuals. C. Egher If you love my work, I would love your support of this project. Even one
picture helps! (Fast, Bipolar Happens!, September 27, 2017) If you love my work, I would love your support of this project. Even one
picture helps! (Fast, Bipolar Happens!, September 27, 2017) Both excerpts suggest that one of the ways in which this new type of
stakeholder can make themselves interesting for medical professionals to
want to collaborate with them is by using their popularity among people
diagnosed with bipolar disorder to encourage them to provide the data
scientists need for research. While medical professionals acquire through
such collaborations much needed data, the bloggers profit from the cred
ibility and legitimacy of research findings obtained through collabora
tions with officially recognized experts, as Tracy notes I don’t have the credibility behind me in terms of getting published in a
journal. So it’s very important that I collaborate with someone who does,
so that the results of something can be published in a way that not just
people on my blog are going to see, but actually doctors are going to see,
and doctors are going to respect. Because I can be the best writer in the
world or the worst writer in the world … if I run a blog, doctors aren’t
necessarily going to pay attention to that whatsoever. It’s understandable. But publishing something in a journal, that’s going to get their attention. And I, just Natasha Tracy, can’t really expect to do that very reasonably. (Tracy, March 29, 2021, online interview) More such collaborations may develop in the future, given that the
expertise of these bloggers about bipolar disorder has been publicly
acknowledged by medical professionals. For instance, Ronald Pies, M.D.,
wrote about Tracy: As a specialist in bipolar disorders, I can say that Natasha’s understanding
of this illness is more accurate and sophisticated than that of many physi
cians I have encountered over the past 30 years. But more than that: she
shows uncommon wisdom and deep compassion, when it comes to dis
cussing psychiatrists and psychiatry. (Pies, Psychiatric Times, May
24, 2012). C. Egher Notable in this
sense are the research activities of Portia Iversen (Iversen, 2007) and
Sharon Terry (Terry & Boyd, 2001), who have directly contributed to the
development of new therapeutic approaches for autism, and to the iden
tification of the gene mutation causing pseudoxanthoma elasticum 170 C. Egher (PXE), respectively. Yet, whereas Iversen and Terry had control over
important resources as the leaders of two influential patient groups and
were not themselves diagnosed with the conditions they studied, Tracy
and Fast are bipolar patients and have managed to acquire the resources
mentioned above individually, through their skillful use of the Internet. Interactional Expertise and the Use
of a Specific Medium In their conceptualization of interactional expertise, Collins and Evans
do not consider the effects of the medium through which interactional
expertise is produced. I expand this notion by showing that the Internet
has importantly shaped how Tracy and Fast have performed their interac
tional expertise. Studying how the Internet shapes the performance of
interactional expertise is particularly important, since “in the context of
the digital shift, the demarcation between certified experts and lay people
is blurring” (Dickel & Franzen, 2016:3). This topic has generated a lot of
interest among scholars in the field of science education and science com
munication, who have studied how the public responds to or engages
with scientific knowledge provided via different media. Important in this
sense is a study conducted by Shanahan (2010) on how scientific and
personal expertise about health was expressed and discussed in the online
comments section of a newspaper. Her study showed that even in peer-
to-peer interactions, the most appreciated comments were those of con
tributors who claimed (some level of) scientific rather than personal
expertise. Even though the online exchanges between the blog authors
studied here and their readers may be conceived as peer-to-peer interac
tions due to the shared diagnosis of bipolar disorder and certain embod
ied experiences, there are important differences that need to be considered. Unlike the contributors scrutinized by Shanahan (2010), Tracy and Fast
are individuals with a well-established public persona, who have to fur
ther demonstrate the interactional expertise displayed in their posts by
(not) engaging with their readers’ comments. While their audience may
include contributory and interactional experts, an important difference
from Shanahan is that such exchanges already take place in conditions of 5 Online Expert Mediators: Expanding Interactional Expertise 171 inequality, since as authors and owners, the bloggers speak to their read
ers, as some of the quotes provided above indicate. Shanahan’s findings
are nevertheless relevant, showing that online scientific expertise is not
determined based on the invocation of credentials, but on one’s ability to
take up scientific practices, such as the provision of evidence and the cita
tion of relevant sources, thereby revealing one’s familiarity with the scien
tific norms and culture. i
Such approaches were adopted by Tracy and Fast as a means to articu
late and reinforce their online standing. Interactional Expertise and the Use
of a Specific Medium For instance, comments from
readers are used as opportunities to display their expertise by giving addi
tional medical information and by correctly identifying specific interven
tions. Since people with experiential expertise display growing tendencies
toward scientization in their contributions (Shanahan, 2010), these blog
gers do not merely invoke scientific claims, but carefully select, apply, and
interpret them. This is how Tracy reacts to a vague comment about a new
test meant to determine the effectiveness of medical treatments for bipo
lar disorder: “I believe you’re talking about the cytochrome P450
(CYP450) tests which I know are offered at the Mayo Clinic. (Also used
in cancer treatment)” (Tracy, Breaking Bipolar, November 5, 2012). Thus, apart from having sufficient knowledge to understand what the
contributor is referring to, Tracy also contextualizes the test, linking it to
other medical disciplines. The bloggers further use their readers’ com
ments as indicative of their informational needs and as sources of inspira
tion for some of their posts. From this perspective, comments help
bloggers retain their popularity and influence by addressing topical issues. l
Yet, the Internet also poses challenges to the display of interactional
expertise, as the information they provide is open to the scrutiny of peo
ple with different levels of education, different views, and at different
moments in time. To become and remain credible mediators, Tracy and
Fast therefore need to show that the knowledge they share is authoritative
while staying open to different perspectives. One way in which they man
age such contradictory expectations is by using the Internet’s multiplicity,
giving different nuances to their messages on different platforms. They
further use the asynchronous and selective character of comment
exchanges to react advantageously to their readers’ unexpected questions
or reactions. Since Tracy and Fast are at liberty to choose when they react 172 C. Egher C. Egher to comments, they can take the time to acquire more information or to
work on a reply until it has a satisfactory shape. In the meantime, other
readers may come to their “help,” by sharing their knowledge and experi
ences. The bloggers’ successful display of interactional expertise is also
informed by the wise selection of instances when they interact with their
readers. Thus, while they choose to intervene in situations where their
knowledge, empathy, and relatability are emphasized, they remain silent
in front of provocations which may alienate their audiences. Interactional Expertise and the Use
of a Specific Medium Comments
rules are another important instrument through which the bloggers may
contain their readers’ challenges and avoid controversy. For instance, ini
tially Tracy did not allow commentators to provide the exact names and
dosage combination of medicines. While this approach was meant to
prevent readers from trying medicines without medical approval, it also
weakened the epistemic claims and challenges they could bring against her.h p
g
y
g g
The technology of blogs also enables Tracy and Fast to display their
interactional expertise using images and hyperlinks. Their blog entries are
often accompanied by images which either illustrate the main message of
the post or bring an additional dimension to the information provided in
writing. Depending on the topic, the bloggers choose for different ratios
between written material and images. For instance, when discussing
alternative ways of ensuring mood stability, Fast only writes a few lines
but provides numerous images depicting relaxing activities. When the
effects of particular medications are discussed, however, the written text
dominates. At the same time, both bloggers provide videos of themselves
on the blog, where they talk about certain experiences or advise their
audiences. While it may be that their use of videos is informed by curios
ity and by the desire to experiment with new technologies and opportu
nities available to update their blogs, such videos also serve to enhance
the authenticity of their accounts, and to strengthen the bond between
themselves and their readers. Through the use of video, the person behind
the text of many posts, books, and magazine articles becomes a three-
dimensional being, who moves and talks in particular ways, whose
appearance may reveal the presence of bipolar disorder, or who may be
the embodiment of its successful management. Hyperlinks reveal important alliances as well as power relations. Both
bloggers use them in order to show that the information they provide is 5 Online Expert Mediators: Expanding Interactional Expertise 173 based on reliable sources. They refer mainly to articles available in medi
cal databases such as PubMed and Medscape or to posts by medical pro
fessionals on platforms where they collaborate. Tracy and Fast thus
position themselves as trustworthy mediators between reliable sources of
medical knowledge and interested audiences. Hyperlinks are also used by
bloggers to emphasize their vast body of work. For instance, Tracy uses
them to direct readers to her older posts. Interactional Expertise and the Use
of a Specific Medium Interestingly, the bloggers gen
erally refrain from using these affordances to share knowledge produced
by other people lacking accreditations or to introduce their readers to
projects initiated by “citizen scientists.” This indicates that the high stand
ing these bloggers enjoy is not due to a subversive use of the Internet, but
rather due to their alliances with powerful stakeholders.
5.1 On Tracy’s blog, the number of comments each post acquires is listed
below the title and a hyperlink is provided, so that interested readers can
directly access them rather than read the post. The hyperlink also draws
attention to the comments visually, since it is provided in blue whereas
the remainder of the information about a specific post is typed in black. i
5.2
C. Egher 174 careful in their use of hyperlinks. Thus, their display of interactional
expertise is importantly shaped by their use of blog affordances. careful in their use of hyperlinks. Thus, their display of interactional
expertise is importantly shaped by their use of blog affordances. 5 Online Expert Mediators: Expanding Interactional Expertise 5 Online Expert Mediators: Expanding Interactional Expertise
In general, both bloggers adapt the combination of medical and expe
riential knowledge, so that it is in line with the type of platform they
contribute on, they react to comments strategically, and they are very 175 Discussion The bloggers discussed here can be seen as a particular and highly success
ful form of entrepreneurial selves (Petersen & Lupton, 1996). While this
new type of stakeholder—online expert mediators—may fulfill a com
plementary or additional function to social movements, it also represents
a move away from them and a focus upon exceptional patient figures,
who have been able to use various resources and the opportunities and
limitations the Internet has made available to become highly influential. This stakeholder category emerges thus at the intersection between a
(mental) health condition, the acquisition of particular types of knowl
edge, and the use of a specific medium. By combining personal 176 C. Egher C. Egher experiences with medical knowledge, Tracy and Fast have gone beyond
the average illness blog, where one’s personal experiences are conveyed in
an intimate, diary-like fashion, and have come closer to issue-based blogs,
where different types of information considered relevant about a particu
lar topic are provided and discussed using arguments and multiple per
spectives (O’Neil, 2005). The interactional expertise that they develop
and articulate to various degrees has a strong bi-directionality, as they
need to be fluent in the language of medical knowledge of bipolar disor
der as well as to retain their experiential knowledge in a format which
allows them to relate to readers diagnosed with bipolar disorder and their
families. Thus, in their acquisition and articulation of interactional exper
tise, online expert mediators are reminiscent of journalists, who “develop
different degrees of bipolar ‘interactional expertise’, specializing in inter
actions with their sources on the one hand and audiences on the other”
(Reich, 2012:339). Furthermore, the online and offline activities of these
bloggers foreground the importance of focusing on the multiple shifting
identities that stakeholders can call upon in their development and per
formance of expertise. Their highly influential position was achieved
through their ability to skillfully switch between their identity as indi
viduals diagnosed with bipolar disorder, as successful blog owners, as rep
resentatives of many people diagnosed with bipolar disorder, and so on. Discussion I
have taken up these insights in the conceptualization of expertise I put
forward, where expertise is approached as a practical achievement realized
through coordination and affective labor among stakeholders who occupy
multiple and shifting positions within a complex ecosystem.h The rise of these stakeholders takes place in a context in which the
informational and health imperatives require people to assume responsi
bility about their health (Kivits, 2013), yet the difficulties of living with
a particular condition may lead them to prefer to follow someone else’s
lead (Lemire et al., 2008). Since the expertise of medical professionals has
been challenged over the last few decades, many people diagnosed may
seek to resolve this tension by following the advice of this new stake
holder type, by using such expert bloggers as arbiters. At the same time,
the rise of this new stakeholder is also due to patients and their families
requiring, apart from medical information, also encouragement and
guidance. Nevertheless, these new stakeholders are also confronted with 5 Online Expert Mediators: Expanding Interactional Expertise 177 suspicion given the varying quality of the health information available
online and the growing awareness that many public speakers and opinion
setters represent particular groups of interest. To be successful, online
expert mediators therefore need to convince their readers to develop dif
ferent types of trust: they must trust the bloggers; they must trust certain
online spaces or platforms; they must trust (at least) the branches of sci
ence the bloggers themselves rely upon (Harris et al., 2011). This also
shows that the type of expertise these new stakeholders have acquired and
perform online is a practical achievement, which they have realized by
moving back and forth between relevant groups within the particular
healthcare ecosystem in which they operate, by choosing to highlight
particular aspects of their identity depending on the context and their
goals, and by being caring but also careful toward other people and other
types of knowledge. Importantly, this chapter has indicated that the medium plays an
important role in how interactional expertise is performed, thereby
extending Collins and Evans’ conceptualization of this notion. In so
doing, it has also brought into relief some problematic aspects concern
ing the development of this new stakeholder category. While interac
tional expertise is necessary for this new type of stakeholdership, a strong
medium is also needed. Discussion Developing interactional expertise has enabled
Tracy and Fast to gain access and to develop close contacts with medical
professionals, yet it is their online popularity, which has provided them
with the resources necessary to engage in substantial exchanges with the
latter. The Internet has therefore allowed them to convincingly position
themselves as representatives of people diagnosed with bipolar disorder in
their relations with medical scientists in a way that is reminiscent of the
approach taken up by the American AIDS activists described by Epstein
(1996). Epstein problematized the position “lay experts” occupy in rela
tion to the “lay lay,” highlighting that the acquisition of competence into
a new type of knowledge impacts how one understands and relates to the
other types of knowledge with which one is endowed as well as on one’s
relations to others. Thus, he warned that by “learning the language and
culture of medical science” (Epstein, 1995:417) people diagnosed risk
distancing themselves from other people diagnosed with the same condi
tion, from their views and interests. From this perspective, the close 178 C. Egher C. Egher collaborations the bloggers develop with medical professionals may lead
to a further obfuscation of the differences in experience, interests, needs,
and values that exist among the people diagnosed with bipolar disorder
(Rowland et al., 2017) who follow their blogs. (
)
g
While blogs have been acknowledged as technologies with a democra
tizing potential (Huovila & Saikkonen, 2016), the findings presented
here show that online expert mediators acquire such high standing by
developing close ties with “traditional” experts. Thus, rather than con
tributing to opening the field of scientific knowledge production to more
people who lack official credentials, online expert mediators might inad
vertently contribute to the refinement of existing hierarchies in the rela
tions between medical professionals and patients. From this perspective,
it is regrettable that the interactions between these bloggers and medical
professionals occur most of the time offline or through private commu
nication, so that it is not possible to observe how they negotiate partici
pation in various projects and support for various initiatives. Since the
bloggers’ interactional expertise is limited to particular areas of medical
knowledge on bipolar disorder and does not exclude personal prefer
ences, online expert mediators also risk presenting their readers a skewed
perspective on the use and effectiveness of the currently available forms of
treatment. Discussion This view is sup
ported by the fact that even when famous medical professionals decided
to share their views on blogs, these were not accompanied by a comment
function. The fact that such medical professionals had become famous
through their activities on radio and television suggests that rather than
using the Internet to become influential, in France people use it as an
additional medium, to reach more audiences or to convey the image of
someone who is also up-to-date regarding online technologies.l Internet, this is a rather puzzling finding. It is all the more surprising
since the results described in Chap. 4 revealed that French online con
tributors were supporters of active forms of patienthood, and tried to
actively manage their condition and to contribute to new knowledge
about treatment effectiveness. The absence of this new type of stakeholder
in France might be linked to particular social and cultural elements,
which shape the use of the Internet and how people relate to their condi
tion. While numerous French blogs on bipolar disorder could be identi
fied, either they were read by few people or they had a very limited
interactive character, receiving five comments or less for most posts. There were also blogs on bipolar disorder which enjoyed greater visibility,
as they were authored occasionally on the online platforms of reputed
French newspapers, such as L’Avventura, a caricature-based blog authored
by Fiamma Luzzati for Le Monde, or La Vie d’un Bipolaire, authored by
W. on the website of L’Express. Nevertheless, even in these cases, the level
of interactivity was low. This might be informed by specific French cul
tural understandings and approaches to blogs, which conceive of them as
online spaces where different types of information can be shared in a
concise manner rather than as interactive platforms. This view is sup
ported by the fact that even when famous medical professionals decided
to share their views on blogs, these were not accompanied by a comment
function. The fact that such medical professionals had become famous
through their activities on radio and television suggests that rather than
using the Internet to become influential, in France people use it as an
additional medium, to reach more audiences or to convey the image of
someone who is also up-to-date regarding online technologies. Discussion On a more positive note, the online expert mediators studied
here may help bridge the digital divide when it comes to medical literacy
by sharing medical knowledge in an accessible manner, by making people
diagnosed and their families aware of the options at their disposal, and by
helping them get in touch with support groups and other organizations. Having achieved a highly influential position, in the future they might
harness their creativity and various skills to contribute in novel ways to
the proliferation and diversification of collaborations between people
diagnosed and medical professionals. Ironically, whereas Fast started her online career after she moved to
France, no French online bloggers enjoying similar standing to her and
Tracy were identified. Since the use of the Internet for mental health–
related purposes has been promoted by French authorities, as Chap. 3 has
indicated, and since many people living in France have access to the 5 Online Expert Mediators: Expanding Interactional Expertise 179 Internet, this is a rather puzzling finding. It is all the more surprising
since the results described in Chap. 4 revealed that French online con
tributors were supporters of active forms of patienthood, and tried to
actively manage their condition and to contribute to new knowledge
about treatment effectiveness. The absence of this new type of stakeholder
in France might be linked to particular social and cultural elements,
which shape the use of the Internet and how people relate to their condi
tion. While numerous French blogs on bipolar disorder could be identi
fied, either they were read by few people or they had a very limited
interactive character, receiving five comments or less for most posts. There were also blogs on bipolar disorder which enjoyed greater visibility,
as they were authored occasionally on the online platforms of reputed
French newspapers, such as L’Avventura, a caricature-based blog authored
by Fiamma Luzzati for Le Monde, or La Vie d’un Bipolaire, authored by
W. on the website of L’Express. Nevertheless, even in these cases, the level
of interactivity was low. This might be informed by specific French cul
tural understandings and approaches to blogs, which conceive of them as
online spaces where different types of information can be shared in a
concise manner rather than as interactive platforms. Discussion A
h
l
i
i
h
h
b
f
h i fl
i l i di id
l p
g
g
g
Another explanation is that the absence of such influential individuals
diagnosed with bipolar disorder or with any other condition, for that
matter, may be due to the fact that the imperative for people to become
active patients and assume responsibility for their health may have led in
France to the development of entrepreneurial subjectivities that manifest
themselves differently. An example in this sense is Bipote, the administra
tor and founder of Le Forum des Bipotes (LFB), mentioned in the previous
chapter, who was also diagnosed with bipolar disorder. Even though as 180 C. Egher C. Egher forum administrator, he had significant power and control, his position
there was not as prominent and as influential as that of the bloggers stud
ied here, despite his substantial knowledge about bipolar disorder. His
preference for a forum rather than a blog may denote a preference for
collective enterprises rather than individual approaches, and it may be
that more people in France share this attitude.h The lack of this new type of stakeholder in the French landscape may
also be informed by the fact that patient associations remain highly influ
ential there, as they are active mediators between medical professionals,
individuals diagnosed, and their families. As such, there may be little
need among people diagnosed for this new type of stakeholdership to
develop, whereas researchers and other official institutions may prefer to
engage in collaborations with patient representatives they are familiar
with and who are endowed with multiple types of knowledge and vast
resources to mobilize people. To conclude, the analysis of the activities of Tracy and Fast has pro
vided important insights regarding some of the conditions necessary to
become online expert mediators. Thus, next to an official diagnosis, peo
ple’s health needs to be stable enough for them to engage in various activ
ities requiring a lot of time and energy. They also need to be able to
communicate in ways which can capture and retain the interest of differ
ent stakeholders. Furthermore, those interested need either to financially
afford giving up their jobs to dedicate themselves to the development of
blogs or to be willing to accept sponsorship or another form of payment,
thereby running the risk of losing their social benefits. 5 Online Expert Mediators: Expanding Interactional Expertise 181 Discussion More research is
needed to understand the ways in which other kinds of knowledge and
online skills shape the acquisition and performance of interactional
expertise, and into the differences and similarities concerning the media
tion work undertaken by this new stakeholder category across different
conditions. This chapter showed how individuals diagnosed with bipolar
disorder responded to pronounced tendencies toward patient engage
ment by developing interactional expertise and used the Internet to
become highly influential, thereby turning themselves into a new stake
holder category, what I called online expert mediators. The next chapter
will describe a different response to such exhortations, as it focuses on
solidarity and the development of a new type of community. References Adams, S. (2013). Maintaining the Collision of Accounts: Crowdsourcing Sites
in Health Care as Brokers in the Co-Production of Pharmaceutical
Knowledge. Information, Communication & Society, 17(6), 657–669. Armstrong, D. (1995). The Rise of Surveillance Medicine. Sociology of Health &
Illness, 17(3), 393–404. Barak, A. (1999). Psychological Applications on the Internet: A Discipline on
the Threshold of a New Millennium. Applied & Preventive Psychology,
8, 231–245. Barak, A., & Grohol, J. (2011). Current and Future Trends in Internet-
Supported Mental Health Interventions. Journal of Technology in Human
Services, 29(3), 155–196. Barak, A., Hen, L., Boniel-Nissim, M., & Shapira, N. (2008). A Comprehensive
Review and a Meta-Analysis of the Effectiveness of Internet-Based
Psychotherapeutic Interventions. Journal of Technology in Human Services,
26(2/4), 109–160. Barbot, J. (2006). How to Build An “Active” Patient? The Work of AIDS
Associations in France. Social Science & Medicine, 62, 538–551.ih Barbot, J., & Dodier, N. (2002). Multiplicity in Scientific Medicine: The
Experience of HIV-Positive Patients. Science, Technology & Human Values,
27, 404–440. Barello, S., Graffigna, G., Vegni, E., & Bosio, A. (2014). The Challenges of
Conceptualizing Patient Engagement in Health Care: A Lexicographic
Literature Review. Journal of Participatory Medicine, 6(11), 1–15. Barnes, M., & Shardlow, P. (1997). From Passive Recipient to Active Citizen:
Participation in Mental Health User Groups. Journal of Mental Health,
6(3), 289–300. Bottles, K. (2013). Health Hackers and Citizen Scientists Shake Up Medical
Research. Physician Executive Journal, 88–90.h Brown, P. (1988). The Transfer of Care: Psychiatric Deinstitutionalization and Its
Aftermath. Routledge Kegan & Paul. Carlbring, P., & Andersson, G. (2006). Internet and Psychological Treatment. How Well Can They Be Combined? Computers in Human Behavior,
22, 545–553. Collins, H., & Evans, R. (2002). The Third Wave of Science Studies. Studies of
Expertise and Experience. Social Studies of Science, 32(2), 235–296. 182 C. Egher Collins, H., & Evans, R. (2007). Re-thinking Expertise. The University of
Chicago Press. Collins, H., & Evans, R. (2007). Re-thinking Expertise. The University of
Chicago Press. Collins, H., & Evans, R. (2015). Expertise Revisited, Part I — Interactional
Expertise. Studies in History and Philosophy of Science, 54, 113–123. Collins, H., Evans, R., Ribeiro, R., & Hall, M. (2006). Experiments with
Interactional Expertise. Studies in History and Philosophy of Science,
37, 656–674. Collins, H., Evans, R., & Weinel, M. (2017). Interactional Expertise. In U. Felt,
R. Fouché, C. Miller, & L. Smith-Doerr (Eds.), The Handbook of Science and
Technology Studies (4th ed., pp. 765–792). References The MIT Press. Crossley, M., & Crossley, N. (2001). “Patient” Voices, Social Movements and
the Habitus: How Psychiatric Survivors “Speak Out”. Social Science &
Medicine, 52, 1477–1489. Davidson, L. (2005). Recovery, Self Management and the Expert Patient-
Changing the Culture of Mental Health from a UK Perspective. Journal of
Mental Health, 14(1), 25–35. De Boer, M., & Slatman, J. (2014). Blogging and Breast Cancer: Narrating
One’s Life, Body and Self on the Internet. Women’s Studies International
Forum, 44(1), 17–25. Dickel, S., & Franzen, M. (2016). The “Problem of Extension” Revisited: New
Modes of Digital Participation in Science. Journal of Science Communication,
15(1), 1–15. Director, D. (2005). Using an Action Research Approach to Involving Service
Users in The Assessment of Professional Competence. European Journal of
Social Work, 8(2), 165–179.h Epstein, S. (1995). The Construction of Lay Expertise: AIDS Activism and the
Forging of Credibility in the Reform of Clinical Trials. Science, Technology &
Human Values, 20(4), 408–437. Epstein, S. (1995). The Construction of Lay Expertise: AIDS Activism and the
Forging of Credibility in the Reform of Clinical Trials. Science, Technology &
Human Values, 20(4), 408–437. Epstein, S. (1996). Impure Science: AIDS, Activism, and the Politics of Knowledge. University of California Press. F
N W d K & O’R
k
A (2005) Th “E
P
” E Epstein, S. (1996). Impure Science: AIDS, Activism, and the Politics of Knowledge. University of California Press. Fox, N., Ward, K., & O’Rourke, A. (2005). The “Expert Patient”: Empowerment
or Medical Dominance? The Case of Weight Loss, Pharmaceutical Drugs and
the Internet. Social Science & Medicine, 69, 1299–1309. Gillard, S., Simons, L., Turner, K., Lucock, M., & Edwards, C. (2012). Patient
and Public Involvement in the Coproduction of Knowledge: Reflection on
the Analysis of Qualitative Data in a Mental Health Study. Qualitative Health
Research, 22(8), 1126–1137. 5 Online Expert Mediators: Expanding Interactional Expertise 183 Goddiksen, M. (2014). Clarifying Interactional and Contributory Expertise. Studies in History and Philosophy of Science, 47, 111–117. Godfrey, M., Callaghan, G., Johnson, L., & Waddington, E. (2003). Supporting
People: A Guide to User Involvement for Organizations Providing Housing
Related Support Services. Office of the Deputy Prime Minister. Gowen, K., Deschaine, M., Gruttadara, D., & Markey, D. (2012). Young
Adults with Mental Health Conditions and Social Networking Websites:
Seeking Tools to Build Community. Psychiatric Rehabilitation Journal,
35(3), 245–250. Grob, G. (2005). The Transformation of Mental Health Policy in Twentieth-
Century America. In M. Gijswijt-Hofstra, H. Oosterhuis, J. Vijselaar, &
H. Freeman (Eds.), Psychiatry and Mental Health Care in the Twentieth-
Century: Comparisons and Approaches (pp. 141–161). Amsterdam
University Press. Harris, A., Wyatt, S., & Kelly, S. (2011). Conceptualizing Trust in Digital
Environments. Health-e skepticism: Trust in the Age of the Internet. In: A
Decade in Internet Time: Symposium on the Dynamics of the Internet and Society
(OII-ICS Symposium), Oxford, UK, 21–24 September 2011, 1–11.h Heilferty, C. (2009). Toward A Theory of Online Communication in Illness:
Concept Analysis of Illness Blogs. Journal of Advanced Nursing, 65(7),
1539–1547. Hewlett, S., Wit, M., Richards, P., Quest, E., Hughes, R., Heiberg, T., &
Kirwan, J. (2006). Patients and Professionals as Research Partners: Challenges,
Practicalities, and Benefits. Arthritis Care & Research, 55(4), 676–680. i
Hickey, G., & Kipping, C. (1998). Exploring the Concept of User Involvement
in Mental Health Through a Participation Continuum. Journal of Clinical
Nursing, 7, 83–88. Hogg, C. (2009). Citizens, Consumers and the NHS: Capturing Voices. Palgrave
Macmillan. Huovila, J., & Saikkonen, S. (2016). Establishing Credibility, Constructing
Understanding: The Epistemic Struggle Over Healthy Eating in the Finnish
Dietetic Blogosphere. Health, 20(4), 383–400. Iversen, P. (2007). Strange Son: Two Mothers, Two Sons, and the Quest to Unlock
the Hidden World of Autism. Riverhead Books. Kallinikos, J., & Tempini, N. (2014). Patient Data as Medical Facts: Social
Media Practices as a Foundation for Medical Knowledge Creation. Information
Systems Research, 25(4), 817–833. Huovila, J., & Saikkonen, S. (2016). Establishing Credibility, Constructing
Understanding: The Epistemic Struggle Over Healthy Eating in the Finnish , P. (2007). Strange Son: Two Mothers, Two Sons, and the Quest to Unlock
Hidden World of Autism. Riverhead Books. 184 C. Egher Kemp, P. (2010). The Creative Involvement of Service Users in the Classroom. In J. Weinstein (Ed.), Mental Health, Service User Involvement and Recovery
(pp. 15–29). Jessica Kingsley Publishers. 5 Online Expert Mediators: Expanding Interactional Expertise Kivits J (2013) E-Health and Renewed Sociological Approaches to Health and
Illness. In Orton-Johnson K, Prior N (eds.) Digital Sociology. Critical
Perspectives. Houndmills: Palgrave Macmillan, 213-226. Klawiter, M. (1999). Racing for the Cure, Walking Women, and Toxic Touring:
Mapping Cultures of Action within the Bay Area Terrain of Breast Cancer. Social Problems, 46(1), 104–126. Kopelson, K. (2009). Writing Patients’ Wrongs: The Rhetoric and Reality of
Information Age Medicine. Journal of Advanced Composition, 29, 353–404. Kraus, R., Stricker, G., & Speyer, C. (Eds.). (2010). Online Counseling: A
Handbook for Mental Health Professionals (2nd ed.). Elsevier Academic Press. Kushner, H. (2004). Competing Medical Cultures, Patient Support Groups,
and the Construction of Tourette’s Syndrome. In R. Packard, P. Brown,
R. Berkelman, & H. Frumkin (Eds.), Emerging Illnesses and Society: Negotiating
the Public Health (pp. 71–101). John Hopkins University Press. Landzelius, K. (2006). Introduction: Patient Organization Movements and
New Metamorphoses in Patienthood. Social Science & Medicine,
62(3), 529–537. Latour, B. (2005). Re-assembling the Social. An Introduction to Actor-Network
Theory. Oxford University Press. h
Lemire, M., Sicotte, C., & Paré, G. (2008). Internet Use and The Logics of
Personal Empowerment in Health. Health Policy, 88, 130–140. Lerner, B. (2001). No Shrinking Violet: Rose Kushner and the Rise of American
Breast Cancer Activism. Western Journal of Medicine, 174(5), 362.h Lucivero, F., & Prainsack, B. (2015). The Lifestylization of Healthcare? Consumer Genomics’ and Mobile Health as Technologies for Healthy
Lifestyle. Applied & Translational Genomics, 4, 44–49. Lupton, D. (1995). The Imperative of Health: Public Health and the Regulated
Body. Sage Publications.hih Lupton, D. (2014). The Commodification of Patient Opinion: The Digital
Patient Experience Economy in the Age of Big Data. Sociology of Health &
Illness, 36(6), 856–869. Marks, I., Cavanagh, K., & Gega, L. (2007). Hands-On Help. Computer-Aided
Psychotherapy. Psychology Press. 5 Online Expert Mediators: Expanding Interactional Expertise 5 Online Expert Mediators: Expanding Interactional Expertise 185 Mazanderani, F., Locock, L., & Powell, J. (2012). Being Differently the Same:
The Mediation of Identity Tensions in the Sharing of Illness Experiences. Social Science & Medicine, 74(4), 546–553. McLean, A. (2000). From Ex-Patient Alternatives to Consumer Options:
Consequences of Consumerism for Psychiatric Consumers and the Ex-Patient
Movement. International Journal of Health Services, 30(4), 821–847. Mulveen, R., & Hepworth, J. (2006). An Interpretative Phenomenological
Analysis of Participation in a Pro-Anorexia Internet Site and Its Relationship
with Disordered Eating. Journal of Health Psychology, 11(2): 283, 296. Naslund, J., Aschbrenner, K., Marsch, L., McHugo, G., & Barteks, S. (2015). Crowdsourcing for Conducting Randomized Trials of Internet Delivered
Interventions in People with Serious Mental Illness: A Systematic Review. Contemporary Clinical Trials, 44, 77–88.h Nettleton, S. (2004). The Emergence of E-scaped Medicine? Sociology,
38(4), 661–679. Nicholas, J., Larsen, M., Proudfoot, J., & Christensen, H. (2015). Mobile Apps
for Bipolar Disorder: A Systematic Review of Features and Content Quality. Journal of Medical Internet Research, 17(8), e198.h Novas, C. (2006). The Political Economy of Hope: Patients’ Organizations,
Science and Biovalue. BioSocieties, 1, 289–305. O’Neil M (2005) Weblogs and Authority. Selected Works. University Paris IV
Sorbonne. Accessed November 18, 2014, from https://works.bepress.com/
mathieu_oneil/12/ Orsini, M., & Smith, M. (2010). Social Movements, Knowledge and Public
Policy: The Case of Autism Activism in Canada and the US. Critical Policy
Studies, 4(1), 38–57.h Petersen, A., & Lupton, D. (1996). The New Public Health. Health and Self in
the Age of Risk. Sage. Pickersgill, M. (2012). What is Psychiatry? Co-Producing Complexity in Mental
Health. Social Theory & Health, 10, 328–347. h
y
Plaisance, K., & Kennedy, E. (2014). A Pluralistic Approach to Interactional
Expertise. Studies in History and Philosophy of Science, 47, 60–68. Expertise. Studies in History and Philosophy of Science, 47, 60–68. Proudfoot, J. (2004). Computer-Based Treatment for Anxiety and Depression: f p
y
p y f
, 7,
Proudfoot, J. (2004). Computer-Based Treatment for Anxiety and Depression: f Proudfoot, J. (2004). Computer-Based Treatment for Anxiety and Depression:
IsItFeasible?IsItEffective?NeuroscienceandBehavioral Reviews 28 353 363 Is It Feasible? Is It Effective? Neuroscience and Behavioral Reviews, 28, 353–363. Rabeharisoa, V., Moreira, T., & Akrich, M. (2013). Evidence-based Activism: Patients’ Organizations, Users’ and Activist’s Groups in Knowledge Society. CSI Working Papers Series, 033, 1–27. 186 C. Egher Reich, Z. (2012). Journalism as Bipolar Interactional Expertise. Communication
Theory, 22, 339–358.h h
Rose, N. (2007). Ybarra, M., & Eaton, W. (2005). Internet-Based Mental Health Interventions.
Mental Health Services Research, 7(2), 75–87. Wyatt, S., Harris, A., Adams, S., & Kelly, S. (2013). Illness Online: Self-
Reported Data and Questions of Trust in Medical and Social Research.
Theory, Culture & Society, 30(4), 131–150. Whitley, R. (2012). The Antipsychiatry Movement: Dead, Diminishing, or
Developing? Psychiatric Services, 63(10), 1039–1041. h
Wynne, B. (1992). Misunderstood Misunderstanding: Social Identities and
Public Uptake of Science. Public Understanding of Science, 1, 281–304. Whitley, R. (2012). The Antipsychiatry Movement: Dead, Diminishing, or
Developing? Psychiatric Services, 63(10), 1039–1041.
Wyatt, S., Harris, A., Adams, S., & Kelly, S. (2013). Illness Online: Self-
Reported Data and Questions of Trust in Medical and Social Research.
Theory, Culture & Society, 30(4), 131–150. 5 Online Expert Mediators: Expanding Interactional Expertise The Politics of Life Itself. Biomedicine, Power and Subjectivity in
the Twenty-First Century. Princeton University Press. Rowland, P., McMillan, S., McGillicuddy, P., & Richards, J. (2017). What Is
“The Patient Perspective” in Patient Engagement Programs? Implicit Logics
and Parallels to Feminist Theories. Health, 21(1), 76–92. Schaffer, R., Kuczynski, K., & Skinner, D. (2008). Producing Genetic
Knowledge and Citizenship Through the Internet: Mothers, Pediatric
Genetics, and Cybermedicine. Sociology of Health and Illness, 30(1), 145–159. Shanahan, M. (2010). Changing the Meaning of Peer-To-Peer? Exploring
Online Comment Spaces as Sites of Negotiated Expertise. Journal of Science
Communication, 9(1), 1–13.i Sharon, T. (2017). Self-tracking for Health and the Quantified Self:
Re-articulating Autonomy, Solidarity and Authenticity in an Age of
Personalized Healthcare. Philosophy & Technology, 30(1), 93–121.fi Smith, D. J., Griffiths, E., Poole, R., di Fiorio, A., Barnes, E., Kelly, M.,
Craddock, N., Hood, K., & Simpson, S. (2011). Beating Bipolar: Exploratory
Trial of a Novel Internet-Based Psychoeducational Treatment for Bipolar
Disorder. Bipolar Disorders, 13, 571–577. Speed, E. (2006). Patients, Consumers, and Survivors: A Case Study of Mental
Health Service User Discourses. Social Science & Medicine, 62, 28–38. Taussig, K.-S., Rapp, R., & Heath, D. (2003). Flexible Eugenics: Technologies
of the Self in the Age of Genetics. In A. Goodman et al. (Eds.), Genetic
Nature/Culture: Anthropology and Science Beyond the Two-Culture Divide
(pp. 58–76). University of California Press. Terry, S., & Boyd, C. (2001). Researching the Biology of PXE: Partnering in the
Process. American Journal of Medical Genetics, 106, 177–184.h Treichler, P. (1999). How to Have Theory in an Epidemic: Cultural Chronicles of
AIDS. Duke University Press. T
B (1995) M di l P
d S i l K
l d
(2 d d ) S y
Turner, B. (1995). Medical Power and Social Knowledge (2nd ed.). Sage. Tutton, R., & Prainsack, B. (2011). Enterprising or Altruistic Selves? Making
Up Research Subjects in Genetics Research. Sociology of Health & Illness,
33(7), 1081–1095. Wathen, N., Wyatt, S., & Harris, R. (Eds.). (2008). Mediating Health
Information: The Go-Betweens in a Changing Socio-Technical Landscape. Palgrave Macmillan. 5 Online Expert Mediators: Expanding Interactional Expertise 5 Online Expert Mediators: Expanding Interactional Expertise 187 Whitley, R. (2012). The Antipsychiatry Movement: Dead, Diminishing, or
Developing? Psychiatric Services, 63(10), 1039–1041. Wyatt, S., Harris, A., Adams, S., & Kelly, S. (2013). Illness Online: Self-
Reported Data and Questions of Trust in Medical and Social Research. Theory, Culture & Society, 30(4), 131–150. h
Wynne, B. (1992). Misunderstood Misunderstanding: Social Identities and
Public Uptake of Science. Public Understanding of Science, 1, 281–304. Ybarra, M., & Eaton, W. (2005). Internet-Based Mental Health Interventions. Mental Health Services Research, 7(2), 75–87. Open Access This chapter is licensed under the terms of the Creative Commons
Attribution 4.0 International License (http://creativecommons.org/licenses/
by/4.0/), which permits use, sharing, adaptation, distribution and reproduction
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right holder. © The Author(s) 2023
C. Egher, Digital Healthcare and Expertise, Health, Technology and Society,
https://doi.org/10.1007/978-981-16-9178-2_6 Digital Biocommunities: Solidarity
and Lay Expertise About Bipolar
Disorder I had already taken the anxiolytics… I had already taken the anxiolytics… I had already taken the anxiolytics… But I’ve managed to ask someone to help me on a forum because I
couldn’t take it anymore. Someone reacted and we’re talking via private
messages. I think this will help me a bit. Thanks. (Derek21, March 18, 2013) Through their online activities, people diagnosed with bipolar disorder
often engage in solidaristic behaviors, providing help and support to sim
ilar others in need, as the quote above illustrates. This draws attention to
another important aspect of expertise, which is not only shaped by the
means through which it is acquired and performed and by the goals it
aims to achieve, but also by the values that motivate and support such
processes. In recent years, expertise about bipolar disorder has been
shaped by the rise of personalized and precision medicine (Evers, 2009;
Ozomaro et al., 2013; Shin et al., 2016), which many believe will lead to
highly individualized approaches to health and will thus have important
moral consequences. Whereas autonomy has featured prominently in
these debates, in recent years scholars have started to investigate how
personalized and precision medicine might affect solidaristic practices. In
so doing, influential commentators have challenged the dominant belief 189 © The Author(s) 2023
C. Egher, Digital Healthcare and Expertise, Health, Technology and Society,
https://doi.org/10.1007/978-981-16-9178-2_6 190 C. Egher that these approaches would necessarily lead to radical forms of individu
alism, arguing, instead, that they could also prompt solidaristic approaches
to healthcare (Prainsack & Buyx, 2017). In this chapter, I join this group
of researchers by studying the tensions between the appeals to solidarity
and individualization in mental healthcare triggered by personalized and
precision medicine and by considering how these tensions are taken up
and reflected in the online exchanges of American and French contribu
tors diagnosed with bipolar disorder. In so doing, I engage in the explora
tion of the one remaining aspect of the conceptualization of expertise
that I put forward in this book, namely its collective nature and the role
of affective labor. Based on the analysis of the empirical materials, I argue
that solidaristic practices underlie numerous online interactions among
people diagnosed with bipolar disorder, thereby contributing to the
development of a new type of collectivity—what I have called “digital
biocommunities”—and promoting the development of lay expertise. 1 This institution was established in 2005 to distribute and oversee the national provision of finan
cial help and assistance to people with disabilities and the elderly. The Individualization of Healthcare: Solidarity
Under Threat
2004
2016
2014
2018
2017 6 Digital Biocommunities: Solidarity and Lay Expertise… 191
1 How the individualizing tendencies underlying personalized and pre
cision medicine affect solidarity has also been the object of vigorous
debates among scholars. Supporters have welcomed these tendencies as
leading to better and more efficient ways to provide healthcare, which
they claimed would ultimately benefit both the individual and society at
large. Thus, by tailoring clinical investigations and therapeutic approaches
to the specific needs and circumstances of every person (Wium-Anderesen
et al., 2017), people would be spared unnecessary tests or therapeutic
approaches less likely to be successful. This would enable the more effec
tive attribution of funds in healthcare, thereby addressing and redressing
a state of precarity triggered by a growing number of people diagnosed
with (mental) health conditions and insufficient funds. Proponents of
personalized and precision medicine have also invoked the language of
empowerment, arguing that the widespread adoption of digital technolo
gies and self-tracking enable people to gain more knowledge and control
over their health (Knoppers & Chadwick, 2005; Steinhubl et al., 2013). In turn, this could contribute to the democratization of the relations
between individuals and medical professionals or even to a hierarchical
reversal thereof, as titles such as “Patient-Driven Health Care Models”
(Swan, 2009) or The Patient Will See You Now (Topol, 2015) suggest. 192 C. Egher C. Egher In contrast, critics have argued that by addressing individuals as unique
from certain points of view, people may end up focusing more on what
distinguishes them from others rather than on what binds them together,
which may lead to “radical” differences. According to Dickenson (2013),
such approaches could bring about a shift from “We Medicine” to “Me
Medicine,” a concern which is eloquently echoed by Prainsack and Buyx
(2017:127): Because every patient is different, as this new version of personalized medi
cine assumes, their health and their diseases are different as well: individual
differences in our genetic makeup, in our gene expression, in the microor
ganisms inhabiting our guts and bodies, in our lifestyles, diets and so forth
render each of us, as well as our physiologies and pathologies, a unique
expression of a particular state of health and disease in any given moment
in time. (Prainsack & Buyx, 2017:127) These approaches thus threaten solidarity and may lead to new forms
of inequality and discrimination (Prainsack & Buyx, 2012), as people
engaging in seemingly preventable individual behaviors, such as smoking
or the consumption of sugar and fats, may be required to pay higher
insurance rates, and/or may be denied access to some medical treatment
and social provisions. These critics warn this way that individual freedom
and responsibility can be invoked in such instances to mask systemic
forms of economic and social inequality, and may even help to perpetuate
them. Challenging what they consider to be the “tyranny of autonomy”
(Foster, 2009) in Western healthcare and the understanding of individu
als as autonomous, rational, self-interested beings, such commentators
(Baylis et al., 2008; Prainsack & Buyx, 2012) have argued instead in
favor of a relational approach. From this point of view, individual identi
ties, values, needs, and perspectives are not dictated by self-interest alone,
but importantly shaped by the other people in one’s life and by the socio-
political context in which one lives. There are indications that such per
spectives are supported by practices on the ground, as exemplified by the
shift from “The Quantified Self” to “The Quantified Us” (Lupton, 2016)
among proponents of health endeavors generally thought to be highly 6 Digital Biocommunities: Solidarity and Lay Expertise… 193 individualistic and individualizing, and by empirical studies that have
identified solidaristic practices at the heart of self-tracking (Sharon, 2017). C. Egher i
p
g (
)
Since I have argued that expertise is a collective notion, requiring the
concerted efforts of numerous stakeholders, the expectation of “radical”
individualization in healthcare raised important questions about its
future and the new shapes that it may take. This was particularly the case
for lay expertise, a collective notion whose meaning and relevance are
rendered uncertain in a healthcare context marked by a focus on indi
vidual differences. The data used in this chapter were therefore initially
approached with the expectation of encountering numerous instances
confirming the idea that individual needs, preferences, and approaches in
mental healthcare have become dominant to the detriment of more col
lective challenges and concerns. Yet, on many blogs and fora people diag
nosed with bipolar disorder continued to seek to understand their
condition collectively and displayed substantial concern for others. For
instance, they tried to make sense of the symptoms they experienced by
placing them in the broader context of their lives, by considering how
their behaviors affected their families, friends, and colleagues, and by
comparing their experiences with those of others with the same diagno
sis. It thus became obvious that solidarity is a value that online contribu
tors diagnosed with bipolar disorder perform online, which shifted the
analytical focus onto its relation to lay expertise, thereby turning this
chapter into a contribution to calls made by scholars to study how values
manifest themselves in practice (Swierstra, 2013; van de Werff, 2018). The Meaning of Solidarity Despite solidarity’s re-appearance in debates about health policy, the
meaning of this concept remains evasive. While it is often defined as “the
glue that keeps people together” (Komter, 2005:2), different perspectives
have been put forward to explain how such social cohesion is achieved. Thus, some scholars approach solidarity as a particular set of feelings and
emotions (Mayhew, 1971), as moral (Etzioni, 1988) and “affective ties”
(Parsons, 1952:157) which inform people’s commitment to others. In
such cases, solidarity is intertwined with the human capacity to 194 C. Egher C. Egher experience and express sympathy, care, and concern for people in their
immediate surroundings. It is thus thought to spring into being rather
automatically, informed by common attachments (instead of rational
considerations) among a relatively small number of people. Others
understand solidarity as a characteristic of groups and societies (Durkheim,
1964; Weber, 1947), regulating the interactions between the individual
and the community (Bayertz, 1998), and potentially furthering the com
mon good. Van Oorschot and Komter noted in this sense that “[t]he
main source of solidarity is a mutual sharing of each other’s fate” (1998,
8), thereby largely conceiving of solidarity as a result of rational choices
and calculations (Hechter, 1987), of the acknowledgment of “shared
identity” and “shared utility” (Van Oorschot & Komter, 1998). Yet other
scholars approach solidarity as a moral, universal, “inclusive” ideal (Dean,
1995), prescribing specific sets of orientations and behaviors which peo
ple should take up in order to increase social bonds in the heterogeneous
societies we currently live in. y
The study of solidarity in this chapter is based, however, on the con
ceptualization put forward by Prainsack and Buyx (2012, 2017), which
has the advantage of being concrete and practice-oriented. In their view
(2012:346), “[s]olidarity signifies shared practices reflecting a collective
commitment to carry ‘costs’ (financial, social, emotional, or otherwise) to
assist others,” and this conceptualization is underpinned by three impor
tant elements. First, it relies upon a relational understanding of person
hood, as these scholars see the individuals’ concerns, values, and
preferences as emerging in interaction with those surrounding them and
as shaped by the socio-cultural environment in which they find them
selves. This allows for solidarity to be distinguished from altruism, as
people are approached as simultaneously self-interested and concerned
for the well-being of others. The Meaning of Solidarity Second, solidarity is based upon the recogni
tion of a relevant similarity, upon people’s acknowledgment that they
share a commonality with others in respect of interest. This makes it pos
sible to distinguish it from charity, as solidaristic practices are understood
to emerge among individuals or groups in symmetrical relations to each
other in regard to the similarity that is relevant in a given context. Third,
while feelings and emotions may play an important role in its develop
ment, solidarity is something that is done, performed. It is manifested 6 Digital Biocommunities: Solidarity and Lay Expertise… 195 through “enacted commitments” (Prainsack & Buyx, 2017:42), which
may vary in scope and impact, ranging from a document or piece of
policy to individual actions undertaken by private citizens. Attention to
these three dimensions made it possible to study the provision of online
texts as informed by solidarity and to focus on how online contributors
diagnosed with bipolar disorder relate to or distinguish themselves from
others rather than approach them as a homogeneous group. As this
involves inclusions and exclusions, this conceptualization also has the
advantage of precluding an approach to solidarity as something exclu
sively positive (Dean, 1995) and encourages a focus on how the affor
dances of online platforms may be implicated in such practices. Solidarity and Idioms of Practice In studying how solidarity relates to lay expertise online, I build upon
multiple studies which have shown that a common diagnosis (Epstein,
2007; Rabeharisoa & Callon, 2002) and similarities in one’s genetic pro
file and potential health risks facilitate the formation of collectives
(Rabeharisoa et al., 2013) and can even contribute to “genomic solidar
ity" (Van Hoyweghen & Rebert, 2012). The analysis is particularly
indebted to Rabinow’s (1996) view that developments in genetics have
led to the emergence of biosociality; that is, they have enabled the forma
tion of new group and individual identities based on genetic and molecu
lar insights. While new types of knowledge transform the ways in which
people understand their condition and relate to others, online interac
tions are importantly shaped by the digital technologies they use, by the
affordances of the social media where they seek and provide information. Thus “its [the internet’s] interactivity and the interaction it allows for can i In studying how solidarity relates to lay expertise online, I build upon
multiple studies which have shown that a common diagnosis (Epstein,
2007; Rabeharisoa & Callon, 2002) and similarities in one’s genetic pro
file and potential health risks facilitate the formation of collectives
(Rabeharisoa et al., 2013) and can even contribute to “genomic solidar
ity" (Van Hoyweghen & Rebert, 2012). The analysis is particularly
indebted to Rabinow’s (1996) view that developments in genetics have
led to the emergence of biosociality; that is, they have enabled the forma
tion of new group and individual identities based on genetic and molecu
lar insights. While new types of knowledge transform the ways in which
people understand their condition and relate to others, online interac
tions are importantly shaped by the digital technologies they use, by the
affordances of the social media where they seek and provide information. Thus “its [the internet’s] interactivity and the interaction it allows for can
facilitate the formation of specific points of view and new ways of articu
lating individual experience to collective positions” (Akrich et al.,
2008:2). Online exchanges may therefore contribute to “fostering com
munity and mutual support, and negotiating medical relationships”
(Sosnowy, 2014:325). Solidarity and Idioms of Practice They may also prompt transformations in the very
meaning and practice of sociality (van Dijck, 2013), as people figure out
what aspects of a technology they use and how they use it in practice, by f
y
p
Thus “its [the internet’s] interactivity and the interaction it allows for can
facilitate the formation of specific points of view and new ways of articu
lating individual experience to collective positions” (Akrich et al.,
2008:2). Online exchanges may therefore contribute to “fostering com
munity and mutual support, and negotiating medical relationships”
(Sosnowy, 2014:325). They may also prompt transformations in the very
meaning and practice of sociality (van Dijck, 2013), as people figure out
what aspects of a technology they use and how they use it in practice, by 196 C. Egher C. Egher tinkering with it as they interact with others. Thus, not only do people
use such technologies for social activities, but their very use is social, in
that people “develop their beliefs about media and ways of using media
within idioms of practice” (Gershon, 2010: loc 117). According to
Gershon, “[i]dioms of practice point to how people have implicit and
explicit intuitions about using different technologies, which they have
developed with their friends, family members, and coworkers” (ibid.) and
which “emerge out of collective discussions and shared practices” (ibid.).h g
p
The concept of idiom of practice underlines the multiple meanings
that a technology can have, depending on its users and on the context of
their engagement with it, on the ways in which those around them use it,
and on the prevailing social norms and values that delineate what it
should and should not be used for. In this sense, Gershon describes how
the development of social media led to the development of various idi
oms of practice regarding acceptable forms of breakup. While some peo
ple considered breaking up via an e-mail a more acceptable approach,
because it was more personal and private, others found that it resembled
too much a monologue, and preferred being notified about such an
occurrence on social media, where turn-taking could unfold faster and
dynamic exchanges could easily occur. While in the early days of a tech
nology, multiple idioms of practice can exist, in time certain practices
may “solidify,” as some uses become widespread in specific contexts. Solidarity and Idioms of Practice The
analysis described in this chapter is based upon a theoretical framework
where this concept is combined with the understanding of solidarity
developed by Prainsack and Buyx (2017). This framework allowed for a
better understanding of the roles that online platforms and their affor
dances play in the performance of this value, of the new forms of sociality
that can thus be developed, and of how they relate to lay expertise. Lay Expertise and Affective Labor Lay expertise is typically developed as people diagnosed become better
informed about the medical knowledge available about their condition,
by learning to interpret their own embodied experiences in light of this
knowledge and by engaging in various tinkering practices to better 6 Digital Biocommunities: Solidarity and Lay Expertise… 197 manage their symptoms in their daily lives. While acquiring medical
knowledge is an activity that in theory one may conduct individually, the
other processes at the heart of lay expertise generally require multiple
social interactions, as people diagnosed encounter others with the same
condition and start making sense of their experiences by comparing
symptoms, treatment reactions, and life circumstances. Importantly, lay
expertise is developed in conditions where people who are brought
together by virtue of the same diagnosis come to experience feelings of
trust, care, and concern for each other. This means that such processes are
importantly underpinned by affective practices, by the various strategies
through which people diagnosed manage their affects and seek to pro
duce specific affects in those they interact with. Nevertheless, previous
studies on lay expertise have mainly focused on the epistemic processes
through which people diagnosed become very knowledgeable about their
condition, and have generally neglected the affective practices, which
support the processes of knowledge acquisition, exchange, and develop
ment. While such a lack of attention may be informed by the age-old
dichotomy between ratio and affect, it is regrettable in a context where
scholars have highlighted the epistemic value of emotions (Nussbaum,
2003). This is especially relevant in regard to online practices, as growing
calls have been made to acknowledge them not only as communicative
activities, but also as forms of labor, through which particular identities
are claimed and networks are developed (Clough, 2008; McCosker,
2018; McCosker & Darcy, 2013). To determine the role affective practices can play in the development
of lay expertise online, it is important to understand how emotions have
come to be associated with the sphere of labor. Psychologists have played
an important role in this sense, as in the first decades of the twentieth
century they highlighted the relevance of emotions for professional prac
tices through their engagements with the army and corporations (Illouz,
2008). Lay Expertise and Affective Labor Thus, during the First World War, intelligence tests were devel
oped, followed in the decades thereafter by personality tests and experi
ments on corporate productivity, which came to be increasingly applied
in personnel recruitment and management (Lussier, 2018). Under the
influence of mental healthcare professionals, the ability to control one’s
emotions and to manage those of others became the mark of rational and 198 C. Egher C. Egher self-interested individuals and started to be seen as important compe
tences, which could significantly further one’s professional career (Illouz,
2008). As Hochschild (2012) showed in here studies on emotional labor,
this trend has become all the more pronounced with the rise of the ser
vice economy, as the display of particular emotions is now integral part of
various jobs. Work on and through emotions has not been reserved, how
ever, only to the professional realm, but has also become integral to the
development and management of the successful self in the realm of pri
vate life (Illouz, 2008). Illouz (2018:148) importantly remarked in this
sense that “the growing focus on emotions in the psy-industries and their
rising economic value in corporations and consumer culture (…) are
intertwined with the rising cultural value of emotions in the constitution
of self-identity, social relations and well-being.” Writing and reading have
been at the core of such developments, as they allowed individuals to
decontextualize and fix what had hitherto been transient emotions, to
reflect upon them, and, in so doing, to manage them. This is important
for the analysis described in this chapter, because while such practices
have generally been reserved for private diaries, online platforms allow
these days for “networked public intimacy” (Kitzman, 2004), facilitating
new approaches for online contributors to manage their selves and to lay
claims to particular identities online. p
Since online exchanges involve not only the management of one’s
emotions, but also those of others, the concept of affective labor is used
to study how affective practices contribute to the development of lay
expertise. Affective labor is understood as “labor that produces or manip
ulates affects such as feelings of ease, well-being, satisfaction, excitement,
or passion” (Hardt & Negri, 2004:108), which take place at a pre-visceral
stage of experience. Lay Expertise and Affective Labor Particularly relevant here is Hardt’s (1999:89) view
that affective labor is indicative of “processes whereby our laboring prac
tices produce collective subjectivities, produce sociality, and ultimately
produce society itself.” This perspective allows me to focus on the per
sonal and social value their online engagements may have for people
diagnosed with bipolar disorder. Whereas a growing amount of value is
nowadays generated from the cognition, communication, affect, and the
immaterial actions of online “prosumers,” the debate among scholars
about the role of immaterial labor in digital media economics is still 6 Digital Biocommunities: Solidarity and Lay Expertise… 199 ongoing. Thus, Hardt and Negri (2004) join many others who have criti
cized users’ engagement with digital technologies as a form of free labor
(Lupton, 2014; Mitchell & Waldby, 2010; Terranova, 2000; Waldby &
Cooper, 2008). More recently, however, a number of scholars (Andersson,
2017; Kneese, 2017; McCosker & Darcy, 2013) have shown that other
forms of value or gratification that users of digital technologies may
derive by engaging in immaterial labor need to be considered. This chap
ter builds upon the views of this latter group of researchers, as I argue that
affective practices are an important, even though tacit, element of lay
expertise, shaping it both directly and indirectly, through the collectives
it supports into being. The data underlying this analysis were collected from one French
forum, Troubles Bipolaires, hosted on the online platform Doctissimo, and
from one American forum, bp Hope. Two threads were selected from the
first two pages of thread titles on the Troubles Bipolaires, which means
that they had been among the most recently contributed to when the
selection occurred. They were initiated in 2013 and 2014, respectively,
and by February 20, 2018, one had gathered 1829 replies and the other
17,102. Fifteen threads from the bp Hope forum which had received at
least 30 comments were selected. This selection criterion was determined
by the need for numerous interactions in order to study the development
of community. There is a considerable difference between the number of
interactions studied on the French forum, which were also atypical for
Troubles Bipolaires, and the ones on the American forum. Nevertheless, I
decided to compare the two, in order to understand whether there was
something specific about sociality on these two threads and whether the
content, the contributors, and/or particular uses of online affordances
explained this difference. While Chap. Lay Expertise and Affective Labor 4 focused on the treatment experi
ences of people diagnosed with bipolar disorder, for this chapter data
were collected about two other important aspects in their lives—the lived
experiences of the symptoms of this condition and personal and social life
with/despite bipolar disorder. The data were analyzed using thematic
analysis combined with approaches derived from conversation analysis,
thus following in the footsteps of researchers who approach online inter
actions as forms of naturally occurring exchanges, given that they resem
ble offline dialogue in terms of turn-taking, action, and reaction
(Armstrong et al., 2012; Kaufman & Whitehead, 2016). 200 C. Egher Relevant Similarities Once I have my bearings, it's
ok, but I need to get used to the place. Sometimes this only happens late …. (georgette393, August 20, 2015) Same here, but that's why I often go to places I know. The adaptation
can take long for me … Decidedly the bipos [people diagnosed with bipo
lar disorder] really tend to function the same way …. (+Vie, August 20, 2015) The identification of such commonalities contributed to the contribu
tors’ feeling part of a community to such an extent that it prompted some
of them to make inferences about all people diagnosed with this condi
tion, as +Vie’s comment suggests. The development of a shared idiom of practice further assisted online
contributors to identify commonalities. For instance, frequent contribu
tors on one of the Troubles Bipolaires threads developed the habit of shar
ing and updating elaborate personal descriptions on a separate location
on the forum. This approach helped them discover similarities in terms
of family circumstances, favorite pets, or places where they had lived. It
also had the disadvantage, however, of rendering one’s newcomer status
more obvious, when online contributors did not use these distinct spaces
on the forum as was customary. On bp Hope, the discovery of additional
commonalities was assisted through the development of threads with a
playful, socially informative character, such as “where were you when…”
or “Sharing quotations.” Next to the structured provision of such per
sonal information, online contributors could identify similarities based
on their profile photos, their motto, or online signatures, which conveyed
through words and/or images their interests, hobbies, or political views. Relevant Similarities Online contributors were initially brought together on the fora studied
by one important similarity: they had all been diagnosed with bipolar
disorder. Behind this rather obvious commonality, many other similari
ties were conflated, such as a similar orientation toward bipolar disorder
and similar approaches in trying to make sense of it and to address it
effectively. Thus, long-lasting interactions developed among people who
understood bipolar disorder as a biological condition, determined by
genetic and neurological factors, and which could be managed through
medication. This shared perspective was apparent, for instance, among
online contributors who joked about not having children to prevent the
transmission of their “bipolar genes,” or referred to neural activity and
faulty circuits in their brain to explain some of their behaviors.fi Another commonality online contributors shared was the difficulty to
narrow down the meaning and influence of bipolar disorder on other
aspects of their health. For instance, while in terrible pain because of tri
geminal neuralgia, a chronic pain condition that affects the trigeminal
nerve, Sylvana confessed to feeling uncertain regarding the source of her
pain. Since none of the procedures undertaken had been very successful,
she had started doubting whether the pain she was experiencing was
solely caused by the trigeminal nerve or whether her diagnosis of bipolar
disorder also played a role, either by rendering her more sensitive to the
experience of pain or more resistant to the effects of the medications pre
scribed. In a similar vein, elaine43, a contributor on the forum bp Hope,
confessed to being uncertain whether the loss of memory she was experi
encing was due to aging, hormonal changes induced by the menopause,
neurological changes bipolar disorder had produced in her brain, or the
long-term effect of the medications she had taken for its management. Such common uncertainties were often underlined by similarities in cer
tain aspects of identity, such as age, gender, and level of education.i Online contributors identified additional similarities in the form that
certain symptoms took for them or in the adjustments they required, 6 Digital Biocommunities: Solidarity and Lay Expertise… 201 such as the adaptation to a new location while on holiday, as the exchange
below illustrates: such as the adaptation to a new location while on holiday, as the exchange
below illustrates: Whether I go far away or not, it’s the same. Performing Solidarity Having identified such similarities, online contributors performed soli
darity by sharing personal strategies to better manage bipolar disorder in C. Egher 202 C. Egher daily life, by informing others about the results of their self-experiments,
and by creating a safe environment where concerns, preferences, and
challenges could be expressed. The following exchange is illustrative in
this sense: The part about psychosis resonates with me. People don’t understand it and
are frightened by it. I find that I can’t talk about it with my loves ones
because it just creates more worry. It’s the most isolating part of my illness. j
g
I would add thoughts of self harm to the list. We all deal with it but it’s
not something we can talk about. (beyondblue, March 7, 2015) 1 user thanked author for this post: Mary Beyondblue, Self harm does seem to be a taboo subject, even on here. I understand
the trigger it is for most but I think it’s important to admit when those
feelings are breathing over our shoulders. Not only for our own well being
but so others know they are not alone. MO (midnightowl, March 7, 2015) MO (midnightowl, March 7, 2015) beyondblue’s comment shows that he feels comfortable enough to
accept the thread initiator’s invitation to contribute to a list of less-talked-
about symptoms experienced by people diagnosed with bipolar disorder. The first paragraph is important because it highlights the relational way
in which this contributor experiences his condition as well as the affective
labor he performs, as he takes into account the impact certain topics may
have on his family and acts accordingly. The contrast between such avoid
ance behaviors toward one’s family and the openness of one’s online con
tributions highlights the important social function fulfilled by online
platforms. The switch from “I” to “we” in the second paragraph indicates
that beyondblue feels solidarity with the other online contributors based
on a common, even though rather taboo, symptom. midnightowl’s reply to beyondblue confirms the solidaristic ethos under
lying such sharing practices, as she encourages him to continue to talk
about self-harm as a form of support for others. Performing Solidarity While she does not dwell
upon it, midnightowl acknowledges that such sharing practices also fur
ther the well-being of the contributor, which supports the view put 6 Digital Biocommunities: Solidarity and Lay Expertise… 203 forward by Prainsack and Buyx (2017) that people act simultaneously
out of self-interest and concern for others when engaging in solidaristic
behaviors. Even though it is a light form of participation, Mary’s appre
ciation of beyondblue’s comment suggests that the online affordances on
the forum ensured a minimal degree of reciprocity among information
providers and information seekers and thus contributed to the develop
ment of relationships. Online contributors also performed solidarity by putting time and
effort into identifying reliable sources of information for those with
whom they frequently interacted. As Sylvana was worried about a surgical
procedure she was due to undergo, online contributors answered her
invitation to help: you make me think that I should look for a very specific forum for “people
in my case”. if one of you is willing to do a search for me, I'm interested. (Sylvana,
April 13, 2015)
*
so…. on docti [N.B.link provided]
next
a discussion on vulgaris [N.B.link provided]
then
a forum [N.B.link provided]
and afterwards
a positive testimony [N.B.link provided]
That done, you’ll still need to look around…
Right now I got to go pick up my son…. (Rianne, April 13, 2015) you make me think that I should look for a very specific forum for “people
in my case”. Sylvana’s first sentence highlights the tendency among online contrib
utors to seek interactions with others with whom they share relevant
similarities, and indicates that individuals may be simultaneously mem
bers of multiple online communities, where they focus on different issues
of interest. Rianne’s reply makes it obvious that she invests time in the
context of a busy schedule and uses her online experience and personal
knowledge of Sylvana to identify online sources of information that she
believes would be of help to her online friend. The small description 204 C. Egher C. Egher Rianne provides about the online platforms she selected suggests that for
these online contributors, interactive online platforms where people can
engage in dialogue are important sources of lay expertise, which they find
useful in case of doubt or anxiety. Performing Solidarity The list can also be understood as the
result of affective labor, as Rianne keeps her list short and easily legible,
and includes in it a positive testimony, to further reassure Sylvana. As already alluded to in some of the examples provided, online con
tributors also performed solidarity by engaging in affective labor, by dis
playing emotional availability in their interactions with other people
diagnosed with bipolar disorder, and by listening to them with respect
and empathy over extended periods of time, judging by the dates and
frequency of the comments. At the same time, they showed consideration
for the effects their reactions might have upon their interlocutors, or paid
attention to the latter’s needs and preferences to personalize their advice
and render it more appealing. Online contributors also performed soli
darity as they sought to motivate people who were going through a dif
ficult time and offered support to those who were experiencing serious
mood episodes, as the following exchange illustrates: 6 Digital Biocommunities: Solidarity and Lay Expertise… There were, however, also important “costs” to the performance of soli
darity. Thus, considerable time was necessary to provide advice and sup
port through well-balanced and carefully considered comments, as was
evidenced by replies where contributors acknowledged other people’s
requests for input, but mentioned that they needed to reflect before pro
viding them with an answer. The provision of information about the
effects and side effects of medications that online contributors had taken
at some point along their bipolar trajectories required at times rather
painful journeys into their past, a revival of periods marked by pain and
suffering. Furthermore, people diagnosed with bipolar disorder shared
with others with whom they acknowledged certain similarities strategies
to manage their condition at the level of daily life, which were often the
result of extensive tinkering. While these “costs” were considerable, online
contributors underwent them as the insights they put forward not only
benefitted others, but also themselves, as they became better aware of
their own behaviors and reactions. The identification of important simi
larities facilitated the development of a new type of collectivity, what I
call “digital biocommunities,” which I discuss below. I had already taken the anxiolytics… I had already taken the anxiolytics… But I’ve managed to ask someone to help me on a forum because I
couldn’t take it anymore. Someone reacted and we’re talking via private
messages. I think this will help me a bit. Thanks. (Derek21, March
18, 2013)
* O.K. If I can also be of any help, it would be my pleasure, even if we
haven’t talked much…. (Liane, March 18, 2013) O.K. If I can also be of any help, it would be my pleasure, even if we
haven’t talked much…. (Liane, March 18, 2013) The importance of the help online contributors provide each other is
highlighted here, as Derek21 frames the interaction with another person
diagnosed with bipolar disorder as an additional therapeutic means to
manage anxiety. While Liane describes herself as “pathologically pathetic”
in her online signature, her reaction suggests that engaging in solidaristic
practices online may constitute a way to claim a different identity, that of
someone strong and capable enough to support another person with
whom she shares an important similarity in a dark moment. 205 6 Digital Biocommunities: Solidarity and Lay Expertise… Digital Biocommunities and Their Roles As the insights provided above illustrated, the online contributors studied
here understood their condition in relational terms, by discovering
important similarities with others and by making sense of their various
experiences through interactions on the fora. The recognition of these
commonalities and the performance of solidarity led to an atmosphere of
shared intimacy, which made online contributors feel at ease and
prompted them to give more detailed and personal information about
themselves. This facilitated the development of digital biocommunities, a
new type of subgroup that emerged based on increasingly more specific
commonalities, including a shared idiom of practice regarding the use of
digital technologies. Based on the analysis, it became apparent that the
coming into being of this new type of collectivity was underlined by
engagements in three types of affective labor: the management of per
sonal affects, the artful display of affective responses, and the careful 206 C. Egher C. Egher orchestration of empathy and distance. This way, relations among a grow
ing number of contributors emerged and were maintained and new
knowledge could be produced, as people diagnosed with bipolar disorder
were able to further their self-knowledge, to perform lay expertise, and to
contribute to its collective development. The attachments and sharing practices developed among the members
of these digital biocommunities enabled them to approach their online
engagements as reliable ill-health indicators, as the following excerpt
illustrates: But anyways, I have the feeling that over the last weeks it’s been less bad
going down [N.B. becoming depressed], so it should be less bad going up
[N.B. becoming manic]h Though when I think about it, I was in such bad shape that I didn’t
come here anymore… It’s crazy how much we forget as time goes by… It’s crazy how much we forget as time goes by… What are you up to now? (Rianne, February 23, 2015) What are you up to now? (Rianne, February 23, 2015) Thus, Rianne appreciated the severity of her depressive episode by
ascribing a considerable weight to her inability to join the forum, as it
prompted her to reassess her initial evaluation. For this contributor, par
ticipation in this digital biocommunity had become part of how she
experienced bipolar disorder, which signals the strength of the social
bonds she had developed there. Digital Biocommunities and Their Roles By developing digital biocommunities, online contributors increas
ingly related to the digital technologies they used as particular means to
act upon disease, as the relative permanence of their posts and the close
ness of their interactions with others enabled them to further their self-
knowledge and to better manage their condition. This was facilitated by
the affordances of fora and by a shared idiom of practice, which allowed
for the interpretation of certain online behaviors as markers of particular
(ill-health) states. Thus, online contributors could heighten their self-
knowledge through their engagements with the posts they had made on
the fora over extended periods of time. Since these posts were accompa
nied by details regarding the time and date when they were made, they
functioned as a form of public online diaries, from which people 6 Digital Biocommunities: Solidarity and Lay Expertise… 207 diagnosed with bipolar disorder discerned specific patterns. This allowed
them to identify triggers for certain mood episodes, or to improve their
assessment of the mood state they experienced, as the following excerpt
illustrates: it’s been the most agonizing thing i’ve ever experienced. i’d prefer to go
through labor and childbirth, because as least when that’s over, it’s
OVER. and besides, it’s way less painful than feeling like your soul is being
tortured and set on fire. it’s been the most agonizing thing i’ve ever experienced. i’d prefer to go
through labor and childbirth, because as least when that’s over, it’s
OVER. and besides, it’s way less painful than feeling like your soul is being
tortured and set on fire. i
it will usually begin with a general feeling of anxiety for no discernable
reason maybe because i’m bored and don’t feel distrated wnough from my
evil thoughts. OR something extremely minor will make me IRATE, such
as getting curly fries when i asked for regular. by then, it’s too late, and i’m
angrily yelling and/or throwing my food. my head starts buzzing with a feeling of electricity/energy, and it feels
like a fly is zipping around my brain, bouncing off the inside of my skull. there’s an unbearable roar in my brain and i cover my ears, shake my head,
and scream/cry. i want to jump out of my skin. i curl up in a ball, in a dark,
quiet, small room, and i’m paralyzed there, totally unable to function (…). Digital Biocommunities and Their Roles I want to knock myself unconscious to get rid of the pain, when they are
REALLY bad (…) they are very hard to get out of. and now i’m so manic i’m misspelling
evry other word, so i know those aren’t even close to all of what is going on
inside, but i will surely upset myself if i try to slow down here and think
anymore. :-/” (noone31, November 6, 2013) noone31 provides a thick description of her experiences of mixed states
by mobilizing highly evocative as well as more broadly relatable compari
sons, which help make her state intelligible to others. The last part of her
post indicates that elements of online communication, such as misspell
ing, function for this contributor as markers of a severe manic episode. Furthermore, the way in which the evocative description of her states is
organized, its rhythm and punctuation suggest that the post can also be
understood as a digital enactment of this mood state. Such practices can
therefore be seen as important steps toward achieving self-management
and self-change, as they allow disease experiences to be “defined, labeled,
and categorized” (Illouz, 2008:196). 208 C. Egher C. Egher C. Egher Other contributors enhanced their self-knowledge through the sub
stantial knowledge other members of the community had acquired about
them, which allowed the latter to mobilize the shared idiom of practice
to interpret “deviant” engagements with the technologies of fora as dis
ease markers. For instance, very short replies or the absence of any emoti
cons across several contributions provided by the same person was seen as
a mark of flat affect, and thus indicative of a depressive episode. Similarly,
in a context where forum interactions tended to be rather short and to
succeed each other quickly, the provision of very long comments, some
times stretching over the equivalent of six to seven pages, was seen as
indicative of a manic episode. The following exchange is illustrative in
this sense: Vana…are you in good shape or is it just an effect of the screen??? (Rianne,
March 4, 2015)
* Vana…are you in good shape or is it just an effect of the screen??? (Rianne,
March 4, 2015)
* the optical effect conveys a true reality! I have been in an up [in a manic
state] for some time now; I'm even starting to think it's my normal state
and nothing will upset it ( )…. (Sylvana, March 4, 2015) The community-building function of the shared idiom of practice
comes into relief here through the use of the euphemism “to be in good
shape,” which for the members of this digital community denoted a
manic state, and through the emoticons and brackets at the end of
Sylvana’s post, which were appropriately interpreted by these contribu
tors. It is important to note the distinct functions fulfilled by the two
emoticons and brackets. Through the Red Face emoticon, characteristic
for Doctissimo (Lombart, 2018), Sylvana conveys her anger and exaspera
tion at not being able to manage her feelings, whereas the second emoti
con fulfills a relational function, as Sylvana uses it to connect with Rianne,
to express regret about the impact the state she finds herself in may have
upon her. This illustrates how affective labor can contribute to the main
tenance of one’s online network. C. Egher Self-knowledge was furthered among the members of digital biocom
munities also through the consultations they engaged in, as they actively 6 Digital Biocommunities: Solidarity and Lay Expertise… 209 invited others to help them interpret their experiences and to determine
the mood states they were in: invited others to help them interpret their experiences and to determine
the mood states they were in: in favor of the up [manic episode]: in favor of the up [manic episode]: in favor of the up [manic episode]: I get up every day around 4 pm I started to put on a lot of jewels whereas for months I had only been
wearing my wedding ring and the one of my deceased mother I started to put on a lot of jewels whereas for months I had only been
wearing my wedding ring and the one of my deceased mother against the up [manic episode]: against the up [manic episode]: I don’t feel excited I don’t do compulsive shopping I am not aggressive (Sylvana, October 1, 2015) Sylvana interpellates the other online contributors as experts, who not
only have substantial experiential knowledge on bipolar disorder, but also
know her very well. She invites them to perform lay expertise by replicat
ing to a certain extent the activities of medical professionals when seeking
to establish a diagnosis. Thus, she describes her online and offline behav
iors as clues which they can use toward the correct identification of her
state. To assist the other online contributors, she places her behaviors in
context, providing information about their frequency and about her own
emotions in regard to them. Through intensive online interactions with
others, Sylvana and other contributors like her could bring in relation to
bipolar disorder aspects of their behaviors they had not previously con
sidered to be shaped by it, or to identify certain patterns which in time
enabled them to better manage this condition. This allowed online con
tributors to further their self-knowledge, as aspects of the self which may
have been opaque or ambiguous to the individual diagnosed with bipolar
disorder were clarified relationally. Furthermore, such exchanges may
have (had) performative effects on each contributor in ways similar to the
narratives disseminated through other media, as signaled by Illouz
(2008:185), who argued that one’s public illness account “compels him
or her to change and to improve his or her condition (…) It makes one
responsible for one’s future but not for one’s past.” In Chap. 4, I have argued that people diagnosed with bipolar disorder
could contribute to the development of new insights about the effects
and side effects of medications through their online engagements on 210 C. Egher blogs and fora. in favor of the up [manic episode]: The findings described above have cast light upon a differ
ent dimension of their contributions, as they indicate that fora can be
used to enhance the knowledge online contributors acquire about them
selves and others in regard to the manifestations of bipolar disorder and
how it shapes their personhood. Such exchanges also enable them to per
form lay expertise or to contribute to its collective development, which I
will discuss in more detail below. 1.Hi Quickjeanie, (and welcome), and Gill, I am diagnosed with BP2,
rapid cycling, mixed 2.states,
3 and I’ve definitely experienced those days with the crying jags that 6 Digital Biocommunities: Solidarity and Lay Expertise… 5.It’s defiantly frustrating and confusing. 6.For me its usually something triggers me or I’m under stress when this
happens. Or 7.I’m under a medications change or even hormones can do it. 6.For me its usually something triggers me or I’m under stress when this
happens. Or 7.I’m under a medications change or even hormones can do it. 8.I think the best idea for learning about these shifts is to keep a daily
journal. You don’t 9.have to write full diary entries, but keeping track of
your moods, stressors, triggers, 10.medications, even the weather all help
you to establish patterns to help you learn to 11.combat these quick shifts. Its also a good tool to take to your Pdoc to be able to 12.discuss these issues
with them. I think coping skills you can learn in therapy are a big 13.help
as well. Learning some deep breathing exercises, how to identify those trig
gers, 14.etc. goes a long way to helping the medications.fi 15.Just know your not alone, and although it’s difficult, try not to be to
hard on yourself. MO (midnightowl, April 5, 2015) 15.Just know your not alone, and although it’s difficult, try not to be to
hard on yourself. MO (midnightowl, April 5, 2015)
* 1.Jeanie, 2.it sounds like the severity of this particular mixed episode was very
unexpected. 3.I believe when something this terrible happens, if we’re not at all pre
pared, it’s even 4.worse. 3.I believe when something this terrible happens, if we’re not at all pre
pared, it’s even 4.worse. 5.How could you prepare for such a thing when you’ve never had this
happen before. 5.How could you prepare for such a thing when you’ve never had this
happen before. 6.I need to make a safety plan for the unexpected episode that could put
me in harm’s 7.way. 6.I need to make a safety plan for the unexpected episode that could put
me in harm’s 7.way. 8.Anyone of us could experience what happened to you. Bipolar is
unpredictable. Meds 9.and therapy and a host of other wellness skills can
not completely protect us. For me 10.this is why a safety plan is so
important. 8.Anyone of us could experience what happened to you. Bipolar is
unpredictable. Meds 9.and therapy and a host of other wellness skills can
not completely protect us. For me 10.this is why a safety plan is so
important. Solidarity and Lay Expertise The analysis of the data indicated that lay expertise on bipolar disorder
developed as an effect of the solidaristic practices which prompted online
contributors to share their embodied, experiential knowledge and the
medical insights they had acquired on this condition. The exchange
below is a good example in this sense as it illustrates how different con
tributors came to discover common elements, which moved them to
share effective strategies, but also to assume different epistemic positions: 1.Hi everyone, I have bipolar I disorder and 2.have recently experienced being in mixed state the worst I have ever
been. It was3.easily the scariest thing I have ever gone through. I was crying
uncontrollably at my4.friends house and couldn’t stop. 2.have recently experienced being in mixed state the worst I have ever
been. It was3.easily the scariest thing I have ever gone through. I was crying
uncontrollably at my4.friends house and couldn’t stop. 5.I can’t explain it to other people very well. 6.My feelings were SO up and down back and forth all at once. The cry
ing wouldn’t7.stop. 8.My friends try to be understanding about having bipolar disorder but
they struggle to 9.really relate. 10.How can I blame them? I 7.am a bit embarrassed about what hap
pened last week. 11.Does anyone have any tips for me?—Jeanie (Quickjeanie, April
5, 2015)
* 11.Does anyone have any tips for me?—Jeanie (Quickjeanie, April
5, 2015)
* 1.Hi Quickjeanie, (and welcome), and Gill, I am diagnosed with BP2,
rapid cycling, mixed 2.states,
3.and I’ve definitely experienced those days with the crying jags that
accompany an 4.ordinary or slightly hypomanic day. 211 6 Digital Biocommunities: Solidarity and Lay Expertise… 6 Digital Biocommunities: Solidarity and Lay Expertise… 11.When I have a mixed episode (most all of my bipolar is mixed and
also rapid cycle) I 12.don’t cry. Pretty much I never cry, even when I want
to. My symptoms are extreme 13.agitation and irritability combined with
depression.14.There are two things that help: #1. Exercise (this is my first
line of defense) #2. 15.Watching a movie (preferably after I’ve exercised so I’m calm downed
enough to enjoy) (elaine43, April 5, 2015) 15.Watching a movie (preferably after I’ve exercised so I’m calm downed
enough to enjoy) (elaine43, April 5, 2015) This exchange follows a two-part sequence often encountered in the
interactions of psychotherapists with their patients (Wynn & Bergvik,
2010). Thus, a first “troubles-talk” (Jefferson, 1988) sequence, where 212 C. Egher C. Egher Quickjeanie describes her feelings, thoughts, and states to indicate the
difficult situation she finds herself in, is followed by a second sequence
where midnightowl provides a supportive response. Another second
sequence is provided, as elaine43 reacts to Quickjeanie’s post before the
latter has the opportunity to engage with midnightowl’s statement. While
Quickjeanie opens her sequence in similar ways to other contributors on
the thread, a significant feature of this post is the question at the end,
which serves as a direct request for advice based on the same diagnosis
and similar experiences. It also indicates that Quickjeanie positions her
self as a non-expert in regard to the management of this group of symp
toms, and considers other forum contributors to be more knowledgeable. By reacting to her post and thereby responding to her interpellation, mid
nightowl and elaine43 situate themselves as experts in this context, and
their posts include various elements meant to justify it. Interesting about
the way in which Quickjeanie organizes her post is the new theme she
introduces in the middle of her description of experienced symptoms
(lines 2–4 and 6–7). Through it, this contributor both acknowledges her
communicational difficulties and suggests that people who lack experien
tial knowledge of the symptoms she describes may have a hard time prop
erly understanding them. This is further reinforced by her expectation
that people on the forum would be able to provide her with advice others
in her immediate surroundings were not able to give her, as denoted by
her question. 6 Digital Biocommunities: Solidarity and Lay Expertise… q
midnightowl seeks to convey alignment with the experiences recounted
by Quickjeanie by mirroring to a large extent the organization the latter
opted for in her post. Like Quickjeanie, she begins her sequence with a
greeting, followed by information about her diagnosis, and a description
of her experiences with mixed states. This serves both to legitimate her
knowledge and to highlight this as an important element she and
Quickjeanie have in common. This exchange illustrates the careful orches
tration of empathy and distance that online contributors engage in to
perform lay expertise, as midnightowl responds reassuringly to the latter’s
expectation of empathy (line 5), but moves on to the provision of knowl
edge, by showing her awareness of particular triggers and by using medi
cal terms. The next and more extensive part of her reply is the response to
Quickjeanie’s direct question, and consists of various suggestions on how 6 Digital Biocommunities: Solidarity and Lay Expertise… 213 the latter could better manage her mixed states. The order of these ele
ments in midnightowl’s post is important, as the move from personal dif
ficulties to strategies serves to establish her expertise. The authority of her
claims thus significantly derives from her ability to successfully, albeit
temporarily, address the challenging symptoms she describes and to man
age the emotions arising along with them. midnightowl concludes her
post with a display of solidarity, as she encourages Quickjeanie to think of
herself as part of a community and provides a caring suggestion in reac
tion to the latter’s statement that she was “a bit embarrassed” by her
behavior. The similarity midnightowl presumes to exist between her and
Quickjeanie is further predicated upon common emotions. This is indi
cated in this part by the preemptive statement “although it’s difficult,”
which signals that midnightowl recognizes this affective state, and is aware
both of how the contributor might react to this suggestion and of the
actual effort required to follow up on it. Such affective labor legitimates
the emotions and experiences described by others and lends greater epis
temic authority to the advice provided. y
p
elaine43 organizes her reply to Quickjeanie in a different way, dedicat
ing a large part of her contribution to the expression of empathy and the
display of solidarity. The first sentence is meant to authenticate
Quickjeanie’s experiences as well as to soothe the feelings of embarrass
ment the latter described. 6 Digital Biocommunities: Solidarity and Lay Expertise… The switch from “I” to “we” in line 3 is impor
tant in relation to solidarity, as it shows that elaine43 thinks of herself,
Quickjeanie, and presumably other people experiencing difficulties with
the management of their symptoms as part of a community, herewith
echoing the last part of midnightowl’s post. At the same time, elaine43
distinguishes among people diagnosed with bipolar disorder based on
their familiarity with the condition, as she pleads to Quickjeanie not to
feel guilty by framing her as an inexperienced novice. This is a perspective
that she nuances by distinguishing between the agency she ascribes peo
ple diagnosed with bipolar disorder and the condition itself, as the under
standing of bipolar disorder as “unpredictable” and capable of catching
off guard any person diagnosed supports her suggestion of creating a
safety plan.h This perspective on bipolar disorder contrasts the one advocated by
midnightowl, who enumerated various options to manage one’s condition 214 C. Egher which she considered effective (lines 8–14), as emphasized through the
use of the superlative adjective “best” and of qualifying adjectives with a
positive (contextual) value in assessments such as “big help,” “good tool,”
“long way.” Without directly interpellating midnightowl, elaine43 engages
with the elements in her enumeration, resisting the largely optimistic
tone of her message. This move suggests that elaine43 conceives of soli
darity in ways which allow one to have distinct individual experiences
while still being part of a large community of sufferers. This can be noted
in the positioning of “us” and “for me” next to each other in line 9. It is
further reinforced in lines 11–13, where even though the mixed states
elaine43 describes are the opposite of those experienced by Quickjeanie
and midnightowl, she still shares her own coping strategies. This contribu
tor thus seems to base her solidaristic practices on the same diagnosis and
to consider this a sufficient commonality for the same strategies to be
effective, even when the condition manifests itself differently. Since these
elements mirror through their position the location of Quickjeanie’s
request for advice in her post, they also serve to provide a sense of
completion. In the examples provided above, the performance of lay expertise was
achieved through the careful combination of empathy and distance, and
the efforts the online contributors were making to manage the flow of
affects triggered by the experiences described by others were understated. 6 Digital Biocommunities: Solidarity and Lay Expertise… There were, however, also numerous instances, where the intensity of
these affects was in full display, as the following quote illustrates: It is true that you are courageous it's amazing I had tears in my eyes [when
reading your account]. I would love to be able to help you but I don’t know what to say to you
I swear I'm sad for you Vana [N.B.Sylvana] I have always said that I didn’t want anybody else to know the pains I’m
experiencing and now it happens to you and it makes me sad and I feel
your pain and I don’t know what to do. Know that violent noises, fatigue, fear, sadness, anger, anxiety, panic will
accentuate your pain. Also the cold as well as burning things. Unlike them,
what is soft will relieve your pain… 6 Digital Biocommunities: Solidarity and Lay Expertise… 6 Digital Biocommunities: Solidarity and Lay Expertise… 215 Don’t take too many analgesics because the sleepier you’ll feel, the more
your muscles will tense. The brain takes it as a signal, like, saying: “Beware! I won’t let go of anything!” Have you been advised to take cortisone in low
dosages? On my face it works well but on my legs it never led to any results. Good and sweet night. (Lera, April 5, 2015) This comment thus highlights the affective labor through which the
negative affective reactions triggered by Sylvana’s post—one of the emo
tional costs of contributing online for people diagnosed with bipolar dis
order—are turned into means through which Lera can relate to her, while
the memory of the latter’s own suffering serves to validate Sylvana’s expe
riences. Furthermore, Lera engages in “caring work,” a key form of affec
tive labor (McCosker & Darcy, 2013), to alleviate Sylvana’s state by
expressing empathy, by encouraging and reassuring her. Building upon
these affective practices enables her to perform lay expertise, as she advises
Sylvana on the emotional and physical states that she should avoid to bet
ter manage the pain by combining embodied knowledge with medical
information. The personal insights people diagnosed with bipolar disorder shared
online and the detailed descriptions of their states and behaviors enabled
others to increase their knowledge about this condition in regard to
aspects that they did not personally experience, as the quote below
illustrates: How the illness transforms a person…. I know the mixed mood state through you Ria…. I could write volumes about it! I had even strongly thought of it as an
outlet it's not bad except that you have to stick to it. And at the moment concentration is not one of my strengths. (georgette393, January 18, 2016) While no individual diagnosed with bipolar disorder can have experi
ential knowledge about all the symptoms of this condition, through their
frequent interactions with other people diagnosed, online contributors
come to develop lay expertise about it and to enrich their personal knowl
edge through other people’s first-hand accounts. This is important, 216 C. Egher C. Egher because it shows that these contributors are not only interested in grasp
ing the individual manifestations of their condition, but they want to
acquire a thorough understanding of bipolar disorder, which is only pos
sible by accumulating different types of knowledge and by relating their
experiences to those of others. 6 Digital Biocommunities: Solidarity and Lay Expertise… Overall, the epistemic relevance of these
insights was often publicly acknowledged, as the following excerpt
illustrates: Thank you for your personal experiences you have helped me understand a
lot more about myself. I only wish my clinical psych was as clear about this
as the information I’ve managed to understand here. (Polar1, May 18, 2016) Discussion This chapter has shown that despite individualizing tendencies in person
alized and precision medicine, solidarity remains an important value for
people diagnosed with bipolar disorder and it underlies the performance
and collective development of lay expertise. Thus, rather than focusing
on the distinctions between themselves and others, the online contribu
tors studied here identified important similarities with each other, which
prompted them to incur personal costs in order to provide others with
help and support. To account for this innovative coming together, I put
forward the concept of digital biocommunities to denote the develop
ment of (sub)groups based on numerous commonalities of experience
and similar engagements with the technologies of fora. By developing
digital biocommunities, online contributors related to the digital tech
nologies they used as particular means to act upon disease. While such
statements are nowadays often made in relation to digital mental health
applications, which provide quantified insights or visualizations, this
chapter has illustrated that people’s ability to manage bipolar disorder is
enhanced through the narratives, thick descriptions, and dialogue that
fora and similar interactive online platforms allow for. Online contribu
tors can further their self-control and better navigate daily life through
the practices of self-revelation/clarification and collective consultation in
which they engage. In so doing, they also contribute to the development 6 Digital Biocommunities: Solidarity and Lay Expertise… 217 of lay expertise about this condition, as a more unified and comprehen
sive image of bipolar disorder and its manifestations at the personal level
emerges through frequent online exchanges.h g
g
q
g
The development of lay expertise traced here depended not only on
epistemic practices, but also on the ability of online contributors to
appeal to the considerations, emotions, and perspectives of their inter
locutors, and on their display of sympathy and empathy. The sharing of
experiential and other types of knowledge required for the development
of lay expertise was also informed by the feelings of well-being that online
contributors experienced in so doing, as they could temporarily position
themselves as knowledgeable, capable, and supportive rather than frail,
vulnerable, and in need of help. Thus, for knowledge to be shared, circu
lated, and produced, it was not enough for people diagnosed to identify
relevant similarities, but they also needed to engage in the affective labor
required when interacting publicly with multiple individuals. Discussion Affective
practices and engagements play therefore an important role in the pro
duction of knowledge, even though these aspects have been thus far
largely neglected in social studies of science. Furthermore, the findings
discussed here illustrated that affective labor is more than unpaid work,
as, through it, online contributors could perform a value they found
important; they acquired self-knowledge and contributed to the develop
ment of collective knowledge on the management of bipolar disorder.hi The close link between solidarity and lay expertise that these findings
illustrated is important in the current context where knowledge is increas
ingly referred to as a resource that can be privately owned (Newell, 2015)
and is thus more often related to other values, such as competitiveness
and efficiency. Nevertheless, it is important to bear in mind that solidar
ity is not in itself a positive value (Dean, 1995). As people come together
with others with whom they share important similarities and are willing
to incur costs in order to assist them, they also distinguish themselves
from those with whom they do not share such similarities. Such tenden
cies could also be noted in this chapter, as some online contributors dis
tinguished in essential ways between people who were diagnosed with
bipolar disorder and those who were not. While such processes of inclu
sion and exclusion may not be prevented, for digital biocommunities to
continue to have positive effects, it is important that their members 218 C. Egher C. Egher reflect upon the criteria they use to include and exclude others and upon
the consequences such practices may have. q
p
y
Digital biocommunities bear some resemblance to self-help groups,
which in the past have facilitated the development of a common identity
among people diagnosed with contested conditions, such as the fibromy
algia syndrome (Barker, 2002). This is in line with previous findings that
have shown that online communities share with their offline predecessors
similar objectives, work practices, modes of approach, and orientation
toward cognitive resources (Akrich, 2010). The development of digital
biocommunities can be interpreted as indicative of a growing need among
people diagnosed to come together, share experiences, and support each
other in a context marked by the increased deregularization of mental
healthcare services. This is supported by the fact that both in the US and
in France the number of self-help and support groups, described in more
detail in Chap. 6 Digital Biocommunities: Solidarity and Lay Expertise… The solidaristic practices described in this chapter were identified at a
time when the pronounced individualization of responsibility brought
about by personalized and precision medicine has led many scholars to
approach solidarity as a value that is under threat and in need of protec
tion (Aarden et al., 2010). The resilience of solidarity in this context indi
cates that it is a very important value to people, who find solace in
knowing that they are not alone in experiencing specific issues. The con
cept of digital biocommunities suggests that as people come together
based on increasingly more specific commonalities of experience, they
might form part of multiple dynamic (sub)groups, depending on the
similarities they focus upon and the solidaristic practices they engage in. This has consequences for the ways in which personhood and “personal
ization” are understood, as it strengthens the idea that they are defined
and re-defined through social interactions and practices which are mean
ingful to people diagnosed. Hopefully, through their multitude and
diversity, the development of digital biocommunities will provide people
diagnosed with bipolar disorder with safe havens, where they can feel at
ease and where they can become better aware of their talents, strengths,
and knowledge, and of the important values they uphold as they share
them with others. Discussion 2, has been increasing over the last few decades (Fox,
2011; Girard, 2008). Since background conditions can further or deter
solidaristic practices (Prainsack & Buyx, 2017), more research is needed
to understand how they affect online engagements, and what role the dif
ferent affordances and designs of fora and other interactive online plat
forms play in such developments and what types of solidarity are thereby
encouraged. g
While self-help groups have been historically less influential in France
than in the US, solidarity is considered by many to be a national value in
France, which might explain the more numerous and frequent exchanges
to support others in need on the Troubles Bipolaires threads. The role of
cultural and social factors in explaining such distinct online behaviors
was further reinforced by the fact that on two other American fora, which
were consulted to compare the number of participants and their interac
tions, few threads exceeded 30 comments, let alone reach hundreds or
thousands. The distinct online landscape available for both countries may
have been another influencing element, as infrastructural, economic, and
institutional factors have shaped the development of a dispersed online
environment in the US and a more centralized one in France. Future
studies are therefore needed to acquire a better understanding of the spe
cific factors that inform such differences in online participation and sup
port between contributors from the US and France. 219 6 Digital Biocommunities: Solidarity and Lay Expertise… References Aarden, E., van Hoyweghen, I., & Horstman, K. (2010). Solidarity in Practices
of Provision: Distributing Access to Genetic Technologies in Health Care in
Germany, the Netherlands and the United Kingdom. New Genetics and
Society, 29(4), 369–388. Akrich, M. (2010). From Communities of Practice to Epistemic Communities:
Health Mobilizations on the Internet. Sociological Research Online, 15(2), 10. Available at http://www.socresonline.org.uk/15/2/10.html Akrich, M., Méadel, C., & Rémy, C. (2008). Building Collectives via the Web? Information and Mobilization on Cancer Websites. Virtually Informed,
2008, 13–40. Andersson, Y. (2017). Blogs and the Art of Dying: Blogging With, and About,
Severe Cancer in Late Modern Swedish Society. OMEGA-Journal of Death
and Dying, 79, 1–20. https://doi.org/10.1177/0030222817719806 220 C. Egher Armstrong, N., Koteyko, N., & Powell, J. (2012). “Oh Dear, Should I Really Be
Saying That on Here?” Issues of Identity and Authority in an Online Diabetes
Community. Health, 16(4), 347–365. Barker, K. (2002). Self-Help Literature and the Making of an Illness Identity:
The Case of Fibromyalgia Syndrome (FMS). Social Problems, 49(3), 279–300. h
Bayertz, K. (Ed.). (1998). Solidarität: Begriff und Problem. Suhrkamp. Baylis, F., Kenny, N., & Sherwin, S. (2008). A Relational Account of Public
Health Care Ethics. Public Health Ethics, 1(3), 196–209.hf Clough, P. (2008). The Affective Turn. Political Economy, Biomedia and Bodies. Theory, Culture & Society, 25(1), 1–22. Clough, P. (2008). The Affective Turn. Political Economy, Biomedia and Bodies. Theory, Culture & Society, 25(1), 1–22. Dean, J. (1995). Reflective Solidarity. Constellations, 2(1), 114–140. Dickenson, D. (2013). Me Medicine vs. We Medicine: Reclaiming Biotechnology
for the Common Good. Columbia University Press.h Durkheim, E. (1964). The Division of Labor in Society. Free Press.hf Epstein, S. (2007). Inclusion. The Politics of Difference in Medical Research. The
University of Chicago Press. Etzioni, A. (1988). The Moral Dimension. Toward a New Economics. Free Press. y
g
ioni, A. (1988). The Moral Dimension. Toward a New Economics. Free Press Evers, K. (2009). Personalized Medicine in Psychiatry: Ethical Challenges and
Opportunities. Dialogues in Clinical Neuroscience, 11(4), 427–434.h Foster, C. (2009). Choosing Life, Choosing Death: The Tyranny of Autonomy in
Medical Ethics and Law. Hart. Fox, S. (2011). Peer-to-peer healthcare. California Healthcare. G
h
I (2010) Th B
k
2 0 Di
i
N Fox, S. (2011). Peer-to-peer healthcare. California Healthcare. Gershon, I. (2010). The Breakup 2.0. Disconnecting over New Gershon, I. (2010). The Breakup 2.0. Disconnecting over New Media. Cornell
University Press. E-book. Girard, V. (2008). References Dossier n° 13- Auto Support en Santé Mentale en France. Bulletin Amades, 75, 1–9.f Hardt, M. (1999). Affective Labor. Boundary, 26(2), 89–100. Hardt, M., & Negri, A. (2004). Multitude. War and Democracy in the Age of
Empire. The Penguin Press. h
Hechter, M. (1987). Nationalism as Group Solidarity. Ethnic and Racial Studies,
10(4), 415–426. Hedgecoe, A. (2004). The Politics of Personalised Medicine: Pharmacogenetics in
the Clinic. Cambridge University Press. Hochschild, A. (2012). The Managed Heart. Commercialization of Human
Feeling (3rd ed.). University of California Press. Hogle, L. (2016). Data-intensive Resourcing in Healthcare. BioSocieties,
11(3), 372–393. Illouz, E. (2008). Saving the Modern Soul. Therapy, Emotions, and the Culture of
Self-Help. University of California Press. 6 Digital Biocommunities: Solidarity and Lay Expertise… 6 Digital Biocommunities: Solidarity and Lay Expertise… 221 Illouz, E. (2018). Emotions as Commodities. Capitalism, Consumption and
Authenticity. Routledge. Kaufman, S., & Whitehead, K. (2016). Producing, Ratifying, and Resisting
Support in an Online Support Forum. Health, 1–17. Advance online publi
cation. https://doi.org/10.1177/1363459315628043 Kitzman, A. (2004). Saved from Oblivion: Documenting the Daily from Diaries to
Web Cams. Peter Lang. Kneese, T. (2017). Mediating Mortality: Transtemporal Illness Blogs and Digital
Care Work. In S. Boret, S. Long, & S. Kan (Eds.), Death in the Early Twenty-
First Century (pp. 179–213). Palgrave Macmillan. Knoppers, B., & Chadwick, R. (2005). Human Genetic Research: Emerging
Trends in Ethics. Nature, 6, 75–79. Komter, A. (2005). Social Solidarity and the Gift. Cambridge University Press. b
Th
h Lombart, E. (2018). The Emoticon Functions in Antiphrasis Interpretation on
Doctissimo’s Forums. In L.-A. Cougnon, B. De Cock, & C. Fairon (Eds.),
Language and the New (instant) Media (pp. 1–17). Presses Universitaires
de Louvain. Lupton, D. (2014). The Commodification of Patient Opinion: The Digital
Patient Experience Economy in the Age of Big Data. Sociology of Health &
Illness, 36(6), 856–869.hi Lupton, D. (2016). The Quantified Self. A Sociology of Self-Tracking. Polity Press. ton, D. (2018). Digital Health. Routledge. Lupton, D. (2018). Digital Health. Routledge. Lussier, K. (2018). Temperamental Workers: Psychology, Business, and the
Humm-Wadsworth Temperament Scale in Interwar America. History of
Psychology, 21(2), 79–99. Mayhew, L. (1971). Society: Institutions and Activity. Columbia Universi Mayhew, L. (1971). Society: Institutions and Activity. Columbia University Press. McCosker, A. (2018). Engaging Mental Health Online: Insights from beyond
blue’s forum influencers. New Media & Society, 20(12), 4748–4764. y
y
McCosker, A. (2018). Engaging Mental Health Online: Insights from beyond
blue’s forum influencers. New Media & Society, 20(12), 4748–4764.f McCosker, A., & Darcy, R. (2013). Living with Cancer. Affective Labour, Self-
Expression and the Utility of Blogs. Information, Communication & Society,
16(8), 1266–1285. Mitchell, R., & Waldby, C. (2010). National Biobanks: Clinical Labor, Risk
Production, and the Creation of Biovalue. Science, Technology & Human
Values, 35(3), 330–355. Newell, S. (2015). Managing Knowledge and Managing Knowledge Work:
What We Know and What the Future Holds. Journal of Information
Technology, 30, 1–17. Nussbaum, M. (2003). Upheavals of Thought: The Intelligence of Emotions. Cambridge University Press. 222 C. Egher Ozomaro, U., Wahlestedt, C., & Nemeroff, C. (2013). Personalized Medicine
in Psychiatry: Problems and Promises. BMC Medicine, 11, 132.h Parsons, T. (1952). The Social System. Tavistock. Prainsack, B. (2017). Personalized Medicine. Empowered Patients in the 21st
Century? 6 Digital Biocommunities: Solidarity and Lay Expertise… New York University Press. Prainsack, B., & Buyx, A. (2012). Solidarity in Contemporary Bioethics —
Towards a New Approach. Bioethics, 26(7), 343–350. Prainsack, B., & Buyx, A. (2017). Solidarity in Biomedicine and Beyond. Cambridge University Press. Rabeharisoa, V., & Callon, M. (2002). The Involvement of Patients’ Associations
in Research. ISSJ, 171, 57–65. Rabeharisoa, V., Moreira, T., & Akrich, M. (2013). Evidence-based Activism:
Patients’ Organizations, Users’ and Activist’s Groups in Knowledge Society. CSI Working Papers Series, 033, 1–27. g
p
Rabinow, P. (1996). Essays on the Anthropology of Reason. Princeton
University Press. Sharon, T. (2017). Self-tracking for Health and the Quantified Self:
Re-articulating Autonomy, Solidarity and Authenticity in an Age of
Personalized Healthcare. Philosophy & Technology, 30(1), 93–121. Shin, C., Han, C., Pae, C., & Patkar, A. (2016). Precision Medicine for
Psychopharmacology:
A
General
Introduction. Expert
Review
of
Neurotherapeutics, 16(7), 831–839. Sosnowy, C. (2014). Practicing Patienthood Online: Social Media, Chronic
Illness, and Lay Expertise. Societies, 4, 316–329. Steinhubl, S., Muse, E., & Topol, E. (2013). Can Mobile Health Technologies
Transform Health Care? JAMA, 310(22), 2395–2396. Swan, M. (2009). Emerging Patient-Driven Health Care Models: An
Examination of Health Social Networks, Consumer Personalized Medicine
and Quantified Self-Tracking. International Journal of Environmental Research
and Public Health, 6(2), 492–525. Swierstra, T. (2013). Nanotechnology and Techno-Moral Change. Ethics &
Politics, 15(1), 200–219. Terranova, T. (2000). Free Labor: Producing Culture for the Digital Economy. Social Text 63, 18(2), 33–58.hh Topol, E. (2015). The Patient Will See You Now. The Future of Medicine Is in Your
Hands. Basic Books.f Van de Werff, T. (2018). Practicing the Plastic Brain. Popular Neuroscience and
the Good Life. Datawyse. 6 Digital Biocommunities: Solidarity and Lay Expertise… © The Author(s) 2023
C. Egher, Digital Healthcare and Expertise, Health, Technology and Society,
https://doi.org/10.1007/978-981-16-9178-2_7 6 Digital Biocommunities: Solidarity and Lay Expertise… 223 Van Dijck, J. (2013). The Culture of Connectivity. A Critical History of Social
Media. Oxford University Press. Van Hoyweghen, I., & Rebert, L. (2012). Your Genes in Insurance: From
Generic Discrimination to Genomic Solidarity. Personalized Medicine,
9(8), 871–877.hh Van Oorschot, W., & Komter, A. (1998). What Is That Ties…? Theoretical
Perspectives on Social Bond. Sociale Wetenschappen, 41(3), 4–24.h Waldby, C., & Cooper, M. (2008). The Biopolitics of Reproduction. Australian
Feminist Studies, 23(55), 57–73. Weber, M. (1947). The Theory of Social and Economic Organization. Free Press. Translated by A.M. Henderson & Talcott Parsons. Weber, G., Mandl, K., & Kohane, I. (2014). Finding the Missing Link for Big
Biomedical Data. JAMA, 311(24), 2479–2480. Wium-Anderesen, I., Vinberg, M., Kessing, L., & McIntyre, R. (2017). Personalized Medicine in Psychiatry. Nordic Journal of Psychiatry, 71(1), 12–19.h Wynn, R., & Bergvik, S. (2010). Studying Empathy as an Interactional Three-
Part Sequence. Patient Education and Counseling, 80(1), 150. Open Access This chapter is licensed under the terms of the Creative Commons
Attribution 4.0 International License (http://creativecommons.org/licenses/
by/4.0/), which permits use, sharing, adaptation, distribution and reproduction
in any medium or format, as long as you give appropriate credit to the original
author(s) and the source, provide a link to the Creative Commons licence and
indicate if changes were made.h The images or other third party material in this chapter are included in the
chapter’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the chapter’s Creative Commons
licence and your intended use is not permitted by statutory regulation or exceeds
the permitted use, you will need to obtain permission directly from the copy
right holder. Expertise in the Age of Big Data The study described in this book was motivated by important debates
about the meaning and the effects of major transformations that expertise
has undergone in Western societies. Over the last few decades, expertise
has come to occupy a rather paradoxical position. On the one hand, we
are surrounded by more expertise because ever more domains of our lives
have come under the authority of “experts” and because expertise has
been increasingly claimed by “non-experts,” by people lacking official
accreditations. On the other hand, the right and authority of experts to
make decisions that impact the lives of many and the grounds upon
which such decisions are made have been called into question, as the rise
in anti-elitist and populist feelings over the last decade indicates. We live,
therefore, at a time, when expertise is both ubiquitous and very much
challenged. The Internet has played an important role in these develop
ments, as it has provided new avenues for the production, dissemination,
and evaluation of knowledge. Since this medium has been widely used by
a broad range of stakeholders in the field of mental health, throughout
this book I have scrutinized the different ways in which it has shaped the
performance of expertise about bipolar disorder. The use of the Internet in mental healthcare has given rise to numerous
fears and expectations. Some have hailed this medium’s potential to help 225 226 C. Egher C. Egher improve the accessibility and affordability of mental health therapies and
have also welcomed the possibility it affords people diagnosed to come
together and exchange insights with others with the same diagnosis. Others have worried about the varying quality of online mental health-
related information and about the ways in which such insights may affect
relations between medical professionals and people diagnosed, which
have been rather strained historically. These fears and hopes are to a
certain extent well founded, yet they neglect the multiple, dynamic
character the Internet can have as well as the different forms of engagement
users can take up online, depending on their personal interests, skills, and
goals, as well as on the social and cultural perspectives which shape their
understanding of specific mental health conditions. In this book, I have
tried to reconcile the agency of users with a perspective which sees online
technologies as value-laden and capable of actively influencing people’s
behaviors. Expertise in the Age of Big Data This way I have been able to compare the ways in which
different stakeholders seek to make their expertise manifest by using
different online technologies and I have scrutinized how the design and
online affordances available on different online platforms shape such
performances. I have also investigated the transformations that such
online engagements may lead to in the relations between people diagnosed
and medical professionals, and have probed the individualizing or
collectivity-generating effects the Internet can have in regard to the online
performance of expertise. Furthermore, I have also focused on how
different cultural norms and values inform these transformations. f
The main finding of this book is that the online performance of exper
tise is not a straightforward process by which offline practices, tools, and
approaches are transferred online, but involves additional skills and com
plex negotiations, which sometimes lead to unexpected configurations. Despite expectations that the availability of health-related information
would lead to patient empowerment and would allow people diagnosed
to re-position themselves in relation to medical professionals, the findings
presented here have revealed a more complex picture, where individuals
diagnosed with bipolar disorder can become highly influential through
their skillful use of the Internet and by developing and cultivating
important alliances with “traditional” experts. Contrary to expectations
that the focus on “radical” differences in personalized and precision f
The main finding of this book is that the online performance of exper
tise is not a straightforward process by which offline practices, tools, and
approaches are transferred online, but involves additional skills and com
plex negotiations, which sometimes lead to unexpected configurations. f
The main finding of this book is that the online performance of exper
tise is not a straightforward process by which offline practices, tools, and
approaches are transferred online, but involves additional skills and com
plex negotiations, which sometimes lead to unexpected configurations. Despite expectations that the availability of health-related information
would lead to patient empowerment and would allow people diagnosed
to re-position themselves in relation to medical professionals the findings 7 Expertise in the Age of Big Data 227 medicine would lead to increased individualization, online contributors
diagnosed with bipolar disorder performed solidarity with others with
whom they shared numerous similarities, which prompted them to
engage in epistemic practices as well as affective labor. Expertise in the Age of Big Data These findings
prompted the development of a new conceptualization of expertise,
understood as a practical and collective achievement, realized through
coordination and affective labor among stakeholders who occupy multiple
and shifting positions within a complex ecosystem. g p
p
Each of the empirical chapters has highlighted different ways in which
expertise on bipolar disorder was performed online by various stakeholders. In this chapter, these findings are brought together and reflected upon to
consider what they can tell us about the meaning and relevance of
expertise in the context where promises generated by big data analytics
suggest that substantial knowledge no longer describes (solely) a set of
human capabilities and may no longer require human intervention in
order to be applied in a variety of contexts. I highlight what the findings
presented in the empirical chapters mean in relation to the key questions
this book has addressed. Different Stakeholder’s Use of the Internet One of the unambiguous conclusions of this study is that different stake
holders used online technologies differently and that the choice thereof
was not only informed by the resources at their disposal, but also by their
goals and priorities. Thus, the analysis revealed that even though the
National Institute of Mental Health (NIMH) and La Haute Autorité de
Santé (HAS), the two governmental institutions studied in this book,
have vast financial and technical resources at their disposal, they are
reluctant Internet users, who have opted for non-interactive platforms
and for conservative online technologies. I have argued that such an
approach allowed them to push perspectives and research orientations
that were no longer popular to the background, and it also enabled them 228 C. Egher C. Egher to obscure from view the disagreements and struggles between different
medical professionals who study or provide medical care for this
condition. Furthermore, the choice for non-interactive platforms made it
possible for them to share their perspectives about bipolar disorder
authoritatively and without directly addressing the challenges brought to
psychiatry by people diagnosed, their families, and even medical
professionals. These governmental agencies used, however, different
online tools, which were better aligned with their main goals and
audiences. For instance, through the affordances available on the pdf files
HAS provided, such as the audio option and the ruler, I have argued that
this governmental agency used its platform for very specific educational
purposes, meant to facilitate information retention and decision-making
in clinical practice. The insights acquired from the empirical analyses
discussed in the other chapters of this book suggest, however, that these
governmental agencies need to consider the heterogeneity of the
population they address in their online performance of expertise about
bipolar disorder. While no access could be secured to find out how the
information posted online was developed and by whom, studying how
aspects of the identity of those designing and implementing this
information shape their work and affect the envisaged users would be a
fruitful direction for future research. Unlike the governmental agencies discussed, people diagnosed with
bipolar disorder used blogs and fora, where they could share their doubts
and insights, either by initiating forum threads or by commenting on
blog posts or threads written by others. 7 Expertise in the Age of Big Data 229 sharing practices among the members of such groups. This way members
of such communities came to use these online platforms as public diaries,
through which they and their readers could keep track of developments
in their mental, physical, and emotional states. The different reasons people diagnosed with bipolar disorder chose to
use the Internet also led to different dynamics. In general, online
contributors interested in specific information shared their treatment
experiences across one or several exchanges within a brief period of time
and returned sometimes after a long interval to share new insights. In
contrast, people interested in developing closer ties with others put a lot
of time and effort into sharing their experiences online and reacting to
those of others, either on the main page of the fora and/or through private
messages with specific contributors. Only a very limited number of
medical professionals have shared their insights on the platforms studied
here, which has prevented the development of clear perspectives on how
they use the Internet to acquire or share information about bipolar
disorder. Since the prevalence of this condition suggests that a considerable
number of medical professionals must also be diagnosed with bipolar
disorder, it would be relevant to find out more about their perspectives in
this regard and on the ways in which they call upon their different types
of knowledge to perform expertise about bipolar disorder online. Even
though the concept of performance implies that meaning comes into
being through the efforts of the actors and of the audience, through the
interactions between users and the various elements of online interfaces
(Drucker, 2010), it was not possible to conduct interviews or to observe
users within the same physical space. Addressing this aspect in future
studies would lead to important contributions. Different Stakeholder’s Use of the Internet It was shown that this allows for
a bottom-up accumulation of perspectives and for a dialogical performance
of expertise, as various treatment experiences, the advantages and
disadvantages of various therapeutic approaches, and the results of self-
experiments can be extensively discussed among numerous contributors. Whereas the people diagnosed studied in Chap. 4 used the Internet to
share their treatment experiences and to acquire specific information, the
online contributors studied in Chap. 6 used fora to come together and
support others with whom they shared important commonalities. I have
shown that how they engaged with the affordances available on these
online platforms played an important role in the development of digital
biocommunities, as it constituted a relevant similarity and reinforced 7 Expertise in the Age of Big Data People Diagnosed, Medical Professionals,
and the Internet The findings discussed in this book also indicated that the Internet is not
a neutral medium through which expertise about bipolar disorder can be
performed, but that it shapes in some notable ways the position people 230 C. Egher C. Egher diagnosed can occupy in relation to medical professionals. Through the
use of blogs and fora, people diagnosed with bipolar disorder were able to
engage in processes of knowledge production about this condition,
thereby acquiring more influence and agency. As discussed in Chap. 4,
through their online affordances which allow for the longitudinal accu
mulation in the same spaces of numerous insights, blogs and fora facili
tated the development of what I have called “digitally informed
hypotheses” about the effects and side effects of medications. This may
have immediate empowering effects for individual users, by confirming
the importance of their insights and thereby encouraging them to assume
a more confident position in their interactions with medical profession
als. In other cases, online platforms seemed to represent therapeutic
approaches in themselves, as getting in touch with other people diag
nosed and talking to them about their issues was often framed as contrib
uting to one’s well-being and mental stability. Also from this point of
view, the Internet appeared to empower some online contributors, as it
assisted them in developing more agency over bipolar disorder. If recog
nized by relevant others, such as family members, medical professionals,
and current or potential employers, this ability of people diagnosed to
better manage their condition could, in turn, improve their quality of life
by leading to more equal relations and more collaborative exchanges.f g
q
g
As people with different understandings of bipolar disorder, who had
different relations with their medical professionals and different needs
and possibilities in their daily life, came together online, even in brief
exchanges, the Internet may also have contributed toward a more open
dialogue about this condition. In time, this may lead to the development
of new standards to determine what accounts for reliable information at
a more general level. Importantly, while at the moment the “digitally
informed hypotheses” described in Chap. 4 require the assistance of med
ical professionals to become clinical evidence, in time this may no longer
be necessary. People Diagnosed, Medical Professionals,
and the Internet As the integration of different types of data fueled by per
sonalized and precision medicine may develop further, the insights pro
vided by people diagnosed through their online interactions may come to
be recognized as clinical evidence, even in the absence of significant inter
ventions from medical professionals. Should this occur, it will be difficult
to deny the contribution of blogs and fora in enabling people diagnosed 7 Expertise in the Age of Big Data 7 Expertise in the Age of Big Data 231 with bipolar disorder to re-position themselves more authoritatively in
relation to medical professionals, as these platforms facilitated the pro
duction of new knowledge and will have thus shaped the prescription of
treatment for this condition. Nevertheless, Chap. 5 showed that while the Internet does not always
favor the powerful, only a small number of individuals are able to re-
position themselves and to acquire a high standing. The two bloggers
studied in this chapter managed through the skillful use of this medium
to become highly influential. Through their popularity, visibility, and
credibility, Tracy and Fast not only shape how their readers understand
and approach bipolar disorder, but also influence the production of
knowledge about this condition, as they engage in productive collabora
tions with medical professionals. Yet, these bloggers did not use their
standing to further democratize participation in the production of knowl
edge. Rather than promoting research projects developed by citizen sci
entists or various crowdsourced initiatives, Tracy and Fast informed their
readers about projects undertaken by medical professionals with whom
they were familiar, or with whom they personally collaborated. Through
the efforts they made to familiarize readers with scientific methodology
and through their advice on how to best approach medical professionals,
these bloggers may, nonetheless, have already helped their readers develop
more collaborative relations with the latter and may have facilitated the
participation of a greater number of people diagnosed with bipolar disor
der in research. The data used in the study described in this book were collected from
blog posts and forum threads with many comments; thus, the insights
provided on the selected online platforms may be more representative of
people’s experiences with certain therapeutic approaches than others. Focusing on the content made available on online platforms with limited
(public) interactions and comparing differences in online interactivity in
relation to various forms of treatment would therefore be a promising
avenue for future research. Furthermore, since the results presented here
are based on the experiences of readers who were motivated enough to
contribute online, they do not represent all people diagnosed with bipo
lar disorder. 7 Expertise in the Age of Big Data More studies are therefore needed to understand the perspec
tives of people who use interactive online platforms for information The data used in the study described in this book were collected from
blog posts and forum threads with many comments; thus, the insights
provided on the selected online platforms may be more representative of
people’s experiences with certain therapeutic approaches than others. C. Egher 232 C. Egher purposes but refrain from contributing and those of people who refuse to
use the Internet for health-related purposes or who do not have access to
it. Studying the impact online contributions have on readers and how
lasting their effects are among other online participants would also pro
vide valuable contributions to the literature. To better understand how
people diagnosed with bipolar disorder re-position themselves in relation
to medical professionals through their use of the Internet, offline ethno
graphic studies on how medical professionals make sense of such online
engagements, how online expertise is brought to medical settings, and
the consequences this has are needed. Cultural Markers and Expertise on Bipolar
Disorder Online The findings presented in this book suggest that local norms and values
play an important role in how expertise on bipolar disorder is performed
online. Content-wise, the differences between the contributions on
American and French online platforms were largely shaped by the ways
in which mental healthcare was organized in these countries and by the
preferred scientific approach to bipolar disorder. Thus, differences in the
insurance system lead to different uses and engagements with the tech
nologies of online platforms. For (temporarily) uninsured American con
tributors, online platforms constituted valuable alternatives or stand-ins
for medical professionals, as these contributors relied upon the advice of
other people diagnosed to identify affordable and effective medications
and to determine some alternative practices they could take up, to
heighten their chances to remain stable. Furthermore, discussions about
generic drugs and their different effects were very popular on American
blogs and fora, but did not occupy a prominent position on French plat
forms. In contrast, many French contributors worried about overmedica
tion in France, as surveys positioned France among the countries with the
highest consumption rates in Europe. Another noteworthy difference
concerns the social impact ascribed to bipolar disorder. Whereas most
American contributors framed this condition as a disability which 7 Expertise in the Age of Big Data 7 Expertise in the Age of Big Data 233 prevented them from gaining or maintaining meaningful employment,
many French contributors were employed, at times in highly demanding
positions. p
The relations between medical professionals and people diagnosed
seemed fairly balanced on French platforms, with some contributors con
fessing to more strained relations and denouncing the practice of forced
hospitalization, while many others expressed trust in their medical pro
fessionals and described their interactions with them as collaborations,
even though between unequal partners. In contrast, on American plat
forms there were more pronounced tendencies for people diagnosed to
complain about the quality of medical care they received, to highlight the
lack of trust medical professionals displayed toward their experiences
with medications. While some contributors explained such strained rela
tions by invoking the close financial links between psychiatrists and phar
maceutical companies, there were also many who ascribed them to the
different levels of expertise of different medical professionals. Furthermore,
in the US, the antipsychiatry movement seemed to have remained influ
ential enough to be taken up on many of the blogs and fora studied. In
France, psychoanalysis was still considered a possible therapeutic
approach, making its presence felt through the use of its terminology in
discussions about the causes of bipolar disorder and about the elements
that may affect people’s response to treatment.hf f
There were also important differences regarding the preferred types of
online technologies people diagnosed with bipolar disorder from these
countries used. Whereas in the US, blogs acquired more readers and
more comments, in France, fora were by far the most popular, with an
impressive number of readers and online contributions. There was also a
notable difference regarding the extent to which online and offline prac
tices were integrated in the US and France. In contrast to French con
tributors, most American online contributors rarely used blogs and fora
to organize offline events or to inform readers about them. More studies
are needed to acquire a better understanding of the factors that account
for such differences in the use of online platforms and their affordances
among American and French online contributors, and of the standing
the latter may come to occupy online. 234 C. Egher The Internet and its Individualizing or
Collectivity-Generating Effects in Relation
to Expertise This book has shown that the Internet has both individualizing and col
lectivity-generating effects in relation to expertise, as people diagnosed
with bipolar disorder shared their insights on this condition online. The
analysis in Chap. 4 showed that the Internet can contribute to the devel
opment of new individual-group configurations. The online contributors
studied shared their treatment experiences as individuals rather than rep
resentatives of certain groups, and their interactions with others were
most of the time too fleeting for actual communities to develop. Nevertheless “light” forms of collectives did come into being. Such col
lectives did not develop, however, through the agency of the online con
tributors alone, but were facilitated by the online affordances of blogs
and fora, which allowed for the accumulation of their insights in the
same spaces. Importantly, even though online contributors could decide
never to return to a specific online platform again, unless they deleted
their contribution, they continued to be part of that collective long after
their last visit, through the insights they had shared. Furthermore, certain
online affordances available on these platforms may further serve as
reminders of their participation, as notifications about new comments or
about specific reactions to their own contributions may allow people
diagnosed to keep track of reactions on that platform, even when they no
longer participate actively. Closely knit collectives could also develop online, as was shown in
Chap. 6. I have termed such (sub)groups “digital biocommunities” to
highlight the fact that they are not only brought together by increasingly
more specific commonalities of experience, but also by shared approaches
to the digital technologies used. Thus, online contributors were drawn
together by similar interests, as they reacted to specific forum threads, by
common attitudes about their condition and the social-political circum
stances they were living in, which they could refer to in brief or more
elaborate forms through the use of specific online affordances available
on these platforms. In contrast, Chap. 5 revealed that the use of the 7 Expertise in the Age of Big Data 235 Internet contributes to the development of a new type of individual
stakeholder, what I have called “online expert mediators.” This new stake
holder category emerges at the intersection between the diagnosis of
bipolar disorder, the acquisition of particular types of knowledge, and an
individual’s skillful use of the Internet. The Internet and its Individualizing or
Collectivity-Generating Effects in Relation
to Expertise It has also been made possible by
the limited trust some of the people diagnosed and their families have
toward medical professionals due to various scandals over the last two
decades regarding the close ties between psychiatrists and pharmaceutical
companies. The rise of online expert mediators may mark a turn from
community activism to exceptional entrepreneurial selves. This book has
therefore described a new form of individualization as well as a new form
of collectivization that the Internet contributed to, and has shown that
both forms exist simultaneously online. By using the concept of interactional expertise to study the activities of
these online expert mediators, expertise about bipolar disorder was
approached as the property of individuals. In so doing, however, the role
of medical professionals and other people from whom these bloggers
learned before starting their own blog and the inspiration and new
insights they acquired from the contributors on their platforms were not
taken into account. While it may well be that Fast is right and that read
ers “appreciate quality above all else” (e-mail interview, 2016), more stud
ies on the behavior of different online audiences and their online
preferences are needed, to understand what such popularity is based on. Given that socioeconomic inequalities have been shown to influence the
quality of (professional) care one receives when healthcare is provided
through public–private partnerships (Engel & Van Lente, 2014), it is
necessary to understand whether online expert mediators represent a
cheap(er) way to access medical information for people with limited
resources or an additional means through which those sufficiently well-
off may seek to manage their health. As the concerns discussed in Chap. 2 in regard to digital and AI-based technologies highlighted, the Internet
could also contribute to harming online contributors on “free” platforms,
if the platform owners sell such data to employers, insurance companies,
banks, and other institutions that importantly shape one’s quality of life. 236 C. Egher Knowledge Production in the Digital
Age: Contributions In this section, the significance of the findings described above is dis
cussed by considering their relevance in regard to processes of knowledge
production in the digital age and to the role the Internet plays in them. In so doing, I argue that we need to move beyond rather simplistic
approaches which see the Internet either as a quick technological fix or as
a postmodern version of Pandora’s box.hi p
The findings presented in this book are the result of a qualitative study,
which focused on the narrative accounts provided by various stakeholders
on different types of online platforms. Yet, as was highlighted in Chap. 2,
this research unfolded against a background where developments in digital
technologies and data analytics have led many to believe that the days of
the relevance of human knowledge as we know it are counted. As we have
seen, enthusiastic about the capacities and potential of high-performing
intelligent machines, such commentators—often developers or owners of
digital companies—believe the acquisition of huge amounts of data from
numerous individuals (will) enable computers and algorithms to provide
better, more relevant solutions to all sorts of problems (Mayer-Schönberger
& Cukier, 2013). Such processes are underlined by a shift from causation
to correlation in the development of knowledge, as such technologies can
process at great speed previously unimaginable volumes of data, and iden
tify relevant patterns. The insights thus acquired are appealing to many,
especially to those who find themselves at sufficient distance from these
technologies, not only because of the remarkable calculations upon which
they are based, but also because of their apparent objectivity, as human bias
is (mistakenly) thought to be largely removed from them. Such digital
technologies are thought to become better alternatives to human expertise
because of the many flaws that are imputed to human professionals, such
as their insufficient or fragmented access to information, their limited
capacities to process all relevant data and to make accurate predictions
about complex phenomena, and their difficulty to overcome personal or
collective ideologies and interests (Topol, 2019). In this context, knowl
edge produced by digital technologies and algorithms is made to shine
brighter, due to its perceived neutrality and objectivity. 237 7 Expertise in the Age of Big Data 7 Expertise in the Age of Big Data This book told, however, a different story, as it emphasized the impor
tance of several typically human competencies, such as sensitivity to con
text, norms, and values, for the performance of expertise. By studying
how governmental agencies and people diagnosed with bipolar disorder
performed expertise online, I argued that expertise could better be
approached as a practical achievement, realized through coordination
and affective labor among stakeholders with different types of knowledge,
who occupy multiple shifting positions across a complex ecosystem. One
of the merits of this conceptualization is that it focuses on affective labor,
showing that it plays an important role in epistemic practices. This is
particularly important in the current context, where big data enthusiasts
have started to invoke affective practices to argue in favor of using artificial
intelligence (AI) in medical practice. Thus, Topol (2019) claims that the
use of AI as diagnostic tools would free up time for medical professionals
to be more “present,” “humane,” and “empathetic” during their encounters
with patients. Furthermore, he argues that “it is essential that we upgrade
diagnosis from an art to a digital-driven science” (Topol, 2019: Loc. 893). As this last statement shows, such visions do not consider affective
labor and practices as part of (medical) expertise, of epistemic practices,
but rather as something different, that can be separated from the former,
and that can be called upon and managed. p
g
The findings presented in this book have shown, however, that affec
tive labor is closely tied to the development and performance of expertise
and that this relation is not limited to people diagnosed (Chap. 6) but
also mobilized in the development and translation of knowledge among
different communities of practice (Chap. 5). Thus, affective practices,
creativity, and adaptability are typically human capabilities that have
been shown to play a significant role in the production of knowledge, as
well as in its accurate interpretation and successful implementation. This
is in line with arguments developed by Collins (2018) in his study on
artificial intelligence, where he highlights the importance of context
sensitivity and language acquisition in regard to expertise. In his opinion,
such abilities can only be acquired through acculturation in a given
society and are importantly tied, from a collective point of view, to
embodiment, to the ways in which one makes sense of the world with
and through one’s body. Furthermore, Collins emphasizes that in their 238 C. 7 Expertise in the Age of Big Data Egher C. Egher interactions with others, people constantly engage in substantial, highly
complex forms of “repair work,” as they make sense of, adapt, fill in, and
modify the broken and incomplete information they receive, in order to
develop appropriate responses. The findings presented in Chap. 4 of this
book support this perspective, because they highlighted the adaptative
character of expertise, as general views on the effectiveness of medications
or on the manifestations of bipolar disorder were enriched by being
assessed and applied to the specific circumstances, needs, and preferences
of various individuals. That epistemic practices are also affective in various
ways is important not only from a scientific point of view, but also
politically, especially given the skewed gendered distribution and income
inequality characterizing professions where care rather than knowledge is
highlighted (Hochschild, 1983/2003). g
g
Another problematic aspect is that affective labor is framed by big data
enthusiasts as something that occurs without the mediation of technology,
when doctors can take their eyes away from computers and scans, and
look instead at the patient. The findings presented here have shown,
however, that digital technologies are an integral part of certain affective
practices, shaping how people perform affective labor, and who the
performers and recipients of affective labor can be. Studying affective
labor online is important also because of the growing amount of value
that is nowadays generated from the cognition, communication, affect,
and the immaterial actions of online “prosumers” (Berardi, 2009; Hardt
& Negri, 2004) and because scholars are divided about the role of such
labor in digital media economics. Thus, many have criticized users’
engagement with digital technologies as a form of free labor (Lupton,
2014; Mitchell & Waldby, 2010; Terranova, 2000; Waldby & Cooper,
2008), particularly since people are typically required to give up ownership
over their data and any claims over potential profits that can be made
from them. The findings presented in this book provide, however, a more
nuanced perspective, as they show that online contributors perform
affective labor in order to assist others, but they also benefit in various
ways from these efforts, either because such practices contribute to their
well-being or because they receive similar assistance from others, when
they need it. 239 7 Expertise in the Age of Big Data 7 Expertise in the Age of Big Data This book also showed that norms and values play an important role in
the performance of expertise, not as sources of bias that need to be
overcome, but as factors that motivate people to contribute in specific
ways to knowledge production. Thus, Chap. 4 highlighted the role
personal values and preferences play in how people assess treatment
effectiveness and the therapeutic improvements or new treatments that
they seek to contribute to, by sharing their personal experiences and
insights. Whereas currently dominant neoliberal imperatives encourage
individuals to assume responsibility for their health, the findings in Chap. 6 showed that lay expertise is importantly tied to the value of solidarity. These insights are aligned with the work of several philosophers of
expertise (Goldman, 2018; Quast, 2018), who have highlighted the
moral dimension of expertise. These authors have argued that people
endowed with expertise are expected to behave responsibly, reflexively,
and virtuously, and to assist others to the best of their abilities. While
solidarity is largely neglected from such considerations, the findings
presented in Chap. 6 suggest that it is a value that is worth paying more
attention to in relation to expertise. By revealing the importance of
affective labor for epistemic practices, this book helps expand the category
of behaviors people endowed with expertise engage in and need to be
aware of. The findings of this book which highlighted the importance of com
munities for knowledge production are also important, as we currently
“live in an environment in which datafied individualization of health
responsibility appears like an inevitability” (McFall, 2019: 61). From this
point of view, the Internet could be seen to finally live up to some of the
visions it generated in its early days, when many expected it to lead to the
development of virtual communities, as people with the same diagnosis
could thus easily come together and talk about matters of interest, regard
less of where they lived (Eysenbach, 2005; Hardey, 1999). By showing
that people diagnosed with bipolar disorder perform expertise online,
this book contributed to a growing body of literature (McFall, 2019;
Prainsack & Buyx, 2017; Sharon, 2017; Van Hoyweghen & Rebert,
2012) that seeks to highlight the importance of this value in (mental)
healthcare practices. 240 7 Expertise in the Age of Big Data 241 considering the online knowledge thus generated as mainly the merit of
the technologies used would be a serious mistake, neglecting the active
ways in which the latter do shape the content produced would be a
danger that STS scholars have repeatedly warned against (Swierstra,
2016; Wyatt et al., 2016). y
This book showed that different stakeholders performed expertise
about bipolar disorder in different ways, depending on their skills and
resources, on the ways in which they (could) chose to react to broader
social transformations, and on the values that underpinned their
engagements. The ways in which science and knowledge production are
currently structured and organized increasingly require experts to make
bold claims and to issue clear, unwavering recommendations about the
problems they are called to solve. The varied and numerous accounts,
doubts, and uncertainties expressed by the people diagnosed with bipolar
disorder studied here constitute important reminders, however, that
modesty, diversity of opinions, and constructive criticism are essential
elements in the development of sound knowledge. By cultivating and
enhancing the Internet’s potential toward inclusive participation and
(self)reflection, inquiries about the meaning and relevance of expertise
will continue to generate enthusiasm, excitement, and heated debates. Many more exhilarating questions are thankfully opening up. C. Egher C. Egher The conceptualization of expertise that I put forward highlights the
complexity and diversity which characterize the production of knowledge
in the digital age and the dynamic ways in which authority and influence
can be (re)distributed among relevant stakeholders. This is important
because in many situations a priori agreements about the identity of the
“experts” or of the stakeholders involved in the development of expertise
no longer hold, the relevance and impact of their input differ depending
on the problem they are called to solve, and their role may change as new
tools or practices are introduced (Waardenburg et al., 2018). From this
point of view, the findings presented here constitute a contribution to the
growing body of expertise studies, which emphasize the fact that nowadays
individuals or groups socialized within different epistemic cultures have
to intensely negotiate the relevance of their insights and the reach of their
influence (Holst & Molander, 2018). By focusing on the knowledge with
which different stakeholders are endowed, the new approach I put
forward also highlights the importance not only of combining different
types of theoretical knowledge, but of bringing together scientific insights
with relevant local factors that only specific stakeholders may have
knowledge of. This new conceptualization of expertise is thus in line with
arguments recently provided by Barrotta and Montuschi (2018: 395),
who state that “local knowledge coming from ‘other sources’ is often
necessary to fill the gap between experts’ knowledge and correct judgement
calls.” Another merit of this approach to expertise is that it draws attention
to the importance of the ecosystem within which relevant stakeholders
are based, as even sound recommendations may be thwarted or lead to
less desirable effects depending on the available infrastructures, policies,
legal provisions, and dominant political climate. Through the findings presented, this book is therefore a contribution
to the work of STS scholars who have cautioned against the hype
surrounding digital technologies and their epistemic potential (Neff,
2013) and have argued, instead, that certain typically human capabilities
continue to be very much needed in processes of knowledge production. These findings are thus important reminders of the value of nuanced
perspectives, developed through rich, detailed studies, for a better
understanding of the different functions and roles that people and digital
technologies fulfill in processes of knowledge production. Whereas 7 Expertise in the Age of Big Data References Barrotta, P., & Montuschi, E. (2018). Expertise, Relevance and Types of
Knowledge. Social Epistemology, 32(6), 387–396.h ,
,
u
,
(
)
p
,
d
yp
Knowledge. Social Epistemology, 32(6), 387–396. Berardi, F. (2009). The Soul at Work: From Alienation to Autonomy. Semiotext (e). Collins, H. (2018). Artifictional Intelligence: Against Humanity’s Surrender to
Computers. Polity Press. E-book.h Berardi, F. (2009). The Soul at Work: From Alienation to Autonomy. Semiotext (e). Collins, H. (2018). Artifictional Intelligence: Against Humanity’s Surrender to
Computers. Polity Press. E-book. Drucker, J. (2010). Graphesis. Paj: The Journal of the Initiative for Digital
Humanities, Media, and Culture, 2(1), 1–50. Engel, N., & Van Lente, H. (2014). Organizing Innovation and Control
Practices: The Case of Public-Private Mix in Tuberculosis Control in India. Sociology of Health and Illness, 36(6), 917–931. 242 C. Egher Eysenbach, G. (2005). Patient-to-Patient Communication: Support Groups
and Virtual Communities. In D. Lewis, G. Eysenbach, R. Kukafka, P. Stavri,
H. Jimison, & W. Slack (Eds.), Consumer Health Informatics: Informing
Consumers and Improving Health Care (pp. 97–106). Springer. Goldman, A. (2018). Expertise. Topoi. An International Review of Philosophy,
37(1), 3–10.h p
g
pp
p
g
Goldman, A. (2018). Expertise. Topoi. An International Review of Philosophy,
37(1), 3–10. Hardey, M. (1999). Doctor in the House: The Internet as A Source of Lay
Health Knowledge and the Challenge to Expertise. Sociology of Healthy &
Illness, 21, 820–835. Hardt, M., & Negri, A. (2004). Multitude. War and Democracy in the Age of
Empire. The Penguin Press. h
Hochschild A (2003/1983). The Managed Heart. Commercialization of Human
Feeling. University of California Press. Holst, C., & Molander, A. (2018). Asymmetry, Disagreement and Biases:
Epistemic Worries about Expertise. Social Epistemology, 32(6), 358–371. Van Hoyweghen, I., & Rebert, L. (2012). Your Genes in Insurance: From
Generic Discrimination to Genomic Solidarity. Personalized Medicine,
9(8), 871–877. Lupton, D. (2014). The Commodification of Patient Opinion: The Digital
Patient Experience Economy in the Age of Big Data. Sociology of Health &
Illness, 36(6), 856–869.h Mayer-Schönberger, V., & Cukier, K. (2013). Big Data. A Revolution That Will
Transform How We Live, Work and Think. Houghton Mifflin Harcourt. E-book.h McFall, L. (2019). Personalizing Solidarity? The Role of Self-Tracking in Health
Insurance Pricing. Economy and Society, 48(1), 52–76. Mitchell, R., & Waldby, C. (2010). National Biobanks: Clinical Labor, Risk
Production, and the Creation of Biovalue. Science, Technology & Human
Values, 35(3), 330–355. Neff, G. (2013). Why Big Data Won’t Cure Us. References Big Data, 1, 117–123. PMID:25161827. Prainsack, B., & Buyx, A. (2017). Solidarity in Biomedicine and Beyond. Cambridge University Press. Quast, C. (2018). Towards a Balanced Account of Expertise. Social Epistemology,
32(6), 397–419. Sharon, T. (2017). Self-tracking for Health and the Quantified Self:
Re-articulating Autonomy, Solidarity and Authenticity in an Age of
Personalized Healthcare. Philosophy & Technology, 30(1), 93–121. Swierstra, T. (2016). Introduction to the Ethics of New and Emerging Science
and Technology. In R. Nakatsu, M. Rauterberg, & P. Ciancarini (Eds.), 7 Expertise in the Age of Big Data Wyatt, S., Harris, A., & Kelly, S. (2016). Controversy Goes Online: Schizophrenia
Genetics on Wikipedia. Science & Technology Studies, 29(1), 13–29. 7 Expertise in the Age of Big Data 243 Handbook
of
Digital
Games
and
Entertainment
Technologies
(pp. 1271–1295). Springer. Handbook
of
Digital
Games
and
Entertainment
Technologies
(pp. 1271–1295). Springer. Terranova, T. (2000). Free Labor: Producing Culture for the Digital Economy. Social Text 63, 18(2), 33–58. Topol, E. (2019). Deep Medicine. How Artificial Intelligence Can Make Healthcare
Human Again. Basic Books. E-book. Waardenburg, L., Sergeeva, A., & Huysman, M. (2018). Hotspots and
Blindspots: A Case of Predictive Policing in Practice. In U. Schultze,
M. Mähring, L. Riemer, C. Østerlund, & M. Aanestad (Eds.), IFIP Advances
in Information and Communication Technology (543) (pp. 96–109). Springer. Waldby, C., & Cooper, M. (2008). The Biopolitics of Reproduction. Australian
Feminist Studies, 23(55), 57–73. Wyatt, S., Harris, A., & Kelly, S. (2016). Controversy Goes Online: Schizophrenia
Genetics on Wikipedia. Science & Technology Studies, 29(1), 13–29. Open Access This chapter is licensed under the terms of the Creative Commons
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© The Author(s) 2023
C. Egher, Digital Healthcare and Expertise, Health, Technology and Society,
https://doi.org/10.1007/978-981-16-9178-2 1 Note: Page numbers followed by ‘n’ refer to notes. © The Author(s) 2023
C. Egher, Digital Healthcare and Expertise, Health, Technology and Society,
https://doi.org/10.1007/978-981-16-9178-2 1 Egher, Digital Healthcare and Expertise, Health, Technology and Society,
https://doi.org/10.1007/978-981-16-9178-2 246 Index Emotions, 15, 135, 193, 194, 197,
198, 209, 213, 217 Emotions, 15, 135, 193, 194, 197, 198, 209, 213, 217 198, 209, 213, 217 Capitalism, 19
Chatbot, 59
Collins, H., 112
Complexity, 4, 6, 92, 118, 119, 122,
123, 126, 128, 129, 131, 136,
138, 147
Contributory expertise, 9,
151, 163–165
Controversies, 81
Conversation analysis, 26, 199
Coordination, 6, 13, 14, 16,
17, 29, 72, 93, 110,
137, 176
Credentials, 97, 151, 159,
171, 178 Chatbot, 59 Empathy, 172, 204, 206, Collins, H., 112 212–215, 217 ,
Empowerment, 78, 190, 191, 226
Entrepreneurial selves, 175
Epstein, S., 113, 115, 177
Etiology, 42, 43, 47
Evidence, 55, 87, 101, 111,
117–119, 121–125, 130, 132,
138, 171
Expectations, 14, 37, 50, 55, 135,
153, 171, 225, 226 Complexity, 4, 6, 92, 118, 119, 122,
123, 126, 128, 129, 131, 136, Empowerment, 78, 190, 191, 226 Entrepreneurial selves, 175
Epstein, S., 113, 115, 177 138, 147 Contributory expertise, 9,
151, 163–165 Contributory expertise, 9, Contributory expertise, 9,
6
6 151, 163–165 151, 163–165 Evidence, 55, 87, 101, 111, Evidence, 55, 87, 101, 111, Controversies, 81 Conversation analysis, 26, 199 138, 171 Coordination, 6, 13, 14, 16,
17, 29, 72, 93, 110,
137, 176
Credentials 97 151 159 Coordination, 6, 13, 14, 16, Expectations, 14, 37, 50, 55, 135, Expectations, 14, 37, 50, 55, 135, 17, 29, 72, 93, 110, 17, 29, 72, 93, 110, 153, 171, 225, 226 137, 176 Eyal, G., 9, 10 Eyal, G., 9, 10 Credentials, 97, 151, 159, Credentials, 97, 151, 159,
171, 178 171, 178 F
Foucault, M., 10, 40
Free labor, 21, 111, 199
Freud, S., 45, 46 D Deinstitutionalization,
50–52, 54, 110 Democratization, 5, 22, 191 G
Genetic studies, 44
Genomics, 190
Gershon, I., 29, 196
Goffman, E., 17, 82, 92
Governmental agencies, 71, 72,
74, 76, 80, 81, 88, 102, 128 Dickenson, D., 192 Diagnostic and Statistical Manual
(DSM), 19, 46
Digital biocommunities, 29, 190,
205, 206, 208, 216–219,
228, 234
Digitally-informed hypotheses,
28, 109–139
Di i l h
i
57 62 Diagnostic and Statistical Manual
(DSM), 19, 46
Digital biocommunities, 29, 190,
205, 206, 208, 216–219,
228, 234
Digitally-informed hypotheses,
28, 109–139
Di i l h
i
57 62 228, 234 Governmental agencies, 71, 72,
74, 76, 80, 81, 88, 102, 128 Digitally-informed hypotheses,
28, 109–139 Digital phenotyping, 57, 62 Digital phenotyping, 57, 62 La Haute Autorité de Santé (HAS),
25, 27, 72, 76, 82,
83, 93–102
Heredity, 27, 40, 41, 44, 45
The Human Genome Project,
19, 47, 118 E
Electronic Freedom of Information
Act (E-FOIA), 74
eMEN, 73
Emotional labor, 198 Electronic Freedom of Information
Act (E-FOIA), 74 1 Alienists, 39–44, 46
Antipsychiatry movement, 51, 233
Artificial intelligence (AI), 27, 48,
55–64, 85
Asylums, 38, 39, 42–44, 49,
51, 53, 56
Authenticity, 172
Authority, 2, 8, 10, 37, 46, 76, 81,
84, 87, 89, 91, 94, 98, 100,
102, 117, 128, 129, 138, 147,
156, 158, 159, 164, 165, 213 Alienists, 39–44, 46 A Accountability, 61, 97, 102f Antipsychiatry movement, 51, 233i Affective labor, 6, 13, 15, 16, 29,
176, 190, 196–199, 202, 204,
205, 208, 213, 215, 217,
227, 237–239f 55–64, 85 Asylums, 38, 39, 42–44, 49, Asylums, 38, 39, 42–44, 49, 51, 53, 56 Affective practices, 16, 197, 198,
215, 237, 238
Affects, 83, 197, 198, 200, 205, 214
Affordances, 6, 16, 23, 24, 28, 76,
82, 83, 86, 89, 91–93,
99–101, 110, 122, 137, 150,
153, 173, 175, 195, 196, 199,
203, 206, 218
Agency, 25, 28, 29, 74n2, 84, 94,
95, 97, 101, 138, 146, 213,
226, 228, 230, 234
Akrich, M., 5, 137, 195, 218
Alcoholics Anonymous (AA), 49, 131
Algorithms, 22, 56, 60, 236 Affective practices, 16, 197, 198,
215, 237, 238 Authority, 2, 8, 10, 37, 46, 76, 81,
84, 87, 89, 91, 94, 98, 100,
102, 117, 128, 129, 138, 147,
156, 158, 159, 164, 165, 213 Affects, 83, 197, 198, 200, 205, 214f Affects, 83, 197, 198, 200, 205, 214f Affordances, 6, 16, 23, 24, 28, 76,
82, 83, 86, 89, 91–93,
99–101, 110, 122, 137, 150,
153, 173, 175, 195, 196, 199,
203, 206, 218 Autonomy, 6, 40, 80, 189, 191, 192 Agency, 25, 28, 29, 74n2, 84, 94,
95, 97, 101, 138, 146, 213,
226, 228, 230, 234
Akrich, M., 5, 137, 195, 218 Alcoholics Anonymous (AA), 49, 131
Biosociality, 137, 195 Algorithms, 22, 56, 60, 236
Bipolar disorder, 4, 17–20, 85, 86, 148 Algorithms, 22, 56, 60, 236
Bipolar disorder, 4, 17–20, 85, 86 245 © The Author(s) 2023
C. Electronic Freedom of Information
Act (E-FOIA), 74 (
)
eMEN, 73
Emotional labor, 198 eMEN, 73
Emotional labor, 198 19, 47, 118 247 207, 209 Narratives, 112, 209, 216h Immaterial labor, 198 The National Institute of Mental
Health (NIMH), 27, 72, 76,
82–95, 89n5, 97,
100–102, 126 Individualization, 29, 118, 120, 122,
123, 132–134, 136, 138, 158,
190–193, 219, 227, 235, 239
Inequalities, 54, 55, 61, 156 Inequalities, 54, 55, 61, 156 Institutions, 4, 8, 10, 11, 22, 24, 25,
27, 39–42, 45, 48, 49, 51, 64,
78, 81, 82, 84, 88, 93, 100,
101, 110, 115, 180, 227, 235 O
Online expert mediators, 28, 146,
151, 154, 156, 161, 162, 165,
166, 169, 175, 177, 178, 180 O
Online expert mediators, 28, 146,
151, 154, 156, 161, 162, 165,
166, 169, 175, 177, 178, 180 O
Online expert mediators, 28, 146,
151, 154, 156, 161, 162, 165,
166, 169, 175, 177, 178, 180 Interactional expertise, 6, 9, 11, 28,
145–180, 235i Index Index M
Mediators, 28, 29, 151, 152, 154,
161, 163, 166, 171, 173, 176,
178, 180, 235
Mol, A., 13
Monitoring devices, 59, 60 Identity, 2, 13, 15, 17, 19, 77, 78,
117, 146, 149, 176, 177, 194,
198, 200, 204, 218 161, 163, 166, 171, 173, 176,
178, 180, 235 178, 180, 235 Mol, A., 13 Idiom of practice, 196, 201, 205,
206, 208
Illness blogs, 149, 156
Illouz, E., 45, 46, 48, 49, 51, 197, Idiom of practice, 196, 201, 205,
206, 208 Monitoring devices, 59, 60 Monitoring devices, 59, 60 Illness blogs, 149, 156 Illouz, E., 45, 46, 48, 49, 51, 197, N
Narratives, 112, 209, 216h International Classification of
Disease (ICD), 19 International Classification of
Disease (ICD), 19 Internet, 1, 2, 4, 6, 15, 17, 20–24,
27–29, 55, 61, 71–76, 79, 88,
91, 100, 109, 111, 115, 136,
139, 145–150, 154–156, 170,
171, 173, 175, 178, 180, 195,
225–232, 234–236, 239, 241 Patient engagement, 109–114,
147–150, 180 g g
147–150, 180 Personalized medicine, 4, 121,
158, 192 Personhood, 194, 210, 219 Prainsack, B., 1, 5, 21, 29, 101, 111,
137, 146, 190, 192, 194, 196,
218 239 K
Kivits, J., 5, 146, 149, 176 K
Kivits, J., 5, 146, 149, 176 Precision medicine, 5, 19, 21, 48, 111, 118, 189–191, 216, 219, 111, 118, 189–191, 216, 219, 226–227, 230 Precision Medicine Initiative, 191 Latour, B., 81, 86 Production of knowledge, 1, 3, 10, Production of knowledge, 1, 3, 10, 109–111, 136, 217, 231, 109–111, 136, 217, 231, Lay expertise, 6, 15, 28, 29, 110, Lay expertise, 6, 15, 28, 29, 110,
111, 113, 116, 135, 136, 146,
189–219, 239 237, 240 189–219, 239 Prognosis, 18, 42, 43 Prognosis, 18, 42, 43 Lupton, D., 175 Psychiatric genetics, 45, 47 248 Index Solidaristic, 29, 189, 193, 194, 202,
204, 210, 214, 218, 219
Solidarity, 191, 195–196,
200–205, 210–216
Stigma, 20
Stigmatization, 148
Support groups, 27, 48–50, 110,
178, 218
Surveillance, 21, 57n2, 59, 63, 149 Solidaristic, 29, 189, 193, 194, 202,
204, 210, 214, 218, 219 Solidaristic, 29, 189, 193, 194, 202,
204, 210, 214, 218, 219 Psychiatry, 3, 27, 37–64, 71,
76–80, 88, 168 Psychiatry, 3, 27, 37–64, 71, Solidaristic, 29, 189, 193, 194, 202,
204, 210, 214, 218, 219
Solidarity, 191, 195–196,
200–205, 210–216
Stigma, 20
Stigmatization, 148
Support groups, 27, 48–50, 110,
178, 218
Surveillance, 21, 57n2, 59, 63, 149 204, 210, 214, 218, 219 76–80, 88, 168 Solidarity, 191, 195–196,
200–205, 210–216 Solidarity, 191, 195–196, Psychoanalysis, 45, 51, 53, 233 200–205, 210–216 200–205, 210–216 Psychoanalytic approaches, Psychoanalytic approaches,
46, 49, 131 Stigma, 20 Stigma, 20 Stigmatization, 148 Psychology, 7, 27, 148, 154 g
Support groups, 27, 48–50, 110,
178, 218 Q
The Quantified Self, 111, 192 Surveillance, 21, 57n2, 59, 63, 149 T
Tactics, 28
Tinkering, 129, 196, 205
Transparency, 61, 97 T
Tactics, 28
Tinkering, 129, 196, 205
Transparency, 61, 97 R
Rhetoric, 86, 89
Rose, N., 3, 10, 19, 37, 39, 43,
44, 46, 47, 76, 78,
111, 146 Rhetoric, 86, 89 Rose, N., 3, 10, 19, 37, 39, 43, Rose, N., 3, 10, 19, 37, 39, 43,
44, 46, 47, 76, 78,
111, 146 Uncertainty, 2, 14, 41, 118, 119,
122–126, 136, 138, 147 S
Self-help, 27, 48–50, 84, 110,
155, 218
Self-knowledge, 135, 206, 208,
209, 217
Self-monitoring, 109, 111
Sharon, T., 169
Similarities, 6, 7, 18, 20, 27, 119,
122, 134, 138, 180, 194, 195,
200–201, 203–205, 213, 216,
217, 219
Sociality, 195, 196, 198, 199 S
Self-help, 27, 48–50, 84, 110,
155, 218
Self-knowledge, 135, 206, 208,
209, 217
Self-monitoring, 109, 111
Sharon, T., 169
Similarities, 6, 7, 18, 20, 27, 119,
122, 134, 138, 180, 194, 19 S
Self-help, 27, 48–50, 84, 110,
155, 218
Self-knowledge, 135, 206, 208,
209, 217 Self-help, 27, 48–50, 84, 110, Self-help, 27, 48–50, 84, 110, Self-help, 27, 48–50, 84, 110,
155, 218 V
Visibility, 22, 123, 137, 149, 156,
161, 162, 173, 179, 231 Self-knowledge, 135, 206, 208,
209, 217 Self-knowledge, 135, 206, 208, Self-monitoring, 109, 111 Self-monitoring, 109, 111 Sharon, T., 169 Similarities, 6, 7, 18, 20, 27, 119,
122, 134, 138, 180, 194, 195,
200–201, 203–205, 213, 216,
217, 219
Sociality, 195, 196, 198, 199 Similarities, 6, 7, 18, 20, 27, 119,
122, 134, 138, 180, 194, 195,
200–201, 203–205, 213, 216,
217, 219
Sociality, 195, 196, 198, 199 W
Web Content Accessibility
Guidelines (WCAG),
75, 89, 98 Sociality, 195, 196, 198, 199 Sociality, 195, 196, 198, 199
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„ICH FORDRE SO VIEL FREIHEIT UND MUSSE, ALS NÖTIG IST, ÜBER MICH SELBST, ÜBER MEINE BESTIMMUNG UND MEINEN WERTH ALS MENSCH ZU DENKEN“.1 ZUR KUNST- UND FREIHEITSKONZEPTION BEI KARL PHILIPP MORITZ
|
Literatura/Literatūra
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cc-by
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4 Der Held des autobiographischen Romans von
Moritz heißt nicht zufällig Anton Reiser. Die Vorliebe
des Autors fürs Reisen findet ihren Ausdruck in
zahlreichen Reiseberichten. Zu nennen wären an
dieser Stelle u.a.: Reisen eines Deutschen in England
im Jahre 1782, biographischer Bericht über die zehn
letzten Lebensjahre unter dem Titel Mein Freund
Anton Reiser. Aus dem Leben des Karl Philipp Moritz
aus dem Jahre 1794 sowie eine dreiteilige Reise-
beschreibung Reisen eines Deutschen in Italien in den
Jahren 1786 bis 1788. 5 Moritz war unter seinen Zeitgenossen von
Goethe bis zu Jean Paul sehr hoch geschätzt. An seinen
Ästhetik-Vorlesungen nahmen unter anderem
Alexander von Humboldt, Ludwig Tieck und Wilhelm
Heinrich Wackenroder teil. 2 Johann Wolfgang von Goethe, „Brief an Char-
lotte von Stein vom 14. Dezember 1786“, Goethes
Briefe, II, Hamburg: Wegner, 1968, 28-30. f
g
g
3 Das Magazin war als eine Sammlung von
Lebensgeschichten, Therapiebeispielen und Beo-
bachtungen uns als ein „Lesebuch für Gelehrte und
Ungelehrte“ gedacht. ISSN 0258–0802. LITERATÛRA 2008 50(5) ISSN 0258–0802. LITERATÛRA 2008 50(5) 1 Karl Philipp Moritz, „Das Edelste in der Natur“,
Schriften zur Ästhetik und Poetik. Kritische Ausgabe,
hrsg. von Hans Joachim Schrimpf, Tübingen:
Niemeyer, 1962, 17. 6 Moritz arbeitete oft parallel an mehreren Texten,
daher kann man in seinen verschiedenen Arbeiten
ähnlich oder sogar gleich klingende Textausschnitte
entdecken. In Reisen eines Deutschen in Italien in den
Jahren 1786 bis 1788 finden wir die Beschreibungen
von Aurora und Fortuna von Guido Reni, die man auch
in seinem drei Jahre früher (1789) publizierten Aufsatz
„Über die Allegorie“ finden kann. Die Arbeit
Vorbegriffe zu einer Theorie der Ornamente (1793)
beinhaltet Passagen aus beiden genannten Texten. In
Die Signatur des Schönen. In wie fern Kunstwerke
beschrieben werden können (1788/1789) finden wir die
Kritik der Winckelmannschen Beschreibung des
Apollo im Belvedere, welche den wesentlichen Teil
des Textes Apollo in Belvedere. Kritik an Winckelmann „ICH FORDRE SO VIEL FREIHEIT UND MUSSE,
ALS NÖTIG IST, ÜBER MICH SELBST, ÜBER MEINE
BESTIMMUNG UND MEINEN WERTH ALS MENSCH
ZU DENKEN“.1 ZUR KUNST- UND
FREIHEITSKONZEPTION BEI KARL PHILIPP MORITZ Krzysztof Tkaczyk Krzysztof Tkaczyk
Wissenschaftlicher Mitarbeiter, Institut für Germanistik, Universität Warschau Wissenschaftlicher Mitarbeiter, Institut für Germanistik, Universität Warschau Redakteur der „Vossischen Zeitung“ (1784-
85), England-, Deutschland- und Italien-
reisender4, seit 1789 Professor für die
Theorien der schönen Künste an der Akade-
mie der Künste in Berlin5 und seit 1791
Mitglied der Akademie der Wissenschaften. In einem Brief aus Rom an Charlotte von
Stein schrieb Johann Wolfgang von Goethe
über ihn: „Er ist wie ein jüngerer Bruder von
mir, von derselben Art, nur da vom Schicksal
verwahrlost und beschädigt, wo ich begünstigt
und vorgezogen bin“.2 Gemeint war Karl
Philipp Moritz (1756 – 1793), deutscher
Schriftsteller und Kunsttheoretiker, Erfor-
scher der griechischen Mythologie, Ästhetiker
und Poesietheoretiker, Vorläufer der moder-
nen Psychologie und Schöpfer des psycho-
logischen Romans, Kinderbuchautor, Grün-
der und Redakteur einer der ersten psycho-
logischen Zeitschriften Deutschlands: des
„Magazins für Erfahrungsseelenkunde“
(1783-1793)3 , Gymnasialprofessor (1784), Kennen gelernt haben sich Moritz und
Goethe in Rom im Jahre 1786 und nach einem
Ausflug ans Meer, an dem auch der bekannte
Maler Johann Heinrich Wilhelm Tischbein
teilnahm, fanden die beiden Männer zueinan- 85 der. Was die materielle und gesellschaftliche
Position Moritz‘ anbetrifft, hat Goethe kon-
kret bezogen auf das Jahr 1786 gewiss Recht,
will man jedoch die Worte Goethes auf die
geistige Dimension der Moritzschen Werke
beziehen, muss man feststellen, dass der große
Meister sich geirrt hat. Moritz ist in seiner
Ästhetik viel fortschrittlicher und inte-
ressanter als der „ältere Bruder“, im sprach-
kritischen Gestus konsequenter als Schiller
und im politischen Denken radikaler als Kant. übertreffen strebt.“7 Der menschliche Geist
sei also suchend und schöpfend, künstlerisch
angelangt, er ringe mit der Natur, wolle sie
nicht nur nachahmen, sondern übertreffen. In „Über die bildende Nachahmung des
Schönen“ (1788)8, dem wichtigsten und
zugleich umfangreichsten Moritzschen Auf-
satz zur Ästhetik (etwa dreißig Druckseiten),
lesen wir über den moralischen Wert des
Schönen: Jede schöne Handlung aber muß nothwen-
dig auch edel seyn: das Edle ist bei ihr die Basis
oder der Fonds des Schönen, durch welches sie
in unser Auge leuchtet. Durch den Mittelbegriff
des Edlen also wird der Begriff des Schönen
wieder zum Moralischen hinübergezogen und
gleichsam daran festgekettet. […] Das Schöne
und Edle unterscheidet sich eben dadurch vom
Guten, daß es nicht nützlich seyn darf, um
schön zu seyn. (1792-1793) ausmacht und auch in dem schon
genannten Reisebericht aus Italien zu finden ist. Die
Arbeiten Versuch einer Vereinigung aller schönen Künste
und Wissenschaften unter dem Begriff des in sich
Vollendeten (1785) und Die metaphysische Schön-
heitslinie (1793) ergänzen dagegen einander und
stehen in enger Beziehung zu dem grundlegenden
Moritzschen Aufsatz zur Ästhetik Über die bildende
Nachahmung des Schönen (1788). Vgl. Egon Menz,
Die Schrift Karl Philipp Moritzens „Über die bildende
Nachahmung des Schönen“, Göppingen: Kümmerle,
1968. 7 Moritz, 1962, 13-19. 8 Karl Philipp, Moritz, „Über die bildende
Nachahmung des Schönen“, Schriften zur Ästhetik und
Poetik. hrsg. von Hans Joachim Schrimpf, Tübingen:
Niemeyer, 1962, 63-93. (1792-1793) ausmacht und auch in dem schon
genannten Reisebericht aus Italien zu finden ist. Die
Arbeiten Versuch einer Vereinigung aller schönen Künste
und Wissenschaften unter dem Begriff des in sich
Vollendeten (1785) und Die metaphysische Schön-
heitslinie (1793) ergänzen dagegen einander und
stehen in enger Beziehung zu dem grundlegenden
Moritzschen Aufsatz zur Ästhetik Über die bildende
Nachahmung des Schönen (1788). Vgl. Egon Menz,
Die Schrift Karl Philipp Moritzens „Über die bildende
Nachahmung des Schönen“, Göppingen: Kümmerle,
1968. 7 Moritz, 1962, 13-19.
8 Karl Philipp, Moritz, „Über die bildende
Nachahmung des Schönen“, Schriften zur Ästhetik und
Poetik. hrsg. von Hans Joachim Schrimpf, Tübingen:
Niemeyer, 1962, 63-93.
9 Moritz, 1962, 67-70. „ICH FORDRE SO VIEL FREIHEIT UND MUSSE,
ALS NÖTIG IST, ÜBER MICH SELBST, ÜBER MEINE
BESTIMMUNG UND MEINEN WERTH ALS MENSCH
ZU DENKEN“.1 ZUR KUNST- UND
FREIHEITSKONZEPTION BEI KARL PHILIPP MORITZ […] Das Nützliche muß offenbar
an dem Schönen als überflüssig und wenn es
sich daran befindet, doch als zufällig und als
nicht dazugehörig betrachtet werden, weil die
wahre Schönheit, ebenso wie das Edle in der
Handlung, durch das Nützliche dabei weder
vermehrt noch durch den Mangel desselben auf
irgendeine Weise vermindert werden kann.9 Die Moritzsche Ästhetik ist alles andere
als einfache Kost. Viele seiner Texte scheinen
fragmentarisch zu sein, einander ergänzend,
aber auch in einem gewissen Widerspruch
zueinander stehend.6 Moritz will den Leser,
für den er ein Labyrinth von nicht selten
unklaren und abstrakten Begriffen geschaffen
hat, zu einer langsamen Lektüre zwingen. Und
das macht er ohne Scheu, weil er voller
aufklärerischen Optimismus auf den Men-
schen und seinen schöpfenden und suchenden
Geist hofft. In „Das Edelste in der Natur“
(1786) schreibt er: „Was gibt es Edleres und
Schöneres in der ganzen Natur als den Geist
des Menschen, auf dessen Vervollkommnung
alles übrige unablässig hinarbeitet und in
welchem sich die Natur gleichsam selbst zu 86 Und gleich danach finden wir folgende
Bemerkung: „Eine Sache wird nämlich
dadurch noch nicht schön, daß sie nicht
nützlich ist sondern dadurch, daß sie nicht
nützlich zu seyn braucht.”10 Was bedeutet
aber, dass sie nicht nützlich zu sein braucht? Um die Frage zu beantworten, „wie denn eine
Sache beschaffen seyn müsse, damit sie nicht
nützlich zu seyn brauche”11, versucht Moritz
den Begriff des Nutzens näher zu bestimmen: steigt also selbst zum Nützlichen herab, und
fließt mit ihm zusammen. – Es giebt seine
Ansprüche mit seinem Namen auf; tritt in
gemessene Schranken, wird zur bescheidnen
Zierde, zur simplen Eleganz.13 Somit bricht Moritz mit der tradierten
Wirkungsästhetik. Jedes Kunstwerk sei ein
autonomes Werk, unabhängig von seinen
potentiellen außerästhetischen sozialen,
historischen, religiösen oder politischen
Inhalten, die man von ihm erwarten bzw. nach
denen man in ihm suchen möchte. Es darf
nicht nach seiner Nützlichkeit betrachtet
werden. Im Bereich der Kunst lässt Moritz
lediglich die „ästhetische Nützlichkeit“ zu: Unter Nutzen denken wir uns nehmlich die
Beziehung eines Dinges als Teil betrachtet, auf
einen Zusammenhang von Dingen, den wir uns
als ein Ganzes denken. Diese Beziehung muß
nehmlich von der Art seyn, daß der Zusammen-
hang des Ganzen beständig dadurch gewinnt
und erhalten wird: je mehrere solcher Bezie-
hungen nun eine Sache auf den Zusammen-
hang, worinn sie sich befindet, hat, um desto
nützlicher ist dieselbe. 13 Ebd., 72.
14 Moritz, 1962, 122. 10 Ebd., 71.
11 Ebd., 71.
12 Ebd., 71. 3. jedes schöne Kunstwerk ist mehr oder
weniger ein Abdruck des uns umgebenden
großen Ganzen der Natur, muß es auch als ein
für sich bestehendes Ganze von uns betrachtet
werden, welches, wie die große Natur, seinen
Endzweck in sich selber hat und um sein selbst
willen da ist. Und nur auf diese Weise be-
trachtet, kann „ICH FORDRE SO VIEL FREIHEIT UND MUSSE,
ALS NÖTIG IST, ÜBER MICH SELBST, ÜBER MEINE
BESTIMMUNG UND MEINEN WERTH ALS MENSCH
ZU DENKEN“.1 ZUR KUNST- UND
FREIHEITSKONZEPTION BEI KARL PHILIPP MORITZ [...] Hieraus sehen wir
also daß eine Sache um nicht nützlich seyn zu
dürfen, nothwendig ein für sich bestehendes
Ganze seyn müsse und daß also mit dem Begriff
des Schönen der Begriff von einem für sich
bestehenden Ganzen unzertrennlich verknüpft
ist.12 3. jedes schöne Kunstwerk ist mehr oder
weniger ein Abdruck des uns umgebenden
großen Ganzen der Natur, muß es auch als ein
für sich bestehendes Ganze von uns betrachtet
werden, welches, wie die große Natur, seinen
Endzweck in sich selber hat und um sein selbst
willen da ist. Und nur auf diese Weise be-
trachtet, kann 4. das Schöne wahrhaft nützlich werden, in
dem es unser Wahrnehmungsvermögen für
Ordnung und Übereinstimmung schärft und
unsern Geist über das Kleine erhebt, weil es alles
Einzelne uns stets im Ganzen und in Beziehung
auf das Ganze deutlich erblicken läßt.14 Das nur Nützliche wird von Moritz als
Gegenteil des Schönen verstanden. Es ist
minderwertig und unwürdig, kann aber auch
gefährlich sein. Verbindet sich das Schöne mit
dem Nützlichen, tritt es in eine enge Be-
ziehung mit ihm, wird es sofort von ihm
annektiert und ihm untergeordnet. In der
Welt der bloß nützlichen Erscheinungen
verliert sich das Schöne entgültig: Die Definition des Kunstwerks als eines
an sich geschlossenen Ganzen und einer
unabhängigen Welt, eines eben autonomen
Werkes, ermöglicht Moritz einen Schritt über
das Ästhetische hinaus zu machen: in die
politische und soziale Sphäre. Moritz befreit
den Menschen von dem nur Nützlichen, der
Mensch sei auch ein edles Wesen, und somit
ein Selbstzweck: Wenn das Schöne sich an dem Nützlichen
befindet, muß es sich auch dem Nützlichen
unterordnen – es ist nicht um sein selbst willen
da – es dient das Nützliche aufzuschmücken – Der einzelne Mensch muß schlechterdings
niemals als ein bloß nützliches, sondern zugleich 87 geordnetes Rad in die Maschine eingreifen –
aber der Geist des Menschen kann durch nichts
untergeordnet werden, er ist ein in sich selbst
vollendetes Ganze.19 als ein edles Wesen betrachtet werden, das
seinen eigenthümlichen Werth in sich selber
hat, wenn auch das ganze Gebäude der
Staatsverfassung, wovon er ein Theil ist, um ihn
her wegfiele.15 Im Irrtum wäre man jedoch, wenn man
annähme, das Moritz die Kantschen Ideen
blind übernimmt. Moritz ist viel radikaler als
Kant. Kant postuliert doch nicht eine unein-
geschränkte Freiheit des Individuums. 15 Ebd., 16.
16 Ebd., 65.
17 Ebd., 65-66.
18 Immanuel Kant, „Beantwortung der Frage: Was
ist Aufklärung?“, Werke in zwölf Bänden, 11, Frankfurt
am Main: Suhrkamp, 1977, 53-61. 19 Moritz, 1962, 16.
20 Kant, 1977, 55.
21 Ebd., 55.
22 Ebd., 55-56. 26 Ebd., 61.
27 Vgl. Moritz, 1962, 17: „Eins der größten Übel,
woran das Menschengeschlecht krank liegt, ist die
schädliche Absonderung desselben, wodurch es in zwei
Teile zerfällt, von welchen man den einen, der sich
erstaunliche Vorzüge vor dem andern anmaßt, den
gesitteten Theil nennt.” und ders. Das menschliche Elend,
S. 25: „Muß Eigennutz und Selbstsucht nothwendig in
der Welt sein – wie soll denn je die allgemeine Quelle
des menschlichen Elends verstopft werden? – Solange
es Unterdrücker gibt, muß es auch Unterdrückte geben.
– Die menschlichen Kräfte wollen freien Spielraum
haben, hat nun die Kraft eines einzigen unter Tausenden
einen zu großen Spielraum, so sind Tausende nicht so
glücklich, wie sie es seyn könnten“. „ICH FORDRE SO VIEL FREIHEIT UND MUSSE,
ALS NÖTIG IST, ÜBER MICH SELBST, ÜBER MEINE
BESTIMMUNG UND MEINEN WERTH ALS MENSCH
ZU DENKEN“.1 ZUR KUNST- UND
FREIHEITSKONZEPTION BEI KARL PHILIPP MORITZ Er
unterscheidet zwischen dem öffentlichen
Gebrauch und dem Privatgebrauch der
Vernunft, wobei „der öffentliche Gebrauch
(der) Vernunft jederzeit frei sein muß, [...] der
Privatgebrauch derselben aber darf öfters sehr
enge eingeschränkt sein, ohne doch darum
den Fortschritt der Aufklärung sonderlich zu
hindern“20. Unter dem öffentlichen Gebrauch
der eigenen Vernunft versteht er „denjenigen,
den jemand als Gelehrter von ihr vor dem
ganzen Publikum der Leserwelt macht. Den
Privatgebrauch (nennt er) denjenigen, den er
in einem gewissen ihm anvertrauten bürger-
lichen Posten, oder Amte, von seiner Vernunft
machen darf“21. Weiter erklärt Kant: Die Selbstbefreiung des Menschen von
dem bloß Nützlichen ist für Moritz die
Voraussetzung und der erste Schritt auf dem
Wege zum neuen, edlen Menschen. Zwischen
dem einen und dem anderen gibt es einen
qualitativen Unterschied. Ein nützlicher
Mensch, schreibt er, ist jener, „der nicht
sowohl an und für sich selbst, als vielmehr nur
in Beziehung auf irgend einen Zusammen-
hang von Dingen außer ihm, unsre Aufmerk-
samkeit verdienet“16. Der edle Mensch zieht
dagegen „für sich ganz allein, unsre ganze
Aufmerksamkeit und Bewunderung auf sich;
ohne alle Rücksicht auf irgend etwas ausser
ihm, oder auf irgendeinen Vortheil, der uns
für unsre eigne Person aus seinem Daseyn
erwachsen könnte.“17 Somit eröffne sich der
Weg zu einer Gesellschaf, die Gemeinschaft
freier selbständiger Wesen sein könnte. Eindeutig aus Immanuel Kant und seiner
Auffassung der Aufklärung, nicht als Epoche
sondern Geisteszustand („Was ist Auf-
klärung?“, 178318) schöpfend, definiert Moritz
die Beziehung zwischen dem Individuum und
der Gesellschaft bzw. dem Bürger und dem
Staat als Beziehung eines denkenden, selbst-
bewussten und frei handelnden Wesens zu
einem Ganzen, das da zum Wohl eben dieses
Wesens ist: Nun ist zu manchen Geschäften, die in das
Interesse des gemeinen Wesens laufen, ein
gewisser Mechanism notwendig, vermittelst
dessen einige Glieder des gemeinen Wesens
sich bloß passiv verhalten müssen, um durch
eine künstliche Einhelligkeit von der Regierung
zu öffentlichen Zwecken gerichtet, oder
wenigstens von der Zerstörung dieser Zwecke
abgehalten zu werden. Hier ist es nun freilich
nicht erlaubt, zu räsonnieren; sondern man
muß gehorchen.22 Laut Kant muss also jeder Bürger, der im
Staat auch den kleinsten Posten belegt, die
unbedeutendste Rolle spielt, an erster Stelle Der Staat kann eine Weile seine Arme,
seine Hände brauchen, daß sie wie ein unter- 88 gehorchen, für die Kritik kommt Zeit „nach
der Arbeit“. „ICH FORDRE SO VIEL FREIHEIT UND MUSSE,
ALS NÖTIG IST, ÜBER MICH SELBST, ÜBER MEINE
BESTIMMUNG UND MEINEN WERTH ALS MENSCH
ZU DENKEN“.1 ZUR KUNST- UND
FREIHEITSKONZEPTION BEI KARL PHILIPP MORITZ Paradoxerweise stellt Kant die
politische Freiheit, oder genauer gesagt die
politische Unfreiheit, der geistigen Freiheit
gegenüber, was nichts anderes ist als die
Akzeptanz der Freiheitseinschränkung über-
haupt, die sich bei Kant überdies mit einem
ungerechten Urteil über die Bürger verbindet: gehoben hat und die Ordnung im Staat sichert. Für Kant sind also ein aufgeklärter Despot
ein idealer Herrscher und der aufgeklärte
Absolutismus die beste Staatsform: Aber auch nur derjenige, der, selbst
aufgeklärt, sich nicht vor Schatten fürchtet,
zugleich aber ein wohldiszipliniertes zahlrei-
ches Heer zum Bürgen der öffentlichen Ruhe
zur Hand hat, – kann das sagen, was ein
Freistaat nicht wagen darf: räsonniert, so viel
ihr wollt, und worüber ihr wollt; nur gehorcht!26 Ein größerer Grad bürgerlicher Freiheit
scheint der Freiheit des Geistes des Volks
vorteilhaft, und setzt ihr doch unübersteigliche
Schranken; ein Grad weniger von jener
verschafft hingegen diesem Raum, sich nach
allem seinen Vermögen auszubreiten.23 Moritz widmet dagegen seine ganze
Aufmerksamkeit dem Individuum (wenn in
seinen Texten der Staat, oder das soziale
System zur Sprache kommen, dann werden
sie heftig kritisiert27), er schreibt aus der
Perspektive dieses Individuums und nur an
dieser Perspektive findet er Interesse: Für Moritz dagegen ist jeder Versuch, die
Freiheit des Individuums einzuschränken,
unakzeptabel: Der Mensch soll keinen Gran von den
Vorzügen seines Wesens verlieren, um in
irgendein Ganzes, das außer ihm ist, gepaßt zu
werden, da er selbst für sich das edelste Ganze
ausmacht. [...] Ich fordre so viel Freiheit und
Muße, als nötig ist, über mich selbst, über
meine Bestimmung und meinen Werth als
Mensch zu denken.24 Daß nun jeder einzelne Mensch, wenn er
seinen Antheil von Kräften zur Erhaltung des
Ganzen aufgewandt hat, sich auch als den
Zweck dieses Ganzen betrachten lerne und
auch von jedem andern so betrachtet werde –
darin besteht eigentlich die wahre Aufklärung,
welche nothwendig allgemein verbreitet seyn
muß, wenn sie nicht als bloße Täuschung und
Blendwerk betrachtet werden soll.28 In einem sind Moritz und Kant zwei-
felsohne einig: die Menschheit befindet sich
erst auf dem Wege zur allgemeinen Auf-
klärung. Die Schlussfolgerungen, die beide
aus dieser Feststellung ziehen, sind jedoch
diametral anders. Kant nennt das Zeitalter der
Aufklärung „das Jahrhundert Friederichs”25
und vertritt die Meinung, dass die meisten
Menschen noch nicht reif genug sind, um ihr
Schicksal selbst und ohne fremde Leitung zu
bestimmen. 23 Ebd., 61.
24 Moritz, 1962, 16-17.
25 Kant, 1977, 59. 26 Ebd., 61. 29 Karl Philipp Moritz, „Aus dem Tagebuche eines
Geistersehers“, Dichtungen und Schriften zur
Erfahrungsseelenkunde, hrsg. von Heide Hollmer und
Albert Meier, Frankfurt am Main: Deutscher
Klassiker Verlag, 2006, 746-747. „ICH FORDRE SO VIEL FREIHEIT UND MUSSE,
ALS NÖTIG IST, ÜBER MICH SELBST, ÜBER MEINE
BESTIMMUNG UND MEINEN WERTH ALS MENSCH
ZU DENKEN“.1 ZUR KUNST- UND
FREIHEITSKONZEPTION BEI KARL PHILIPP MORITZ Sie sollten dem aufgeklärten
Monarchen dankbar sein, dass er die Reli-
gionsfreiheit garantiert, die Zensur auf- Die Aufklärung und die Freiheit stellen
jedoch auch ihre Forderungen, und diese 28 Moritz, 1962, 18 89 Auch hier ist Moritz radikaler als Kant und
überdies klingt er mit seiner Vorstellung der
Eigenverantwortung des Menschen immer
noch äußerst aktuell. nimmt Moritz nicht nur wahr, sondern er
akzeptiert sie: Wer nicht auf eine vernünftige Weise tätig
ist, der braucht auch nicht frei zu sein. – Der
untätige Mensch sei sein ganzes Leben hin-
durch in einem Kerker eingesperrt – die Welt
wird dabei nichts verlieren.29 “ICH FORDRE SO VIEL FREIHEIT UND MUSSE, ALS NÖTIG IST, ÜBER MICH SELBST,
ÜBER MEINE BESTIMMUNG UND MEINEN WERTH ALS MENSCH ZU DENKEN”. ON KARL
PHILIPP MORITZ’S CONCEPT OF ART AND FREEDOM Krzysztof Tkaczyk S u m m a r y Goethe was wrong. In his aesthetic, Moritz is far more
interesting and progressive than his “elder brother”;
in his literary criticism – more consistent than Schiller,
and in perceiving society and politics – more radical
than Kant. This article is mainly on key concept
regarding Moritz’s aesthetic works: the concept of
beauty and its functionalism. Definition of “the work
of art” as a closed, specified unity enables Moritz to
get out of the frame of aesthetics itself and to enter
the sphere of politics. Moritz liberates man from the
restraints of utility and treats him as a noble individual
who possesses the artistic value in himself. In 1786 in Rome, Johann Wolfgang Goethe wrote
about him: “He is my younger brother; we art cut
from the same mould. However, fate was against him,
whereas I was fortune’s favourite.” He meant Karl
Phillip Moritz (1756-1793), a German writer and art
critic, Greek mythology historian, precursor of modern
psychology and the author of psychological novel. If
we assume that Goethe’s comments concern Moritz’s
material and social status only, we would have to admit
that Goethe was right. However, if we apply these
judgments to the quality of his (Moritz’s) works, we
would be inclined to saying that in this case the great Anschrift des Verfassers:
Instytut Germanistyki, Uniwersytet Warszawski
ul. Browarna 8/10, 00-311 Warszawa, Polen
E-Mail: krzysztof_tkaczyk@o2.pl Gauta 2008-11-05
Priimta 2008-11-27 90
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https://openalex.org/W4319349085
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https://bajangjournal.com/index.php/IJSS/article/download/4930/3659
|
English
| null |
OPPORTUNITIES AND CHALLENGES OF THE IMPLEMENTATION OF ISLAMIC LAW IN INDONESIA
|
International Journal of Social Science
| 2,023
|
cc-by
| 9,338
|
Article Info Islamic law only became an authoritative source (a legal source that has legal force)
in constitutional law when the Jakarta Charter was placed in the Republic of
Indonesia's Presidential Decree dated 5 July 159 as can be seen in the preamble to the
Decree as follows: "That we believe that the Jakarta Charter is dated 22 June 1945
animates the 1945 Constitution and constitutes a series of units in the Constitution.”
The word "animating" negatively means that it is not permissible to make laws in the
Indonesian state that are contrary to Islamic law for its adherents. Even though Islamic
law has failed to be obtained by Islamic parties, the opportunity to make laws based
on or inspired by Islamic law through the mechanism of representative democracy in
Indonesia is still wide open. Challenges faced by Islamic law The toughest challenge
at this time does not lie in the constitutional logic of accommodating Islamic law in
national law, but in the dialectics between secular currents of Western thought and
expressions of the diversity of Muslims which sometimes creates phobias. This is an open access article under the CC BY-SA license. Keywords:
Application, Law, Islam y
p
This is an open access article under the CC BY-SA license. This is an open access article under the CC BY-SA license. Corresponding Author:
Samsidar Jamaluddin
Universitas Islam Negeri, Alauddin Makassar, Indonesia
E-mail: samsidarjamaluddin@staiddimaros.ac.id Corresponding Author:
Samsidar Jamaluddin E-mail: samsidarjamaluddin@staiddimaros.ac.id OPPORTUNITIES AND CHALLENGES OF THE
IMPLEMENTATION OF ISLAMIC LAW IN INDONESIA By
Samsidar Jamaluddin1, Muhrim Djakat2, Hidayatussalam3, Sabri Samin4, Misbahuddin5
1,2,3,4,5Universitas Islam Negeri, Alauddin Makassar, Indonesia
E-mail: 1samsidarjamaluddin@staiddimaros.ac.id By
Samsidar Jamaluddin1, Muhrim Djakat2, Hidayatussalam3, Sabri Samin4, Misbahuddin5
1,2,3,4,5Universitas Islam Negeri, Alauddin Makassar, Indonesia
E-mail: 1samsidarjamaluddin@staiddimaros.ac.id Article Info
Article history:
Received Nov 09, 2022
Revised Dec 17, 2022
Accepted Jan 29, 2023
Keywords:
Application, Law, Islam 2177 2177 International Journal of Social Science (IJSS)
Vol.2 Issue.5 Februari 2023, pp: 2177-2186
ISSN: 2798-3463 (Printed) | 2798-4079 (Online)
DOI: https://doi.org/10.53625/ijss.v2i5.4930 1 Abdul Ghofur, Demokrasi dan Prospek Hukum Islam di Indonesia Studi Atas Pemikiran Gus Dur (Yogyakarta:
Pustaka Pelajar, 2002). …………………………………………………………………………………………………………………………………………………………………..
Journal homepage: https://bajangjournal com/index php/IJSS
1 Abdul Ghofur, Demokrasi dan Prospek Hukum Islam di Indonesia Studi Atas Pemikiran Gus Dur (Yogyakarta:
Pustaka Pelajar, 2002).
2 Ghofur.
3 Ghofur. 2 Ghofur. 1. INTRODUCTION This attempt was not successful due to strong
tensions between secular nationalists and Islamic nationalists.7 Islamic law only became an authoritative source (a legal source that has legal force) in constitutional law when
the Jakarta Charter was placed in the Republic of Indonesia's Presidential Decree dated 5 July 159 as can be seen in
the preamble to the Decree as follows: "That we believe that the Jakarta Charter is dated 22 June 1945 animates the
1945 Constitution and constitutes a series of units in the Constitution.” The word "animating" negatively means that it is not permissible to make laws in the Indonesian state that are
contrary to Islamic law for its adherents. Positively it means that adherents of Islam are required to carry out Islamic
law. Therefore, a law must be made that will enforce Islamic law in national law. This opinion is in accordance with
the statement of Prime Minister Juanda in 1959 which reads: Recognition of the existence of the Jakarta Charter as a
historical document for the government also means recognition of its influence on the 1945 Constitution. g
g
Although the Jakarta Charter which would give constitutional status to the application of Islamic law was not
accommodated by the New Order government as part of the 1945 Constitution, several aspects relating to the
application of Islamic law have been legislated in the national legal system. At least, there are seven legal rules that
are legislated into Indonesian positive law. Namely First Law No. 1 of 1971 concerning Marriage; Second, Law No. 7
of 1989 concerning the Religious Courts;8 Third, PP No. 28 of 1977 concerning Waqf; Fourth Law No. 7 of 1992 jo. Law No. 10 of 1998 and Law Number 23 of 1999 concerning the National Banking System which permits the operation
of Islamic Banks; Fifth, Presidential Decree No. 1 of 1991 concerning Compilation of Islamic Law; Sixth Law No. 17
of 1999 concerning Organizing Hajj; and Seventh, Law Number. 38 of 1999 concerning Management of Zakat. All of these legal products accommodate elements of Islamic law that are applied without having to include any
reference to the Jakarta charter.9 The tug-of-war to turn the values of Islamic law into positive law often occurs,
between nationalist and Islamic political forces which have become the two currents of power that determine the
stability of the management of state power. 1. INTRODUCTION Practically speaking, in the context of the formal life of
the state, especially as reflected in court practice, it seems that the scope is greatly reduced, and generally only concerns
family law.4 It is not surprising, therefore, that when the Dutch arrived in the archipelago in 1605, Islamic law was
ingrained in the souls of its adherents in such a way as it was confirmed by a number of existing Islamic kingdoms.5 law, to matters relating to human relations on a broader scale. Practically speaking, in the context of the formal life of
the state, especially as reflected in court practice, it seems that the scope is greatly reduced, and generally only concerns
family law.4 It is not surprising, therefore, that when the Dutch arrived in the archipelago in 1605, Islamic law was
ingrained in the souls of its adherents in such a way as it was confirmed by a number of existing Islamic kingdoms.5 In the Indonesian context, the term Islamic law refers to a meaning given to fiqh provisions in force in Indonesia
which at the same time displays its distinctive Indonesian character. So that Indonesian values and culture are reflected
in it, both in terms of legal principles and the patterns of thought that underlie them, showing differences from the fiqh
that applies in several other countries, such as Sudan and the Northern Nigeria region, and several other countries that
are still trying to implement the scope Islamic law as a whole, including Islamic penal law.6 In the struggle for the early days of independence, the Islamic elites fought with all their might to include a
phrase in the Preamble of the 1945 Constitution, which obliges Muslim residents to always carry out their religious
obligations. This is what is known as the Jakarta Charter which includes 7 words, which are believed to give
constitutional basis for the application of Islamic law in Indonesia. 1. INTRODUCTION Background to the Problem History has noted that Islamic law (fiqh) has existed and applies to its adherents
(living law) since the arrival of Islam in Indonesia.1 Peacefully in the 7th century AD or coinciding with the 1st century
Hijri. When the archipelago was controlled by the Sultans at that time, Islamic law was even integrated into life (the
royal state). Even though during the time of the Sultanate Islamic law had been adopted as formal and material law,
especially in the fields of marriage, grants, waqf and inheritance, in general they were still guided by the first
abstraction and spread throughout the archipelago, namely Sirat-al-mustaqim written by Nuruddin ar -Raniry in the
17th century. According to Hamka, the description of this book and its Syarah were expanded and used as a standard
for resolving disputes between Islamic communities in the Sultanates of Palembang and Banten. The same thing was
also found in Islamic kingdoms in Java. Therefore, it is very regrettable, even though during the Sultanate he had
played the role of advisor and judge, no abstract positive law had been drawn up from the content of fiqh doctrines. In the original Malacca law which is also the territory of the Archipelago, as stated by Hooker, quoted by Abdul
Gafur, even though it recognizes customary law, elements of Islamic law are clearly found in various places.2 For
example regarding the punishment for the killing of free people or slaves, the punishment for cutting off the hand for
theft and the punishment for adultery. In marriage law, the elements of Islamic law can be seen even more clearly,
because the legal requirements are regulated according to the teachings of fiqh, especially based on the Shafi'i school
of thought.3 Based on this fact, it can be said that the Islamic legal tradition is the law that applies in the archipelago. Normatively, Islamic law covers almost all aspects of life, starting from matters of religious rituals, civil law, criminal Journal homepage: https://bajangjournal.com/index.php/IJSS 2178 International Journal of Social Science (IJSS)
Vol.2 Issue.5 February 2023, pp: 2177-2186
ISSN: 2798-3463 (Printed) | 2798-4079 (Online) law, to matters relating to human relations on a broader scale. 4 Muhammad Abdun Nasir, Positivisasi Hukum Islam di Indonesia (Mataram: IAIN Mataram Press, 2004).
5 Juhaya S. Praja, Hukum Islam di Indonesia Perkembangan dan Pembentukan (Bandung: Remaja Rosda Karya,
1991). aja.
9 Azyumardi Azra Arskal Salim, No TitleNegara Dan Syariat Dalam Perspektif Politik Hukum Indonesia, dalam
Syariat Islam Padangan Muslim Liberal, editor (Jakarta: Sembrani Aksara Nusantra, 2003). )
6 Nasir, Positivisasi Hukum Islam di Indonesia. 7 Praja, Hukum Islam di Indonesia Perkembangan dan Pembentukan.
8 Praja. …………………………………………………………………………………………………………………………………………………………………
4 Muhammad Abdun Nasir, Positivisasi Hukum Islam di Indonesia (Mataram: IAIN Mataram Press, 2004).
5 Juhaya S. Praja, Hukum Islam di Indonesia Perkembangan dan Pembentukan (Bandung: Remaja Rosda Karya,
1991).
6 Nasir, Positivisasi Hukum Islam di Indonesia.
7 Praja, Hukum Islam di Indonesia Perkembangan dan Pembentukan.
8 Praja.
9 Azyumardi Azra Arskal Salim, No TitleNegara Dan Syariat Dalam Perspektif Politik Hukum Indonesia, dalam
Syariat Islam Padangan Muslim Liberal, editor (Jakarta: Sembrani Aksara Nusantra, 2003). 2. RESEARCH METHODS This research is a type of library research. What is called library research or often also called literature study,
is a series of activities related to methods of collecting library data, reading and taking notes and processing research
materials 10. In this study, the author applies the library research method because at least there are several underlying reasons. First, that data sources cannot only be obtained from the field. Sometimes data sources can only be obtained from
libraries or other documents in written form, either from journals, books or other literature. Second, literature study is
needed as a way to understand new symptoms that occur that cannot be understood, then with this literature study it
will be possible to understand these symptoms. So that in overcoming a symptom that occurs, the author can formulate
a concept to solve a problem that arises. The third reason is that library data remains reliable to answer the research
questions.11 However, information or empirical data that has been collected by other people, whether in the form of
books, scientific reports or research reports, can still be used by library researchers. Even in certain cases the field data
is still not significant enough to answer the research questions that will be carried out. The stages that must be taken by the author in library research are as follows: a. Collect research materials. Because this research is a library research, the material collected is in the form of information or empirical data sourced
from books, journals, results of official and scientific research reports and other literature that supports the theme of
this research. b. Read bibliography. Reading activities for research purposes is not a passive job. Readers are asked to
simply absorb all "knowledge" information in reading material, but rather a "hunt" activity that requires active and
critical involvement of the reader in order to obtain maximum results.12 In reading research materials, readers must dig
deeply into reading materials that allow them to find new ideas related to the research title. c. Make research notes. The activity of recording research materials is arguably the most important stage and perhaps also the heaviest
culmination of the entire series of library research 13. Because in the end all the material that has been read must be
drawn a conclusion in the form of a report. d. Processing research notes. 1. INTRODUCTION Even though Islamic law has failed to be obtained by Islamic parties, the
opportunity to make laws based on or inspired by Islamic law through the mechanism of representative democracy in
Indonesia is still wide open. However, that does not mean without challenges that must be faced. In this challenge,
there seems to be a concern that secular Western thought and religious expressions of Muslims sometimes give rise to
phobias and on the other hand, Islamic activists will use democracy to kill democracy. This article tries to describe the
long journey of the struggle to incorporate Islamic law into the Indonesian constitution and laws, the extent of the
opportunities and challenges. With the various descriptions described above, several main issues can be formulated,
how are the opportunities for implementing Islamic law in Indonesia and what are the challenges faced by Islamic law
in Indonesia. Praja. 9 Azyumardi Azra Arskal Salim, No TitleNegara Dan Syariat Dalam Perspektif Politik Hukum Indonesia, dalam
Syariat Islam Padangan Muslim Liberal, editor (Jakarta: Sembrani Aksara Nusantra, 2003). Journal homepage: https://bajangjournal.com/index.php/IJSS 2179 International Journal of Social Science (IJSS)
Vol.2 Issue.5 Februari 2023, pp: 2177-2186
ISSN: 2798-3463 (Printed) | 2798-4079 (Online)
DOI: https://doi.org/10.53625/ijss.v2i5.4930 2. RESEARCH METHODS All materials that have been read are then
processed or analyzed to get a conclusion drawn up in the form of a research report. Content analysis or content study is a research methodology that utilizes a set of procedures to draw valid
conclusions from a book or document 14. Meanwhile, Harold D. Lasswell stated that content analysis is research that
is an in-depth discussion of the content of written or printed information in the mass media. From the explanation
above, it can be concluded that content analysis is a research method with certain stages to extract the essence of an
idea or information and then draw a conclusion. The author uses data analysis techniques in the form of content analysis
because this type of research is a type of library research, where the data source is in the form of books and documents
as well as literature in other forms. …………………………………………………………………………………………………………………………………………………………………..
Journal homepage: https://bajangjournal.com/index.php/IJSS
10 Mestika Zed, Metode Penelitian Kepustakaan (Jakarta: Yayasan Obor Indonesia, 2008).
11 Zed.
12 Zed.
13 Zed.
14 J Moleong Lexy, Metodologi Penelitian Kualitatif (Bandung: PT. Remaja Rosdakarya, 2008).
15 Zaini Rahman, Fiqh Nusantara dan Sistem Hukum Nasional dan Sistem Hukum Nasional Perspektif Kemaslahatan
Kebangsaan (Yogyakarta: Pustaka Pelajar, 2016). Zed.
14 J Moleong Lexy, Metodologi Penelitian Kualitatif (Bandung: PT. Remaja Rosdakarya, 2008).
15 Zaini Rahman, Fiqh Nusantara dan Sistem Hukum Nasional dan Sistem Hukum Nasional Perspektif Kemaslahatan
Kebangsaan (Yogyakarta: Pustaka Pelajar, 2016). Because this research is a library research, the material collected is in the form of information or empirical
data sourced from books, journals, results of official and scientific research reports and other literature that
supports the theme of this research. Because this research is a library research, the material collected is in the form of information or empirical
data sourced from books, journals, results of official and scientific research reports and other literature that
supports the theme of this research. 0 Mestika Zed, Metode Penelitian Kepustakaan (Jakarta: Yayasan Obor Indonesia, 2008).
1 Zed. 16 Rahman.
17 Marzuki Wahid & Rumadi, Fiqh Madzhab Negara Kritik Atas Politik Hukum Islam di Indonesia (Yogyakarta:
LKiS, 2001).
18 Eklektisisme dan Hukum Nasional Kompetensi Antara Hukum Islam dan Hukum Umum, A. Qodri Azizy
(Yogyakarta: Gama Media, 2002).
19 Abdul Halim, Peradilan Agama dalam Politik Hukum Islam di Indonesia dari Otoriter Konservatif Menuju
Konfigurasi Demokrasi Responsif (Jakarta: Raja Grafindo Persada, 2000).
20 Bustanul Arifin, Pelembagaan Hukum Islam Di Indonesia Akar Sejarah, Hambatan dan Prospeknya (Jakatra:
Gema Insani Press, 1996). Research Results and Discussion A. Opportunities for Islamic Law to Enter the National Legal System pp
g
y
Islamic law is constitutionally very strong because it has become an integral part of the national legal system. The legal system in this context means the unified whole of an order which consists of parts or elements that are closely
related to one another. The Indonesian legal system adheres to the principle of pluralism as a logical consequence of
the social and political conditions of a pluralistic society. Broadly speaking, the development of national law originates
from three legal systems, namely customary law, Islamic law and Western law. All three are a unified system, the parts
of which can work together and are interrelated according to certain patterns and plans. In a legal system, there should
not be contradictions or overlapping between the existing parts.15 pp g
g p
When viewed from the legal principle, respectively, the legal system which seems to stand alone without ties,
is actually bound by several meanings that are more general in nature, namely that both express an ethical claim. In Journal homepage: https://bajangjournal.com/index.php/IJSS 2180 International Journal of Social Science (IJSS)
Vol.2 Issue.5 February 2023, pp: 2177-2186
ISSN: 2798-3463 (Printed) | 2798-4079 (Online) this case as part of the national legal system, Islamic law has its own uniqueness, especially in its attachment to the
legitimacy of how to understand and issue it from the sources of the Qur'an and Hadith. The ethical norms or values
displayed by the Islamic legal system certainly have different essences and perspectives from those in the Western
customary and legal systems. this case as part of the national legal system, Islamic law has its own uniqueness, especially in its attachment to the
legitimacy of how to understand and issue it from the sources of the Qur'an and Hadith. The ethical norms or values
displayed by the Islamic legal system certainly have different essences and perspectives from those in the Western
customary and legal systems. Within the framework of the national legal system, what is meant by Islamic law is not the Koran and Hadith
themselves, but rather the product of understanding these two sources. As a product of understanding, ideally it must
adapt to the demands of the locality and state system. Research Results and Discussion According to the principles and principles of the state, the
existence and enforceability of Islamic law is tied to the legal framework for the formation of national law. Because
the norms of Islamic law cannot be carried out without legitimacy and governmental authority, especially those relating
to public law. In the Islamic doctrine itself, the need for state institutions or government authorities is obligatory both
in reason and syara.16 In the Indonesian context, with Pancasila as the basis of the state, the values of Islamic law which were
originally individually rooted in the individuals of the Muslim community can be transformed in such a way as to
become a unified whole with the national legal system. Therefore, the existence of demands for the formulation of
Islamic law seen from the point of view of positive legal theory is logical. After Indonesia became independent, and
Pancasila and the 1945 Constitution were established as a source of law, then in the context of the implementation of
Islamic law it emerged as a counter theory to the theories of the colonial period. There are at least three theories that
can be noted, namely the theory of receptive exit, receptive a contrario and the theory of existence. The three theories
essentially refute the previous theoretical arguments. At the same time, he acknowledged and emphasized the existence
of Islamic law and Pancasila and the 1945 Constitution.17 Throughout the New Order, Islamic law was never a separate policy in particular. There is not a single pointer
in the legal political texts of the New Order concerning the existence of Islamic law. However, this does not mean that
Islamic law does not receive attention. In practice, in practical terms, Islamic law has a place in the national legal
system, and even formally, its position is better than in previous times. In this context, it seems that the New Order regarded Islamic law as part of religion, not as an autonomous law,
which could be developed independently as long as it referred to its basic sources. This assumption is not wrong, but
there can be a narrowing of meaning if religion is understood in a secularistic frame of mind, such as ongoing
perception. This can be seen clearly in the division of the judicial area into four neighborhoods. The first is the General
Court. Second, the Religious Courts. Research Results and Discussion 1 of 1974 (concerning
Marriage), Law no. 7 of 1989 (concerning the Religious Courts), and Presidential Instruction. No. 1 of 1991
(concerning the dissemination of the Compilation of Islamic Law). But also included in the legal provisions of PP No. 9 of 1975 (implementation of Law No. 1 of 1974) PP. No. 28 of 1977 concerning Waqf of Owned Land), PP. No. 72
of 1992) concerning Banks based on the Principle of Profit Sharing) and Law no. No. 4 of 1979 (regarding child
welfare).23 Thus, the dynamics of Islamic law in Indonesia experience a pattern of renewal in the history of its practical
empirical development as positive law into the juridical institutionalization model. Therefore, the Islamic world, for
example, has very diverse experiences related to efforts made to maintain the existence of its religious laws, ranging
from the most "extreme left" to the extreme right..24 Abdullah Ahmed Anna'im, in detail explained the pattern of
renewal in the Islamic world in general in the field of inheritance and family law in the Islamic world which he
considered unsatisfactory.: 1. Takshih al-Qadhi (the right of the ruler to decide and uphold court decisions), is used as a procedure to limit
the application of sharia to matters of civil law for Muslims. The same procedure is also used to prevent courts
from applying sharia in specific circumstances without changing the relevant sharia rules. For example, to
prevent child marriage, Egyptian law in 1931 denied matrimonial assistance through the courts by blocking
the court from claiming marriage if the husband was not yet 19 years old or the wife was not 16 years old at
the time of the proceedings. p
g
2. Takhayyur, namely selecting various schools of thought in an eclectic manner such as Sudan through fatwas
(judicial directives) that allow courts to deviate from the rules of the Hanafi school of thought.25 p
g
2. Takhayyur, namely selecting various schools of thought in an eclectic manner such as Sudan through fatwas
(judicial directives) that allow courts to deviate from the rules of the Hanafi school of thought.25 3. A form of reinterpretation is used to limit a man's freedom of divorce and polygamy for example, the Tunisian
Law of personal status of 1956. 21 Praja, Hukum Islam di Indonesia Perkembangan dan Pembentukan.
22 Rumadi, Fiqh Madzhab Negara Kritik Atas Politik Hukum Islam di Indonesia.
23 Nasir, Positivisasi Hukum Islam di Indonesia.
24 Rumadi, Fiqh Madzhab Negara Kritik Atas Politik Hukum Islam di Indonesia.
25 Abdullah Ahmed An-Nai‟m, Dekonstruksi Syariah Wacana Kebasan Sipil, Hak Asasai Manusia dan Hubungan
Internasional, terj (Yogyakarta: LKiS, 1994). Research Results and Discussion Third, Military Court, Fourth, State Administrative Court.18 g
y
This division of the judiciary indicates the existence of religious and non-religious areas, so that by itself it
forms the terminology of religious law and state (non-religious) law. Within this perceptional framework that tends to
be secularistic, Islamic law gets its justification from the New Order's legal politics. This justification is actually a
continuation of previous periods, namely Law no. 22 of 1946 jo. UU no. 32 of 1954 and PP no. 45 of 1957.19 p
p
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Islamic law as positive law (lex positive constitutum) was first enacted in Law no. 1 of 1974 article 2 paragraph
(1) is still general in nature (religious law), not specifically Islamic law, the article reads "marriage is valid, if it is
carried out according to the laws of each religion and belief". Then article 63 paragraph 1 emphasized that the Religious
Courts as contained in article 10 of Law no. 14 of 1970 is specifically for people who are Muslims.20 This is based on
the history of the Religious Courts in Indonesia which has been running for more than one hundred years, and on the
premise that only Islam brings law in its purest sense (aqidah wa shari'ah). Therefore, Muslims need a court where
they seek legal justice. Therefore, the existence of the Religious Courts (Islam) in Indonesia, is not because Muslims
constitute the majority in Indonesia, but because the Religious Courts are a legal necessity for Muslims. In other words,
Islamic law insofar as it concerns the areas of authority of the Religious Courts is the national law in the Republic of
Indonesia. Indonesian national law is a collection of legal norms in society derived from elements of Islamic law,
customary law, and Western law (general law science). If this is limited, it is actually not progress. Since the VOC era, Islamic marriage and inheritance laws have
become positive law and are used as material law in determining court decisions for native Muslims. During the Old Journal homepage: https://bajangjournal.com/index.php/IJSS 2181 International Journal of Social Science (IJSS)
Vol.2 Issue.5 Februari 2023, pp: 2177-2186
ISSN: 2798-3463 (Printed) | 2798-4079 (Online)
DOI: https://doi.org/10.53625/ijss.v2i5.4930 Order era, Law no. 5 of 1960, which was accused of being communist, also brought awareness to the validity of
agrarian law over religious law. Research Results and Discussion It is a necessity for the existence of Islamic law within the ius constitutum framework,
with the enactment of Law no. 7 of 1989 concerning the Religious Courts and Presidential Instruction No. 1 of 1991
concerning Compilation of Islamic Law. The existence of these two laws, Islamic law is not only recognized for its
existence, but definitively it has become part of national law and a pillar of state justice, both materially and formally. It has become part of the restructuring and reform of national law. p
g
The existence of the reality of the world of Indonesian politics and the content of legislation in Indonesia reflects
the application of Islamic law to be reckoned with. The proof is that there are seven legal regulations that have been
legislated into the positive law of the State of Indonesia. Namely First Law No. 1 of 1971 concerning Marriage; Second,
Law No. 7 of 1989 concerning the Religious Courts;21 Third, Government Regulation Number 28 of 1977 concerning
Waqf; Fourth Law No. 7 of 1992 jo. Law No. 10 of 1998 and Law Number. 23 of 1999 concerning the National
Banking System which permits the operation of Islamic Banks; Fifth, Presidential Decree No. 1 of 1991 concerning
Compilation of Islamic Law; Sixth Law No. 17 of 1999 concerning Organizing Hajj; and Seventh, Law Number. 38
of 1999 concerning Management of Zakat. It's just that, as a material law, we still haven't fully received political wiil from the state apparatus, we're still
at a crossroads. And its legal area is still limited to the law of inheritance, marriage and endowments, wills, grants and
sadaqah, not much with the actual capabilities of Islamic law.22 q
p
Meanwhile, in terms of value, the provisions of Islamic law do not only color Law no. 1 of 1974 (concerning
Marriage), Law no. 7 of 1989 (concerning the Religious Courts), and Presidential Instruction. No. 1 of 1991
(concerning the dissemination of the Compilation of Islamic Law). But also included in the legal provisions of PP No. 9 of 1975 (implementation of Law No. 1 of 1974) PP. No. 28 of 1977 concerning Waqf of Owned Land), PP. No. 72
of 1992) concerning Banks based on the Principle of Profit Sharing) and Law no. No. 4 of 1979 (regarding child
welfare).23 Meanwhile, in terms of value, the provisions of Islamic law do not only color Law no. Research Results and Discussion It states that divorce does not occur except by court decision, and courts are
permitted to oblige the husband to pay a sum of money as compensation if the court feels that the husband is
not treating his wife fairly or is looking for reasons to divorce. The same thing also happened in Indonesia
which is reflected in Law no. 1 of 1974 and the Compilation of Islamic Law (KHI). Islamic law has enormous prospects and potential in the development of national law. There are several
considerations that make Islamic law worthy of being a reference in the formation of national law. When viewed from
the political reality and legislation in Indonesia it seems that the values of Islamic law are increasingly deserving of
reckoning as seen in several laws and regulations whose presence further strengthens Islamic law, namely: Marriage
Law Law No. 1 of 1974 concerning Marriage was ratified and promulgated in Jakarta on January 2, 1974 (State Gazette
of 1974 No. Supplement to State Gazette Number. 3019). Law on Religious Courts Law no. 7 of 1989 concerning the Journal homepage: https://bajangjournal.com/index.php/IJSS 2182 International Journal of Social Science (IJSS)
Vol.2 Issue.5 February 2023, pp: 2177-2186
ISSN: 2798-3463 (Printed) | 2798-4079 (Online) International Journal of Social Science (IJSS)
Vol.2 Issue.5 February 2023, pp: 2177-2186
ISSN: 2798-3463 (Printed) | 2798-4079 (Online) Religious Courts passed and promulgated in Jakarta on December 29, 1989 (State Gazette of the Republic of Indonesia
of 1989 No. 49, Supplement to the State Gazette of the Republic of Indonesia No. 34). Then on March 20, 2006 Law Religious Courts passed and promulgated in Jakarta on December 29, 1989 (State Gazette of the Republic of Indonesia
of 1989 No. 49, Supplement to the State Gazette of the Republic of Indonesia No. 34). Then on March 20, 2006 Law
No. 3 of 2006 concerning Amendments to Law no. 7 of 1989 concerning the Religious Courts. What is a relief from
this law is the wider authority of the Religious Courts, especially the authority to resolve cases in the field of sharia
economics. To explain the various sharia issues above, the National Sharia Council (DSN) has issued a number of
fatwas relating to the sharia economy, which to date have reached 53 fatwas. The fatwa can be the main material in preparing the compilation. Research Results and Discussion However, it encounters
several obstacles, including the following: ,
g
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1. The source of Islamic tasyri': the source of tasyri' is a sacred thing. In doing ijtihad they are afraid of making
mistakes that hinder the implementation of codification. 1. The source of Islamic tasyri': the source of tasyri' is a sacred thing. In doing ijtihad they are afraid of making
mistakes that hinder the implementation of codification. 2. Freedom of ijtihad: to have ijtihad for those who are entitled is a human right. If the results of ijtihad have
been codified, it means that they no longer accept other ijtihad, even though in the case of new issues it is
obligatory to have ijtihad. g
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3. Freedom of belief: Islam does not force people to embrace religion. Therefore, if this fiqh is codified, it means
limiting the freedom of belief for others. g
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Freedom of belief: Islam does not force people to embrace religion. Therefore, if this fiqh is codified g
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3. Freedom of belief: Islam does not force people to embrace religion. Therefore, if this fiqh is codi
limiting the freedom of belief for others. Freedom of belief: Islam does not force people to embrace religion. Therefore, if this fiqh is codified
limiting the freedom of belief for others. The method adopted in carrying out the codification was carried out in stages in the first century, namely; First,
determine the state's official school of thought. Abu Jafar al-Mansur asked Imam Malik bin Anas (d. 179 H./796 AD)
that his school contained in the book al-Muwata' be determined by the caliph as the official school (state) as the only
standard book for reference to Islamic law.28 Imam Malik rejects and asserts that there is no exclusive claim to divine
truth, How does Imam Malik insist that there is no valid reason to force a certain set of legal opinions to be applied in
a country, because it would be contrary to other sets of legal opinions.29 I am an ordinary human being who has
limitations in understanding the hadith of the Prophet. What I have collected in my book is only part of Islamic law. The companions of the Prophet had died and the knowledge of Islamic law had also gone with them. Research Results and Discussion In connection with the addition of quite a lot
of authority to the religious courts as stated in Law no. 3 of 2006 concerning sharia economics, while Islamic law
regarding sharia economics is still scattered in the books of fiqh and fatwa of the National Sharia Council, the existence
of the Compilation of Sharia Economic Law (KHES) which is based on PERMA Number. 2 of 2008, September 10,
2008.26 In addition, there are several regulations containing Islamic law and their level below the law, among others,
in the era of democracy, especially after the reforms and amendments to the 1945 Constitution, the opportunity for the
application of Islamic law in a wider area is very large. To see the extent of the visibility of these opportunities can be
analyzed through three approaches; namely the legal system and political approach, the socio-cultural approach, as
well as the paradigm of Islamic legal thought which will be built in the Indonesian context. These three approaches
can be models for studying the actualization of Islamic law in the state context, not only in Indonesia but in countries
with a majority Muslim population and not Islamic countries.27
B
Ch ll
h I
l
i
f I l
i L
i I d
i B. Challenges to the Implementation of Islamic Law in Indonesia The Challenges Faced by Muslims in Establishing Islamic Shari'ah Through State Institutions Since the
beginning of the historical period for the formation of Islamic law, debates about whether Islamic law needs to be
codified or legislated by the state have emerged. Ottoman rule began in the early 14th century in Anatoli Turkey and
lasted approximately four centuries. The Ottoman state initially did not adhere to a particular school, then followed the
Hanafi school officially for fatwas and trials. The Ottoman government intended to codify laws. However, it encounters
several obstacles, including the following: The Challenges Faced by Muslims in Establishing Islamic Shari'ah Through State Institutions Since the
beginning of the historical period for the formation of Islamic law, debates about whether Islamic law needs to be
codified or legislated by the state have emerged. Ottoman rule began in the early 14th century in Anatoli Turkey and
lasted approximately four centuries. The Ottoman state initially did not adhere to a particular school, then followed the
Hanafi school officially for fatwas and trials. The Ottoman government intended to codify laws. …………………………………………………………………………………………………………………………………………………………………..
26 Rahman, Fiqh Nusantara dan Sistem Hukum Nasional dan Sistem Hukum Nasional Perspektif Kemaslahatan
Kebangsaan.
27 Rahman.
28 H. Rachamt Djatmika, Perkembangan Fiqh di Dunia Islam, dalam Tjun Sudarman, Hukum Islam Hukum Islam di
Indonesia Perkembangan dab Pembentukan (Jakarta: Remaja Rosda Karya, 1991).
29 Khaled M. Abou El Fadhl, Melawan Tentara Tuhan Yang Berwewenang dan dan yang kesewenang-wenangan
dalam Wacana Islam (Jakarta: Serambi, 2001).
30 Nurrohman, “Hukum Islam Di Era Demokrasi: Tantangan dan Peluang bagi Formalisasi Politik Syari‟at Islam di
Indonesia,” ADDIN Vol. 9, No (2015): 170. 26 Rahman, Fiqh Nusantara dan Sistem Hukum Nasional dan Sistem Hukum Nasional Perspektif Kemaslahatan
Kebangsaan.
27 R h Indonesia Perkembangan dab Pembentukan (Jakarta: Remaja Rosda Karya, 1991).
29 Khaled M. Abou El Fadhl, Melawan Tentara Tuhan Yang Berwewenang dan dan yang kesewenang-wenangan
dalam Wacana Islam (Jakarta: Serambi, 2001). Research Results and Discussion Therefore, my
book is inadequate if used as a reference.30 Then Sultan Salim 1 (1512-1520), the ninth Sultan Uthman) saw the need
to establish an official state school, so a Sultan decree was issued (Qararan Sultaniyyan/firman) announcing the Hanafi
school of thought as the official state school that must be followed in matters of "judicial and fatwa" . This way was
followed in Egypt. After Egypt was controlled by the Turks, the system of four qadi in Egypt after the reign of Mahmud Journal homepage: https://bajangjournal.com/index.php/IJSS Rahman.
28 H. Rachamt Djatmika, Perkembangan Fiqh di Dunia Islam, dalam Tjun Sudarman, Hukum Islam Hukum Islam d
Indonesia Perkembangan dab Pembentukan (Jakarta: Remaja Rosda Karya, 1991).
29 Kh l d M Ab
El F dhl M l
T
t
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Y
B
d
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k 30 Nurrohman, “Hukum Islam Di Era Demokrasi: Tantangan dan Peluang bagi Formalisasi Politik Syari‟at Islam di
Indonesia,” ADDIN Vol. 9, No (2015): 170. Rahman.
28 H. Rachamt Djatmika, Perkembangan Fiqh di Dunia Islam, dalam Tjun Sudarman, Hukum Islam Hukum Islam di
Indonesia Perkembangan dab Pembentukan (Jakarta: Remaja Rosda Karya, 1991).
29 Khaled M. Abou El Fadhl, Melawan Tentara Tuhan Yang Berwewenang dan dan yang kesewenang-wenangan
dalam Wacana Islam (Jakarta: Serambi, 2001). Journal homepage: h The tradition and history of Islamic
law is closer to the common law tradition than to continental law. There has never been a codification of law in the
social history of Islamic law until the end of the Ottoman dynasty. The codification of Islamic law at the end of the
Ottoman dynasty was the result of contonantal European influence. In the Indonesian context, Islamic law that is suitable for certain areas is not necessarily suitable for other
regions. Islamic law formulated in classical books is also not necessarily suitable for today's conditions. Therefore
qanunization or the formalization of Islamic law has the potential to curb changes in Islamic law which are always
developing with the opening of the door to ijtihad. Because of this, Islamic law, which is enacted by state authorities
who have the authority to form qanun regulations, will quickly become obsolete.33 Challenges also come from those who do not agree with the formalization of Islamic law through state laws. With the promulgation of Islamic law through state laws, the implementation of Islamic law seems to depend on the
state. Views like this become a debate related to the relationship between religion and the state which drains so much
of this nation's energy. From the early days of preparation for independence to more than half a century after
independence. However, during that long period of time, a common platform has not been found even though during
the New Order era this debate "as if" was considered finished. In fact, in the 2000 MPR annual session on the basic
issues of the state, or at least the desire to include the word "Islam" in one of the articles of the 1945 Constitution, was
still on the agenda of certain Islamic groups represented by the PPP and Crescent Star factions. g
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To see what the position of Islamic law is in the course of Indonesian history, at least there is a theory related
to the relationship between religion and the state that characterizes the lives of Muslims. The terms religion and state
referred to here are religion and state in their current institutionalized form. Religion in its basic sense is a system of
values/teachings or spiritual moral awareness that is believed to be true by its adherents to be used as a view and guide
in life. Journal homepage: h 2183 International Journal of Social Science (IJSS)
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ISSN: 2798-3463 (Printed) | 2798-4079 (Online)
DOI: https://doi.org/10.53625/ijss.v2i5.4930 International Journal of Social Science (IJSS)
Vol.2 Issue.5 Februari 2023, pp: 2177-2186
ISSN: 2798-3463 (Printed) | 2798-4079 (Online)
DOI: https://doi.org/10.53625/ijss.v2i5.4930 Ali Pasha, the Ottoman government, issued the Word (Sultan's decree) that the fatwa and the Court must be based on
the fatwa of Imam Hanafi.31 Second, the preparation of an opinion (madzhab). The preparation of a school of thought to be enforced in the
territory of the Ottoman Empire, namely by compiling the Ottoman civil law (al-Qanun al-Madani al-Uthmani), which
is known as Mjallatul Ahkam al-Adliyah, and other Ottoman laws taken from the Hanafi school. The government
(caliph) determines its validity and orders all people to obey it, by including in the last paragraph of article 1801 that
judges must follow the sultan's orders and may not follow other Mujtahid. Third, make a compilation from different sects. Apart from being based on the Hanafi school of thought as the
official school of thought, when drafting laws that would apply to the entire Ottoman territory, other schools were also
taken from other schools if the other schools were more suitable for human benefit. This method was also followed in
Egypt, Syria, Iraq, Tunisia, and others. For example, in legislation regarding al-ahwalusy-syakhsiyyah waqf,
inheritance, and wills that are adapted to current needs and the changing social conditions that surround them. (al-
ahwal wa al dhuruf). )
Fourth, taking modern legislation, the fourth stage in drafting legislation in the Ottoman Arab State was taking
several laws that were in accordance with Islamic sharia from modern legislation such as civil law (civil), trade law
(tijari), and criminal law ( jana'i).32 Since then, Islamic law has lived and developed independently of state mechanisms. Each judge has the freedom
to choose the Islamic law that he will apply and the state does not have the authority to interfere in their affairs. Establishing Islamic sharia norms through state institutions or qanunization contains both positive and negative
aspects. The positive aspect can provide a relatively uniform standard of Islamic law. But the negative aspect can
reduce the freedom of the judge in choosing the provisions of Islamic law that are most suitable for the particular case
he is facing. In the classical Islamic tradition, it is the judge who makes the law. 31 Djatmika, Perkembangan Fiqh di Dunia Islam, dalam Tjun Sudarman, Hukum Islam Hukum Islam di Indonesia
Perkembangan dab Pembentukan.
32 Djatmika.
33 Rumadi, Fiqh Madzhab Negara Kritik Atas Politik Hukum Islam di Indonesia.
34 Rumadi. Journal homepage: h It seems that al-Mawardy (w.1058 M)
and al-Ghazali as one of the Islamic political theorists who support this paradigm. 3. The secularistic paradigm This paradigm rejects the two paradigms above. Instead, the secularistic paradigm
proposes a separation (disparity) of religion over the state and the separation of the state over religion. The
concept of addunya al-akhirah, ad, din ad dawlah or age ad dunya, age ad-din is dichotomized separately. In
the context of Islam, this paradigm rejects the basis of the state on Islam, or at least rejects the determination
of Islam in a certain form of state. The supporter of this secularistic paradigm is Aliy Abdul ar-Raziq (1887-
1966), a Muslim scholar from Egypt. 33 And it was practiced in earnest by Mustafa Kemal Ataturk when he
came to power in Turkey in the 20s of this century..36 p
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What about the reality of Islamic politics in Indonesia. The paradigm of thought mentioned above in the context
of the Indonesian state, still has adherents of the secularistic paradigm, adherents of the first two paradigms whose
performance appears to be more prominent and therefore always identified as a cultural Islamic movement. It seems
that Muslims who struggle outside of secular Islamic groups/organizations are not seen as an Islamic political
constituency. Din Syamsuddin quoted M. Arskal Salim G.P. trying to map the mainstream of Islamic political thought
in Indonesia tends to ignore adherents of the secularist paradigm. He mentioned that there are at least three schools of
Islamic political thought in Indonesia, namely formalistic, fundamentalistic, militant, extremist, radical, fanatical,
jihadist and substantive. The formalistic school (Islamic scriptualism) which places more emphasis on symbolic-
legalistic expressions, and the fundamentalistic school which places more importance on the revivalism of classical
Islamic culture are presumably loyal adherents of the integralistic paradigm above. Meanwhile, the subtantivistic
school offers a religious understanding that places more emphasis on the substance of the teachings. Rather than a
formal legal form of teaching, it is an adherent of the symbiotic paradigm.37 g
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In the history of Islamic politics in Indonesia, the formalistic school that adheres to the Integralistic paradigm
above was once dominant, especially during the Old Order Government. However, the dominance of this formalistic
school collapsed when the New Order came to power in Indonesia. Journal homepage: h For Islamic teachings sourced from the revelation of Allah SWT, namely the Qur'an and Al-Sunnah. but in its
development to this day, religion is not only a system of spiritual values/teachings and moral awareness as it originated,
but has turned into an institution or organizational body. As a result of this change, at the empirical practical level, bad
relations often occur, such as inter-sect conflict of the same religion, between religions, and between religion and the
state.34 Islamic political theorists sociologists formulate several theories about the relationship between religion and
the state. These theories are broadly divided into three paradigms of thought: Journal homepage: https://bajangjournal.com/index.php/IJSS 2184 International Journal of Social Science (IJSS)
Vol.2 Issue.5 February 2023, pp: 2177-2186
ISSN: 2798-3463 (Printed) | 2798-4079 (Online) 1. Integralistic Paradigm (Unified Paradigm) In the integralistic paradigm, religion and the state are integrated
(integrated) the area of religion includes politics or the state. The state is both a political and a religious
institution. Therefore, according to this paradigm, the head of state is the holder of religious power and
political power. Its government is organized on the basis of "divine sovereignty", because supporters of this
paradigm believe that sovereignty originates and is in the "hands of God." This paradigm is adopted by the
Shia group and the Jamaat Islami Fundamentalist group in Pakistan to call the state (ad-dawlah) replaced by
imamate (leadership)..35 1. Integralistic Paradigm (Unified Paradigm) In the integralistic paradigm, religion and the state are integrated
(integrated) the area of religion includes politics or the state. The state is both a political and a religious
institution. Therefore, according to this paradigm, the head of state is the holder of religious power and
political power. Its government is organized on the basis of "divine sovereignty", because supporters of this
paradigm believe that sovereignty originates and is in the "hands of God." This paradigm is adopted by the
Shia group and the Jamaat Islami Fundamentalist group in Pakistan to call the state (ad-dawlah) replaced by
imamate (leadership)..35 p
2. Symbiotic Paradigm (Symbiotic Paradigm) Religion and the state, according to this paradigm, are related
symbiotically, namely a relationship that is reciprocal and requires each other. In this case, religion requires
a state because with the state, religion can develop. Conversely, the state also needs religion, because with
religion the state can develop in spiritual ethical and moral guidance. 38 Arskal Salim. 35 Rumadi.
36 Arskal Salim, No TitleNegara Dan Syariat Dalam Perspektif Politik Hukum Indonesia, dalam Syariat Islam
Padangan Muslim Liberal, editor.
37 Arskal Salim.
38 Arskal Salim 35 Rumadi. 37 Arskal Salim. 39 Arskal Salim. Journal homepage: h Contemporary thinkers criticize the formalization of Islamic law in
Aceh that the implementation of conservative Islamic law in Aceh should be a lesson for everyone. We should not
repeat our failures and allow conservative groups to dictate Islamic law as happened in Aceh. Many people in the
community do not realize how Islam in Aceh has been exploited in such a way by conservative groups to promote
something new, namely a type of Islam that suppresses women, restricts freedom of speech, applies strict rules of
conduct which are actually contrary to local traditions and the character of Islam. alone.40 Procontracts in response to this paradigm, for example between NU and Muhammadiyah do not support Islamic
sharia legislation projects in the regions because they prefer national law. This is because society in general realizes
that Islamic law is actually more of an ethical norm that originates from religious teachings. As an ethical norm,
Muslims both individually and collectively can actually carry out all the ethical norms contained in Islamic law without
any interference or coercion from the authorities. Contemporary thinkers criticize the formalization of Islamic law in
Aceh that the implementation of conservative Islamic law in Aceh should be a lesson for everyone. We should not Aceh that the implementation of conservative Islamic law in Aceh should be a lesson for everyone. We should not
repeat our failures and allow conservative groups to dictate Islamic law as happened in Aceh. Many people in the
community do not realize how Islam in Aceh has been exploited in such a way by conservative groups to promote
something new, namely a type of Islam that suppresses women, restricts freedom of speech, applies strict rules of
conduct which are actually contrary to local traditions and the character of Islam. alone.40 The toughest challenge at this time does not lie in the constitutional logic of accommodating Islamic law in
national law, but in the dialectics between secular currents of Western thought and expressions of the diversity of
Muslims which sometimes creates phobias. The fact is that law in Indonesia is dominantly patterned with the legal
system inherited from the Netherlands which has long been the mainstream of legal thought in Indonesia. This situation
objectively according to the logic of legal democracy is not something that needs to be questioned. Journal homepage: h With the concept of depoliticization of religion,
through the application of the principle of unity, the formalistic school that manifested itself in the form of Islamic
political parties was "dominated" by the New Order government so that it lost its relevance. The thoughts of Nurcholis
Madjid who are famous for the Islamic jargon Yes, Islamic Party No also helped eliminate the significance of this
formalistic school.38 As a substitute, during the New Order, the substantive school with its symbiotic paradigm became dominant. This flow reinforces a new perspective in viewing the relationship between religion and the State as follows: itute, during the New Order, the substantive school with its symbiotic paradigm became dominant
ces a new perspective in viewing the relationship between religion and the State as follows: p
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1. There is no clear evidence that the Qur'an and al-Sunnah oblige the Islamic community to establish an Islamic
State; 2. Islam is not a political ideology. Islam contains only political principles or ethics; not a political ideology. Islam contains only political principles or ethics; 3. Understanding of Islamic political teachings should be based on a contextual and comprehensive
interpretation according to the demands of the times; 4. Understanding of Islamic political doctrine is relative and therefore opens up opportunities for multiple
interpretations and can change; 5. Islamic political struggle is more directed to build commitment to Islamic values and not to institutions or
organizations that use Islamic symbols.39 6 Arskal Salim, No TitleNegara Dan Syariat Dalam Perspektif Politik Hukum Indonesia, dalam Syariat Islam
Padangan Muslim Liberal, editor. 7 Arskal Salim 38 Arskal Salim. 39 Arskal Salim. Journal homepage: https://bajangjournal.com/index.php/IJSS Journal homepage: https://bajangjournal.com/index.php/IJSS Journal homepage: https://bajangjournal.com/index.php/IJSS Journal homepage: h 2185 International Journal of Social Science (IJSS)
Vol.2 Issue.5 Februari 2023, pp: 2177-2186
ISSN: 2798-3463 (Printed) | 2798-4079 (Online)
DOI: https://doi.org/10.53625/ijss.v2i5.4930 Procontracts in response to this paradigm, for example between NU and Muhammadiyah do not support Islamic
sharia legislation projects in the regions because they prefer national law. This is because society in general realizes
that Islamic law is actually more of an ethical norm that originates from religious teachings. As an ethical norm,
Muslims both individually and collectively can actually carry out all the ethical norms contained in Islamic law without
any interference or coercion from the authorities. 40 Rahman, Fiqh Nusantara dan Sistem Hukum Nasional dan Sistem Hukum Nasional Perspektif Kemaslahatan
Kebangsaan.
41 Rahman. Journal homepage: h On the contrary,
awareness must be awakened that Islamic law cannot simply appear with religious symbols without the emergence of
substance and legal concepts that take into account the principles of national pluralism, culture, customs, and other
religious legal values. This is where the importance of a collection of thoughts and methods of establishing adequate Islamic law in
the Indonesian context. This is because the classical fiqh formulations that have been the reference for Indonesian
Muslims in particular still display old legal statuses that may only be suitable for the area and time of the author, but
as a whole are not necessarily in accordance with the socio-political conditions in Indonesia. That is, before talking
about constitutional opportunities for accommodation of Islamic law in the development of national law, it is first
necessary to formulate the substance and style of Islamic law which will be applied in Indonesia, of course which will
not damage the pillars of nationality.41 4. CONCLUSION Based on the results of the study above, in this paper several core points can be concluded, namely: 1). The
opportunity for Muslims is actually guaranteed by the constitution to formalize Islamic law, but this hope is
increasingly unclear. However, over time, the opportunity to apply the values and norms of Islamic law teachings into
the constitutional system of Indonesian legislation continues to gain a significant portion. The legal systems that color
the national legal system are the western legal system, customary law and the legal system. Islam. Of these three legal
systems, Islamic law has the opportunity to provide input for the formation of national law. On the other hand, even
though Islamic law does not gain legitimacy from the State, the values and norms of Islamic law still exist and are
fully implemented by Indonesian Muslims based on Islamic teachings contained in the Qur'an and the Sunnah of the
Prophet. 2). Challenges faced by Islamic law The toughest challenge at this time does not lie in the constitutional logic
of accommodating Islamic law in national law, but in the dialectics between secular currents of Western thought and
expressions of the diversity of Muslims which sometimes creates phobias. The fact is that law in Indonesia is
dominantly patterned with the legal system inherited from the Netherlands which has long been the mainstream of
legal thought in Indonesia. REFERENCES REFERENCES
[1]
An-Nai‟m, Abdullah Ahmed. Dekonstruksi Syariah Wacana Kebasan Sipil, Hak Asasai
Manusia dan Hubungan Internasional, terj. Yogyakarta: LKiS, 1994. [2]
Arifin, Bustanul. Pelembagaan Hukum Islam Di Indonesia Akar Sejarah, Hambatan dan
Prospeknya. Jakatra: Gema Insani Press, 1996. [3]
Arskal Salim, Azyumardi Azra. No TitleNegara Dan Syariat Dalam Perspektif Politik
Hukum Indonesia, dalam Syariat Islam Padangan Muslim Liberal, editor. Jakarta: Sembrani
Aksara Nusantra, 2003. [1]
An-Nai‟m, Abdullah Ahmed. Dekonstruksi Syariah Wacana Kebasan Sipil, Hak Asasai
Manusia dan Hubungan Internasional, terj. Yogyakarta: LKiS, 1994. p
y
[3]
Arskal Salim, Azyumardi Azra. No TitleNegara Dan Syariat Dalam Perspektif Politik
Hukum Indonesia, dalam Syariat Islam Padangan Muslim Liberal, editor. Jakarta: Sembrani
Aksara Nusantra, 2003. Journal homepage: https://bajangjournal.com/index.php/IJSS 2186 International Journal of Social Science (IJSS)
Vol.2 Issue.5 February 2023, pp: 2177-2186
ISSN: 2798-3463 (Printed) | 2798-4079 (Online) [4]
Djatmika, H. Rachamt. Perkembangan Fiqh di Dunia Islam, dalam Tjun Sudarman, Hukum
Islam Hukum Islam di Indonesia Perkembangan dab Pembentukan. Jakarta: Remaja Rosda
Karya, 1991. [5]
Eklektisisme, dan Hukum Nasional Kompetensi Antara Hukum Islam dan Hukum Umum. A. Qodri Azizy. Yogyakarta: Gama Media, 2002. [6]
Fadhl, Khaled M. Abou El. Melawan Tentara Tuhan Yang Berwewenang dan dan yang
kesewenang-wenangan dalam Wacana Islam. Jakarta: Serambi, 2001. [7]
Ghofur, Abdul. Demokrasi dan Prospek Hukum Islam di Indonesia Studi Atas Pemikiran
Gus Dur. Yogyakarta: Pustaka Pelajar, 2002. [8]
Halim, Abdul. Peradilan Agama dalam Politik Hukum Islam di Indonesia dari Otoriter
Konservatif Menuju Konfigurasi Demokrasi Responsif. Jakarta: Raja Grafindo Persada,
2000. [9]
Lexy, J Moleong. Metodologi Penelitian Kualitatif. Bandung: PT. Remaja R [9]
Lexy, J Moleong. Metodologi Penelitian Kualitatif. Bandung: PT. Remaja Rosdakarya, 2008. [10] Nasir, Muhammad Abdun. Positivisasi Hukum Islam di Indonesia. Mataram: IAIN Mataram
Press, 2004. [10] Nasir, Muhammad Abdun. Positivisasi Hukum Islam di Indonesia. Mataram: IAIN Mataram
Press, 2004. [11] Nurrohman. “Hukum Islam Di Era Demokrasi: Tantangan dan Peluang bagi Formalisasi
Politik Syari‟at Islam di Indonesia.” ADDIN Vol. 9, No (2015): 170. [12] Praja, Juhaya S. Hukum Islam di Indonesia Perkembangan dan Pembentukan. Bandung:
Remaja Rosda Karya, 1991. [13] Rahman, Zaini. Fiqh Nusantara dan Sistem Hukum Nasional dan Sistem Hukum Nasional
Perspektif Kemaslahatan Kebangsaan. Yogyakarta: Pustaka Pelajar, 2016. [14] Rumadi, Marzuki Wahid &. Fiqh Madzhab Negara Kritik Atas Politik Hukum Islam di
Indonesia. Yogyakarta: LKiS, 2001. gy
[15] Zed, Mestika. Metode Penelitian Kepustakaan. Jakarta: Yayasan Obor Indonesia, 2008. Journal homepage: https://bajangjournal.com/index.php/IJSS gy
Mestika. Metode Penelitian Kepustakaan. Jakarta: Yayasan Obor Indonesia, 2008.
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VIRTUAL CONNECTION BETWEEN DIGITAL AND PHYSICAL WORLD BASED ON SIXTH SENSE TECHNOLOGY
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International Journal of Computational Science and Information Technology (IJCSITY) Vol.4,No.1,February 2016 International Journal of Computational Science and Information Technology (IJCSITY) Vol.4,No.1,February 2016 KEYWORDS Six sense technology, Hand gestures, c++, JAVA programing, Digital World, Camera, Smartphone, Projector, Mirror. ABSTRACT The Sixth Sense ‘An Extrasensory Perception’ has turned into the new called ‘The Sixth Sense Technology’
which has emerged in few years. Sixth Sense Technology is a mini-projector coupled with a camera and a
cell phone—which acts as the computer and connected to the Cloud, all the information stored on the web. In this time we have seen lots of technology devices which are mind blowing inventions for human’s .As a
latest technology called six sense technologies. Six sense technology is a kind of device which does not
need to carry any heavy technology device such as camera, watch & computers etc. Here if we have to take
picture of any beautiful things or any things then we do not need a camera. Six sense technology is a new & interesting type of technology, which is easiest using for all type of people. The main factor of six sense devices is the distance, distance between the gesture and camera neither very
large nor very small. This technology is all about interacting to the digital world in most efficient & direct
way .Human has many type of senses to perceive the information around us whenever we come across a
thing, a person or a place. That information helps us make judgment the appropriate action to be taken. But arguably, the most useful information that can help us take the right decision and judgments is not
naturally cognoscible information with the help of our five senses, namely the data, information and
knowledge that mankind has amalgamated but, rather it’s the ‘Sixth Sense Technology.’ This journel
focuses on and makes us aware with the sixth sense technology which provides an integration of the digital
world with the physical world; it helps us understand how the sixth sense device had overpowered the five
natural senses; it also pours light over its various applications, its security related issues and further
implications. VIRTUAL CONNECTION BETWEEN DIGITAL AND
PHYSICAL WORLD BASED ON SIXTH SENSE
TECHNOLOGY Sneha Verma And S.Sugumaran School of Electrical Electronics and Communication, Galgotias University, NCR, U.P 1.INTRODUCTION ‘Sixth Sense’ is a wearable gestural interface that augments the physical world around us with
digital information and lets us use natural hand gestures to interact with that information. Every
one of us is aware of the five basic senses – seeing, feeling, smelling, tasting and hearing. These
senses have evolved through millions of years. When these senses are not able to do any type of
thing then six sense technologies came and this is totally based on the Indian mythology. i.e. it
just depend on our thinking, in this technology the information’s are stored traditionally on a
paper or a digital storage device. Sixth sense technology helps to bridge this gap between tangible
and non-tangible world. Six sense devices contain pocket projector, camera, web enabled smart
phone, and coloured markers which are used to track the hand gestures by the user hands. This
six sense technology can also obey hand gestures in the infamous minority report. This is a magic
each & everywhere we want, it’s a gateway between digital & real world. 1 1 DOI :10.5121/ijcsity.2016.4101 International Journal of Computational Science and Information Technology (IJCSITY) Vol.4,No.1,February 2016 International Journal of Computational Science and Information Technology (IJCSITY) Vol.4,No.1,February 2016 3.HARDWARE COMPONENT OF SIX SENSE DEVICE In six sense technology uses following devices – 1.camera 2.mobile component 3.mirror
component 4.projector 5.colored marker. 2.WHAT IS SIX SENSE? Sixth Sense in scientific or non-scientific terms is defined as Extra Sensory Perception or in short
ESP. It involves the reception of information not gained through any of the five senses. Nor is it
taken from any experiences from the past or known. Sixth Sense aims to more seamlessly
integrate online information and tech into everyday life. By making available information needed
for decision-making beyond what we have access to with our five senses, it effectively gives
users a sixth sense. [2] 3.1.Mobile component: - Consist of a web enabled smart phone which process the data send
by the camera. 3.1.Mobile component: - Consist of a web enabled smart phone which process the data send
by the camera. 3.2.Camera: - It captures the image of the object in view & tracks the users hand gestures. It
sends data to smart phone for further processing. Basically it act as a digital eye which connect to
the world of digital information. 3.3.Mirror component: - The uses of the mirror are significant as the projector, dangles
pointing down wards from the neck. 3.3.Mirror component: - The uses of the mirror are significant as the projector, dangles
pointing down wards from the neck. 3.4.Projector: - The projector is the key output device of the six sense system the projector
visually augments surfaces walls and physical object the user is interacting with by projecting
digital information and graphical user interface the projector itself contains a battery inside with
three hours of battery life. Suggested product of projectors is LED or LASER. 3.4.Projector: - The projector is the key output device of the six sense system the projector
visually augments surfaces walls and physical object the user is interacting with by projecting
digital information and graphical user interface the projector itself contains a battery inside with
three hours of battery life. Suggested product of projectors is LED or LASER. 3.5.Colored marker: - Color marker is used to marking the user’s finger with color red, green,
yellow & blue. It’s placed at the tip of user’s fingers here web cam to recognize the hand
gestures. [3] There is no boundation of taking a color, we can choose any color. 2 2 ional Journal of Computational Science and Information Technology (IJCSITY) Vol.4,No.1,February 2016 International Journal of Computational Science and Information Technology (IJCSITY) Vol.4,No.1,February 2016 Figure no- 2 hardware component
Figure no- 3 original device
Figure no- 4 model of six sense devices Figure no- 2 hardware component Figure no- 2 hardware component Figure no- 3 original device Figure no- 3 original device Figure no- 3 original device Figure no- 4 model of six sense devices Figure no- 4 model of six sense devices 5.SOFTWARE USED Software program processes the video stream data captured by the camera, the Sixth Sense
software will be open source. As far as this seems to be a little set of items, there will not be user
interfaces or much advanced programs for the users. There will be much harder and secured
coding inside the device to make sure the security of the software. It will be interesting to know
the new language for coding for a Sixth Sense device, as for as this seems to be a little set of
items there will not be user interfaces or much advances program for the users. There will be
much harder & secured coding inside the device to make sure the security of the software. The
prototype system runs on windows platform & majority of the code is written in C++ & C#. Uploading newer versions as it is being developed this will also
include a mobile version. [4] 4.WORKING OF SENSE DEVICES 3
The sixth sense device uses different technologies like gesture recognition, image processing, etc. At present the commercial product is not launched but the prototype is prepared. The sixth sense
prototype is made by using very common and easily available equipment’s like pocket projector, 3 International Journal of Computational Science and Information Technology (IJCSITY) Vol.4,No.1,February 2016 a mirror, mobile components, color markers and a camera. The projector projects visual images
on a surface. This surface can be wall, table, book or even your hand. Thus, the entire world is
just like a screen now. When user moves their hands to form different movements with colored
markers on the finger tips, the camera captures these movements. Both the projector and the
camera are connected to the mobile computing device in the user’s pocket. Recognition is made
using computer vision technique. These markers act as visual tracking fiducials. The software
program processes this video stream data and interprets the movements into gestures. The
gestures are different from one another and are assigned some commands. These gestures can act
as input to application which is projected by the projector. The projector is aligned downwards
for compactness; therefore images would be formed at the user’s feet if mirror wasn’t used. The
mirror reflects the image formed by the projector to front. The entire hardware is fabricated in the
form of a pendent and helmet also. The entire product cost around $ 350 and that also because of
projector. It works very similar like a touch screen phone with entire world as the screen. Figure-no 5 working diagram Figure-no 5 working diagram 6.WHY DISCOVER SIX SENSE TECHNOLOGY 4
Basically humans take decisions after acquiring inputs from the senses. But the information is not
sufficient in humans than its effect to the right decisions. But in this time with the help of
Internet, we collect a huge number of information which making a good decision on a few
minutes. This six sense technology provides us with the freedom of interacting with digital world 4 International Journal of Computational Science and Information Technology (IJCSITY) Vol.4,No.1,February 2016 with hand gestures .This technology has a wide application in the field of Artificial Intelligence. This methodology can aid in synthesis of bots that will be able to interact with humans. [5]
Figure no- 6 Internet access huge number of information with hand gestures .This technology has a wide application in the field of Artificial Intelligence. This methodology can aid in synthesis of bots that will be able to interact with humans. [5] Figure no- 6 Internet access huge number of information Figure no- 6 Internet access huge number of information 7.BACKGROUND OF THIS TECHNOLOGY The history behind the six sense technology goes back into 1990s when Steve Mann first attempt
to propose a neck worn projector & a camera combination. The idea of implementing computer
technologies to daily task as six senses was further developed by pranav mistry who also appears
to be an MIT lab Phd student as well as Steve man. Pranav Mistry is an Indian computer scientist. At present, he is the head of Think Tank team Director and researcher of Samsung. He is best
known for his work on six sense technology .[6] The first prototype of the six sense technology
was actually bigger then what it looks like today & it was not working properly to use in daily
life. Pranav Mistry first tried out his idea on a simple computer mouse. First he put two rollers
into one mouse and see if he could obtain data and guide the movements of the mouse. Then two
rollers did not work properly so he decided to use four rollers and see if it could work better four
rollers gave him the idea that he could use the same idea on fingers & that is what the next moved
on to. [7] Figure no-7 mouse Figure no-7 mouse 8.1.Making a call We can use the Sixth Sense technology to project a keypad onto your hand, and then use that
virtual keypad to make a call. Calling a number also will not be a great task with the introduction
of Sixth Sense Technology. No mobile device will be required, just type in the number with your 5 5 International Journal of Computational Science and Information Technology (IJCSITY) Vol.4,No.1,February 2016 palm acting as the virtual keypad. The keys will come up on the fingers. The fingers of the other
hand will then be used to key in the number and call. Figure no- 8 making a call Figure no- 8 making a call 8.4.Create video on the paper The Sixth Sense system also augments physical objects the user is interacting with by projecting
more information about these objects projected on them. For example, a newspaper can show live
video news or dynamic information can be provided on a regular piece of paper. Thus a piece of
paper turns into a video display. Figure no- 11 create multimedia reading Figure no- 11 create multimedia reading 8.2.Map Making The sixth sense also implements map which lets the user display the map on any physical surface
and find his destination and he can use his thumbs and index fingers to navigate the map, for
example, to zoom in and out and do other controls. Figure no- 9 map making Figure no- 9 map making 8.3.Making Virtual Wrist Watch Sixth Sense all we have to do is draw a circle on our wrist with our index finger to get a virtual
Watch that gives us the correct time. The computer tracks the red marker cap or piece of tape,
recognizes the gesture, and instructs the projector to flash the image of a watch onto his wrist. 6 International Journal of Computational Science and Information Technology (IJCSITY) Vol.4,No.1,February 2016 Figure no- 10 checking time in virtual wrist watch
8.4.Create video on the paper Figure no- 10 checking time in virtual wrist watch
8.4.Create video on the paper 8.5.Drawing On Desired Surface The drawing application lets the user draw on any surface by tracking the fingertip
Movements of the user’s index finger. Figure no- 12 drawing Figure no- 12 drawing 7 International Journal of Computational Science and Information Technology (IJCSITY) Vol.4,No.1,February 2016 8.6.Zooming features 8.6.Zooming features
Figure no- 13 Zoom in and Zoom out Figure no- 13 Zoom in and Zoom out Figure no- 13 Zoom in and Zoom out The user can zoom in or zoom out using intuitive hand movements. The user can zoom in or zoom out using intuitive hand movements. 8.7.Get Product Information Maes says Sixth Sense uses image recognition or marker technology to recognize
products you pick up, and then feeds you information on those products. For example, if
you're trying to shop "green" and are looking for paper towels with the least amount of
bleach in them, the system will scan the product you pick up off the shelf and give you
guidance on whether this product is a good choice for you. Figure no- 14 product information Figure no- 14 product information 8.8.Getting book information Maes says Sixth Sense uses image recognition or marker technology to recognize products you
pick up, and then feeds you information on books. The system can project AmazonRatings on
that book, as well as reviews and other relevant information. 8 International Journal of Computational Science and Information Technology (IJCSITY) Vol.4,No.1,February 2016 Figure no- 15 book information Figure no- 15 book information 8.9.Taking a pictures If we fashion our index fingers and thumbs into a square (the typical "framing" gesture), the
system will snap a photo. After taking the desired number of photos, we can project them onto a
surface, and use gestures to sort through the photos, and organize and resize them with the help of
zooming feature. Figure no- 16 taking pictures Figure no- 16 taking pictures 9.1.Mouse less Mouse less is a concept which is developed in order to avoid the usage of mouse in computers. It
helps to provide a familiarity of the physical mouse without actually requiring a physical mouse. Even through the computer hardware technology has been developed to a greater extent, still
there has been no proper substitute for a physical mouse. This new invention aims to remove the
requirement of having a physical mouse altogether but still it provides the feeling of a physical
mouse that users are familiar with. It basically consists of an infrared laser beam and infrared
camera. Both laser and camera are embedded together with computer. The laser beam module is
modified with a line cap and placed such that it creates a plane of IR laser just above the surface
the computer sits on. The user cups their hand, as if a physical mouse was present underneath,
and the laser beam lights up the hand which is in contact with the surface. The IR camera detects
those bright IR blobs using computer vision. The change in the position and arrangements of
these blobs are interpreted as mouse cursor movement and mouse clicks. 9.WAY FORWARD Upcoming projects making use of sixth sense technology. 8.10.Get Flight Updates The system will recognize your boarding pass and let you know whether your flight is on time
and if the gate has changed. Figure no- 17 Flight updates Figure no- 17 Flight updates 9 International Journal of Computational Science and Information Technology (IJCSITY) Vol.4,No.1,February 2016 Getting The Of Information On People
Figure no- 18 Information on people 8.11.Getting The Of Information On People 8.11.Getting The Of Information On People
Figure no- 18 Information on people
Sixth Sense also is capable of "a more controversial use”. When you go out and meet someone,
projecting relevant information such as what they do, where they work, and also m it could
display tags about the person floating on their shirt. It could be handy if it displayed their
Facebook relationship status so that you knew not to waste your time. Figure no- 18 Information on people Sixth Sense also is capable of "a more controversial use”. When you go out and meet someone,
projecting relevant information such as what they do, where they work, and also m it could
display tags about the person floating on their shirt. It could be handy if it displayed their
Facebook relationship status so that you knew not to waste your time. International Journal of Computational Science and Information Technology (IJCSITY) Vol.4,No.1,February 2016 International Journal of Computational Science and Information Technology (IJCSITY) Vol.4,No.1,February 2016 International Journal of Computational Science and Information Technology (IJCSITY) Vol.4,No.1,February 2016 9.4.TaPuma TaPuMa is the short form of tangible public map. It is another attempt to bring the digital world
nearer to the real world by exploring the techniques of six sense technology. Key word based
search mechanism has vastly influenced and modified the ways in which we search information’s. The keyword based search allows the user to filter out the required data from the vast data that are
available on search engines such as yahoo and Google. TaPuMa allows people to use their own
belongings, the objects they usually carry with them to access relevant and just-in-time
information and locations of places from a public map. The project also explores and analyzes the
advantages and challenges of this novel interface for information acquirement. The broad concept
behind the project TaPuMa is objects Amelioration, where the functions of everyday objects can
be expanded by using their affordances or functionalities in a variety of different contexts. 9.3.Inktuitive Even though there has been a tremendous development of computer aided designing tools, still
paper and pencil is the preferred by most of designers, especially in the earlier stages of
designing. The project ‘inktuitive’ in aimed to combine the process of creation that is inherent in
paper and pencil with the power of computing that the digital design tools provide. It also extends
the natural work-practice of using physical paper by giving the pen the ability to control the
design in physical three-dimensional space, freeing it from its tie to the paper. There always
remains an intuition of pen and paper , but the lines that have been drawn are be captured and
translated into shapes in the digital strokes. The platform provides a novel interaction mechanism
for drawing and designing using above the surface pen movements. In other words ‘inkuitive’ can
be considered as an intuitive physical design workspace that aims to bridge the gap and bring
together the conventional design tools such as paper and pencil with the power and convenience
of the digital tool for design. 9.2.Third eye Third eye is new technology that enables multiple viewers to see different things on a same
display screen at the same time. With third eye, we can have a public sign board where a Japanese
tourist sees all the instructions in Japanese and an American in English. We don’t need to have
the split screen in games now. Each player can see his/her personal view of game on the TV. screen. Two people watching TV can watch their favorite channel on a single TV. Screen. A
public display can show secret messages or patterns. In the same movie theater, people can see
different end of suspense movie. 10 10.CONCLUSION The sixth sense technology concept is an effort to connect this data in the digital world in to the
real world. With the help or hand gestures we can draw any picture and zoom in or zoom out of
that picture and another exciting application, when our Five senses not able to do anything then
six sense arises, there are many application of six sense technology. the main thing of this
technology, we can directly connect the digital world without help of any digital device, this
technology makes a bridge between the physical world and digital world. The sixth sense
technology and devices are definitely way forward .sixth sense is a quite efficient way compared
to the text and graphic based user interface. It has the potential to form the transparent user
interface for accessing the information around us. Even through the sixth sense devices are
currently in development stages and have not been used in large now, it’s quite predictable that
this technology have the potential revolutionize the way in which we interacts with the digital
word. And there are another very exciting application of six sense technology to store a data in our
fingertip, it will be possible with the help of six sense technology, memory card , P.D, is not
necessary to store the data, we can easily store in our body and display on the desired surface. 11 11 International Journal of Computational Science and Information Technology (IJCSITY) Vol.4,No.1,February 2016 11.ADVANTAGES 1. Six sense technology is an open source.[7] gy
p
2. six sense technology is a gadget that could be work as jewellery & can do various things
like take pictures, given direction (like GPS) Browse the web etc.[1] 3. Camera can act as a eye for the blind person.[9] 4. Six sense technology is a user friendly device. 5. Six sense technology is so light, easy to use & easy to implement 6. Six sense technology is not heavy to carry around 7. Six sense technology is not that expensive (cost about $350 or 17000 /-) It uses hand gestures to interact with digital in 9. Data access directly from machine in real time y
10. It is gesture-controlled wearable computing device that feeds our relevant information
and turns any surface into an interactive display. 10. It is gesture-controlled wearable computing device that feeds our relevant information
and turns any surface into an interactive display. ACKNOWLEDGEMENT We would like to thank Dr. Sibaram Khara, Dean(Academics), Dr.Dilip Debnath, Dean,
SEECE, Galgotias University, NCR, UP for their kind guidance and support in compilation of
the paper. 12.LIMITATION 1. There is no easy way to fix if six sense device is break. 2. Six sense device cannot be used for long period of time 3. Privacy is a major issue; if we open up the gadget on a busy street then everyone would
be able to see what we are doing 4. This device is not projecting a hard surface. 5. This technology is not attach to all the time with human body parts, because high waves
radiation emitted may cause some disease 6. Projection is better in the night time & dark areas rather than mornings & bright areas. 7. Software does support the ability to use real time video streams in order to produce
augmented reality 8. Hardware limitation of the devices that we currently carry around with us for example
many phones will not allow the external camera feed to be manipulated in real time 9. Post processing can occur however [8] REFERENCES [1]
Suryavanshi et al., International Journal of Advanced Research in Computer Science and Software
Engg. 3(11), November - 2013, pp. 744-748
[2]
www.blendernation.com/sixth-sense-technology/
[3]
http:// dspace.cusat.ac.in
[4]
http:// code.google.com/ six sense/ wiki/ hardware/software
[5]
Www. Engineersgarage.com/articles/ six sense technology [6] en.wikipedia.org/ wiki/ pranav_mistry
[7]
Misy 3310dogukan.weebly.com/ six sense history
[8]
http:// six sense technology. hit103.blogspot.in
[9]
www.scribd.com/ doc/ 29354104 / six sense technology. [10] http://gizmodo.com/5167790/sixth-sense-technology-may-change-how-we-lookat-the-world-forever
[11] http://theviewspaper.net/sixth-sense-technology-will-revolutionize-the-world/ [1]
Suryavanshi et al., International Journal of Advanced Research in Computer Science and Software
Engg. 3(11), November - 2013, pp. 744-748 [2]
www.blendernation.com/sixth-sense-technology/ [2]
www.blendernation.com/sixth-sense-technolog [3]
http:// dspace.cusat.ac.in [4]
http:// code.google.com/ six sense/ wiki/ hardware/software p
g
g
[5]
Www. Engineersgarage.com/articles/ six sense technology [6] en.wikipedia.org/ wiki/ pranav_mis [7]
Misy 3310dogukan.weebly.com/ six sense history [8]
http:// six sense technology. hit103.blogspot.in 0] http://gizmodo.com/5167790/sixth-sense-technology-may-change-how-we-lookat-the-world-forever
1] h
// h
i
/ i h
h
l
ill
l i
i
h
ld/ [10] http://gizmodo.com/5167790/sixth-sense-technology-may-change-how-we-look [10] http://gizmodo.com/5167790/sixth-sense-technology-may-change-how-we-lookat-the-world-forever
[11] http://theviewspaper.net/sixth-sense-technology-will-revolutionize-the-world/ 0] http://gizmodo.com/5167790/sixth-sense-technology-may-change-how-we-lookat-the-world-forever
1] http://theviewspaper.net/sixth-sense-technology-will-revolutionize-the-world/ [11] http://theviewspaper.net/sixth-sense-technology-will-revolutionize-the-world/ 12 Authors
SNEHA VERMA is a student of GALGOTIAS UNIVERSITY, Greater Noida. She is
currently pursuing M.Tech degree in Communication engineering. Her main areas of
interest are optical Communication,computer networks and optical networks. SUGUMARAN.S was born in Pallikonda,Vellore District, Tamil Nadu, India in 1976. He received his Bachelors of Engineering Degree from the Department of Electronics
and Communication Engineering from Bharathiyar University, Coimbatore, India and
Master’s Degree in Optical Communication from CEG, Anna University, Chennai in
1998 and 2004 respectively. After working as Faculty in different designations in VIT
University, Vellore, he is currently working as Assistant Professor Gr.-III in School of
Electrical,Electronics and Communication Engineering, Galgotias University, Greater Noida,NCR. He is
currently pursuing the PhD degree in Optical communication and networks. His research include nonlinear
effects (FWM, CPM) on WDM networks and FTTH application . Authors SNEHA VERMA is a student of GALGOTIAS UNIVERSITY, Greater Noida. She is
currently pursuing M.Tech degree in Communication engineering. Her main areas of
interest are optical Communication,computer networks and optical networks. SUGUMARAN.S was born in Pallikonda,Vellore District, Tamil Nadu, India in 1976. He received his Bachelors of Engineering Degree from the Department of Electronics
and Communication Engineering from Bharathiyar University, Coimbatore, India and
Master’s Degree in Optical Communication from CEG, Anna University, Chennai in
1998 and 2004 respectively. After working as Faculty in different designations in VIT
University, Vellore, he is currently working as Assistant Professor Gr.-III in School of
Electrical,Electronics and Communication Engineering, Galgotias University, Greater Noida,NCR. He is
currently pursuing the PhD degree in Optical communication and networks. His research include nonlinear
effects (FWM, CPM) on WDM networks and FTTH application . SUGUMARAN.S was born in Pallikonda,Vellore District, Tamil Nadu, India in 1976. He received his Bachelors of Engineering Degree from the Department of Electronics
and Communication Engineering from Bharathiyar University, Coimbatore, India and
Master’s Degree in Optical Communication from CEG, Anna University, Chennai in
1998 and 2004 respectively. After working as Faculty in different designations in VIT
University, Vellore, he is currently working as Assistant Professor Gr.-III in School of
Electrical,Electronics and Communication Engineering, Galgotias University, Greater Noida,NCR. He is
currently pursuing the PhD degree in Optical communication and networks. His research include nonlinear
effects (FWM, CPM) on WDM networks and FTTH application . 13 13
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The role of thought-content and mood in the preparative benefits of upward counterfactual thinking
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Motivation and emotion
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1 1 COUNTERFACTUAL THINKING 1 Preprint, in press, Motivation and Emotion The Role of Thought-Content and Mood in the Preparative Benefits of
Upward Counterfactual Thinking
Andrea L. Myers
University of Konstanz
Sean M. McCrea
Maurissa P. Tyser
University of Wyoming
Correspondence:
Sean M. McCrea
Department of Psychology
University of Wyoming
Dept. 3415
1000 E. University Ave.
Laramie, WY, 82071, USA
Telephone: +1 307 766-6149
FAX: +1 307 766-2926
smccrea@uwyo.edu Correspondence:
Sean M. McCrea
Department of Psychology
University of Wyoming
Dept. 3415
1000 E. University Ave. Laramie, WY, 82071, USA
Telephone: +1 307 766-6149
FAX: +1 307 766-2926
smccrea@uwyo.edu COUNTERFACTUAL THINKING 2 2 Abstract Abstract Upward counterfactual thoughts identify how a prior outcome could have been better and have
been shown to improve subsequent performance. Both the identification of corrective actions
(content-specific effects) and the more general mobilization of effort as a result of negative affect
(content-neutral effects) have been suggested to underlie performance benefits. The results of
three experiments presented here indicate that counterfactual thoughts have broad benefits for
performance, independent of their content and beyond the effects of planning. These benefits
were consistently dependent upon the experience of negative affect, but were eliminated when
negative affect could be (mis)attributed to an intervening task. This misattribution effect is
consistent with the operation of a mood-as-input process in which affect informs judgments of
goal progress. Overall, the findings suggest that the benefits of upward counterfactual thinking
reflect more broad attempts to improve following a subjectively unsatisfactory performance. KEYWORDS: Counterfactual thinking, motivation, planning, self-regulation, implementation
intentions COUNTERFACTUAL THINKING 4 of which we are aware have compared the consequences of counterfactual thinking to those of
forming such a plan. Third, the notion that negative affect resulting from counterfactual thinking
influences judgments of goal progress has not been directly examined. The present research
seeks to address these important questions. of which we are aware have compared the consequences of counterfactual thinking to those of
forming such a plan. Third, the notion that negative affect resulting from counterfactual thinking
influences judgments of goal progress has not been directly examined. The present research
seeks to address these important questions. COUNTERFACTUAL THINKING 3 3 The Role of Thought-Content and Mood in the Preparative Benefits of
Upward Counterfactual Thinking The Role of Thought-Content and Mood in the Preparative Benefits of Upward Counterfactual Thinking Upward counterfactual thoughts identify how a prior outcome could have been better (e.g., “I
could have done better if I had studied more”, Kahneman & Miller, 1986; Markman, Gavanski,
Sherman, & McMullen, 1993; Roese, 1994). These thoughts have been shown in both laboratory
and field settings to play an important role in self-regulation following failure, increasing task
preparation, persistence, and performance (Markman et al., 1993; Markman, McMullen, &
Elizaga, 2008; Nasco & Marsh, 1999; Roese, 1994). In their Functional Theory of Counterfactual Thinking, Epstude and Roese (2008) argue that
there are two pathways by which counterfactuals influence task performance: a content-specific
pathway, and a content-neutral pathway. In the content-specific pathway, the corrective action
suggested by the counterfactual is translated into a behavioral intention or plan (see Figure 1, top
panel). In the content-neutral pathway, any benefits to performance are independent of the
behaviors mentioned in the counterfactual thought. For example, upward counterfactuals tend to
increase negative affect (Markman et al., 1993; Roese, 1994), which in turn can be interpreted by
the individual as indicating that his or her performance was inadequate (Gendolla & Brinkman,
2005; Martin, Ward, Achee, & Wyer, 1993). Dissatisfaction with performance will then lead to
greater task persistence and performance (Markman et al., 2008). Despite the important
distinction between content-specific and content-neutral pathways made by Epstude and Roese
(2008), researchers have only rarely controlled for the strategy specified in the counterfactual
thought (see Roese, 1994, for a notable exception). Thus, it is difficult to know how specific the
benefits of these thoughts actually tend to be. Second, though the content-specific pathway
suggests that counterfactuals are translated into corresponding behavioral intentions, no studies Content-Specific Pathway To the extent that a counterfactual thought identifies a controllable cause for a poor
performance, individuals can form an intention to change their behavior (Epstude & Roese,
2008). The thought “If I had done x, I would have done better” can easily be reformulated into
the intention “I will do x!” (Johnson & Sherman, 1990; Roese, 1994). In other words,
counterfactual thinking should encourage planning of subsequent behavior. Indeed, recent
studies by Smallman and colleagues (Smallman & McCulloch, 2012; Smallman & Roese, 2009)
indicate that considering a counterfactual possibility facilitates the formation of a corresponding
intention to engage in this behavior in the future. For example, the counterfactual thought “could
have worn sunscreen” made after experiencing a bad sunburn facilitates the formation of the
behavioral intention “now I will wear sunscreen” (Smallman & McCulloch, 2012). Once a behavioral intention is formed, one should be more likely to carry out the specified
behavior. Epstude and Roese (2008) propose that the intentions resulting from counterfactual
thinking have effects similar to those of implementation intentions. Implementation intentions
are simple plans that specify a critical situation or cue and connect it to a specific goal-directed
response, taking the form “When x occurs, then I will perform behavior y” (Gollwitzer, 1999). These plans can be differentiated from goal intentions (e.g, “I intend to reach x”) that specify a
certain goal or outcome to be achieved. Implementation intentions form an automatic association
between the critical situation and the specific response (Gollwitzer, 1999; Webb & Sheeran, COUNTERFACTUAL THINKING 5 5 2007), thereby promoting goal attainment. Numerous studies have documented the benefits of
forming these intentions in a variety of domains, including healthy diet (Achtziger, Gollwitzer, &
Sheeran, 2008), exercise (de Vet, Oenema, & Brug, 2011), academic and athletic performance
(Achtziger et al., 2008; Bayer & Gollwitzer, 2007), and prospective memory (Craig McFarland
& Glisky, 2012). Direct evidence that counterfactual thoughts lead to specific corrective actions is sparse. Roese (1994, Study 3) observed that individuals who generated counterfactuals concerning a
particular behavior (i.e., the use of clues on an anagram task) were more likely to engage in that
behavior in a subsequent performance. However, the specific behavior mentioned in these
thoughts was not experimentally manipulated. As a result, it is not clear whether thought content
was causally related to subsequent behavior. Likewise, a study by Markman et al. (2008) found
that the usefulness of the strategy contained in the counterfactual (as rated by judges) predicted
improved performance. A stronger test of the content-specific hypothesis would involve
experimentally manipulating the content of the counterfactual or comparing the effects of
counterfactual thinking to those of forming a plan. Content-Neutral Pathway and the Role of Affect One manner in which upward counterfactual thoughts could affect performance in a content-
neutral manner is by increasing effort mobilization. This proposition is most notably espoused by
Markman and McMullen‟s (2003; Markman et al., 2008) Reflection and Evaluation Model
(REM). According to the REM, the affective and motivational consequences of counterfactual
thinking depend on whether individuals reflect upon the counterfactual alternative in isolation
(i.e., vividly imagining the alternative outcome), or evaluate the actual outcome relative to the
standard of the counterfactual alternative. When individuals evaluate an upward counterfactual, COUNTERFACTUAL THINKING 6 the comparison of the outcome to a better alternative increases negative affect, whereas
reflecting upon a better alternative outcome increases positive affect. Affect is then presumed to
serve as input (Cervone, Kopp, Schaumann, & Scott, 1994; Gendolla & Brinkman, 2005; Martin
et al., 1993) into decisions concerning whether the goal has been completed and whether
additional action is necessary. When holding a “do your best” goal (i.e., a task of unfixed
difficulty), experiencing positive affect should lead to the conclusion that one is satisfied with
the performance and can cease goal pursuit, whereas experiencing negative affect should lead to
the conclusion that one is dissatisfied with the performance and goal pursuit is worth continuing
(Cervone et al., 1994; Gendolla & Brinkman, 2005; Markman & McMullen, 2003). Thus,
upward counterfactual thoughts increase task effort only when the individual experiences
negative affect due to evaluative processing. In other words, resultant affect should moderate the
consequences of upward counterfactual thinking. Several studies have found support for this perspective. Markman et al. (2008) asked
participants to generate their own upward counterfactuals after an anagram task. They found that
improved persistence and performance increased for individuals who engaged in evaluative (but
not reflective) processing of upward counterfactuals. Furthermore, these effects were explained
by increased negative affect. Conversely, upward counterfactual thoughts that serve to excuse
performance by identifying a self-handicap (and thus decrease negative state self-esteem)
undermine subsequent preparative effort and performance (McCrea, 2008; Tyser, McCrea, &
Knüpfer, 2012). However, there is only indirect evidence for the mood-as-input explanation of
these findings. A stronger test would involve manipulating the ability of affect to serve as a cue
to judgments of goal progress. COUNTERFACTUAL THINKING 7 Although the content-specific and content-neutral pathways are conceptually distinct, the
question arises whether they are completely independent of each other. Epstude and Roese
(2008) suggested that the pathways may at times interact. For example, a counterfactual mindset
could increase the strength of intentions formed as a result of subsequent counterfactual thinking. We sought to pose a more basic question, namely, whether these two pathways represent two
phases of goal pursuit rather than truly independent processes. Simply put, our view is that
content-specific planning is likely to be dependent upon underlying “content-neutral” Although the content-specific and content-neutral pathways are conceptually distinct, the
question arises whether they are completely independent of each other. Epstude and Roese
(2008) suggested that the pathways may at times interact. For example, a counterfactual mindset
could increase the strength of intentions formed as a result of subsequent counterfactual thinking. We sought to pose a more basic question, namely, whether these two pathways represent two
phases of goal pursuit rather than truly independent processes. Simply put, our view is that
content-specific planning is likely to be dependent upon underlying “content-neutral”
motivational considerations. For example, would individuals form intentions to change their
behavior as a result of counterfactual thinking when they are lacking the motivation to continue
goal pursuit? Conversely, if highly motivated to improve on a task as a result of counterfactual
thinking, would individuals limit themselves to the corrective actions suggested by the thought? The Action-Phase Theory of goal-pursuit (Heckhausen, 2003; Heckhausen & Gollwitzer,
1987) is instructive in answering these questions. The Action-Phase model holds that individuals
first decide upon a goal, then make plans for how they will pursue the goal, strive towards the
goal, and finally evaluate their progress toward the goal. Counterfactual thinking is most likely to
take place in this final, post-actional phase of goal pursuit (see also Epstude & Roese, 2008). In
this phase, the individual must assess whether the goal was reached, determine whether The Action-Phase Theory of goal-pursuit (Heckhausen, 2003; Heckhausen & Gollwitzer,
1987) is instructive in answering these questions. The Action-Phase model holds that individuals
first decide upon a goal, then make plans for how they will pursue the goal, strive towards the
goal, and finally evaluate their progress toward the goal. Counterfactual thinking is most likely to
take place in this final, post-actional phase of goal pursuit (see also Epstude & Roese, 2008). COUNTERFACTUAL THINKING In
this phase, the individual must assess whether the goal was reached, determine whether
continued goal pursuit is worthwhile, and identify what, if any, changes in behavior should be
made. Importantly, the Action-Phase model suggests that changes will be made only when the
individual has determined that continued striving toward the goal is necessary and worthwhile. Similarly, many models of motivation (e.g., Bandura, 1991; Vroom, 1964) suggest action is
dependent on the expected value of pursuing the goal. Thus, specific behavioral intentions will
only be formed and influence behavior when the individual is motivated to improve on past continued goal pursuit is worthwhile, and identify what, if any, changes in behavior should be
made. Importantly, the Action-Phase model suggests that changes will be made only when the
individual has determined that continued striving toward the goal is necessary and worthwhile. Similarly, many models of motivation (e.g., Bandura, 1991; Vroom, 1964) suggest action is
dependent on the expected value of pursuing the goal. Thus, specific behavioral intentions will
only be formed and influence behavior when the individual is motivated to improve on past COUNTERFACTUAL THINKING 9 they may identify and pursue additional means of achieving the goal. This proposed content-
neutral pathway to performance improvement is presented in Figure 1 (bottom panel). they may identify and pursue additional means of achieving the goal. This proposed content-
neutral pathway to performance improvement is presented in Figure 1 (bottom panel). COUNTERFACTUAL THINKING 8 8 performance. In studies demonstrating that counterfactuals facilitate the formation of behavioral
intentions (Smallman & McCulloch, 2012; Smallman & Roese, 2009), individuals were first
provided with an objectively negative event (e.g., bad sunburn). What if no objective information
concerning success or failure is available? In this case, according to the REM (Markman &
McMullen, 2003), affect will be used as input into judgments of goal progress. In either case, the
corrective actions mentioned in the counterfactual are likely to influence subsequent behavior
only when individuals judge their performance as inadequate (Carver & Scheier, 1990). Having decided to continue pursuing the goal, individuals in the post-actional phase will turn
to plan for the future (Heckhausen, 2003). Such plans are likely to be influenced immediately by
prior thoughts concerning “what went wrong” (Smallman & McCulloch, 2012; Smallman &
Roese, 2009). However, with ample time and opportunity, individuals highly motivated to
improve might consider alternative means. Evidence for this proposition comes from recent
studies by Myers (2009), which found no effect of the opportunity to consider counterfactual
thoughts on the subsequent formation of plans. Given the time to reflect, participants generated
plans suggested by the counterfactual and plans not suggested by the counterfactual in equal
measure. To summarize, we view content-neutral and content-specific processes as consecutive steps
in the post-actional phase of goal-pursuit rather than as two separate pathways (see also
Heckhausen, 2003; Markman & McMullen, 2003). Particularly when goal progress is more
subjectively defined (i.e., “do your best”), counterfactual thinking will influence task effort via a
mood-as-input process (Markman & McMullen, 2003). Next, counterfactual thinking can
suggest corrective actions to be taken. However, it may be difficult to detect content-specific
effects on behavior if individuals are highly motivated to improve. Provided the opportunity, Goals of the Present Work We have discussed two pathways by which counterfactual thoughts might improve
subsequent performance. The present studies were designed to systematically examine which of
these views better accounts for the consequences of counterfactual thinking. Crucial points in
addressing this question are 1) whether the benefits of counterfactual thinking are dependent
upon perceiving past performance as insufficient, 2) whether mood serves as input into these
judgments, and 3) whether counterfactual benefits for subsequent performance reflect the
application of a specified strategy or a broader effort to improve. According to a strict content-specific view (Figure 1, top panel), any benefits of
counterfactual thinking should be a) due to changes in the behavior specified by the thought, and
b) similar to the benefits of forming an implementation intention. In contrast, according to a
strict content-neutral view (Figure 1, bottom panel), any benefits of counterfactual thinking
should be a) dependent on negative affect serving as a valid cue to goal progress, and b) largely
independent of strategic content of the thought, with quite different consequences for behavior
relative to an implementation intention. Based on the theorizing discussed above, we expected
counterfactual thoughts to influence performance in a more content-neutral manner, as follows. Based on the REM (Markman & McMullen, 2003) and the mood-as-input perspective
(Gendolla & Brinkman, 2005; Martin et al., 1993), we expected that counterfactual effects would
be limited to those experiencing relatively more negative affect following counterfactual
thinking. Study 3 directly tested the mood-as-input hypothesis by manipulating whether affect
could serve as a valid cue to evaluations of goal progress (see also Gendolla & Krüsken, 2002; COUNTERFACTUAL THINKING 10 Schwarz & Clore, 1983). When negative affect could be (mis)attributed to an intervening task,
we expected that counterfactual benefits for the focal task would be eliminated. Schwarz & Clore, 1983). When negative affect could be (mis)attributed to an intervening task,
we expected that counterfactual benefits for the focal task would be eliminated. We addressed the question of the relative breadth of counterfactual benefits for performance
in two ways. First, by controlling the content of the counterfactual thought, the specificity of
consequences for performance could be directly observed. Second, we assessed whether the
consequences of considering a counterfactual were the same as those of forming an
implementation intention. In Studies 1 and 2, we fully crossed the manipulation of upward
counterfactual thoughts and implementation intentions in a 2 x 2 design and examined strategy-
specific and strategy-unrelated behaviors. In summary, the present work represents an advance over past studies in that we
systematically controlled for the strategic content of the counterfactual thought, compared the
effects of counterfactual thinking to that of forming a plan, and manipulated the informational
value of negative mood. Study 1: Improving Performance on an Anagram Task We conducted an initial study to test these predictions, adapting the anagram paradigm
developed by Roese (1994, Study 3). Participants attempted to solve anagrams under time
pressure. They could try to solve an item, skip over the item (minimizing the loss of points) or
“buy” a clue (making it easier to solve the item correctly, but with a penalty). Participants
received feedback and were then given a chance to complete a second set of items. The
counterfactual and implementation intention provided to participants both mentioned the strategy
of using a clue after 60 seconds. Performance on a second set of anagrams was then measured. We expected that implementation intentions would result in the application of the specified
strategy, whereas counterfactual thoughts would result in more broad performance improvement. Participants and Design Participants were 95 (39 male, 56 female) students at a German university.1 Two male
participants mentioned either implementation intentions or “if-then intentions” during debriefing
and were therefore excluded from the analyses. Participants were either paid 4€ or were given
research participation credit upon their completion of the study. They were randomly assigned to
conditions in a 2 (counterfactual vs. no counterfactual) x 2 (implementation intention vs. no
implementation intention) between-subjects design. COUNTERFACTUAL THINKING 11 However, consistent with past work (Markman et al., 2008; McCrea, 2008), we predicted that
counterfactual thoughts would only benefit performance when individuals experienced more
negative affect. Specifically: H1. The counterfactual thought to use more clues will increase performance in set 2,
compared to the control condition, but only among individuals reporting more negative affect. Moreover, this effect should hold even when controlling for clue use. H2. The implementation intention to purchase a clue after 60 seconds will increase the use of
this strategy in set 2, relative to no implementation intention. Moreover, any benefits of
implementation intentions for performance will be explained by changes in clue use. Materials The anagram task was adapted from Roese (1994, Study 3). In that study, participants could
purchase a clue which provided the middle letter of the anagram in the correct position at a cost
of 30 points (i.e., equivalent to 30 seconds of time). Because clue use was actually found to be
harmful to performance, we conducted a pretest to determine whether providing the initial letter
of the anagram would improve performance. Twenty-one participants were assigned to complete COUNTERFACTUAL THINKING 12 20 eight-letter anagram items. Anagrams were created by listing the letters alphabetically. For
half of the items, the first letter was provided. Inclusion of this clue was counterbalanced and the
order of presentation randomized. Participants solved more items correctly when provided with
the clue (M = 7.76, SD = 1.09) than when not provided with the clue (M = 2.71, SD = 1.76), t(20)
= 10.40, p <.001, and did so faster, Mdifference = -44.34 sec, t(20) = 9.19, p < .001. Thus, the clues
were objectively helpful in solving the items. In the main experiment, participants were allotted two minutes to provide a solution to an
item before the computer automatically advanced to the next anagram. The order of the
anagrams was randomized within each ten-item set, and the sets counterbalanced. Scoring was as
follows. Participants gained 120 points for a correct answer, with a point deducted for each
second spent on the item. They lost a point for each second spent on an incorrect or unsolved
item. It was also possible to skip an item, although participants were unable to return to it later. Thus, participants were advised to skip over anagrams that were too difficult to minimize the loss
of points. Participants also had the option to buy a clue (in which the first letter of the correct
solution was presented) at the cost of 30 points. Participants were advised that it would behoove
them to buy clues on anagrams they were close to solving. Based on the pretest, the cost of the
clue (equivalent to 30 seconds of time) did not outweigh the time-savings provided by the clue. COUNTERFACTUAL THINKING 13 Next, an example item and scoring procedure for the anagram task was provided. To ensure
that participants understood the task and scoring, participants were required to answer a few
comprehension questions (e.g., “A person tries to solve an anagram for 110 seconds and then
decides to skip over this anagram. How many points is this person awarded?”) and were
provided the correct answer. To ensure that all participants held the same goal intention, they
were asked to write the goal “I want to achieve as many points as possible” three times. Next, an example item and scoring procedure for the anagram task was provided. To ensure
that participants understood the task and scoring, participants were required to answer a few
comprehension questions (e.g., “A person tries to solve an anagram for 110 seconds and then
decides to skip over this anagram. How many points is this person awarded?”) and were
provided the correct answer. To ensure that all participants held the same goal intention, they
were asked to write the goal “I want to achieve as many points as possible” three times. Participants then attempted the first set of anagrams. At the conclusion of the first set, they were
provided feedback including total score, number of items correctly solved, number of items
skipped, number of clues used, and number of items in which the time limit was exceeded. Participants then attempted the first set of anagrams. At the conclusion of the first set, they were
provided feedback including total score, number of items correctly solved, number of items
skipped, number of clues used, and number of items in which the time limit was exceeded. At this point, participants were randomly assigned to one of two counterfactual conditions. Those in the counterfactual condition read a statement supposedly made by a past participant,
indicating that his or her performance would have been improved by purchasing a clue for the
difficult items (“If I had bought a clue after 60 seconds for anagrams that were difficult to solve,
then my total score on this test would have been better.”). Participants were asked to write the
thought three times and relate this thought to their own past performance. Those in the no
counterfactual condition proceeded to the next part of the procedure. As before, participants in all conditions were asked to write the goal statement three times. Procedure Participants were told that the experiment dealt with problem solving under time pressure
and to what extent experience impacts performance. The task was described as a “word
discovery” task: The study would involve completing two versions of the same task, and
participants would receive feedback after each. Participants then completed demographic items. COUNTERFACTUAL THINKING Because participants had already completed a similar set of items, received feedback, and formed
a “do your best” goal, the second block of items represented a task of unfixed difficulty
(Gendolla & Brinkman, 2005). Participants were then randomly assigned to one of two implementation intention conditions. Those in the implementation intention condition read a statement supposedly made by a past
participant concerning his or her future performance, and indicating a plan to purchase a clue for Results Results COUNTERFACTUAL THINKING 14 difficult items (“Whenever I cannot solve an anagram within 60 seconds, then I will buy a
clue.”). Participants were then asked to write this thought three times and relate it to their
upcoming performance. Those in the no implementation intention condition proceeded to the
next part of the procedure. difficult items (“Whenever I cannot solve an anagram within 60 seconds, then I will buy a
clue.”). Participants were then asked to write this thought three times and relate it to their
upcoming performance. Those in the no implementation intention condition proceeded to the
next part of the procedure. They were then informed that the researchers were interested in the role of mood in
performance. Participants completed a 32-item mood measure which included nine negative
mood adjectives (worried, upset, guilty, scared, hostile, jittery, ashamed, anxious, inadequate) as
well as positive mood and self-esteem related items (see also Cathy McFarland & Ross, 1982;
Watson, Clark, & Tellegen, 1988). Participants were asked how strongly they felt each item at
the present time, using a five-point scale (1 = not at all to 5 = very strong). Participants
completed the second anagram set and received feedback. They were then fully debriefed and
received compensation for their participation. Overview We examined effects of negative mood and condition on set 2 scores, controlling for set 1
scores.2, 3 Analyses were conducted on specified strategy use (clues used after approximately 60
seconds) and accuracy (number of anagrams solved). Accuracy and points scored were largely
redundant (rs > .83) and so we focus on the former in our presentation of the results. Regression
analyses were conducted due to the inclusion of continuous negative mood scores. Negative
mood scores (M = 14.16, SD = 4.96, Cronbach‟s α = .84) were positively skewed, and therefore
log transformed (M = 2.60, SD = 0.31). A 2 (counterfactual vs. no counterfactual) x 2
(implementation intention vs. no implementation intention) ANOVA failed to reveal any
significant differences in negative mood by condition, Fs < 1.30, ps > .25. Following the COUNTERFACTUAL THINKING 16 than in the no counterfactual control condition, simple-slope = 0.92, t(88) = 2.49, p < .02, 95%
CI: 0.18 to 1.65. The counterfactual effect was not significant among those low in negative
mood, simple-slope = -0.15, t(88) = 0.42, p > .67, 95% CI: -0.85 to 0.56. There were no
significant effects of implementation intentions. Test of content-specificity. There were no significant effects of counterfactual thought on
clue use. We nonetheless conducted an additional analysis to examine whether the clue use. We nonetheless conducted an additional analysis to examine whether the
Counterfactual x Negative mood interaction on accuracy was even partly due to the specified
strategy of using clues. We therefore specified a bootstrap model (Hayes, 2012) predicting set 2
accuracy with counterfactual thought condition as the predictor, negative mood as the moderator,
set 2 clue use in the 50-70 sec. time period as the mediator, and set 1 accuracy as a covariate. We
observed that the indirect effect through clue use was not significant, B = 0.07, 95% CI = -0.19
to 0.42. Set 2 clue use was not predictive of accuracy in set 2, β = .076, t(87) =0.71, p = .48, f2 <
.01, and the Counterfactual x Negative mood interaction was largely unchanged, β = .212, t(87)
= 1.91, p = .06, f2 = .04. Moreover, the effect of counterfactual thought remained significant
among those reporting high negative mood when controlling for clue use, simple-slope = 0.90,
t(87) = 2.43, p < .02, 95% CI: 0.16 to 1.64. COUNTERFACTUAL THINKING 15 recommendations of Aiken and West (1991), mood score was mean-centered, and effects coding
was utilized for the counterfactual (no counterfactual = -1; counterfactual = 1) and
implementation intention (no implementation intention = -1; implementation intention = 1)
conditions. Set 1 scores, negative mood, implementation intention condition, and counterfactual
thought condition were entered into the model in an initial step, and all two-way interactions of
negative mood and the condition variables were entered into the model in a second step. The
three-way interaction of negative mood and the condition variables did not approach significance
in any analysis, and so this term was trimmed from the model to increase power (Judd & Kenny,
1981). Significant interactions were plotted and probed using simple-slope tests at +/-1 SD from
the mean (Aiken & West, 1991). Results of the regression analyses are presented in Table 1. recommendations of Aiken and West (1991), mood score was mean-centered, and effects coding
was utilized for the counterfactual (no counterfactual = -1; counterfactual = 1) and Clue use. Given that the strategy mentioned in the implementation intention and
counterfactual thought involved using a clue only after 60 seconds had elapsed, we examined the
effects of negative mood and condition on the number of clues that were used between 50 and 70
seconds in set 2 (M = 0.91, SD = 1.88), controlling for the number of clues that were used
between 50 and 70 seconds in set 1 (M = 0.45, SD = 0.83). Only the effect of implementation
intention condition was significant. Individuals in the implementation intention condition (M =
1.47, SD = 2.31) used more clues in the 50-70 sec. time period than did those in the no
implementation intention condition (M = 0.40, SD = 1.16).4 Accuracy. We next examined the effects of negative mood and condition on the number of
anagrams solved in set 2 (M = 3.78, SD = 2.40), controlling for the number solved in set 1 (M =
3.10, SD = 2.13). Those reporting more negative mood performed better, and this effect was
qualified by a Counterfactual x Negative mood interaction, see Figure 2. Simple-slope tests
revealed that individuals high in negative mood performed better in the counterfactual condition COUNTERFACTUAL THINKING Discussion Counterfactual thoughts did not significantly affect the specified strategy of using clues after
60 seconds. Nonetheless, counterfactual thought led to greater improvement in performance
among participants high in negative mood. These individuals improved to a greater extent
compared to those in the control condition or who reported less negative mood. In summary, the
results were consistent with the hypothesis that counterfactuals would more broadly influence
performance, independent of specified strategy, and only when accompanied by negative affect. COUNTERFACTUAL THINKING 17 Although it is possible that the lack of a counterfactual effect on clue use reflects a Type II error,
the benefits of counterfactual thinking among those high in negative mood held even when
controlling for this behavior. Thus, the results are inconsistent with a content-specific pathway to
performance. The effects of counterfactual thinking were in sharp contrast to the effects of forming an
implementation intention. Forming an if-then plan led to increased use of the specified strategy,
but did not significantly affect performance. These findings are consistent with past work on
implementation intentions showing benefits to specified behavior, but not other aspects of
performance (Parks-Stamm, Gollwitzer, & Oettingen, 2007). It is also important to note that
implementation intentions had benefits over and above the effects of counterfactual thoughts. That is, there were additional benefits to forming a behavioral intention even when participants
had already considered a related counterfactual thought. Taken as a whole, the results of Study 1 are consistent with the notion that counterfactuals
lead to more broad, content-neutral, performance benefits (Markman & McMullen, 2003;
McCrea, 2008; McMullen & Markman, 2000). Specifically, negative affect is a signal that goal
progress has been insufficient. Corrective actions are more likely to be taken, potentially
including an increase in effortful persistence. Although counterfactuals may suggest specific
corrective actions that should be taken, they may also motivate the individual to consider
additional means to the goal not explicitly mentioned. Study 2 – Persistence on a Word-Completion Task In Study 1, we observed that counterfactuals benefited performance even when tightly
controlling the strategic content of the thought and the specified behavior. These findings are
consistent with the notion that dissatisfaction with goal progress leads to increased effort COUNTERFACTUAL THINKING 18 mobilization (Martin et al., 1993). However, we focused exclusively on performance, and not
directly on effort put forth on the task. Study 2 was therefore designed to assess persistence of
effort (Bandura, 1977), measuring the amount of time participants invested in an insolvable task
(see also Markman et al., 2008; Vohs & Heatherton, 2000). A second issue concerns the nature of our manipulations. Because we wished to tightly
control the content of the statements individuals considered, we did not allow individuals to
freely generate these thoughts. Rather, the strategies mentioned in these statements were
provided. One potential drawback of this method is that there may have been more experimental
demand for individuals to pursue the implementation intention, particularly due to the “whenever
x happens, I will do y” phrasing. Counterfactuals on the other hand, with their “if only…” A second issue concerns the nature of our manipulations. Because we wished to tightly
control the content of the statements individuals considered, we did not allow individuals to
freely generate these thoughts. Rather, the strategies mentioned in these statements were
provided. One potential drawback of this method is that there may have been more experimental
demand for individuals to pursue the implementation intention, particularly due to the “whenever
x happens, I will do y” phrasing. Counterfactuals on the other hand, with their “if only…”
phrasing, are more similar to a suggestion and thus may have created less experimental demand
to pursue the mentioned strategy. Another potential issue along these lines is that participants
may not have accepted the premise of the counterfactual (Petrocelli, Percy, Sherman, & Tormala,
2011). That is, they may not have believed that their performance would have been better using
the mentioned strategy, and so they made other plans. To address these concerns in Study 2,
participants were asked to generate their own counterfactuals and implementation intentions. In
this manner, we could be assured the selected strategy was likely to be believable to participants
and freely chosen. We could then examine whether the benefits of upward counterfactual
thinking were still independent of their content. provided. One potential drawback of this method is that there may have been more experimental
demand for individuals to pursue the implementation intention, particularly due to the “whenever
x happens, I will do y” phrasing. COUNTERFACTUAL THINKING COUNTERFACTUAL THINKING 19 H2: Implementation intentions that refer to task persistence should predict greater
persistence, compared to a control thought. Due to the inclusion of insolvable items, we did not make predictions concerning
improvement in performance. Participants and Design Seventy-nine (54 female) students at a German university were recruited to participate in the
study. Participants received 3€ or research participation credit as compensation. Participants
were randomly assigned to one of four conditions in a 2 (counterfactual vs. no counterfactual) x
2 (implementation intention vs. no implementation intention) between-subjects design. Materials COUNTERFACTUAL THINKING Counterfactuals on the other hand, with their “if only…”
phrasing, are more similar to a suggestion and thus may have created less experimental demand
to pursue the mentioned strategy. Another potential issue along these lines is that participants
may not have accepted the premise of the counterfactual (Petrocelli, Percy, Sherman, & Tormala,
2011). That is, they may not have believed that their performance would have been better using
the mentioned strategy, and so they made other plans. To address these concerns in Study 2,
participants were asked to generate their own counterfactuals and implementation intentions. In phrasing, are more similar to a suggestion and thus may have created less experimental demand
to pursue the mentioned strategy. Another potential issue along these lines is that participants
may not have accepted the premise of the counterfactual (Petrocelli, Percy, Sherman, & Tormala,
2011). That is, they may not have believed that their performance would have been better using
the mentioned strategy, and so they made other plans. To address these concerns in Study 2, participants were asked to generate their own counterfactuals and implementation intentions. In
this manner, we could be assured the selected strategy was likely to be believable to participants
and freely chosen. We could then examine whether the benefits of upward counterfactual
thinking were still independent of their content. Our predictions were as follows: Our predictions were as follows: H1: Counterfactual thinking, regardless of strategic content, should lead to greater
persistence compared to a control thought, but only when individuals report more negative
affect. COUNTERFACTUAL THINKING 20 deducted from their score. For example, if they correctly completed a word and bought two
clues, then they would be awarded four points. No points were awarded for incorrect or
incomplete solutions. After each set of ten items, participants were provided with accurate
feedback on their performance, including how many words were correctly solved, how many
clues were bought, and total score. They did not have the possibility of seeing which words were
correctly solved. deducted from their score. For example, if they correctly completed a word and bought two
clues, then they would be awarded four points. No points were awarded for incorrect or
incomplete solutions. After each set of ten items, participants were provided with accurate
feedback on their performance, including how many words were correctly solved, how many
clues were bought, and total score. They did not have the possibility of seeing which words were
correctly solved. Materials The word-completion task consisted of eight incomplete German words and two non-words. Participants were instructed to successfully complete as many words as possible. Each word was
comprised of eight letters. Participants were also provided with a category to which the word
belonged and two letters to assist them in solving the items. There was only one possible solution
for each word, whereas non-word items had no correct solution. At any point, participants had
the option of attempting to complete the item, buying a clue, skipping over the item, or quitting
the task altogether. Each time that a clue was bought, a single missing letter appeared. The word-completion task consisted of eight incomplete German words and two non-words. Participants were instructed to successfully complete as many words as possible. Each word was
comprised of eight letters. Participants were also provided with a category to which the word
belonged and two letters to assist them in solving the items. There was only one possible solution
for each word, whereas non-word items had no correct solution. At any point, participants had
the option of attempting to complete the item, buying a clue, skipping over the item, or quitting
the task altogether. Each time that a clue was bought, a single missing letter appeared. Participants could purchase up to four clues (totalling six of the eight letters). The unsolved items
were presented in a loop, such that one could return to any skipped items. Participants were
allowed to use as much time as they wished to complete the ten items. Participants could purchase up to four clues (totalling six of the eight letters). The unsolved items
were presented in a loop, such that one could return to any skipped items. Participants were
allowed to use as much time as they wished to complete the ten items. Scores were based on the number of correctly solved items. For each correctly completed
word, participants were awarded with six points; however, for every clue bought, one point was Procedure Participants were told the experiment involved problem solving, and the effects of practice. Thus, they were told that they would be completing two versions of the same task, receiving
feedback each time. They were then assigned a do-your-best goal (“I want to achieve as many
points as possible”) and were asked to write this goal three times. Next, participants completed
several manipulation checks concerning their understanding and motivation on the task, using a
seven-point scale (1 = not at all to 7 = very much). They indicated to what extent they were
willing to put effort into the task, they were motivated to perform well, and they understood what
to do on the task. Participants then began the first block of the word completion task, and time
spent on the task was recorded. Items presented in the first and second block were
counterbalanced. After quitting the first block, participants received feedback on their performance. They were
then randomly assigned to one of two counterfactual conditions. Those in the counterfactual
condition were told that people often have different thoughts when considering a past
performance and that people often think “If I had only done …, then I would have performed
better.” Participants were asked to generate such a thought concerning their performance on the
first block of trials. They were given the sentence stem: “If only I had …, then I would have COUNTERFACTUAL THINKING 21 achieved more points” and asked to complete the statement. They were then asked to write this
same thought two additional times. This procedure was therefore similar to that of Study 1, with
the exception that thought content was generated spontaneously. Those in the control thought condition were told that people often have different thoughts
when considering the past and that people often think “What if…” about the past. Participants
were asked to generate such a thought about their activities on the previous day. They were given
the sentence stem: “If only yesterday I had…” and asked to complete this statement. They were
then asked to write this same thought two additional times. Prior to beginning the second set of items, participants were asked to again write the goal
intention (“I want to achieve as many points as possible”) three times. Thus, the second block of
word-completion items represented a task of unfixed difficulty (Gendolla & Brinkman, 2005). Half of the participants were then assigned to an implementation intention condition. They
were told that people have different thoughts when considering a future performance, and that
these thoughts often take the form of “Whenever … happens, then I will...” They were asked to
generate a thought of this form concerning how they could perform better on the upcoming task. They were given the sentence stem: “Whenever … happens, then I will…” They were then asked
to repeat this thought an additional two times. Next, participants completed a measure of mood that included the same nine negative mood
adjectives as in Study 1, along with eight positive mood items. Next participants completed the
motivation and understanding measures again. They then completed the second block of the
word-completion task and time-spent was again recorded. Participants received feedback and
were debriefed. Results and Discussion Performance We examined accuracy in set 2 (M = 5.14, SD = 2.04), controlling for accuracy in set 1 (M =
5.04, SD = 1.98). However, the regression analysis revealed no significant effects of mood or
condition on performance, all ts < 1, ps > .34. COUNTERFACTUAL THINKING 22 Overview Participants indicated that participants were motivated to perform well (Mset1 = 6.09, Mset2 =
5.37) and that they understood what to do on the task (Mset1 = 5.11, Mset2 = 5.54). Regression
analyses were performed as in Study 1.5 Negative mood scores (Cronbach‟s α = .88, M = 14.06,
SD = 5.63) were positively skewed. Analyses were therefore conducted using log-transformed
scores (M = 2.58, SD = 0.34). A 2 (counterfactual vs. no counterfactual) x 2 (implementation
intention vs. no implementation intention) ANOVA conducted on negative mood scores revealed
an unexpected Counterfactual x Implementation intention interaction, F(1,75) = 4.21, p < .05, η2
= .05. Simple-effects analyses revealed that, among those in the implementation intention
condition, reported negative mood was lower in the counterfactual condition (M = 2.49, SD =
0.26) than in the no counterfactual condition (M = 2.69, SD = 0.41), t(37) = 2.16, p < .05, η2 =
.11. Within the no implementation intention condition, the counterfactual condition (M = 2.63,
SD = 0.35) did not differ from the control condition (M = 2.56, SD = 0.32), t(38) < 1 , p > .48, η2
= .01. There were no other effects, Fs < 1.13, ps > .29. Importantly, diagnostics conducted on the
analyses reported below revealed that multicollinearity was not problematic.6 COUNTERFACTUAL THINKING 23 in set 1 (M = 5.95 sec, SD = 1.07), see Table 2. The Counterfactual x Negative mood interaction
was marginally significant, see Figure 3. Among those high in negative mood, willingness to put
forth effort was nonsignificantly higher in the counterfactual condition than in the control
condition, simple-slope = 0.20, t(74) = 0.89, p = .38, 95% CI: -0.25 to 0.64. Among those low in
negative mood, willingness to put forth effort was nonsignificantly lower in the counterfactual
condition than in the control condition, simple-slope = -0.16, t(74) = 0.71, p = .48, 95% CI: -0.60
to 0.28. Thus, individuals reporting high negative mood and assigned to the counterfactual
condition were highly motivated to put forth effort on the task at least as much as participants in in set 1 (M = 5.95 sec, SD = 1.07), see Table 2. The Counterfactual x Negative mood interaction
was marginally significant, see Figure 3. Among those high in negative mood, willingness to put
forth effort was nonsignificantly higher in the counterfactual condition than in the control
condition, simple-slope = 0.20, t(74) = 0.89, p = .38, 95% CI: -0.25 to 0.64. Among those low in
negative mood, willingness to put forth effort was nonsignificantly lower in the counterfactual
condition than in the control condition, simple-slope = -0.16, t(74) = 0.71, p = .48, 95% CI: -0.60
to 0.28. Thus, individuals reporting high negative mood and assigned to the counterfactual
condition were highly motivated to put forth effort on the task, at least as much as participants in
an other condition Reported effort To ensure that increased persistence on the task did not reflect slower responding due to low
motivation, we examined the effects of negative mood and condition on reported willingness to
put forth effort in set 2 (M = 5.46 sec, SD = 1.46), controlling for willingness to put forth effort Persistence We next examined the effects of negative mood and condition on the time spent in set 2 (M =
474.34 sec, SD = 303.24), controlling for the time spent in set 1 (M = 775.26 sec, SD = 457.67). Time spent on each set (in sec) was square-root transformed to reduce skewness (set 1 M =
26.74, SD = 7.82; set 2 M = 20.81, SD = 6.46). The model predicting time spent in set 2 is
presented in Table 2. The Counterfactual x Negative mood interaction was significant, see Figure
4. Among those reporting high negative mood, the counterfactual led to significantly more
persistence relative to the control condition, simple-slope = 1.88, t(74) = 2.22, p = .03, 95% CI:
0.19 to 3.56. There was no effect of counterfactual condition among those reporting low negative
mood, simple-slope = -0.42, t(74) = 0.50, p = .62, 95% CI: -2.09 to 1.25. Consistent with the
previous study, counterfactual thoughts affected persistence among those high in negative mood,
independent of the opportunity to form implementation intentions. Importantly, these effects are
unlikely to represent slower responding due to a lack of motivation in light of results on the self-
report measure of willingness to put forth effort. We next examined the effects of negative mood and condition on the time spent in set 2 (M =
474.34 sec, SD = 303.24), controlling for the time spent in set 1 (M = 775.26 sec, SD = 457.67). Time spent on each set (in sec) was square-root transformed to reduce skewness (set 1 M =
26.74, SD = 7.82; set 2 M = 20.81, SD = 6.46). The model predicting time spent in set 2 is
presented in Table 2. The Counterfactual x Negative mood interaction was significant, see Figure 4. Among those reporting high negative mood, the counterfactual led to significantly more
persistence relative to the control condition, simple-slope = 1.88, t(74) = 2.22, p = .03, 95% CI:
0.19 to 3.56. There was no effect of counterfactual condition among those reporting low negative
mood, simple-slope = -0.42, t(74) = 0.50, p = .62, 95% CI: -2.09 to 1.25. Consistent with the
previous study, counterfactual thoughts affected persistence among those high in negative mood,
independent of the opportunity to form implementation intentions. Persistence Importantly, these effects are
unlikely to represent slower responding due to a lack of motivation in light of results on the self-
report measure of willingness to put forth effort. COUNTERFACTUAL THINKING 24 COUNTERFACTUAL THINKING 25 variable contrasted those who generated a thought unrelated to time with those in the control
condition (1 and -1, respectively). Similarly, participants in the implementation intention
condition had a score of 1 on either the time-related thought and 0 on the unrelated thought
dummy variables, or vice versa. Those in the control condition were assigned a score of -1 on
both variables. Because these dummy variables sum to the original condition variable, this
analysis allows for tests of mediation (i.e., including both variables in the model explains the
same variance as the original condition variable). We then conducted a regression analysis
replacing the implementation intention and counterfactual thought terms with these dummy
variables. The decomposed Counterfactual x Implementation intention interactions were not
included, as preliminary analyses revealed no significant effects of these terms. The resulting
model can be seen in Table 3. variable contrasted those who generated a thought unrelated to time with those in the control
condition (1 and -1, respectively). Similarly, participants in the implementation intention
condition had a score of 1 on either the time-related thought and 0 on the unrelated thought
dummy variables, or vice versa. Those in the control condition were assigned a score of -1 on
both variables. Because these dummy variables sum to the original condition variable, this
analysis allows for tests of mediation (i.e., including both variables in the model explains the
same variance as the original condition variable). We then conducted a regression analysis
replacing the implementation intention and counterfactual thought terms with these dummy
variables. The decomposed Counterfactual x Implementation intention interactions were not
included, as preliminary analyses revealed no significant effects of these terms. The resulting
model can be seen in Table 3. Neither the time-related counterfactual vs. control nor its interaction with negative mood
significantly predicted persistence. Only the interaction of Unrelated counterfactual vs. control x
Negative mood interaction predicted persistence. Specifically, counterfactuals unrelated to time
led to increased persistence when individuals reported more negative mood, simple-slope = 3.18,
t(74) = 3.20, p < .01, 95% CI: 1.20 to 5.15. Counterfactuals unrelated to time did not predict
persistence when individuals reported less negative mood, simple-slope = -1.06, t(74) = 1.03, p =
.31, 95% CI: -3.10 to 0.99. In addition, the Time-related implementation intentions vs. control x Negative mood
interaction was significant. Controlling for thought content Two independent judges unaware of the hypotheses coded each statement listed within the
counterfactual and implementation intention conditions. Judges assigned each statement to one
of two categories: those concerned with patience and time spent on the task, and all other
thoughts (e.g., purchasing clues, using a dictionary). Agreement for the counterfactual thoughts
was very high (95%, κ = .90). Frequencies and example counterfactual thoughts in these
categories were as follows: time (f = 20) “If only I would have given myself more time”; other (f
= 21) “If only I would have made riskier guesses”. Agreement for the implementation intentions
was also good (80%, κ = .60). Frequencies and example implementation intentions in these
categories were as follows: time (f = 18) “Whenever I do not know a word, then I will consider
longer”; other (f = 21) “Whenever I cannot see a solution, I will buy a clue”. Opportunity to
generate counterfactual thoughts did not impact the content of generated implementation
intentions. In the no counterfactual condition (n = 19), 42% of participants generated an
implementation intention concerning time, and in the counterfactual condition (n = 20), 50% of
participants generated an implementation intention concerning time, χ2(1) = 0.24, p = .62, φ =
.08. In order to examine whether the effects of implementation intentions and counterfactual
thoughts on persistence were specific to their content, we decomposed each condition variable
into two dummy variables based on the thought coding. Participants in the counterfactual
condition had a score of 1 on the time-related thought and 0 on the unrelated thought dummy
variables, or vice versa. Those in the control condition were assigned a score of -1 on both
variables. Thus, the first dummy variable contrasted those who generated a time-related thought
(given a score of 1) with those in the control condition (given a score of -1). The second dummy COUNTERFACTUAL THINKING 26 time-related implementation intentions predicted nonsignificantly less persistence relative to the
control condition, simple-slope = -0.93, t(74) = 0.88, p = .38, 95% CI: -3.02 to 1.17 time-related implementation intentions predicted nonsignificantly less persistence relative to the
control condition, simple-slope = -0.93, t(74) = 0.88, p = .38, 95% CI: -3.02 to 1.17 In summary, the results of Study 2 supported hypothesis 1. Even when participants were able
to generate their own thoughts, counterfactuals appeared to influence effort in a content-neutral
manner, and only when accompanied by more negative affect. Hypothesis 2 was partially In summary, the results of Study 2 supported hypothesis 1. Even when participants were able
to generate their own thoughts, counterfactuals appeared to influence effort in a content-neutral
manner, and only when accompanied by more negative affect. Hypothesis 2 was partially
supported. Only implementation intentions concerning time predicted time spent on the task. However, this effect was moderated by affect, such that implementation intentions tended to
predict subsequent behavior only when accompanied by more negative mood. This finding
suggests that implementation intentions may also be subject to motivational consideration. More
importantly for our purposes, the benefits of forming implementation intentions did appear to be
tied to their content. Only when controlling for thought content did any implementation intention
effect emerge. Finally, forming an implementation intention had benefits for task persistence
over and above generating a counterfactual thought. COUNTERFACTUAL THINKING Among those high in negative mood, time-related implementation
intentions predicted nonsignificantly greater persistence relative to the control condition, simple-
slope = 1.30, t(74) = 1.17, p = .25, 95% CI: -.91 to 3.52. Among those low in negative mood, COUNTERFACTUAL THINKING Study 3 – Mood Discounting Thus far, we have observed that counterfactuals influence subsequent performance and
persistence only when accompanied by relatively more negative affect. According to the REM
(Markman & McMullen, 2003), negative affect resulting from counterfactual thinking is a signal
of insufficient goal progress and the need to increase effort. Study 3 was designed to test this
explanation for our findings. One way to test the mood-as-input hypothesis is to manipulate the
validity of mood as a cue to goal progress (Gendolla & Krüsken, 2002; Schwarz & Clore, 1983). In other words, if mood can be attributed to other sources in the environment (e.g., pills, lighting,
or an intervening task) then individuals should discount current feelings as an indicator of goal
progress. We therefore used a misattribution paradigm (see also Gendolla & Krüsken, 2002; COUNTERFACTUAL THINKING 27 Schwarz & Clore, 1983) to determine whether the mood moderation effects we observed in
Studies 1 and 2 were due to participants using mood-as-input into judgments of goal progress. We therefore introduced an additional task that supposedly primed neutral or negative feelings. We predicted a three-way interaction of counterfactual condition, negative mood, and this
misattribution cue. In the control cue (neutral prime) condition, we expected to replicate the
Counterfactual thought x Negative mood interaction observed in previous studies. That is, Schwarz & Clore, 1983) to determine whether the mood moderation effects we observed in
Studies 1 and 2 were due to participants using mood-as-input into judgments of goal progress. We therefore introduced an additional task that supposedly primed neutral or negative feelings. We predicted a three-way interaction of counterfactual condition, negative mood, and this
misattribution cue. In the control cue (neutral prime) condition, we expected to replicate the
Counterfactual thought x Negative mood interaction observed in previous studies. That is,
counterfactuals should increase performance relative to a control thought only among individuals
reporting more negative affect. In the misattribution cue (negative prime) condition, we expected
that the Counterfactual thought x Negative mood interaction would be eliminated, because
individuals would assume their negative mood was due to the influence of the subliminal primes
rather than their task performance. counterfactuals should increase performance relative to a control thought only among individuals
reporting more negative affect. In the misattribution cue (negative prime) condition, we expected
that the Counterfactual thought x Negative mood interaction would be eliminated, because
individuals would assume their negative mood was due to the influence of the subliminal primes
rather than their task performance. Participants and Design Participants were 87 (58 female) students at a German university, 88.5% of whom were age
25 or younger. They received 4€ as compensation, regardless of performance on the tasks. One
male participant was excluded from the analyses as an outlier on the mood, effort, and Participants were 87 (58 female) students at a German university, 88.5% of whom were age
25 or younger. They received 4€ as compensation, regardless of performance on the tasks. One
male participant was excluded from the analyses as an outlier on the mood, effort, and
understanding measures. Participants were randomly assigned to conditions in a 2
(counterfactual vs. no counterfactual) x 2 (misattribution cue vs. control cue) between-subjects
design. understanding measures. Participants were randomly assigned to conditions in a 2
(counterfactual vs. no counterfactual) x 2 (misattribution cue vs. control cue) between-subjects
design. COUNTERFACTUAL THINKING 28 were told that they would receive one point for every correct answer, and would receive 0.50€
for every ten points. were told that they would receive one point for every correct answer, and would receive 0.50€
for every ten points. Lexical decision task. Participants were told the task was a pretest for a future study. On
each trial, a fixation cross was presented for 250ms. Next, a letter-string was presented for 70ms,
followed by a mask. Participants were asked to identify the letter-string as a word („D‟-key) or
non-word („K‟-key). There were a total of 50 trials, including 25 words and 25 non-words. The
words included eight positive (e.g., friend, sunshine), eight negative (e.g., danger, cockroach),
and nine neutral (e.g., table, pencil) words. Materials Anagram task. The anagram task consisted of two blocks of twenty trials. On each trial,
participants could choose whether to solve a six-letter anagram or skip the item. Participants COUNTERFACTUAL THINKING COUNTERFACTUAL THINKING 29 presented on the screen. As a result, participants were warned that they may notice that their
mood became more negative as a result of the task, but that this effect usually dissipates after ten
minutes. Those assigned to the control cue condition were not given any information about the
effects of the task on their mood. presented on the screen. As a result, participants were warned that they may notice that their
mood became more negative as a result of the task, but that this effect usually dissipates after ten
minutes. Those assigned to the control cue condition were not given any information about the
effects of the task on their mood. Next, participants were randomly assigned to one of two thought conditions. Those assigned
to the counterfactual condition were asked to consider the thought “if only I had concentrated
during the anagram task, I would have done better“. They were asked to write this thought three
times. Those assigned to the control thought condition were asked to consider the thought “I
would like to know how my friends would have done on this task”. They were asked to write this
thought three times. Following this task, they again wrote the goal “I want to earn as many points
as possible!” three times. The second set of items thus represented a task of unfixed difficulty. Participants next completed the 17-tem measure of mood as in Study 2. They again rated their
motivation, willingness to put forth effort, and understanding of the task. They then completed
the second block of the anagram task. Procedure Participants were told the study involved problem-solving under time pressure, and that they
would complete two versions of an anagram task, receiving feedback after each part. After
providing demographic information, the anagram task was explained. Participants were given the
goal “I want to earn as many points as possible!” and asked to write this goal three times. They
completed the measures of motivation, willingness to put forth effort, and understanding of the
task as in Study 2. They then completed the first block of 20 anagrams. All participants were
then given accurate feedback concerning the number of items correctly solved, number of items
skipped, and total number of points earned. Next, participants were asked to complete the lexical decision task. Following this task,
participants were randomly assigned to one of two cue conditions. Those in the misattribution
cue condition were told that, during the lexical decision task, negative words had been presented
between the fixation cross and the target letter-string. These primes were supposedly presented
so briefly as to be subliminal. Participants were told the purpose of the study was to examine
whether these subliminal primes would facilitate the identification of negative target words Results and Discussion Participants indicated that they were motivated to perform well (Mset1 = 5.76, Mset2 = 5.36),
were willing to put forth effort (Mset1 = 5.77, Mset2 = 5.51), and that they understood what to do
on the task (Mset1 = 5.05, Mset2 = 4.43). Effect coding was used for the thought (counterfactual =
1, no counterfactual = -1) and cue (control = 1, misattribution = -1) variables. Negative mood
scores (M = 14.14, SD = 5.45, Cronbach‟s α = .85) were positively skewed, and so scores on this
measure were log-transformed (M = 2.59, SD = 0.34). A 2 (counterfactual vs. no counterfactual)
x 2 (misattribution cue vs. control cue) ANOVA on negative mood scores did not reveal any COUNTERFACTUAL THINKING COUNTERFACTUAL THINKING 31 The results of Study 3 strongly supported the mood-as-input (Cervone et al., 1994; Gendolla
& Brinkman, 2005; Martin et al., 1993) and REM (Markman & McMullen, 2003) perspectives
on the role of affect in determining the behavioral consequences of counterfactual thoughts. As
in prior studies, counterfactual thoughts led to better performance only when accompanied by
negative affect. However, when negative affect was said to be the result of an intervening task, it
could no longer be used to inform judgments of goal progress (Gendolla & Krüsken, 2002;
Schwarz & Clore, 1983). As a result, the counterfactual no longer improved performance. COUNTERFACTUAL THINKING 30 significant differences, Fs < 2.22, ps > .14. Preliminary analyses revealed an order effect on
performance, and so this variable was included as a covariate in the analysis. significant differences, Fs < 2.22, ps > .14. Preliminary analyses revealed an order effect on
performance, and so this variable was included as a covariate in the analysis. We examined accuracy in set 2 (M = 6.19, SD = 4.02) controlling for accuracy in set 1 (M =
5.42, SD = 4.03). These scores were then regressed onto the following model: order, negative
mood scores, counterfactual condition, and cue condition were entered in the first step, all two-
way interactions of negative mood scores, thought condition, and cue condition were entered in
the second step, and the three-way interaction of negative mood scores, thought condition, and
cue condition was entered in the final step.7 The results of this analysis are presented in Table 5. In addition to effects of order and negative mood, the predicted Counterfactual x Negative mood
x Cue interaction was significant, see Figures 5a and 5b. The Counterfactual x Negative mood
interaction was calculated within each cue condition in order to decompose this significant three-
way interaction. Within the control cue condition, the Counterfactual x Negative mood interaction observed in
previous studies was replicated, β = .453, t(76) = 2.23, p = .03. Performance was marginally
better in the counterfactual condition relative to the control condition among participants
reporting more negative mood, simple-slope = 1.65, t(76) = 1.92, p = .06, 95% CI = -0.06 to
3.37. Counterfactual thought had no effect among those reporting less negative mood, simple-
slope = -0.93, t(76) = 1.32, p = .19, 95% CI = -2.33 to 0.47. This pattern was eliminated in the misattribution cue condition, β = -.314, t(76) This pattern was eliminated in the misattribution cue condition, β = -.314, t(76) = 1.64, p =
.11. When told their negative mood may be due to the lexical decision task, counterfactual
thoughts did not improve performance relative to the control condition among those low in
negative mood, simple-slope = 1.12, t(76) = 1.46, p = .15, 95% CI = -0.41 to 2.65, or among
those high in negative mood, simple-slope = -0.67, t(76) = 0.90, p = .37, 95% CI = -2.14 to 0.81. General Discussion The present work sought to examine two differing explanations of the benefits of
counterfactual thinking for subsequent performance. The content-specific view holds that
counterfactuals are translated into an intention to engage in a specific behavior in the future
(Smallman & Roese, 2009). Thus, the benefits of counterfactual thinking should be quite
comparable to those of forming a plan (Epstude & Roese, 2008). According to the REM
(Markman & McMullen, 2003), counterfactuals indirectly influence judgments of progress
toward a goal, particularly when no objective information exists. When individuals experience
more negative affect as a result of counterfactual thinking, they should judge their performance
as inadequate (Carver & Scheier, 1990; Cervone et al., 1994; Gendolla & Brinkman, 2005;
Martin et al., 1993). Effort on the task is increased, leading to more broad improvement
(Markman et al., 2008). Based on the REM and Action-Phase Theory (Heckhausen, 2003;
Heckhausen & Gollwitzer, 1987), we argued that any content-specific benefits should be
dependent on subjective evaluations of goal progress. The present studies therefore had two
aims: determining the specificity of counterfactual benefits for performance relative to those of COUNTERFACTUAL THINKING 32 forming a plan, and examining the role of mood in moderating these effects. The design of the
studies allowed us to directly compare the explanatory power of these two accounts. forming a plan, and examining the role of mood in moderating these effects. The design of the
studies allowed us to directly compare the explanatory power of these two accounts. The findings of three studies were largely supportive of the REM (Markman & McMullen,
2003) and Action-Phase Theory (Heckhausen, 2003; Heckhausen & Gollwitzer, 1987). First, we
observed benefits of counterfactual thinking only when these thoughts were accompanied by
more negative affect. Second, the effects of counterfactual thinking were eliminated when
negative affect could be misattributed to an unrelated task. Third, the benefits of counterfactual
thought for performance were largely unrelated to their strategic content. That is, we found that
counterfactuals significantly improved performance even in the absence of significant changes in
the specified behavior (Study 1), and that counterfactuals with unrelated strategic content
significantly influenced behavior (Study 2). Finally, the effects of counterfactual thinking were
unique and additive to the benefits of forming a corresponding plan in the form of an
implementation intention. These results held for a variety of tasks, specified strategies, and
manipulations of counterfactual thinking. Counterfactual thoughts seem to have broad benefits
for performance, but only when individuals are dissatisfied with their prior efforts. We discuss
the implications of these findings below. Role of Mood in Moderating Benefits of Counterfactual Thinking Role of Mood in Moderating Benefits of Counterfactual Thinking Consistent with the REM (Markman et al., 2008) and mood-as-input (see also Gendolla &
Brinkman, 2005; Martin et al., 1993) views, we found that counterfactual thoughts only led to
corrective action and performance improvement when accompanied by negative affect. Consistent with the REM (Markman et al., 2008) and mood-as-input (see also Gendolla &
Brinkman, 2005; Martin et al., 1993) views, we found that counterfactual thoughts only led to
corrective action and performance improvement when accompanied by negative affect. Moreover, when affect could be (mis)attributed to the context rather than one‟s performance,
these benefits were eliminated. These findings are consistent with past work showing that
counterfactuals that result in negative affect are most likely to increase intentions to improve Moreover, when affect could be (mis)attributed to the context rather than one‟s performance,
these benefits were eliminated. These findings are consistent with past work showing that
counterfactuals that result in negative affect are most likely to increase intentions to improve COUNTERFACTUAL THINKING 33 (Markman et al., 2008), as well as with work showing that counterfactuals used to excuse poor
performance reduce subsequent effort (McCrea, 2008; Tyser et al., 2012). Future work should
continue to test the mood-as-input explanation for the role of affect in determining the
consequences of counterfactual thinking. For example, the tasks in our studies featured “do your
best” goals (i.e., tasks of unfixed difficulty), which are inherently subjective. The role of affect
may be minimized in tasks with more objective standards (e.g., a percentile ranking or point total
that must be reached). In addition, it should be noted that individuals in our studies held conscious goals and affect
was likely used to inform decisions about whether and how to pursue these goals. In other work,
the co-activation of negative affect and nonconscious (i.e., subliminally primed) goals has been
shown to undermine motivation (Aarts, Custers, & Holland, 2007). The impact of counterfactual
thinking when pursuing goals nonconsciously thus remains an interesting area for exploration. Breadth of Benefits of Counterfactual Thinking The REM (Markman & McMullen, 2003) proposes that counterfactuals increase persistence
as a result of judgments (informed by negative affect) of insufficient goal progress. Consistent
with this view, we found that counterfactual thinking influenced persistence and performance
independent of the strategic content of the thought. These effects were in sharp contrast to the
consequences of forming an implementation intention. Replicating past work (Parks-Stamm et
al., 2007), implementation intentions benefitted the specified behavior, but did not affect other
goal-relevant behaviors. Thus, counterfactual thinking had effects quite different from those of
forming an if-then plan. lman and colleagues (Smallman & McCulloch, 2012; Smallman & Roese, 2009) Smallman and colleagues (Smallman & McCulloch, 2012; Smallman & Roese, 2009)
previously demonstrated that counterfactual thinking increases the accessibility of intentions to COUNTERFACTUAL THINKING 34 engage in the specified behavior (i.e., “In the future, I will do X”). One may then ask why we did
not more consistently observe content-specific benefits of counterfactual thought. Although we
expected counterfactuals would have benefits to performance beyond those specified in the
thought, this does not preclude engaging in the specified behavior at all. We suggest several
possible reasons for this finding. As discussed earlier, individuals in our studies could have formed additional intentions not
suggested by the counterfactual thought. In the short timeframes (i.e., a few seconds) assessed in
previous studies (Smallman & McCulloch, 2012; Smallman & Roese, 2009), counterfactual
thoughts might increase the accessibility of corresponding intentions. However, when individuals
have the opportunity to deliberate they could spontaneously consider other strategies to improve. Consistent with this view, we found in Study 2 that the opportunity to generate a counterfactual
thought did not significantly affect the content of subsequently generated implementation
intentions (replicating Myers, 2009). Thus, given a few moments to form an implementation
intention, individuals may form the same plan regardless of the content of prior counterfactual
thoughts. A second possibility is that counterfactual thinking does not result in well-encoded future
intentions. In a recent study by McCrea, Penningroth, and Tyser (2012), it was observed that
implementation intentions forged a strong cue-response link in memory and improved A second possibility is that counterfactual thinking does not result in well-encoded future
intentions. In a recent study by McCrea, Penningroth, and Tyser (2012), it was observed that
implementation intentions forged a strong cue-response link in memory and improved
prospective memory performance on a go/no-go task. Participants who formed a counterfactual
or a plan that did not take an “if-then” format did not significantly differ from a control group on
these measures. Thus, it is possible that counterfactuals facilitate intentions that are poorly
encoded or that occasionally fail to take the “if-then” format of an implementation intention. A
I
t
t R l f
Th
ht C
t
t? COUNTERFACTUAL THINKING 35 Even if the specified strategy is not enacted, the content of a counterfactual thought is still
important in determining its consequences for behavior. In addition to influencing mood and
thereby the perceived need for additional effort, upward counterfactuals also affect self-efficacy
and feelings of control for subsequent performance (Nasco & Marsh, 1999; Tal-Or, Boninger, &
Gleicher, 2004). That is, the corrective actions suggested by the counterfactual may be helpful in
so far as they help the individual feel as if he or she is capable of completing the task. Thus, Even if the specified strategy is not enacted, the content of a counterfactual thought is still
important in determining its consequences for behavior. In addition to influencing mood and
thereby the perceived need for additional effort, upward counterfactuals also affect self-efficacy
and feelings of control for subsequent performance (Nasco & Marsh, 1999; Tal-Or, Boninger, &
Gleicher, 2004). That is, the corrective actions suggested by the counterfactual may be helpful in
so far as they help the individual feel as if he or she is capable of completing the task. Thus,
another critical feature of upward counterfactuals may be that they tend to focus on controllable
actions (Markman, Gavanski, Sherman, & McMullen, 1995). Thoughts that specify external
(e.g., “If only I was luckier”) or uncontrollable (e.g., “If only I was smarter”) causes for poor
performance may not have the same benefits for the mobilization of effort as do those specifying
controllable actions. The combination of dissatisfaction with prior performance and feelings of
control should be highly motivating. We are currently examining this question in our laboratory. another critical feature of upward counterfactuals may be that they tend to focus on controllable
actions (Markman, Gavanski, Sherman, & McMullen, 1995). Thoughts that specify external
(e.g., “If only I was luckier”) or uncontrollable (e.g., “If only I was smarter”) causes for poor
performance may not have the same benefits for the mobilization of effort as do those specifying
controllable actions. The combination of dissatisfaction with prior performance and feelings of
control should be highly motivating. We are currently examining this question in our laboratory. I
li
ti
f
B h
i
Ch
d Eff
ti
S lf R
l ti COUNTERFACTUAL THINKING 36 counterfactual thinking can increase motivation upon receiving feedback (see also Gollwitzer,
Wieber, Myers, & McCrea, 2010). Implementation intentions can then direct this energy into
productive behavior. counterfactual thinking can increase motivation upon receiving feedback (see also Gollwitzer,
Wieber, Myers, & McCrea, 2010). Implementation intentions can then direct this energy into
productive behavior. Implications for Behavior Change and Effective Self-Regulation Implementation intentions have been shown to improve goal striving in a variety of domains
(Gollwitzer & Sheeran, 2006) and therefore are of great interest to applied researchers. The
finding that counterfactual thinking can have additional beneficial effects beyond planning is
therefore exciting and should be investigated in other research contexts. Specifically, upward
counterfactual thinking seems to more broadly increase performance, whereas implementation
intentions promote specific behaviors. In similar work examining simulations of future events,
Oettingen and colleagues (2001; Stadler, Oettingen, & Gollwitzer, 2009) have found that
mentally contrasting a desired outcome with a current state of reality increases the mobilization
of effort, and that doing so also enhances the impact of subsequently-formed implementation
intentions. Thus, much like mental contrasting can increase motivation prior to a task, Conclusion Counterfactuals are ubiquitous and powerful thoughts that not only determine our affective
reactions to events, but also our subsequent behaviors. Particularly after negative events, we
often think about what went wrong and try to learn from our mistakes. The present research
indicates that these kinds of thoughts may often be beneficial because we become committed to
improvement more generally, rather than because of any one particular “lesson learned.” Counterfactuals are ubiquitous and powerful thoughts that not only determine our affective
reactions to events, but also our subsequent behaviors. Particularly after negative events, we
often think about what went wrong and try to learn from our mistakes. The present research
indicates that these kinds of thoughts may often be beneficial because we become committed to
improvement more generally, rather than because of any one particular “lesson learned.”
Certainly, such flexibility points to the functionality of these thoughts and the need for more
research into how they are translated into subsequent action. Certainly, such flexibility points to the functionality of these thoughts and the need for more
research into how they are translated into subsequent action. COUNTERFACTUAL THINKING 37 References Aarts, H., Custers, R., & Holland, R. W. (2007). The nonconscious cessation of goal pursuit:
When goals and negative affect are coactivated. Journal of Personality and Social
Psychology, 92, 165-178. Achtziger, A., Gollwitzer, P. M., & Sheeran, P. (2008). Implementation intentions and shielding
goal striving from unwanted thoughts and feelings. Personality and Social Psychology
Bulletin, 34, 381-393. Aiken, L. S., & West, S. G. (1991). Multiple regression: Testing and interpreting interactions. Thousand Oakes, CA: Sage Publications. Bandura, A. (1977). Self-efficacy: Toward a unifying theory of behavioral change. Psychological Review, 54, 191-215. Bandura, A. (1991). Social cognitive theory of self-regulation. Organizational Behavior &
Human Decision Processes, 50, 248-287. Bayer, U. C., & Gollwitzer, P. M. (2007). Boosting scholastic test scores by willpower: The role
of implementation intentions. Self and Identity, 6, 1-19. Carver, C. S., & Scheier, M. F. (1990). Origins and functions of positive and negative affect: A
control-process view. Psychological Review, 97, 19-35. Cervone, D., Kopp, D. A., Schaumann, L., & Scott, W. D. (1994). Mood, self-efficacy and
performance standards: Lower moods induce higher standards for performance. Journal
of Personality & Social Psychology, 67, 499-512. de Vet, E., Oenema, A., & Brug, J. (2011). More or better: Do the number and specificity of
implementation intentions matter in increasing physical activity? Journal of Sport &
Exercise Psychology, 12, 471-477. COUNTERFACTUAL THINKING 38 Epstude, K., & Roese, N. J. (2008). The functional theory of counterfactual thinking. Personality
& Social Psychology Review, 12, 168-192. Epstude, K., & Roese, N. J. (2008). The functional theory of counterfactual thinking. Personality
& Social Psychology Review, 12, 168-192. cardiovascular response: The diagnostic value of mood counts. Emotion, 2, 251-262. Gollwitzer, P. M. (1999). Implementation intentions and effective goal pursuit: Strong effects of
simple plans. American Psychologist, 54, 493-503. Gollwitzer, P. M., & Sheeran, P. (2006). Implementation intentions and goal achievement: A
meta-analysis of effects and processes. Advances in Experimental Social Psychology, 38,
69-119. Gollwitzer, P. M., Wieber, F., Myers, A. L., & McCrea, S. M. (2010). How to maximize
implementation intention effects. In C. R. Agnew, D. E. Carlston, W. G. Graziano & J. R. Kelly (Eds.), Then a miracle occurs: Focusing on behavior in social psychological theory
and research. Newy York: Oxford Press. Hayes, A. F. (2012). PROCESS: A versatile computational tool for observed variable
moderation, mediation, and conditional process modeling. Manuscript submitted for
publication. Heckhausen, H. (2003). Motivation und handeln (2nd ed.). Berlin: Springer. Heckhausen, H., & Gollwitzer, P. M. (1987). Thought contents and cognitive functioning in
motivational versus volitional states of mind. Motivation & Emotion, 11, 101-120. Johnson M K & Sherman S J (1990) Constructing and reconstructing the past and the future Heckhausen, H., & Gollwitzer, P. M. (1987). Thought contents and cognitive functioning in
motivational versus volitional states of mind. Motivation & Emotion, 11, 101-120. Johnson, M. K., & Sherman, S. J. (1990). Constructing and reconstructing the past and the future
in the present. In E. T. Higgins & R. M. Sorrentino (Eds.), Handbook of motivation and COUNTERFACTUAL THINKING 39 cognition: Foundations of social behavior, Vol. 2 (pp. 482-526). New York, NY:
Guilford Press. Judd, C. M., & Kenny, D. A. (1981). Estimating the effects of social interventions. Cambridge:
Cambridge University Press. Kahneman, D., & Miller, D. T. (1986). Norm theory: Comparing reality to its alternatives. Psychological Review, 93(2), 136-153. Markman, K. D., Gavanski, I., Sherman, S. J., & McMullen, M. N. (1993). The mental
simulation of better and worse possible worlds. Journal of Experimental Social
Psychology, 29(1), 87-109. Markman, K. D., Gavanski, I., Sherman, S. J., & McMullen, M. N. (1995). The impact of
perceived control on the imagination of better and worse possible worlds. Personality &
Social Psychology Bulletin, 21(6), 588-595. Markman, K. D., & McMullen, M. N. (2003). A reflection and evaluation model of comparative
thinking Personality & Social Psychology Review 7 244 267 Markman, K. D., & McMullen, M. N. (2003). A reflection and evaluation model of comparative thinking. Personality & Social Psychology Review, 7, 244-267. thinking. Personality & Social Psychology Review, 7, 244-267. Markman, K. D., McMullen, M. N., & Elizaga, R. A. (2008). Counterfactual thinking, persistence, and performance: A test of the Reflection and Evaluation Model. Journal of
Experimental Social Psychology, 44, 421-428. Martin, L. L., Ward, D. W., Achee, J. W., & Wyer, R. S. (1993). Mood as input: People have to
interpret the motivational implications of their moods. Journal of Personality & Social
Psychology, 64, 317-326. McCrea, S. M. (2008). Self-handicapping, excuse-making, and counterfactual thinking:
Consequences for self-esteem and future motivation. Journal of Personality & Social
Psychology, 95, 274-292. McCrea, S. M. (2008). Self-handicapping, excuse-making, and counterfactual thinking: Consequences for self-esteem and future motivation. Journal of Personality & Social
Psychology, 95, 274-292. COUNTERFACTUAL THINKING 40 McCrea, S. M., Penningroth, S. L., & Tyser, M. P. (2012). Intention strategies: Implementation
intentions uniquely boost prospective memory performance. Manuscript in preparation. University of Wyoming. McCrea, S. M., Penningroth, S. L., & Tyser, M. P. (2012). Intention strategies: Implementation
intentions uniquely boost prospective memory performance. Manuscript in preparation. University of Wyoming. McFarland, C., & Glisky, E. (2012). Implementation intentions and imagery: Individual and
combined effects on prospective memory among young adults. Memory and Cognition,
40, 62-69. McFarland, C., & Ross, M. (1982). Impact of causal attributions on affective reactions to success McFarland, C., & Ross, M. (1982). Impact of causal attributions on affective reactions to success
and failure. Journal of Personality & Social Psychology, 43(5), 937-946. McMullen, M. N., & Markman, K. D. (2000). Downward counterfactuals and motivation: The
wake-up call and the Pangloss effect. Personality & Social Psychology Bulletin, 26(5),
575-584. Myers, A. L. (2009). Can the performance effects of upward counterfactual thoughts be attributed to effort mobilization? Dissertation, University of Konstanz, Konstanz,
Germany. Nasco, S. A., & Marsh, K. L. (1999). Gaining control through counterfactual thinking. Personality & Social Psychology Bulletin, 25(5), 556-568. Oettingen, G., Pak, H., & Schnetter, K. (2001). Self-regulation of goal-setting: Turning free
fantasies about the future into binding goals. Journal of Personality & Social Psychology,
80, 736-753. Parks-Stamm, E. J., Gollwitzer, P. M., & Oettingen, G. (2007). Action control by
implementation intentions: Effective cue detection and efficient response initiation. Social Cognition, 25, 248-266. COUNTERFACTUAL THINKING 41 Petrocelli, J. V., Percy, E. J., Sherman, S. J., & Tormala, Z. L. (2011). Counterfactual potency. Journal of Personality and Social Psychology, 100, 30-46. Petrocelli, J. V., Percy, E. J., Sherman, S. J., & Tormala, Z. L. (2011). Counterfactual potency. Journal of Personality and Social Psychology, 100, 30-46. Roese, N. J. (1994). The functional basis of counterfactual thinking. Journal of Personality &
Social Psychology, 66(5), 805-818. Schwarz, N., & Clore, G. L. (1983). Mood, misattribution, and judgments of well-being: Informative and directive functions of affective states. Journal of Personality and Social
Psychology, 45, 513-523. Smallman, R., & McCulloch, K. C. (2012). Learning from yesterday's mistakes to fix tomorrow's
problems: When functional counterfactual thinking and psychological distance collide. Smallman, R., & McCulloch, K. C. (2012). Learning from yesterday's mistakes to fix tomorrow's Smallman, R., & McCulloch, K. C. (2012). Learning from yesterday's mistakes to fix tomorrow's
problems: When functional counterfactual thinking and psychological distance collide. European Journal of Social Psychology, 42, 383-390. Smallman, R., & Roese, N. J. (2009). Counterfactual thinking facilitates the formation of
intentions: Evidence for a content-specific pathway in behavioral regulation. Journal of
Experimental Social Psychology, 45, 845-852. Stadler, G., Oettingen, G., & Gollwitzer, P. M. (2009). Physical activity in women. Effects of a
self-regulation intervention. American Journal of Preventive Medicine, 36, 29-34. Tabachnik, B. G., & Fidell, L. S. (1996). Using multivariate statistics (3rd ed.). New York:
HarperCollins. Tal-Or, N., Boninger, D. S., & Gleicher, F. (2004). On becoming what we might have been: Tal-Or, N., Boninger, D. S., & Gleicher, F. (2004). On becoming what we might have been:
Counterfactual thinking and self-efficacy. Self and Identity, 3, 5-26. Counterfactual thinking and self-efficacy. Self and Identity, 3, 5-26. Tyser, M. P., McCrea, S. M., & Knüpfer, K. (2012). Pursuing perfection or pursuing protection?:
Self-evaluation motives moderate the behavioral consequences of counterfactual
thoughts. European Journal of Social Psychology, 42, 372-382. COUNTERFACTUAL THINKING 42 Vohs, K. D., & Heatherton, T. F. (2000). Self-regulatory failure: A resource-depletion approach. Psychological Science, 11, 249-254. Vohs, K. D., & Heatherton, T. F. (2000). Self-regulatory failure: A resource-depletion approach. Psychological Science, 11, 249-254. (1964). Work and motivation. New York: Wiley. Watson, D., Clark, L. A., & Tellegen, A. (1988). Development and validation of brief measures
of positive and negative affect: The PANAS scales. Journal of Personality & Social
Psychology, 54, 1063-1070. Webb, T. L., & Sheeran, P. (2007). How do implementation intentions promote goal attainment? A test of component processes. Journal of Experimental Social Psychology, 43, 295-302. COUNTERFACTUAL THINKING 43 Author Note Author Note Andrea L. Myers, Department of Psychology, University of Konstanz, Kontanz, Germany. Sean M. McCrea and Maurissa P. Tyser, Department of Psychology, University of Wyoming,
Laramie, Wyoming, USA. The authors wish to thank Katja Paessler, Alena Friedrich, and Sandra Paul who served as
experimenters, and Alex Jaudas for his helpful suggestions. This research was supported by
grants to the second author from the University of Konstanz (AFF 13/05) and the German
Research Foundation (DFG MC68/1-1; DFG MC68/2-1). Correspondence concerning this article
should be addressed to Sean M. McCrea, Department of Psychology, University of Wyoming,
Dept. 3415, 1000 E. University Ave., Laramie, WY 82071, USA. Electronic mail may be sent
via the Internet to smccrea@uwyo.edu. COUNTERFACTUAL THINKING 44 Footnotes 1. We report all data exclusions, conditions, and hypothesis-relevant measures. Data collection
proceeded until the end of the semester or a predetermined cell size was reached. 2. Our hypotheses centered on the effects of negative mood. Principle-components analysis was
utilized to identify mood dimensions. Preliminary analyses revealed no interactions of positive
mood or self-esteem with either condition variable (ps > .07). Thus, we do not discuss these
other variables further. 3. Analyzing change scores did not alter the pattern of results in any of the three studies. 3. Analyzing change scores did not alter the pattern of results in any of the three studies. 4. When examining selection of clues at all other time points (prior to 50 sec. or after 70 sec.),
there was only an effect of implementation intention condition, β = -.227, t(85) = 2.64, p = .01. Those in the implementation intention condition were actually less likely to select clues at these
times, consistent with the notion that they carried out the specified behavior only at the specified
time. There were no effects of counterfactual thought condition (all ts < 1, ps > .70). 5. Preliminary analyses revealed no effects of positive mood (ps > .20), and so we do not discuss
this variable further. 5. Preliminary analyses revealed no effects of positive mood (ps > .20), and so we do not discuss
this variable further. 6. For the primary analyses, the condition index was < 12 and variance inflation factor (VIF) <
1.22. For the mediation analysis, the condition index was <9 and VIF < 2.39. A condition index
< 30 and VIF < 5 is considered acceptable (see Tabachnik & Fidell, 1996). Moreover, the results
remained stable when dropping the negative mood term or the Counterfactual x Implementation
intention term from the analysis. 7. Preliminary analyses revealed no effects of positive mood (ps > .52), and so we do not discuss
this variable further. 7. Preliminary analyses revealed no effects of positive mood (ps > .52), and so we do not discuss
this variable further. COUNTERFACTUAL THINKING 45 Table 1 Table 1
Strategy use and performance in set 2 (Study 1)
Clue use (50-70 sec)
β
t
p
f2
Clue use (50-70 sec) in set 1
.097
0.95
.35
.01
Negative mood
.209
1.93
.06
.04
Counterfactual
-.005
0.05
.96
<.01
Implementation intention
.296
2.93
<.01
.10
Counterfactual x Negative mood
.108
1.01
.32
.01
Implementation intention x Negative
mood
.093
0.92
.36
<.01
Counterfactual x Implementation-
intention
-.073
0.72
.47
<.01
Accuracy
β
t
p
f2
Accuracy in set 1
.138
1.24
.22
.02
Negative mood
.038
0.33
.75
<.01
Counterfactual
.169
1.61
.11
.03
Implementation intention
.047
0.44
.66
<.01
Counterfactual x Negative mood
.230
2.02
<.05
.05
Implementation intention x Negative
mood
.058
0.55
.59
<.01
Counterfactual x Implementation-
intention
.026
0.24
.81
<.01 COUNTERFACTUAL THINKING COUNTERFACTUAL THINKING 46 Table 2
Reported effort and time spent on set 2 (Study 2)
Willingness to put forth effort in set 2
β
t
p
f2
Willingness to put forth effort in set 1
.344
3.11
<.01
.14
Negative mood
.115
1.03
.31
.02
Counterfactual
.050
0.46
.65
<.01
Implementation intention
.155
1.37
.18
.03
Counterfactual x Negative mood
.202
1.76
.08
.04
Implementation intention x Negative
mood
.135
1.17
.24
.02
Counterfactual x Implementation-
intention
.130
1.18
.24
.02
Time spent on set 2
β
t
p
f2
Time spent on set 1
.545
5.81
<.001
.48
Negative mood
-.132
1.38
.17
.03
Counterfactual
.149
1.56
.12
.03
Implementation intention
.027
0.28
.78
<.01
Counterfactual x Negative mood
.228
2.29
.03
.07
Implementation intention x Negative
mood
.164
1.63
.11
.04
Counterfactual x Implementation-
intention
.017
0.18
.86
<.01 COUNTERFACTUAL THINKING 47 Table 3
Decomposed model predicting time spent in set 2
Time spent on set 2
β
t
p
f2
Time spent on set 1
.532
5.87
<.001
.51
Negative mood
.046
0.43
.67
<.01
Time-related counterfactual vs. control
-.068
0.56
.58
<.01
Unrelated counterfactual vs. control
.213
1.85
.07
.05
Time-related implemenation intention
vs. control
.158
1.33
.19
.03
Unelated implemenation intention vs. control
-.123
1.07
.29
.02
Time-related counterfactual vs. control x
Negative mood
-.180
1.36
.18
.03
Unrelated counterfactual vs. control x
Negative mood
.424
3.20
<.01
.15
Time-related implementation intention
vs. control x Negative mood
.262
2.14
.04
.07
Unrelated implementation intention vs. Table 1 control x Negative mood
-.103
0.84
.40
<.01 48 COUNTERFACTUAL THINKING COUNTERFACTUAL THINKING Table 4
Accuracy in set 2 (Study 3)
Accuracy in set 2
β
t
p
f2
Accuracy in set 1
.601
6.18
<.001
.50
Order
.285
3.00
<.01
.12
Negative mood
.083
0.82
.42
<.01
Counterfactual
.074
0.80
.43
<.01
Cue
-.059
0.64
.53
<.01
Counterfactual x Negative mood
.049
0.50
.62
<.01
Counterfactual x Cue
.017
0.18
.86
<.01
Negative mood x Cue
-.035
0.37
.72
<.01
Counterfactual x Negative mood
x Cue
.266
2.78
<.01
.10 49 COUNTERFACTUAL THINKING Counterfactual thought
Implementation intention
Performance
Counterfactual thought
Planning
Performance
Figure 1. Content-specific pathway (top panel) and content-neutral pathways (bottom panel) to
performance improvement (adapted from Epstude & Roese, 2008; Markman & McMullen,
2003). Motivation to
improve
Related and
unrelated
plans
“If only I had
done x in
situation y…”
Negative
affect
Related and
unrelated
behaviors
“When y, I
will do x!”
Behavior x
“If only I had
done x in
situation y…” Counterfactual thought Performance Counterfactual thought Figure 1. Content-specific pathway (top panel) and content-neutral pathways (bottom panel) to
performance improvement (adapted from Epstude & Roese, 2008; Markman & McMullen,
2003) 50 COUNTERFACTUAL THINKING Figure 2. Counterfactual x Negative mood interaction on set 2 accuracy (Study 1). 2
2.5
3
3.5
4
4.5
5
5.5
6
No Counterfactual
Counterfactual
Number correct
Low Negative
Mood
High Negative
Mood 2
2.5
3
3.5
4
4.5
5
5.5
6
No Counterfactual
Counterfactual
Number correct
Low Negative
Mood
High Negative
Mood Counterfactual No Counterfactual Figure 2. Counterfactual x Negative mood interaction on set 2 accuracy (Study 1). 51 COUNTERFACTUAL THINKING 4
4.5
5
5.5
6
6.5
7
No Counterfactual
Counterfactual
Willingness to put forth effort
Low Negative
Mood
High Negative
Mood 4
No Counterfactual
Counterfactual Figure 3. Counterfactual x Negative mood interaction on willingness to put forth effort in set 2
(Study 2). Figure 3. Counterfactual x Negative mood interaction on willingness to put forth effort in set 2
(Study 2). 52 COUNTERFACTUAL THINKING Figure 4. Counterfactual x Negative mood interaction on time spent in set 2 (Study 2). 15
16
17
18
19
20
21
22
23
24
25
No Counterfactual
Counterfactual
Time spent in sec. (square-root transformed)
Low Negative
Mood
High Negative
Mood Figure 4. Counterfactual x Negative mood interaction on time spent in set 2 (Study 2). 53 COUNTERFACTUAL THINKING Figure 5a and 5b. Table 1 Counterfactual x Negative mood interaction on difference in number co
within the (a) control cue condition and (b) the misattribution cue condition (Study
3
4
5
6
7
8
9
No Counterfactual
Counterfactual
Number correct
Low Negative
Mood
High Negative
Mood
3
4
5
6
7
8
9
No Counterfactual
Counterfactual
Number correct
Low Negative
Mood
High Negative
Mood 3
4
5
6
7
8
9
Number correct
Low Negative
Mood
High Negative
Mood 3
No Counterfactual
Counterfactual 3
4
5
6
7
8
9
No Counterfactual
Counterfactual
Number correct
Low Negative
Mood
High Negative
Mood Low Negative
Mood
High Negative
Mood No Counterfactual
Counterfactual Figure 5a and 5b. Counterfactual x Negative mood interaction on difference in number correct
within the (a) control cue condition and (b) the misattribution cue condition (Study 3). Figure 5a and 5b. Counterfactual x Negative mood interaction on difference in number correct
within the (a) control cue condition and (b) the misattribution cue condition (Study 3). Figure 5a and 5b. Counterfactual x Negative mood interaction on difference in number correct
within the (a) control cue condition and (b) the misattribution cue condition (Study 3).
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https://openalex.org/W3135758644
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https://research.rug.nl/files/169891084/Antimicrobial_loading_of_nanotubular_titanium_surfaces_favoring_surface_coverage_by_mammalian_cells_over_bacterial_colonization.pdf
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English
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Antimicrobial loading of nanotubular titanium surfaces favoring surface coverage by mammalian cells over bacterial colonization
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Materials science & engineering. C, Biomimetic materials, sensors and systems
| 2,021
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cc-by
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Antimicrobial loading of nanotubular titanium surfaces favoring surface
coverage by mammalian cells over bacterial colonization nny C. van der Mei a,*, Yijin Ren b, Henk J. Busscher a, Brandon W. Peterson a ingen and University Medical Center of Groningen, Department of Biomedical Engineering, Antonius Deusinglaan 1, 9713 AV Groningen, the b University of Groningen and University Medical Center of Groningen, Department of Orthodontics, Hanzeplein 1, 9700 RB Groningen, the Netherlands A R T I C L E I N F O Keywords:
Silver nanoparticles
Co-cultures
Biomaterial-associated infection
Biomaterials implants
Local drug delivery
Gentamicin Titanium is frequently used for dental implants, percutaneous pins and screws or orthopedic joint prostheses. Implant surfaces can become peri-operatively contaminated by surgically introduced bacteria during implan
tation causing lack of surface coverage by mammalian cells and subsequent implant failure. Especially implants
that have to function in a bacteria-laden environment such as dental implants or percutaneous pins, cannot be
surgically implanted while being kept sterile. Accordingly, contaminating bacteria adhering to implant surfaces
hamper successful surface coverage by mammalian cells required for long-term functioning. Here, nanotubular
titanium surfaces were prepared and loaded with Ag nanoparticles or gentamicin with the aim of killing
contaminating bacteria in order to favor surface coverage by mammalian cells. In mono-cultures, unloaded
nanotubules did not cause bacterial killing, but loading of Ag nanoparticles or gentamicin reduced the number of
adhering Staphylococcus aureus or Pseudomonas aeruginosa CFUs. A gentamicin-resistant Staphylococcus epi
dermidis was only killed upon loading with Ag nanoparticles. However, unlike low-level gentamicin loading,
loading with Ag nanoparticles also caused tissue-cell death. In bi-cultures, low-level gentamicin-loading of
nanotubular titanium surfaces effectively eradicated contaminating bacteria favoring surface coverage by
mammalian cells. Thus, care must be taken in loading nanotubular titanium surfaces with Ag nanoparticles,
while low-level gentamicin-loaded nanotubular titanium surfaces can be used as a local antibiotic delivery
system to negate failure of titanium implants due to peri-operatively introduced, contaminating bacteria without
hampering surface coverage by mammalian cells. high housing capacity for therapeutic agents, including antimicrobials
and can be easily fabricated via electrochemical anodization [7]. Contents lists available at ScienceDirect Contents lists available at ScienceDirect Available online 10 March 2021
0928-4931/© 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
* Corresponding author at: Department of Biomedical Engineering, University Medical Center Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, the
Netherlands.
E-mail address: h.c.van.der.mei@umcg.nl (H.C. van der Mei).
https://doi.org/10.1016/j.msec.2021.112021
Received 6 December 2020; Received in revised form 17 February 2021; Accepted 4 March 2021 * Corresponding author at: Department of Biomedical Engineering, University Medical Center Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, the
Netherlands.
E-mail address: h.c.van.der.mei@umcg.nl (H.C. van der Mei).
https://doi.org/10.1016/j.msec.2021.112021
Received 6 December 2020; Received in revised form 17 February 2021; Accepted 4 March 2021 * Corresponding author at: Department of Biomedical Engineering, University Medical Center Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, the
Netherlands.
E-mail address: h.c.van.der.mei@umcg.nl (H.C. van der Mei). University of Groningen
Antimicrobial loading of nanotubular titanium surfaces favoring surface coverage by
mammalian cells over bacterial colonization
Ren, Xiaoxiang; van der Mei, Henny C.; Ren, Yijin; Busscher, Henk J.; Peterson, Brandon W.
Published in:
Materials science & engineering c-Biomimetic and supramolecular systems
DOI:
10.1016/j.msec.2021.112021
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from
it. Please check the document version below.
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2021
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Ren, X., van der Mei, H. C., Ren, Y., Busscher, H. J., & Peterson, B. W. (2021). Antimicrobial loading of
nanotubular titanium surfaces favoring surface coverage by mammalian cells over bacterial colonization.
Materials science & engineering c-Biomimetic and supramolecular systems, 123, 1-9. Article 112021.
https://doi.org/10.1016/j.msec.2021.112021 University of Groningen
Antimicrobial loading of nanotubular titanium surfaces favoring surface coverage by
mammalian cells over bacterial colonization
Ren, Xiaoxiang; van der Mei, Henny C.; Ren, Yijin; Busscher, Henk J.; Peterson, Brandon W. Published in:
Materials science & engineering c-Biomimetic and supramolecular systems
DOI:
10.1016/j.msec.2021.112021
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from
it. Please check the document version below. Document Version
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Publication date:
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Citation for published version (APA):
Ren, X., van der Mei, H. C., Ren, Y., Busscher, H. J., & Peterson, B. W. (2021). Antimicrobial loading of
nanotubular titanium surfaces favoring surface coverage by mammalian cells over bacterial colonization. Materials science & engineering c-Biomimetic and supramolecular systems, 123, 1-9. Article 112021. https://doi.org/10.1016/j.msec.2021.112021 University of Groningen Published in:
Materials science & engineering c-Biomimetic and supramolecular systems DOI:
10.1016/j.msec.2021.112021 Link to publication in University of Groningen/UMCG research database Citation for published version (APA):
Ren, X., van der Mei, H. C., Ren, Y., Busscher, H. J., & Peterson, B. W. (2021). Antimicrobial loading of
nanotubular titanium surfaces favoring surface coverage by mammalian cells over bacterial colonization. Materials science & engineering c-Biomimetic and supramolecular systems, 123, 1-9. Article 112021. https://doi.org/10.1016/j.msec.2021.112021 Copyright
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Antimicrobial loading of nanotubular titanium surfaces favoring surface coverage by
mammalian cells over bacterial colonization
Ren, Xiaoxiang; van der Mei, Henny C.; Ren, Yijin; Busscher, Henk J.; Peterson, Brandon W.
Published in:
Materials science & engineering c-Biomimetic and supramolecular systems
DOI:
10.1016/j.msec.2021.112021
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from
it. Please check the document version below.
Document Version
Publisher's PDF, also known as Version of record
Publication date:
2021
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Ren, X., van der Mei, H. C., Ren, Y., Busscher, H. J., & Peterson, B. W. (2021). Antimicrobial loading of
nanotubular titanium surfaces favoring surface coverage by mammalian cells over bacterial colonization.
Materials science & engineering c-Biomimetic and supramolecular systems, 123, 1-9. Article 112021.
https://doi.org/10.1016/j.msec.2021.112021 Materials Science & Engineering C 123 (2021) 112021
Contents lists available at ScienceDirect
Materials Science & Engineering C
journal homepage: www.elsevier.com/locate/msec Materials Science & Engineering C 123 (2021) 112021 1. Introduction Other studies report a negative impact on viability of
human mesenchymal-stem cells [17] and osteoblast [20] over a Ag
nanoparticle loading range of 3.3–14.5 at.% [18] or around 9.7 wt%
[17]. [13–22] over a loading range between 1.6 and 15.1 at.% Ag by X-ray
photoelectron spectroscopy (XPS) [19,22] up to 14.9 wt% by Energy
Dispersive X-ray Spectroscopy (EDS) [17]. In order to better control the
loading process of nanotubes, nanotubes have also been loaded with Ag
nanoparticles incorporated in polydopamine (PDA) [23]. The joint effect
of cell wall damage by nanotube edges and dopamine-incorporated Ag
nanoparticles at a loading of 0.4–1.6 at.% Ag yielded killing of Gram-
positive Staphylococcus aureus and Gram-negative Escherichia coli [24]. Integration by tissue cells yields the best, natural protection of a bio
materials implant against colonizing bacteria [12,25]. This dictates that
antimicrobial loaded nanotubular titanium surfaces should be tissue
compatible, but reports on tissue compatibility of nanotubular titanium
surfaces differ. Nanotubular titanium surfaces loaded with 4.1–4.9 at.%
[21] or around 1.3 wt% [26] Ag nanoparticles stimulated proliferation
of osteoblasts. Other studies report a negative impact on viability of
human mesenchymal-stem cells [17] and osteoblast [20] over a Ag
nanoparticle loading range of 3.3–14.5 at.% [18] or around 9.7 wt%
[17]. nanotubular titanium surfaces have been demonstrated to kill adhering
bacteria [35,37–39], these high-level loadings may block the pathway of
gentamicin loaded nanotubular titanium surfaces towards clinical
translation by hampering tissue integration and due to the oto- and
nephrotoxicity of gentamicin. Ideally, release rates should be restricted
to the same rates as clinically occurring from e.g. gentamicin loaded
bone cements. Tissue integration and bacterial colonization are two simultaneous,
competing processes that have been pictured as a “race for the surface”
[25]. During surgical implantation, it is hard to avoid bacterial
contamination of an implant surface [40]. This makes peri-operative
contamination one of the pathways through which biomaterial-
associated infection can develop [41]. Particularly, dental implants
and percutaneous pins that have to be placed, heal and function in a
bacteria-laden environment, are prone to peri-operative bacterial
contamination and especially dental implants require effective surface
coverage by mammalian cells for long-term functioning. This makes it
imperative to study antimicrobial coatings in bi-culture models [42] in
which surface coverage by mammalian cells is studied on bacterially
contaminated surfaces (see Fig. 1), in addition to mono-culture studies
on either bacterial killing or surface coverage by mammalian cells. 1. Introduction i Nanotubular titanium surfaces loaded with Ag nanoparticles (no
loading level given) in combination with systemically administered
vancomycin, rifampin, gentamicin or levofloxacin, enhanced antibiotic
efficacy against an S. aureus infection in the medullary cavity under
neath the tibia plateau in rats [27]. Insertion of a titanium rod with Ag
nanoparticles filled nanotubules in combination with systemic applica
tion of antibiotics was demonstrated to yield synergistic efficacy against
the infection, as supported by histological and immunohistochemical
analyses, but not by culturing, i.e. the gold standard in clinical micro
biology [28]. Moreover, gentamicin e.g., was daily administered by tail
vein injection at a dose of 5 mg/kg every 24 h, which is at the limit of
safe veterinary application in rodents [29] and humans (3–5 mg/kg
every 24 h [30]). Although the study concludes that combination of Ag
loaded nanotubular titanium with systemically applied gentamicin may
be promising for the treatment of biomaterial-associated infection,
systemically applied gentamicin can have severe side-effects [31] due to
its oto- and nephrotoxicity [32]. Yet, gentamicin remains ideal for local
delivery of an antibiotic and is clinically applied in bone cements and
porous beads [31,33] for anchoring of titanium implants in bone for the
treatment of osteomyelitis, respectively. Accordingly, nanotubular tita
nium surfaces have been loaded with different antibiotics [34],
including gentamicin [35]. Commercial and clinically applied, genta
micin loaded bone cements have clinically safe, initial gentamicin
release rates of around 5 μg/(cm2 h) [36]. These clinically approved
release rates are much lower than gentamicin release rates from
experimental, gentamicin loaded nanotubular titanium surfaces,
ranging from 15 to 212 μg/(cm2 h) [35,37–39] to a potentially harmful
value of 1060 μg/(cm2 h) [39]. Although these high level loaded Therefore, we here first compare the effects of Ag nanoparticle or
gentamicin loading of nanotubular titanium surfaces with respect to the
killing of three frequently occurring clinical implant pathogens
(S. aureus, Staphylococcus epidermidis (gentamicin-resistant) and Pseu
domonas aeruginosa) and the adhesion, spreading and growth of human
gingival fibroblasts and osteoblasts in mono-cultures. Antimicrobial
loadings that passed mono-culture evaluations with a positive outcome,
were subsequently evaluated in bi-cultures, in which mammalian cell
adhesion, spreading and growth were evaluated in the presence of
contaminating implant pathogens adhering to the surface (peri-opera
tive infection model). 1. Introduction Nanostructured surfaces are called “promising” to control bacterial
adhesion and biofilm formation and can be distinguished based on pe
riodic- or random-occurrence of nanostructured features [1]. Nano
structured surfaces generally yield low bacterial adhesion and those
bacteria that adhere have multiple contact sites with a substratum sur
face. Accordingly, bacteria adhering to nanostructured surfaces suffer
highly localized cell wall deformation and sometimes even rupture
causing bacterial death upon adhesion to sharp contact points [2,3]. Other promising features of nanostructured surfaces include thermal
effects [4], photo-induced reactive oxygen species production [5] and
increased antimicrobial housing [6]. Nanotubular titanium surfaces
constitute an example of a highly periodic nanostructured surface with a Many biomaterials implants and devices, such as dental implants,
percutaneous pins and screws or orthopedic joint prostheses, are made
of titanium or its alloys and prone to failure due to colonization by
infecting bacteria [3,8]. An immune system frustrated by the presence of
the biomaterial [9], the biofilm mode of growth limiting deep pene
tration of an antimicrobial in the infectious biofilm [10] and an
increasing number of bacterial strains and species that are resistant to all
known antibiotics [11], have made infection the number one cause of
biomaterials implant failure [12]. Antimicrobial-loaded nanotubular
titanium surfaces might be ideally suitable to combat frequently
occurring titanium-associated infection. Highly periodic nanotubular
titanium surfaces have been loaded mainly with Ag nanoparticles (NPs) Materials Science & Engineering C 123 (2021) 112021 X. Ren et al. [13–22] over a loading range between 1.6 and 15.1 at.% Ag by X-ray
photoelectron spectroscopy (XPS) [19,22] up to 14.9 wt% by Energy
Dispersive X-ray Spectroscopy (EDS) [17]. In order to better control the
loading process of nanotubes, nanotubes have also been loaded with Ag
nanoparticles incorporated in polydopamine (PDA) [23]. The joint effect
of cell wall damage by nanotube edges and dopamine-incorporated Ag
nanoparticles at a loading of 0.4–1.6 at.% Ag yielded killing of Gram-
positive Staphylococcus aureus and Gram-negative Escherichia coli [24]. Integration by tissue cells yields the best, natural protection of a bio
materials implant against colonizing bacteria [12,25]. This dictates that
antimicrobial loaded nanotubular titanium surfaces should be tissue
compatible, but reports on tissue compatibility of nanotubular titanium
surfaces differ. Nanotubular titanium surfaces loaded with 4.1–4.9 at.%
[21] or around 1.3 wt% [26] Ag nanoparticles stimulated proliferation
of osteoblasts. 2.1. Preparation of nanotubular arrays on a titanium surface and Ag
nanoparticle loading Titanium discs (14 mm of diameter, 1 mm in thickness, 99.6% purity,
Alfa Aesar, Ward Hill, MA, USA) were degreased in methanol (99%),
acetone (99%) and absolute ethanol using an ultrasonic cleaner for 10
min and subsequently rinsed in demineralized water. In order to equip
the titanium discs with a nanotubular array, discs were anodized in an
ethylene glycol solution (>99.5%) containing NH4F (0.3 wt%) and H2O
(2 wt%) for 1 h at 30 V. Titanium foil was used as the anode and a
platinum electrode was used as the cathode. The two electrodes were Fig. 1. Schematic presentation of the in vitro mono- and bi-culture models used to evaluate the impact of antimicrobial-loaded, nanotubular titanium surfaces on
colonization by contaminating bacteria and surface coverage by mammalian cells. (A) Schematic presentation of the mono-culture bacterial (left) and mammalian
cell (right) models used. (B) Schematic presentation of a peri-operative bi-culture model, in which a bacterially contaminated implant surface (left) is subsequently
exposed to mammalian cells for adhesion and spreading on the surface (right). 2.3. Characterization of different titanium surfaces Field emission scanning electron microscopy (Lyra FIB-SEM dual-
beam, Tescan, Brno, Czechia) was used to observe the topography of the
nanotubular structure. Energy dispersive X-ray spectroscopy (EDS) was
done on the same microscope to analyze the chemical composition of the
different titanium samples. Water contact angles were measured using 1–1.5 μL water droplets. Water droplets were applied to the surfaces with a micro-syringe and
contact angles derived from the droplet contours after black-white
thresholding and analyzed using MATLAB software. To observe killing of bacteria on the different surfaces, discs with
adhering bacteria, were placed in a 3M™ Petrifilm® Rapid Aerobic
Count (RAC) Plate (3 M Microbiology, St. Paul, Minnesota, USA). Before
use, the nutrient loaded gel side of the Petrifilm® was hydrated with 1
mL of sterilized demineralized water for 60 min. Next 10 μL of a bac
terial suspension in PBS (5 × 106 bacteria/mL) was placed onto the disc
surface and the disc was placed in the Petrifilm® system to contact the
nutrient loaded gel. After incubation at 37 ◦C for 48 h, the numbers of
colony forming units (CFUs) were enumerated. Killing efficacy was
expressed with respect to the number of CFUs observed on uncoated,
smooth titanium discs. For X-ray photoelectron spectroscopy (XPS), a Surface Science in
strument (Mountain View, CA, USA), equipped with an aluminum anode
(10 kV, 22 mA) and a quartz monochromator was applied. The angle of
photoelectron collection was 55◦with the sample surfaces and the
electron flood gun was set at 14 eV. A wide-scan over a binding energy
range of 1–1200 eV was made with a 1000 × 250 μm spot and a pass
energy of 150 eV. Binding energies were determined by setting the
binding energy of the C1s peak (carbon bound to carbon) at 284.8 eV. XPS was carried out in duplicate on two independently prepared sam
ples of each titanium surface. 2.2. Loading of titanium samples with gentamicin Gentamicin (G) was introduced either onto the surface of Ti, Ti NT or
Ti NT-PDA discs. To this end, discs were immersed in 1 mL of a 200 μg/
mL gentamicin solution (Sigma-Aldrich, St. Louis, MO, USA) for 2 h
under gentle shaking. After 2 h, the samples were taken out carefully and
washed with demineralized water. These discs were labeled as Ti-G, Ti
NT-G and Ti NT-PDA-G. Antibacterial efficacy was measured using a membrane damage
assay, based on SYTO 9 and propidium iodide (Thermo Fisher Scientific,
Waltham, MA) staining and a culture-based assay. To allow initial
bacterial adhesion, 1 mL of the bacterial suspension in PBS (5 × 106/mL)
was put on the sample discs for bacterial sedimentation, after which the
suspension was replaced by TSB and adhering bacteria were grown for
24 h at 37 ◦C. After 24 h, the culture medium was removed, and the discs
were rinsed with PBS. 10 μL of a SYTO 9 solution and 10 μL of a pro
pidium iodide solution were mixed in 7 mL PBS to stain the biofilm
adhering to the discs. After staining for 15 min at room temperature in
the dark, the staining solution was removed and bacteria were imaged
using fluorescence microscopy (Leica DM4000, Leica Microsystems Ltd.,
Wetzlar, Germany) to visualize cell wall damaged bacteria. Fig. 1. Schematic presentation of the in vitro mono- and bi-culture models used to evaluate the impact of antimicrobial-loaded, nanotubular titanium surfaces on
colonization by contaminating bacteria and surface coverage by mammalian cells. (A) Schematic presentation of the mono-culture bacterial (left) and mammalian
cell (right) models used. (B) Schematic presentation of a peri-operative bi-culture model, in which a bacterially contaminated implant surface (left) is subsequently
exposed to mammalian cells for adhesion and spreading on the surface (right). Fig. 1. Schematic presentation of the in vitro mono- and bi-culture models used to evaluate the impact of antimicrobial-loaded, nanotubular titanium surfaces on
colonization by contaminating bacteria and surface coverage by mammalian cells. (A) Schematic presentation of the mono-culture bacterial (left) and mammalian
cell (right) models used. (B) Schematic presentation of a peri-operative bi-culture model, in which a bacterially contaminated implant surface (left) is subsequently
exposed to mammalian cells for adhesion and spreading on the surface (right). 2 Materials Science & Engineering C 123 (2021) 112021 X. Ren et al. gentamicin and the gentamicin concentrations determined as described
above. gentamicin and the gentamicin concentrations determined as described
above. separated at a distance of 4.5 cm. During the electrochemical process,
the solution was stirred at 450 rpm. After anodizing, nanotubular tita
nium (Ti NT) discs were rinsed in demineralized water to remove
possible residues of the electrolyte solution. 2.5. Antibacterial assays in mono-cultures p
y
To introduce a polydopamine layer onto the surface of the nano
tubular titanium discs, the discs were immersed in 2 mg/mL dopamine
hydrochloride (Sigma-Aldrich, St. Louis, MO) (10 mM Tris buffer, pH
8.5), followed by shaking for 18 h. These discs were labeled as Ti NT-
PDA. Subsequently, Ti NT-PDA discs were immersed in 1 mM AgNO3
solutions (Sigma-Aldrich, St. Louis, MO, USA) during 6 h to allow re
action of AgNO3 with PDA and form Ag nanoparticles in the nano
tubules. The discs obtained were labeled as Ti NT-PDA-Ag discs. Alternatively, nanotubular titanium discs were immersed in a 1 mM
AgNO3 solution for 6 h and then immersed in 2 mg/mL dopamine hy
drochloride, followed by shaking for 18 h. The obtained samples were
labeled as Ti NT-Ag-PDA. S. aureus ATCC 12600, P. aeruginosa ATCC 27853 and gentamicin-
resistant S. epidermidis ATCC 35984 were used in this study to assess
the antibacterial effect of the modified titanium samples. The minimum
inhibitory concentration (MIC) of these strains to gentamicin amounted
1 μg/mL, 2 μg/mL, and 32 μg/mL, respectively. The bacteria were
inoculated onto blood agar plates and incubated at 37 ◦C. After 24 h, one
colony was transferred in 10 mL of Tryptone Soya Broth (TSB, OXOID,
Basingstoke, UK) and incubated for 24 h at 37 ◦C. Subsequently, this
bacterial pre-culture was added to 200 mL growth medium and incu
bated for another 16 h at 37 ◦C. Then, bacteria were harvested by
centrifugation at 6500g for 5 min at 10 ◦C and washed twice with sterile
PBS and suspended in PBS. Finally, the staphylococcal suspension was
sonicated, while cooling in an ice bath (3 × 10 s at 30 W) in order to
break bacterial aggregates. Bacterial suspensions were enumerated in a
Bürker-Türk counting chamber and further diluted in PBS to a concen
tration of 5 × 106 bacteria/mL. i Table 1 Elemental composition of the different titanium samples, determined using EDS
and expressed both in weight and atom percentages. Elemental composition of the different titanium samples, determined using EDS
and expressed both in weight and atom percentages. Elemental composition of the different titanium samples, determined using EDS
and expressed both in weight and atom percentages. Sample
Weight percentage (atom percentage)
C
N
O
Ti
Ag
Ti
7.0 (23.1)
93.0 (76.9)
Ti NT
30.2 (56.5)
69.8 (43.5)
Ti NT-PDA
9.5 (18.9)
3.2 (5.4)
32.4 (48.3)
55.0 (27.4)
Ti NT-PDA-Ag
5.9 (12.6)
1.9 (3.4)
33.6 (53.7)
55.4 (29.6)
3.3 (0.8)
Ti NT-Ag-PDA
5.8 (12.6)
2.3 (4.4)
32.4 (52.5)
57.8 (31.6)
2.4 (0.6)
Ti-G
2.7 (7.4)
6.4 (14.8)
11.3 (23.0)
78.4 (53.3)
Ti NT-Ga
3.2 (7.2)
3.3 (6.3)
28.5 (47.4)
60.9 (33.9)
Ti NT-PDA-Ga
7.5 (15.1)
2.4 (4.1)
31.1 (46.6)
50.9 (25.5)
a Trace amounts of Si, F, P, Na and K were found (not presented). To mimic peri-operative contamination prior to surface coverage by
mammalian cells, 1 mL of a S. aureus, P. aeruginosa or S. epidermidis
suspension (5 × 104/mL) in PBS was added on the different discs in a 24
well-plate and bacteria allowed to sediment for 1 h. After 1 h, the discs
were transferred to a new well, washed three times with PBS yielding
approximately 1 × 103 bacteria/cm2 on the implant surface and again
transferred to a new well. Subsequently, 1 mL of a HGF (2 × 104/mL) or
U2OS (5 × 104/mL) in DMEM-HG with FBS was added and incubated at
37 ◦C in 5% CO2. After 24 h of growth, cells were stained and analyzed,
as described above. 2.8. Statistical analyses surface compositions for uncoated Ti and Ti NT surfaces. Ti and O
occurred in a ratio of between 1:3 and 1:4, indicative of the presence of
an oxide skin composed of different titanium oxides. Similar as in EDS,
less Ag (only 0.5 at.%) was found in Ti NT-Ag-PDA than in Ti NT-PDA-Ag
samples, possessing 2.7 at.% Ag (Table 2). Importantly, the high-
resolution XPS spectra of Ti NT-Ag-PDA and Ti NT-PDA-Ag samples
demonstrated an Ag binding energy peak with a gap of 6 eV in the 368.3
eV (Ag 3d5/2) and 374.3 eV (Ag 3d3/2) doublet (Fig. S2D), suggesting
that metallic silver Ag(0) was formed [22]. All data were plotted in GraphPad Prism and Origin. One-way
ANOVA with a Bonferroni post-hoc test was employed using GraphPad
Prism version 8.0 software. A value of p < 0.05 was deemed statistically
significant. 3.1. Characterization of nanotubular titanium surfaces with Ag
nanoparticle loading Uncoated titanium was relatively hydrophobic with a water contact
angle of 62◦(Fig. 2A). Application of nanotubular structures on the ti
tanium surface enhanced the spreading of water and reduced water
contact angles to between 20 and 22◦, regardless of a PDA, PDA-Ag or
Ag-PDA coating. Scanning electron microscopy revealed that the nano
tubules were evenly distributed over the entire surface. After immersion
in a AgNO3 solution, nanotubules were preserved and clearly observable
(Fig. 2B) possessing a diameter of 64 ± 10 nm. The diameters of the
nanotubes became smaller after coating with polydopamine (55 ± 8 nm)
and immersion in a AgNO3 solution (Ti NT-PDA-Ag and Ti NT-Ag-PDA
had diameters of 53 ± 8 nm and 52 ± 10 nm, respectively). EDS indi
cated that Ag was successfully applied (Table 1). Less Ag was measured
in Ti NT-Ag-PDA (2.4 wt%) than in Ti NT-PDA-Ag (3.3 wt%). The depth
of information of EDS is too high to detect the relatively thin oxide skin
on the uncoated Ti surface. More surface sensitive XPS data (see Fig. S2,
for wide-scan binding energy spectra) showed nearly identical elemental 3. Results 3.1. Characterization of nanotubular titanium surfaces with Ag
nanoparticle loading 2.4. Measurement of the amount of gentamicin loaded and released Human gingival fibroblasts (HGF) (HGF-1, ATCC-CRL-2014) and
U2OS osteosarcoma cells (ATCC HTB-94) were obtained from the
American Type Culture Collection (Manassas, USA) and grown in Dul
becco’s modified Eagle’s medium (DMEM-HG) supplemented with 10%
Fetal Bovine Serum (FBS, Invitrogen, Breda, The Netherlands) at 37 ◦C
in 5% CO2. Cells from passages 5–10 were used. HGFs (1 mL, 2 × 104/
mL) and U2OS cells (1 mL, 5 × 104/mL) were seeded on the different
disc surfaces in a 24 well-plate and incubated at 37 ◦C in 5% CO2. After
24 h of growth, the cells were stained with phalloidin-TRITC and DAPI
and analyzed using a fluorescent microscope (Leica DM4000). Surface
coverage and number of cells were subsequently derived from the im
ages using image J software. Gentamicin loading and release were measured using complexing of
gentamicin with o-phthaldialdehyde [43]. First o-phthaldialdehyde re
agent was prepared by mixing 2.5 g o-phthaldialdehyde (OPA), 62.5 mL
methanol and 3 mL 2-mercaptoethanol to 560 mL 0.04 M sodium borate
solution. The reagent was stored in a brown bottle in a dark chamber and
settled for at least 24 h before use. The amount of gentamicin loaded was
determined from the amount of residual gentamicin in solution. To this
end, 1 mL of the gentamicin solution left after loading of a sample was
mixed with 1 mL of OPA reagent and 1 mL isopropanol for 45 min. UV–Vis absorbance at 333 nm of the gentamicin-OPA complexes in the
different solutions was measured to determine amounts of gentamicin
loaded using a calibration curve over the concentration range between 1
and 200 μg/mL (Fig. S1). Alternatively, gentamicin loaded discs were immersed in 1 mL of
phosphate buffered saline (PBS, 10 mM potassium phosphate, 0.15 M
NaCl, pH 7) with gently shaking to allow 24 h and 48 h release of 3 Materials Science & Engineering C 123 (2021) 112021 X. Ren et al. 2.7. Surface coverage by mammalian cells upon bacterial challenges in bi-
cultures 3.4. Surface coverage of different coating surfaces by HGFs and U2OS
cells in mono-cultures After the Ti, Ti NT and Ti NT-PDA discs were prepared, gentamicin
was loaded on the samples by a simple immersion assay. Gentamicin
loading showed a reduction in water contact angles to 20◦both for Ti
NT-G and Ti NT-PDA-G surfaces. In XPS binding energy spectra, genta
micin loading was evident by an increased N1s signal (Table 2). A
smooth titanium surface hardly adsorbed any gentamicin (Fig. 3A)
compared with nanotubular titanium. Gentamicin loading of the Ti NT-
PDA sample was less than in absence of PDA but still higher than of
smooth titanium. In line, gentamicin release was highest from Ti NT-G
surfaces (Fig. 3B). The increase in cumulative release between 24 and
48 h was relatively small compared to release over the first 24 h, indi
cating near-complete release of the gentamicin in around two days. Assuming an initially linear release kinetics over at least 24 h, it can be
calculated that for our low-level gentamicin loaded nanotubular tita
nium surface, the release rate varied between 0.20 μg/(cm2 h) (Ti NT-G)
and 0.13 μg/(cm2 h) (Ti NT PDA-G). These release rates are at the lower
end of the gentamicin loading ranges observed in the literature for nano-
tubular titanium surfaces [35,37–39] and lower than gentamicin release
from clinically approved bone cements [36]. Mammalian cells spread and adhered well on titanium surfaces,
regardless of the presence of nanotubules with or without a PDA layer,
both with respect to surface coverage for HGF and U2OS cells as well as
with respect to cell numbers (Fig. 5). Despite its known oto- and neph
rotoxicity [44], our low-level gentamicin loading had no negative effects
on tissue spreading and adhesion, but loading with Ag nanoparticles
(0.5 at.% and 2.7 at.% Ag) was detrimental over the two concentrations
applied and caused a reduction in cell surface coverage to well below the
critical limit of 40%, required for tissue cells to win the race for the
surface from colonizing bacteria [45]. Therefore, Ag nanoparticle
loading was not further pursued in bi-culture studies. 3.5. Surface coverage by mammalian cells on bacterially contaminated
titanium surfaces in bi-cultures In a peri-operative contamination model, the presence of S. aureus
(Figs. S5A and S6A) or P. aeruginosa (Figs. S5B and S6B) adhering on
titanium surfaces in absence of a gentamicin loading, led to a strongly
reduced surface coverage and number of adhering HGFs (Fig. 6A and B
for S. aureus and P. aeruginosa, respectively) and U2OS cells (Fig. 7A and
B for S. aureus and P. aeruginosa, respectively) as compared with tissue
cell spreading and adhesion on titanium surfaces without contaminating
bacteria. Attesting to the relatively low virulence of S. epidermidis [46]
and despite the gentamicin-resistance of the strain used, the presence of
contaminating S. epidermidis (Figs. S5C and S6C) yielded no reductions
in cell surface coverage or numbers (Figs. 6C and 7C for HGF and U2OS
cells, respectively). Gentamicin loading of smooth titanium surfaces did
not enhance spreading and adhesion of tissue cells on surfaces
contaminated with S. aureus or P. aeruginosa. However, due to the
increased gentamicin housing capacity of nanotubular titanium sur
faces, tissue cell spreading and adhesion in presence of contaminating
S. aureus or P. aeruginosa were enhanced to the same level as observed on
uncontaminated titanium surfaces. Due to the low virulence of
S. epidermidis, further improvement in cell surface coverage or numbers
upon gentamicin loading was impossible. Importantly however in this
case, gentamicin loading had no negative effects on surface coverage by
mammalian cells. 3.3. Antibacterial activity assays in mono-cultures Fluorescence images of S. aureus, P. aeruginosa and S. epidermidis
biofilms after 24 h of growth showed the adhesion of bacteria on
different titanium samples (Fig. S3). Nanotubules did not cause
demonstrable cell wall damage, but the incorporation of Ag nano
particles and gentamicin clearly prevented growth of adhering S. aureus
and P. aeruginosa and virtually no bacteria could be observed. For the
gentamicin-resistant S. epidermidis only Ag nanoparticles could prevent
the growth. Fluorescence images furthermore show that gentamicin
release from smooth titanium surface is insufficient to cause demon
strable cell wall damage. When bacteria were applied to the surfaces in low numbers, typical
of peri-operative contamination, 48 h growth within the Petrifilm®
plates yielded between 104 and 105 CFU/cm2, neither application of
nanotubules with or without PDA, nor gentamicin adsorption to smooth
titanium surfaces caused a decrease in the viability of contaminating
bacteria, regardless of the strain considered (Fig. 4). However, when Ti
NT was loaded with gentamicin or Ag nanoparticles, bacterial killing
was more than 3 log units for both S. aureus and P. aeruginosa. For the
gentamicin-resistant S. epidermidis only loading with Ag nanoparticles
showed killing of more than 4 log units. Ag nanoparticles covered with
PDA (0.5 at.% Ag) were less effective in reducing the number of CFUs
than when applied on top of an existing layer of PDA (2.7 at.% Ag). 3.2. Gentamicin loading and release from different titanium surfaces 3.2. Gentamicin loading and release from different titanium surfaces 3.4. Surface coverage of different coating surfaces by HGFs and U2OS
cells in mono-cultures Table 2 Elemental composition of the different titanium surfaces, determined using XPS
(see Fig. S2 for wide-scan binding energy spectra and high-resolution spectra of
silver). Data represent averages with ± signs denoting SD over duplicate mea
surements with separately prepared samples. Surface
Atom percentagea
C
N
O
Ti
Ag
Ti
26.7 ± 0.1
1.5 ± 0.2
51.7 ± 0.2
14.4 ± 0.0
Ti NT
20.8 ± 2.2
0.5 ± 0.0
47.6 ± 4.5
16.7 ± 1.7
Ti NT-PDA
52.7 ± 0.6
6.2 ± 0.1
30.5 ± 1.0
6.0 ± 0.8
Ti NT-PDA-Ag
50.9 ± 1.3
5.7 ± 0.7
30.6 ± 0.9
6.1 ± 0.4
2.7 ± 0.4
Ti NT-Ag-PDA
55.4 ± 1.7
6.1 ± 0.1
29.8 ± 1.8
5.2 ± 0.5
0.5 ± 0.0
Ti-G
26.2 ± 2.4
2.9 ± 0.6
49.8 ± 6.7
14.4 ± 0.2
Ti NT-G
24.4 ± 0.1
4.0 ± 0.7
46.4 ± 0.2
13.6 ± 0.6
Ti NT-PDA-G
53.0 ± 1.4
6.1 ± 0.1
30.7 ± 0.2
6.4 ± 0.3
a Trace amounts of Si, F, P, Ca were found (not presented). Fig. 2. Physicochemical characterization of different, AgNP loaded nanotubular titanium surfaces. (A) Schematics of the different nanotubular Ti surfaces together
with their water contact angles (WCA). (B) SEM micrographs of the different titanium surfaces with and without nanotubules. The bright spots in the SEM of Ti NT-
PDA-Ag represent AgNPs. Fig. 2. Physicochemical characterization of different, AgNP loaded nanotubular titanium surfaces. (A) Schematics of the different nanotubular Ti surfaces together
with their water contact angles (WCA). (B) SEM micrographs of the different titanium surfaces with and without nanotubules. The bright spots in the SEM of Ti NT-
PDA-Ag represent AgNPs. X. Ren et al. Materials Science & Engineering C 123 (2021) 112021 3.2. Gentamicin loading and release from different titanium surfaces 4. Discussion Titanium and its alloys are widely used as implant materials. How
ever, around 10% failures of titanium implants result from bacterial
infection within a year after implantation [47], presumably for a large Fig. 3. Gentamicin loading and release from different nanotubular titanium surfaces. (A) The amount of gentamicin loaded on smooth titanium surfaces (Ti) and in nanotubular titanium surfaces in absence (Ti NT) and presence of a PDA overlayer (Ti
NT-PDA) after 2 h immersion in 1 mL (200 μg/mL) gentamicin solution. (B) The cumulative amount of gentamicin released from the different titanium surfaces after
24 h and 48 h immersion in 1 mL PBS solution. Error bars denote SEM over three experiments. * denotes a significant difference from smooth titanium samples (p
< 0.05). Fig. 3. Gentamicin loading and release from different nanotubular titanium surfaces. (A) The amount of gentamicin loaded on smooth titanium surfaces (Ti) and in nanotubular titanium surfaces in absence (Ti NT) and presence of a PDA overlayer (Ti
NT-PDA) after 2 h immersion in 1 mL (200 μg/mL) gentamicin solution. (B) The cumulative amount of gentamicin released from the different titanium surfaces after
24 h and 48 h immersion in 1 mL PBS solution. Error bars denote SEM over three experiments. * denotes a significant difference from smooth titanium samples (p
< 0.05). g
g
(A) The amount of gentamicin loaded on smooth titanium surfaces (Ti) and in nanotubular titanium surfaces in absence (Ti NT) and presence of a PDA overlayer (Ti
NT-PDA) after 2 h immersion in 1 mL (200 μg/mL) gentamicin solution. (B) The cumulative amount of gentamicin released from the different titanium surfaces after
24 h and 48 h immersion in 1 mL PBS solution. Error bars denote SEM over three experiments. * denotes a significant difference from smooth titanium samples (p
< 0.05). 5 Materials Science & Engineering C 123 (2021) 112021 X. Ren et al. Materials Science & Engineering C 123 (2021) 112021
Fig. 4. Killing of contaminating bacteria growing on smooth and different antimicrobial-loaded, nanotubular titanium surfaces. (A) Numbers of S. aureus CFUs
retrieved per unit surface area from the different titanium surfaces. (B) Same as panel (A), now for P. aeruginosa. (C) Same as panel (A), now for gentamicin-resistant
S. epidermidis. Error bars denote SEM over three experiments with separately cultured bacteria. * denotes a significant difference from smooth titanium samples
without antimicrobial loading (p < 0.05). X. 4. Discussion Ren et al. Fig. 4. Killing of contaminating bacteria growing on smooth and different antimicrobial-loaded, nanotubular titanium surfaces. (A) Numbers of S. aureus CFUs
retrieved per unit surface area from the different titanium surfaces. (B) Same as panel (A), now for P. aeruginosa. (C) Same as panel (A), now for gentamicin-resistant
S. epidermidis. Error bars denote SEM over three experiments with separately cultured bacteria. * denotes a significant difference from smooth titanium samples
without antimicrobial loading (p < 0.05). Fig. 5. Interaction of mammalian cells after 24 h of
growth on smooth and different antimicrobial-
loaded, nanotubular titanium surfaces (data repre
sent a quantification of the fluorescence images pre
sented in Fig. S4). (A) Surface coverage by adhering
HGFs after growth on the different titanium surfaces. (B) Number of adhering HGFs (per cm2) after growth
on the different titanium surfaces. (C) Same as panel
(A), now for U2OS cells. (D) Same as panel (B), now
for U2OS cells. Error bars denote SEM over three
experiments with separately cultured cells. * denotes
a significant difference from smooth titanium sur
faces without antimicrobial loading (p < 0.05). Fi
gr
loa
sen
sen
HG
(B
on
(A
for
ex
a
fac Fig. 5. Interaction of mammalian cells after 24 h of
growth on smooth and different antimicrobial-
loaded, nanotubular titanium surfaces (data repre
sent a quantification of the fluorescence images pre
sented in Fig. S4). (A) Surface coverage by adhering
HGFs after growth on the different titanium surfaces. (B) Number of adhering HGFs (per cm2) after growth
on the different titanium surfaces. (C) Same as panel
(A), now for U2OS cells. (D) Same as panel (B), now
for U2OS cells. Error bars denote SEM over three
experiments with separately cultured cells. * denotes
a significant difference from smooth titanium sur
faces without antimicrobial loading (p < 0.05). Fig. 5. Interaction of mammalian cells after 24 h of
growth on smooth and different antimicrobial-
loaded, nanotubular titanium surfaces (data repre
sent a quantification of the fluorescence images pre
sented in Fig. S4). (A) Surface coverage by adhering
HGFs after growth on the different titanium surfaces. (B) Number of adhering HGFs (per cm2) after growth
on the different titanium surfaces. (C) Same as panel
(A), now for U2OS cells. (D) Same as panel (B), now
for U2OS cells. Error bars denote SEM over three
experiments with separately cultured cells. 4. Discussion * denotes
a significant difference from smooth titanium sur
faces without antimicrobial loading (p < 0.05). Fig. 5. Interaction of mammalian cells after 24 h of
growth on smooth and different antimicrobial-
loaded, nanotubular titanium surfaces (data repre
sent a quantification of the fluorescence images pre
sented in Fig. S4). (A) Surface coverage by adhering
HGFs after growth on the different titanium surfaces. (B) Number of adhering HGFs (per cm2) after growth
on the different titanium surfaces. (C) Same as panel
(A), now for U2OS cells. (D) Same as panel (B), now
for U2OS cells. Error bars denote SEM over three
experiments with separately cultured cells. * denotes
a significant difference from smooth titanium sur
faces without antimicrobial loading (p < 0.05). part due to peri-operative bacterial contamination. Ag nanoparticle or
gentamicin loaded nanotubular titanium surfaces have been described
to discourage infectious biofilm formation of titanium surfaces, but
often without consideration of clinical constraints or collateral damage
to tissue cells surrounding an implant. Literature studies report effective
killing of bacteria on nanotubular surfaces at high-levels (up to 14.9 wt
%) of Ag nanoparticle loading [18,20,22], while we here demonstrate
(Tables 1 and 2) that loading (up to 3.3 wt%) suffices for killing of
bacteria adhering to titanium surfaces in numbers typical of peri-
operative contamination [48]. Previously, Ag nanoparticle loading of
nanotubular titanium surfaces has also been described to kill pathogenic
S. aureus and E. coli [24]. Unfortunately, we found severe reductions in
surface coverage by HGF and U2OS cells and numbers even upon Ag nanoparticle loading of nanotubular titanium surfaces (Fig. 5). Litera
ture has been conflicting with regards to effects of Ag nanoparticle
loading of nanotubular titanium surfaces [18,20,22,49,50]. Based on
our results of Ag nanoparticle loading of nanotubular titanium surfaces,
we decided that the combined effects of nanotubular titanium and
loading of nanotubules with Ag nanoparticles is too damaging towards
mammalian cells covering the surface to make it suitable as a coating for
biomaterials implants. Literature also described, that high-level loading of nanotubular ti
tanium surfaces with gentamicin yielded killing of several pathogenic
bacterial strains, but gentamicin release rates were extremely high
around 133–1060 μg/(cm2 h) [38,39]. Mostly, these gentamicin con
centrations have not been reported to present a negative impact on 6 Materials Science & Engineering C 123 (2021) 112021 X. Ren et al. Materials Science & Engineering C 123 (2021) 112021
Fig. 6. 4. Discussion Therefore,
our work is uniquely based on low-level gentamicin loading, in which
“low” is defined as yielding smaller or equal gentamicin release rates as
from clinically-applied gentamicin loaded bone cements [36] used to fix
titanium implants in bone. Approval of gentamicin loaded bone cement
has been quite a struggle in the past between clinicians wanting to
protect their patients against bacterial infections and regulatory bodies
disapproving clinical use of gentamicin-loaded cements because of oto-
and nephrotoxicity [52]. Particularly in the USA, the FDA has been
lingering longest with the approval of gentamicin loaded bone cement. Based on recently published, so-called “enrichment principles” proposed
by a Trans-Atlantic consortium [53], an already-approved combination
of implants or devices with an antimicrobial coating, replacement of that
coating with another coating or application to an implant or device with
different designs or geometries should not require the same type and
extent of regulatory assessment of risk/benefit as for the already
approved device. By comparison with bone cement, this principle, called “enrichment-from-component”, would be applicable to our low-level
gentamicin loaded, nanotubular titanium surfaces and make its pros
pects for downward clinical translation more likely than of high-level
gentamicin loaded variants. Low-level gentamicin-loaded nanotubular titanium surfaces would
be particularly useful as a prophylactic coating to prevent negative ef
fects of peri-operatively introduced bacteria on a biomaterials implant
surface because in presence of gentamicin loading the balance between
surface coverage by mammalian cells and bacterial colonization is
clearly shifted in favor of cell surface coverage. 4. Discussion S5). (A) Percentage
surface coverage of a substratum surface by adhering
HGF and the number of adhering HGF (per cm2) in
absence (dotted line) and presence of S. aureus
contaminated surfaces. (B) Same as panel (A), now
for P. aeruginosa contaminated surfaces. (C) Same as
panel (A), now for surfaces contaminated by a
gentamicin-resistant S. epidermidis strain. The dotted
lines indicate the percentage surface coverage and
number of cells per cm2 of a smooth titanium surface
in absence of bacterial contamination. Error bars
denote SEM over three experiments with separately
cultured bacteria and tissue-cells. * denotes a signif
icant difference from smooth titanium surface, while
# indicates a significant difference from correspond
ing titanium surfaces without antimicrobial loading
(p < 0.05). mammalian cell behavior [35,37,39], although few papers did report a
negative impact of high-level gentamicin loading of nanotubular tita
nium surfaces on osteoblast cells [51]. In general unfortunately, the in
vitro evaluations applied [35,37–39] and their results, negate the clin
ical observations of oto- and nephrotoxicity of gentamicin. Therefore,
our work is uniquely based on low-level gentamicin loading, in which
“low” is defined as yielding smaller or equal gentamicin release rates as
from clinically-applied gentamicin loaded bone cements [36] used to fix
titanium implants in bone. Approval of gentamicin loaded bone cement
has been quite a struggle in the past between clinicians wanting to
protect their patients against bacterial infections and regulatory bodies
disapproving clinical use of gentamicin-loaded cements because of oto-
and nephrotoxicity [52]. Particularly in the USA, the FDA has been
lingering longest with the approval of gentamicin loaded bone cement. Based on recently published, so-called “enrichment principles” proposed
by a Trans-Atlantic consortium [53], an already-approved combination
of implants or devices with an antimicrobial coating, replacement of that
coating with another coating or application to an implant or device with
different designs or geometries should not require the same type and
extent of regulatory assessment of risk/benefit as for the already
approved device. By comparison with bone cement, this principle, called mammalian cell behavior [35,37,39], although few papers did report a
negative impact of high-level gentamicin loading of nanotubular tita
nium surfaces on osteoblast cells [51]. In general unfortunately, the in
vitro evaluations applied [35,37–39] and their results, negate the clin
ical observations of oto- and nephrotoxicity of gentamicin. 4. Discussion The race for the surface: growth of HGF cells
on different, bacterially contaminated nanotubular
titanium surfaces with and without gentamicin-
loading
(peri-operative
contamination
model). Contaminating bacteria were sedimented from sus
pension, yielding approximately 1 × 103 bacteria/
cm2 (data represent a quantification of the fluores
cence images presented in Fig. S5). (A) Percentage
surface coverage of a substratum surface by adhering
HGF and the number of adhering HGF (per cm2) in
absence (dotted line) and presence of S. aureus
contaminated surfaces. (B) Same as panel (A), now
for P. aeruginosa contaminated surfaces. (C) Same as
panel (A), now for surfaces contaminated by a
gentamicin-resistant S. epidermidis strain. The dotted
lines indicate the percentage surface coverage and
number of cells per cm2 of a smooth titanium surface
in absence of bacterial contamination. Error bars
denote SEM over three experiments with separately
cultured bacteria and tissue-cells. * denotes a signif
icant difference from smooth titanium surface, while
# indicates a significant difference from correspond
ing titanium surfaces without antimicrobial loading
(p < 0.05). X. Ren et al. Fig. 6. The race for the surface: growth of HGF cells
on different, bacterially contaminated nanotubular
titanium surfaces with and without gentamicin-
loading
(peri-operative
contamination
model). Contaminating bacteria were sedimented from sus
pension, yielding approximately 1 × 103 bacteria/
cm2 (data represent a quantification of the fluores
cence images presented in Fig. S5). (A) Percentage
surface coverage of a substratum surface by adhering
HGF and the number of adhering HGF (per cm2) in
absence (dotted line) and presence of S. aureus
contaminated surfaces. (B) Same as panel (A), now
for P. aeruginosa contaminated surfaces. (C) Same as
panel (A), now for surfaces contaminated by a
gentamicin-resistant S. epidermidis strain. The dotted
lines indicate the percentage surface coverage and
number of cells per cm2 of a smooth titanium surface
in absence of bacterial contamination. Error bars
denote SEM over three experiments with separately
cultured bacteria and tissue-cells. * denotes a signif
icant difference from smooth titanium surface, while
# indicates a significant difference from correspond
ing titanium surfaces without antimicrobial loading
(p < 0.05). Fig. 6. The race for the surface: growth of HGF cells
on different, bacterially contaminated nanotubular
titanium surfaces with and without gentamicin-
loading
(peri-operative
contamination
model). Contaminating bacteria were sedimented from sus
pension, yielding approximately 1 × 103 bacteria/
cm2 (data represent a quantification of the fluores
cence images presented in Fig. CRediT authorship contribution statement Conception and design of study: all authors
Acquisition of data: Xiaoxiang Ren
Analysis and interpretation of data: Xiaoxiang Ren, Henny C. van
der Mei, Henk J. Busscher, Brandon W. Peterson
Drafting the manuscript: Xiaoxiang Ren, Brandon Peterson
Revising the manuscript: Henny C. van der Mei, Yijin Ren, Henk J. Busscher
Supervision: Brandon W. Peterson, Henny C. van der Mei, Henk J. Busscher
Approval final version of the manuscript: all authors. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.msec.2021.112021. Acknowledgements XR likes to thank the China Scholarship Council and W.J. Kolff
Institute, UMCG, Groningen, The Netherlands for financial support. Authors were employed by their own organizations. [2] E.P. Ivanova, J. Hasan, H.K. Webb, V.K. Truong, G.S. Watson, J.A. Watson, V.
A. Baulin, S. Pogodin, J.Y. Wang, M.J. Tobin, C. L¨obbe, R.J. Crawford, Natural [1] Y. Luan, S. Liu, M. Pihl, H.C. van der Mei, J. Liu, F. Hizal, C.H. Choi, H. Chen,
Y. Ren, H.J. Busscher, Bacterial interactions with nanostructured surfaces, Curr,
Opin. Colloid Interface Sci. 38 (2018) 170–189. 5. Conclusions Low-level loading of nanotubular titanium surfaces with gentamicin
favors surface coverage by mammalian cells over colonization by peri-
operatively introduced bacteria. Loading of nanotubular titanium with
Ag nanoparticles should be avoided because of adverse effects on
mammalian cells due to the combination of Ag nanoparticles and a
nanostructured surface that together yield a negative impact of surface
coverage by mammalian cells. This combination remains useful however
to kill contaminating bacteria in applications of titanium in which 7 Materials Science & Engineering C 123 (2021) 112021
Fig. 7. The race for the surface: growth of U2OS cells
on different, bacterially contaminated nanotubular
titanium surfaces with and without gentamicin-
loading
(peri-operative
contamination
model). Contaminating bacteria were sedimented from sus
pension, yielding approximately 1 × 103 bacteria
cm−2 (data represent a quantification of the fluores
cence images presented in Fig. S6). (A) Percentage
surface coverage of a substratum surface by adhering
U2OS cells and the number of adhering U2OS cells
(per cm2) in absence (dotted line) and presence of
S. aureus contaminated surfaces. (B) Same as panel
(A), now for P. aeruginosa contaminated surfaces. (C)
Same as panel (A), now for surfaces contaminated by
gentamicin-resistant S. epidermidis. The dotted lines
indicate the percentage surface coverage and number
of cells per cm2 of a smooth titanium surface in
absence of bacterial contamination. Error bars denote
SEM over three experiments with separately cultured
bacteria and tissue-cells. * denotes a significant dif
ference from smooth titanium surface, while # in
dicates a significant difference from corresponding
titanium surfaces without antimicrobial loading (p <
0.05). X. Ren et al. Materials Science & Engineering C 123 (2021) 112021 X. Ren et al. surface coverage by mammalian cells is not required. CRediT authorship contribution statement
and/or publication of this a
herein are those of the auth
representing views of the fun
Fig. 7. on dif
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dicates
titaniu
0.05). X. Ren et al. Fig. 7. The race for the surface: growth of U2OS cells
on different, bacterially contaminated nanotubular
titanium surfaces with and without gentamicin-
loading
(peri-operative
contamination
model). Contaminating bacteria were sedimented from sus
pension, yielding approximately 1 × 103 bacteria
cm−2 (data represent a quantification of the fluores
cence images presented in Fig. S6). [1] Y. Luan, S. Liu, M. Pihl, H.C. van der Mei, J. Liu, F. Hizal, C.H. Choi, H. Chen,
Y. Ren, H.J. Busscher, Bacterial interactions with nanostructured surfaces, Curr,
Opin. Colloid Interface Sci. 38 (2018) 170–189.
[2] E.P. Ivanova, J. Hasan, H.K. Webb, V.K. Truong, G.S. Watson, J.A. Watson, V.
A. Baulin, S. Pogodin, J.Y. Wang, M.J. Tobin, C. L¨obbe, R.J. Crawford, Natural 5. Conclusions (A) Percentage
surface coverage of a substratum surface by adhering
U2OS cells and the number of adhering U2OS cells
(per cm2) in absence (dotted line) and presence of
S. aureus contaminated surfaces. (B) Same as panel
(A), now for P. aeruginosa contaminated surfaces. (C)
Same as panel (A), now for surfaces contaminated by
gentamicin-resistant S. epidermidis. The dotted lines
indicate the percentage surface coverage and number
of cells per cm2 of a smooth titanium surface in
absence of bacterial contamination. Error bars denote
SEM over three experiments with separately cultured
bacteria and tissue-cells. * denotes a significant dif
ference from smooth titanium surface, while # in
dicates a significant difference from corresponding
titanium surfaces without antimicrobial loading (p <
0.05). Fig. 7. The race for the surface: growth of U2OS cells
on different, bacterially contaminated nanotubular
titanium surfaces with and without gentamicin-
loading
(peri-operative
contamination
model). Contaminating bacteria were sedimented from sus
pension, yielding approximately 1 × 103 bacteria
cm−2 (data represent a quantification of the fluores
cence images presented in Fig. S6). (A) Percentage
surface coverage of a substratum surface by adhering
U2OS cells and the number of adhering U2OS cells
(per cm2) in absence (dotted line) and presence of
S. aureus contaminated surfaces. (B) Same as panel
(A), now for P. aeruginosa contaminated surfaces. (C)
Same as panel (A), now for surfaces contaminated by
gentamicin-resistant S. epidermidis. The dotted lines
indicate the percentage surface coverage and number
of cells per cm2 of a smooth titanium surface in
absence of bacterial contamination. Error bars denote
SEM over three experiments with separately cultured
bacteria and tissue-cells. * denotes a significant dif
ference from smooth titanium surface, while # in
dicates a significant difference from corresponding
titanium surfaces without antimicrobial loading (p <
0.05). surface coverage by mammalian cells is not required. and/or publication of this article. Opinions and assertions contained
herein are those of the authors and are not construed as necessarily
representing views of the funding organizations or their employer(s). References The authors declare the following financial interests/personal re
lationships which may be considered as potential competing interests:
HJB is also director-owner of a consulting company SASA BV. The au
thors declare no potential conflicts of interest with respect to authorship 8 Materials Science & Engineering C 123 (2021) 112021 X. Ren et al. bactericidal surfaces: mechanical rupture of Pseudomonas aeruginosa cells by cicada
wings, Small 8 (2012) 2489–2494. bactericidal surfaces: mechanical rupture of Pseudomonas aeruginosa cells by cicada
wings, Small 8 (2012) 2489–2494. [28] G.E. Pfyffer, H.M. Welscher, P. Kissling, C. Cieslak, M.J. Casal, J. Gutierrez,
S. Rüsch-Gerdes, Comparison of the mycobacteria growth indicator tube (MGIT)
with radiometric and solid culture for recovery of acid-fast bacilli, J. Clin. Microbiol. 35 (1997) 364–368. [3] Y. Cao, B. Su, S. Chinnaraj, S. Jana, L. Bowen, S. Charlton, P. Duan, N. S. Jakubovics, J. Chen, Nanostructured titanium surfaces exhibit recalcitrance
towards Staphylococcus epidermidis biofilm formation, Sci, Rep. 8 (2018) 1071. i [29] C. Chareul, J. Faccini, A. Monro, J. Nachbaur, A second peak in uptake of
gentamicin by rat kidney after cessation of treatment, Biopharm. Drug Dispos. 5
(1984) 21–24. i
[4] M. Pihl, E. Bruzell, M. Andersson, Bacterial biofilm elimination using gold nanorod
localised surface plasmon resonance generated heat, Mater. Sci. Eng. C 80 (2017)
54–58. [30] G. Kahlmeter, J.I. Dahlager, Aminoglycoside toxicity - a review of clinical studies
published between 1975 and 1982, J. Antimicrob. Chemother. 13 (1984) 9–22. [5] D. Campoccia, L. Montanaro, C.R. Arciola, A review of the biomaterials
technologies for infection-resistant surfaces, Biomaterials 34 (2013) 8533–8554. [31] D. Neut, H. van de Belt, J.R. Van Horn, H.C. van der Mei, H.J. Busscher, The effect
of mixing on gentamicin release from polymethylmethacrylate bone cements, Acta
Orthop, Scand 74 (2003) 670–676. [6] F. Hizal, I. Zhuk, S. Sukhishvili, H.J. Busscher, H.C. van der Mei, C.H. Choi, Impact
of 3D hierarchical nanostructures on the antibacterial efficacy of a bacteria-
triggered self-defensive antibiotic coating, ACS Appl. Mater. Interfaces 7 (2015)
20304–20313. [32] A. Lakhera, A. Ganeshpurkar, D. Bansal, N. Dubey, Chemopreventive role of
Coriandrum sativum against gentamicin-induced renal histopathological damage in
rats, Interdiscip, Toxicol. 8 (2015) 99–102. ¨ [7] S. Mei, H. Wang, W. Wang, L. Tong, H. Pan, C. Ruan, Q. Ma, M. Liu, H. Yang,
L. Zhang, Y. Cheng, Y. Zhang, L. Zhao, P.K. References Chu, Antibacterial effects and
biocompatibility of titanium surfaces with graded Ag incorporation in titania
nanotubes, Biomaterials 35 (2014) 4255–4265. [33] N. Müller, P. Schmidlin, M. ¨Ozcan, Adhesive durability of bone cements containing
gentamicin or gentamicin/clindamycin-based antibiotics on titanium used for oral
implants, J. Adhes. Sci. Technol. 30 (2016) 2130–2145. [8] C. Yue, B. Zhao, R. Kuijer, H.C. van der Mei, H.J. Busscher, E.T.J. Rochford, The
implant infection paradox: why do some succeed when others fail? Eur. Cells
Mater. 29 (2015) 303–313. p
[34] H. Zhang, Y. Sun, A. Tian, X. Xue, L. Wang, A. Alquhali, X. Bai, Improved
antibacterial activity and biocompatibility on vancomycin-loaded TiO2 nanotubes:
in vivo and in vitro studies, Int. J. Nanomedicine 8 (2013) 4379–4389. [9] B.N. Brown, S.F. Badylak, Expanded applications, shifting paradigms and an
improved understanding of host-biomaterial interactions, Acta Biomater. 9 (2013)
4948–4955. i [35] T. Kumeria, H. Mon, M.S. Aw, K. Gulati, A. Santos, H.J. Griesser, D. Losic,
Advanced biopolymer-coated drug-releasing titania nanotubes (TNTs) implants
with simultaneously enhanced osteoblast adhesion and antibacterial properties,
Colloids Surfaces B Biointerfaces 130 (2015) 255–263. [10] D. Davies, Understanding biofilm resistance to antibacterial agents, Nat. Rev. Drug
Discov. 2 (2003) 114–122. [36] D. Neut, R.J.B. Dijkstra, J.I. Thompson, H.C. van der Mei, H.J. Busscher,
Antibacterial efficacy of a new gentamicin-coating for cementless prostheses
compared to gentamicin-loaded bone cement, J. Orthop. Res. 29 (2011)
1654–1661. [11] S.B. Levy, M. Bonnie, Antibacterial resistance worldwide: causes, challenges and
responses, Nat. Med. 10 (2004) S122–S129. [12] H.J. Busscher, H.C. van der Mei, G. Subbiahdoss, P.C. Jutte, J.J.A.M. van den
Dungen, S.A.J. Zaat, M.J. Schultz, D.W. Grainger, Biomaterial-associated infection:
locating the finish line in the race for the surface, Sci. Transl. Med. 4 (2012)
153rv10. [37] W.T. Lin, H.L. Tan, Z.L. Duan, B. Yue, R. Ma, G. He, T.T. Tang, Inhibited bacterial
biofilm formation and improved osteogenic activity on gentamicin-loaded titania
nanotubes with various diameters, Int. Nanomedicin. 9 (20144) 1215–1230. [13] L. Juan, Z. Zhimin, M. Anchun, L. Lei, Z. Jingchao, Deposition of Ag NP on titanium
surface for antibacterial effect, Int. J. Nanomedicine 5 (2010) 261–267. [38] W. Feng, Z. Geng, Z. Li, Z. Cui, S. Zhu, Y. Liang, Y. Liu, R. Wang, X. Yang,
Controlled release behaviour and antibacterial effects of antibiotic-loaded titania
nanotubes, Mater. Sci. Eng. C 62 (2016) 105–112. [14] K. Das, S. Bose, A. Bandyopadhyay, B. Karandikar, B.L. References van der Mei, Mammalian cell growth
versus biofilm formation on biomaterial surfaces in an in vitro post-operative
contamination model, Microbiology 156 (2010) 3073–3078. h l
d
d
h
d
l
h [19] L. Zhao, H. Wang, K. Huo, L. Cui, W. Zhang, H. Ni, Y. Zhang, Z. Wu, P.K. Chu,
Antibacterial nano-structured titania coating incorporated with Ag NP,
Biomaterials 32 (2011) 5706–5716. [46] M. Otto, Staphylococcus epidermidis — the ‘accidental’ pathogen, Nat. Rev. Microbiol. 7 (2009) 555–567. [20] J. Chen, M.L. Mei, Q.L. Li, C.H. Chu, Mussel-inspired Ag-nanoparticle coating on
porous titanium surfaces to promote mineralization, RSC Adv. 6 (2016)
104025–104035. [47] S.H. Choi, Y.S. Jang, J.H. Jang, T.S. Bae, S.J. Lee, M.H. Lee, Enhanced antibacterial
activity of titanium by surface modification with polydopamine and silver for
dental implant application, J. Appl. Biomater. Funct. Mater. 17 (2019), https://doi. org/10.1177/2280800019847067. [21] M. Li, Q. Liu, Z. Jia, X. Xu, Y. Shi, Y. Cheng, Y. Zheng, Polydopamine-induced
nanocomposite Ag/CaP coatings on the surface of titania nanotubes for
antibacterial and osteointegration functions, J. Mater. Chem. B 3 (2015)
8796–8805. [48] F.L. Bowling, D.S. Stickings, V. Edwards-Jones, D.G. Armstrong, A.J.M. Boulton,
Hydrodebridement of wounds: effectiveness in reducing wound bacterial
contamination and potential for air bacterial contamination, J. Foot Ankle Res. 2
(2009) 1–8. [22] Z. Jia, P. Xiu, P. Xiong, W. Zhou, Y. Cheng, S. Wei, Y. Zheng, T. Xi, H. Cai, Z. Liu,
C. Wang, W. Zhang, Z. Li, Additively manufactured macroporous titanium with
silver-releasing micro-/nanoporous surface for multipurpose infection control and
bone repair - a proof of concept, ACS Appl. Mater. Interfaces 8 (2016)
28495–28510. [49] B.S. Necula, J.P.T.M. van Leeuwen, L.E. Fratila-Apachitei, S.A.J. Zaat, I. Apachitei,
J. Duszczyk, In vitro cytotoxicity evaluation of porous TiO2-Ag antibacterial
coatings for human fetal osteoblasts, Acta Biomater. 8 (2012) 4191–4197. [23] J. Xu, N. Xu, T. Zhou, X. Xiao, B. Gao, J. Fu, T. Zhang, Polydopamine coatings
embedded with silver nanoparticles on nanostructured titania for long-lasting
antibacterial effect, Surf, Coatings Technol. 320 (2017) 608–613. [50] R.G. Contreras, H. Sakagami, H. Nakajima, J. Shimada, Type of cell death induced
by various metal cations in cultured human gingival fibroblasts, Vivo (Brooklyn)
24 (2010) 513–517. [51] A. Ince, N. Schütze, C. Hendrich, R. Thull, J. Eulert, J.F. L¨ohr, In vitro investigation
of orthopedic titanium-coated and brushite-coated surfaces using human
osteoblasts in the presence of gentamycin, J. Arthroplast. 23 (2008) 762–771. References Gibbins, Surface coatings
for improvement of bone cell materials and antimicrobial activities of Ti implants,
J. Biomed. Mater. Res. - Part B Appl. Biomater. 87 (2008) 455–460. [39] K.C. Popat, M. Eltgroth, T.J. LaTempa, C.A. Grimes, T.A. Desai, Decreased
Staphylococcus epidermidis adhesion and increased osteoblast functionality on
antibiotic-loaded titania nanotubes, Biomaterials 28 (2007) 4880–4888. [15] L. Liu, R. Cai, Y. Wang, G. Tao, L. Ai, P. Wang, M. Yang, H. Zuo, P. Zhao, H. He,
Polydopamine-assisted silver nanoparticle self-assembly on sericin/agar film for
potential wound dressing application, Int. J. Mol. Sci. 19 (2018) 2875. [40] A.D. Pye, D.E.A. Lockhart, M.P. Dawson, C.A. Murray, A.J. Smith, A review of
dental implants and infection, J. Hosp. Infect. 72 (2009) 104–110. [
]
b
i
h l
b
h
i d
i [16] X. Ding, Y. Zhang, J. Ling, C. Lin, Rapid mussel-inspired synthesis of PDA-Zn-Ag
nanofilms on TiO2 nanotubes for optimizing the antibacterial activity and
biocompatibility by doping polydopamine with zinc at a higher temperature,
Colloids Surfaces B Biointerfaces 171 (2018) 101–109. [41] M. Abu-Ta’a, M. Quirynen, W. Teughels, D. Van Steenberghe, Asepsis during
periodontal surgery involving oral implants and the usefulness of peri-operative
antibiotics: a prospective, randomized, controlled clinical trial, J. Clin. Periodontol. 35 (2008) 58–63. [42] G. Subbiahdoss, R. Kuijer, D.W. Grijpma, H.C. van der Mei, H.J. Busscher,
Microbial biofilm growth vs. tissue integration: “the race for the surface”
experimentally studied, Acta Biomater. 5 (2009) 1399–1404. [17] S. Amin Yavari, L. Loozen, F.L. Paganelli, S. Bakhshandeh, K. Lietaert, J.A. Groot,
A.C. Fluit, C.H.E. Boel, J. Alblas, H.C. Vogely, H. Weinans, A.A. Zadpoor,
Antibacterial behavior of additively manufactured porous titanium with
nanotubular surfaces releasing silver ions, ACS Appl. Mater. Interfaces 8 (2016)
17080–17089. [43] X. Zhang, U.P. Wyss, D. Pichora, M.F.A. Goosen, Biodegradable controlled
antibiotic release devices for osteomyelitis: optimization of release properties,
J. Pharm. Pharmacol. 46 (1994) 718–724. [18] A.P.R. Alves Claro, R.T. Konatu, A.L. do A. Escada, M.C. de Souza Nunes, C. V. Maurer-Morelli, M.F. Dias-Netipanyj, K.C. Popat, D. Mantovani, Incorporation of
Ag NP on Ti7.5 Mo alloy surface containing TiO2 nanotubes arrays for promoting
antibacterial coating, vitro and in vivo study, Appl. Surf. Sci. 455 (2018) 780–788. [44] R. Reimschuessel, D. Williams, Development of new nephrons in adult kidneys
following gentamicin-induced nephrotoxicity, Ren, Fail. 17 (1995) 101–106. [45] G. Subbiahdoss, R. Kuijer, H.J. Busscher, H.C. References d ff
h k
b
l
¨
b
k
l b l [24] A. Gao, R. Hang, X. Huang, L. Zhao, X. Zhang, L. Wang, B. Tang, S. Ma, P.K. Chu,
The effects of titania nanotubes with embedded silver oxide nanoparticles on
bacteria and osteoblasts, Biomaterials 35 (2014) 4223–4235. [25] A.G. Gristina, P.T. Naylor, Q.N. Myrvik, Biomaterial-centered infections: microbial
adhesion versus tissue integration, Science 237 (1987) 1588–1595. [52] D.O. Kendoff, T. Gehrke, P. Stangenberg, L. Frommelt, H. B¨osebeck, Bioavailability
of gentamicin and vancomycin released from an antibiotic containing bone cement
in patients undergoing a septic one-stage total hip arthroplasty (THA) revision: a
monocentric open clinical trial, HIP Int. 26 (2016) 90–96. [26] M. Sarraf, A. Dabbagh, B. Abdul Razak, R. Mahmoodian, B. Nasiri-Tabrizi, H.R. M. Hosseini, S. Saber-Samandari, N.H. Abu Kasim, H. Abdullah, N.L. Sukiman,
Highly-ordered TiO2 nanotubes decorated with Ag2O NP for improved
biofunctionality of Ti6Al4V, Surf, Coatings Technol. 349 (2018) 1008–1017. h [53] H.J. Busscher, V. Alt, H.C. van der Mei, P.H. Fagette, W. Zimmerli, T.F. Moriarty,
J. Parvizi, G. Schmidmaier, M.J. Raschke, T. Gehrke, R. Bayston, L.M. Baddour, L. C. Winterton, R.O. Darouiche, D.W. Grainger, A trans-atlantic perspective on
stagnation in clinical translation of antimicrobial strategies for the control of
biomaterial-implant-associated infection, ACS Biomater. Sci. Eng. 5 (2019)
402–406. [27] N. Xu, H. Cheng, J. Xu, F. Li, B. Gao, Z. Li, C. Gao, K. Huo, J. Fu, W. Xiong, Ag-
loaded nanotubular structures enhanced bactericidal efficiency of antibiotics with
synergistic effect in vitro and in vivo, Int. J. Nanomedicine 12 (2017) 731–743. 9
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Improving maternal and child nutrition in China: An analysis of nutrition policies and programs initiated during the 2000-2015 Millennium Development Goals era, and implications for achieving the Sustainable Development Goals
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RESEARCH ARTICLE Open Access Improving maternal and child nutrition in
China: an analysis of nutrition policies and
programs initiated during the 2000–2015
Millennium Development Goals era and
implications for achieving the Sustainable
Development Goals Xin Huang1†, Bo Yang1†, Qin Liu1*
, Ruilin Zhang2, Shenglan Tang3,4 and Mary Story5 (2020) 39:12 (2020) 39:12 Huang et al. Journal of Health, Population and Nutrition (2020) 39:12
https://doi.org/10.1186/s41043-020-00221-y Huang et al. Journal of Health, Population and Nutrition (2020) 39:12
https://doi.org/10.1186/s41043-020-00221-y Abstract Background: Although good progress was made in maternal and child nutrition during the Millennium
Development Goals (MDGs) era, malnutrition remains one of the major threats on global health. Therefore, the
United Nation set several nutrition-related goals in the Sustainable Development Goals (SDGs). There is much to be
learned from individual countries in terms of efforts and actions taken to reduce malnutrition. China, as a
developing country, launched a number of nutrition improvement policies and programs that resulted in dramatic
progress in improving maternal and child nutrition during the MDGs era. This study explored the impact,
experiences, and lessons learned from the nutrition policies and programs initiated in China during the MDGs era
and implications to achieve the SDGs for China and other developing countries. Method: The CNKI database and official websites of Chinese government were searched for reviews on nutrition-related
policies and intervention programs. A qualitative study was conducted among key informants from the Chinese government,
non-governmental organizations (NGOs), and universities for two major national nutrition intervention programs. (Continued on next page) Correspondence: liuqin81622@163.com
†Xin Huang and BoYang contributed equally as first authors. 1School of Public Health and Management, Research Center for Medicine
and Social Development, Collaborative Innovation Center of Social Risks
Governance in Health, Chongqing Medical University, No.1 Yixueyuan Road,
Yuzhong District, Chongqing 400016, P. R. China
Full list of author information is available at the end of the article Background to undernutrition [9]. Every country in the world is af-
fected by malnutrition. WHO indicates [9] combating
malnutrition in all its forms is one of the greatest global
health
challenges,
diverse
multi-sector
interventions
aimed at changing behaviors at the individual level com-
bined with policies, systems and environmental changes
need to be considered for implementation at a national
scale to eliminate malnutrition. Therefore, the Sustain-
able Development Goals
(SDGs) set six specific goals
for nutrition, including by 2025 (1) 40% reduction in
children younger than 5 years who are stunted, (2) 50%
reduction of anemia in women of reproductive age, (3)
30% reduction of low birth weight, (4) no increase in
childhood overweight, (5) increase the rate of exclusive
breastfeeding in the first 6 months up to at least 50%,
and (6) reduce and maintain childhood wasting to < 5%
[10, 11]. There is much to be learned from individual
countries in terms of efforts and actions taken to reduce
maternal and child malnutrition, in order to achieve the
nutrition-related 2030 SGDs [10, 11]. g
The Millennium Development Goals (MDGs), estab-
lished by the United Nations (UN) in 2000, set out eight
ambitious goals to be achieved by 2015. Two of them
were directly linked to the health of children and
women: (1) reduce by two thirds the mortality rate
among children under five and (2) reduce by three quar-
ters the maternal mortality ratio. During the MDGs era,
maternal and child nutrition has been improved greatly
in many developing countries through various interven-
tion and policy strategies. The Global Nutrition Policy
Review (2013) based on 119 World Health Organization
(WHO) Member States indicated that most countries
had a range of policies and regulations on nutrition [1],
including breastfeeding and complementary feeding im-
provement strategies, micronutrient supplements for
pregnant women, infants, and young children [2], school
feeding programs [3], and child obesity interventions [4]. These interventions played an important role in improv-
ing child and maternal nutrition in many developing
countries. © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if
changes were made. The images or other third party material in this article are included in the article's Creative Commons
licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons
licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the
data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 13 Huang et al. Journal of Health, Population and Nutrition (2020) 39:12 (Continued from previous page) Results: The literature review documented that during the MDGs era, six nutrition policies and eight trans-province and
nationwide nutrition intervention programs collectively made good progress in improving maternal and child nutrition in
China. Nutrition policies tended to be targeted at infants and children, with less attention on reproductive and maternal
nutrition. Nutrition intervention programs focused primarily on undernutrition and have achieved positive results, while for
breastfeeding improvement and prevention and control on overweight and obesity were limited. Results from the qualitative
study indicated that effective nutrition program implementation was facilitated through the cooperation of multiple sectors
and by the government and NGO partnerships, however, still face challenges of insufficient operational funds from local
governments and inadequacy of program monitoring and management. Conclusion: Nutrition policies and intervention programs promulgated in China during the MDGs era have made major
contributions to the rapid decline of undernutrition and are in line to achieve the SDGs related to child wasting, stunting, low
birth weight, and anemia in reproductive-age women. However, appropriate policies and program implementation are needed
to improve exclusive breastfeeding rates and reduce obesity to achieve the SDGs in years to come. Keywords: China, Nutrition, Maternal and child health, MDG, SDG Background China is a country with regional and
the socioeconomic differences across provinces, which
can be divided roughly into three regions: The Western
part is a less developed region, the Middle is average,
and the Eastern part is the most developed region. Yunnan province, located in Western China, was chosen
to represent a less developed region and Hubei province
(located in Middle China) to represent an average devel-
oped region. No province in the Eastern region was in-
cluded since the nutrition intervention program was not
be implemented in this region. Interviews with stakeholders involved in developing and
implementing nutrition intervention programs are im-
portant to better understand programs’ successes, chal-
lenges, and improvements needed in China to achieve
the nutrition-related SDGs [10, 11]. For this study, the reviews of literature and policy doc-
uments evaluating nutrition-related policies and inter-
vention programs in China during the MDGs (2000–
2015) were conducted, and qualitative interviews were
undertaken with key informants from the Chinese gov-
ernment, non-governmental organizations (NGOs), and
universities for two major national nutrition programs. The aim of the study was to better understand the
nutrition-related
experiences
and
best
practices
in
China, in order to achieve the 2030 nutrition-related
SDGs [10, 11]. Further, experiences and lessons learned
from China have implications for other developing coun-
tries facing similar nutrition and health problems that
China faced two decades ago and could provide insights
into improving maternal and child nutrition to achieve
the nutrition-related SDGs. g
A total of 23 key informants participated in the inter-
views with 10 stakeholders from national sectors, six at
the provincial level from Hubei, and seven from Yunnan. These informants had experience and expertise in ma-
ternal and child nutrition and included the chief leader
from Chinese Centers for Disease Control and Preven-
tion (CDCs), HFPC, MoE, maternal and child hospitals,
All-China Women’s Federation (ACWF) and national
foundations (e.g., UNICEF, China Children and Teen-
ager’s
Fund
(CCTF),
China
Development
Research
Foundation (CDRF)), and nutrition intervention pro-
grams and experts from the top universities/institutions
in China (see Table S1 for details). Qualitative interviews Qualitative interviews with key informants knowledgeable
on the maternal and child nutrition intervention programs
were conducted at the national and provincial levels dur-
ing June and July, 2017. The in-depth interviews were conducted in-person by
two Chinese researchers (two of the co-authors). All in-
terviews were recorded anonymously with informed con-
sent. Each interview lasted about 45–60 min. This study
was conducted according to the guidelines laid down in Review of literature and policy documents This study utilized the China National Knowledge Infra-
structure (CNKI) database to search and collect the
studies and documents on nutrition policies and inter-
ventions programs for Chinese women and children
from January 2000 to December 2015. Studies were
searched using the following terms: maternal nutrition,
infant nutrition, child nutrition, nutrition policies, and
intervention programs. We also checked references of
included studies and documents in order to collect more
information. Searches were also conducted of official
websites of the Chinese government and related depart-
ments, such as Ministry of Education (MoE, http://www. moe.gov.cn) of China, National Health and Family Plan-
ning Commission of China (HFPC, http://www.nhc.gov. cn), and United Nations International Children’s Fund
(UNICEF, https://www.unicef.org). In addition, grey doc-
uments were included in our study, and we also con-
sulted with related experts and institutions for more
detailed information about the nutrition policies and
intervention programs for Chinese women and children. Interview methods The interviews followed a semi-structured topic inter-
view guide conducted in Chinese. The topic guide was
formulated based on the literature reviews of interven-
tion programs and consultation from nutrition experts,
which was then adjusted according to the specialties of
the interviewees, in order to obtain as much detailed in-
formation as possible. The interview guide mainly in-
cluded three themes of each nutrition intervention
program: (1) enabling factors, (2) challenges in program
implementation, and (3) stakeholders’ views on sustain-
ability. To ensure the authenticity and effectiveness of
the interview, researchers contacted the high-level inter-
viewees in advance, providing information on the con-
text of the study and interview questions that would be
asked during the interview. Background Counseling or educational interventions in-
creased exclusive breastfeeding (EBF) rates [5], supple-
mentation with folate and iron reduced the risk of
anemia and maternal mortality [6, 7], providing micro-
nutrient supplementation to pregnant women reduced
the risk of low birth weight [2], school feeding programs
reduced the rate of stunting, wasting, and anemia [3],
and school-based obesity interventions [4] and nutrition
and agricultural policies [8] reduced the incidence of
child obesity. China, as a developing country, launched a number of
nutrition improvement policies and programs that re-
sulted in dramatic progress and impact in improving ma-
ternal and child nutrition during the MDGs period from
2000 to 2015. It is reported that in China from 2002 to
2013 for children under 6 years, the rates of stunting were
reduced in half from 16.3 to 8.1% and underweight from
5.7 to 2.5% [12]. Anemia rates for pregnant women de-
creased from 28.9 to 9.3% and among lactating mothers
from 30.7 to 17.2% [12]. The mortality rate of children
under 5 years of age dropped from 61.0 per 1000 in 1991
to 10.7 per 1000 in 2015 [13]. The national maternal mor-
tality rate decreased from 88.8 per 100,000 in 1990 to 23.2
per 100,000 in 2013 [13]. While good progress was made in maternal and child
nutrition during the MDGs era, malnutrition remains
one of the major threats on global health. According to
the WHO, in 2016, an estimated 155 million children
under the age of 5 were suffering from stunting, while
41 million were overweight or obese, roughly 45% of
deaths among children under 5 years of age were linked Policy and program analysis enables us to better
understand and strengthen the policy environment and
the implementation of the intervention programs [14]. Page 3 of 13 Page 3 of 13 Huang et al. Journal of Health, Population and Nutrition (2020) 39:12 Steering Committee members from the Gates-funded
grant “Achieving Health-Related SDGs in China: Devel-
oping Evidence-based Options for Actions” (led by Duke
Global Health Institute) helped with the selection of key
informants. To better understand the implementation of
nutrition policies and programs across the mainland of
China, interviews with key stakeholders from both the
national level and provincial level (Yunnan and Hubei)
were conducted. Qualitative data analysis y
All the analysis stages were conducted independently by
two researchers. A six-stage Framework Approach [15]
was used to guide the qualitative analysis: (1) Transcrip-
tion: all audio-taped interviews were transcribed verba-
tim by one researcher and kept anonymous and checked
by another researcher. (2) Familiarization: all interviews
were read in full text by 2 independent researchers sim-
ultaneously. Notes were made in the interview transcrip-
tions if any important segments were identified. (3)
Development
of
an
analytical
framework:
thematic
framework was developed based on the objectives, topic
guide, and new themes from the interviewees. (4)
Coding: MAXQDA (version 12) was used to manage
transcriptions. Coding
was
performed
by
two
re-
searchers, one responsible for coding and the other re-
sponsible for checking. (5) Charting: a case display was
formulated per interviewee based on thematic frame-
work. Each case consisted of a short interview answer of
each question. (6) Interpretation: summarize the main
findings and present the results with key quotations in
the text. Overview of nutrition intervention policies during the
MDGs (2000–2015) The results of the literature review documented that
during the MDGs, the Chinese government enacted sev-
eral national nutrition policies to improve maternal and
child nutrition, and six policies showed the most impact
(Table 1). Of those, three were issued by State Council,
two by MoH, and one by MoE. Four of the six focused
on children under 5 years of age and school-aged youth,
one was aimed at women (the Outline for the Develop-
ment of Chinese woman) and one (the Outline for the
Development of food and nutrition) was for all Chinese
residents, with emphasis on priority populations (e.g., in-
fants and children, adolescents, women, and the elderly). For nutrition areas, two of the policies focused on
undernutrition improvement of woman or school-age
children without specific goals, the Outline for the Qualitative analysis on selected nutrition programs
Of th
ight
t iti
i t
ti
g
th N Qualitative analysis on selected nutrition programs
Of the eight nutrition intervention programs, the Nutrition
Package for 6-24 months Infants Program (Nutrition Pack-
age Program) and the Nutrition Improvement Program for
Rural Compulsory Education Students (Nutrition Improve-
ment Program) were the two most extensively implemented
as well as effective nutrition interventions in China and
played an essential role in the improvement of child nutri-
tion (see Table 2 for details). Based on the qualitative inter-
views, we analyzed the enabling factors, challenges, and
stakeholders’ views on sustainable development of the two
programs. Literature/policy document review and analysis Literature/policy document review and analysis
We systematically summarized for each article/report:
the maternal and child nutrition-related policies (issued
department, duration of implementation, target popula-
tion, nutrition-related content) and intervention pro-
grams
(target
population,
implementation
area
and
duration, organizer, contents and effects) in China. Sampling and participants Purposive sampling, as well as snowball sampling, was
used to select the key informants. Consultants and Page 4 of 13 Page 4 of 13 Huang et al. Journal of Health, Population and Nutrition (2020) 39:12 Huang et al. Journal of Health, Population and Nutrition the Declaration of Helsinki, and all procedures involving
research study participants were approved by the Ethics
Committee of Duke University (2017-1359) Development of Chinese Children, Infant feeding strategy
and Technical specification and guidance on child feed-
ing, addressed the child undernutrition including low
birth weight, stunting, anemia, micronutrient deficiency,
and breastfeeding or complementary feeding; two of
them had specific nutrition goals. The Outline for the
Development of Food and Nutrition in China is the most
comprehensive nutrition policy, which focused on im-
provement for undernutrition and infant feeding, but
also took overweight and obesity into account. Priority
was given to geographic regions (e.g., rural and western
areas) and the food industry (e.g., dairy industry, soybean
industry and food processing). Development of Chinese Children, Infant feeding strategy
and Technical specification and guidance on child feed-
ing, addressed the child undernutrition including low
birth weight, stunting, anemia, micronutrient deficiency,
and breastfeeding or complementary feeding; two of
them had specific nutrition goals. The Outline for the
Development of Food and Nutrition in China is the most
comprehensive nutrition policy, which focused on im-
provement for undernutrition and infant feeding, but
also took overweight and obesity into account. Priority
was given to geographic regions (e.g., rural and western
areas) and the food industry (e.g., dairy industry, soybean
industry and food processing). Overview of the nutrition intervention programs during
the MDGs era Findings from the literature review indicated that during
the MDGs period, eight trans-province and national ma-
ternal and (or) child nutrition intervention programs were
implemented. Most of these programs were organized by
multiple government sectors and NGOs such as ACWF
and UNICEF. Six of the programs aimed to improve child
undernutrition, including wasting, stunting, underweight,
micronutrient deficiency, and anemia. Among them, two
focused on both maternal and child health, two were tar-
geted at infants under 3 years, and two programs to bene-
fit primary and middle school students. The other two
programs focused on breastfeeding promotion and child
obesity interventions respectively. Table 2 summarizes the
target population, implementation area, content, and
intervention effects of these programs. The Nutrition Package for 6-24 months Infants Program Enabling factors According to the respondents, the suc-
cessful implementation of the nutrition package program
was attributed largely to (1) the efficient execution of a
three-tiered network consisting of a county-township-
village system, (2) cooperation between government de-
partments and NGOs, and (3) support from national
policies and funding. Huang et al. Journal of Health, Population and Nutrition (2020) 39:12 Page 5 of 13 Huang et al. The Nutrition Package for 6-24 months Infants Program Journal of Health, Population and Nutrition (2020) 39:12 Huang et al. Journal of Health, Population and Nutrition Table 1 Overview of nutrition intervention policies during the MDGs (Continued)
Nutrition intervention
policies
Issued
department
Implemental
duration
(year)
Target
population
Nutrition-related contents of the policy
• Strengthen supervision of infant supplementary foods
• Prevent child micronutrient deficiencies
Technical specification and
guidance on child feeding
and nutrition [22]
MoH
2012~
Children under 7
and their parents
Provide guidance for parents on breastfeeding, food
conversion, reasonable diet, and dietary behavior. Plan of the national medium-
and long-term education
reform and development
(2010–2020) [23]
MoE
2010–2020
Chinese students
(mainly)
Improve the nutritional status of students, in particular,
launch the implementation of the nutritional
improvement plan for rural primary and middle school
students in minority and poverty-stricken areas. Nutrition Improvement Program for Rural Compulsory
Education Students Enabling
factors
Findings
from
the
qualitative
interviews
indicated
three
main
factors
that
were
effective
in
promoting
the
implementation
of
the
nutrition improvement program: (1) active promotion,
(2)
technical
guidance
from
government,
and
(3)
collaboration among multi-sectors. The “low price” bidding principle: “Many companies bid to make
nutritional packages, the principle that local governments must choose the
company with the lowest bid causing price competition reduced the
quality of nutrition packages.” (informant from CDRF) The “low price” bidding principle: “Many companies bid to make
nutritional packages, the principle that local governments must choose the
company with the lowest bid causing price competition reduced the
quality of nutrition packages.” (informant from CDRF) Active promotion: “MoE of Yunnan province have been trying to
publicize the regulations and objectives in schools, and some media has
also reported the project.” (informant from MoE of Yunnan province) Funding shortages of local government#: “The funds for publicity, stuff
training, quality control and evaluation of the program, which need local
budget paid in this project, is insufficient, as the local government is too
poor to afford that.” (informant from CDRF) Funding shortages of local government#: “The funds for publicity, stuff
training, quality control and evaluation of the program, which need local
budget paid in this project, is insufficient, as the local government is too
poor to afford that.” (informant from CDRF) Technical guidance from government: “Chinese CDC has developed an
‘electronic nutritionist’ for guiding school canteens to conduct balanced
diet, the Yunnan government also formulate recipes for the school based
on Yunnan local ingredients.” (informant from MoE of Yunnan province) Floating children: “A large number of floating children who migrant to
urban with their rural-to-urban migrant worker parents cannot get nutri-
tion packages as the packages were issued according to household regis-
tration.” (informant form CDRF) Collaboration between government sectors: “we (Chinese CDC) were
responsible for the monitoring and evaluation of the program. We
organized monitoring and evaluation, collected data such as height and
weight, and detect hemoglobin, VA and VD every year.” (informant from
Nutrition Department of Chinese CDC) “We (MoE) were mainly
responsible for safety of food and funds.” (informant from MoE) #The implementation Plan of The Nutrition Package for 6-24 months Infants
Program stated that the national finance pays for producing nutrition package,
and the local finance support funds for project publicity, stuff training, quality
control, and evaluation [39]. The Nutrition Package for 6-24 months Infants Program M E Mi i
f Ed
i
M H Mi i
f H
l h Table 1 Overview of nutrition intervention policies during the MDGs (Continued) Central finance supporting for poor local government: “The national
budget should arrange a sum of money to help some poor local
government for stuff training, publicity, evaluation etc...” (informant from
MoH of Yunnan province) Central finance supporting for poor local government: “The national
budget should arrange a sum of money to help some poor local
government for stuff training, publicity, evaluation etc...” (informant from
MoH of Yunnan province) Cooperation of government sectors and NGOs: “MoH issued
documents and notices to stipulate tendering and procurement
requirements, and hold annual meetings for the promotion of nutrition
improvement projects.” (informant from MoH) “ACWF mainly undertook
the work of mobilization, publicity and family education.” (informant from
ACWF) Strengthen bidding management: “Scientific and technical bidding
principle are needed, apart from the price, the quality of nutrition packages
should also be taken into consideration.” (officer of CCTF) Support from national policies and funding: “National finance
allocated 100 million financial support for purchasing the nutrition
packages, while many enterprises provide financial support for the
promotion of nutrition packages.” (informant from MoH) Strengthen evaluation: “Sufficient evaluations are needed for finding and
solving problems and adjusting implementation plan in time.” (professor
from Wuhan University) Challenges in program implementationThe qualitative
analysis found four major challenges: (1) the “low price”
bidding
principle
reduced
the
quality
of
nutrition
packages, (2) funding shortages of local government, (3)
nutrition packs stored in hot environments, resulting in
fatty acids degradation, and (4) floating children (children
migrating to urban with their rural-to-urban migrant
worker parents) receive limited nutrition packages. The Nutrition Package for 6-24 months Infants Program Journal of Health, Population and Nutrition Table 1 Overview of nutrition intervention policies during the MDGs
Nutrition intervention
policies
Issued
department
Implemental
duration
(year)
Target
population
Nutrition-related contents of the policy
Outline for the development of
Chinese children [16, 17]
State
Council
2001–2010
Children
Set five specific goals for child nutrition improvement:
By 2010,
• Moderate and severe malnutrition rates of children
under 5 years: fell by 1/4 on the basis of 2000
• Low birth weight: less than 5%
• Prevalence of infant parents’ knowledge of scientific
feeding: more than 85%
• Infants’ breastfeeding rate reaches 85% in provincial
units, add supplementary foods timely and reasonably
• Reduce child vitamin A deficiency
2011–2020
Children
Set four goals specific goals for child nutrition
improvement:
By 2020,
• Low birth weight: less than 4%
• Exclusive breastfeeding in the first 6 months: at
least 50%
• Anemia of children under 5 years: less than 12%
• Stunting and underweight of children under 5 years:
less than 7% and 5% respectively
Outline for the development
of Chinese woman [18]
State
Council
2011–2020
Woman
Set one goal for woman nutrition improvement:
• Reduce the prevalence of moderate and severe
anemia in pregnant women
Outline for the development
of food and nutrition in
China [19, 20]
State
Council
2001–2010
Chinese residents
Give priority to the development of
• Three key food industries (dairy industry, soybean
industry, and food processing industry)
• Two key areas (rural and western areas)
• Three key groups (adolescents, women and children,
the elderly)
Set three goals for improvement on maternal and
child nutrition:
By 2010,
• Stunting and underweight of children under 5 years:
less than 15% and 5% respectively
• Anemia of pregnant woman and children under
5 years: less than 20% and 15% respectively
• Promote the breastfeeding of infants within 4 months,
and gradually supplement various supplementary foods
for infants over 4 months old
2014–2020
Chinese residents
Give priority to the development of
• Three key products (high quality agricultural products,
convenient nutrient processed foods, dairy and soy foods)
• Three key areas (poor areas, rural areas, floating population
gathering, and new urbanization areas)
• Three key groups (maternal and infants, children and
adolescents, the elderly)
Set four goals for improvement on maternal and child nutrition
By 2020,
• The stunting of children under 5 years: less than 7%
• Anemia rate of the whole population: less than 10%
• Anemia of pregnant and lactating woman, and children under
5 years: less than 17% and 12% respectively
• Reduce the growth rate of overweight, obesity, and
dyslipidemia of Chinese residents
Infant feeding strategy [21]
MoH
2007~
Infants
• Protect, promote, and support breastfeeding and increase
the rate of exclusive breastfeeding in first 6 months
• Add supplementary foods timely and reasonably intervention policies during the MDGs
ssued
epartment
Implemental
duration
(year)
Target
population
Nutrition-related contents of the policy
tate
ouncil
2001–2010
Children
Set five specific goals for child nutrition improvement:
By 2010,
• Moderate and severe malnutrition rates of children
under 5 years: fell by 1/4 on the basis of 2000
• Low birth weight: less than 5%
• Prevalence of infant parents’ knowledge of scientific
feeding: more than 85%
• Infants’ breastfeeding rate reaches 85% in provincial
units, add supplementary foods timely and reasonably
• Reduce child vitamin A deficiency
2011–2020
Children
Set four goals specific goals for child nutrition
improvement:
By 2020,
• Low birth weight: less than 4%
• Exclusive breastfeeding in the first 6 months: at
least 50%
• Anemia of children under 5 years: less than 12%
• Stunting and underweight of children under 5 years:
less than 7% and 5% respectively
tate
ouncil
2011–2020
Woman
Set one goal for woman nutrition improvement:
• Reduce the prevalence of moderate and severe
anemia in pregnant women
tate
ouncil
2001–2010
Chinese residents
Give priority to the development of
• Three key food industries (dairy industry, soybean
industry, and food processing industry)
• Two key areas (rural and western areas)
• Three key groups (adolescents, women and children,
the elderly)
Set three goals for improvement on maternal and
child nutrition:
By 2010,
• Stunting and underweight of children under 5 years:
less than 15% and 5% respectively
• Anemia of pregnant woman and children under
5 years: less than 20% and 15% respectively
• Promote the breastfeeding of infants within 4 months,
and gradually supplement various supplementary foods
for infants over 4 months old
2014–2020
Chinese residents
Give priority to the development of
• Three key products (high quality agricultural products,
convenient nutrient processed foods, dairy and soy foods)
• Three key areas (poor areas, rural areas, floating population
gathering, and new urbanization areas)
• Three key groups (maternal and infants, children and
adolescents, the elderly)
Set four goals for improvement on maternal and child nutrition:
By 2020,
• The stunting of children under 5 years: less than 7%
• Anemia rate of the whole population: less than 10%
• Anemia of pregnant and lactating woman, and children under
5 years: less than 17% and 12% respectively
• Reduce the growth rate of overweight, obesity, and
dyslipidemia of Chinese residents
MoH
2007~
Infants
• Protect, promote, and support breastfeeding and increase
the rate of exclusive breastfeeding in first 6 months
• Add supplementary foods timely and reasonably
• Increase breastfeeding rate of infants within 6 months
• Prohibit selling formulate milk in hospitals
• Strengthen public education on breastfeeding
• Strengthen the standardized management of baby-friendly
hospitals
• Guide the rational addition of supplementary foods for
infants over 6 months Table 1 Overview of nutrition intervention policies during the MDGs Infant feeding strategy [21]
MoH
2007~
Infants Infant feeding strategy [21]
MoH
2007~
Infants • Guide the rational addition of supplementary foods for
infants over 6 months Page 6 of 13 Huang et al. Nutrition Improvement Program for Rural Compulsory
Education Students Challenges
in
program
implementation Since
the
project covered a wide geographical area, involved a
large number of departments, and had no precedent to
follow, it has faced several problems: (1) misconceptions
from parents, (2) shortage of operational funding, (3)
lack of nutrition professionals, and (4) poor quality of
monitoring. Additionally, the program has excluded Stakeholders’ views on sustainable development In
order to achieve sustainability of the programs, key
informants
from
across
departments
proposed
improvements in cost-effectiveness, a larger population
reach, and practical strategies. Page 7 of 13 Huang et al. Journal of Health, Population and Nutrition (2020) 39:12 Table 2 Overview of nutrition intervention programs during the MDGs
Program
Target
population
Implementation
area
Implementation
duration (year)
Organizer
Content
Effect on nutrition improvement
Nutrition package for 6–
24 months infants [24,
25]
Infants aged 6–24
months in rural
areas
341 national
poverty counties
in 21 provinces*
Since 2012
MoH and ACWF
Providing a “nutrition package”
(complementary food supplement
powder, including various nutrient such
us protein and micronutrients) free of
charge to children aged 6–24 months per
day and provision of nutrition knowledge
and skills for parents on infant feeding. • One year after the implementation of
the project, the anemia rate of infants in
the monitored areas dropped from 32.9
to 26.0%, stunting rate dropped from
10.1 to 8.4%, incidence of diarrhea in
infants dropped from 14.2 to 9.4%, and
average cost of diarrhea in infants aged
6–24 months dropped from 98.8 yuan to
74.4 yuan. • Parents’ nutrition knowledge significantly
improved. Integrated Early
Childhood
Development (IECD)
[26]
Infants aged 0–3
years in rural areas
160 poverty
villages in Shanxi
and Guizhou
province
Since 2013
UNICEF, PAOSC HFPC,
ACWF
Providing comprehensive interventions,
including nutrition supplementation,
health service delivery, and child
protection to infants aged 0–3 years, to
explore intervention models for integrated
early childhood development and child
protection. The project is still under implementation,
and the results of the evaluation have not
been released yet. Nutrition Improvement Program for Rural Compulsory
Education Students Improving nutrition,
food safety and food
security for China’s most
vulnerable women and
children (CFSN) [27, 28]
The high-risk
population of 1.2
million children
and women of
childbearing age
In six of the
poorest counties
in western China
2009–2013
WHO, FAO, ILO, UNDP,
UNESCO, UNICEF,
UNIDO, and WFP, over
20 Chinese ministries at
the central and local
level
• Providing a basis for policy formulation
by providing reliable and timely
information on the scale, distribution,
type, and causes of malnutrition in
China. • Increasing emphasis on exclusive
breastfeeding, provision of nutritional
supplements, and school-based interven-
tions to develop comprehensive pro-
grams to alleviate hunger and
malnutrition among children. • Making production, processing, and
production of infant and young child
food safer through a regulation of
shared responsibility. • The rate of underweight and stunting
for 6 to 23-month-infants decreased by
58.2% and 35.9% respectively. • The consumed proportion of
micronutrient food in the pilot area
increased from 21.3 to 30.1%. • Different groups such as women,
children, and teachers have greatly
improved their knowledge of food safety
knowledge. • There was a 33.8% decrease in the
prevalence of anemia and a 46%
decrease in vitamin A deficiency and
insufficiency in the pilot areas. Supplementation of
folic acid to prevent
neural tube defects [29–
31]
Women in the
period of 3
months before
pregnancy to the
first trimester of
pregnancy
Rural areas in
China
Since 2009
MoH
Providing folic acid supplements to
women in the period of 3 months before
pregnancy to the first trimester of
pregnancy for free and promotes folic
acid knowledge to the gravidas and their
families. • After the implementation of this project,
the incidence of neonatal neural tube
defects in China decreased significantly,
from 11.96/10000 in 2000 to 2.18/10000
in 2015. • The incidence of neural tube defects in
China dropped from the 3rd highest
incidence of birth defects to 9th. Nutrition improvement
program for rural
compulsory education
students [32–34]
Compulsory
education
students (grades 1
to 9) in rural area
Of 699 poverty
counties in 21
provinces and
corps#
Since 2011
MoE, MoF, MoH, and
other 16 departments
The government provided nutritional
dietary subsidies to rural compulsory
education students in poverty areas, with
a standard of 3 yuan per person per day
(excluding weekend and holiday), which
increased to 4 yuan since October 2014. Nutrition Improvement Program for Rural Compulsory
Education Students • Data monitored by the China CDC
(2012–2015) showed that average height
of boys and girls in 2015 was 1.2–1.4 cm
higher than that of 2012, and the
average weight increased by 0.7 kg and
0.8 kg, which was higher than the Table 2 Overview of nutrition intervention programs during the MDGs
Program
Target
population
Implementation
area
Implementation
duration (year)
Organizer
Nutrition package for 6–
24 months infants [24,
25]
Infants aged 6–24
months in rural
areas
341 national
poverty counties
in 21 provinces*
Since 2012
MoH and A Page 8 of 13 Huang et al. Journal of Health, Population and Nutrition (2020) 39:12 g
height. Baby-friendly hospital
[37]
Pregnant women,
lactating women,
and baby
Whole country
Since 1992
HFPC
In 1992, China has begun to establish
baby-friendly hospitals in accordance with
10 measures to protect, promote, and
support breastfeeding by the WHO and
UNICEF, which aims to give full play to
the important role of hospitals in promot-
ing breastfeeding. No nationwide data for the effects on
baby-friendly hospital. Happy ten minutes [38]
Primary students
Whole country
Since 2012
Chinese CDC and ILSI
Focal Point in China
Except for the daily physical education
curriculum set up in the school, teachers
organize a 10-min simple and interesting
exercise activity for students on each
learning day. • After the implementation of the project,
the BMI-Z score of primary school stu-
dents was improved. MoH Ministry of Health, ACWF All-China Women’s Federation, PAOSC Poverty Alleviation Office of the State Council, HFPC Health and Family Planning Commission, FAO Food and Agriculture Organization, ILO
International Labor Organization, UNDP United Nations Development Program, UNESCO United Nations Educational Scientific and Cultural Organization, UNICEF United Nations International Children’s Emergency Fund,
UNIDO United Nations Industrial Development Organization, WFP World Food Program, GAIN Global Alliance for Improved Nutrition, MoE Ministry of Education, MoF: Ministry of Finance, MoA Ministry of Agriculture, ILSI
International Life Sciences Institute
*21 provinces: Hubei, Shanxi, Inner Mongolia, Jilin, Heilongjiang, Anhui, Jiangxi, Henan, Hubei, Hunan, Guangxi, Chongqing, Sichuan, Guizhou, Yunnan, Tibet, Shaanxi, Gansu, Qinghai, Ningxia, Xinjiang
#21 provinces and crops: Hubei, Shanxi, Inner Mongolia, Jilin, Heilongjiang, Anhui, Jiangxi, Henan, Hubei, Hunan, Guangxi, Chongqing, Sichuan, Guizhou, Yunnan, Tibet, Shaanxi, Gansu, Qinghai, Ningxia, Xinjiang, crops le 2 Overview of nutrition intervention programs during the MDGs (Continued)
gram
Target
population
Implementation
area
Implementation
duration (year)
Organizer
Content
Effect on nutrition improvement
average growth rate of rural students in
China. Implementation of nutrition-related policies Shortage of operational funding: “The national finances did not
allocate the services and work expenses, which need to be supported by
the local authorities, so that the project monitoring, personnel training
work is difficult to carry out due to the shortage of local finance.”
(informant from MoE of Yunnan province). Implementation of nutrition related policies
During the MDGs, China promulgated six nutrition
policies and regulations to promote nutrition and health
conditions of Chinese woman and children. These
nutrition policies focused on the nutritional needs of
women and children at certain age and economic
conditions. For
example,
breastfeeding
and
complementary feeding improvement of infants and the
nutrition of school children from poor rural families. Additionally, the nutritional policies put forward specific
nutritional goals to be achieved in certain years and
identified and prioritized key areas and populations, as
well
as
relevant
technical
guidance
for
nutrition
improvement. Lack of nutrition professionals: “The canteen staff knew very little about
nutrition knowledge, making it hard to provide students with a balanced
meal.” (informant from Nutrition Department of Chinese CDC) Poor monitoring: “The monitoring sample size is too large, work fund
and manpower cannot meet the needs of the monitoring work, staff lacks
regular physical examination and questionnaire survey training, which
make it is difficult to guarantee the quality of monitoring data.” (informant
from Hubei CDC) Unfairness and inequities: “In fact, children in preschool, was not
covered by the program.” (informant from Peking University) “Student
participating in the program is based on household registration, only rural
students could be included, without taking into account the urban poor
students.” (Committee of experts on the Nutrition Improvement Program) While progress was made, we also found a number of
shortcomings of nutrition policies. First, the issued
policies put most of the focus on undernutrition,
complementary feeding, and breastfeeding improvement,
while goals and guidance for prevention of overweight
and obesity were rare. Secondly, currently, there are no
laws
to
guarantee
the
effective
implementation
of
relevant
policies
throughout
provinces,
cities,
and
regions in China. For example, the China Nutrition
Improvement Action Plan issued by the State Council in
1997 has not been implemented widely until 2003 [40]. It is not possible to clarify the responsibilities of various
departments
and
guarantee
the
cultivation
and
maintenance of nutrition professionals. In contrast,
Japan and the USA have made major breakthroughs in
nutrition improvement programs. Nutrition Improvement Program for Rural Compulsory
Education Students • The anemia rate decreased from 17.0%
in 2012 to 7.8% in 2015. plan for student in
a [35, 36]
Primary and
middle school
students in the
urban area
Urban area
Since 2000
Chinese MoA, HGPC,
MoE, MoF, etc. Providing “student milk” to primary and
middle school students. • It improved the nutritional status of
primary and middle school students,
expanded the publicity and education of
drinking milk and health, and created a
good atmosphere for the development
of the dairy industry. • Children’s BMI improved obviously, but
the short-term height growth is small;
the long-term continuous drinking of
student milk has a greater effect on
height. -friendly hospital
Pregnant women,
lactating women,
and baby
Whole country
Since 1992
HFPC
In 1992, China has begun to establish
baby-friendly hospitals in accordance with
10 measures to protect, promote, and
support breastfeeding by the WHO and
UNICEF, which aims to give full play to
the important role of hospitals in promot-
ing breastfeeding. No nationwide data for the effects on
baby-friendly hospital. py ten minutes [38]
Primary students
Whole country
Since 2012
Chinese CDC and ILSI
Focal Point in China
Except for the daily physical education
curriculum set up in the school, teachers
organize a 10-min simple and interesting
exercise activity for students on each
learning day. • After the implementation of the project,
the BMI-Z score of primary school stu-
dents was improved. Ministry of Health, ACWF All-China Women’s Federation, PAOSC Poverty Alleviation Office of the State Council, HFPC Health and Family Planning Commission, FAO Food and Agriculture Organization, ILO
national Labor Organization, UNDP United Nations Development Program, UNESCO United Nations Educational Scientific and Cultural Organization, UNICEF United Nations International Children’s Emergency Fund,
O United Nations Industrial Development Organization, WFP World Food Program, GAIN Global Alliance for Improved Nutrition, MoE Ministry of Education, MoF: Ministry of Finance, MoA Ministry of Agriculture, ILSI
national Life Sciences Institute
provinces: Hubei, Shanxi, Inner Mongolia, Jilin, Heilongjiang, Anhui, Jiangxi, Henan, Hubei, Hunan, Guangxi, Chongqing, Sichuan, Guizhou, Yunnan, Tibet, Shaanxi, Gansu, Qinghai, Ningxia, Xinjiang
rovinces and crops: Hubei, Shanxi, Inner Mongolia, Jilin, Heilongjiang, Anhui, Jiangxi, Henan, Hubei, Hunan, Guangxi, Chongqing, Sichuan, Guizhou, Yunnan, Tibet, Shaanxi, Gansu, Qinghai, Ningxia, Xinjiang, crops Huang et al. Journal of Health, Population and Nutrition (2020) 39:12 Page 9 of 13 preschool children and poor students in the urban cities,
which caused unfairness and inequities. Experiences of nutrition programs and implications for
achieving the Sustainable Development Goals China implemented eight nationwide maternal and child
nutrition
intervention
programs
during
the
MDGs
period. Several government sectors and NGOs were
involved in developing and implementing each of the
programs, for example, MoH, collaborated with MoF for
Nutrition Package Program, and MoE, MoF, MoH, and
CDC worked collaboratively together for the Nutrition
Improvement
Program. Multi-sectoral
collaboration
among government sectors including health, finance,
and education sectors built a comprehensive implemen-
tation
system
for
nutrition
interventions. The Scientific evaluation: “Current evaluation indicators of height and
weight are not only related to diet, but also affected by other factors
including sleep, exercise, hormones and many other factors. So, this
monitoring design should be more reasonable.” (Committee of experts on
the Nutrition Improvement Program) Implementation of nutrition-related policies The USA enacted a
series of federal nutrition-related laws in the 1960s and
1970s to reduce hunger and to guarantee implementa-
tion, such as the National School Nutrition Lunch Act
[41], and in Japan the school feeding law, nutrition im-
provement law, and Nutritionist Law [42]. Stakeholders’ views on sustainable development In
response to these challenges, informants from various
sectors
(Nutrition
Department
of
Chinese
CDC,
UNICEF, CDRF, Hubei CDC, MoE of Hubei
and
Kunming
Medical
University)
put
forward
recommendations for sustainability of the project as
presented in the following table. Stakeholders’ views on sustainable development In
response to these challenges, informants from various
sectors
(Nutrition
Department
of
Chinese
CDC,
UNICEF, CDRF, Hubei CDC, MoE of Hubei
and
Kunming
Medical
University)
put
forward
recommendations for sustainability of the project as
presented in the following table. Reconsider the inclusion criteria of target children: “The inclusion
criteria should be changed, for example, to include children from urban
poor families. Rural household registration should not be used as a
standard of entry.” (Committee of experts on the Nutrition Improvement
Program) Pay attention to child obesity: “At present, undernutrition is not
completely solved, while the problem of overweight and obesity has
emerged. In this case, the project should pay attention to both
undernutrition and overweight and obese children.” (informant from CDRF) Develop adequate legislation: “The government could introduce a law
on nutritional protection for students, and establish a sound nutritional
feeding system.” (Director from MoE of Hubei) Develop adequate legislation: “The government could introduce a law
on nutritional protection for students, and establish a sound nutritional
feeding system.” (Director from MoE of Hubei) Enhance the nutrition knowledge and skill of students, teachers,
parents and canteen staff: “Promoting nutrition and health education of
students, teachers and parents, to cultivate good diet habits for students.”
(informant from Child & adolescent nutrition, UNICEF) “It is important to
train the canteen staff to improve their nutritional knowledge and catering
skills.” (informant from Nutrition Department of Chinese CDC) Nutrition Improvement Program for Rural Compulsory
Education Students learned from the leading national nutrition intervention
projects to understand the experiences of maternal and
child nutrition strategies in China that could help
achieve the nutrition-related 2030 SDGs in China. Misconceptions from parents: “Some parents simply regarded the
program as free lunch, causing ‘crowding-out effects’ in many regions.”
(informant from MoE) Misconceptions from parents: “Some parents simply regarded the
program as free lunch, causing ‘crowding-out effects’ in many regions.”
(informant from MoE) Misconceptions from parents: “Some parents simply regarded the
program as free lunch, causing ‘crowding-out effects’ in many regions.”
(informant from MoE) Misconceptions from parents: “Some parents simply regarded the
program as free lunch, causing ‘crowding-out effects’ in many regions.”
(informant from MoE) Discussion This study analyzed the main content of nutrition-
related policies, successes, and challenges and lessons Huang et al. Journal of Health, Population and Nutrition (2020) 39:12 Page 10 of 13 Page 10 of 13 collaboration between health-related NGOs including
UNICEF, ACWF, CDRF, and WHO who had inter-
national experience and innovative ideas was one of
strongest enabling factors for maternal and child nutri-
tion improvement interventions in China. Additionally,
we found that China prioritized and focused heavily on
improvements to reduce child undernutrition. collaboration between health-related NGOs including
UNICEF, ACWF, CDRF, and WHO who had inter-
national experience and innovative ideas was one of
strongest enabling factors for maternal and child nutri-
tion improvement interventions in China. Additionally,
we found that China prioritized and focused heavily on
improvements to reduce child undernutrition. established to evaluate the effectiveness of BFHI, which
may be one of the reasons why the EBF rate of infants
aged 6 months in China have been low, despite the large
number of hospitals participating and that the program
was established in the early 1990s. Studies have shown
that education and consults in communities are effective
actions for EBF improvement [48]. Comprehensive evalua-
tions of BFHI, and more targeted interventions combined
with government regulations and NGO support, including
community and field technical assistance should be initi-
ated in China to achieve the EBF SDG goal. For reducing undernutrition, six programs have been
launched
by
the
Chinese
government
and
NGOs,
achieving positive
results. These interventions
have
reduced the incidence and prevalence of maternal and
child
undernutrition
including
anemia,
stunting,
underweight, and neural tube defects (Table 2). But
more progress needs to be made in undernutrition
interventions, for instance, the target population has
been mainly infants aged 6–36 months and primary and
middle school students, with less attention on preschool
children 36–48 months of age. Further, as shown in the
qualitative analysis, the two key nutrition interventions
in China continue to face challenges such as insufficient
local operational funds and lack of monitoring. There
are four SDGs related to undernutrition, including low
birth
weight,
stunting
and
wasting
in
in
children
younger than 5 years, and anemia in reproductive-age
women. In China, the low birth weight rate was 3.3% in
2013, in a low level [12]. The wasting rate of children
under 5 years was 2.7%, which has already reached the
SDG target in 2016 [43]. Discussion For child stunting and anemia
in women, policies and intervention programs have
achieved positive results (Table 2). The stunting preva-
lence of children under 5 years from 2000 to 2016 has
achieved 41% reduction [43] and 70% and 40% reduction
for anemia in lactating women and pregnant women
from 2002 to 2013, respectively [12]. Therefore, the SDG
nutrition targets of child stunting, low birth rate, and
anemia in women in China could likely be achieved if
the challenges mentioned above could be addressed with
multi-sector approaches. g
Regarding overweight and obesity, although China’s
childhood overweight and obesity rates were already at a
high level (the prevalence of overweight for children
aged 1–4 years in 2016 was 11.9% and 6.9% for obesity
[43]), few regulation and targets have been issued to
prevent and control obesity, and nationwide intervention
programs on prevention or treatment of child obesity
are rare. The school-based program “Happy ten minutes”
is the only nationwide intervention on child obesity. Fur-
thermore, this program only targeted at primary stu-
dents, without obesity intervention actions for children
in pre- and middle school. From 2000 to 2016, the rate
of overweight and obesity of children under 1–4 years
has been increasing, from 9.5% and 3.9% to 11.9% and
6.9% respectively [43]; the SDG of no increase in child-
hood overweight has not been achieved. Therefore, more
coordinated national overweight and obesity polices and
intervention actions for pre- and middle school children
are urgently needed in order for China to achieve the
SDG goal of no increase in childhood overweight. Opti-
mal nutrition is related to many factors, such as appro-
priate
complementary
feeding,
lack
of
infectious
diseases, access to health care and a nutritious and af-
fordable food supply, and a healthy environment, all of
which also should be considered as priorities by govern-
ment to achieve the SDGs. Authors’ contributions S.T., M.S., and Q.L. designed the research study. Q.L. and R.Z. performed the
research. Q.L., X.H., and B.Y. analyzed the data and wrote the manuscript. All
authors read and approved the final manuscript Acknowledgements This paper as part of the outputs emanating from the project entitled
“Achieving health related sustainable development goals in China:
developing evidence-based options for actions” is funded by the Bill & Me-
linda Gates Foundation (grant number: OOP1148464) and led by Duke Glo-
bal Health Institute with a partnership of several Chinese universities and
organizations. The authors wish to thank Dr. Jiahui Zhang from the Develop-
ment Research Center of the State Council of China, Prof. Jing Fang from
Kunming Medical University, Prof. Hao Xiang from Wuhan University, and
Jianduan Zhang from UNICEF for their assistance in contacting the key inter-
viewees. We also gratefully acknowledge the officials, nutrition project man-
agers, and professors who accepted our interviews. Abbreviations MDGs: The Millennium Development Goals; UN: United Nations; WHO: World
Health Organization; EBF: Exclusive breastfeeding; SGDs: Sustainable
Development Goals; NGOs: Non-governmental organizations; CNKI: China
National Knowledge Infrastructure database; MoE: Ministry of Education;
HFPC: National Health and Family Planning Commission of China;
UNICEF: United Nations International Children’s Fund; CDCs: Centers for
Disease Control; CCTF: China Children and Teenager’s Fund; CDRF: China
Development Research Foundation; ACWF: All-China Women’s Federation;
PAOSC: Poverty Alleviation Office of the State Council; FAO: Food and
Agriculture Organization; ILO: International Labor Organization; UNDP: United
Nations Development Program; UNESCO: United Nations Educational
Scientific and Cultural Organization; UNIDO: United Nations Industrial
Development Organization; WFP: World Food Program; GAIN: Global Alliance
for Improved Nutrition; MoA: Ministry of Agriculture; ILSI: International Life
Sciences Institute; BFHI: Baby-friendly hospital There were several potential limitations that should be
acknowledged in this study. Although we conducted a
comprehensive review of maternal and child nutrition
policies implemented during the MDGs era in China, we
found the evaluation of policies are insufficient, and
therefore, we cannot adequately assess effectiveness or
impact. Also, we only assessed the effects of nutrition
intervention policies on nutrition improvement and
evaluation of implementation and cost-effectiveness of
the two projects is lacking. Our qualitative interviews
were only for project leaders and managers. We did not
interview the target populations of those two projects. Still, our findings will be beneficial in informing efforts
to achieve the SDG nutrition goals in China and provide
lessons learned for other developing countries in redu-
cing the prevalence of malnutrition. Funding This work was supported by the Bill & Melinda Gates Foundation [grant
number: OOP1148464]. Lessons learned from China for other developing
countries A monitoring system is essential for
nutrition interventions, not only to evaluate trends and
progress, but also to help identify gaps, problems, and
any unintended consequences. In addition, the chal-
lenges that China is facing, such as lack of local budgets,
problems with monitoring systems, lack of a nutrition
workforce, and large number of floating children, may
alert other developing countries that when planning and
implementation nutrition interventions, more attention
needs
to
be
paid
to
minimize
and
avoid
similar
problems. 24-month-old children and women are important for
improving nutrition. Further more specific nutritional
goals and technical guidance for nutrition policy are also
essential. (3) Nutrition interventions play an essential
role in improving maternal and child nutrition and re-
quire multi-sectoral collaboration of related government
sectors and NGOs and a comprehensive monitoring sys-
tem. The implementation of nutrition intervention in
China involves multiple sectors, such as finance, health,
and education; only by clarifying their respective respon-
sibilities and cooperating with each other can the pro-
grams be implemented successfully. The NGOs such as
UNICEF and ACWF are important collaborators in con-
ducting nutrition intervention pilots, disseminating nu-
trition knowledge, and monitoring intervention effects. Governments need to establish public-private partner-
ships with NGOs. A monitoring system is essential for
nutrition interventions, not only to evaluate trends and
progress, but also to help identify gaps, problems, and
any unintended consequences. In addition, the chal-
lenges that China is facing, such as lack of local budgets,
problems with monitoring systems, lack of a nutrition
workforce, and large number of floating children, may
alert other developing countries that when planning and
implementation nutrition interventions, more attention
needs
to
be
paid
to
minimize
and
avoid
similar
problems. limited attention on overweight. Overall, China has
made major contributions to the rapid decline of
undernutrition and is likely with continued efforts to
achieve the SDGs of child wasting, stunting, low birth
weight, and anemia in reproductive-age women. How-
ever, appropriate policies and interventions are needed
to improve breastfeeding rates and to control and pre-
vent childhood obesity in years to come. Supplementary Information
Supplementary information accompanies this paper at https://doi.org/10. 1186/s41043-020-00221-y. Supplementary Information
Supplementary information accompanies this paper at https://doi.org/10. 1186/s41043-020-00221-y. Supplementary Information
Supplementary information accompanies this paper at https://doi.org/10. 1186/s41043-020-00221-y. Additional file 1: Table S1. Information sheet of interview participants. Availability of data and materials The datasets used and/or analyzed during the current study are available
from the corresponding author on reasonable request. The datasets used and/or analyzed during the current study are available
from the corresponding author on reasonable request. Our findings document that the nutrition policies and
nutrition
intervention
programs
the
Chinese
government promulgated during the MDGs period have
improved
maternal
and
child
nutrition,
especially
undernutrition in children. Collectively, the nutrition
policies have created a strong supportive environment
for nutrition improvement in China; however, policies
have put the main focus on reducing undernourished
children and women and improving breastfeeding, with Ethics approval and consent to participate This study was conducted according to the guidelines laid down in the
Declaration of Helsinki, and all procedures involving research study
participants were approved by the Ethics Committee of Duke University (Ref
number: 2017-1359). Verbal informed consent was obtained from all subjects. Verbal consent was witnessed and formally recorded. Lessons learned from China for other developing
countries For EBF improvements, we found although several
policies and guidance for EBF improvement were issued,
they have not been implemented well. The EBF rate for
infants aged 6 months in China in 2013 was only 20.8%
[12], which is far away from the SDG goal of 50% [10, 11]. On one hand, intervention actions on EBF improvement
were few. The baby-friendly hospital is the only nation-
wide intervention program in China. On the other hand,
although China started to establish baby-friendly hospitals
(BFHI) in 1992, and had a large number (6711) of BFHI
[44], few evaluations have been conducted on its impact
for EBF improvement. In our literature search, we only
found two hospital-based studies [45, 46] and one survey
[47] evaluating the effects improvement on breastfeeding
rates, and no nationwide evaluations have been done. Sound monitoring and evaluation system has not been China’s positive experience on reducing undernutrition
provides several important lessons for other developing
countries with high rates of undernutrition: (1) achieving
rapid reductions in undernutrition clearly requires high-
level political attention. The Chinese government has re-
alized the importance of nutrition, especially for children
and women. During the MDGs era, a large number of
nutrition policies were promulgated, not only at the na-
tional level, but also poverty-stricken and rural areas. These nutrition policies are the basis for nutrition work. (2) Policies with priority targets are another reason for
the progress and achievement made in China through
the nutrition interventions. Prioritized nutrition, espe-
cially in poverty and remote areas, and the focus on 0– Page 11 of 13 Page 11 of 13 Huang et al. Journal of Health, Population and Nutrition (2020) 39:12 24-month-old children and women are important for
improving nutrition. Further more specific nutritional
goals and technical guidance for nutrition policy are also
essential. (3) Nutrition interventions play an essential
role in improving maternal and child nutrition and re-
quire multi-sectoral collaboration of related government
sectors and NGOs and a comprehensive monitoring sys-
tem. The implementation of nutrition intervention in
China involves multiple sectors, such as finance, health,
and education; only by clarifying their respective respon-
sibilities and cooperating with each other can the pro-
grams be implemented successfully. The NGOs such as
UNICEF and ACWF are important collaborators in con-
ducting nutrition intervention pilots, disseminating nu-
trition knowledge, and monitoring intervention effects. Governments need to establish public-private partner-
ships with NGOs. References Jin X. Strategies for survival and development of children aged 0~6 in China:
from evidence to action. Beijing: Peking University Medical Press; 2018. 6. Pena-Rosas JP, Viteri FE. Effects of routine oral iron supplementation with or
without folic acid for women during pregnancy. Cochrane Database of Syst
Rev. 2006;19:CD004736. 6. Pena-Rosas JP, Viteri FE. Effects of routine oral iron supplementation with or
without folic acid for women during pregnancy. Cochrane Database of Syst
Rev. 2006;19:CD004736. 31. Xinhua News Agency. China’s maternal mortality rate will fall by three-
quarters in 25 years, reaching the UN Millennium Development Goals one
year ahead of schedule. 2015. http://www.gov.cn/xinwen/2015-06/10/
content_2877596.htm. Accessed 24 July 2018. 7. Gera T, Sachdev HP, Nestel P, et al. Effect of iron supplementation on
hemoglobin response in children: systematic review of randomized
controlled trials. J Pediatr Gastroenterol Nutr. 2007;44:468–86. 32. Ma GS. Nutritional status of students in China and relevant nutrition
improvement policy. Chin J Sch Health. 2013;34:641–3. 8. Gortmaker SL, Swinburn BA, Levy D, et al. Changing the future of obesity:
science, policy, and action. Lancet. 2011;378:838–47. 8. Gortmaker SL, Swinburn BA, Levy D, et al. Changing the future of obesity:
science, policy, and action. Lancet. 2011;378:838–47. 33. Ministry of Education of the People’s Republic of China. Report on the
implementation of nutrition improvement plan for compulsory education
students in rural areas. 2017. http://www.moe.edu.cn/jyb_xwfb/gzdt_gzdt/
s5987/201703/t20170302_297934.html. Accessed 25 July 2018. 9. WHO. Malnutrition. 2018. www.who.int/news-room/fact-sheets/detail/
malnutrition. Accessed 3 June 2018. 10. The United States. Goal 2: Zero Hunger. Goal 2 target. 2015. https://www. un.org/sustainabledevelopment/hunger/#tab-6f500e1aa274b23d96d. Accessed 8 June 2018. 34. Ma SP, Li JD. More than 100 billion national investment, covering thirty million
students: this rural student meal is enough for full, but how to for nutritional. 2017. http://www.cdrf.org.cn/mtgz/4185.jhtml. Accessed 25 July 2018. 11. McGuire S. World Health Organization. Comprehensive implementation plan
on maternal, infant, and young child nutrition. Geneva, Switzerland, 2014. Adv Nutr. 2015;6:134–5. 35. Li GW, Liao WK, Fang Y. Working together: the head of the member unit of
the National Student Drinking Milk Program Inter-Ministerial Coordination
Group talks about the student milk plan. China Dairy. 2008;4:28–30. 12. Chang JL. Comprehensive monitoring report on the nutritional and health
status of Chinese residents (2010-2013). Beijing: Peking University Medical
Press; 2016. 12. Chang JL. Comprehensive monitoring report on the nutritional and health
status of Chinese residents (2010-2013). Beijing: Peking University Medical
Press; 2016. 36. Pu YY, Zhang F, Wang H, et al. References 26. China Development Gateway. Pilot project on integrated early childhood
development in poverty-stricken areas. 2013. http://cn.chinagate.cn/
povertyrelief/2013-08/07/content_29645768.htm. Accessed 8 July 2018. 1. WHO. Global nutrition policy review: what does it take to scale up nutrition
action?. 2013. http://www.who.int/nutrition/publications/policies/global_
nut_policyreview/en/.Accessed 3 June 2018. 1. WHO. Global nutrition policy review: what does it take to scale up nutrition
action?. 2013. http://www.who.int/nutrition/publications/policies/global_
nut_policyreview/en/.Accessed 3 June 2018. 27. Wang HY, Chen B, Xia ZY, et al. Evaluation research of joint program
“Improving nutrition, food safety and food security for China’s most
vulnerable women and children”. Chin Health Resour. 2014;17:5–7. 2. Bhutta ZA, Ahmed T, Black RE, et al. What works? Interventions for maternal
and child undernutrition and survival. Lancet. 2008;371:417–40. 2. Bhutta ZA, Ahmed T, Black RE, et al. What works? Interventions for maternal
and child undernutrition and survival. Lancet. 2008;371:417–40. 2. Bhutta ZA, Ahmed T, Black RE, et al. What works? Interventions for maternal
and child undernutrition and survival. Lancet. 2008;371:417–40. 28. WHO Representative Office in China. Improving nutrition, food safety and
food security for China’s most vulnerable women and children. 2014. http://
www.wpro.who.int/china/mediacentre/releases/2014/20140627/en/. Accessed 23 July 2018. 3. Neervoort F, Rosenstiel IV, Bongers K, et al. Effect of a school feeding
programme on nutritional status and anaemia in an urban slum: a
preliminary evaluation in Kenya. J Trop Pediatr. 2013;59:165–74. 3. Neervoort F, Rosenstiel IV, Bongers K, et al. Effect of a school feeding
programme on nutritional status and anaemia in an urban slum: a
preliminary evaluation in Kenya. J Trop Pediatr. 2013;59:165–74. 4. Verrotti A, Penta L, Zenzeri L, et al. Childhood obesity: prevention and
strategies of intervention. A systematic review of school-based interventions
in primary schools. J Endocrinol Invest. 2014;37:1155–64. 4. Verrotti A, Penta L, Zenzeri L, et al. Childhood obesity: prevention and
strategies of intervention. A systematic review of school-based interventions
in primary schools. J Endocrinol Invest. 2014;37:1155–64. 29. Chinese Ministry of Health. Circular of the Ministry of Health on the issuance
of the Program for the Management of the Supplementary folic Acid
Program for the Prevention of Neural Tube defects. 2010. http://www.gov. cn/zwgk/2010-06/28/content_1639533.htm. Accessed 23 July 2018. y
5. Sarah H, Das JK, Salam RA, et al. Breastfeeding promotion interventions and
breastfeeding practices: a systematic review. BMC Public Health. 2013;
13(Suppl 3):S20. 5. Sarah H, Das JK, Salam RA, et al. Breastfeeding promotion interventions and
breastfeeding practices: a systematic review. BMC Public Health. 2013;
13(Suppl 3):S20. 30. Received: 27 December 2019 Accepted: 19 November 2020 Received: 27 December 2019 Accepted: 19 November 2020 25. Sun J, Li J, Cai KX, et al. Monitoring and evaluation of child nutrition
improvement projects in poor areas [Abstract]. Paper presented at The
Twelfth National Conference on Nutrition Science. Beijing; 2015. May 16-18. The authors declare that they have no competing interests. The authors declare that they have no competing interests. Consent for publication
ll
h
d h All authors reviewed this manuscript and provided consent for publication. Page 12 of 13 Page 12 of 13 Page 12 of 13 Huang et al. Journal of Health, Population and Nutrition (2020) 39:12 Huang et al. Journal of Health, Population and Nutrition (2020) 39:12 Competing interests 19. State Council of the People’s Republic of China. Outline for the
development of food and nutrition in China (2001-2010). 2001. http://www. gov.cn/gongbao/content/2001/content_61214.htm. Accessed 3 July 2018. Author details
1 20. State Council of the People’s Republic of China. Outline for the
development of food and nutrition in China (2014-2020). 2014. http://www. gov.cn/zwgk/2014-02/10/content_2581766.htm. Accessed 3 July 2018. 1School of Public Health and Management, Research Center for Medicine
and Social Development, Collaborative Innovation Center of Social Risks
Governance in Health, Chongqing Medical University, No.1 Yixueyuan Road,
Yuzhong District, Chongqing 400016, P. R. China. 2Information Center, The
First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, P. R. China. 3Department of Population Health Science and Duke Global Health
Institute, Duke University, Durham, NC 27708, USA. 4Duke Kunshan University,
Kunshan, Jiangsu 215316, P. R. China. 5Department of Family Medicine and
Community Health and Duke Global Health Institute, Duke University,
Durham, NC 27708, USA. 21. Ministry of Health of the People’s Republic of China. Infant feeding strategy. 2007. http://www.gov.cn/fwxx/jk/2007-08/01/content_703104.htm. Accessed
5 July 2018. 22. Ministry of Health of the People’s Republic of China.Technical specification
and guidance on child feeding and nutrition. Chin J Child Heal Care. 2012;
20:763–6. 23. Guo FJ, He SG. Chinese child malnutrition interventions and
implementation experience. Chin J Child Heal Care. 2015;23:724–6. 23. Guo FJ, He SG. Chinese child malnutrition interventions and
implementation experience. Chin J Child Heal Care. 2015;23:724– 24. Chen SY. Story of “Nutritional Package”. Outlook Weekly (newspaper) 2015;
40-41. Received: 27 December 2019 Accepted: 19 November 2020 References Systematic review of the implementation of
school milk program in China and its impact on growth and development
in school age children. Jo China Med Univ. 2015;44:978–982+986. 13. Ministry of Foreign Affairs People’s Republic of China, United Nations
System in China. Report on China’s implementation of the Millennium
Development Goals (2000-2015). Beijing: the United Nations; 2015. 13. Ministry of Foreign Affairs People’s Republic of China, United Nations
System in China. Report on China’s implementation of the Millennium
Development Goals (2000-2015). Beijing: the United Nations; 2015. 37. Cai JY. Research on the nutritional problems and its influencing factors of
the children under five in China (Dissertation). Beijing: Peking Union
Medical College; 2013. 14. Walt G, Gilson L. Reforming the health sector in developing countries: the
central role of policy analysis. Health Policy Plan. 1994;9:353–70. 38. Ma GS, Mi J, Ma J. Report on childhood obesity in China. Beijing: People’s
Health Publishing House; 2017. 15. Gale NK, Heath G, Cameron, et al. Using the framework method for the
analysis of qualitative data in multi-disciplinary health research. BMC Med
Res Methodol. 2013;13:1–8. 39. National Health and Family Planning Commission of China. Notice on the
Issuance of the 2014 Implementation Plan for The Nutrition Package for 6-
24 months Infants Program. 2014. http://www.nhc.gov.cn/fys/s3585/2
01411/254523446f9241a3a3553e19dec77421.shtml. Accessed 25 Oct 2020. 16. State Council of the People’s Republic of China. Outline for the development
of Chinese children (2001-2010). 2011. http://www.scio.gov.cn/ztk/xwfb/46/11/
Document/978177/978177_1.htm. Accessed 15 June 2018. 40. Yin SA. National nutrition and health improvement needs legal guarantee. Chin Prev Med. 2003;37:325–6. 17. State Council of the People’s Republic of China. Outline for the
development of Chinese children (2011-2020). 2011. http://www.scio.gov. cn/ztk/xwfb/46/11/Document/976030/976030.htm. Accessed 20 June 2018. 18
State Council of the People’s Republic of China Outline for the 41. Zhang J, Zhang B, Zhang GJ, et al. Summary of US nutrition regulations and
policies. Chin J Health Educ. 2011;27:921–3. 42. Zeng HY, National Nutrition Planning Research Group. The situation of
nutritional legislation in the United States and Japan and its enlightenment
to China. Econ Res Ref. 2005;59:9–16. 18. State Council of the People’s Republic of China. Outline for the
development of Chinese woman (2011-2020). 2011. http://www.scio.gov.cn/
ztk/xwfb/46/11/Document/976066/976066.htm. Accessed 2 July 2018. Page 13 of 13 Huang et al. Journal of Health, Population and Nutrition (2020) 39:12 43. Yang B, Huang X, Liu Q, et al. References Child nutrition trends over the past two
decades and challenges for achieving nutrition SDGs and national targets in
China. Int J Environ Res Public Health. 2020;17:1129. 44. Baby Friendly Hospital Initiative Development Fund. National list of baby-
friendly hospitals. 2020. http://www.aiyingyiyuan.com/HospitalMap.html. Accessed 22 Oct 2020. 45. Zhu HL, Fan PL. Evaluation of the effect of establishing baby-friendly
hospital from the change of breast-feeding rate. Matern Child Health Care
China. 1997;4:225. 46. Mo ZD, Shen LJ, Su BL, et al. The role of creating baby friendly hospital to
improve breastfeeding rate. Int Med Health Guid News. 2004;10:195–7. 46. Mo ZD, Shen LJ, Su BL, et al. The role of creating baby friendly hospital to
improve breastfeeding rate. Int Med Health Guid News. 2004;10:195–7. 47. Chen AL, Yuan SY, Cao ZL. Evaluation of the effect of establishing a baby-
friendly hospital on breastfeeding. Pract Prev Med. 1999;6:381. 47. Chen AL, Yuan SY, Cao ZL. Evaluation of the effect of establishing a baby-
friendly hospital on breastfeeding. Pract Prev Med. 1999;6:381. 48. Renfrew MJ, Craig D, Dyson L, et al. Breastfeeding promotion for infants in
neonatal units: a systematic review. Child Care Health Dev. 2010;36:165–78. 48. Renfrew MJ, Craig D, Dyson L, et al. Breastfeeding promotion for infants in
neonatal units: a systematic review. Child Care Health Dev. 2010;36:165–78. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in
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Locomotion in Extinct Giant Kangaroos: Were Sthenurines Hop-Less Monsters?
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Christine M. Janis1*, Karalyn Buttrill1¤, Borja Figueirido2 volutionary Biology, Brown University, Providence, Rhode Island, United States of America, 2 Departamento de Ecologı´a y Geologı´a
d de Ma´laga Ma´laga Spain 1 Department of Ecology and Evolutionary Biology, Brown University, Providence, Rhode Island, United States of America, 2 Departamento de Ecologı´a y Geologı´a,
Facultad de Ciencias, Universidad de Ma´laga, Ma´laga, Spain Abstract Copyright: 2014 Janis et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its
Supporting Information files (tables S5 and S6). Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data a
Supporting Information files (tables S5 and S6). Funding: CJ was supported by The Bushnell Research and Teaching Grant (private foundation grant to Brown University). The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: Christine_Janis@Brown.edu Competing Interests: The authors have declared that no competing interests exist. * Email: Christine_Janis@Brown.edu ¤ Current address: Hampden Family Pet Hospital, Englewood, CO, United States of America ¤ Current address: Hampden Family Pet Hospital, Englewood, CO, United States of America Introduction hopping gait [3]. Three different lineages of macropodids, two of
them extinct, attained masses of greater than any extant kangaroo
(i.e., .90 kg); kangaroos larger than extant kangaroos are
commonly referred to as ‘‘giant kangaroos’’ [2]. In this paper we
specifically address the locomotor abilities of the extinct subfamily
Sthenurinae, and propose that they employed a bipedal striding
type of gait (see Figure 1). We propose that this gait would have
been used at only at slow speeds in the smaller sthenurines, with
hopping employed at faster speeds, but in the very large sthenurine
species this may have been their sole mode of locomotion. Locomotion in Extinct Giant Kangaroos: Were
Sthenurines Hop-Less Monsters? Christine M. Janis1*, Karalyn Buttrill1¤, Borja Figueirido2 October 2014 | Volume 9 | Issue 10 | e109888 Abstract Sthenurine kangaroos (Marsupialia, Diprotodontia, Macropodoidea) were an extinct subfamily within the family
Macropodidae (kangaroos and rat-kangaroos). These ‘‘short-faced browsers’’ first appeared in the middle Miocene, and
radiated in the Plio-Pleistocene into a diversity of mostly large-bodied forms, more robust than extant forms in their build. The largest (Procoptodon goliah) had an estimated body mass of 240 kg, almost three times the size of the largest living
kangaroos, and there is speculation whether a kangaroo of this size would be biomechanically capable of hopping
locomotion. Previously described aspects of sthenurine anatomy (specialized forelimbs, rigid lumbar spine) would limit their
ability to perform the characteristic kangaroo pentapedal walking (using the tail as a fifth limb), an essential gait at slower
speeds as slow hopping is energetically unfeasible. Analysis of limb bone measurements of sthenurines in comparison with
extant macropodoids shows a number of anatomical differences, especially in the large species. The scaling of long bone
robusticity indicates that sthenurines are following the ‘‘normal’’ allometric trend for macropodoids, while the large extant
kangaroos are relatively gracile. Other morphological differences are indicative of adaptations for a novel type of locomotor
behavior in sthenurines: they lacked many specialized features for rapid hopping, and they also had anatomy indicative of
supporting their body with an upright trunk (e.g., dorsally tipped ischiae), and of supporting their weight on one leg at a
time (e.g., larger hips and knees, stabilized ankle joint). We propose that sthenurines adopted a bipedal striding gait (a gait
occasionally observed in extant tree-kangaroos): in the smaller and earlier forms, this gait may have been employed as an
alternative to pentapedal locomotion at slower speeds, while in the larger Pleistocene forms this gait may have enabled
them to evolve to body sizes where hopping was no longer a feasible form of more rapid locomotion. itor: Brian Lee Beatty, New York Institute of Technology College of Osteopathic Medicine, United States of America Editor: Brian Lee Beatty, New York Institute of Technology College of Osteopathic Medicine, United States of America Received April 29, 2014; Accepted September 11, 2014; Published October 15, 2014 Received April 29, 2014; Accepted September 11, 2014; Published October 15, 2014 Copyright: 2014 Janis et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Kangaroo diversity past and present ‘‘Giant’’ forms can be
found within the Sthenurinae (among the genera Sthenurus,
Simosthenurus, and Procoptodon) [6], and within the Macro-
podinae in the extant genus Macropus (extinct species M. titan and
M. ferragus), and the extinct genus Protemnodon (P. brehus, P. roechus, and P. anak). Figure 2. Skeletons of (A) Sthenurus stirlingi and (B) Macropus
giganteus. Modified from Wells and Tedford, 1995. Original artist
Lorraine Meeker, American Museum of Natural History (reproduced
here by permission). doi:10.1371/journal.pone.0109888.g002 Figure 2 shows the difference in the skeletons of Pleistocene
sthenurines and large macropodines. A few smaller sthenurines are known from the Miocene, and
these forms retained the fifth toe. The middle Miocene Wanburoo
hilarus ([13]), originally described as a bulungamayine, was the
smallest known sthenurine [4]), with an estimated body mass of 7–
8 kg [14]. Unfortunately, postcranial materials from this animal
are unknown. The slightly larger (9–15 kg [14]) middle [15] and
early late Miocene Rhizosthenurus flanneryi [16] is known from a
partial skeleton as well as cranial material. The late late Miocene
[17] Hadronomas had estimated body mass of around 30 kg [4]. Note that Prideaux [6] has determined that several species of
Sthenurus are on the stem lineage of the genus Procoptodon,
although lacking the specialties of this highly derived taxon, and
has renamed them as ‘‘Procoptodon’’. These include two smaller
species (body mass of around 50 kg [2]) included here; ‘‘P. gilli
and ‘‘P’’. browneorum. The genus Macropus has species of a diversity of body sizes, the
smallest today being Macropus parma (the parma wallaby) with an
average body mass of around 4 kg [8]. Macropus titan (related to
the extant grey kangaroo) had an estimated body mass of up to
150 kg [2]. A similar estimate has been obtained for M. ferragus
(related to the extant red kangaroo) [9], although this animal
appears to have been somewhat larger in linear dimensions than
M. titan. Within the genus Protemnodon, all species were fairly
large, although not all were larger than extant kangaroos. Protemnodon hopei had an estimated mass of 45 kg [2]. Other
smaller Protemnodon species, such as P. otibandus and P. snewini,
were of a similar size (as estimated by measurements of foot bones). The largest species was P. roechus, with an estimated mass of
around 166 kg [2]. Protemnodon spp. Kangaroo diversity past and present Kangaroos are famous for their style of locomotion – bipedal
hopping (also known as ricochetal or saltatory locomotion), which
is unique among relatively large mammals (i.e., over around 5 kg
in body mass). While the popular notion of a kangaroo is of a fairly
large animal, such as the grey kangaroo (Macropus [Macropus]
giganteus) or the red kangaroo (Macropus [Osphranter] rufus),
members of the superfamily Macropodoidea (‘‘kangaroos’’ in the
broadest sense) contain animals of a diversity of sizes and habits,
including the secondarily arboreal tree-kangaroos (Dendrolagus
spp.). However, the recent past diversity of kangaroos, persisting to
perhaps as recently as 30,000 years ago, included several kinds of
kangaroos much larger than any known at present. The largest of
the extant kangaroos (red kangaroo males) can weigh up to 90 kg,
although the average weight for males of this species is only
around 55 kg, with females averaging around 25 kg [1,2]. However, Pleistocene kangaroos existed that weighed up to 240 kg
[2], a size that calls into question their biomechanical abilities for a There are three families within the Macropodoidea (taxonomy
following Prideaux and Warburton [4], see Table S1): the
Balbaridae (an extinct family, considered to be basal to both
extant families [5]); the Hypsiprymnodontidae (the extant musky
rat-kangaroo, Hypsiprymnodon moschatus, plus a number of
extinct genera); and the Macropodidae. The family Macropodidae
is usually divided into four subfamilies: Potoroinae (rat-kangaroos),
Lagostrophinae (containing the extant banded hare-wallaby,
Lagostrophus fasciatus, and the extinct genus Troposodon), October 2014 | Volume 9 | Issue 10 | e109888 1 PLOS ONE | www.plosone.org Locomotion in Sthenurine Kangaroos Figure 1. Reconstruction of Sthenurus stirlingi. By Brian Regal. doi:10.1371/journal.pone.0109888.g001 Figure 2. Skeletons of (A) Sthenurus stirlingi and (B) Macropus
giganteus. Modified from Wells and Tedford, 1995. Original artist
Lorraine Meeker, American Museum of Natural History (reproduced
here by permission). doi:10.1371/journal.pone.0109888.g002 Figure 1. Reconstruction of Sthenurus stirlingi. By Brian Regal. doi:10.1371/journal.pone.0109888.g001 Macropodinae (containing all other extant macropodids, olus
several extinct genera including Protemnodon), and Sthenurinae
(containing
the
extinct
genera
Archaeosimus,
Hadronomas,
Metasthenurus,
Procoptodon,
Rhizosthenurus,
Sthenurus,
Si-
mosthenurus, and Wanburoo, see Prideaux [6]). A possible fifth
subfamily is the Bulungamayinae, which is a paraphyletic
assemblage of extinct taxa basal to the Macropodinae plus
Sthenurinae [5]). Molecular phylogenies (e.g., [7]) show similar
relationships among the living taxa, although the higher level
taxonomic terminology differs (e.g., the rat-kangaroos are consid-
ered to be a separate family, Potoroidae). Kangaroo diversity past and present are commonly referred to as
‘‘giant wallabies’’: but while they might be somewhat wallaby-like
in their skull and dentition, their postcranial elements are unlike
any other terrestrial macropodid; they have relatively short tibiae,
short feet and (at least in P. anak) a relatively long neck. There has
been speculation that at least some may have been secondarily
quadrupedal in their locomotion [10,11]. October 2014 | Volume 9 | Issue 10 | e109888 Locomotion in Sthenurine Kangaroos These rodents, like small macropodids, use quadrupedal
gaits at slow speeds [18]. However, the ankle extensor tendons of
hopping rodents are more robust for their size than in kangaroos,
which may reflect the need to withstand relatively high forces
during acceleration. Hopping can also be seen in some lemuriform
primates (e.g., sifakas moving on the ground) and occasionally in
Arctic hares (Lagomorpha): again, these animals are no more than
around 5 kg in weight, below the weight where hopping becomes
efficient in terms of storage of elastic energy in the extensor
tendons (see below). However, larger kangaroos pay a price for their locomotor
efficiency in terms of bone and tendon stress. Placental mamma-
lian cursors change their posture with increasing body size,
thereby reducing the torque of the ground reaction force around
their limbs [30], but large kangaroos hop with the same flexed
limb posture as smaller ones. There is no evidence that there is any
difference in the properties of the ankle extensor muscles and their
tendons that power hopping in kangaroos (gastrocnemius, flexor
digitorum longus [ =
profundus], and plantaris [ =
flexor
digitorum superficialis]) in comparison with similar placentals,
although enzyme levels in these muscles in kangaroos may indicate
higher levels of aerobic work, and 86% of the total body
mitochondrial volume in M. rufus is in the upper hind limb
musculature [28]. In addition, bone stresses on the tibia appear to
be greater in large kangaroos (but not in smaller kangaroos) than
in similar-sized placentals, although there is some evidence that
tibial bone cortical thickness increases with increasing body size in
kangaroos [3]. It is well known that, in larger kangaroos (e.g., Macropus rufus),
hopping is an extremely efficient gait: unlike the situation in
placentals, where the energy costs of locomotion increase linearly
with speed, in the larger kangaroos energy costs and speed become
decoupled, and thus the daily expenditure of locomotor foraging is
much less for the large kangaroos than for similarly-sized cursorial
placentals. (Note: although the term ‘‘cursorial’’ usually refers to
quadrupedal locomotion, we use the term here in relation to
kangaroo locomotion, where ‘‘more cursorial’’ equals ‘‘more
specialized for fast hopping’’.) Even in a medium-sized wallaby,
the energy savings from elastic energy storage during hopping
have been estimated at around 40% [27]. Locomotion in Sthenurine Kangaroos However, while much of
the research focus has been on the spectacular performance of the
large kangaroos, this cannot explain the initial reason for adopting
a hopping gait. This decoupling of energy costs and speeds is only
true in kangaroos above around 6 kg, and the smaller potoroines
(e.g., Potorous, Bettongia) show no such advantage, although
hopping in Bettongia (at least at relatively high speeds, above 3 m/
sec) is nevertheless less expensive than locomotion in a quadruped
of similar size [28]. However, one distinct advantage of hopping,
over and above any storage of elastic energy, is that speed can be
attained simply by increasing stride length without concomitant
increase in stride frequency, which reduces the energetic costs of
limb recycling [28]. Certain allometric scaling relationships differ between kanga-
roos and placental mammals, which likely relates to the fact that
large kangaroos maintain the crouched posture of smaller ones,
rather than altering their posture with increasing body size, see
[30]. The overall hind limb length of macropodoids scales with
positive allometry, largely due to strong positive allometry of tibia
length [3]. The size of the hind limb, the limb muscles, and the
cross sectional area of the foot extensors all scale with isometry in
placentals. All of the extensor muscles of the hind limb in
macropodines, with the exception of the sartorius, scale with
strong positive allometry, while fascicle length tends to scale with
negative allometry, resulting in extremely strong positive allometry
for hind limb muscle physiological cross sectional area [3]. However, note that in potoroines, elastic energy saving is primarily
in the plantaris muscle, with the gastrocnemius and flexor
digitorum longus being more involved in joint control and
acceleration capacity [3]. This greater muscle power in kangaroos
is required to offset the relatively greater torques around the ankle
joint in the absence of changing posture with increasing size. In
contrast, the tendon cross sectional areas of the gastrocnemius and
flexor digitorum longus scale with negative allometry, although the
cross sectional areas of the plantaris tendon scales with positive
allometry [3]. Locomotion in Sthenurine Kangaroos Locomotion in Sthenurine Kangaroos placed together on the ground, the hind feet are lifted, and the tail
is used as a ‘‘fifth leg’’ to propel the animal through the space
between the fore feet [22]. A quadrupedal bound is seen in the
quokka (Setonix brachyurus) and tree-kangaroos (Dendrolagus
spp.), and tree-kangaroos uniquely perform (along tree branches) a
quadrupedal walk, with alternate movement of the limbs [21]. Tree-kangaroos have also been observed walking bipedally along
branches [23]. kangaroos: in bipedal or quadrupedal running there is no net
torsion on the body that would cause the head to pitch, as the
angular momentum of the legs cancel each other out. But in
bipedal hopping, where the legs act together in phase, their action
creates a moment around the center of mass, such that the body
would tend to pitch with each hop. Motion of the tail reduces this
tendency: the tail swings forwards as the hind legs swing
backwards, thus cancelling out the moment produced by the
limbs, and reducing the effective pitch of the head to around ten
degrees [27]. kangaroos: in bipedal or quadrupedal running there is no net
torsion on the body that would cause the head to pitch, as the
angular momentum of the legs cancel each other out. But in
bipedal hopping, where the legs act together in phase, their action
creates a moment around the center of mass, such that the body
would tend to pitch with each hop. Motion of the tail reduces this
tendency: the tail swings forwards as the hind legs swing
backwards, thus cancelling out the moment produced by the
limbs, and reducing the effective pitch of the head to around ten
degrees [27]. The adaptive reasons for adopting the hopping gait are not
entirely clear. Hopping is clearly a very efficient form of
locomotion in larger kangaroos (see discussion below), but it
would have first evolved in relatively small forms, likely of a mass
of less than 5 kg [24]. Baudinette [25], Bennett [26], and
McGowan et al. [3] summarize much recent information about
kangaroo hopping, and the discussion below is derived from their
papers. Hopping has evolved several times in rodents, but most of
these are very small, with the largest being the springhare (Pedetes
capensis) with a body mass of around 4 kg (the size of a small
wallaby). October 2014 | Volume 9 | Issue 10 | e109888 Locomotion in macropodoids Hopping is the quintessential kangaroo gait, seen in all extant
macropodoids with the exception of the musky rat-kangaroo
(Hypsiprymnodon moschatus), where it is considered to be
primarily absent (although this is conjectural because there are
inadequate data on this animal locomoting at high speeds [18]. Some earlier, extinct macropodoids (Balbaridae) may have been
quadrupedal bounders rather than hoppers [11]. All extant
kangaroos have a digitigrade hind foot posture while locomoting,
although they may rest with the hind feet in a plantigrade stance. Among the potoroines (rat-kangaroos), potoroos (Potorous spp.)
habitually bound and only hop at high speed when alarmed [19],
while bettongs (Bettongia spp.) habitually hop like macropodines
[20]. Within the macropodines, all species use the form of slow,
pentapedal progression while foraging on the ground [21], a gait
that is actually used more frequently than hopping during the
course of the day [22]. In the pentapedal ‘‘walk’’, the forefeet are Sthenurines are the kangaroos that are usually spoken of as
being the ‘‘giant kangaroos’’. They are distinguished not only by
their size, but by many other features, such as their relatively short
faces, the loss of the fifth digit in the foot in the Pleistocene forms,
and their specialized arms and hands that have been interpreted as
an adaptation for browsing [12]. Sthenurines have also been noted
as having especially robust limb bones, but the bones of the larger
species of the extinct genus Protemnodon are similarly robust (see
later discussion). Sthenurines, like Protemnodon spp., were all
fairly large, but not all species were larger than extant kangaroos. October 2014 | Volume 9 | Issue 10 | e109888 PLOS ONE | www.plosone.org 2 Locomotion in Sthenurine Kangaroos issue. How does this relate to the condition in kangaroos? While
larger species will be better able to use elastic energy recovery to
assist in their locomotion, the larger the animal, the lower the
tendon safety factor, and the greater the danger of tendon rupture:
while smaller kangaroos have estimated tendon safety factors of
around ten (typical for non-hopping mammals), the estimated
safety factor of the Achilles tendon in large kangaroos approaches
one. McGowan et al. [3] estimate that, at a projected body mass of
around 140 kg, a fast-hopping kangaroo would have a tendon
safety factor of less than one. However, a large male red kangaroo
(Macropus rufus), weighing around 80–90 kg, would still be
operating with a very low tendon safety factor of 1.1, and many
extinct kangaroos have body mass estimates well in excess of 140
kg (although this is around the mass estimate for the largest extinct
species of Macropus). Few extant kangaroo individuals can be
found with a body mass of greater than around 50 kg, and most
kangaroos weigh considerably less than this, see [1]. Extrapola-
tions from the data of McGowan et al. [3] would predict that the
sthenurine Procoptodon goliah, at a body mass of as much as 250
kg, would have had a tendon safety factor of around 0.89; they
note that hopping would have been severely limited in this animal,
especially during acceleration, if indeed possible at all. However,
McGowan et al. [3] propose that relatively thicker tendons in these
larger macropodids (as also seen in rock wallabies, Petrogale spp.)
would have enabled them to exert higher forces at larger sizes. McGowan [32] presented evidence that the greatly enlarged site of
attachment of the gastrocnemius (i.e., the Achilles tendon) on the
calcaneum in sthenurines was indicative of tendons sufficiently
large to withstand the forces of hopping locomotion. However, it is
also true that a relatively thicker tendon has a lesser capacity for
elastic energy storage and, as noted below, the moment arm for
the gastrocnemius in sthenurines is much shorter than in large
macropodines. Obviously large sthenurines would have had to
have relatively large gastrocnemius muscles to support and propel
their body mass during locomotion, but this does not necessarily
mean that they could hop well, if at all. apparent difference in proportions is due to a shorter trunk
rather than to longer hind limbs. Locomotion in Sthenurine Kangaroos (See [34], which
concludes that sthenurines have a relatively shorter
vertebral column than macropodines, despite the same
number of vertebrae, due to a shorter length of the lumbar
vertebrae.) (iii) Pleistocene sthenurines have reduced the fifth digit in the
hind limb to a vestigial nubbin of the metatarsal. There is
little evidence that they retained the syndactylous small
second and third digits in the hind limb typical of other
kangaroos (although a remnant of metatarsal III is known
in a couple of specimens [12]). Pleistocene sthenurines have reduced the fifth digit in the
hind limb to a vestigial nubbin of the metatarsal. There is
little evidence that they retained the syndactylous small
second and third digits in the hind limb typical of other
kangaroos (although a remnant of metatarsal III is known
in a couple of specimens [12]). (iv) (iv)
The tail of Sthenurus is slightly shorter than that of
Macropus. In addition, the anterior caudal vertebrae have
shorter (although robust) centra, but with shorter diapoph-
yses and metapophyses, and vestigial anapophyses. This
may indicate a reduced capacity for lateral extension of the
tail [12]. (iv)
The tail of Sthenurus is slightly shorter than that of
Macropus. In addition, the anterior caudal vertebrae have
shorter (although robust) centra, but with shorter diapoph-
yses and metapophyses, and vestigial anapophyses. This
may indicate a reduced capacity for lateral extension of the
tail [12]. (v) (v)
The lumbar vertebrae of Sthenurus are massive, with huge
metapophyses, but the transverse processes have been
reduced or lost, and the backbone appears to have been
relatively rigid with limited flexion in the dorsoventral
plane. (vi)
And, finally, in general the limb elements of Sthenurus,
especially in the hind limbs, are much more robust than
those of Macropus: Wells and Tedford [12] note that the
cross-sectional area of the femur of Sthenurus is almost
double that of a Macropus of similar linear dimensions. We present here a description of the hindimb locomotor
anatomy of sthenurine kangaroos, which is essential for an
understanding of the analyses performed. Much of this anatomical
description is drawn from Wells and Tedford [12], but some novel
features are also included. Note that the anatomical illustrations
here are designed to show features not emphasized in Wells and
Tedford [12], and the reader is referred to this publication for
additional details. Descriptive anatomy of sthenurine kangaroos While sthenurine locomotor performance has rarely been
considered as markedly different from that of modern large
kangaroos (genus Macropus), there are many anatomical differ-
ences that reflect the fact that these animals had a rather different
functional biology. The classic work on sthenurine anatomy is that
of Wells and Tedford [12], in their comparison of species of
Sthenurus (mainly S. stirlingi and S. tindalai) with the exant grey
kangaroo, Macropus giganteus): they note that, while the overall
postcranial proportions of Sthenurus resembles those of Macropus,
there are some key differences. Vertebral column. The number of precaudal vertebrae is
similar in sthenurines and macropodines (7 cervical, 13 thoracic
and 6 lumber), but the overall appearance of the vertebral column
is very different. The overall appearance in sthenurines is for
massive vertebrae in comparison to macropodines, especially in
the lumbar region, where large and laterally expanded metapo-
physes give the impression of rigidity. Wells and Tedford [12] note
that the anticlinal vertebra in Sthenurus is more posterior (L1 or
L2, versus T10 or T11 in Macropus), with no modification of the
nature of the zygapophyses (as in Macropus). Large metapophyses
appear on T11, and increase in size dramatically in the more
posterior portion of the trunk. While metapophyses are also
present in the posterior trunk of Macropus, they are smaller, and
less
deflected
medially. The
neural
arches
are
shorter
in
Sthenurus, although broader in the anteroposterior direction,
and are directed caudally (while those of Macropus are directed
cranially). The lumbar vertebrae of Sthenurus are also markedly
different in having greatly reduced transverse processes, and in the
vestigial nature or absence of diapophyses and anapophyses (that
are prominent on the lateral surfaces of the lumbar vertebrae in
macropodines), and the almost platycoelus centra indicate limited
mobility. (i)
While the forelimbs of Macropus and Sthenurus are of
similar overall length, the manus of sthenurines is longer,
and the radius and ulna are shorter. Modifications of the
sthenurine forelimb allow for specialized grasping, and the
scapular morphology may allow for elevation of the
forelimb over the head, as in humans. Somewhat similar
modifications of the forelimb, especially in the scapula, are
seen in tree- kangaroos, which do extensive reaching with
the forelimbs [33]. (ii) The hind limbs are of similar proportions in both, but
Wells and Tedford [12] claim that the limbs are
proportionally longer in relation to the vertebral column
in Sthenurus. Locomotion in Sthenurine Kangaroos The supplementary information contains a
number of bivariate plots (Figures S1–S3), which are separate from
the multivariate analyses discussed later, and provide a visual
impression of some of the anatomical differences between
sthenurines and other kangaroos. Locomotion in Sthenurine Kangaroos While this anatomy allows for greater proportional
muscle power and elastic energy storage in larger kangaroos,
accounting in part for their superior locomotor performance, it
appears that larger macropodids operate with unusually high
musculoskeletal stresses, and tendon safety factor (estimated from
the ratio of the tendon cross sectional area to the physiological
cross sectional area of the attached muscle (see [3]: at safety factors
below one tendon failure becomes likely [31]) might be a limiting
factor for body mass and/or locomotor performance. The pentapedal walk, which is employed at low speeds in
kangaroos, is energetically very expensive, more so than hopping
at higher speeds [28]. Kangaroos have been shown to progress
from a pentapedal gait to hopping at a Froude number of 0.5 [25],
which is similar to the transition to the gallop in quadrupeds (the
Froude number relates size, speed, and stride length, and is used in
the analysis of vertebrate gaits). There must thus be some
biomechanical or energetic reason why hopping cannot be
employed at low speeds: Dawson [29] proposed (p. 68) that, due
to the specialized limb morphology of kangaroos, hopping would
likely be even more expensive than pentapedal locomotion at slow
speeds. The role of the tail is also important in hopping in The mechanical tensile stress of tendons plays a limiting factor
in body size and speed in all mammals. In placental mammals
maximum locomotor performance peaks at a body mass of around
50 kg (cheetah or pronghorn size), and there is evidence that this is
the size at which strain on the locomotor tendons becomes an PLOS ONE | www.plosone.org October 2014 | Volume 9 | Issue 10 | e109888 3 Locomotion in Sthenurine Kangaroos Descriptive anatomy of sthenurine kangaroos Wells and Tedford
[12] note only two sacral vertebrae in Sthenurus, as in Macropus
(and most other marsupials), but the senior author has observed
sthenurine sacra that are composed of three vertebrae, involving
the incorporation of the first caudal vertebra (e.g., Simosthenurus
occidentalis SAM P18308). Wells and Tedford [12] note a more
rigid sacrolumbar connection in Sthenurus stirlingi than in
Macropus (whereas the smaller S. tindalei is more like Macropus). The sthenurine sacral anatomy is again indicative of a resistance to
rotational torsion. Macropodines in general also have a fairly narrow tip of the
ilium (tuber coxa), while this is broad in sthenurines, and narrower
in species of Macropus than in most other kangaroos. The
sthenurine condition is approached only by tree-kangaroos
(Dendrolagus spp.) among macropodines, while other more
open-habitat, fast hopping kangaroos such as the nail-tail wallabies
(Onychogalea spp.) and hare-wallabies (Lagorchestes spp.) parallel
the Macropus species in having narrow tuber coxae (see Figure
S1A). While there is debate about the systematic position of the
nail-tail wallabies, the molecular data [7] show that both
Onychogalea and Lagorchestes are independent radiations to
Macropus within the Macropodinae: they can thus serve as a
comparison to Macropus species for considerations of morpho-
logical features related to greater cursoriality. The tuber coxae
serve as the area of origin of the gluteus superficialis and the
cranial head of the caudofemoralis in Macropus [36], but not in
Setonix and Dendrolagus, where these muscles originate from the
thoracolumbar fascia [38]. The gluteal and caudofemoralis
muscles act to extend the hip: they may be important in elevating
the body over the hip when the feet are on the ground, and would
also enable supporting the body over a single hind leg. In all
kangaroos the sartorious muscle originates from the tuber coxa,
and the medial and deep gluteals originate from the iliac blade
[38]. Pelvis (Figure 3). The ilium of both Sthenurus and Macro-
pus is long, but in Sthenurus the ilia flare more laterally, and have
a greatly expanded blade. Elftman [35] interprets flared ilia as
allowing for a greater volume of erector spinae musculature. Wells
and Tedford [12] note that the areas of insertion for the gluteal
muscles (on the lateral side of the blade) and the iliacus muscle (on
the dorsomedial side of the blade) in Sthenurus are, respectively,
1.8 and 1.6 times the amount of insertion area in Macropus. October 2014 | Volume 9 | Issue 10 | e109888 Descriptive anatomy of sthenurine kangaroos However, this is not apparent in their figures
(see Figure 2): Sthenurus appears to have a somewhat
shorter trunk than Macropus, which would mean that this October 2014 | Volume 9 | Issue 10 | e109888 PLOS ONE | www.plosone.org 4 Locomotion in Sthenurine Kangaroos Figure 3. Pelvis. (A) Simosthenurus occidentalis (SAM: P17358). (B)
Dendrolagus dorianus (SAM: M9190) (C). Macropus robustus (SAM:
M3695). Left lateral view. Scale bar = 5 cm. doi:10.1371/journal.pone.0109888.g003 Wells and Tedford [12] interpret this anatomy as indicating
rigidity to resist rotational stress on the backbone. In Sthenurus
resistance would have been effected not only by the zygapophyses,
but also by by the epaxial muscles (multifidi) and ligaments
attached to the greatly enlarged metapophyses. They [12] note
that the loss of diapophyses and anapophyses indicate the
reduction of the longissimus dorsi component of the epaxial
musculature. They [12] interpret the massive dorsal epaxial
components (the multifidi) as being used to elevate the front half of
the body for the proposed browsing posture. But this interpreta-
tion is problematical. The notion of the ‘‘elevation of the body’’
seems to be derived from the function proposed by Elftman [35] in
kangaroos for the erector spinae muscles (this being the lumbar
region merging of multifidus and longissimus dorsi muscles); but
this proposed function was in the context of preventing the front
end of the body collapsing under the force of gravity during
locomotion. It would be impossible for the erector spinae to raise
the body up over the hip, as they insert cranially to the hip joint
(along the dorsomedial border of the ilium). The muscles that
would be capable of exerting this action of raising the body would
be ones that span the hip joint dorsally: the gluteus superficialis
and the cranial head of the caudofemoralis ( = gluteobiceps)
muscles (see [36]), that originate from the tuber coxae of the ilium
(and/or the thoracolumbar fascia, see below) and run dorsally over
the hip socket to insert on the femur. Figure 3. Pelvis. (A) Simosthenurus occidentalis (SAM: P17358). (B)
Dendrolagus dorianus (SAM: M9190) (C). Macropus robustus (SAM:
M3695). Left lateral view. Scale bar = 5 cm. doi:10.1371/journal.pone.0109888.g003 Sacrum. The sacrum of sthenurines is broader and shorter
than in macropodines, with more pronounced sacral wings (alae
sacrales) for the articulation with the ilium. Descriptive anatomy of sthenurine kangaroos Flared iliac blades, indicating enlarged gluteal muscles, are
common among large mammals that engage in bipedal browsing
(see [37]). They [12] also note that the iliopectineal tuberosity (at
the base of the iliac spine), the area of origin of the rectus femoris,
is larger in Sthenurus: the acetabulum is also larger in Sthenurus,
and the acetabulae are placed further apart, resulting in a more
wide-legged stance. The larger species in the genus Macropus have elongated ischia
(dorsal length of ischium around 65–70% of the length of the
ilium), with a pronounced posteroventral projection to the bone. This long ischium provides an elongated moment arm for the
muscles that retract the femur, both the hamstring complex and
the adductor complex. Elftman [35] notes that the elongation of
the ischia effectively turns the action of the adductors into femoral
retractors (i.e., hip extenders). This is obviously advantageous for
powerful hip extension during rapid locomotion, paralleled among
placentals by the extension of the ischia in the cheetah (Acinonyx
jubatus) in comparison with less cursorial felids [39]. However, this October 2014 | Volume 9 | Issue 10 | e109888 October 2014 | Volume 9 | Issue 10 | e109888 PLOS ONE | www.plosone.org 5 Locomotion in Sthenurine Kangaroos Figure 4. Femur. (A) Simosthenurus occidentalis (SAM: P17259). (B)
Macropus sp. (SAM: P17270). All left side: upper = proximal articular
view; lower = lateral distal view. Scale bar = 5 cm. doi:10.1371/journal.pone.0109888.g004 condition is derived in Macropus among the other macropodoids,
which have a relatively shorter ischium (less than 60% the length
of the ilium), although the nail-tail wallabies (Onychogalea spp.)
have also elongated their ischia, to an even greater percentage of
the ilium (.80%) than in Macropus (see Figure S1B). This
increased length of the pelvis caudal to the acetabulum can be seen
even more clearly in a comparison of the length of the
puboischiatic symphysis (Figure S1C). However, sthenurines have ischia that are markedly different to
those of other macropodids, approached (convergently) only
among the tree-kangaroos. The ischia are only somewhat shorter
than in the regular macropodid condition, but are tipped dorsally:
Wells and Tedford [12] note that the angle between the ilium and
the ischium is 170o in Macropus, but 145o in Sthenurus. Descriptive anatomy of sthenurine kangaroos The shape of the femoral head is round in sthenurines, rather than
ovoid as in Macropus (see Figure 4). The more ovoid morphology
of Macropus is the derived one among macropodoids, and is likely
related to restricting femoral motion to a parasaggital plane, as
also seen among cursorial bovids [41]. The neck of the femur is
also elongated in Macropus, which may increase the moment arm
for the gluteal muscles, again reflecting cursorial adaptations. As noted by Wells and Tedford [12] the adductor scar on the
posterior part of the femur in Sthenurus is placed more distally
than in Macropus. This marks the insertion point of the quadratus
femoris ( = ischiofemoralis), which originates from the ischium and
is essentially the most proximal component of the adductor
musculature complex. Hopwood and Butterworth [36] note that
this muscle is tendinous in Macropus, and suggest that it acts as a
form of check ligament to prevent overflexion of the hip during
jumping (i.e., to limit the forward motion of the femur). Without
knowing the more general condition for this muscle in all
macropodids it is impossible to even guess whether or not this
muscle was tendinous in sthenurines. The longer moment arm
afforded by the more distal insertion on the femur might be
indicative of more powerful limb retraction and abduction, or it
might simply be the case that the different morphology of the
sthenurine ischium (see above) changed the previous nature of the
moment arm, and the more distal placement of the insertion is
merely compensating for this. Tree-kangaroos (Dendrolagus spp.)
also have a relatively distally placed adductor scar (see Figure
S2C), and a correspondingly large quadratus femoris muscle [38]. Wells and Tedford [12] suggested that the articular facets on the
Sthenurus distal femoral condyles would allow for a greater range
of knee motion than in Macropus. They also noted that the lateral
femoral condyle is markedly larger than the medial one in both
Macropus and Sthenurus. However, both condyles are elongated The femoral shaft is curved in both Macropus and sthenurines,
but the orientation of the femur is slightly different, so that the
knee points medially in Macropus and laterally in sthenurines [42]. The greater trochanter, the major point of insertion of the gluteal
muscles, is large in both Macropus and the larger sthenurines: this
may relate partly to body size, as smaller sthenurines (e.g., S. Descriptive anatomy of sthenurine kangaroos The head of the femur is proportionally
large in sthenurines (matching the enlarged acetabulum, see
above): tree-kangaroos (Dendrolagus spp.) also have relatively
large femoral heads, as do Protemnodon spp. (see Figure S2A). The shape of the femoral head is round in sthenurines, rather than
ovoid as in Macropus (see Figure 4). The more ovoid morphology
of Macropus is the derived one among macropodoids, and is likely
related to restricting femoral motion to a parasaggital plane, as
also seen among cursorial bovids [41]. The neck of the femur is
also elongated in Macropus, which may increase the moment arm
for the gluteal muscles, again reflecting cursorial adaptations. The femoral shaft is curved in both Macropus and sthenurines,
but the orientation of the femur is slightly different, so that the
knee points medially in Macropus and laterally in sthenurines [42]. The greater trochanter, the major point of insertion of the gluteal
muscles, is large in both Macropus and the larger sthenurines: this
may relate partly to body size, as smaller sthenurines (e.g., S. andersoni) have a proportionally smaller greater trochanter [12]. However, Wells and Tedford [12] also note that the greater
trochanter is relatively longer, and more closely aligned with the
axis of the femoral shaft, in Sthenurus. This echos the point made
above, that a larger volume of gluteal musculature would enable
sthenurines to balance their body weight over one leg: in humans,
larger superficial gluteal muscles are important in preventing
collapse of the body medially when the weight is borne on one leg
[43]. Femur (Figure 4). The head of the femur is proportionally
large in sthenurines (matching the enlarged acetabulum, see
above): tree-kangaroos (Dendrolagus spp.) also have relatively
large femoral heads, as do Protemnodon spp. (see Figure S2A). The shape of the femoral head is round in sthenurines, rather than
ovoid as in Macropus (see Figure 4). The more ovoid morphology
of Macropus is the derived one among macropodoids, and is likely
related to restricting femoral motion to a parasaggital plane, as
also seen among cursorial bovids [41]. The neck of the femur is
also elongated in Macropus, which may increase the moment arm
for the gluteal muscles, again reflecting cursorial adaptations. Femur (Figure 4). The head of the femur is proportionally
large in sthenurines (matching the enlarged acetabulum, see
above): tree-kangaroos (Dendrolagus spp.) also have relatively
large femoral heads, as do Protemnodon spp. (see Figure S2A). Descriptive anatomy of sthenurine kangaroos This
difference in anatomy results in a markedly different shape of the
obturator foramen, which is elongated and ovoid in Macropus,
moderately oval in most other macropodoids, and circular/
triangular in sthenurines (and also in Dendrolagus spp.) (see
Figure 3). This dorsal tipping of the ischium markedly repositions
the moment arm for the hamstring muscles, especially for the
biceps femoris, which originates from the ischial tuberosities [36]. Wells and Tedford [12] note that the ischial anatomy in
sthenurines would increase the area of origin of the quadratus
femoris, which acts as a femoral adductor, and this could be
important in preventing the legs from spreading when standing
upright. Figure 4. Femur. (A) Simosthenurus occidentalis (SAM: P17259). (B)
Macropus sp. (SAM: P17270). All left side: upper = proximal articular
view; lower = lateral distal view. Scale bar = 5 cm. doi:10.1371/journal.pone.0109888.g004 A notable difference in the pelvic area is in the size of the
epipubic bones (see Figure 3). The epipubics of Sthenurus are
almost as long as the ilium, but are no more than half the length of
the ilium in Macropus [12]. The epipubics of Macropus are
possibly somewhat reduced over the primitive macropodoid
condition, but in Dendrolagus spp. the epipubics are of similar
relative size to those of sthenurines (see Figure S1D). The prime
function of the marsupial epipubic bones, to which the pectineus,
pyramidalis and hypaxial muscles attach, is to stiffen the trunk
during locomotion: the epipubics act part of a kinetic linkage
between the femur and the hypaxial muscles, resisting torsion and
diagonal stress across the trunk [40]. Wells and Tedford [12] note that the lesser trochanter is
‘‘weaker’’ in Sthenurus, but it is placed more distally on the
femoral shaft in sthenurines than in other macropodoids (although
tree-kangaroos are more like sthenurines), increasing the moment
arm for the iliopsoas (see Figure S2B). Note that the area for the
origin of the iliacus on the medial surface of the iliac blade is also
greater in sthenurines. It is not completely clear what the function
might be of a more powerful iliopsoas in sthenurines: it may relate
to different mechanics of femur protraction if the femur is being
held in a more vertical position (i.e., with an upright trunk),
because the moment arm of the iliopsoas would be less favorable
with a limb in this position. g
[
]
Femur (Figure 4). Descriptive anatomy of sthenurine kangaroos andersoni) have a proportionally smaller greater trochanter [12]. However, Wells and Tedford [12] also note that the greater
trochanter is relatively longer, and more closely aligned with the
axis of the femoral shaft, in Sthenurus. This echos the point made
above, that a larger volume of gluteal musculature would enable
sthenurines to balance their body weight over one leg: in humans,
larger superficial gluteal muscles are important in preventing
collapse of the body medially when the weight is borne on one leg
[43]. Wells and Tedford [12] suggested that the articular facets on the
Sthenurus distal femoral condyles would allow for a greater range
of knee motion than in Macropus. They also noted that the lateral
femoral condyle is markedly larger than the medial one in both
Macropus and Sthenurus. However, both condyles are elongated October 2014 | Volume 9 | Issue 10 | e109888 PLOS ONE | www.plosone.org 6 Locomotion in Sthenurine Kangaroos position. Wells and Tedford [12] note a longer and more robust
medial malleolus in Sthenurus than in Macropus, and an articular
groove that is more of an ‘‘oblate cup’’ in shape than the ‘‘shallow,
arcuate’’ form in Macropus. They comment that this morphology
would mean a more constrained tibioarticular articulation in
Sthenurus, but they do not specifically note a unique morphology
of the sthenurine distal tibia: that is, of a plantar process that fits in
a tongue-in-groove linkage into the astragalar trochlea (see
Figure 5). This morphology can also be observed in the Miocene
sthenurine Hadronomas (NT 2469: personal observation of senior
author). in the anteroposterior direction in sthenurines, giving them a more
elliptical shape than in Macropus (see Figure 4). More important-
ly, sthenurines have a greater width across the distal femoral
condyles than do macropodines: that is, they have relatively bigger
knees, as well as relatively bigger hip joints (see Figure S2D). Tree-
kangaroos also have relatively broader knees than other macro-
podines (see Figure S2D). in the anteroposterior direction in sthenurines, giving them a more
elliptical shape than in Macropus (see Figure 4). More important-
ly, sthenurines have a greater width across the distal femoral
condyles than do macropodines: that is, they have relatively bigger
knees, as well as relatively bigger hip joints (see Figure S2D). Tree-
kangaroos also have relatively broader knees than other macro-
podines (see Figure S2D). Tibia and Fibula (Figure 5). Descriptive anatomy of sthenurine kangaroos All macropodids have rela-
tively long tibia, up to twice the length of the femur. Tibial length
scales with positive allometry in macropodids in general [3,11]. Tibia lengths are shorter in Dendrolagus spp. and taxa that rely on
more quadrupedal (pentapedal) locomotion, such as the New
Guinea forest-wallabies (Dorcopsis and Dorcopsulus spp.). Sthe-
nurines
have
tibiae
of
comparable
lengths
to
generalized
macropodids, but the extinct Protemnodon spp. have relatively
short tibiae [11]. With regards to the fibula, Wells and Tedford [12] note that
Sthenurus lacks the distinctive posterior process of the head of the
fibula seen in Macropus, which apparently allows for greater
flexion of the knee. They conclude that this morphology may
relate a longer groove in the proximal tibia for the insertion of the
fibula, and interpret this as allowing for a greater internal rotation
of the lower limb about the knee. They relate this to the notion of
to sthenurines needing to achieve greater limb rotation to position
their feet medial when landing while hopping, as their wider pelvis
would otherwise result in more lateral placement of the feet. However, this could also relate to a rotation of the body around
the knee when the foot was placed on the ground, as would be
experienced with bipedal striding. [
]
On the proximal tibia, Wells and Tedford [12] note that the
lateral and medial condyles are of approximately equal size in
Sthenurus and Macropus, but did not comment on the elongation
of the tibial tuberosity in Macropus (which is derived relative to
other macropodoids) (see Figure S3A). The enlarged tibial
tuberosity of Macropus goes along with the greater size of the
proximal portion of the tibial (cnemial) crest. Murray [42] notes
that the macropodines and sthenurines differ in tibia diaphysis:
macropodines have a sharply defined tibial crest that is limited to
the proximal quarter of the bone, terminating in a distinct notch,
and the anterior profile of the tibia is straight; sthenurines have a
elongated crest that is convex in profile, and blends into the more
distal shaft, and the anterior profile of the tibia tends to be sinuous
(especially in Procoptodon). By comparison with the potoroine
condition, Murray [42] concluded that it is the macropodine
condition that is the derived one. Locomotion in Sthenurine Kangaroos The fibular facet on the calcaneum is more prominent in
Macropus than in Sthenurus, and is also located in a more medial
position on the tarsus (see Figure 6). The generalized macropodoid
condition appears to be for a facet that is less prominent, but
located in a medial position as in Macropus. The facet is more
prominent in sthenurines than in many macropodids, which
suggests that the facet has been displaced laterally in sthenurines. Bishop [44] interprets the position of the sthenurine fibular facet as
enabling greater ability to pronate the foot, which could be
important in terms of bearing weight on the medial side of the
foot, and notes that this motion would necessitate the observed
sthenurine morphology indicating powerful ligaments binding
together the astragalus and calcaneum. Murray [42] also interprets
a suite of astragalocalcaneal features as relating to bearing weight
more on the medial side of the foot (including the plantar crest of
the calcaneum being more elongated on the medial side), seen in
Hadronomas as well as in more derived sthenurines. However, if
both feet were habitually landing on the ground at the same time,
as in almost all other macropodoids, there would be little need to
favor one side of the foot for weight-bearing. Humans (as opposed
to apes) have a suite of morphological adaptations related to the
shifting of their weight to the medial side of the foot during
locomotion [43]. This feature of sthenurines may again be
indicative of weight bearing on one leg at a time. Macropodines in general also have a fairly broad medial
malleolar process (for the reception of the medial malleolus of the
tibia). This process is large in Dendrolagus spp., but more
restricted in Macropus, and narrower still in sthenurines: this goes
along with the derived sthenurine feature of a larger and more
posteriorly-directed navicular facet [42]. This morphology appears
to be related to intratarsal mobility, being greater in Dendrolagus
spp., somewhat restricted in Macropus, and even more restricted
in sthenurines. Murray [42] also notes the derived condition in
sthenurines of a more mesially directed and proximally short
astragalar head. In the calcaneum, Macropus has a much longer calcaneal tuber
than that of Sthenurus, and the orientation differs: the calcaneal
tuber in Macropus is orientated straight up and slightly posteriorly,
while in Sthenurus it is curved slightly forwards (see Figure 6). Locomotion in Sthenurine Kangaroos Locomotion in Sthenurine Kangaroos than in Sthenurus. A greater degree of rotation of the axis of the
astragalus is seen in other macropodoids, in particular Dendrola-
gus spp. (see [44,45]). Both the morpohology and the degree of
angulation of the astragalus in sthenurines appear to be derived
features among macropodids, also seen in the Miocene sthenurine
Hadronomas [42], and to a certain extent in the smaller and more
primitive Miocene sthenurine Rhizosthenurus [16]. The sthenur-
ine orientation of the axis of the astragalus is likely related to a foot
more restricted to anteroposterior motion, with compressive
stresses being directed more anteroventrally [12]. Bishop [44]
also notes a much greater prominence in sthenurines of the points
of insertion of the ligaments binding the astragalus and calcaneum. Dendrolagus spp., but is more constricted in sthenurines (see [42]). The transverse plantar sulcus, on the lateral side of the calcaneum,
is relatively narrow in sthenurines, reflecting the generalized
macropodid condition. This sulcus is broader in Macropus and
other cursorial kangaroos such as Onychogalea; it houses the
tendon of the peroneus longus, which runs from the craniolateral
shaft of the fibula, as it passes over the calcaneum to the plantar
surface of the foot to insert on the lateral surface of the first
metatarsal. Its action appears to be to both flex the ankle and to
counteract the action of the tibialis anterior in preventing evulsion
of the pes [46]. The convergent morphology between Macropus
and Onychogalea indicates that this anatomy relates functionally to
more rapid and/or sustained hopping, and may reflect increasing
need for the control of the foot position on landing with frequent
and rapid limb return. Macropus also has an astragalus that is elongated in the
proximodistal direction, which may relate to a greater excursion of
the tibia over the foot during rapid hopping. This is not paralleled
in other more cursorial kangaroos. Sthenurines have an especially
highly raised medial trochlear ridge, paralleling the condition in
horses, and a long lateral trochlear ridge (see Figure 6) (both noted
as being derived features by Murray [42]). The fibular facet on the
astragalus in sthenurines, which is on the side of the lateral
trochlear ridge, is also broader in sthenurines than in other
macropodids (see Figures 6, S3B). Locomotion in Sthenurine Kangaroos A
relatively short calcaneal tuber is the generalized condition in
macropodines, and longer tubers have been evolved convergently
in more cursorial kangaroos, such as Petrogale, Lagorchestes, and
Onychogalea (see Figure S3C). A deep body to the tuber (in the
dorsoventral plane) is also a sthenurine feature: Murray [42] notes
that most macropodoids have a tuber that is either shallow at the
base or, as in Macropus, broad at the base but tapering towards
the tip, but is unable to determine the polarity of this feature. However, a very distinctive and derived sthenurine feature is the
great broadening of the tip of the tuber in the mediolateral
direction (which is paralleled to a certain extent in Dendrolagus
spp.) (see Figure 6). In Dendrolagus, this calcaneal morphology is
associated with a short Achilles tendon [45]. Bishop [44] notes this
feature, but also notes that the more proximal portion of the tuber
in sthenurines is relatively narrow in the mediolateral plane, and
concludes that this indicates forces acting upon the calcaneal tuber
as being largely in the sagittal plane, implying less adaptation to
fast locomotion in sthenurines than in Macropus. The sustentaculum tali, on the medial side of the calcaneum, is
where the weight of the animal’s body is transmitted from the
astragalus to the calcaneum, and from there to the foot. The
sustentaculum is taller in the dorso-plantar direction in both
Macropus and Sthenurus than in other macropodids, giving the
calcaneum an asymmetric ‘‘hunched shoulders’’ appearance when
viewed from the plantar side. A larger sustentaculum would
indicate greater capacity for weight transmission, either from a
larger body size and/or from greater forces encountered in rapid
locomotion. However, the shape of the sustentaculum is notably
different in sthenurines than in macropodines. Bishop [44] notes
that it is narrower (in the mediolateral direction) in sthenurines,
and proposes this as part of a suite of adaptations that allow for
plantar flexion when the foot is internally rotated. In medial
aspect, the sustentaculum is dorsoplantarly deep and right-angled
in sthenurines, while in macropodines it is narrower with the distal
border orientated at a 45o angle. Descriptive anatomy of sthenurine kangaroos The tibial crest serves as the
insertion point both for the tibalis anterior (which flexes the foot)
and for the gracilis, which abducts the leg [36]. A shorter, more
prominent tibial crest would concentrate the origin of the tibialis
anterior proximally, and may relate to the ability for more rapid
foot flexion. Astragalus and Calcaneum (Figure 6). In concert with the
tongue-in-groove fit of the distal tibia into the astragalus, the
astragular trochlea groove is much deeper in sthenurines than in
any other macropodoid, with high medial and lateral ridges (see
Figure 6). Wells and Tedford [12] note that the axis of the
astragalus differs between Sthenurus and Macropus: the astragalus
is at a right angle to the longtitudinal axis of the pes in Sthenurus,
but is rotated medially in Macropus by as much as ten degrees,
and with a less robust connection of astragalus and calcaneum Figure 6. Astragalus and calcaneum. (A) Simosthenurus occidentalis
(SAM: P17258 [reversed]). (B) Macropus giganteus (SAM: P17523
[reversed]). All left side: upper = proximodorsal view; lower = lateral
view. Scale bar = 2 cm. doi:10.1371/journal.pone.0109888.g006 On the distal tibia, Murray [42] notes that the tibioastragalar
joint is rotated in an anteriomedial direction in sthenurines, which
he ascribes to a compensation for the outwardly-rotated knees, and
a morphology which would rotate the feet to be in a more medial Figure 5. Tibia. (A) Procoptodon goliah (NMV2010). (B) Macropus
giganteus (AMNH 2390). All left side: upper = distal articular view
(plantar side upwards); lower = posterior (plantar) view, showing
articulation with tarsus. Scale bar = 5 cm. doi:10.1371/journal.pone.0109888.g005 Figure 5. Tibia. (A) Procoptodon goliah (NMV2010). (B) Macropus
giganteus (AMNH 2390). All left side: upper = distal articular view
(plantar side upwards); lower = posterior (plantar) view, showing
articulation with tarsus. Scale bar = 5 cm. doi:10.1371/journal.pone.0109888.g005 Figure 6. Astragalus and calcaneum. (A) Simosthenurus occidentalis
(SAM: P17258 [reversed]). (B) Macropus giganteus (SAM: P17523
[reversed]). All left side: upper = proximodorsal view; lower = lateral
view. Scale bar = 2 cm. doi:10.1371/journal.pone.0109888.g006 October 2014 | Volume 9 | Issue 10 | e109888 October 2014 | Volume 9 | Issue 10 | e109888 PLOS ONE | www.plosone.org 7 October 2014 | Volume 9 | Issue 10 | e109888 Locomotion in Sthenurine Kangaroos In Dendrolagus the
ventromedian facet is indistinct and merged with the dorsolateral
facet, a morphology that Warburton and Prideaux [45] interpret
as allowing for a greater amount of inversion and eversion of the
foot than in terrestrial kangaroos. In sthenurines all of the facets,
and especially the ventromedian facet, are elongated in the
dorsoplantar direction (i.e., a large measure C21 as seen in
Figure 9K), resulting in a square profile of the distal surface. Interestingly, the large species of Protemnodon did not broaden the
ventromedian facet in this fashion (e.g., in Flinders University
specimen 1611; personal observation of senior author), suggesting
that large size alone is not the reason for this change in
morphology. The distal articular surface of the fourth metapodial is fairly
square-shaped in most macropodoids, but in sthenurines not only
is the distal surface proportionally larger, but it is more
mediolaterally elongated (see Figure 7). This is reflected in the
rectangular profile of the proximal articular surface of the
proximal phalanx of the fourth pedal digit [42]. Sthenurines also
have a less prominent distal metapodial keel than Macropus [42]. This might reflect a greater amount of movement of the phalanges
on the metapodial (see [49]), although the potential functional
reasons for this are unclear. Metatarsals and Phalanges (Figure 7). The fourth meta-
tarsal is long and curved in Macropus, with a prominent posterior
bulge (the plantar crest) that extends down the proximal third of
the bone. The metatarsals are also curved in Sthenurus, but
appear to be proportionally somewhat shorter, and Wells and
Tedford [12] note that the cross-sectional area in Sthenurus is
from 1.2 to 1.5 times larger than would be predicted for a
Macropus of similar size. The plantar crest is deeper in sthenurines
and extends further down the length of the bone. This crest serves
as the insertion for the origin of interosseus muscles, and is reduced
in placentals with an unguligrade stance where these muscles have
been reduced to ligaments [48]. The phalanges of sthenurines are distinctive: the proximal and
middle phalanges appear ‘‘waisted’’, with expanded proximal and
distal ends, but a narrower median portion. The proximal phalanx
of Macropus is relatively long, almost twice the length of the
second phalanx, while in Sthenurus the second phalanx is around
80% of the length of the first. Locomotion in Sthenurine Kangaroos The generalized macropodine condition, as seen for example in
Dorcopsulus and Setonix, is for relatively short metatarsals; the
metatarsals of sthenurines are elongated
over this general
condition, but they tend to be shorter than those of Macropus
and other more cursorial macropodines such as Petrogale,
Onychogalea, and Lagorchestes (see Figure S3D), where this
morphology has evolved convergently several times [42]. The
metatarsals of tree-kangaroos (Dendrolagus spp.) have been
shortened, more so in the New Guinea species than in the
Australian ones [45]. Protemnodon spp. also have fairly short
metatarsals. morphology has been considered important as an adaptation for
hopping, in limiting movement between the calcaneum and the
cuboid [44]; but note that this morphology is also seen in the
musky rat-kangaroo (Hypsiprymnodon moschatus), which is con-
sidered to be a primarily non-hopping form [21], and is retained
(although reduced) in Dendrolagus spp. [44,45]. A stepped
calcaneum is also seen among the balbarids, supposedly non-
hopping macropodoids [47]. Sthenurines retain the more gener-
alized macropodoid condition, while in Macropus the ‘‘stepping’’
is more pronounced and the dorsolateral facet is wider in the
mediolateral direction. The proximal articular surface of the fourth metapodial
(articulating with the cuboid) has a fairly flat anterior profile in
most macropodoids, while in sthenurines this surface curves
posteriorly both medially and laterally, resulting in a more plantar
(versus more lateral) positioning of the intra-articular groove [42]. The facets for the articulation with the ectocuneiform in the tarsus
are larger in sthenurines than in macropodines (see Figure 7A). For a similarly sized bone, the proximal articular surface is about
50% larger in Sthenurus than in Macropus [12], and the plantar
eminence forms around one third of the width of the posterior
proximal surface, but less than 20% in Macropus. Sthenurines
exhibit a derived condition for macropodids for all of these
features (see Figure 7). All of these differences between sthenurines
and macropodines indicate a relatively larger ankle joint in
sthenurines. In distal view, the generalized macropodine condition of the
calcaneum (as seen, for example, in Dorcopsulus and Setonix) is for
equal-sized dorsolateral and dorsomedial facets, with a relatively
narrow (in the dorsoplantar direction) ventromedian facet giving
the distal surface a rectangular profile. Locomotion in Sthenurine Kangaroos Bishop [44] interprets this
morphology as preventing the medial dislodgement of the tendon
of the flexor digitorum longus (which passes over the top of the
sustentaculum), which could be important in the act of elevating
the foot to stand on the toes (i.e., in moving from a plantigrade,
resting, posture to a digitigrade, locomotor posture). The calcaneal tuber serves as the insertion of the gastrocnemius
muscle, and also for a portion of the semitendinosus (at least in
Macropus giganteus [36]), via the Achilles tendon. A longer
calcaneal heel will increase the moment arm of the gastrocnemius:
this will not only provide more power for the hop, but will also
allow for a greater amount of elastic energy storage in the
gastrocnemius tendon. On the distal calcaneum, all macropodoids have a ‘‘stepped’’
cubonavicular facet, with the dorsolateral facet being projected
more ventrally than the dorsomedial or ventromedian facets. This The sulcus on the medial side of the calcaneum for the flexor
digitorum longus is fairly broad in most macropodids, especially in October 2014 | Volume 9 | Issue 10 | e109888 October 2014 | Volume 9 | Issue 10 | e109888 PLOS ONE | www.plosone.org 8 Speculations on sthenurine locomotor anatomy Despite the potential biomechanical problems of hopping
locomotion in large kangaroos, the ability of sthenurines to use
hopping as a mode of locomotion has rarely been questioned (but
see [2,31]). Sthenurines possess the elongated hind limbs (with an
especially long tibia) seen in extant large kangaroos, which have
been interpreted as an adaptation for hopping [11,12], but this
anatomy was inherited from their macropodid ancestry: many
sthenurine species were of such large size that the biomechanics of
hopping are rendered implausible [2,31]. While Wells and Tedford [12] did not question the notion of
hopping in sthenurines, they did note that the slow, pentapedal
gait of macropodines was likely impossible. As a consequence of
the modification of the forelimb for browsing in sthenurines, there
was limited ability for dorsiflexion of the hand, so they would have
had difficulty in placing their hand on the ground in the requisite
palmigrade position. The hands are also highly specialized, with
extremely long third phalanges, and they appear unsuited to
weight bearing. Sthenurus also has a smaller olecranon process for
the insertion of the triceps than large species of Macropus, which
Wells and Tedford [12] interpreted as limiting the ability to
support the anterior body weight over the hands, or to provide
propulsion with the forelimbs. The anterior caudal vertebrae in
Sthenurus have reduced processes for muscle attachment in
comparison with Macropus [12], implying less tail musculature
and perhaps a tail that is no longer used to propel the body as in
pentapedal locomotion. Also note that pentapedal locomotion
involves considerable flexion of the backbone, which appears to be
limited in sthenurines (see below). A problem thus arises when
considering sthenurine locomotion over a variety of speeds. If they
just had a ‘‘greater dependence on bipedal saltation’’, as proposed
by Wells and Tedford [12], p. 85, then how did they manage to
locomote at slow speeds? As discussed previously, hopping is
difficult if not impossible at slow speeds, and large modern
macropodines employ pentapedal locomotion up to speeds of
around 3 m/sec [18,28]. One possible reason for the lack of questioning about the mode
of locomotion of sthenurines is the issue of monodactyly. This has
been seen as analogous to the attainment of monodactyly in the
equid lineage, and thus indicative of a highly cursorial lifestyle,
rendering sthenurines even more specialized hoppers than the
large macropodines (see [52], p. Speculations on sthenurine locomotor anatomy 59), although Wells and Tedford
[12] later emended that conclusion to perceiving sthenurines as
being slow hoppers. However, despite their monodactyl pes, other
skeletal
modifications
of
sthenurines
are
less
indicative
of
adaptations for faster locomotion: we later propose an alternative,
non-locomotor, explanation for monodactyly in sthenurines. The apparent ‘‘robusticity’’ of sthenurines has often been noted,
but is not well understood. Wells and Tedford [12] proposed that
the robusticity relates to them having to support their ‘‘great
weight’’ in a bipedal browsing posture, where they are envisaged
to have risen up on their hind legs, gerenuk-style, and reached
over their heads with their arms. However, why should it be the
case that sthenurines were so much more heavily build than
macropodines of similar linear dimensions? A speculation that has
been raised is that, because sthenurines were browsers, they had to
support a massive gut (and this reason is also given for the larger
distance between the pelvic acetabulae and for the massive
epipubic bones in sthenurines) (e.g., Wells and Tedford [12]). But
this hypothesis is problematic: not only are kangaroos foregut
fermenters (so any ‘‘massively enlarged’’ portion of their gut would
be well anterior to the pelvic region) but, in ungulate placental
mammals at least, it is the gut fermentation areas of grazers, rather
than browsers, that are the more massive [53]. Consider the black
rhino
(Diceros
bicornis,
a
browser)
and
the
white
rhino
(Ceratotherium simum, a grazer): they are of a similar size
(although the black rhino is slightly smaller), but there is no
indication that there is any difference in the relative robusticity of
their skeletal framework. Thus, on the basis of diet alone, one
would expect the large grazing species of Macropus to be the more
robust forms, which is clearly not the case. In addition modern
kangaroos show little capacity for gut expansion [54]. We propose here that sthenurine kangaroos employed a novel
type of gait, certainly at slow speeds and likely also at faster speeds
in larger species: that of bipedal striding with a relatively upright
trunk. This gait is not unique among macropodids, as it is
occasionally seen in tree-kangaroos, walking along a branch [23]. This is not to imply that sthenurines, especially the smaller ones,
never employed a hopping gait, but that the addition of this gait to
their locomotor repertoire can explain many of the anatomical
peculiarities of these animals. Locomotion in Sthenurine Kangaroos Locomotion in Sthenurine Kangaroos and powerful than those in other macropodoids (and also apparent
in Hadronomas), represent more torque being placed on the first
phalanx. why would this feeding posture necessitate more robust limbs? The
weight of the animal would be the same whatever its posture
(although admittedly some of the directional forces would be
different). It seems more likely that some sort of dynamic forces
applied during locomotion would be the issue necessitating more
sturdy support. The hopping gaits of extant kangaroos mean that
both hind feet are always applied to the ground at the same time. In human running the vertical ground reaction forces applied to
each foot on landing are between two and three times the body
weight [55]. Does kangaroo-style hopping mean that this reaction
force is evened out over both hind feet? If so, perhaps more robust
limbs reflect a locomotor shift to bearing weight on one foot at a
time. Speculations on sthenurine locomotor anatomy In the previous section we discussed
the anatomical differences between sthenurines and large macro-
podines, and proposed that the differences in sthenurine anatomy
from other large kangaroos can be related to supporting the body
weight during locomotion over a single hind leg. In following
sections we present analyses of anatomical data comparing
sthenurines with other kangaroos (both extant and extinct), and
discuss how our results support this hypothesis. A clue to this issue of ‘‘robusticity’’ may be found in the
proportions of a third lineage of kangaroos to reach ‘‘giant’’
proportions: species of the extinct macropodine Protemnodon. Large species of Protemnodon showed similar robusticity to the
sthenurines, despite a very different postcranial anatomy to either
sthenurines or large species of Macropus (and with craniodental
anatomy indicative of a mixed-feeding diet). This raises the issue of
what is the normal allometric scaling for kangaroos: we will discuss
later the likelihood that the real issue is that it is the large species of
Macropus that are relatively gracile, with sthenurines and
Protemnodon spp. representing the ‘‘normal’’ condition. Locomotion in Sthenurine Kangaroos In comparison with other
macropodids, this appears to represent a relative enlargement of
the medial phalanx in Sthenurus, in addition to the lack of
lengthening of the proximal one as seen in Macropus. The
generalized macropodid condition is for a relatively short proximal
phalanx, with elongation seen convergently in more cursorial
kangaroos, such as Petrogale, Onychogalea, and Lagorchestes,
while the
proximal
phalanx
is shorter
and
broadened
in
Dendrolagus spp. A lengthened proximal phalanx is also seen in
more cursorial ungulates (hoofed placentals), where it apparently
relates to a lengthening of the plantar tendons, increasing the
‘‘springiness’’ of the foot [50]. Figure 7. Fourth metatarsal. (A) Sthenurus stirlingi (AMNH 117496). (B) Macropus giganteus (AMNH 2390 [reversed]). All left side: upper =
proximal articular view; lower = distal articular view (plantar side
downwards). Scale bar = 2 cm. doi:10.1371/journal.pone.0109888.g007 The ungual phalanx is curved and rather claw-shaped in most
macropodoids, being narrow in the mediolateral direction. More
distinctive claws are seen in the species of Dendrolagus. In
contrast, the ungual phalanx in sthenurines is more blunt and
rounded, described as ‘‘hoof-like’’ by Kear et al. [51]. Similar
ungual phalangeal morphology is seen in the earlier (Miocene)
sthenurines Hadronomas and Rhizosthenurus, and convergently
among Protemnodon spp. [51]. Figure 7. Fourth metatarsal. (A) Sthenurus stirlingi (AMNH 117496). (B) Macropus giganteus (AMNH 2390 [reversed]). All left side: upper =
proximal articular view; lower = distal articular view (plantar side
downwards). Scale bar = 2 cm. doi:10.1371/journal.pone.0109888.g007 Wells and Tedford [12], and Murray [42], note well-developed
scars for the plantar cruciate ligaments of the pes in Sthenurus:
Murray [42] postulates that these scars, that are more separated October 2014 | Volume 9 | Issue 10 | e109888 October 2014 | Volume 9 | Issue 10 | e109888 PLOS ONE | www.plosone.org 9 Locomotion in Sthenurine Kangaroos Locomotion in Sthenurine Kangaroos Natural History (New York, NY, USA); The Australian Museum
(Sydney, NSW, Australia); the University of New South Wales
(Sydney, NSW, Australia); the Queensland Museum (Brisbane,
QLD, Australia); Museum Victoria (Melbourne, VIC, Australia);
the Western Australian Museum (Perth, WA, Australia); the
Northern Territory Museum and Art Galleries (Alice Springs, NT,
Australia); the South Australian Museum (Adelaide, SA, Austra-
lia); and Flinders University (Bedford Park, SA, Australia). a few other forms. For the purposes of seeing if the morphology of
modern kangaroo species fell within the range of the smaller
extinct taxa, we included information from the late Oligocene/
early Miocene balbarid Nambaroo gillespieae, the late Oligocene/
early Miocene Ngamaroo archeri (Macropodidae incertae sedis,
possibly basal to macropodids above the level of the potoroines),
and the late Miocene macropodine Dorcopsoides sp. Taxonomy
was taken from Prideaux and Warburton [4]. For the purposes of
comparing sthenurines with other large kangaroos, we included
information from several species of the Pleistocene macropodine
Protemnodon. We also included information from two ‘‘giant’’ (i.e.,
larger than living forms) Pleistocene species of the extant genus
Macropus: M. titan and M. ferragus. The details of the specimens
measured are presented in Table S3, S4. Some of the specimens
used in the multivariate analyses are represented by a composite of
different individuals (see Table S3 and figure captions). );
y (
,
,
)
The measurements included 22 from the pelvis, 24 from the
femur, 15 from the tibia, 10 from the astragalus, 9 from the
calcaneum (plus an additional 16 for a further analysis), and 14
from the pes (see Table S2 and Figures 8, 9; the original
measurements are available in Table S5). We did not take
measurements of the fibula because of the rarity of preservation of
complete fibulae in the fossil record. Measurements of the fifth
metatarsal were taken, but not included in the analyses (to avoid
the possibility that sthenurines would be grouped merely on the
fact that their fifth metatarsal is vestigial). However, we did
determine that there is no reduction of the fifth metatarsal with
increasing cursoriality in extant macropodids. In a few instances of
missing data from extant taxa (usually involving the distal phalanx
of the fourth pedal digit) measurements were added from related
and similarly sized individuals (to prevent these incomplete
individuals from being excluded from the multivariate analyses;
details provided in Table S3). Locomotion in Sthenurine Kangaroos We also created a separate data set
with 16 additional calcaneal measurements (with only some
overlap with the first one in terms of the individuals sampled). This
included 70 individuals: 44 extant forms (including 33 species,
again sampling all extant genera), and 26 extinct forms (including
10 sthenurines, eight Protemnodon spp., and representatives of the
smaller fossil forms mentioned below). (Details of the included taxa
are in Table S4, and the original measurements are available in
Table S6). Data We took linear measurements, taken with digital calipers, of 66
extant kangaroo individuals (belonging to 45 species) and 78
extinct kangaroo individuals (belonging to 18 genera) (see Tables
S2–4). All of the specimens measured were housed in accredited
museum collections: these include the American Museum of Another hypothesis for the more massive hind limb structures in
sthenurines is related to the supposed browsing posture, where the
animal must raise its body up over the hips (see [12], p. 78]. But October 2014 | Volume 9 | Issue 10 | e109888 PLOS ONE | www.plosone.org 10 Statistical analyses We performed both bivariate and multivariate analyses on the
linear measurements using the statistical package SPSS v. 19. We
created a number of bivariate plots of the different bony elements
of the skeleton, primarily to confirm the visual observations
discussed in the ‘‘Descriptive Anatomy’’ sections, and these are to
be found in the supplementary information, along with some
discussion of the distribution of taxa (Figures S1–S3). Certain
anatomical variables were plotted against a measurement of that
same bone that appeared to correlate best with body mass (as
determined from the PCA scores). Thus pelvic elements were
plotted again the iliac blade length, femoral elements (plus
metatarsal length) against femur length, tibial elements against
tibia average cross sectional diameter, and tarsal measurements
against astragalus width. These plots are presented for visual
inspection, and we have not attempted to demonstrate any
statistical significance. However, they clearly show the differences
between sthenurines and other macropodoids. Every genus and almost every species of extant macropodoid
was measured. Extinct taxa included not only sthenurines, but also Figure 8. Measurements used in analyses-1. Drawings, all of left side elements, primarily from photographs of Macropus fuliginosus, AMNH
2390. (A) Pelvis, lateral view. (B) Pelvis, ventral view. (C) Pelvis, dorsal view. (D) Femur, anterior view. (E) Femur, posterior view. (F) Femur, proximal
articular view (anterior of shaft downwards). (G) Distal femur, medial view. (H) Distal femur, lateral view. A detailed description of the measurements is
provided in Table S2. doi:10.1371/journal.pone.0109888.g008 Figure 8. Measurements used in analyses-1. Drawings, all of left side elements, primarily from photographs of Macropus fuliginosus, AMNH
2390. (A) Pelvis, lateral view. (B) Pelvis, ventral view. (C) Pelvis, dorsal view. (D) Femur, anterior view. (E) Femur, posterior view. (F) Femur, proximal
articular view (anterior of shaft downwards). (G) Distal femur, medial view. (H) Distal femur, lateral view. A detailed description of the measurements is
provided in Table S2. doi:10.1371/journal.pone.0109888.g008 October 2014 | Volume 9 | Issue 10 | e109888 PLOS ONE | www.plosone.org 11 Locomotion in Sthenurine Kangaroos Figure 9. Measurements used in analyses-2. Drawings, all of left side elements, primarily from photographs of Macropus fuliginosus, AMNH
2390, Calcaneum from Macropus giganteus, AMNH 74753). (A) Tibia, lateral view (fibula removed). (B) Tibia, proximal articular view (plantar side
downwards). (C) Tibia, distal articular view (plantar side upwards). (D) Astragalus, anterior (dorsal) view. (E) Astragalus, medial view. (F) Astragalus,
lateral view. (G) Calcaneum, anterior (dorsal) view. Statistical analyses (H) Calcaneum, posterior (plantar) view. (I) Calcaneum, lateral view. (J) Calcaneum (head only)
medial view. (K) Calcaneum, distal articular view (plantar side upwards). (L) Pes, anterior (dorsal) view. A detailed description of the measurements is
provided in Table S2. doi:10.1371/journal.pone.0109888.g009 Figure 9. Measurements used in analyses-2. Drawings, all of left side elements, primarily from photographs of Macropus fuliginosus, AMNH
2390, Calcaneum from Macropus giganteus, AMNH 74753). (A) Tibia, lateral view (fibula removed). (B) Tibia, proximal articular view (plantar side
downwards). (C) Tibia, distal articular view (plantar side upwards). (D) Astragalus, anterior (dorsal) view. (E) Astragalus, medial view. (F) Astragalus,
lateral view. (G) Calcaneum, anterior (dorsal) view. (H) Calcaneum, posterior (plantar) view. (I) Calcaneum, lateral view. (J) Calcaneum (head only)
medial view. (K) Calcaneum, distal articular view (plantar side upwards). (L) Pes, anterior (dorsal) view. A detailed description of the measurements is
provided in Table S2. doi:10.1371/journal.pone.0109888.g009 The multivariate analyses included both Principal Components
Analysis (PCA) of log-transformed variables and Linear Discrim-
inant Analysis. These were performed on the following sets of data. (i) All hind limb bones combined: this did not allow for the
inclusion of many extinct taxa, but three sthenurine species (of the
genera Sthenurus, Simosthenurus and ‘‘Procoptodon’’) could be
included, two of which were composite specimens (see Table S3
for details). (ii) All hind limb bones except the pelvis: this allowed
us to include many more extinct taxa, including species of
Protemnodon, and the sthenurine genera Hadronomas and
Procoptodon. (iii) On the calcaneum alone (using the separate
dataset). The calcaneum is a highly informative bone in terms of
locomotor behavior [44,56], but it is usually to be found bound to
the astragalus and distal tarsal bones in both extant and fossil
specimens: hence our separate analysis from specimens where an
isolated calcaneum was available. Two of the bivariate plots (Figure 10, showing the scaling of
femur and tibia diameter against the length of the same bones),
relate to the issue of ‘‘robusticity’’ in larger kangaroos discussed
previously, and are shown in the results section. They are also
included in Text S1 with a degree of taxa identification (Figure
S4), but are shown here without those labels for reasons of clarity. Here three different regression lines were created to examine
differences in scaling relationships (in all cases the extinct ‘‘giant’’
species of the extant genus Macropus [M. titan and M. October 2014 | Volume 9 | Issue 10 | e109888 Statistical analyses ferragus]
were omitted, as their placement in the analyses was more like that
of the sthenurines than that of the large extant species of
Macropus): (i) all of the taxa; (ii) extant species only; (iii) all of the
taxa except the extant species of the genus Macropus. As our interest here was in testing for differences in the
regression slopes for pair of groups (see above), we performed a
Student’s t-test between the coefficients (b) of the three regression
lines. The null hypothesis (of no difference between the slopes) will
be rejected if the regression slopes of each adjusted model are not
statistically significant different from each other. In addition, in
order to explore if different scaling relationships follow an
allometric trend or rather an isometric one, we performed a
Student’s t-test between the coefficients (b) of each regression line
and the theoretical value of a slope equals to one (i.e., the expected
coefficient for isometry when two linear measurements are
regressed). The null hypothesis (of isometric scaling) will be
rejected if the slope of a given bivariate regression model
significantly departs from unity, thus suggesting a significant
allometric trend. All the discriminant analyses were performed by the stepwise
approach. This approach was preferred over the direct method
because it only uses the best set of variables for discriminating
among the groups compared (e.g., [57–59]). The selection
criterion in the stepwise model was the inclusion of variables with
F probability between ,0.05–0.01 (depending on sample size and
the number of variables), and the exclusion of variables with F
probability.0.1. The first analysis was run with an F probability
,0.05 of inclusion and, if this analysis included too many variables
for the sample size of each specific analysis (see above), we
modified the F probability up to ,0.01. The F probability for
excluded variables was not modified in all of the analyses October 2014 | Volume 9 | Issue 10 | e109888 PLOS ONE | www.plosone.org 12 Locomotion in Sthenurine Kangaroos Figure 10. Scaling of long bone length versus diameter (i.e., robusticity). (A) Femur length versus average femur cross-sectional diameter
(B) Tibia length versus average tibia midshaft cross sectional diameter. The regression line and its 95% confidence interval (dotted lines) are also
shown (A r2 = 0.96, B r2 = 0.933). Statistical analyses Key: Open diamond = Hypsiprymnodon moschatus; filled circles = potoroines; open circles = extant species of
Macropus; half tone circles = extinct (‘‘giant’’) species of Macropus (M. titan or M. ferragus); filled diamonds = extant macropodines (other than
Macropus or Dendrolagus) and lagostrophines; open squares = species of Dendrolagus; filled stars = sthenurines; open stars = other extinct taxa. The regression line and its 95% confidence interval (dotted lines) are also shown. doi:10.1371/journal.pone.0109888.g010 Figure 10. Scaling of long bone length versus diameter (i.e., robusticity). (A) Femur length versus average femur cross-sectional diameter
(B) Tibia length versus average tibia midshaft cross sectional diameter. The regression line and its 95% confidence interval (dotted lines) are also
shown (A r2 = 0.96, B r2 = 0.933). Key: Open diamond = Hypsiprymnodon moschatus; filled circles = potoroines; open circles = extant species of
Macropus; half tone circles = extinct (‘‘giant’’) species of Macropus (M. titan or M. ferragus); filled diamonds = extant macropodines (other than
Macropus or Dendrolagus) and lagostrophines; open squares = species of Dendrolagus; filled stars = sthenurines; open stars = other extinct taxa. The regression line and its 95% confidence interval (dotted lines) are also shown. doi:10.1371/journal.pone.0109888.g010 t = 20.657; P-value = 0.5119): that is the inclusion of the extinct
kangaroos does not greatly affect the regression line that fits the
extant taxa. However, slope 2 is different from both other slopes at
the 99% level of significance (slope 1 vs. slope 2: n = 69; t = 3.294;
P-value = 0.001; slope 1 vs. slope 3: n = 69; t = 3.776; P-
value = 0.0002); that is, with the exclusion of the large extinct
species, the slope is significantly less steep. We interpret this to
mean that the femoral proportions of sthenurines and Protemno-
don spp. are following the ‘‘normal’’ allometric relationships for
kangaroos, and that the larger species of Macropus are acting to
pull the slope down to a lower level. performed. The power of the discriminant functions was evaluated
from the value of the Wilks’ lambda statistic (l), which measures
the proportion of the total variance explained by the within-groups
differences in the discriminant scores [60]. Results Tibia length versus tibia diameter (Figure 10B). Here
the scatter around the regression line is considerably greater than
for the femur proportions, and the 95% confidence limits of the
slope are much broader. (See Figure S4B for the confidence limits
for the regression model and the identification of some of the taxa). The slope for all of the taxa (excluding the extinct Macropus
species M. titan and M. ferragus, as previously discussed) is 0.983
(slope 1); the slope for the extant species only is 0.842 (slope 2); the
slope for all species excluding species of Macropus is 1.078 (slope
3). Slope 1 is not different from isometry at the 95% level of
significance (slope 1: n = 92; t = 20.595; P-value = 0.552). Both
slopes 2 and 3 are different from isometry at the 95% level of
significance (slope 2: n = 66; t = 24.7215; P-value,0.0001; slope
3: n = 69; t = 2.777; P-value = 0.0071): that is, slope 2 represents
negative isometry, and slope 3 positive isometry. Statistical analyses However, as this
statistic provides little information on the effectiveness of the
discriminant function for reclassifying the specimens, we assessed
the significance of this value by comparing it with the percentage
of correct assignments using the leave-one-out cross-validation
approach described in Mendoza et al. [57]. October 2014 | Volume 9 | Issue 10 | e109888 Multivariate analyses The three included sthenurine species have, unsurprisingly, high
scores on the first component, due to their large size. But what is of
great interest is that they also have high scores on the second
component, falling with the extant macropodoids that rarely or
never hop. (In fact, their negative scores put them within the
morphospace of the tree-kangaroos.) Principal Components Analysis of all hind limb bones
(Figure 11A, Table 1). The PCA performed from the dataset
including all the bones yielded two eigenvectors with eigenvalues
higher than 1.0, which jointly explained the 93% of the original
variance. This analysis proved excellent for distinguishing between
extant macropodoids that are more specialized hoppers from those
that are rare, or less specialized hoppers (such as tree kangaroos,
the non-hopping Hypsiprymnodon moschatus, and the forest and
woodland dwelling wallabies). The first PC (l = 79.263; 91.10% of
variance explained) is interpreted as a size vector because all the
variables had positive loadings and fairly high values (Table 2). However,
the
second
PC
(l = 1.543;
1.774%
of
variance
explained) is interpreted as a shape vector because not all the
loadings of the variables on this eigenvector were positive
(Table 1). The morphospace depicted from the scores of the
specimens on these two PCs is shown in Figure 11A and the factor
loadings of the variables on each eigenvector are shown in
Table 1. Table 1 shows the variables that are responsible for the
distribution of the taxa along the second principal component. The pelvis features prominently in the placement of the taxa with
negative scores: of prime importance are the width of the tuber
coxa (the dorsal tip of the ilium, indicative of a large origin of the
superficial gluteals and the cranial head of the caudofemoralis) and
the length of the epipubic bone. Also important are the size of the
ischial tuberosity (indicative of a large area for the origin of the
hamstrings), a large femoral head, a broad calcaneal tuber, and a
deep fibula facet on the astragalus. The only length measurement
with high loadings on the second component is the length of the
third phalanx of the fourth pedal digit: this seems to be reflecting
the long claws on the hind limbs of the tree-kangaroos. Locomotion in Sthenurine Kangaroos This is not merely an allometric issue as
the species of nail-tail wallabies (Onychogalea spp.), which are of a
similar size to the small species of Macropus, and which have
evolved more cursorial anatomy convergently with Macropus (see
previous discusson), have similarly negative scores on the second
component as the large species of Macropus. The only specimen
which appears to be ‘‘out of place’’ here is the brush-tailed bettong
or woylie, Bettongia penicillata: this potoroine is not notably
cursorial in its anatomy or behavior, but may be falling with low
scores on the second component because of relatively small
epipubic bones (see later discussion): note that, when the analysis is
performed without the pubis (Figure 11B) B. penicillata clusters
with the other potoroines. pull the regression line down to an overall negative allometric
scaling: that is, that large extant kangaroos have tibia that are
proportionally more slender in comparison with their length. Summary of bivariate analyses. We interpret these differ-
ent scaling relationships as follows: that it is not really the case that,
compared with other kangaroos in general, that the larger extinct
forms were ‘‘more robust’’; rather, it is the extant large species of
Macropus that are relatively gracile. To present this in a allegorical
fashion: if the only medium-to-large sized felid that survived today
was the cheetah, this would appear to be the ‘‘normal’’ form of a
large cat, and the bones of an extinct leopard would be seen as
being unexpectedly ‘‘robust’’. But with the known diversity of
extant felids, it is apparent that cheetah is a specialized gracile
form, probably representing morphological adaptations for speed
at a larger size. We propose that the same is true of the large
extant kangaroos: they are the ones who do not follow the
‘‘normal’’ kangaroo scaling relationships – they are the ones who
are exceptionally gracile while the sthenurines and Protemnodon
spp. have the expected proportions. We propose that modern large
species of Macropus are ‘‘cheetahs’’: they do not represent the
‘‘norm’’ for kangaroos in general, and their gracility is probably an
adaptation to allow them to maintain rapid hopping at a larger
body size than optimal for this type of locomotion. October 2014 | Volume 9 | Issue 10 | e109888 Locomotion in Sthenurine Kangaroos Locomotion in Sthenurine Kangaroos Macropus (i.e., ‘‘regular’’ kangaroos and wallabies). The species of
Macropus on Figure 11A can be identified as follows: small species
(M. parma, M. eugenii, and M. dorsalis), which cluster amongst
the other terrestrial macropodines; medium-sized species (M. rufogriseus, M. agilis, and M. parryi), which have slightly more
positive scores on the first component than any of the other
macropodines; and large species with the highest scores on the first
component (M. antilopinus, M. fuliginosus, M. giganteus, M. robustus, and M. rufus). Note how the scores of Macropus species
on the second component change with increasing size, with the
smaller species having the least negative scores, and the larger ones
the most negative scores. This is not merely an allometric issue as
the species of nail-tail wallabies (Onychogalea spp.), which are of a
similar size to the small species of Macropus, and which have
evolved more cursorial anatomy convergently with Macropus (see
previous discusson), have similarly negative scores on the second
component as the large species of Macropus. The only specimen
which appears to be ‘‘out of place’’ here is the brush-tailed bettong
or woylie, Bettongia penicillata: this potoroine is not notably
cursorial in its anatomy or behavior, but may be falling with low
scores on the second component because of relatively small
epipubic bones (see later discussion): note that, when the analysis is
performed without the pubis (Figure 11B) B. penicillata clusters
with the other potoroines. Macropus (i.e., ‘‘regular’’ kangaroos and wallabies). The species of
Macropus on Figure 11A can be identified as follows: small species
(M. parma, M. eugenii, and M. dorsalis), which cluster amongst
the other terrestrial macropodines; medium-sized species (M. rufogriseus, M. agilis, and M. parryi), which have slightly more
positive scores on the first component than any of the other
macropodines; and large species with the highest scores on the first
component (M. antilopinus, M. fuliginosus, M. giganteus, M. robustus, and M. rufus). Note how the scores of Macropus species
on the second component change with increasing size, with the
smaller species having the least negative scores, and the larger ones
the most negative scores. Bivariate analyses Femur length versus femur diameter (Figure 10A). The
slope for all the taxa (excluding the extinct Macropus species M. titan and M. ferragus, as previously discussed) is 1.334 (slope 1);
the slope for the extant species only is 1.179 (slope 2); the slope for
all species excluding species of Macropus is 1.378 (slope 3). (See
Figure S4A for the confidence limits for the regression model and
the identification of some of the taxa.) All of these lines are
different from isometry (expected slope of 1) at the 95% level of
significance, showing that the larger animals have relatively more
robust femora (slope 1: n = 95; t = 10.790; P-value ,0.0001; slope
2: n = 69; t = 6.3477; P-value ,0.0001; slope 3: n = 71; t = 9.819;
P-value ,0.0001). To a first approximation, slope 2 is the
regression line excluding the extinct ‘‘giant’’ kangaroos (which
tend to fall above the regression line for all of the taxa), and slope 3
is the regression line excluding the large extant, specialized fast-
hopping kangaroos (which tend to fall below the regression line for
all of the taxa). Slopes 1 and 3 are different from each other at the 95% level of
significance, while slope 2 is different from both of the other slopes
at the 99% level of significance. Thus, with the inclusion of the
sthenurines and Protemnodon spp. the relative width of the tibia is
scaling with isometry or slight positive allometry. But exclusion of
the extinct species means that the large living species of Macropus However, the differences between the slopes are interesting. There is no statistical difference between slopes 1 and 3 (n = 71; October 2014 | Volume 9 | Issue 10 | e109888 PLOS ONE | www.plosone.org 13 Multivariate analyses In contrast, length measurements figure prominently in the
negative loadings on the second component: most important is the
length of the fourth metatarsal and the first phalanx, with the
length of the tibia and second phalanx being less important. The
height of the iliopectineal process on the pelvis is an important
element on this axis of the second component, and this can be seen
especially in large species of Macropus. This serves as the area of
origin of the pectineus, a short muscle that would act to rapidly
move the leg forwards from a backwardly positioned femur, and
may be important for rapid recycling of the limb during hopping. Three other variables (length of puboischiatic symphysis, anterior-
posterior width of the obturator foramen, and the dorsal length of
the ischium) relate to an elongated posterior portion of the
ischium, which provides for a longer moment arm for the
hamstring and adductor muscle complexes, important in powerful
limb retraction during hopping (see previous discussion). Another
variable with negative loadings includes the size of the tibial crest,
reflecting a large area for the origin of the tibialis anterior, possibly
reflecting rapid foot flexion (see previous discussion). The most obvious feature of Figure 11A is that, among the
extant macropodoids, there is a division along the second
component between specialized hoppers (with negative scores)
and forms that either do not hop (the musky rat-kangaroo
Hypsiprymnodon moschatus), hop rarely (the tree-kangaroos,
Dendrolagus spp.), or which are unspecialized, forest-dwelling
hoppers (the New Guinea forest-wallabies, Dorcopis spp. and
Dorcopsulus spp., the quokka, Setonix brachyurus, and the
pademelons, Thylogale spp.) The Oligo-Miocene balbarid Nam-
baroo gillespieae also falls within this clustering, supporting the
hypothesis that these animals were not hoppers, or poor hoppers at
best [47]. The tree-kangaroos form a distinct cluster with higher
positive scores than any other macropodines: the one with the
obviously lower scores, Dendrolagus bennettianus, is one of the
more basal, Australian, members of this tribe (see [45]). The macropodoids that have negative scores on the second
component include the more specialized hoppers that are also
more open-habitat forms: these include the swamp wallaby
(Wallabia bicolor), the rock-wallabies (Petrogale spp.) the hare-
wallabies (Lagorchestes spp. Multivariate analyses Note that the position of the sthenurines on
component two is despite their being unlike the other taxa in
this position in many respects, in that they have variables with high
negative loadings on this component, such as a long tibia, and a
relatively long fourth metatarsal and first phalanx. negative loadings on this component. The sthenurines all have less
negative scores on the second component than the large species of
Macropus, including the extinct ‘‘giant’’ species M. titan. The
Miocene sthenurine Hadronomas puckridgi falls relatively close to
Macropus. Two sthenurines, unlike any extant form apart from
Dendrolagus spp., have slightly positive scores on the second
component: the largest, most specialized taxa, Procoptodon sp. (probably = P. goliath) and the somewhat smaller Simosthenurus
occidentalis. Note that Procoptodon sp. and S. occidentalis tend to
cluster together in all other analyses where both are included. A
couple of Protemnodon species are included in this analysis, and
they fall within the range of the less specialized sthenurines, likely
indicating less specialized hopping abilities rather than any
particularly sthenurine qualities. Principal
Components
Analysis
without
the
pelvis
(Figure 11B, Table 2). As the pelvis is rarely completely
preserved in fossil forms, we performed a second PCA, excluding
those variables measured on the pelvis, in order to include more
extinct taxa. Again, while the first PC (l = 60.734; 93.443% of
variance explained) was interpreted as a size vector according to
the loadings of the variables on this axis (see Table 3), the second
PC was interpreted as a shape vector (l = 0.672; 1.034% of
variance explained). Despite the fact that only the first PC had
eigenvalues higher than one, we also extracted the second PC
because it represents aspects of morphological differences among
the specimens. The morphospace depicted from the scores of the
specimens on these two PCs is shown in Figure 11B, and the factor
loadings of the variables on each eigenvector are shown in
Table 2. Table 2 shows the variables that are responsible for the
distribution of taxa along the second component. The most
positively loading variable is the length of the third phalanx of the
fourth digit, reflecting the long claws of the tree-kangaroos. Additional variables loading positively include a number of
measurements of the astragalus that indicate a broader joint
surface with greater rotational abilities of the ankle. Multivariate analyses Variables
which would seem to apply to the sthenurines as well as
Dendrolagus spp. include a larger femoral head, a broader and
wider calcaneal tuber, and the length of the gluteal insertion on the
femur, reflecting the large size of this muscle (which provides
stability over the hip joint) previously indicated by the large tuber
coxae. Despite the fact that the pelvis figured so prominently in the first
analysis, with its exclusion the taxa fall in a similar position within
the morphospace. One notable difference is the more negative
position along component two of the potoroos (Potorous spp.),
which are the least specialized hoppers among the potoroines. However, regarding the scores of the different taxa, only
Dendrolagus spp. have positive scores. Thus this analysis, more
so than the first one, is distinguishing between tree-kangaroos and
other forms. The position of the sthenurines on the second
component, in a similar position to the less-specialized extant
forms, largely reflects low values for the variables that have high New variables with negative loadings include a longer (in the
anterior-posterior direction) proximal articular surface of the tibia
(which reflects a longer tibial tuberosity, incorporated into the
value
of
a
higher
tibial
crest
on
the
tibia),
and
several
measurements of the length of the calcaneal tuber (reflecting the
moment arm for the gastrocnemius muscle, the primary foot
extensor). The most important of the calcaneal tuber length
measurements is the lateral length, which reflects a greater amount
of ‘‘stepping’’ of the articulation of the calcaneus with the Table 1. Factor loadings for Principal Components Analysis: all hind limb bones. Table 1. Factor loadings for Principal Components Analysis: all hind limb bones. Loading Positively on the second component
Loading negatively on the second component
Var. Variable Description
Load. Var. Variable Description
Load. P13
Maximum width of tuber coxa
0.300
M1
Length of fourth metatarsal
20.379
P15
Length of epipubic bone
0.296
Ph1l
Length of first phalanx on digit 4
20.356
P12
Minimum width of tuba coxa
0.255
P17
Height of iliopectineal process
20.316
P19
AP width of ischial tuberosity
0.236
P6
Length of puboischiatic symp. 20.337
Ph3L
Length of third phalanx on digit 4
0.203
T6
Height of tibial crest
20.318
C5
ML (mid width) of calcaneal tuber
0.205
T1
Length of tibia
20.249
A4
Width of fibula facet on lat. Multivariate analyses and Lagostrophus fasciatus), the nail-
tail wallabies (Onychogalea spp.), and the species of the genus The difference between the positive and negative loadings on
the second component is that the variables loading positively October 2014 | Volume 9 | Issue 10 | e109888 October 2014 | Volume 9 | Issue 10 | e109888 PLOS ONE | www.plosone.org 14 Locomotion in Sthenurine Kangaroos Figure 11. Multivariate analyses of hindlimb bones. Key as for Figure 10: taxa included and explanation of any composite fossils are listed and
explained in Tables S3, 4. (A) Principal Components Analysis using all hind limb bones. The dotted line indicates the division between extant taxa that
PLOS ONE | www.plosone.org
15
October 2014 | Volume 9 | Issue 10 | e109888 Figure 11. Multivariate analyses of hindlimb bones. Key as for Figure 10: taxa included and explanation of any composite fossils are listed and
explained in Tables S3, 4. (A) Principal Components Analysis using all hind limb bones. The dotted line indicates the division between extant taxa that PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 15 Locomotion in Sthenurine Kangaroos Locomotion in Sthenurine Kangaroos are more specialized hoppers, and those that are less specialized or that rarely hop. (B) Principal Components Analysis without the pelvis. The dotted
line indicates the division between extant taxa that are more specialized hoppers, and those that are less specialized or that rarely hop. The
placement of taxa along both components is very similar to that shown in Figure 11A, except where otherwise noted. (C). Discriminant Analysis using
all hind limb bones. Sthenurines and extant macropodines (plus Lagostrophus) only. (D). Principal Components Analysis, calcaneum only. Key as for
Figure 10 except for the following additions: half tone diamonds = extinct Miocene macropodine Dorcopsoides; half tone stars = Miocene
sthenurines ( = Hadronomas puckridgi unless otherwise indicated). (E). Discriminant Analysis, calcaneum only. Key as for (D). doi:10.1371/journal.pone.0109888.g011 largely reflect adaptations for stability and power (large joint
surfaces, large superficial gluteal muscles, broad tarsal bones,
strong abdominal muscle support [epipubics]); while those loading
negatively reflect adaptations for speed (long limb segment lengths,
modifications
for
rapid
and
powerful
limb
retraction
and
protraction). October 2014 | Volume 9 | Issue 10 | e109888 Multivariate analyses ridge
0.199
P20
AP width of obturator foramen
0.198
F8
AP width of femoral head
0.140
Ph2L
Length of second phalanx on digit 4
20.188
C7
DP (top width) of calcaneal tuber
0.122
P7
Dorsal length of ischium
20.181
Key: AP = anteroposterior; DP = dorsoplantar; ML = mediolateral. doi:10.1371/journal.pone.0109888.t001 October 2014 | Volume 9 | Issue 10 | e109888 October 2014 | Volume 9 | Issue 10 | e109888 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 16 Locomotion in Sthenurine Kangaroos Table 2. Factor loadings for Principal Components Analysis: without the pelvis. Loading Positively
Loading negatively
Var. Variable Description
Load. Var. Variable Description
Load. Ph3L
Length of third phalanx
0.226
M1
Length of the fourth metatarsal
20.336
C5
ML (mid width) of calcaneal tuber
0.183
Ph1L
Length of first phalanx
20.320
F8
AP width of femoral head
0.112
T6
Height of tibial crest
20.222
P19
AP width of ischial tuberosity
0.236
T1
Length of tibia
20.212
A4
Width of fibula facet on lat. ridge
0.165
Ph2L
Length of second phalanx
20.178
A8
ML width of medial tibial facet
0.108
T7
Length of proximal art. surface
20.117
A2
Max. MW width of astragalus
0.107
C2
Lateral AP length of calcaneum
20.102
C7
DP (top width) of calcaneal tuber
0.122
C3
Plantar AP length of calcaneum
20.092
F11
Length of gluteal insertion
0.100
C1
Medial AP length of calcaneum
20.088
Key: AP = anteroposterior; DP = dorsoplantar; ML = mediolateral. doi:10.1371/journal.pone.0109888.t002 cubonavicular (see previous discussion). Thus the negative loadings
on this axis represent modifications for rapid hopping, as with the
previous analysis. extreme outlier at the opposite end of this function from the
potoroines. This analysis is presented in the Supplementary
Information (Figure S5, Tables A and B in Text S2): here we chose
to do an analysis comparing only sthenurines with macropodines. No pelvis measurements were selected by the analysis with all the
variables, so this analysis was not repeated with the exclusion of
the pelvis. The morphospace depicted from the scores of the
specimens on the two functions is shown in Figure 11C, and the
factor loadings of the variables on each eigenvector are shown in
Table 3. Summary of Principal Components Analyses. Multivariate analyses The PCA
of all the bones together proved excellent for distinguishing
between extant macropodoids that are more specialized hoppers
from those that are rare, or less specialized hoppers (such as tree-
kangaroos, the non-hopping Hypsiprymnodon moschatus, and the
forest and woodland dwelling wallabies), and the loadings of the
variables along the second component made sense in terms of the
functional morphology. In the analysis with all hind limb bones the
Pleistocene sthenurines clustered with the rare hoppers, although
the smallest form, ‘‘Procoptodon’’ gilli, had the lowest scores. With
the pelvis removed, the distinction was not so marked and the
analysis appeared to be mainly separating the tree-kangaroos
(Dendrolagus spp.) from the other forms. The position of the
sthenurines was not so clearly separate from the more specialized
macropodines in this analysis, but all sthenurine individuals had
scores that were more towards the positive end of the component
than did any of the large species of Macropus. Three groups were defined: Group 1 = species of Macropus;
Group 2 = other extant macropodine species (plus Lagostrophus),
excluding Dendrolagus spp.; Group 3 = Dendrolagus spp. The
extinct taxa were added as unknowns. The value of the Wilks’
lambda statistic for the first function was close to zero and highly
significant (l = 0.04; x2 = 137.221; d.f. = 8; P,0.001). Similarly,
the value of the Wilks’ lambda statistic for the second function was
also highly significant (l = 0.299; x2 = 51.317; d.f. = 3; P,0.001). We obtained a 94.2% of correct reclassifications using the leave-
one-out method of cross-validation, which suggests that both
discriminant functions combined a set of skeletal traits that
accurately distinguished the three groups compared. Discriminant Analysis of all hind limb bones (Figure 11C,
Table 3). We originally performed this analysis with the
complete range of taxa, but the results appeared to be somewhat
skewed by the inclusion of the potoroines. While on the first
function the two larger sthenurine species (Sthenurus sterling and
Simosthenurus occidentalis) were clearly separate from all other
macropodoids (apart from Dendrolagus spp.), the second function
appeared to be distinguishing potoroines from other macropo-
doids, with the extinct balbarid Nambaroo gillespeiae being an The first function distinguishes Dendrolagus spp. (with negative
values) from other extant macropodines. The more specialized
sthenurines (Procoptodon sp. October 2014 | Volume 9 | Issue 10 | e109888 Multivariate analyses Perhaps
surprisingly, Hadronomous puckridgi has higher scores on both
this function, and the first one, than ‘‘P.’’ gilli and the presumed
female individuals of Sthenurus stirlingi. The only macropodine to
cluster among the high-scoring sthenurines is the exinct ‘‘giant’’
Macropus titan, possibly reflecting the fact that this function is
largely (but not entirely) a reflecting body size. Note that the
smaller species of Macropus cluster with the other macropodines,
but there is no simple size-sorting among the larger species of
Macropus, and Hadronomas puckridgi is no larger than the large
extant species of Macropus. And, of course, Dendrolagus spp.,
with higher scores than any of the non-Macropus species of
macropodines, are not any larger than all of these taxa. Taxa that have positive values on the second component (the
first component representing body size) comprise most of the
extant macropodoids, with the exception of the tree-kangaroos
(Dendrolagus spp.) Perhaps surprisingly, the basal species of
Dendrolagus, D. bennettianus, is the one here with the highest
negative loadings, falling away from the other kangaroos, whereas
in the PCA for all hind limb bones it was the one Dendrolagus
species that tended to cluster with the other kangaroos. The extant
macropodoids that place with negative scores include mainly the
non-specialized hoppers: Potorous spp. (potoroos), Setonix bra-
chyurus (the quokka), Thylogale spp. (pademelons), and Dorcopsis
spp. (New Guinea forest-wallabies), but not Hypsiprymnodon
moschatus. The Oligo-Miocene taxa (the macropodid Ngamaro
archeri and the balbarid Nambaroo gillespieae) also have negative
scores, but most of specimens of the late Miocene macropodine,
Dorcopsoides sp., have positive scores. Perhaps surprisingly, the
smallest species of Macropus, M. eugenii and M. irma, also have
negative scores, as does the swamp wallaby, Wallabia bicolor. With regard to the variables with high loadings on the second
function: here the mediolateral width of the astragalus has the
highest positive loadings; this may be the variable that is acting to
sort individuals by body size, in part (and the relatively high scores
of Dendrolagus spp. are explained by their more flexible ankle). The maximum width of the greater trochanteric ridge also has
relatively high scores: all of the Macropus species have positive
scores on this function, as they did with this variable on the first
function. Multivariate analyses The larger gluteals
in sthenurines could reflect the balancing of the trunk over the hips
while foraging, and the more stable ankle joint could reflect
balancing the body weight over one leg while striding. The fact
that the width of the astragalus, which featured prominently in
some earlier analyses, is not included here confirms the suspicion
that the high loadings of this variable in other analyses largely
distinguished Dendrolagus spp., with their more flexible ankles,
from other macropodids. The only variable with negative loadings is the mediolateral
width of the base of the astragalus. As notec previously, this feature
may be distinguishing Dendrolagus spp. based on their more
mobile tarsal joint, and Procoptodon sp. may be falling into this
area of the morphospace because of its large size (and hence a
proportionally larger ankle joint). Two measures of the femur have
high positive scores on this axis: the maximum width of the greater
trochanteric ridge, and the length of the medial tibial condyle. The
width of the trochanteric ridge reflects the insertion of the gluteals,
perhaps indicating larger hind limb retractor musculature in the
species of Macropus. The functional significance of the length of
the medial tibial condyle is not clear: as all the macropodids have a
medial condyle that is shorter than the lateral one, this variable
may be reflecting a more symmetrical distal femur, possibly related
to hopping behavior. The length of the fourth metatarsal also has
slight positive loadings, probably reflecting the difference from the
very short metatarsals of Dendrolagus spp. Principal
Components
Analysis
of
the
calcaneum
(Figure 11D, Table 4). The analysis of the calcaneum yielded
a first principal component (l = 22.652; 94.38% of variance
explained), which represents body size, and a second component
(l = 0.301; 1.254% of variance explained), which represents
aspects of morphological differences among the specimens. Despite the fact that only the first PC had eigenvalues higher
than one, which means that body size is responsible for a high
amount of the total shape variation, we also extracted the second
PC because it represents aspects of morphological differences
among the specimens. The morphospace depicted from the scores
of the specimens on these two PCs is shown in Figure 11D and the
factor loadings of the variables on each eigenvector are shown in
Table 4. The second function mostly separates the sthenurines from the
macropodines, with the exception of ‘‘Procoptodon’’ gilli. Multivariate analyses and Simosthenurus occidentalis), and
the presumed male specimen of Sthenurus stirlingi (see [12]) also
have high negative scores on function one, but only Procoptodon
sp. falls into the same level of scores as Dendrolagus spp. The
presumed female specimens of S. stirlingi (see [12]) have Table 3. Coefficient loadings for Discriminant Analysis: all hind limb bones (sthenurines and extant macropodines only). Loading on Factor 1
Loading on Factor 2
Var. Variable Description
Load. Var. Variable Description
Load. F20
Length of medial tibial condyle
0.231
A2
ML width of astragalus
0.399
F12
Max width of greater trochanter ridge
0.212
F12
Max width of greater trochanter ridge
0.187
M1
Length of fourth metatarsal
0.085
M1
Length of fourth metatarsal
20.005
A2
ML width of astragalus
20.723
F20
Length of medial tibial condyle
20.194
Key: ML = mediolateral. doi:10.1371/journal.pone.0109888.t003
PLOS ONE | www.plosone.org
17
October 2014 | Volume 9 | Issue 10 | e109888 Table 3. Coefficient loadings for Discriminant Analysis: all hind limb bones (sthenurines and extant ma ient loadings for Discriminant Analysis: all hind limb bones (sthenurines and extant macropodines only) October 2014 | Volume 9 | Issue 10 | e109888 October 2014 | Volume 9 | Issue 10 | e109888 17 Locomotion in Sthenurine Kangaroos somewhat negative scores in comparison with the Macropus
individuals, but ‘‘Procoptodon’’ gilli has the most positive of scores
of any macropodid. The non-Macropus macropodines (excluding
Dendrolagus spp.) tend to have more negative scores than the
Macropus individuals, possibly reflecting less specialization for
hopping (the non-Macropus individuals that have positive scores
on this function tend to be the more hopping specialists, but there
is no distinct separation within the group). function incorporated only two variables, both loading positively. These were the height of the medial malleolus on the distal tibia
(T14: highest loading) and the width of the greater trochanteric
ridge (F12). On a univariate axis non-Macropus macropodines
have the lowest scores, species of Macropus have intermediate
scores, and the sthenurine species have the highest scores. These
features relate to the relative stability of the ankle joint and to the
relative size of the gluteals. Thus it seems that the Macropus
species have a more stable ankle joint and larger gluteals than non-
Macropus macropodines, perhaps due to greater hopping special-
izations, while the sthenurines are more extreme in these features,
but perhaps for reasons unrelated to hopping. October 2014 | Volume 9 | Issue 10 | e109888 Multivariate analyses However, the sthenurines also have high scores: as
previously discussed, sthenurines have large areas for gluteal origin
and insertion, but here perhaps reflecting balance over an upright
trunk rather than limb retraction. The length of the fourth
metatarsal here has weakly negative scores, again possibly
explained by the position in the morphospace of Dendrolagus
spp. However, the length of the medial tibial condyle has the
highest negative scores. This may reflect the relatively larger
length of the lateral tibial condyle observed in sthenurines, as
previously discussed. Most of the large extinct taxa (sthenurines and Protemnodon
spp.) have negative scores, with the largest forms (e.g., Procoptodon
sp., Protemnodon cf. brevus) having the most negative ones. The
smaller, and/or more gracile species of Protemnodon, P. snewini
and P. anak, have positive scores, clustering with the larger species
of Macropus, as do the several individuals of the Miocene
sthenurine Hadronomas puckridgi. The smaller Miocene sthenur-
ine Rhizosthenurus flanneryi has slightly negative scores, but still
falls within the range of extant hopping macropodines. But
basically, as with the PCA for all hind limb bones, the Pleistocene
sthenurines and the larger species of Protemnodon fall with the To investigate this further we performed a CVA with and
without Dendrolagus spp. (not illustrated): this resulted in just two
predetermined groups (species of Macropus and other macro-
podines [plus Lagostrophus]), with the sthenurines classified as
unknowns. The value of the Wilks’ lambda statistic for the first
function was highly significant (l = 0.266; x2 = 49.019; d.f. = 2;
P,0.001) and the 89.8% of the taxa were correctly classified. The October 2014 | Volume 9 | Issue 10 | e109888 PLOS ONE | www.plosone.org 18 Locomotion in Sthenurine Kangaroos Table 4. Factor loadings for Principal Components Analysis: Calcaneum only. Loading positively on the second component
Loading negatively on the second component
Var. Variable Description
Load. Var. Variable Description
Load. Multivariate analyses C17
Width of sulcus for tendon of peroneus longus
0.284
C5
ML width of midshaft of tuber (dorsal ridge only)
20.187
C25
Length of roughened area on plantar side of tuber
0.138
C18
Width of sulcus for tendon of flexor digitorum longus
20.174
C3
Medial AP length
0.095
C16
ML width of midshaft of tuber (plantar side)
20.165
C12
AP length of tuber
0.094
C15
Length of ectal facet
20.154
C2
Lateral AP length
0.091
C6
DP width of top of tuber
20.078
C11
AP length of CLAJ
0.090
C13
Mediolateral width across CLAJ
20.077
C1
Plantar DV length
0.088
C14
Maximum width of calcaneal head
20.073
Key: AP = anteroposterior; DP = dorsoplantar; ML = mediolateral. CLAJ = continuous lower ankle joint. doi:10.1371/journal.pone.0109888.t004 Loading negatively on the second component Key: AP = anteroposterior; DP = dorsoplantar; ML = mediolateral. CLAJ = continuous lower ankle joint. doi:10.1371/journal.pone.0109888.t004 rarely-hopping extant macropodines, in particular with the tree-
kangaroos (Dendrolagus spp.). the scores of the specimens on both functions is visually displayed
in Figure 11E and the factor loadings of the variables on each
eigenvector are shown in Table 5. The highest positive loading variable on the second component
is the width of the sulcus on the latero-plantar side of the calcaneal
head for the tendon of the peroneus longus muscle. As discussed
previously, this action of this muscle it to both flex the ankle and to
counteract the action of the tibialis anterior in preventing the
evulsion of the pes. Thus this reflects morphology specialized for
rapid hopping, and accounts for the high scores of the larger
species of Macropus (especially M. rufus) on this component. Almost all of the other variables loading positively on the second
component reflect the length of the calcaneal tuber: a long
calcaneal tuber indicates a longer moment arm for the gastroc-
nemius muscle, again indicative of powerful and/or rapid
hopping. Also loading weakly with positive values is the dorso-
ventral length of the CLAJ (continuous lower ankle joint), which
may simply reflect a rather narrow calcaneal head. This analysis picked out four variables: two relating to the size
and shape of the continuous lower ankle joint (where the astragalus
contacts the calcaneum), one relating to the width of the calcaneal
tuber, and one relating to the width of the cubonavicular facet at
the base of the calcaneum, where the calcaneum contacts the
cubonavicular bone. October 2014 | Volume 9 | Issue 10 | e109888 Multivariate analyses Also loading
with high negative values is the width of the sulcus for the tendon
of the flexor digitorum longus, on the medio-plantar side of the
calcaneal head. This loading largely reflects the width of this sulcus
in the tree-kangaroos, and may be related to their climbing ability. Another variable with moderately negative high loadings on the
second component is the size of the ectal facet, which is one of the
places where the astragalus articulates with the calcaneum (the
other being the sustentacular facet). Thus a large ectal facet
represents morphology adapted for weight-bearing and foot
stability. again a feature of the smaller macropodines and Dendrolagus spp. The variable with the highest positive loadings on the second
function is the mediolateral width across the CLAJ: the tree-
kangaroos have high positive scores on this function, and this may
reflect an ankle joint that has some mediolateral mobility, as
employed in climbing. The dorsoventral width of the CLAJ also
has high positive values on this function: the larger species of
Macropus and Protemnodon have positive scores on the second
function (see discussion below), while most of the other extant
macropodoids have negative (or at least less positive) scores. A
narrow CLAJ may reflect a tarsal morphology that is better
adapted for a restricted range of motion of the leg about the foot in
the parasaggital plane, at least in the smaller taxa. The widths of
the calcaneal tuber and the cubonavicular facet have negative
loadings on the second function, reflecting the low scores of the
larger sthenurines. The smaller and/or more gracile Miocene Discriminant Analysis of the calcaneum (Figure 11E,
Table 5). Three groups were defined: Group 1 = non-hopping
or occasionally hopping taxa (species of Dendrolagus plus
Hypsiprymnodon moschatus); Group 2 = regular hoppers (macro-
podines [including Lagostrophus] with the exception of the genera
Dendrolagus and Macropus); Group 3 = specialized hoppers (the
species of Macropus). The extinct taxa were added as unknowns. The analysis yielded two functions that together allow a 93.2% of
correct assignments using the leave-one-out method of cross-
validation, which suggests that both discriminant functions
combined a set of skeletal traits that accurately distinguishes
among the three groups compared. The value of the Wilks’
lambda statistic for the both functions is significant (Function I:
l = 0.092; x2 = 94.067; d.f. = 8; P,0.001; Function II: l = 0.465;
x2 = 30.213 d.f. Multivariate analyses For the first function the positive values largely reflect the dorso-
plantar width of the cubonavicular facets (i.e., across the
lateromedial and ventromedian facets). This function appears to
reflect body size, in part, as among the extant kangaroos the larger
forms (e.g., the larger species of Macropus) have more positive
scores, and most of the Pleistocene sthenurines also have highly
positive scores. However, this function is one of the few that
distinguishes between the tree-kangaroos and the sthenurines. Tree-kangaroos have strong negative scores on this function,
probably as the result of their merging of the ventromedian facet
with the dorsolateral facet, which may allow for greater intratarsal
motion [45], while the high positive scores of the sthenurines
reflect the lengthening of the ventromedian facet. The variable
loading with the greatest negative values on function one is the
mediolateral width of the calcaneal tuber, a variable that is also
large in sthenurines, despite the fact that they cluster with positive
scores on this function: this variable also distinguishes Dendrolagus
spp. from the other macropodoids. The dorsoventral width of the
CLAJ also has high negative values on function one, reflecting a
relatively narrow articulation between astragalus and calcaneum,
again a feature of the smaller macropodines and Dendrolagus spp. The variable with the highest positive loadings on the second
function is the mediolateral width across the CLAJ: the tree-
kangaroos have high positive scores on this function, and this may
reflect an ankle joint that has some mediolateral mobility, as
employed in climbing. The dorsoventral width of the CLAJ also
has high positive values on this function: the larger species of
Macropus and Protemnodon have positive scores on the second
function (see discussion below), while most of the other extant
macropodoids have negative (or at least less positive) scores. A
narrow CLAJ may reflect a tarsal morphology that is better
adapted for a restricted range of motion of the leg about the foot in
the parasaggital plane, at least in the smaller taxa. The widths of
the calcaneal tuber and the cubonavicular facet have negative
loadings on the second function, reflecting the low scores of the
larger sthenurines. The smaller and/or more gracile Miocene The variables loading with high negative loadings on the second
component largely reflect the width of both the calcaneal tuber
and the calcaneal head, especially the CLAJ. A broader calcaneum
reflects foot stability rather than rapid locomotion. Multivariate analyses = 3; P,0.001). The morphospace depicted from Discriminant Analysis of the calcaneum (Figure 11E,
Table 5). Three groups were defined: Group 1 = non-hopping
or occasionally hopping taxa (species of Dendrolagus plus
Hypsiprymnodon moschatus); Group 2 = regular hoppers (macro-
podines [including Lagostrophus] with the exception of the genera
Dendrolagus and Macropus); Group 3 = specialized hoppers (the
species of Macropus). The extinct taxa were added as unknowns. The analysis yielded two functions that together allow a 93.2% of
correct assignments using the leave-one-out method of cross-
validation, which suggests that both discriminant functions
combined a set of skeletal traits that accurately distinguishes
among the three groups compared. The value of the Wilks’
lambda statistic for the both functions is significant (Function I:
l = 0.092; x2 = 94.067; d.f. = 8; P,0.001; Function II: l = 0.465;
x2 = 30.213 d.f. = 3; P,0.001). The morphospace depicted from October 2014 | Volume 9 | Issue 10 | e109888 PLOS ONE | www.plosone.org 19 Locomotion in Sthenurine Kangaroos Table 5. Coefficient loadings Discriminant Analysis: Calcaneum only. Loading on Factor 1
Loading on Factor 2
Var. Variable Description
Load. Var. Variable Description
Load. C21
Dorso-plantar width across surface of
cubonavicular facets
0.933
C13
Medio-lateral width across CLAJ
0.586
C13
Medio-lateral width across CLAJ
0.014
C11
Dorso-ventral width across CLAJ
0.239
C11
Dorso-ventral width across CLAJ
20.533
C5
Medio-lateral width of midshaft of tuber
(main anterior ridge)
20.111
C5
Medio-lateral width of midshaft of tuber
(main anterior ridge)
20.615
C21
Dorso-plantar width across surface of
cubonavicular facets
20641
Key: CLAJ = continuous lower ankle joint. doi:10.1371/journal.pone.0109888.t005 sthenurines, Rhizosthenurus and Hadronomas, tend to cluster with
the extant kangaroos on both functions. nurus are more specialized in their skull and dentition than
Sthenurus [6], and they may also be more specialized in their
postcranial anatomy. The Miocene wallaby-sized sthenurine,
Rhizosthenurus flanneryi, represented here only by the pes, is
not distinguished from similar-sized macropodines. The larger,
grey kangaroo-sized Miocene sthenurine Hadronomus puckridgi,
represented here by all elements except the pelvis, sometimes
clusters with the macropodines (e.g., Figures 11B, D, and most of
the SI bivariate plots), and sometimes with the larger Pleistocene
sthenurines (e.g., Figure 11C). Multivariate analyses An interesting observation is that, while function one seems to
represent a size axis in part, with the large sthenurines having the
highest scores, other ‘‘giant’’ kangaroos do not cluster in this
morphospace: rather, the large species of Protemnodon, and
especially the extinct Macropus ferragus, have relatively low values
on function one, but high values on function two. This may
represent independent evolution of large size and mode of weight
bearing
over
the
foot:
while
the
sthenurines
enlarge
the
ventromedian cubonavicular facet, other large kangaroos have a
larger overall size of the CLAJ. The inclusion of other extinct taxa (Nambaroo gillespieae,
Ngamaroo archeri, and Dorcopsoides sp.) in many of the analyses
shows that they generally cluster with the extant macropodoids,
supporting the hypothesis that it is the sthenurine anatomy that is
distinctive from the general macropodoid bauplan (rather than
representing the primtivie condition). The inclusion of the species
of Protemnodon is interesting: the smaller (P. snewini) or more
gracile (P. anak) forms often cluster with the similarly sized
macropodines, especially on the bivariate plots. However, larger
species tend to cluster with the sthenurines (e.g., Figures 11B, C)
or occupy a different portion of the morphospace from either
sthenurines or extant macropodines (e.g., Figures 11E, S5B). Although the locomotion of Protemnodon spp. is not a subject of
this paper (they were included primarily because they represent a
different lineage of large, robust kangaroos), these results may
indicate that the larger species, at least, were not hopping (or not
deploying hopping as their habitual gait), but did not have a
similar type of locomotion to the sthenurines. Summary
of
the
multivariate
analyses
of
the
calcaneum. The results of the PCA were similar to that of
the other PCAs: the Pleistocene sthenurines were distributed along
the second component in a similar fashion to Dendrolagus spp. and the less specialized terrestrial macropodines. The Miocene
sthenurine Hadronomas puckridgi clustered with the larger species
of Macropus, while the smaller Miocene sthenurine Rhizosthe-
nurus flanneryi occupied a fairly middling position in the
morphospace. The CVA was the only multivariate analysis to
separate the sthenurines from the tree-kangaroos. Here the
Pleistocene sthenurines occupied more or less their own area of
the morphospace along the first function: the distribution of other
large-bodied taxa showed that this was not simply an effect of their
large size. Discussion The large extinct species of Macropus (M. titan and M. ferragus) also behave in an interesting fashion in the analyses. In
terms of the tibia diameter, at least, they are as ‘‘robust’’ as the
other large extinct kangaroos (see Figure 9B). In the other
bivariate plots (Figures S1–3) they tend to follow the trajectory
of the extant species, except in some features of the ankle joint
(Figures S3B, C) where they cluster more with the other large
extinct forms. In the multivariate analyses they sometimes group
with the other species of Macropus (Figures 11B, D, S5B), with the
larger sthenurines (Figure 11C), or in a different portion of the
morphospace to either (Figure 11E). In any event, their inclusion
shows that the difference between the large sthenurines and the
smaller-sized extant large species of Macropus is not simply a
matter of body size. October 2014 | Volume 9 | Issue 10 | e109888 Overview This would limit the ability to hop, but would brace the
backbone against the rotational forces that would be generated by
alternate limb loadings. The large epipubic bones also indicate a
bracing of the trunk. of a specific other type of locomotion, as there are no similar
extant forms with which they could cluster. Using the results, and
the implications of the differences in the descriptive anatomy, we
present an argument based on principles of functional anatomy. We summarize the aspects of sthenurine anatomy that differ
from those of large hopping macropodines as follows: (i) anatomy
of sthenurines that indicates they would have had difficulty with
Macropus-like rapid hopping; (ii) anatomy indicative of a habitual
more upright posture, with the femur at an angle more parallel to
the iliac blade and with less flexed limbs; and (iii) anatomy
indicative of bearing weight on one foot at a time, as would be the
case with a locomotor gait of bipedal walking. While modifications
listed under (ii) could be taken as indicative merely of bipedal
browsing with an upright trunk, in combination with (i) and (iii)
they also lend support to this posture being employed during
locomotion. Important differences in anatomy are presented in
Tables 6, 7. p
g
p
p
y
We summarize the aspects of sthenurine anatomy that differ
from those of large hopping macropodines as follows: (i) anatomy
of sthenurines that indicates they would have had difficulty with
Macropus-like rapid hopping; (ii) anatomy indicative of a habitual
more upright posture, with the femur at an angle more parallel to
the iliac blade and with less flexed limbs; and (iii) anatomy
indicative of bearing weight on one foot at a time, as would be the
case with a locomotor gait of bipedal walking. While modifications
listed under (ii) could be taken as indicative merely of bipedal
browsing with an upright trunk, in combination with (i) and (iii)
they also lend support to this posture being employed during
locomotion. Important differences in anatomy are presented in
Tables 6, 7. Anatomical features indicative of limited hopping at
best. The anatomy of the sthenurine lumbar spine is contra-
indicative to hopping. Hopping in extant kangaroos entails a
considerable amount of flexion of the lumbar vertebral column, The reduction of the sthenurine tail is also a problematical issue. Macropodine kangaroos can use their tail as a support when Table 6. Overview The descriptive anatomy of Pleistocene sthenurines shows that
they have numerous differences from large macropodines, over
and
above
their
greater
‘‘robusticity’’. The
bivariate
and
multivariate analyses show that they tend to cluster away from
the macropodines in the morphospace. The larger species
(Sthenurus stirlingi, Simosthenurus occidentalis, and Procoptodon
sp.) invariably occupy a different portion of the morphospace to
the macropodines (apart from Dendrolagus spp., [tree-kangaroos]
with
which
they
tend
to
group);
and
while
the
smaller
‘‘Procoptodon’’ gilli is usually distinct from the macropodines in
the bivariate analyses (see especially Figures S1A, S1C, S2B, and
S3B) it sometimes clusters with them in the multivariate analyses. In the analyses where both are included, Procoptodon sp. and
Simosthenurus tend to group together, away from the species of
Sthenurus (see Figures 11B, C, S5B), despite the size difference
(Simosthenurus is considerably smaller than both Procoptodon and
Sthenurus stirlingi). However, both Procoptodon and Simosthe- However, while the results here clearly indicate that at least the
larger sthenurines were distinctly different in their hind limb
anatomy from the large extant macropodines, they can at best
support the hypothesis that sthenurines were different in their
locomotion from extant kangaroos: they cannot provide evidence October 2014 | Volume 9 | Issue 10 | e109888 PLOS ONE | www.plosone.org 20 Locomotion in Sthenurine Kangaroos of a specific other type of locomotion, as there are no similar
extant forms with which they could cluster. Using the results, and
the implications of the differences in the descriptive anatomy, we
present an argument based on principles of functional anatomy. especially at the point of the anticlinal ( = diaphragmatic) vertebra
[61], but the morphology of the sthenurine backbone appears to
be specifically designed to limit mobility in this area. Note also
virtual loss of the vertebral transverse processes, indicating great
reduction of the longissimus dorsi musculature, which is essential
for creating dorsiflexion of the spine, and counterbalancing regular
flexion. Wells and Tedford [12] interpret sthenurine lumbar
anatomy as rigidity to resist rotational stress on the backbone,
which they note can be caused by alternate limb loadings in
mammals: but why would sthenurines require a backbone resistant
to rotational stress if they were moving both hind limbs
simultaneously in a kangaroo-like fashion? The anatomy of the
lumbar vertebrae in sthenurines indicates an extremely rigid back,
where in addition the muscles that flex the spine have been greatly
reduced. Key: Italics indicate the primitive condition for the morphology. BS = bipedal striding. FP = foraging posture. RH = rapid hopping. Italics indicate the primitive
condition for the morphology (as determined by the condition in generalized small macropodines such as Dorcopsis).
@ = also seen in tree-kangaroos (Dendrolagus spp.).
# = also seen in smaller, rapid-hopping macropodines (e.g., species of Onychogalea and Lagorchestes).
doi:10.1371/journal.pone.0109888.t006 Overview Summary of important differences between sthenurines and large species of Macropus 1: Lumbar vertebrae, pelvis and
proximal limb bones. Bony element
Morphology in large
macropodines
Morphology in sthenurines
Implications of derived
morphology
Functional relevance
Lumbar vertebrae (general)
Unmodified
Enlarged, massive
Resistance to rotational torsion
BS
Transverse processes
Unmodified
Reduced or absent
Reduction in back flexibility (reduced
longissimus dorsi muscles)
FP and/or BS
Metapophyses
Unmodified
Enlarged and laterally expanded
Reduction in back flexibility (enlarged
multifidus muscles)
FP or BS
Sacrum
Unmodified, two vertebrae
Enlarged, may include three
vertebrae. Resistance to rotational torsion
BS
Iliac blade
Unflared, narrow tuber coxae
Flared laterally, broad, enlarged
tuber coxae@
Greater origin of gluteal & iliacus
muscles
FP and/or BS
Width between acetabulae
Relatively narrow
Relatively broad
More stable bipedal stance
FP
Ischium length
Elongated#
Not elongated
Increased moment arm of hamstring
muscles
RH
Angle between ischium and
ilium
1700
1450 (i.e., tipped dorsally)@
Reorientated moment arm of
hamstring muscles
FP and/or BS
Epipubic bones
Relatively short#
Long and broad@
Stiffening of trunk, resist rotational
torsion
FP and/or BS
Femoral head size
Regular
Enlarged (i.e., larger hips)@
Increased weight-bearing
BS
Femoral head shape
Ovoid
Round
Restricts movement to parasaggital
plane
RH
Femoral neck
Elongated
Regular
Increased moment arm of gluteals
RH
Lesser trochanter of femur
Regular position
Placed distallybroad@
Increased moment arm of iliopsoas
BS and/or FP
Position of femoral adductor
scar
Regular position
Placed distallydistallybroad@
Increased moment arm of m. quadratus femoris
BS and/or FP
Femoral condyle width
Unmodified
Increased (i.e., larger knees)@
Increased weight-bearing
BS
Tibial tuberosity
Long#
Short
Increased area of insertion of tibialis
anterior
RH
Medial malleolus of tibia
Unmodified
Longer and more robust
Stabilization of tibia-astragalar
articulation
BS and/or FP
Tibial distal plantar flange
Absent
Prominent
Stabilization of tibia-astragalar
articulation
BS and/or FP
Key: Italics indicate the primitive condition for the morphology. BS = bipedal striding. FP = foraging posture. RH = rapid hopping. Italics indicate the primitive
condition for the morphology (as determined by the condition in generalized small macropodines such as Dorcopsis). @ = also seen in tree-kangaroos (Dendrolagus spp.). # = also seen in smaller, rapid-hopping macropodines (e.g., species of Onychogalea and Lagorchestes). doi:10.1371/journal.pone.0109888.t006 nt differences between sthenurines and large species of Macropus 1: Lumbar vertebrae, pelvis and Table 6. Summary of important differences between sthenurines and large species of Macropus 1: Lumbar vertebrae, pelvis and
proximal limb bones. Table 6. October 2014 | Volume 9 | Issue 10 | e109888 Overview BS = bipedal striding. FP = foraging posture. RH = rapid hopping. Italics indicate the primitive
condition for the morphology (as determined by the condition in generalized small macropodines). @ = also seen in tree-kangaroos (Dendrolagus spp.). # = also seen in smaller, rapid-hopping macropodines (e.g., species of Onychogalea and Lagorchestes). doi:10.1371/journal.pone.0109888.t007 longus muscle, and the elongated proximal phalanx on the fourth
medal digit. Note also the hoof-like ungual
phalanges in
sthenurines, which Kear et al. [47] interpreted as an adaptation
for ‘‘hopping at slower speeds’’. standing on their hind legs, both when standing plantigrade in
repose, or standing digitigrade to fight [1]. As this digitigrade
standing is the posture proposed for sthenurines browsing, it seems
strange that they would have reduced the size of their tail. In
addition, the tail is important in hopping locomotion: the tail is
swung downwards as the hind limbs swing backwards, thus
cancelling the inertia of the hind limbs [25]. If sthenurines hopped
in a Macropus-like fashion, then their heavier limbs would require
an even more sturdy tail to balance out these inertial forces. However, if they were employing bipedal striding, then a large tail
would no longer be necessary to cancel out inertial forces, and
additionally a large tail could possibly impede such locomotion by
dragging on the ground. The relatively shorter and less muscular
tail of sthenurines may also reflect its lack of use in pentapedal
locomotion. Anatomical features indicative of a habitual upright
posture. That is, with a fairly upright trunk, the femur in a
more vertical position, and the limb joints with more obtuse angles
(i.e., less ‘‘crouched’’). The morphology of the sthenurine ischium, tipped dorsally and
forming a more acute angle to the ilium, repositions the moment
arm for the hamstring muscles, important in limb retraction. This
ischial anatomy is seen in placentals among primates that
locomote with an upright trunk, as it maintains the moment
arm of the hamstrings when the femur is rotated posteriorly in this
posture [62]. Convergence in this feature is seen in tree-kangaroos
(and also koalas, see plate XIV in [35]), supporting the hypothesis
that this anatomy is related to the trunk position relative to the
femur. Overview Summary of important differences between sthenurines and large species of Macropus 1: Lum
proximal limb bones. October 2014 | Volume 9 | Issue 10 | e109888 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 21 Locomotion in Sthenurine Kangaroos Table 7. Summary of important differences between sthenurines and large large species of Macropus II: Pes. Bony element
Morphology in large
macropodines
Morphology in sthenurines
Implications of derived
morphology
Functional relevance
Astragalar trochlear groove
Shallow with low ridges
Deep with high ridges,
esp. medial
Stabilization of tibia-astragalar
articulation
BS
Astragalar fibular facet
Narrow
Broad
Increased weight-bearing
BS
Medial malleolar astragalar
process
Broad
Narrow
Reduced intratarsal mobility
BS and/or FP
Calcaneal tuber
Long, posteriorly reflected#
Shorter, straight or anteriorly
reflected
Increased moment arm of m. gastrocnemius
RH
Calcaneal tuber dorsoventral
width
Unmodified
Deep
Increased weight-bearing
BS
Calcaneal tuber tip
Unmodified
Broadened in mediolateral
plane@
Larger insertion area of Achilles
tendon
BS and/or FP
Transverse plantar sulcus on
calcaneum
Broad#
Narrow
Larger sulcus for peroneus longus
tendon
RH
Fibular facet on calcaneum
More medial position
More lateral position
Greater ability to pronate foot
BS and/or FP
Sustentaculum tali on
calcaneum
Broad medio-laterally
Narrow medio-laterally
Greater ability for plantar flexion
with internally rotated foot
BS and/or FP
Cubonavicular facet on
calcaneum
Pronounced ‘‘stepping’’ of
dorsolateral facet
Moderate ‘‘stepping’’ of
dorsolateral facet
Restriction of motion between
calcaneum and cuboid
RH
Cubonavicular facet on
calcaneum
Relatively narrow
Elongated in dorsoplantar
direction
Increased weight-bearing
BS
Length of fourth metatarsal
Greatly elongated
Moderately elongated
Increased length of distal limb
RH
Proximal articular surface of
fourth metatarsal
Flat anterior profile, small
plantar eminence
Enlarged & curved anterior
profile, large plantar eminence
Increased weight-bearing
BS
Distal articular surface of
fourth metatarsal
Square-shaped
Larger and medio-laterally
broadened. Increased weight-bearing
BS
Metatarsal keels
Moderately prominent
Less prominent
Stabilization of phalanges on
metatarsus
RH
Proximal phalanx of fourth
pedal digit
Elongated#
Regular length
Lengthening of plantar tendons,
more ‘‘springy’’ foot. RH
Middle phalanx of fourth
pedal digit
Regular length
Elongated
Increased weight-bearing
BS
Key: Italics indicate the primitive condition for the morphology. BS = bipedal striding. FP = foraging posture. RH = rapid hopping. Italics indicate the primitive
condition for the morphology (as determined by the condition in generalized small macropodines). @ = also seen in tree-kangaroos (Dendrolagus spp.). # = also seen in smaller, rapid-hopping macropodines (e.g., species of Onychogalea and Lagorchestes). doi:10.1371/journal.pone.0109888.t007 Key: Italics indicate the primitive condition for the morphology. October 2014 | Volume 9 | Issue 10 | e109888 Locomotion in Sthenurine Kangaroos The long tuber in large species of
Macropus reflects an increase in the moment arm for the limb
extensors (the gastrocnemius, in this case), to balance out the large
ground reaction forces occasioned by a crouched posture, which
become relatively larger with larger body size. The shorter tuber in
sthenurines implies a lesser moment arm for the gastrocnemius,
which given their large size would only be biomechanically
possible if the moment arm for the ground reaction force was also
reduced, as would be occasioned by a more upright posture at the
ankle joint. The very broad area on the calcaneum for the
attachment of the Achilles tendon in sthenurines may indicate an
insertion that is fleshy rather than tendinious; if this were the case,
this would definitely be in contradiction to a hopping mode of
locomotion. Aspects of the sthenurine ankle joint have been interpreted as
rotating the feet inwards for a more medial position, and/or for
bearing more weight on the medial side of the foot: these include
the rotation of the tibioastragalar joint in an anteriomedial
direction; the longer groove in the proximal tibia for the insertion
of the fibula, allowing for a greater internal rotation of the lower
limb about the knee; the lateral displacement of the fibular facet as
enabling greater ability to pronate the foot; a sustentaculum tali
that is narrow in the mediolateral direction (allowing for plantar
flexion with internal rotation of the foot); and the plantar crest of
the calcaneum being more elongated on the medial side (see
[12,42]). Humans (as opposed to apes) have a suite of morpho-
logical adaptations related to the shifting of their weight to the
medial side of the foot during locomotion [43]. These features of
sthenurines may again be indicative of weight bearing on one leg
at a time. Another suite of features of the sthenurine ankle joint include
ones relating to a greater stabilization of the tibia on the astragalus,
and ones resisting movement of the astragalus on the calcaneum. Such features could indicate a need to resist greater rotational
forces incurred by bearing weight on one leg at a time. Sthenurines
are unique among macropodoids in having a plantar process on
the distal tibia that fits in a tongue-in-groove linkage into the
astragalar trochlea (also seen in the Miocene Hadronomas). Locomotion in Sthenurine Kangaroos Locomotion in Sthenurine Kangaroos upright trunk, due to the insertion of the hypaxials on the
epipubics. leg [43]. The enlarged gluteus medius and minimus, as well as the
iliacus, as indicated by the expanded iliac blade in sthenurines,
could also aid in this postural support. Enlarged areas of insertion
for the gluteals are also seen in camelids, who balance the body
over pairs of lateral legs during pacing locomotion, and who thus
require large limb abductor muscles to prevent the body from
collapsing medially [49]. The enlarged tuber coxae of the ilium, also echoed to a certain
extent in tree-kangaroos (see Figure S1A), reflect enlarged
superficial gluteal muscles and an enlarged cranial head of the
caudofemoralis [36]. These muscles would be important for
raising the front end of the body up over the hips, as would be
necessary in a bipedal feeding posture. The enlarged femoral head and distal condyles in sthenurines
are reminiscent of the differences between the human and ape
conditions [43], and may be indicative of increased load bearing in
supporting the weight on a single leg. Elliptically shaped femoral
condyles, as seen in sthenurines, are also a new feature in humans,
interpreted as minimizing the load on the knee [43]. The elliptical
shape in humans also increases the moment arm of the quadriceps
femoris, which aids in maintaining balance in a straight-legged
position [43]; this morphology in sthenurines could also reflect
walking with a straighter knee. The more distal placement on the femur of the adductor scar
and the lesser tuberosity would increase the moment arms of the
quadratus femoris and iliopsoas, respectively. This might relate to
more powerful limb adduction and protraction, but might simply
reflect compensation for the altered moment arms of these muscles
occurred by a difference in the position of the femur relative to the
pelvis with an upright trunk. p
p g
The shorter calcaneal tuber in sthenurines might also reflect a
change in posture at the ankle joint. In considering forces acting
over any joint, the moment arm produced by the limb extensors
must balance the ground reaction force. Crouched limb postures
result in a long lever arm for the ground reaction force, and the
postural changes in larger animals reduce this lever arm,
increasing the limb’s effective mechanical advantage [30]. As
noted previously, extant kangaroos do not change their locomotor
posture with increasing size. Locomotion in Sthenurine Kangaroos An
analogous morphology is also seen in cursorial placental mammals
such as canids and horses, and is also present in the thylacine
( =
the ‘‘marsupial wolf’’ Thylacinus cynocephalus; personal
observation of the senior author). In conjunction with this, the
trochlear groove on the astragalus is deeper in sthenurines, with a
raised medial trochlear ridge. There is also evidence of an increase
in the size in the ligaments that bind the astragalus to the
calcaneum [44], and the astragular trochlea has been rotated
medially, now being at more of a right angle to the longtitudinal
axis of the pes; this would restrict the movement of the tibia on the
pes to a more anteroposterior motion, with compressive stresses
being directed more anteroventrally (see [12]). The constriction of
the sulcus for the flexor digitorum longus in sthenurines,
intepreteted by Bishop [44] as preventing the dislodging of this
tendon while elevating the foot, could also indicate greater stress
on one foot at a time. A narrow medial malleolar process, and the
larger and more posteriorly directed cuboid facet, would restrict
any motion between the astragalus and the cuboid. Anatomical features indicative of bearing weight on one
foot at a time. Part of the greater ‘‘robustness’’ of the hind
limbs of sthenurines could be explained by each limb having to
bear the full weight of the animal at some point in the locomotor
cycle, something not experienced by hoppers: as noted earlier, the
real issue here may be that large species of Macropus are unusually
gracile, so that sthenurines appear to be ‘‘robust’’ only in
comparison with these highly specialized hoppers. Many morpho-
logical features of sthenurine hind limbs actually parallel those
seen in humans that distinguish them from other apes. In the
human condition this obviously relates to walking bipedally rather
than quadrupedally, rather than a transition from hopping, but the
same principles apply. The short and broad sacrum of sthenurines could be interpreted
as resisting greater rotational forces, as occasioned by walking with
alternate limbs (as could, as well, the more rigid lumbar spine, as
discussed above). Humans have a broader sacrum than other apes,
which is interpreted as relating to weight support and transmission
during locomotion with a habitual bipedal posture [43]. Overview Tree-kangaroos and koalas also resemble sthenurines in
having extremely large epipubic bones, which Elftman [35]
interpreted as important for supporting the viscera with an Sthenurines also lack many of the features of Macropus that are
specialized for hopping (although this would not rule out the
ability to hop): these features in Macropus include the elongated
ilium, the long tibial tuberosity and prominent tibial crest, the
elongation of the trochlea of the astragalus, the long calcaneal
tuber, the enlarged sulcus on the calcaneum for the peroneus October 2014 | Volume 9 | Issue 10 | e109888 PLOS ONE | www.plosone.org 22 October 2014 | Volume 9 | Issue 10 | e109888 A hypothesis about the sthenurine monodactyl condition A hypothesis about the sthenurine monodactyl condition
None of the scenarios of locomotor evolution in sthenurines
account for the issue of the loss of the fifth pedal digit (the original
idea of Tedford [52], that this represented a greater specialization
for hopping, has now been abandoned; see earlier discussion). Although Murray [42] considers that the weight had already
shifted to the medial side of the foot in the Miocene sthenurine
Hadronomas (see also Kear [16], for Rhizosthenurus), thus rending
the animal functionally monodactyl, this does not explain why the
digit would become vestigial. An explanation is proposed here
based on developmental trajectories. Pigs (Sus scrofa), like other
artiodactyls, have digits three and four as the primary ones on both
fore and hind feet, with the reduction of digits one, two and five. Developmental studies show that this has occurred through
evolutionary modifications in the patterning of their limbs:
developing limb buds of pigs are different from those of a mouse,
with a reduction of the condensations of cartilages that form the
lateral digits [63]. If this is indeed a general developmental pattern
in mammals, with regards to the elongation of certain digits and
the reduction of others, then the sthenurine hand might provide a
clue to the sthenurine foot. The middle to early late Miocene sthenurine Rhizosthenurus
flanneryi was fairly large for a macropodid by today’s standards
(probably around 12–15 kg). While it retained the fifth toe, it also
had some modifications of the ankle joint indicative of shifting its
body weight to the medial side of the foot, a morphology that can
be
interpreted
as
weight-bearing
on
one
leg
at
a
time. Rhizosthenurus had a few modifications of its forelimbs indicative
of the type of specialized behavior proposed for later sthenurines,
such as enlarged epicondylar areas on the humerus for digital
flexors [16]. The larger late late Miocene sthenurine Hadronomas
(estimated body mass of 30 kg) also retained the fifth digit, but had
other hind limb features resembling those of later sthenurines [42]. Hadronomas sometimes clusters with the macropodines in our
analyses, and sometimes with the Pleistocene sthenurines, perhaps
indicating an intermediate functional morphology. Although the
hand of this animal is unknown, it had features of the scapula
resembling those of later sthenurines, including a reduced
supraspinous fossa and an enlarged coracoid process, which have
been
functionally
implicated
in
Pleistocene
sthenurines
for
reaching with the forearms [12]. Locomotion in Sthenurine Kangaroos In the
sthenurine pelvis, the inflated tuber coxae indicate an enlargement
of muscles (gluteus superficialis and caudofemoralis) that would
not only elevate the body, but would also balance the body over a
single leg and prevent medial tipping by their abduction action. This is reminiscent of the human morphology of the enlargement
and repositioning of the gluteus superficialis (via the shorter and
broader iliac blades), interpreted as an adaptation for bipedal
walking in preventing collapse at the hip while balancing on one There are a number of morphological features indicative of
overall greater weight bearing by both the tarsus and the foot. The
lateral trochlear ridge on the astragalus, that forms an articulation
with the fibula, is thicker in sthenurines than in other macro-
podoids. The overall size of the calcaneonavicular articulation is October 2014 | Volume 9 | Issue 10 | e109888 October 2014 | Volume 9 | Issue 10 | e109888 PLOS ONE | www.plosone.org 23 Locomotion in Sthenurine Kangaroos proportionally greater in sthenurines, in particular with a very
deep ventromedian facet, rendering the shape of the joint square
rather than rectangular. Correspondingly, the proximal articular
surface on the fourth metatarsal is proportionally larger, and
rendered more square in shape by an enlarger plantar eminence. The distal articular surface of the fourth metatarsal is also
proportionally larger and more rectangular, matched by a
similarly shaped proximal articulation on the proximal phalanx. Both proximal and distal articular surfaces are enlarged on the
proximal and medial phalanges, giving these bones a ‘‘waisted’’ or
‘‘I-beam’’ shape. developmental program in the hand of sthenurines, resulting in a
developmental reduction of the condensation cartilage for digit
five, may also have found expression in the foot. If an animal like
Hadronomas was already functionally monodactyl, then there
might have been no active selection pressure against such an
anatomical change. October 2014 | Volume 9 | Issue 10 | e109888 Conclusions The following is a scenario for the evolution of bipedal walking
in sthenurines. The key to understanding sthenurine evolution is to
realize that the forelimb and hind limb anatomy are linked in a
way that channels their locomotor mode. Sthenurines inherited
their ‘‘hopping anatomy’’, with a long tibia and digitigrade foot
posture while moving, from their macropodid ancestry; thus any
change in locomotion from bipedal hopping would be within the
constraints of this anatomy as a starting point. One of the
characteristic features of at least the Pleistocene sthenurines is a
forelimb that has been specialized for browsing, with the
concomitant anatomy that would restrict them from putting the
hand on the ground in a palmar stance, or from supporting their
body weight on their forelimbs [12]. Hence the slow, pentapedal
motion seen in extant large macropodines would be difficult or
impossible. This presents a functional problem, because no
mammal that hops today uses hopping for its slowest gait, and it
seems unlikely on energetic and biomechanical terms that an
animal as large as a sthenurine would be able to do this. Finally, there are a number of features indicative of enlarged
plantar ligaments on the pes of sthenurines. These could be
interpreted as being related to springiness of the foot during
hopping (see e.g., [52]), but elastic energy storage in the hind limb
in general is counter-indicated by the constriction of the sulcus for
the flexor digitorum longus and the lesser mechanical advantage of
the gastrocnemius (occasioned by the shorter calcaneal tuber). The
larger plantar crest on the fourth metatarsal in sthenurines is
indicative of larger interosseus muscles, and the well-developed
scars for plantar cruciate ligaments on the plantar side of the
proximal phalanx could as equally well indicate greater torque
being placed on this bone [42], and stabilization of the foot under
greater pressure while bearing weight on one leg at a time. (TIF) Figure S3
Other morphological variables from the tibia
and pes. (A) Tibia average midshaft diameter versus the anterior-
posterior length of the tibia tuberosity. (B) Width (medio-lateral) of
the base of the astragalus versus the width (dorso-plantar) of the
fibular facet on the lateral trochlear ridge of the astragalus. (C)
Width of the base of the astragalus versus the length of the
calcaneal tuber. (D) Length of the femur versus the length of the
fourth metatarsal. Figure S4
Scaling of long bone length versus diameter
(shown in Figure 10) with labeled taxa. (A) Femur length
versus average femur cross-sectional diameter. (B) Tibia length
versus average tibial midshaft cross sectional diameter. (TIF) ,
q
p
A likely reason that hopping locomotion has not been
questioned for sthenurines is their generally kangaroo-like hind
limb, with features such as a long tibia that are characteristic of
hopping mammals. But, remember that this is the anatomy that
they inherited from their macropodid ancestry. And, if sthenurines
were indeed engaging in bipedal walking, then the long tibiae
would give them a long length of stride, which would be
energetically efficient. The loss of the fifth digit is perhaps
counterintuitive for an animal that might require more foot
support over a single leg, but Murray [42] has proposed, based on
pedal
anatomy,
that
Hadronomas
was already
functionally
monodactyl, and we propose here a possible developmental
reason for the reduction of this digit to vestigial remnant. One
puzzling aspect of sthenurine anatomy, in the context of this
hypothesis of bipedal walking, is that the knees that face outwards
rather than inwards. Humans differ from apes in their more
knock-kneed (valgus) stance, which relates to their placing of the
foot in the midline during locomotion, which aids with balance
over the hips in bipedal walking [43]. Sthenurines, on the other
hand, appear to have had a more bow-legged (vargus) stance. The
sthenurine stance may relate to biomechanical issues arising from
the very different anatomical starting point of a kangaroo to a
hominid for adopting such a gait. Perhaps this stance was
originally adopted in the context of foraging behavior: a wider
stance might make walking more clumsy, but would provide a
broader base for balancing while standing. Locomotion in Sthenurine Kangaroos even if they retained hopping as their regular faster gait. Adaptations for bipedal posture for feeding would also preadapt
them for maintaining this posture while moving and balancing their
weight over a single limb (as is already indicated by the foot
modifications shifting the body weight to the medial side of the foot). In conclusion, although a fossilized trackway would be the only
means of completely verifying our hypothesis of sthenurines using
a bipedal striding gait, their anatomy is clearly different from that
of large macropodines. Sthenurines lack the specialized features
for fast hopping seen in macropodines and the differences in
anatomy from the extant forms can be functionally related to
locomotion bearing weight on one leg at a time. g
y
g
)
The spinal anatomy of Rhizosthenurus and Hadronomas is
unknown, but at some point sthenurines would have started to
stabilize the lumbar region of their spine (as described by Wells
and Tedford [12] in the large Pleistocene Sthenurus stirlingi), in
order to support their trunk and stabilize their front end while
foraging with an upright trunk. The lumbar vertebrae with greatly
enlarged metapophyses and reduced transverse processes indicate
an increase in size of the multifidous musculature and, concur-
rently, a decrease in size of the longissimus dorsi. However, this
anatomy would pose a further conflict with locomotion, both
pentapedal walking and bipedal hopping. With a stiff, shorter
lumbar region they would no longer be able to arch the back to
perform pentapedal locomotion (even if they could support their
weight on their hands), and with the stiff lumbar spine and
reduced longissimus musculature they would be unable to dorsiflex
in the region of the anticlinal vertebra, as seen in modern
kangaroo hopping locomotion. (TIF) Figure S5
Additional discriminant analyses. (A) Using all
of the hind limb bones, for all of the taxa. (B) Without the pelvis,
for all of the taxa. (TIF) (TIF) Or perhaps there were
issues relating to carrying large pouch young with a different type
of locomotion, which entailed rotational torsion of the body rather
than the pitching seen in hopping, which necessitated this stance. Supporting Information Figure S1
Ilium length versus other aspects of ilial
morphology. (A) Ilium length versus width of the tuber coxa. (B)
Ilium length versus the dorsal length of the ischium. (C) Ilium
length versus the ventral length of the puboischiatic symphysis. (D)
Ilium length versus the length of the epipubic bone. (TIF) Figure S2
Femur length versus other aspects of femoral
morphology. (A) Femur length versus the anterior-posterior
width of the femoral head. (B) Femur length versus the distance of
femoral head to the base of the lesser trochanter. (C) Femur length
versus the length of the femur from the proximal end to the base of
the adductor scar. (D) Femur length versus the width of the femur
across the distal condyles. The majority of the Pleistocene sthenurines were no larger than
modern large Macropus species [6], still without severe biome-
chanical limitations for hopping (apart from the morphology of the
lumbar spine). Such animals likely would have been able to do
some slow hopping, but may have become increasingly reliant on
bipedal walking for much of their daily locomotor repertoire. But,
as reliance on bipedal walking grew, with concomitant adaptive
morphology, they would have been able to increase their body size
to outside of the range where hopping is biomechanically feasible
(because of the safety factors involved with tendon strength, see
[3]). Whether or not the largest sthenurines (especially the large
species of Procoptodon) abandoned hopping altogether is un-
known, and would require more data to attempt to determine. A hypothesis about the sthenurine monodactyl condition The sthenurine hand shows an elongation of the second, third,
and fourth digit, accompanied by the reduction of digits one and
five, while the digits of the hands of other macropodoids are of
more equal length [12]. If this change in hand anatomy had been
accomplished by the developmental process of suppressing the
formation of the digits one and five (as described in pigs [63]) it is
possible that this process was also transmitted to the hind limb. Sa´nchez-Villagra and Mencke [64] describe a similar echoing in
the hind limb of a developmental change in the forelimb in the
mole, Talpa europa: the transformation of the radial sesamoid into
a prepollux in the hand is accompanied by the transformation of
the tibial sesamoid into a (small) prehallux in the foot. They [64]
propose that this change in foot morphology is not a functional
adaptation (unlike the condition in the hand), but that it reflects a
common epigenetic control of the hand and foot. Pedal digit one is
lost in all macropodids, and digits two and three have been
severely reduced, so in this respect the foot development has
already been shifted from the basic pentadactyl pattern. But a new Thus, the Miocene sthenurines, although still relatively small,
showed postcranial modifications indicative of specialized browsing
using their hands, and weight-bearing on one foot at a time. We
propose that these animals, while likely still hopping for their faster
locomotor gait, were starting to use some bipedal walking for
locomoting at slow speeds: their capacity for slow pentapedal
locomotion would be compromised by the more specialized
forelimbs, and hopping at very slow speeds appears to be
biomechanically impossible. Remember that bipedal walking has
been observed in tree-kangaroos, so is certainly not an impossible
gait for a macropodid. Additionally, sthenurines have been
proposed to have been feeding with an upright trunk, and reaching
up over their heads into tall vegetation. With this feeding posture,
resorting to pentapedal motion to move to the next bush would
mean frequent postural changes and energy expenditure. During
their early evolutionary history sthenurines may have been
performing some bipedal shuffling in order to move short distances, October 2014 | Volume 9 | Issue 10 | e109888 October 2014 | Volume 9 | Issue 10 | e109888 PLOS ONE | www.plosone.org 24 Locomotion in Sthenurine Kangaroos References 9. Prideaux GJ, Long JA, Aycliffe LK, Hellstrom JC, Pillans B, et al. (2007) An
arid-adapted middle Pleistocene vertebrate fauna from south-central Australia. Nature 445: 422–425. Doi:10.1038/nature05471 28. Dawson TJ, Webster KN (2010) Energetic characteristics of macropodoid
locomotion. In: Coulson, Eldridge M, editors. Macropods: The Biology of
Kangaroos, Wallabies, and Rat-Kangaroos. Collingwood, VIC: CSIRO
Publishing. pp. 99–108 10. Flannery TF (2008) Chasing Kangaroos: A Continent, a Scientist, and a -
Search for the World’s Most Extraordinary Creature. New York: Grove Press. 1–272 p. 29. Dawson TJ (1983) Monotremes and Marsupials: the Other Mammals. London:
Edward Arnold. 1–87 p. 11. Kear BP, Lee MSY, Gerdtz WR, Flannery TF (2008) Evolution of hind limb
proportions in kangaroos (Marsupialia: Macropodoidea). In: Sargis EJ, Dagosto
M, editors. Mammalian Evolutionary Morphology: A Tribute to Fred Szalay. Dordrecht; Springer. pp. 25–35. 30. Biewener AA (2005) Biomechanical consequences of scaling. J Exp Biol 208:
1665–1676. Doi: 10.1242/jeb.01520 j
31. Bennett MB, Taylor CR (1995) Scaling of elastic strain energy in kangaroos and
the benefits of being big. Nature 287: 56–59. 12. Wells RT, Tedford RH (1995) Sthenurus (Macropodidae: Marsupialia) from the
Pleistocene of Lake Callabonna, South Australia. Bull Am Mus Nat Hist 225: 1–
111. 32. McGowan CP (2009) Could giant kangaroos hop? Scaling of tendon geometry
and skeletal features. Integr Comp Biol 49(1): e1 (meeting abstract). and skeletal features. Integr Comp Biol 49(1): e1 (meetin 33. Warburton NM, Harvey KJ, Prideaux GJ, O’Shea JE (2011) Functional
morphology of the forelimb of living and extinct tree-kangaroos (Marsupialia:
Macropodidae). J Morphol 272: 1230–1244. Doi: 10.1002/jmor.10979. 13. Cooke BN (1999) Wanburoo hilarus gen. et sp. nov. a lophodont bulungamayine
kangaroo (Marsupialia: Macropodoidea: Bulungamayinae) from the Miocene
deposits of Riversleigh, northwestern Queensland. Rec West Aust Mus,
Supplement 57: 239–253. 34. Chen X, Milne N, O’Higgins P (2005) Morphological variation of the
thoracolumbar vertebrae in Macropodidae and its functional relevance. J Morphol 266: 125–255. Doi: 10.1002/jmor.10370 14. Travouillon KJ, Legendre S, Archer M, Hand SJ (2009) Palaeoecological
analyses of Riversleigh’s Oligo-Miocene Sites: Implications for Oligo-Miocene
climate change in Australia: Palaeogeogr Palaeocl 276: 24–37. Doi: 10.1016/
j.palaeo.2009.02.025. 35. Elftman H (1929) Functional adaptations of the pelvis in marsupials. Bull Am
Mus Nat Hist 58: 189–232. 36. Hopwood PR, Butterfield RM (1976) The musculature of the proximal pelvic
limb of the Eastern Grey Kangaroo Macropus major (Shaw) Macropus giganteus
(Zimm). J Anat 121: 259–277. 15. References 20. Webster KN, Dawson TJ (2003) Locomotion energetics and gait characteristics
of a rat-kangaroo, Bettongia penicillata, have some kangaroo-like features. J
Comp Physiol B 173: 549–557. Doi 10.1007/s00360-003-0364-6 1. Dawson TJ (1995) Kangaroos: Biology of the Largest Marsupials. Ithaca:
Comstock Publishing Associates. 1–62 p. g
,
g
p
,
g
Comp Physiol B 173: 549–557. Doi 10.1007/s00360-003-0364-6 2. Helgen KM, Wells RT, Kear BP, Gerdtz WR, Flannery TF (2006) Ecological
and evolutionary significance of sizes of giant extinct kangaroos. Aust J Zool 54:
293–303. Doi 10.1071/ZO05077 21. Windsor DE, Dagg AI (1971) The gaits of the Macropodinae (Marsupialia). J Zool Lond 163: 165–175. 22. O’Connor SM, Dawson TJ, Kram R, Donelan JM (2014) The kangaroo’s tail
propels and powers pentapedal locomotion. Biol Lett 10: 20140381. Doi: org/
10.1098/rsbl.2014.0381 3. McGowan CP, Skinner J, Biewener AA (2008) Hind limb scaling of kangaroos
and wallabies (superfamily Macropodoidea): implications for hopping perfor-
mance, safety factor, and elastic savings. J Anat 212: 153–163. Doi: 10.1111/
j.1469-7580.2007.00841.x 23. Martin R (2005) Tree-Kangaroos of Australia and New Guinea. Collingwood:
CSIRO Publishing. 1–58 p. 4. Prideaux GJ, Warburton NM (2010) An osteology-based appraisal of the
phylogeny and evolution of kangaroos and wallabies (Macropodidae: Marsupi-
alia). Zool J Linn Soc 159: 954–987. Doi: 10.1111/j.1096-3642.2009.00607.x 24. Burk A, Westerman M, Springer M (1998) The phylogenetic position of the
musky rat-kangaroo and the evolution of bipedal hopping in kangaroos. Syst Biol
47: 457–474. )
J
j
5. Kear BP, Cooke BN (2001) A review of macropodoid systematics with the
inclusion of a new family. Mem Ass Australas Palaeontologists 25: 83–101. 25. Baudinette RV (1989) The biomechanics and energetics of locomotion in
Macropodoidea. In: Grigg G, Jarman P, Hume I, editors. Kangaroo, Wallabies,
and Rat-Kangaroos, Minneapolis: University of Minnesota Press. pp. 245–253. 6. Prideaux GJ (2004) Systematics and evolution of the sthenurine kangaroos. Univ
of Calif Publ Geol Sci 146: 1–623. 26. Bennett MB (2000) Unifying principles in terrestrial locomotion: do hopping
Australian mammals fit in? Physiol Biochem Zool 73: 726–735. Doi: 1522-
2152/2000/7306-99144$03.00 7. Meredith RW, Westerman M, Springer MS (2008) A phylogeny and timescale
for the living genera of kangaroos and kin (Macropodiformes: Marsupialia) based
on nuclear DNA sequences. Aust J Zool 56: 395–410. Doi 10.1071/ZO08044 27. Alexander RMcN, Vernon A (1975) The mechanics of hopping by kangaroos
(Macropodidae). J Zool Lond 177: 265–303. q
J
8. Cronin L (2008) Australian Mammals. Jacana NSW: Allen and Unwin. 1–190 p. Acknowledgments We thank the following people for access to specimens in their collections:
Judy Chupasko (Museum of Comparative Zoology, Harvard University,
USA); Judy Galkin and Eileen Westwig (American Museum of Natural
History, NY, New York, USA); Mike Archer and Sue Hand (University of
New South Wales, NSW, Sydney, Australia); Sandy Ingleby and Anja
Divljan (Australian Museum, Sydney, NSW, Australia); Erich Fitzgerald,
David Pickering, Tom Rich and Karen Roberts (Museum Victoria,
Melbourne, VIC, Australia); Heather Janetzki, Kirsten Spring, Scott
Hocknull, and Andrew Rozefelds (Queensland Museum, Brisbane, QLD,
Australia); Claire Stevenson, Ric How, and Alex Baynes (Western We thank the following people for access to specimens in their collections:
Judy Chupasko (Museum of Comparative Zoology, Harvard University,
USA); Judy Galkin and Eileen Westwig (American Museum of Natural
History, NY, New York, USA); Mike Archer and Sue Hand (University of
New South Wales, NSW, Sydney, Australia); Sandy Ingleby and Anja
Divljan (Australian Museum, Sydney, NSW, Australia); Erich Fitzgerald,
David Pickering, Tom Rich and Karen Roberts (Museum Victoria,
Melbourne, VIC, Australia); Heather Janetzki, Kirsten Spring, Scott
Hocknull, and Andrew Rozefelds (Queensland Museum, Brisbane, QLD,
Australia); Claire Stevenson, Ric How, and Alex Baynes (Western Table S1
Taxonomy of the Superfamily Macropodoi-
dea. Table S2
Measurements taken. (DOC) Table S3
Specimens measured for analyses. (DOC) Table S4
Specimens measured for calcaneum only
analyses. (DOC) Table S5
Data for all of the bones. (PDF) Table S6
Additional data on the calcaneum. (PDF) Table S6
Additional data on the calcaneum. (PDF) Text S1
Description of bivariate plots. (DOC) October 2014 | Volume 9 | Issue 10 | e109888 PLOS ONE | www.plosone.org 25 Locomotion in Sthenurine Kangaroos Australian Museum, Perth, WA. Australia); Adam Yates (Northern
Territories Museum and Art Gallery, Alice Springs, NT, Australia);
Catherine Kemper, David Stemmer, Mary-Anne Binnie, Ben McHenry,
and Mike Lee (South Australian Museum, Adelaide, SA, Australia); and
Gavin Prideaux and Aaron Camens (Flinders University, Bedford Park,
SA, Australia). Australian Museum, Perth, WA. Australia); Adam Yates (Northern
Territories Museum and Art Gallery, Alice Springs, NT, Australia);
Catherine Kemper, David Stemmer, Mary-Anne Binnie, Ben McHenry,
and Mike Lee (South Australian Museum, Adelaide, SA, Australia); and
Gavin Prideaux and Aaron Camens (Flinders University, Bedford Park,
SA, Australia). Text S2
Additional discriminant analyses, using all of
the taxa. Table A, Coefficient loadings for additional Discrim-
inant Analysis for all of the taxa: all hind limb bones. Table B,
Coefficient loadings for additional Discriminant Analysis for all
taxa: without the pelvis. (DOC) We also thank Megan Dawson, Edwardo Garza and Alison Migliori for
data entry; Brian Regal for the restoration of Sthenurus stirlingi (Figure 1),
and Natividad Chen for the other anatomical artwork; Steve Wroe, Natalie
Warburton, and Robin Crompton for comments on an earlier version of
the MS, and Angela Horner, Dan Riskin and Karen Sears for some specific
comments; and Kenny Travouillon for obtaining the measurements for
Nambaroo gillespieae. Natalie Warburton and Karen Black made
comments on the original manuscript that greatly improved its quality. Author Contributions Analyzed the data: BF. Wrote the paper: CJ BF. Collected osteological
data: CJ KB. Locomotion in Sthenurine Kangaroos 42. Murray PF (1995) The postcranial skeleton of the Miocene kangaroo,
Hadronomas puckridgi Woodburne (Marsupiala, Macropodidae). Alcheringa
19: 119–170. 54. Munn AJ, Streich WJ, Hummel J, Clauss M (2008) Modelling digestive
constraints in non-ruminant and ruminant foregut-fermenting mammals. Comp
Biochem Physiol A 151: 78–84. Doi: 10.1016/j.cbpa.2008.06.001 g
g
Biochem Physiol A 151: 78–84. Doi: 10.1016/j.cbpa.2008.06.001 43. Aiello L, Dean C (1990) An Introduction to Human Evolutionary Anatomy. London, Academic Press. 1–596 p. 55. Nilsson J, Thorstensson A (2008) Ground reaction forces at different speeds of
human walking and running. Acta Physiol Scand 136: 217–227. Doi: 10.1111/
j.1748-1716.1989.tb08655.x p
44. Bishop N (1997) Functional anatomy of the macropodid pes. P Linn Soc NSW
117: 17–50. 56. Bassarova M, Janis CM, Archer M (2009) The calcaneum – on the heels of
marsupial locomotion. J Mammal Evol 16: 1–23. Doi: 10.1007/s10914-008-
9093-7 45. Warburton NM, Prideaux GJ (2010) Functional pedal morphology of the extinct
tree-kangaroo Bohra (Diprotodontia: Macropodidae). In: Coulson, Eldridge M,
editors. Macropods: The Biology of Kangaroos, Wallabies, and Rat-Kangaroos. Collingwood, VIC: CSIRO Publishing. pp. 137–151. 57. Mendoza M, Janis CM, Palmqvist P (2002) Characterizing complex cranio-
dental patterns related to feeding behaviour in ungulates: a multivariate
approach. J Zool Lond 258: 223–246. Doi: 10.1017/S0952836902001346 46. Hopwood PR, Butterfield RM (1990) The locomotor apparatus of the crus and
pes the Eastern Grey Kangaroo, Macropus giganteus. Aust J Zool 38: 397–413. 58. Figueirido B, Pe´rez-Claros JA, Torregrosa V, Martı´n-Serra A, Palmqvist P
(2010) "Demythologizing Arctodus simus, the ‘short-faced’" and "long-legged"
predaceous bear that never was. J Vertebr Paleontol 30: 262–275. Doi:
10.1080/02724630903416027 47. Kear BP, Cooke BN, Archer M, Flannery TF (2007) Implications of a new
species of the Oligo-Miocene kangaroo (Marsupialia: Macropodoidea) Nam-
baroo, from the Riversleigh World Heritage Area, Queensland, Australia. J Paleontol 81: 1147–1167. Doi: 0022-3360/07/0081-1147$03.00 59. Samuels JX, Meachen JA, Sakai SA (2013) Postcranial morphology and the
locomotor habits of living and extinct carnivorans. J Morphol 274: 121–146. Doi: 10.1002/jmor.20077 J
48. Clifford AB (2010) The evolution of the unguligrade manus in artiodactyls. J Vertebr Paleontol 30: 1827–1839. Doi: 10.1080/02724634.2010.521216 J
49. Janis CM, Theodor JM, Boisvert B (2002) Locomotor evolution in camels
revisited: a quantitative analysis of pedal anatomy and the acquisition of the
pacing gait. J Vertebr Paleontol 22: 110–121. 60. Davis JC (2002) Statistics and Data Analysis in Geology, 3rd ed. New York:
Wiley. 1–656 p. 61. References Arena RA, Black KH, Archer M, Hand SJ, Godthelp G, et al. (2014)
Reconstructing a Miocene pitfall trap: recognition and interpretation of
fossiliferous Cenozoic palaeokarst. Sediment Geol 304: 28–43. Doi: org/
10.1016/j.sedgeo.2014.01.005. 37. Hooker JJ (2007) Bipedal browsing adaptations of the unusual Late Eocene –
earliest Oligocene tylopod Anoplotherium (Artiodactyla, Mammalia). Zool -
J Linn Soc 151: 609–659. 16. Kear BP (2002) Phylogenetic implications of macropodid (Marsupialia:
Macropodoidea) postcranial remains from Miocene deposits of Riversleigh,
northwestern Queensland. Alcheringa 26: 299–318. Doi.org/10.1080/
03115510208619259 38. Warburton NM, Yakovleff M, Malric A (2012) Anatomical adaptations of the
hind limb musculature of tree-kangaroos for arboreal locomotion (Marsupialia:
Macropodinae). Aust J Zool 60: 246–258. http://dx.doi.org/10.1071/
ZO12059 17. Woodburne M (1967) The Alcoota fauna, central Australia. Australia Bur Min
Resources, Bull 87: 1–185. 39. Hudson PE, Corr SA, Payne-Davis R, Clancy SN, Lane E, et al. (2011)
Functional anatomy of the cheetah (Acinonyx jubatus) hindlimb. J Anat 218:
363–374. Doi: 10.1111/j.1469-7580.2010.01310.x 18. Webster KN, Dawson TJ (2004) Is the energetics of mammalian hopping
locomotion advantageous in arid environments? Aust Mamm 26: 153–160. 40. Reilly SM, White TD (2003) Hypaxial motor patterns and the function of
epipubic bones in primitive mammals. Science 299: 400–402. 19. Baudinette RV, Halpern EA, Hinds DS (1993) Energetic cost of locomotion as a
function of ambient temperature and during growth in the marsupial Potorous
tridactylus. J Exp Biol 174: 81–95. 41. Kappleman J (1988) Morphology and locomotor adaptations of the bovid femur
in relation to habitat. J Morphol 198: 119–130. PLOS ONE | www.plosone.org October 2014 | Volume 9 | Issue 10 | e109888 October 2014 | Volume 9 | Issue 10 | e109888 26 Locomotion in Sthenurine Kangaroos Badoux DM (1965) Some notes on the functional anatomy of Macropus
giganteus Zimm. with general remarks on the mechanics of bipedal leaping. Acta Anat 62: 418–433. 50. O’Sullivan JA (2008) Evolution of the proximal third phalanx in Oligocene-
Miocene equids, and the utility of phalangeal indices in phylogeny reconstruc-
tion. In: Sargis EJ, Dagosto M, editors. Mammalian Evolutionary Morphology:
A Tribute to Fred Szalay. Dordrecht; Springer. pp. 159–165. 62. Fleagle JG, Anapol FC (1992) The indriid ischium and the hominid hip. J Human Evol 22: 285–305. g
J
g
A Tribute to Fred Szalay. Dordrecht; Springer. pp. 159–165. 51. Kear BP, Archer M, Flannery TF (2001) Bulungamayine (Marsupialia:
Macropodidae) postcranial elements from the late Miocene of Riversleigh,
northwestern Queensland. Mem Ass Australas Palaeontologists 25: 103–122. 63. Sears KE, Bormet K, Rockwell A, Powers LE, Cooper LN, et al. (2011)
Developmental basis of mammalian digit reduction: a case study in pigs. Evol
Dev 13: 533–541. Doi: 10.1111/j.1525-142X.2011.00509.x 52. Tedford RH (1966) A review of the macropodid genus Sthenurus. Univ of Calif
Publ Geol Sci 64: 1–71. 64. Sa´nchez-Villagra MR, Menke PR (2005) The mole’s thumb – evolution of the
hand skeleton in talpids (Mammalia). Zoology 108: 3–12. Doi: 10.1016/
j.zool.2004.07.006 53. Clauss M, Lechner-Doll M, Streich WJ (2003) Ruminant diversification as an
adaptation to physicomechanical characteristics of forage. A reevaluation of an
old debate and a new hypothesis. Oikos 102: 253–262. October 2014 | Volume 9 | Issue 10 | e109888 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 27
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Dynamics Finite Element for Ship Damage Collision Analysis
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1Hasanudin, Wasis D. Aryawan, Achmad Zubaydi, and Teguh
Putranto are with Department of Naval Architecture and Shipbuilding
Engineering, Sepuluh Nopember Institute of Technology, Surabaya,
60111, Indonesia. E-mail: hasanudin@na.its.ac.id; wasis@na.its.ac.id;
zubaydi@gmail.com; theories@gmail.com. The 2nd International Seminar on Science and Technology
August 2nd 2016, Postgraduate Program Institut Teknologi Sepuluh Nopember, Surabaya, Indonesia The 2nd International Seminar on Science and Technology
August 2nd 2016, Postgraduate Program Institut Teknologi Sepuluh Nopember, Surabaya, Indonesia The 2nd International Seminar on Science and Technology
August 2nd 2016, Postgraduate Program Institut Teknologi Sepuluh Nopember, Surabaya, Indonesia Hasanudin1, Wasis D. Aryawan1, Achmad Zubaydi1, and Teguh Putranto1 One of the hull form design innovation is to change the
ship structure that will definitely change the internal
energy and the strength of ship. But, it needs to be also
considered about the seakeeping and resistance if the
innovation provide more advantages in ship design [2]. Abstract Along with the growing fleet of vessels, ship
collision accident cases are becoming more frequent. It is
unavoidable that the rapid shipping due to the number of
vessels that operate relatively large and the ship is waiting for
the loading and unloading at the port. This study will
examine the design of the solid bulk cargo ship safe for
shipping operations congested areas. The finite element
method is used to determine the amount of internal energy in
the structure of damaged vessel. The striking ship is assumed
a tanker that have a bulbous bow. Bulk carrier is designed
with space of double skin 1.8 m and the structure of wing
tank is determined previously. The results obtained from this
research is the design of the cargo hold of bulk carrier that
has a double skin to protect the leaking compartment when
the ship was crashed. Penetration in depth 1.8 m will be
occurred at ship if ship crashed is with speed 12 knots and
weight 1400 tons. Figure 1. The midship section of double and single skin Keywordsfinite element, bulk carrier, double skin,
penetration. Figure 1. The midship section of double and single skin I. INTRODUCTION1 Transient procedure during simulation process of ship
collision could be carried out and give the sophisticated
result. Theory, numerical, and experimental method are
compared and analyzed in order to obtain the relation
between the used methods [3]. The collision between
ship and floating ice is analysed to obtain the internal
energy occurs during the collision. Using simple
formulation, the damage structure caused by collision
can be researched [4]. One of the research about ship damage collision have
carried out by Minorsky [1]. The absorbed damaged
energy and kinetic energy are determined by using
simple momentum theory. Interaction between ship and
water around are assumed in constant added mass
condition. Minorsky does also introduce the relation
between deformed volume of structure and absorbed
collision energy. This empirical method can be used for
greater collision (in other word, for more 30 MJ of
absorbed energy). This approach is used generally dan
modified by other researcher. Collision analysis with numerical simulation method of
dynamic finite element so that the energy received by the
vessel can be known [5]. The deeper penetration occurs,
the more energy that can be absorbed. There was a time
that happened was a constant force for the ship that
crashed into the transverse construction of the ship that
was hit so that the construction of this attempt to hold. The energy absorbed by the ship can be converted into
the mass of the ship that rammed so it can be estimated
that hit the ship size [6]. Probability damage stability of ship is depend on the
subdivision of compartment that it have researched by
Jasionowski. The more compartment built, the more time
needed to load and unloading. Study of Formal Safety
Assessment (FSA) of bulk carrier is also carried out by
International Organization that is lead by Royal
Institution of Naval Architects. The main goal of FSA is
to investigate whatever the new requirement about
survivability and structure fulfils the SOLAS Chapter
XII [2]. Dynamics Finite Element for Ship Damage
Collision Analysis Hasanudin1, Wasis D. Aryawan1, Achmad Zubaydi1, and Teguh Putranto1 120 120 II. METHOD This research uses numerical method which Finite
Element Method (FEM) is chosen to solve the problem
of dynamics analysis. Initially, two ships is modeled in
one file or solution. Then, it is applied in boundary
condition so that the collision phenomena are occurred. A. FEM Model of Two Ship There are 2 advantages bulk carrier that uses double
skin: (i) ability to withstand influence collision causing
more penetration than single skin and (ii) maintain the
outer structure from corrosion caused by contact between
steel of ship and load in cargo hold. Therefore, damaged
of plate and profile can be avoided. The lightweight of
double skin is more increase than single skin as shown at
Figure 1. Dynamic modelling for simulation of ship collision
done until penetration of 1.8 m. Modelling adapted to the
model transverse to the ship was hit and models bulbous
bow for ships crashing. Models made in one file so that
the settings for the element type, material properties, etc. performed on the same file. Element types used in this
model are good for Shell 163 ships crashing or the ship
was hit. For the definition of material properties, plastic
material kinematics applied to the ship was hit. It is The 2nd International Seminar on Science and Technology
121
August 2nd 2016, Postgraduate Program Institut Teknologi Sepuluh Nopember, Surabaya, Indonesia
intended to get the deformation and damage to the ship
was hit. Figure 2. FEM model for two ships
B Material Properties
Figure 3. Flowchart
Literature Study
FEM Model for Two Ship
Application of Boundary Condition
Internal Energy of Crashed Ship
Mass of Striking Ship
Width of Double Skin
Finish 121 The 2nd International Seminar on Science and Technology
August 2nd 2016, Postgraduate Program Institut Teknologi Sepuluh Nopember, Surabaya, Indonesia Figure 3. Flowchart
Literature Study
FEM Model for Two Ship
Application of Boundary Condition
Internal Energy of Crashed Ship
Mass of Striking Ship
Width of Double Skin
Finish intended to get the deformation and damage to the ship
was hit. Literature Study Figure 2. FEM model for two ships Figure 2. FEM model for two ships A. Force and Internal Energy During Collision Figure 3 shows a dynamic simulation of the collision
vessel at 1657 mm penetration. If seen ships crashing, no
deformation and stress on the hull of the crash. This is
because the ship that rammed assumed as a rigid body
while the ship was hit by a plastic material kinematics
conditioned as meaning that the material will undergo
elastic and plastic regions dependent on displacement
occurs. The time needed to complete the simulation is 3
days with the CPU capacity Inter Core i5 RAM 16 GB. TABLE 1. MATERIAL PROPERTIES FOR STRIKING AND CRASHED SHIP
No. Input
Value
Dimension
1. Density
7550
kg/m3
2. Elasticity Modulus
220
GPa
3. Poisson Number
0.33
-
4. Yield Stress
320
MPa
5. Tangensial Modulus
630
MPa
6. Hardness Parameter
0
-
7. Strain Rate (C)
3200
1/s
8. Strain Rate (P)
5
-
9. Strain Failure
0.325
- Figure 3. Distribution of stress at the crashed ship C. Boundary Condition C. Boundary Condition C. Boundary Condition The boundary conditions given to the ship was hit by a
breakdown as follows: • Fixed support, applied to corrugated bulkhead
locations. • Fixed support, applied to corrugated bulkhead
locations. • Symmetrical boundary condition, applied to the
location of the center line. It is intended to reduce the
time computer solutions. III. RESULTS AND DISCUSSION III. RESULTS AND DISCUSSION Material used to model ship collision is mild steel that
applied in whole of ship structure. But, for striking ship,
the material properties is added for rigid body it means
that the model does not appear the stress, energy,
displacement, etc. Rigid body material was applied to the
ship crashed. This is meant to avoid deformations and
stresses on the ship that rammed that ship that rammed
assumed completely rigid. Once the modelling is complete, then both models set
the distance is so close together. The tip of the bulbous
bow rammed the ship must be aligned with the plate side
of the boat that was hit. The initial position of the
collision should be right in the middle of the cargo hold
and the higher the position set baseline collision ships
rammed the boat parallel to the baseline hit. A Force and Internal Energy During Collision The most affect factor for analysis of FEM dynamics
is the strain failure meaning that the parameter
determines the failure of material. D. Mass of Striking Ship Figure 3. Distribution of stress at the crashed ship Using simple physics law about the law of
conservation energy, the conversion for internal energy
can be used to obtain the mass of striking ship. It is
based on the assumption that the energy had by ship
structure can stop the ship moving or crashed ship. From Fig. 4 can be seen the relationship between force
against penetration. When a ship strikes a moment
outside the ship hull plates, the speed at the time was 12
knots. 122 The 2nd International Seminar on Science and Technology
August 2nd 2016, Postgraduate Program Institut Teknologi Sepuluh Nopember, Surabaya, Indonesia Settings on the dynamic simulation are assumed
constant speed when the ship hit until the specified
penetration. Style and energy began to arise when the
outer hull plates mashing vessel. Based on the formula
Minorsky, the energy value is influenced by the slab
thickness, length of penetration, penetration width, depth
of penetration. of ship collision. The energy is converted to mass ship
rammed. The width of the double skin should have as
effectively as possible so as not to detract too much load
space. In addition, the probabilistic damage stability can
also be improved from the previous width of the double
skin. Selection of wide double skin in this study is based
on aspects of ship collision damage. Therefore, the width
of the double skin, the ship will be smaller due to
possible damage to the hull in a ship collision. Figure 4. The graph of force and penetration in speed 12 knots
Figure 5. The graph of internal energy and penetration in speed 12
knots Figure 4. The graph of force and penetration in speed 12 knots Figure 6. The graph of striking ship and velocity in speed 12 knots Figure 5. The graph of internal energy and penetration in speed 12
knots Figure 5. The graph of internal energy and penetration in speed 12
knots gure 6. The graph of striking ship and velocity in speed 12 knots Figure 6 shows the mass of the ship that rammed
against the penetration produced with speed variation. From Fig. 6 it can be seen that the greatest gradient of
the line contained in a line speed of 12 knots. From the
results can be analyzed that the mass of the ship that
crashed influenced by the ship speed crashed. CONCLUSION Based on the result and discussion, the conclusion
taken at this research as follows: 1. The force and energy would being increase when the
penetration is deeper. The pattern of force is
increase and decrease along penetration which it
means that the structure of double skin is damaged
caused by the collision of striking ship. 2. Mass of striking ship is 1400 ton would cause the
damage of inner hull when the striking ship moves
speed 12 knots. REFERENCES [1] A. Jasionowski, D. Vassalos., “Ship Vulnerability to Flooding”,
Safety At Sea Ltd., The Ship Stability Research Center,
University of Glasgow Strathclyde, UK, 2009. D. Mass of Striking Ship The
greater of ship mass, the deeper of the penetration. The
mass of striking ship consists of deadweight and
lightweight or relevant with ship displacement. Figure 5. The graph of internal energy and penetration in speed 12
knots Such behaviour is influenced by the law of
conservation of energy and momentum eternity law. In
addition, the material properties also affect the ship was
hit by internal energy. Speed mashing a material other
material will cause a different style although the speed
variation does not significantly affect the results of the
internal energy occurs. knots Graph force versus penetration tends to increase with
increasing penetration caused by contact that occurs
between the bulbous bow with a ship that was hit is also
getting bigger. Therefore, the frictional force and the
contact force between the two create value in Fig. 4 is
likely to increase. Fig. 4 shows the graph up and down
behavior that can be interpreted that the condition
construction damaged by being hit by a bulbous bow. From Fig. 5 can be seen the relationship between internal
energy against penetration. When a ship strikes a
moment outside the ship hull plates, the speed at the time
was 12 knots. Settings on the dynamic simulation are
assumed constant speed when the ship hit until the
specified penetration. Style and energy began to arise
when the outer hull plates mashing vessel. Based on the
formula Minorsky, the energy value is influenced by the
slab thickness, length of penetration, penetration width,
depth of penetration. Graph internal energy versus
penetration tends to increase with increasing penetration
caused by contact that occurs between the bulbous bow
with a ship that was hit is also getting bigger. Therefore,
the frictional force and the contact force between the two
create value in Fig. 5 is likely to increase. Fig. 5 shows
the graph up and down behavior that can be interpreted
that the condition construction damaged by being hit by
a bulbous bow. ACKNOWLEDGEMENTS Researcher would like to express the appreciation for
LPPM ITS that have given the fund and support through
Penelitian Pemula Grant in 2016. [1] A. Jasionowski, D. Vassalos., “Ship Vulnerability to Flooding”,
Safety At Sea Ltd., The Ship Stability Research Center,
University of Glasgow Strathclyde, UK, 2009. B. Mass of Striking Ship B. Mass of Striking Ship Based on the results of the energy and the force
generated by the simulation of ship collision can then be
determined based on the width of the double skin aspects y
g
y
[2] T. Putranto and A. Sulisetyono, “Analisa Numerik Gerakan dan
Kekuatan Kapal Akibat Beban Slamming Pada Kapal Perang 123 The 2nd International Seminar on Science and Technology
August 2nd 2016, Postgraduate Program Institut Teknologi Sepuluh Nopember, Surabaya, Indonesia
Tipe Corvette”, Jurnal Ilmu Pengetahuan dan Teknologi
Kelautan, Vol. 12, No. 3, Universitas Diponegoro, 2015. Tipe Corvette”, Jurnal Ilmu Pengetahuan dan Teknologi
Kelautan, Vol. 12, No. 3, Universitas Diponegoro, 2015. [3] M. J. Petersen, “Dynamics of Ship Collision”, [3] M. J. Petersen, “Dynamics of Ship Collision”, Ocean
Engineering Journal, Vol. 12 (VII), pp. 111-123, 1982. [3] M. J. Petersen, “Dynamics of Ship Collision”, Ocean
Engineering Journal, Vol. 12 (VII), pp. 111-123, 1982. Engineering Journal, Vol. 12 (VII), pp. 111-123, 1982. [4] O. Ozguc, Das. P. K., Baltrop, N., “A Comparative Study on the
Structural Integrity of Single and Double Side Skin Bulk Carrier
Under Collision Damage”, Department of Naval Architecture
and Marine Engineering, University of Glasgow and Stratchlyde,
UK, 2006. [5] MARPOL, “Regulation 18 - Segregrated Ballast Tanks”, 2002. [6] T. Putranto, W. D. Aryawan, A. Zubaydi, “Kajian Penentuan
Lebar Double Skin Bulk Carrier Ditinjau Dari Damage
Collision”, Seminar Nasional Pascasarjana, ITS, 2013.
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https://openalex.org/W4386136742
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https://www.nature.com/articles/s41467-023-40961-z.pdf
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Immune stress suppresses innate immune signaling in preleukemic precursor B-cells to provoke leukemia in predisposed mice
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Nature communications
| 2,023
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cc-by
| 18,388
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Article https://doi.org/10.1038/s41467-023-40961-z Immune stress suppresses innate immune
signaling in preleukemic precursor B-cells to
provoke leukemia in predisposed mice Marta Isidro-Hernández
1,2,13, Ana Casado-García1,2,13, Ninad Oak
3,13,
Silvia Alemán-Arteaga
1,2,13, Belén Ruiz-Corzo1,2, Jorge Martínez-Cano
4,
Andrea Mayado2,5, Elena G. Sánchez6, Oscar Blanco2,7, Ma Luisa Gaspar
8,
Alberto Orfao
2,5, Diego Alonso-López
9, Javier De Las Rivas
2,10,
Susana Riesco
11, Pablo Prieto-Matos
2,11, África González-Murillo
6,
Francisco Javier García Criado2,12, María Begoña García Cenador2,12,
Manuel Ramírez-Orellana
6, Belén de Andrés
8,
Carolina Vicente-Dueñas
2,11,13
, César Cobaleda
4,13
,
Kim E. Nichols
3,13
& Isidro Sánchez-García
1,2,13 Marta Isidro-Hernández
1,2,13, Ana Casado-García1,2,13, Ninad Oak
3,13,
Silvia Alemán-Arteaga
1,2,13, Belén Ruiz-Corzo1,2, Jorge Martínez-Cano
4,
Andrea Mayado2,5, Elena G. Sánchez6, Oscar Blanco2,7, Ma Luisa Gaspar
8,
Alberto Orfao
2,5, Diego Alonso-López
9, Javier De Las Rivas
2,10,
Susana Riesco
11, Pablo Prieto-Matos
2,11, África González-Murillo
6,
Francisco Javier García Criado2,12, María Begoña García Cenador2,12,
Manuel Ramírez-Orellana
6, Belén de Andrés
8,
Carolina Vicente-Dueñas
2,11,13
, César Cobaleda
4,13
,
Kim E. Nichols
3,13
& Isidro Sánchez-García
1,2,13 The initial steps of B-cell acute lymphoblastic leukemia (B-ALL) development
usually pass unnoticed in children. Several preclinical studies have shown that
exposure to immune stressors triggers the transformation of preleukemic B
cells to full-blown B-ALL, but how this takes place is still a longstanding and
unsolved challenge. Here we show that dysregulation of innate immunity plays
a driving role in the clonal evolution of pre-malignant Pax5+/−B-cell precursors
toward leukemia. Transcriptional profiling reveals that Myd88 is down-
regulated in immune-stressed pre-malignant B-cell precursors and in leukemic
cells. Genetic reduction of Myd88 expression leads to a significant increase in
leukemia incidence in Pax5+/−Myd88+/−mice through an inflammation-
dependent mechanism. Early induction of Myd88-independent Toll-like
receptor 3 signaling results in a significant delay of leukemia development in
Pax5+/−mice. Altogether, these findings identify a role for innate immunity
dysregulation in leukemia, with important implications for understanding and
therapeutic targeting of the preleukemic state in children. Childhood B-ALL is considered a clonal blood disorder whose earliest
stages take place during embryonic life. Hematopoietic progenitor cells
carrying germline or somatic alterations affecting transcription factor
genes that regulate early hematopoietic or early B cell development
(such as germline ETV6, PAX5, or IKZF1 mutations, or the somatic ETV6-
RUNX1 fusion) represent what are commonly defined as preleukemic
cells1. These preleukemic cells are compatible with normal hematopoie-
tic development in a large majority of cases. Immune stress suppresses innate immune
signaling in preleukemic precursor B-cells to
provoke leukemia in predisposed mice The “delayed
infection” hypothesis is based on the possibility that an immune sys-
tem unexposed to common infections early in life may function
abnormally when exposed to such infections later in life. Specifically,
the abnormal immune signaling triggered by these infections is pro-
posed to lead to a series of genetic events that culminate in the
development of leukemia. Alternatively, the “population mixing”
hypothesis was postulated to explain the appearance of small clusters
of patients with B-ALL in a localized place and time12. It posits that
childhood B-ALL arises as an uncommon result of exposure to any
otherwise common infection, but this would only become noticeable
as clusters of B-ALL at times of population mixing, when a relatively
large number of susceptible children naïve to this infection mix with a
large number of infected individuals, leading to a (largely subclinical)
widespread epidemics12. In either case, a transient deficiency of the
immune priming could system act together with a gut microbiome
dysbiosis to drive B-ALL. However, little is known about how exposure
to infection promotes such transformation of preleukemic cells. (Fig. 1a, b, Supplementary Fig. S1 and Supplementary Data 1–3). Thus,
predisposing genetic alteration shapes distinct gut microbiota
regardless of whether the alteration is associated with infection-
dependent or infection-independent ALL development. We have previously shown that altering the gut microbiome with
antibiotic treatment elicits leukemia in Pax5+/−mice, even in the
absence of an infectious trigger (i.e., in specific pathogen-free condi-
tions, SPF). Therefore, we next explored whether altering the gut
microbiome under SPF conditions might lead to leukemia induction in
Sca1-ETV6-RUNX1, Sca1-Lmo2, and Sca1-BCR-ABLp190 mice. These mice
were treated with an antibiotic cocktail (ampicillin, vancomycin,
ciprofloxacin, imipenem, and metronidazole) in their drinking water
for 8 weeks, which was started at around 8 weeks of age. In all three
models, leukemia incidence, time to disease onset and the disease
characteristics from both immunophenotypic and histological points
of view were not altered by antibiotic treatment compared to non-
antibiotic treated mice (Fig. 1c, Supplementary Fig. S2). Thus, mod-
ifying the gut microbiome through bacterial depletion impacts B-ALL
development in Pax5+/– mice but not in these other leukemia-
predisposed strains. mmune training by early exposure to infection does not pre-
vent leukemia development in Pax5+/−mice In agreement with the delayed exposure hypothesis, epidemiologic
studies suggest that exposures leading to immune stimulation (i.e.,
breastfeeding, daycare attendance, contact with pets, allergies) during
early childhood are correlated with reduced development of the most
common B-ALL subtypes (ETV6-RUNX1+ and high-hyperdiploid B-
ALL)16–21. To ascertain whether the timing of infection exposure is
relevant for B-ALL development in Pax5+/−mice, heterozygous mutant
and control littermate WT mice were exposed to the same infectious
conditions since pregnancy and birth, and then during the rest of their
lifespan (we named this the “early exposure group”) (Fig. 2a). The
microbiological status in the conventional facility was defined and
controlled for 2 years (Supplementary Data 4). The early exposure to
common pathogens induced a significant decrease in peripheral blood
(PB) B-cells in both WT and Pax5+/−mice. However, this decrease does
not seem to be related to leukemogenesis as WT mice never develop
B-ALL. Under this scenario, specific B-ALL development was observed
in 25% (7 out of 28) of Pax5+/−animals (but none of the WT mice) of the
early exposure group (Fig. 2b), closely resembling the penetrance of
B-ALL development in Pax5+/−mice with delayed exposure3 and in
humans
harboring
a
heterozygous
PAX5
c.547G>A
pathogenic
variant22–24. The leukemias occurring in Pax5+/−early exposure mice
developed between 9 and 17 months of age, similar to those devel-
oping in delayed-exposure mice3, and they exhibited the same leu-
kemic immunophenotype (Fig. 2c and Supplementary Fig. S3–S5). Therefore, the timing of infection does not appear to impact leukemia
development in Pax5+/−mice with exposure to an immune stressor (i.e.,
housing in a conventional, non-SPF, facility) representing the trigger-
ing event. Immune stress suppresses innate immune
signaling in preleukemic precursor B-cells to
provoke leukemia in predisposed mice Taken together, these results suggest that,
although childhood ALL up until now have been speculated to have a
common underlying association with infection, there is etiologic het-
erogeneity regarding the role of gut microbiome dysbiosis in the
development of different genetic subtypes of childhood ALL. Germline mutations affecting the B cell master regulator PAX5 or
the presence of the somatic ETV6-RUNX1 fusion are associated with
B-ALL development in a proportion of human carriers, and this process
can be faithfully recapitulated in Pax5+/−or ETV6-RUNX1 transgenic
mice3,5. Using these and other mouse models, recent studies have
begun to address how preleukemic cells respond to infectious stimuli;
in this way, it has been demonstrated that infection-driven B-ALL
development in Pax5+/−mice is T-cell independent13. Further, the pre-
sence of the Pax5+/−or ETV6-RUNX1 mutations uniquely shapes the gut
microbiota
when
compared
to
the
microbiota
of
wild-type
littermates13. Curiously, the Pax5+/−-specific gut microbiota protects
these predisposed mice from leukemia development, and its alteration
by antibiotic treatment early in life is sufficient to induce leukemia,
even in the absence of exposure to common pathogens13. In the pre-
sent study, we sought to determine how Pax5+/−and ETV6-RUNX1
preleukemic cells switch to a leukemic state using the Pax5+/−and ETV6-
RUNX1 mouse models, which are uniquely suited for the study of the
early stages of malignant transformation2. Herein, we describe how the
temporary malfunction of innate immune signaling plays a key role in
this process in Pax5+/−mice, and we identify how this is mediated by a
molecular mechanism based on the partial downregulation of Myd88,
finally leading to B-ALL in Pax5+/−mice. These findings have important
implications for the understanding and potential therapeutic targeting
of preleukemic cells in susceptible children. Immune training by early exposure to infection does not pre-
vent leukemia development in Pax5+/−mice Immune stress suppresses innate immune
signaling in preleukemic precursor B-cells to
provoke leukemia in predisposed mice However, occasionally they
can transform over time through the acquisition of additional somatic
genetic alterations that lead to the appearance of full-blown B-ALL2–5. The nature of the environmental or intrinsic signals that con-
tribute to this “switch” from the preleukemic state to the leukemic
state remains unclear. The majority of preleukemic genetic alterations
on their own are also insufficient to cause B-ALL in mice, essentially
recapitulating the scenario in humans2. Recent preclinical studies have
shown that exposure to an immune stressor such as common patho-
gens is necessary to provide the selection advantage that allows pre-
leukemic cells to give rise to leukemia in genetically susceptible strains
of mice3,5. Epidemiologic data demonstrating that infection can act as a
trigger for B-ALL development in humans further supports these ears at the end of the paper. e-mail: cvd@usal.es; cesar.cobaleda@csic.es; Kim.Nichols@STJUDE.ORG; isg@usal.es Nature Communications| (2023) 14:5159 Nature Communications| (2023) 14:5159 Nature Communications| (2023) 14:5159 1 https://doi.org/10.1038/s41467-023-40961-z Article findings6–11. However, the nature and order of the events by which
infection triggers leukemia is still a matter of debate. The “delayed
infection” hypothesis is based on the possibility that an immune sys-
tem unexposed to common infections early in life may function
abnormally when exposed to such infections later in life. Specifically,
the abnormal immune signaling triggered by these infections is pro-
posed to lead to a series of genetic events that culminate in the
development of leukemia. Alternatively, the “population mixing”
hypothesis was postulated to explain the appearance of small clusters
of patients with B-ALL in a localized place and time12. It posits that
childhood B-ALL arises as an uncommon result of exposure to any
otherwise common infection, but this would only become noticeable
as clusters of B-ALL at times of population mixing, when a relatively
large number of susceptible children naïve to this infection mix with a
large number of infected individuals, leading to a (largely subclinical)
widespread epidemics12. In either case, a transient deficiency of the
immune priming could system act together with a gut microbiome
dysbiosis to drive B-ALL. However, little is known about how exposure
to infection promotes such transformation of preleukemic cells. findings6–11. However, the nature and order of the events by which
infection triggers leukemia is still a matter of debate. https://doi.org/10.1038/s41467-023-40961-z https://doi.org/10.1038/s41467-023-40961-z b
a
c
Axis 1
(13.20%)
Axis 2
(11.28%)
Axis 1
(13.20%)
Axis 4
(6.77%)
Axis 5
(4.09%)
Axis 3
(8.148%)
ns
p= 0.001
p= 0.001
p= 0.001
Sca1-Lmo2
(n=180)
WT
(n=66)
Sca1-BCR-ABLp190
(n=180)
Pax5+/-
(n=120)
p= 0.001
p= 0.001
p= 0.001
Distances to WT
Sca1-Lmo2
WT
Sca1-BCR-ABLp190
Pax5+/- Axis 1
(13.20%)
Axis 4
(6.77%)
Axis 5
(4.09%)
Sca1-Lmo2
WT
Sca1-BCR-ABLp19
Pax5+/- b
a
c
Axis 1
(13.20%)
Axis 2
(11.28%)
Axis 1
(13.20%)
Axis 4
(6.77%)
Axis 5
(4.09%)
Axis 3
(8.148%)
***
ns
ns
ns
p= 0.001
p= 0.001
p= 0.001
Sca1-Lmo2
(n=180)
WT
(n=66)
Sca1-BCR-ABLp190
(n=180)
Pax5+/-
(n=120)
p= 0.001
p= 0.001
p= 0.001
Distances to WT
Sca1-Lmo2
WT
Sca1-BCR-ABLp190
Pax5+/-
Genetic susceptibility to ALL shapes a specific gut microbiota in pre-
ed mice. a Pairwise Jaccard distances (beta diversity) were computed for all
es. Distance metric was ordinated into 3D via Principal Coordinates Analysis
) and visualized via Emperor. Axes indicate percent of explained variance. wise Permanova tests with 999 permutations were applied to test for dif-
es in beta diversity grouped by mouse genotype (WT n = 12, Sca1-BCR-
90 n = 15, Sca1-Lmo2 n = 15 and Pax5+/−n = 10). Scatter dot plots visualize
d distances between samples of the same genotype. Data are shown as
± SD,“n = ” reports measured distances, “p“ is the p-value of the Permanova
test. c Leukemia specific survival curve of Sca1-BCR-ABLp190 treated (n = 13) or
untreated (n = 7) with antibiotic6s (p-value = 0.6514), Sca1-Lmo2 treated (n = 24)
untreated (n = 14) with antibiotics (p-value = 0.7935), and Sca1-ETV6-RUNX1 treat
(n = 6) or untreated (n = 10) with antibiotics mice p-value > 0.9999), and Pax5+/−
treated (n = 19) or untreated (n = 20) with antibiotics (p-value = 0.0002). None o
them were exposed to common infections. P-values are from Log-rank
(Mantel–Cox) test. Source data are provided as a Source Data file. Abx: treated wi
a cocktail of antibiotics for 8 weeks. Microbiome disruption promotes leukemia development in
Pax5+/−mice but not other genetically prone strains Pax5
mice but not other genetically prone strains
Our group recently found that both Pax5+/−and Sca1-ETV6-RUNX1
transgenic mice harbor distinct genotype-specific gut microbiomes
directly related to the genetic alteration they are carrying13. This work
established an experimental paradigm to evaluate and identify the
factors that modulate the selective advantage of preleukemic hema-
topoietic precursors. Thus, we first asked whether this relationship
between leukemia-predisposition genetic alteration and gut micro-
biome is an inherent characteristic of genetic alterations associated
with infection-driven leukemias or, rather, predisposing genetic
alterations associated with infection-independent leukemias. To test
this possibility, we took advantage of the Sca1-Lmo2 mouse model,
which develops infection-independent T-ALL14, and the Sca1-BCR-
ABLp190 model, which develops infection-independent B-ALL15. We
initially analyzed the impact of these genotypes on the microbiome
using stool samples from Sca1-Lmo2 and Sca1-BCR-ABLp190 mice. Indeed, the effect of each genetic alteration shaped a characteristic gut
microbiome that was distinct between these two mouse strains and
differed from the microbiome in wild-type (WT) and Pax5+/−animals Nevertheless,
it
remained
to
be determined
whether
the
mechanism driving B-ALL susceptibility in Pax5+/−mice under early
exposure to infections was similar to the one driving B-ALL onset in
Pax5+/−mice with delayed exposure. Examination of B cells in pre-
leukemic Pax5+/−early exposure littermates revealed significantly
reduced proportions of total B-cells in the peripheral blood (PB) when
compared to WT littermates housed under similar conditions (Sup-
plementary Fig. S6). This decrease in PB B-cell proportions was similar
to what we observed in the delayed-exposure Pax5+/−group (Supple-
mentary Fig. S6). To identify the somatically acquired second hits Nature Communications| (2023) 14:5159 2 Article https://doi.org/10.1038/s41467-023-40961-z a
Axis 1
(13.20%)
Axis 2
(11.28%)
Axis 1
(13.20%)
Axis 4
(6.77%)
Axis 5
(4.09%)
Axis 3
(8.148%)
Sca1-Lmo2
WT
Sca1-BCR-ABLp190
Pax5+/- b
Axis 1
(13.20%)
Axis 1
(13.20%)
Axis 5
(4.09%)
Axis 3
(8.148%)
p= 0.001
p= 0.001
p= 0.001
Sca1-Lmo2
(n=180)
WT
(n=66)
Sca1-BCR-ABLp190
(n=180)
Pax5+/-
(n=120)
p= 0.001
p= 0.001
p= 0.001
Distances to WT b
p= 0.001
p= 0.001
p= 0.001
Sca1-Lmo2
(n=180)
WT
(n=66)
Sca1-BCR-ABLp190
(n=180)
Pax5+/-
(n=120)
p= 0.001
p= 0.001
p= 0.001
Distances to WT b c
***
ns
ns
ns c
***
ns
ns
ns c Fig. 1 | Genetic susceptibility to ALL shapes a specific gut microbiota in pre- test. c Leukemia specific survival curve of Sca1-BCR-ABLp190 treated (n = 13) or
untreated (n = 7) with antibiotic6s (p-value = 0.6514), Sca1-Lmo2 treated (n = 24) or
untreated (n = 14) with antibiotics (p-value = 0.7935), and Sca1-ETV6-RUNX1 treated
(n = 6) or untreated (n = 10) with antibiotics mice p-value > 0.9999), and Pax5+/−
treated (n = 19) or untreated (n = 20) with antibiotics (p-value = 0.0002). None of
them were exposed to common infections. P-values are from Log-rank
(Mantel–Cox) test. Source data are provided as a Source Data file. Abx: treated with
a cocktail of antibiotics for 8 weeks. disposed mice. a Pairwise Jaccard distances (beta diversity) were computed for all
samples. Distance metric was ordinated into 3D via Principal Coordinates Analysis
(PCoA) and visualized via Emperor. Axes indicate percent of explained variance. b Pairwise Permanova tests with 999 permutations were applied to test for dif-
ferences in beta diversity grouped by mouse genotype (WT n = 12, Sca1-BCR-
ABLp190 n = 15, Sca1-Lmo2 n = 15 and Pax5+/−n = 10). Scatter dot plots visualize
Jaccard distances between samples of the same genotype. Data are shown as
Mean ± SD,“n = ” reports measured distances, “p“ is the p-value of the Permanova 3 Nature Communications| (2023) 14:5159 https://doi.org/10.1038/s41467-023-40961-z Article ading to leukemia development, we next performed whole exome
equencing of four Pax5+/−early exposure tumors and their corre-
ponding germline. Similar to leukemias from the delayed Pax5+/−
roup, early exposure tumors showed recurrent mutations affecting
he remaining WT Pax5 allele, with Pax5 p.P80R occurring in 2 out of 4
nalyzed leukemias (Supplementary Fig. S7). https://doi.org/10.1038/s41467-023-40961-z Moreover, an activating
ak1 p.F837V variant was identified in one of the leukemic samples,
which again highlights the importance of the JAK/STAT-si
pathway in Pax5+/−leukemias, as shown for Jak1 and Jak3 muta
the Pax5+/−delayed-exposure B-ALL3,25. Interestingly, 2 out of
analyzed harbored somatic mutations affecting the RAS-RA
ERK-pathway (Nras p.G13D and Kras p.Q61H) (Supplementary F
Somatic mutations that deregulate this signaling pathway are
the most common genetic aberrations in childhood ALL26. In su
a
BONE MARROW
PERIPHERAL BLOOD
Wild type
Pax5+/- early
e
r
u
s
o
p
x
e
(R324)
IgM
B220
CD19
B220
IgM
B220
CD19
B220
Pax5+/- delay
e
r
u
s
o
p
x
e
(R253)
b
c
*
B-
L
L
A
c
ifi
c
e
p
s
survival
B-ALL specific
survival
*
ns
*
*
Early-
e
r
u
s
o
p
x
e
y
a
l
e
D
-
e
r
u
s
o
p
x
e
Endpoint
E 0-21
2 months
1 month
Birth
PN 0-21
4 months
Endpoint
E 0-21
2 months
1 month
Birth
PN 0-21
4 months
Article
https://doi.org/10.1038/s41467-023-4 a
b
*
B-
L
L
A
c
ifi
c
e
p
s
survival
B-ALL specific
survival
*
ns
*
*
Early-
e
r
u
s
o
p
x
e
y
a
l
e
D
-
e
r
u
s
o
p
x
e
Endpoint
E 0-21
2 months
1 month
Birth
PN 0-21
4 months
Endpoint
E 0-21
2 months
1 month
Birth
PN 0-21
4 months a
Early-
e
r
u
s
o
p
x
e
y
a
l
e
D
-
e
r
u
s
o
p
x
e
Endpoint
E 0-21
2 months
1 month
Birth
PN 0-21
4 months
Endpoint
E 0-21
2 months
1 month
Birth
PN 0-21
4 months a b
*
B-
L
L
A
c
ifi
c
e
p
s
survival
B-ALL specific
survival
*
ns
*
*
e
D b
*
B-
L
L
A
c
ifi
c
e
p
s
survival
B ALL specific
* b B-ALL specific
i
l ns leading to leukemia development, we next performed whole exome
sequencing of four Pax5+/−early exposure tumors and their corre-
sponding germline. Similar to leukemias from the delayed Pax5+/−
group, early exposure tumors showed recurrent mutations affecting
the remaining WT Pax5 allele, with Pax5 p.P80R occurring in 2 out of 4
analyzed leukemias (Supplementary Fig. S7). https://doi.org/10.1038/s41467-023-40961-z Moreover, an activating
Jak1 p.F837V variant was identified in one of the leukemic samples,
which again highlights the importance of the JAK/STAT-signaling
pathway in Pax5+/−leukemias, as shown for Jak1 and Jak3 mutations in
the Pax5+/−delayed-exposure B-ALL3,25. Interestingly, 2 out of 4 mice
analyzed harbored somatic mutations affecting the RAS-RAF-MEK-
ERK-pathway (Nras p.G13D and Kras p.Q61H) (Supplementary Fig. S7). Somatic mutations that deregulate this signaling pathway are among
the most common genetic aberrations in childhood ALL26. In summary,
BONE MARROW
PERIPHERAL BLOOD
Wild type
Pax5+/- early
e
r
u
s
o
p
x
e
(R324)
IgM
B220
CD19
B220
IgM
B220
CD19
B220
Pax5+/- delay
e
r
u
s
o
p
x
e
(R253)
c
Nature Communications| (2023)14:5159
4 BONE MARROW
Wild type
Pax5+/- early
e
r
u
s
o
p
x
e
(R324)
IgM
B220
CD19
B220
IgM
B220
Pax5+/- delay
e
r
u
s
o
p
x
e
(R253)
c BONE MARROW
PERIPHERAL BLOOD
Wild type
Pax5+/- early
e
r
u
s
o
p
x
e
(R324)
IgM
B220
CD19
B220
IgM
B220
CD19
B220
Pax5+/- delay
e
r
u
s
o
p
x
e
(R253)
c c PERIPHERAL BLOOD IgM IgM which again highlights the importance of the JAK/STAT-signaling
pathway in Pax5+/−leukemias, as shown for Jak1 and Jak3 mutations in
the Pax5+/−delayed-exposure B-ALL3,25. Interestingly, 2 out of 4 mice
analyzed harbored somatic mutations affecting the RAS-RAF-MEK-
ERK-pathway (Nras p.G13D and Kras p.Q61H) (Supplementary Fig. S7). Somatic mutations that deregulate this signaling pathway are among
the most common genetic aberrations in childhood ALL26. In summary, leading to leukemia development, we next performed whole exome
sequencing of four Pax5+/−early exposure tumors and their corre-
sponding germline. Similar to leukemias from the delayed Pax5+/−
group, early exposure tumors showed recurrent mutations affecting
the remaining WT Pax5 allele, with Pax5 p.P80R occurring in 2 out of 4
analyzed leukemias (Supplementary Fig. S7). Moreover, an activating
Jak1 p.F837V variant was identified in one of the leukemic samples, Nature Communications| (2023) 14:5159 4 Article https://doi.org/10.1038/s41467-023-40961-z Fig. 2 | Early exposure to infection promotes the development of B-cell pre-
cursor acute lymphoblastic leukemia in Pax5+/−mice. a Experimental setup. Female mice of the “early exposure group” got pregnant already in a conventional
facility (i.e., non-SPF conditions) where they are exposed to common infections
during their lifespan, and the born pups were kept in these conditions for their
lifetime. Reduced expression of Myd88 in immune-stressed preleukemic
Pax5+/−precursor B-cells Innate immunity plays a critical role in shaping the evolution of
cancers with a demonstrated causal association with infection27. Indeed, it has been reported that, in infection-triggered cancers,
Toll-like receptors (TLRs) play a central role in inhibiting as well as
in promoting cancer formation, both at the level of immune cells
within the tumor microenvironment and acting directly on the
cancer cells themselves27. To elucidate whether TLR signaling is
involved in the transition of pre-malignant B cells to B-ALL, we next
performed a Mouse Toll-Like Receptor Signaling Pathway PCR Array
using precursor B cells isolated from the bone marrow (BM) of
Pax5+/−and WT mice kept either under SPF (Specific Pathogen Free)
conditions or exposed to natural infection (i.e., housed in the con-
ventional facility) both for 3 or 6 months. As we previously showed,
Pax5+/−
mice
only
developed
B-ALL
under
natural
infection
exposure3. Analysis of the TLR signaling pathway in preleukemic
precursor B cells of Pax5+/−mice exposed to infections revealed a
decrease in the expression of the adaptors Myd88 and Tirap, as well
as the downstream transcription factor Nfkb1a, (Supplementary
Fig. S8). Using real-time PCR quantification, we observed that low
levels of Myd88 could be detected in a small percentage of Pax5+/−
preleukemic mice, and that this expression was within a similar
range to that observed in WT precursor B-cells (Fig. 3a). However,
Myd88 expression was significantly decreased in Pax5+/−precursor
B-cells from antibiotic-treated mice under SPF conditions and was
equal to the level observed in Pax5+/−leukemic B-cells (Fig. 3a). The
antibiotic treatment of ETV6-RUNX1 mice also induced a reduction
in Myd88 expression in precursor B-cells (Fig. 3a). This finding
suggests that the inability of Abx-treatment to drive leukemia pro-
gression in the Sca1-ETV6-RUNX1 model therefore, does not appear
to be due to a failure to down-regulate Myd88 expression in pre-
cursor B-cells. While the Sca1-ETV6-RUNX1 model might have a dif-
ferent reliance on Myd88 signaling or on the cell intrinsic
developmental stage where molecular alterations should take place,
these results highlight the need to identify downstream mechan-
isms to provide possible explanations for the inability of Abx-
treatment to drive leukemia progression in the Sca1-ETV6-RUNX1
model. Thus, induction of a leukemogenic state is associated with a
decrease of Myd88 expression in Pax5+/−B-ALL blasts. Downregulation of Myd88 increases the penetrance of infection-
driven B-ALL development in Pax5+/−mice Downregulation of Myd88 increases the penetrance of infection-
driven B-ALL development in Pax5+/−mice we obtained in vivo evidence showing that Pax5+/−mice exposed to
infections early in life develop leukemia with similar incidence, latency,
and molecular features as Pax5+/−mice exposed later in life. These
results show that immune training by early exposure to infection does
not have a protective effectagainst the conversion of preleukemic cells
to B-ALL in mice heterozygous for a germline Pax5 mutation. These
findings strongly point toward a cell-autonomous defect within Pax5+/−
preleukemic cells and further reinforce the view of etiologic variation
in leukemia development among different B-ALL molecular subtypes. we obtained in vivo evidence showing that Pax5+/−mice exposed to
infections early in life develop leukemia with similar incidence, latency,
and molecular features as Pax5+/−mice exposed later in life. These
results show that immune training by early exposure to infection does
not have a protective effectagainst the conversion of preleukemic cells
to B-ALL in mice heterozygous for a germline Pax5 mutation. These
findings strongly point toward a cell-autonomous defect within Pax5+/−
preleukemic cells and further reinforce the view of etiologic variation
in leukemia development among different B-ALL molecular subtypes. p
Given the downregulation of Myd88 in preleukemic cells under anti-
biotic treatment as well as in Pax5+/−B-ALL blasts, we next directly
tested the requirement for Myd88 in B-ALL development. First, we
decreased the expression of Myd88 by generating Pax5+/−;Myd88+/−
mice by interbreeding, and examined whether Pax5+/−;Myd88+/−animals
were more prone to infection-induced B-ALL than Pax5+/−mice. Pax5+/
−;Myd88+/−mice were exposed to natural infections, and B-ALL devel-
opment was monitored. We observed that B-ALL development was
significantly increased in Pax5+/−;Myd88+/−mice, where 64% (16 out of
25) developed B-ALL (Fig. 3b), closely resembling the penetrance of
B-ALL development in Pax5+/−animals treated with antibiotics13. To
extend these findings, SPF-housed, two months-old WT and Pax5+/−
mice were intraperitoneally (i.p.) injected with PBS or 35 mg LPS (a
Myd88-dependent TLR ligand) every other day for eight times over 2
weeks. This exposure of SPF-housed Pax5+/−mice to an Myd88-
dependent TLR ligand generated B-ALL in 20% of the mice (Fig. 4). In
contrast, the exposure of SPF-housed Pax5+/−mice to an Myd88-
independent TLR ligand (mice treated with an intravenous injection of
200 μg of poly(I:C) for a total of 2 times, separated by four weeks
between doses) did not generate B-ALL (Fig. https://doi.org/10.1038/s41467-023-40961-z Female mice from the “delayed-exposure group” got pregnant under SPF
conditions, and pups were kept in SPF until one month of age, when they were
moved to the conventional facility. The mice were monitored periodically. The
endpoint was established at 2 years of age (22–24 months) or when the mice
showed signs of disease (clinical signs or presence of blasts in peripheral blood);
the animals were sacrificed and the hematopoietic and non-hematopoietic tissues
were analyzed. E: embryonic period; PN: postnatal period; SPF: specific pathogen
free. b B-ALL specific survival curve of Pax5+/−animals born and kept into non-SPF
conditions (red, n = 28) compared to WT control mice (black, n = 21) showing a significantly (log-rank P value 0.0151) shortened life span and compared to Pax5+/−
animals always kept into SPF conditions (green, n = 20) showing a significantly (log-
rank P value 0.0177) shortened life span (left panel), and B-ALL specific survival
curve of Pax5+/−early exposure group (red, n = 28) compared to Pax5+/−delayed-
exposure group (blue, n = 41) showing similar life span (log-rank P value 0.5491) and
comparing Pax5+/−animals always kept into SPF conditions (green, n = 20) to Pax5+/−
delayed-exposure group (blue, n = 41) showing a significantly (log-rank P value
0.0098) shortened life span (right panel). Source data are provided as a Source
Data file. c Flow cytometric analysis of hematopoietic subsets in diseased Pax5+/−
early exposure mice. Representative plots of cell subsets from the bone marrow
and peripheral blood show accumulation of blast B cells in Pax5+/−early exposure
mice (n = 7, age: 9–17 months) compared to control age-matched littermate WT
mice (n = 4, age: 8–16 months). Pax5+/−early exposure mice show a similar immune-
phenotype than Pax5+/−delayed-exposure mice (n = 9, age: 6–16 months). Downregulation of Myd88 increases the penetrance of infection-
driven B-ALL development in Pax5+/−mice 4), therefore supporting
the direct participation of Myd88 modulation in B-ALL development in
this model. The advent of leukemia in Pax5+/−;Myd88+/−mice under
natural infection exposure occurred at between 7 and 20 months of
age (Fig. 3b), similar to the Pax5+/−group. Thus, the latency in leuke-
mogenesis was not changed. Furthermore, the critical role of Myd88
downregulation for B-ALL development in Pax5+/−mice was further
confirmed by both the lack of B-ALL development in Pax5+/+;Myd88+/−
mice under natural infection exposure (0 out of 26) (Fig. 3b) and the
appearance of B-ALL in Pax5+/−;Myd88+/−mice even in the absence of
exposure to infections (5 out 8, 62.5%) (Supplementary Fig. S9), a
condition where Pax5+/−mice never develop B-ALL3. In all these cases
the characterization of Pax5+/−;Myd88+/−B-ALLs showed that they are
histologically and phenotypically similar to those appearing in the
Pax5+/−group (Fig. 3c, d and Supplementary Figs. S10–S12). Reduced expression of Myd88 in immune-stressed preleukemic
Pax5+/−precursor B-cells S16, S17, Supplementary Data
ns
e
v
it
a
l
e
R
n
o
i
s
s
e
r
p
x
e
8
8
d
y
M
f
o
Preleukemic
Leukemic
Myd88-/-
WT pre-proB
Pax5+/-
Pax5+/- (Abx)
Pax5+/- (Abx)
Pax5+/- Myd88+/-
Sca1-ETV6-RUNX1
Sca1-ETV6-RUNX1 (Abx)
***
* p=0.012
* p=0.0378
***
** p=0.004
***
***
***
***
** p=0.001
a
b
c
d
Peyer’s
patches
Spleen
Lymph
nodes
Myd88+/-
Pax5+/- Myd88+/-
10x
10x
10x
10x
40x
10x
40x
10x
40x
10x
40x
10x
40x
10x
40x
500 µm
500 µm
500 µm
500 µm
500 µm
500 µm
500 µm
100 µm
100 µm
100 µm
100 µm
100 µm
100 µm
40x
40x
40x
500 µm
500 µm
100 µm
100 µm
100 µm
BONE MARROW
IgM
B220
Pax5+/- Myd88+/-
B-ALL
Healthy
Myd88+/-
0
5
10
15
20
25
0
20
40
60
80
100
Months
B-ALL-specific survival
Wild Type (n=23)
Pax5+/-(n=41)
Myd88+/-Pax5+/- (n=30)
Myd88+/- Pax5+/+ (n=26)
*
**
***
**
****
Article
https://doi.org/10.1038/s41467-023-4096 https://doi.org/10.1038/s41467-023-40961-z Article ns
e
v
it
a
l
e
R
n
o
i
s
s
e
r
p
x
e
8
8
d
y
M
f
o
Preleukemic
Leukemic
Myd88-/-
WT pre-proB
Pax5+/-
Pax5+/- (Abx)
Pax5+/- (Abx)
Pax5+/- Myd88+/-
Sca1-ETV6-RUNX1
Sca1-ETV6-RUNX1 (Abx)
***
* p=0.012
* p=0.0378
***
** p=0.004
***
***
***
***
** p=0.001
a
b
0
5
10
15
20
25
0
20
40
60
80
100
Months
B-ALL-specific survival
Wild Type (n=23)
Pax5+/-(n=41)
Myd88+/-Pax5+/- (n=30)
Myd88+/- Pax5+/+ (n=26)
*
**
***
**
**** ns
e
v
it
a
l
e
R
n
o
i
s
s
e
r
p
x
e
8
8
d
y
M
f
o
Preleukemic
Leukemic
Myd88-/-
WT pre-proB
Pax5+/-
Pax5+/- (Abx)
Pax5+/- (Abx)
Pax5+/- Myd88+/-
Sca1-ETV6-RUNX1
Sca1-ETV6-RUNX1 (Abx)
***
* p=0.012
* p=0.0378
***
** p=0.004
***
***
***
***
** p=0.001
a b
0
5
10
15
20
25
0
20
40
60
80
100
Months
B-ALL-specific survival
Wild Type (n=23)
Pax5+/-(n=41)
Myd88+/-Pax5+/- (n=30)
Myd88+/- Pax5+/+ (n=26)
*
**
***
**
**** b a Pax5+/−;Myd88+/−B-ALLs exhibited a gene expression pattern similar
to human B-ALLs, and the gene expression profiles of Pax5+/
;Myd88+/−and Pax5+/−B-ALLs did not show major differences
between them or when compared with gene expression profiles
derived from blast cells from patients with B-ALL (Supplementary
Fig. Reduced expression of Myd88 in immune-stressed preleukemic
Pax5+/−precursor B-cells To explore the relevance of our findings for human leukemia,
we compared the role of Myd88 activity on mRNA expression in
from tumor cells from patients with B-ALL28–31. Using RNA micro-
array gene expression analyses, we identified differentially expres-
sed genes (n = 2463) in leukemic BM from Pax5+/−;Myd88+/−mice
compared with BM precursor B cells from WT mice (Supplementary
Fig. S13 and Supplementary Data 5). When compared with defined
signatures (genesets) previously identified as specifically up- or
down-regulated in human B-ALLs28,29,31, the transcriptional profiles
of mouse B-ALLs behaved similar to the ones derived from human
leukemias (Fig. 5a and Supplementary Fig. S13). Similarly, the genes
differentially expressed between mouse B-ALLs and normal mouse
B cell progenitors showed a parallelism with genesets related to
malignant transformation (Supplementary Fig. S14). Therefore, Nature Communications| (2023) 14:5159 5 Pax5+/−;Myd88+/−B-ALLs exhibited a gene expression pattern similar
to human B-ALLs, and the gene expression profiles of Pax5+/
−;Myd88+/−and Pax5+/−B-ALLs did not show major differences
between them or when compared with gene expression profiles
derived from blast cells from patients with B-ALL (Supplementary
Fig. S15 and Supplementary Data 6), indicating that Myd88 is not
significantly contributing to the leukemic gene expression profile of
infection-driven B-ALL. To identify somatically acquired second hits leading to B-A
development in Pax5+/−;Myd88+/−mice, we performed whole-geno
sequencing of paired tumor and germline tail DNA samples from ei
Pax5+/−;Myd88+/−tumors. The percentage of leukemic cells in
sequenced samples ranged from 88.1% to 98.7%. We identified sev
somatically acquired recurrent mutations and copy-number variati
involving B cell transcription factors in diseased Pax5+/;Myd88+/−m
(Fig. 5b, Supplementary Figs. Reduced expression of Myd88 in immune-stressed preleukemic
Pax5+/−precursor B-cells S15 and Supplementary Data 6), indicating that Myd88 is not
significantly contributing to the leukemic gene expression profile of
nfection-driven B-ALL. To identify somatically acquired second hits leading to B-ALL
development in Pax5+/−;Myd88+/−mice, we performed whole-genome
sequencing of paired tumor and germline tail DNA samples from eight
Pax5+/−;Myd88+/−tumors. The percentage of leukemic cells in the
sequenced samples ranged from 88.1% to 98.7%. We identified several
somatically acquired recurrent mutations and copy-number variations
involving B cell transcription factors in diseased Pax5+/;Myd88+/−mice
(Fig. 5b, Supplementary Figs. S16, S17, Supplementary Data 7). c
d
Peyer’s
patches
Spleen
Lymph
nodes
Myd88+/-
Pax5+/- Myd88+/-
10x
10x
10x
10x
40x
10x
40x
10x
40x
10x
40x
10x
40x
10x
40x
500 µm
500 µm
500 µm
500 µm
500 µm
500 µm
500 µm
100 µm
100 µm
100 µm
100 µm
100 µm
100 µm
40x
40x
40x
500 µm
500 µm
100 µm
100 µm
100 µm
BONE MARROW
IgM
B220
Pax5+/- Myd88+/-
B-ALL
Healthy
Myd88+/-
Nature Communications| (2023) 14:5159
6 d
Peyer’s
patches
Spleen
Lymph
nodes
Myd88+/-
Pax5+/- Myd88+/-
10x
10x
10x
10x
40x
10x
40x
10x
40x
10x
40x
10x
40x
10x
40x
500 µm
500 µm
500 µm
500 µm
500 µm
500 µm
500 µm
100 µm
100 µm
100 µm
100 µm
100 µm
100 µm
40x
40x
40x
500 µm
500 µm
100 µm
100 µm
100 µm c
BONE MARROW
IgM
B220
Pax5+/- Myd88+/-
B-ALL
Healthy
Myd88+/- d c Pax5+/- Myd88+/- IgM Pax5+/−;Myd88+/−B-ALLs exhibited a gene expression pattern similar
to human B-ALLs, and the gene expression profiles of Pax5+/
−;Myd88+/−and Pax5+/−B-ALLs did not show major differences
between them or when compared with gene expression profiles
derived from blast cells from patients with B-ALL (Supplementary
Fig. S15 and Supplementary Data 6), indicating that Myd88 is not
significantly contributing to the leukemic gene expression profile of
infection-driven B-ALL. To identify somatically acquired second hits leading to B-ALL
development in Pax5+/−;Myd88+/−mice, we performed whole-genome
sequencing of paired tumor and germline tail DNA samples from eight
Pax5+/−;Myd88+/−tumors. The percentage of leukemic cells in the
sequenced samples ranged from 88.1% to 98.7%. We identified several
somatically acquired recurrent mutations and copy-number variations
involving B cell transcription factors in diseased Pax5+/;Myd88+/−mice
(Fig. 5b, Supplementary Figs. S16, S17, Supplementary Data 7). https://doi.org/10.1038/s41467-023-40961-z Thus, early Myd88-independent TLR ligation during the preleukemic
phase in Pax5+/−mice resulted in significant delay of B-ALL develop-
ment under oncogenic environmental conditions, supporting the view
that immune modulation during the preleukemic stage can sig-
nificantly alter progression to B-ALL2. Furthermore, recurrent mutations affecting the JAK/STAT and RAS
signaling pathways were detected (Fig. 5b, Supplementary Fig. S16). The same genes are also mutated in Pax5+/−murine leukemias3,13,25 and
human B-ALL samples26. Thus, the drivers of infection-associated B-
ALLs are similar in both Pax5+/−;Myd88+/−and Pax5+/−leukemic cells. Collectively, these observations revealed that downregulation of
Myd88 is associated with increased development of leukemia with a
similar genetic profile as the leukemias observed in Pax5+/−mice and
those from human patients. Pax5+/−;Myd88+/−precursor B cells exhibit an inflammatory
transcriptional profile p
p
We next studied the function of Myd88 downregulation at the pre-
leukemic stages by examining both the accumulation of myeloid cells
and the transcriptional profiles of Pax5+/−;Myd88+/−preleukemic pre-
cursor B-cells in comparison with those from Pax5+/−preleukemic cells
(Supplementary Figs. S18–21 and Supplementary Data 8–10). Myd88
downregulation was not associated with an accumulation of myeloid
cells in Pax5+/−;Myd88+/−mice when compared to Pax5+/−;Myd88+/+ mice
(Supplementary Fig. S18). However, Pax5+/−;Myd88+/−proB cells were
characterized by an increased expression of genes involved in activa-
tion of signaling effectors (for example, Notch, apoptosis and IL2-
STAT5 signaling) associated with cell proliferation and survival
(Fig. 5c). Pax5+/−;Myd88+/−proB cells were also enriched in genes asso-
ciated with pre-B and follicular murine B cell developmental stages
(Supplementary Fig. S21A). Interestingly, the downregulation of Myd88
in Pax5+/−proB cells exacerbated the number of genes differentially
expressed that are regulated by Pax5, like Cd19, Slamf6, Bst1, Foxo1, or
Bcar3 (Supplementary Figure S21B). Myd88 downregulation in Pax5+/−
preleukemic precursor B-cells also augmented the expression of genes
involved in the interferon-α response, TNFα signaling via NFκB and
interferon-γ response (Fig. 5c), in agreement with the increase in
inflammatory cytokines observed in peripheral blood in Pax5+/
−;Myd88+/−mice (Supplementary Fig. S22). These findings suggest that
Myd88-dependent signaling was hyperactive in Pax5+/−cells, and failed
to upregulate feedback inhibitors of the Myd88–NFκB signaling
pathway (Fig. 5c), in agreement with previous results showing that
Myd88 deficiency results in tissue-specific changes in cytokine induc-
tion and inflammation in infected mice32. These results are also in
agreement with in vitro studies showing that pro‐inflammatory cyto-
kines like IL-6/IL-1β/TNFα33 or TGF-β dependent signaling34,35 promote
malignant transformation of pre‐leukemic B cells. https://doi.org/10.1038/s41467-023-40961-z Since Myd88 is a critical component in innate
immunity induction following TLR ligation, we questioned whether
Myd88-independent TLR ligation during the preleukemic phase might
alter progression to B-ALL. Therefore, we have addressed this
hypothesis by prior exposure of Pax5+/−mice to Myd88-independent
TLR agonists, such as the synthetic double-stranded RNA analogue
poly(I:C)36. Specifically, we established a mouse cohort consisting of 37
control littermate WT and 66 Pax5+/−mice. All mice were born in a SPF
facility. Four weeks after birth, mice were transferred to a conventional
facility, and 14 WT and 31 Pax5+/−mice were pre-treated with an intra-
venous injection of 200 μg of poly(I:C) for a total of 2 times, separated
by four weeks between doses, and the remaining 23 WT and 35 Pax5+/−
mice received vehicle (Fig. 6c). The microbiologic status of mice was
monitored before and periodically throughout treatment, with no
differences observed between treated and untreated animals (Sup-
plementary Data 4). As a result of poly(I:C) treatment, we observed a
specific increase in inflammatory cytokines and CD8+ T cells in per-
ipheral blood in both WT and Pax5+/−mice (Fig. 6b and Supplementary
Figs. S23–S25), showing that poly(I:C) administration was having a
similar immune-activating effect in WT and Pax5+/−mice. We next
evaluated the immediate effects of Poly(I:C) treatment on the pre-
leukemic cell population and myeloid cell compartments. Poly(I:C)
treatment does not reduce the preleukemic compartment in the bone
marrow of Pax5+/−mice (Supplementary Fig. S26) but it did induce an
accumulation of myeloid cells in both WT and Pax5+/−mice (Supple-
mentary Fig. S27). However, consistent with our previous findings, 23%
(8 out of 35) of Pax5+/−mice receiving vehicle developed and suc-
cumbed to leukemia (Fig. 6c). The prevalence and the phenotypic and
histological characteristics of B-ALL were similar in animals treated
with poly(I:C) and untreated Pax5+/−mice, while 25% (8 out of 31) of
treated Pax5+/−mice developed B-ALL and died from the disease
(p = 0.7930) (Fig. 6c, and Supplementary Fig. S28, S29). In contrast, the
latency to leukemia in Pax5+/−mice treated with poly(I:C) was sig-
nificantly longer than in the Pax5+/−control group receiving vehicle,
where it was delayed until 12–21 months of age (p = 0.0019) (Fig. 6e). Thus, early Myd88-independent TLR ligation during the preleukemic
phase in Pax5+/−mice resulted in significant delay of B-ALL develop-
ment under oncogenic environmental conditions, supporting the view
that immune modulation during the preleukemic stage can sig-
nificantly alter progression to B-ALL2. https://doi.org/10.1038/s41467-023-40961-z Myd88 expression. Since Myd88 is a critical component in innate
immunity induction following TLR ligation, we questioned whether
Myd88-independent TLR ligation during the preleukemic phase might
alter progression to B-ALL. Therefore, we have addressed this
hypothesis by prior exposure of Pax5+/−mice to Myd88-independent
TLR agonists, such as the synthetic double-stranded RNA analogue
poly(I:C)36. Specifically, we established a mouse cohort consisting of 37
control littermate WT and 66 Pax5+/−mice. All mice were born in a SPF
facility. Four weeks after birth, mice were transferred to a conventional
facility, and 14 WT and 31 Pax5+/−mice were pre-treated with an intra-
venous injection of 200 μg of poly(I:C) for a total of 2 times, separated
by four weeks between doses, and the remaining 23 WT and 35 Pax5+/−
mice received vehicle (Fig. 6c). The microbiologic status of mice was
monitored before and periodically throughout treatment, with no
differences observed between treated and untreated animals (Sup-
plementary Data 4). As a result of poly(I:C) treatment, we observed a
specific increase in inflammatory cytokines and CD8+ T cells in per-
ipheral blood in both WT and Pax5+/−mice (Fig. 6b and Supplementary
Figs. S23–S25), showing that poly(I:C) administration was having a
similar immune-activating effect in WT and Pax5+/−mice. We next
evaluated the immediate effects of Poly(I:C) treatment on the pre-
leukemic cell population and myeloid cell compartments. Poly(I:C)
treatment does not reduce the preleukemic compartment in the bone
marrow of Pax5+/−mice (Supplementary Fig. S26) but it did induce an
accumulation of myeloid cells in both WT and Pax5+/−mice (Supple-
mentary Fig. S27). However, consistent with our previous findings, 23%
(8 out of 35) of Pax5+/−mice receiving vehicle developed and suc-
cumbed to leukemia (Fig. 6c). The prevalence and the phenotypic and
histological characteristics of B-ALL were similar in animals treated
with poly(I:C) and untreated Pax5+/−mice, while 25% (8 out of 31) of
treated Pax5+/−mice developed B-ALL and died from the disease
(p = 0.7930) (Fig. 6c, and Supplementary Fig. S28, S29). In contrast, the
latency to leukemia in Pax5+/−mice treated with poly(I:C) was sig-
nificantly longer than in the Pax5+/−control group receiving vehicle,
where it was delayed until 12–21 months of age (p = 0.0019) (Fig. 6e). Discussion The association of B-ALL incidence with infectious stressors has been
supported by epidemiological data for several decades now; however,
until recently, there were no experimental models available capable of
providing a suitable biological mechanistic explanation for these
findings3–5,13,25. However, leukemogenesis in these animal models
occurs relatively late related to its age peak in humans, and we do not Engagement of Myd88-independent TLR signaling delays nat-
ural infection-driven B-ALL development https://doi.org/10.1038/s41467-023-40961-z value < 0.0001. b B-ALL-specific survival of Pax5+/−and Myd88+/−; Pax5+/−mice (Pax5+/
−, blue line, n = 41), (Myd88+/−;Pax5+/−dark green line, n = 30), (WT, black line, n = 23),
and (Myd88+/−;Pax5+/+, brown line, n = 26) all of them exposed to common infections. Log-rank (Mantel–Cox) test p-value > 0.9999 when comparing WT vs Myd88+/
−:Pax5+/+ mice, p-value = 0.0117 when comparing Pax5+/−vs Myd88+/−;Pax5+/+ mice and
p-value < 0.0001 when comparing Myd88+/−;Pax5+/−vs Myd88+/−;Pax5+/+ mice. c Flow
cytometric analysis of bone marrow showing the accumulation of blast B cells
(B220+ IgM+/−) in the leukemic Myd88+/−;Pax5+/−mice (X012 and W182) and com-
pared with a healthy Myd88+/−mouse (X650), all of them exposed to common
infections. d Haematoxylin and eosin staining of a tumor-bearing Myd88+/−;Pax5+/−
mice showing infiltrating blast cells in the spleen, Peyer’s patches, and lymph
nodes, compared with an age-matched Myd88+/−mouse. Loss of normal archi-
tecture due to leukemic cell infiltration can be seen. A representative example of 16
diseased mice is shown in the figure. Magnification and the corresponding scale bar
are indicated in each case. Source data are provided as a Source Data file. Fig. 3 | Reduction in Myd88 levels significantly increases the development of
leukemias in Pax5+/−mice upon exposure to natural infections. a Q-PCR of
Myd88 expression levels in preleukemic Pax5+/−and Sca1-ETV6-RUNX1 and leukemic
Pax5+/−proB cells. Preleukemic samples: sorted BM proB (B220low IgM-) cells from 3
months-old of wild-type (n = 5), Pax5+/−(n = 3), Sca1-ETV6-RUNX1 (n = 5) and Pax5+/−
(n = 3) and Sca1-ETV6-RUNX1 (n = 5) mice treated with Abx for 8 weeks were ana-
lyzed by Q-PCR to quantified the expression of Myd88. Leukemic samples: BM
leukemic cells from Pax5+/−mice treated with Abx for 8 weeks (n = 3) and Myd88+/
−;Pax5+/−mice (n = 3) were analyzed by Q-PCR to quantify the expression of Myd88. All the mice analyzed were exposed to infections (in the conventional facility). Total
BM from Myd88−/−mice was used as a negative control. Error bars represent the
mean and SD. For the significant differences, unpaired t test p-values (two-tailed)
are indicated in each case. Only relevant comparisons are shown. p = 0.0378 when
comparing Pax5+/−(Abx) vs Sca1-ETV6-RUNX1(Abx); p = 0.001 when comparing
Pax5+/−vs Pax5+/−(Abx); p = 0.012 when comparing Sca1-ETV6-RUNX1 vs Sca1-ETV6-
RUNX1-(Abx); p = 0.004 when comparing Pax5+/−vs Sca1-ETV6-RUNX1 and *** for p- Myd88 expression. Reduced expression of Myd88 in immune-stressed preleukemic
Pax5+/−precursor B-cells C
i
i
| (2023)
9
6 To identify somatically acquired second hits leading to B-ALL
development in Pax5+/−;Myd88+/−mice, we performed whole-genome
sequencing of paired tumor and germline tail DNA samples from eight
Pax5+/−;Myd88+/−tumors. The percentage of leukemic cells in the
sequenced samples ranged from 88.1% to 98.7%. We identified several
somatically acquired recurrent mutations and copy-number variations
involving B cell transcription factors in diseased Pax5+/;Myd88+/−mice
(Fig. 5b, Supplementary Figs. S16, S17, Supplementary Data 7). Pax5+/−;Myd88+/−B-ALLs exhibited a gene expression pattern similar
to human B-ALLs, and the gene expression profiles of Pax5+/
−;Myd88+/−and Pax5+/−B-ALLs did not show major differences
between them or when compared with gene expression profiles
derived from blast cells from patients with B-ALL (Supplementary
Fig. S15 and Supplementary Data 6), indicating that Myd88 is not
significantly contributing to the leukemic gene expression profile of
infection-driven B-ALL. Nature Communications| (2023) 14:5159 6 https://doi.org/10.1038/s41467-023-40961-z Article Engagement of Myd88-independent TLR signaling delays nat-
ural infection-driven B-ALL development c Haematoxylin and eosin staining of a tumor-bearing Pax5+/−mouse injected with
LPS unexposed to common infections showing infiltrating blast cells in the spleen,
liver, and lymph nodes and compared with a healthy WT mouse. Loss of normal
architecture can be seen due to the infiltrating cells morphologically resembling
lymphoblast. Magnification and the corresponding scale bar are indicated in each
case. Source data are provided as a Source Data file. with PBS. b Flow cytometry representative illustration of the percentage of leu-
kemic B cells (B220+IgM+ subsets) in PB, BM, spleen and LN from a diseased Pax5+/−
mouse injected with LPS compared to an age-matched healthy WT mouse. c Haematoxylin and eosin staining of a tumor-bearing Pax5+/−mouse injected with
LPS unexposed to common infections showing infiltrating blast cells in the spleen,
liver, and lymph nodes and compared with a healthy WT mouse. Loss of normal
architecture can be seen due to the infiltrating cells morphologically resembling
lymphoblast. Magnification and the corresponding scale bar are indicated in each
case. Source data are provided as a Source Data file. Fig. 4 | Pax5+/−mice injected with LPS develop B-ALL without infection expo-
sure. a B-ALL-specific survival of Pax5+/−mice injected with LPS (a Myd88- with PBS. b Flow cytometry representative illustration of the percentage of leu-
kemic B cells (B220+IgM+ subsets) in PB, BM, spleen and LN from a diseased Pax5+/−
mouse injected with LPS compared to an age-matched healthy WT mouse. / p
j
(
y
depenedent TLR ligand) (orange line, n = 10), Pax5+/−mice injected with poly(I:C) (a
Myd88-indepenedent TLR ligand) (light blue line, n = 10), Pax5+/−mice injected with
PBS (as control) (green line, n = 10), control wild type (WT) mice injected with LPS
(red line, n = 10), WT mice injected with poly(I:C) (dark blue line, n = 10), and WT
mice injected with PBS (black line, n = 10), all of them housed in an SPF facility
(without exposure to common infections). Log-rank (Mantel–Cox) test p-value =
0,1464 when comparing Pax5+/−mice injected with LPS and Pax5+/−mice injected c Haematoxylin and eosin staining of a tumor-bearing Pax5+/−mouse injected with
LPS unexposed to common infections showing infiltrating blast cells in the spleen,
liver, and lymph nodes and compared with a healthy WT mouse. Loss of normal
architecture can be seen due to the infiltrating cells morphologically resembling
lymphoblast. Magnification and the corresponding scale bar are indicated in each
case. Engagement of Myd88-independent TLR signaling delays nat-
ural infection-driven B-ALL development a B-ALL-specific survival of Pax5+/−mice injected with LPS (a Myd88-
penedent TLR ligand) (orange line, n = 10), Pax5+/−mice injected with poly(I:C) (a
yd88-indepenedent TLR ligand) (light blue line, n = 10), Pax5+/−mice injected with
S (as control) (green line, n = 10), control wild type (WT) mice injected with LPS
ed line, n = 10), WT mice injected with poly(I:C) (dark blue line, n = 10), and WT
ce injected with PBS (black line, n = 10), all of them housed in an SPF facility
ithout exposure to common infections). Log-rank (Mantel–Cox) test p-value =
1464 when comparing Pax5+/−mice injected with LPS and Pax5+/−mice injected
with PBS. b Flow cytometry representative illustration of the percentage of leu-
kemic B cells (B220+IgM+ subsets) in PB, BM, spleen and LN from a diseased Pax5+/−
mouse injected with LPS compared to an age-matched healthy WT mouse. c Haematoxylin and eosin staining of a tumor-bearing Pax5+/−mouse injected with
LPS unexposed to common infections showing infiltrating blast cells in the spleen,
liver, and lymph nodes and compared with a healthy WT mouse. Loss of normal
architecture can be seen due to the infiltrating cells morphologically resembling
lymphoblast. Magnification and the corresponding scale bar are indicated in each
case. Source data are provided as a Source Data file. ature Communications| (2023)14:5159
8 c Fig. 4 | Pax5+/−mice injected with LPS develop B-ALL without infection expo-
sure. a B-ALL-specific survival of Pax5+/−mice injected with LPS (a Myd88-
depenedent TLR ligand) (orange line, n = 10), Pax5+/−mice injected with poly(I:C) (a
Myd88-indepenedent TLR ligand) (light blue line, n = 10), Pax5+/−mice injected with
PBS (as control) (green line, n = 10), control wild type (WT) mice injected with LPS
(red line, n = 10), WT mice injected with poly(I:C) (dark blue line, n = 10), and WT
mice injected with PBS (black line, n = 10), all of them housed in an SPF facility
(without exposure to common infections). Log-rank (Mantel–Cox) test p-value =
0,1464 when comparing Pax5+/−mice injected with LPS and Pax5+/−mice injected with PBS. b Flow cytometry representative illustration of the percentage of leu-
kemic B cells (B220+IgM+ subsets) in PB, BM, spleen and LN from a diseased Pax5+/−
mouse injected with LPS compared to an age-matched healthy WT mouse. Engagement of Myd88-independent TLR signaling delays nat-
ural infection-driven B-ALL development We have shown that exposure to natural infections promotes full-
blown B-ALL in Pax5+/−mice and that the malignant transformation of
preleukemic precursor B-cells is associated with reduced levels of Nature Communications| (2023) 14:5159 7 https://doi.org/10.1038/s41467-023-40961-z Article b
a
c
0
5
10
15
20
25
30
0
20
40
60
80
100
Age (months)
B-ALL specific survival
WT PBS (n=10)
WT Poly(I:C) (n=10)
WT LPS (n=10)
Pax5+/- PBS (n=10)
Pax5+/- Poly(I:C) (n=10)
Pax5+/- LPS (n=10)
Pax5+/- (L643)
Wild type
PERIPHERAL
BLOOD
BONE
MARROW
LYMPH
NODES
SPLEEN
IgM
B220
T
W
l
o
rt
n
o
C
Pax5+/- a
0
5
10
15
20
25
30
0
20
40
60
80
100
Age (months)
B-ALL specific survival
WT PBS (n=10)
WT Poly(I:C) (n=10)
WT LPS (n=10)
Pax5+/- PBS (n=10)
Pax5+/- Poly(I:C) (n=10)
Pax5+/- LPS (n=10) a b
PERIPHERAL
BLOOD
BONE
MARROW
LYMPH
NODES
SPLEEN
IgM
B220
T
W
l
o
rt
n
o
C
Pax5+/- b LYMPH
NODES T
W
l
o
rt
n
o
C c
h
p
m
y
L
e
d
o
n
10x
40x
10x
40x
n
e
e
l
p
S
10x
40x
Pax5+/- (L643)
Wild type
10x
40x
r
e
v
i
L
10x
40x
10x
40x
500 µm
100 µm
500 µm
100 µm
500 µm
100 µm
500 µm
100 µm
500 µm
100 µm
500 µm
100 µm
50 µm
40x
50 µm
40x
50 µm
40x
50 µm
40x
50 µm
40x
50 µm
40x c
h
p
m
y
L
e
d
o
n
10x
40x
10x
40x
n
e
e
l
p
S
10x
40x
Pax5+/- (L643)
Wild type
10x
40x
r
e
v
i
L
10x
40x
10x
40x
500 µm
100 µm
500 µm
100 µm
500 µm
100 µm
500 µm
100 µm
500 µm
100 µm
500 µm
100 µm
50 µm
40x
50 µm
40x
50 µm
40x
50 µm
40x
50 µm
40x
50 µm
40x
g. 4 | Pax5+/−mice injected with LPS develop B-ALL without infection expo-
re. Engagement of Myd88-independent TLR signaling delays nat-
ural infection-driven B-ALL development Source data are provided as a Source Data file. Nature Communications| (2023) 14:5159 8 https://doi.org/10.1038/s41467-023-40961-z Article b
c
Enrichment Score
JURIC_UP (BCR-ABL ALL)
Pax5+/- Myd88+/- (B-ALL)
WT proB and PreB
FDR=0.023
KOHLMANN_UP (BCR-ABL ALL)
WT proB and PreB
FDR=0.048
Pax5+/- Myd88+/- (B-ALL)
LINKA_UP (ETV6-RUNX1 ALL)
WT proB and PreB
FDR=0.090
Pax5+/- Myd88+/- (B-ALL)
a
FDR=0.003
INTERFERON_ALPHA
RESPONSE
Pax5+/- Myd88+/- (Healthy)
Pax5+/- (Healthy)
FDR=0.01
TNFA_SIGNALING
VIA_NFKB
Pax5+/- Myd88+/- (Healthy)
Pax5+/- (Healthy)
FDR=0.011
INTERFERON_GAMMA
RESPONSE
Pax5+/- Myd88+/- (Healthy)
Pax5+/- (Healthy)
FDR=0.018
NOTCH_SIGNALING
Pax5+/- Myd88+/- (Healthy)
Pax5+/- (Healthy)
FDR=0.027
IL2_STAT5_SIGNALING
Pax5+/- Myd88+/- (Healthy)
Pax5+/- (Healthy)
FDR=0.026
INFLAMMATORY_RESPONSE
Pax5+/- Myd88+/- (Healthy)
Pax5+/- (Healthy)
FDR=0.027
APOPTOSIS
Pax5+/- Myd88+/- (Healthy)
Pax5+/- (Healthy)
FDR=0.037
UNFOLDED_PROTEIN
RESPONSE
Pax5+/- Myd88+/- (Healthy)
Pax5+/- (Healthy)
Enrichment Score
ES=0.39
ES=0.59
ES=0.36
ES=0.61
0
5
. 0
=
S
E
2
5
. 0
=
S
E
ES=0.65
5
4
. 0
=
S
E
7
4
. 0
=
S
E
ES=0.46
ES=0.46
Genetic impact of Myd88 downregulation in Pax5+/−infection-driven
a. a GSEA showing that leukemic Pax5+/−;Myd88+/−cells from diseased mice
similar profiles for genesets previously identified in human B-ALL
8,29,31. The genesets shown are significantly enriched with a nominal p-
%. Statistical analysis was done using Gene set enrichment analysis (GSEA)
m. b Whole Genome Sequencing in Pax5+/−;Myd88+/−B-ALLs. Oncoprint of
single nucleotide mutations and copy number alterations across 8 leu-
mples from Pax5+/−;Myd88+/−mice. Somatic alterations are clustered by
gene. Tumor DNA was derived from whole leukemic BM or LN, while tail DNA of
respective mouse was used as reference germline material. Previously reporte
known human or mouse leukemia hotspot mutations are highlighted (red). Me
tumor variant allele fraction (VAF) for each single nucleotide mutation is shown
the dotplot on the right. c GSEA plots showing the 8 gene sets that are significan
enriched at nominal p-value < 1% in Pax5+/−;Myd88+/−proB cells from healthy m
Enrichment analysis was done using the hallmarks collection from MSigDB55. Engagement of Myd88-independent TLR signaling delays nat-
ural infection-driven B-ALL development S
tistical analysis was done using Gene set enrichment analysis (GSEA) algorithm
e
ttps //do o g/ 0 038/s
6
0 3
096 b
Enrichment Score
JURIC_UP (BCR-ABL ALL)
Pax5+/- Myd88+/- (B-ALL)
WT proB and PreB
FDR=0.023
KOHLMANN_UP (BCR-ABL ALL)
WT proB and PreB
FDR=0.048
Pax5+/- Myd88+/- (B-ALL)
LINKA_UP (ETV6-RUNX1 ALL)
WT proB and PreB
FDR=0.090
Pax5+/- Myd88+/- (B-ALL)
a
ES=0.39
ES=0.59
ES=0.36 b b Enrichment Score
JURIC_UP (BCR-ABL ALL)
Pax5+/- Myd88+/- (B-ALL)
WT proB and PreB
FDR=0.023
KOHLMANN_UP (BCR-ABL ALL)
WT proB and PreB
FDR=0.048
Pax5+/- Myd88+/- (B-ALL)
LINKA_UP (ETV6-RUNX1 ALL)
WT proB and PreB
FDR=0.090
Pax5+/- Myd88+/- (B-ALL)
a
ES=0.39
ES=0.59
ES=0.36 a JURIC_UP (BCR-ABL ALL) Enrichment Score c
WT proB and PreB
FDR=0.090
Pax5+/- Myd88+/- (B-ALL)
FDR=0.003
INTERFERON_ALPHA
RESPONSE
Pax5+/- Myd88+/- (Healthy)
Pax5+/- (Healthy)
FDR=0.01
TNFA_SIGNALING
VIA_NFKB
Pax5+/- Myd88+/- (Healthy)
Pax5+/- (Healthy)
FDR=0.011
INTERFERON_GAMMA
RESPONSE
Pax5+/- Myd88+/- (Healthy)
Pax5+/- (Healthy)
FDR=0.018
NOTCH_SIGNALING
Pax5+/- Myd88+/- (Healthy)
Pax5+/- (Healthy)
FDR=0.027
IL2_STAT5_SIGNALING
Pax5+/- Myd88+/- (Healthy)
Pax5+/- (Healthy)
FDR=0.026
INFLAMMATORY_RESPONSE
Pax5+/- Myd88+/- (Healthy)
Pax5+/- (Healthy)
FDR=0.027
APOPTOSIS
Pax5+/- Myd88+/- (Healthy)
Pax5+/- (Healthy)
FDR=0.037
UNFOLDED_PROTEIN
RESPONSE
Pax5+/- Myd88+/- (Healthy)
Pax5+/- (Healthy)
Enrichment Score
ES=0.36
ES=0.61
0
5
. 0
=
S
E
2
5
. 0
=
S
E
ES=0.65
5
4
. 0
=
S
E
7
4
. 0
=
S
E
ES=0.46
ES=0.46
Fig. 5 | Genetic impact of Myd88 downregulation in Pax5+/−infection-driven
leukemia. a GSEA showing that leukemic Pax5+/−;Myd88+/−cells from diseased mice
present similar profiles for genesets previously identified in human B-ALL
samples28,29,31. The genesets shown are significantly enriched with a nominal p-
value < 1%. Statistical analysis was done using Gene set enrichment analysis (GSEA)
algorithm. b Whole Genome Sequencing in Pax5+/−;Myd88+/−B-ALLs. Oncoprint of
somatic single nucleotide mutations and copy number alterations across 8 leu-
kemia samples from Pax5+/−;Myd88+/−mice. Somatic alterations are clustered by
gene. Tumor DNA was derived from whole leukemic BM or LN, while tail DNA of the
respective mouse was used as reference germline material. Previously reported
known human or mouse leukemia hotspot mutations are highlighted (red). Mean
tumor variant allele fraction (VAF) for each single nucleotide mutation is shown on
the dotplot on the right. c GSEA plots showing the 8 gene sets that are significantly
enriched at nominal p-value < 1% in Pax5+/−;Myd88+/−proB cells from healthy mice. Enrichment analysis was done using the hallmarks collection from MSigDB55. Sta-
tistical analysis was done using Gene set enrichment analysis (GSEA) algorithm. Engagement of Myd88-independent TLR signaling delays nat-
ural infection-driven B-ALL development Using these mouse models, here we have provided
showing that gut microbiome dysbiosis is a universal char
0
5
10
15
20
25
0
20
40
60
80
100
Months
B-ALL specific survival
* p=0.0154
** p=0.0034
ns p=0.7930
Pax5+/-
Pax5+/- (polyI:C)
WT
WT (polyI:C)
b
22.86%
25.81%
c
e
n=8
Without
treatment
Pax5+/-
Poly(I:C)
treatment
n=8
2
4
6
8
12
14
0
5
10
15
20
25
% CD8+ T cells in peripheral blood
Months
B-ALL
Healthy
n=35
Without
treatment
Pax5+/-
Poly(I:C)
treatment
n=31
d
B-ALL latency
(months)
0
5
10
15
20
25
** p-valor=0.0019
** p=0.0019
Pax5+/-
Pax5+/- (polyI:C)
WT
WT (polyI:C) b
2
4
6
8
12
14
0
5
10
15
20
25
% CD8+ T cells in peripheral blood
Months
Pax5+/-
Pax5+/- (polyI:C)
WT
WT (polyI:C) b b 0
5
10
15
20
25
0
20
40
60
80
100
Months
B-ALL specific survival
* p=0.0154
** p=0.0034
ns p=0.7930
Pax5+/-
Pax5+/- (polyI:C)
WT
WT (polyI:C)
c
e
2
4
6
8
12
14
0
5
10
% CD8+ T cell
Months
** p-valor=0
** p=0 0019 0
5
10
15
20
25
0
20
40
60
80
100
Months
B-ALL specific survival
* p=0.0154
** p=0.0034
ns p=0.7930
Pax5+/-
Pax5+/- (polyI:C)
WT
WT (polyI:C)
c
e
Months
** p valor 0 00
**
0 0019 c e selection pressure of exposure to a common infectious environ-
ent, murine B cell progenitors need more time to acquire Jak3
utations in comparison with their human counterparts, probably
cause human B cell progenitors are to a large extent independent
IL737. Using these mouse models, here we have provided evidence
owing that gut microbiome dysbiosis is a universal characteristic
e
n=8
Without
treatment
Pax5+/-
Poly(I:C)
treatment
n=8
B-ALL latency
(months)
0
5
10
15
20
25
** p-valor=0.0019
** p=0.0019 e
n=8
Without
treatment
Pax5+/-
Poly(I:C)
treatment
n=8
B-ALL latency
(months)
0
5
10
15
20
25
** p-valor=0.0019
** p=0.0019 e 22.86%
25.81%
B-ALL
Healthy
n=35
Without
treatment
Pax5+/-
Poly(I:C)
treatment
n=31
d d d Pax5+/- the selection pressure of exposure to a common infectious environ-
ment, murine B cell progenitors need more time to acquire Jak3
mutations in comparison with their human counterparts, probably
because human B cell progenitors are to a large extent independent
of IL737. yet have an explanation for this finding2. Engagement of Myd88-independent TLR signaling delays nat-
ural infection-driven B-ALL development c
FDR=0.003
INTERFERON_ALPHA
RESPONSE
Pax5+/- Myd88+/- (Healthy)
Pax5+/- (Healthy)
FDR=0.01
TNFA_SIGNALING
VIA_NFKB
Pax5+/- Myd88+/- (Healthy)
Pax5+/- (Healthy)
FDR=0.011
INTERFERON_GAMMA
RESPONSE
Pax5+/- Myd88+/- (Healthy)
Pax5+/- (Healthy)
FDR=0.018
NOTCH_SIGNALING
Pax5+/- Myd88+/- (Healthy)
Pax5+/- (Healthy)
FDR=0.027
IL2_STAT5_SIGNALING
Pax5+/- Myd88+/- (Healthy)
Pax5+/- (Healthy)
FDR=0.026
INFLAMMATORY_RESPONSE
Pax5+/- Myd88+/- (Healthy)
Pax5+/- (Healthy)
FDR=0.027
APOPTOSIS
Pax5+/- Myd88+/- (Healthy)
Pax5+/- (Healthy)
FDR=0.037
UNFOLDED_PROTEIN
RESPONSE
Pax5+/- Myd88+/- (Healthy)
Pax5+/- (Healthy)
Enrichment Score
ES=0.61
0
5
. 0
=
S
E
2
5
. 0
=
S
E
ES=0.65
5
4
. 0
=
S
E
7
4
. 0
=
S
E
ES=0.46
ES=0.46 c Enrichment Score Pax5+/- (Healthy) Pax5+/- (Healthy) Fig. 5 | Genetic impact of Myd88 downregulation in Pax5+/−infection-driven Fig. 5 | Genetic impact of Myd88 downregulation in Pax5+/−infection-driven
leukemia. a GSEA showing that leukemic Pax5+/−;Myd88+/−cells from diseased mice
present similar profiles for genesets previously identified in human B-ALL
samples28,29,31. The genesets shown are significantly enriched with a nominal p-
value < 1%. Statistical analysis was done using Gene set enrichment analysis (GSEA)
algorithm. b Whole Genome Sequencing in Pax5+/−;Myd88+/−B-ALLs. Oncoprint of
somatic single nucleotide mutations and copy number alterations across 8 leu-
kemia samples from Pax5+/−;Myd88+/−mice. Somatic alterations are clustered by gene. Tumor DNA was derived from whole leukemic BM or LN, while tail DNA of the
respective mouse was used as reference germline material. Previously reported
known human or mouse leukemia hotspot mutations are highlighted (red). Mean
tumor variant allele fraction (VAF) for each single nucleotide mutation is shown on
the dotplot on the right. c GSEA plots showing the 8 gene sets that are significantly
enriched at nominal p-value < 1% in Pax5+/−;Myd88+/−proB cells from healthy mice. Enrichment analysis was done using the hallmarks collection from MSigDB55. Sta-
tistical analysis was done using Gene set enrichment analysis (GSEA) algorithm. impact of Myd88 downregulation in Pax5+/−infection-driven Fig. 5 | Genetic impact of Myd88 downregulation in Pax5+/−infection-driven
leukemia. a GSEA showing that leukemic Pax5+/−;Myd88+/−cells from diseased mice
present similar profiles for genesets previously identified in human B-ALL
samples28,29,31. The genesets shown are significantly enriched with a nominal p-
value < 1%. Statistical analysis was done using Gene set enrichment analysis (GSEA)
algorithm. b Whole Genome Sequencing in Pax5+/−;Myd88+/−B-ALLs. Oncoprint of
somatic single nucleotide mutations and copy number alterations across 8 leu-
kemia samples from Pax5+/−;Myd88+/−mice. Engagement of Myd88-independent TLR signaling delays nat-
ural infection-driven B-ALL development Somatic alterations are clustered by Nature Communications| (2023) 14:5159 9 9 https://doi.org/10.1038/s41467-023-40961-z Article a
b
2
4
6
8
12
14
0
5
10
15
20
25
% CD8+ T cells in peripheral blood
Months
Pax5+/-
Pax5+/- (polyI:C)
WT
WT (polyI:C) a ve an explanation for this finding2. We believe that the time to
mogenesis is, to a large extent, related to the species-specific
nces in the dependence of early B cell progenitors from IL7. e
early
B
cell
progenitors
are
strongly
dependent
on
naling, and when IL7 is removed from cell cultures, these cells
7, when bound to its receptor, activates Jak3 and phosphorylates
We speculate that this may be the cause explaining why, under
the selection pressure of exposure to a common infectiou
ment, murine B cell progenitors need more time to acq
mutations in comparison with their human counterparts,
because human B cell progenitors are to a large extent ind
of IL737. Engagement of Myd88-independent TLR signaling delays nat-
ural infection-driven B-ALL development We believe that the time to
leukemogenesis is, to a large extent, related to the species-specific
differences in the dependence of early B cell progenitors from IL7. Murine
early
B
cell
progenitors
are
strongly
dependent
on
IL7 signaling, and when IL7 is removed from cell cultures, these cells
die. IL7, when bound to its receptor, activates Jak3 and phosphorylates
STAT5. We speculate that this may be the cause explaining why, under Using these mouse models, here we have provided evidence
showing that gut microbiome dysbiosis is a universal characteristic Using these mouse models, here we have provided evidence
showing that gut microbiome dysbiosis is a universal characteristic Nature Communications| (2023) 14:5159 10 Article https://doi.org/10.1038/s41467-023-40961-z https://doi.org/10.1038/s41467-023-40961-z Immune stress
(e g infection exposure antibiotic treatment etc ) can affect both healthy (a) and
only in Pax5+/−preleukemic progenitors will this downregulation interfere with
innate immune signaling in a way that allows the appearance of secondary muta-
tions leading to the development of a full blown leukemia The incidence of B ALL is only in Pax5+/−preleukemic progenitors will this downregulation interfere with
innate immune signaling in a way that allows the appearance of secondary muta-
tions leading to the development of a full-blown leukemia.The incidence of B-ALL is
directly proportional to the percentage of Pax5+/−preleukemic cells having a
downregulation of Myd88 expression. Fig. 7 | Suppression of innate immune signaling by immune stress triggers
leukemia development from Pax5+/−preleukemic precursors. Immune stress
(e.g. infection exposure, antibiotic treatment, etc.) can affect both healthy (a) and
Pax5+/−preleukemic carriers (b) alike, and in both of them it can elicit a partial
downregulation of Myd88 expression in individual B cell progenitors. However, in the clonal evolution of pre-malignant B-cell precursors toward
B-ALL as a result of immune stress. The molecular landscape char-
acterization of innate immune regulators that promote the initiation
of B-ALL has allowed us the identification of Myd88 as one key player
in this process. Indeed, a preleukemic B-cell-dependent role was
evidenced by an increase in B-ALL incidence upon Myd88 down-
regulation in the leukemia-prone Pax5+/−model (Fig. 7). The down-
regulation of Myd88 was shown to function in preleukemic B-cells by
upregulating the expression of genes involved in interferon-α
response, TNFα signaling via NFκB and interferon-γ response and
activation of signaling effectors (for example, Notch, apoptosis and
IL2-STAT5 signaling) associated with cell proliferation and survival. This inflammation-dependent role for Myd88 in triggering B-ALL
within preleukemic B-cells has also been shown for other TLRs in
cancer27. The characterization of the innate immunity signaling
within preleukemic precursor B cells showed that all Pax5+/−pre-
leukemic cells are not identical (Fig. 6). As proposed, this variability
in the expression of Myd88 under immune stress could provide a
means for preleukemic cells to adapt their behavior to the environ-
mental conditions and take advantage of potential fluctuations in the
immune proficiency of the organism under stress conditions2. We
speculate that such adaptation to new conditions might in turn lead
to a rewiring of preleukemic cells themselves, which might affect
immune molecules (such as TLRs and other immunomodulatory
molecules), favouring the acquisition of second hits. https://doi.org/10.1038/s41467-023-40961-z An increase in CD8+ T cells can be observed in the blood of
treated mice, only being statistically significant for the Pax5+/−mice (unpaired t test
(two-tailed), p-value = 0.0087) (for WT mice the unpaired t test has a nonsignificant
p-value = 0.5069). Each point represents the mean of the levels of the CD8+ T cells
in all mice in each group for each of the different time points (n = 31 for Pax5+/−
poly(I:C)-treated mice, n = 8 for Pax5+/−untreated mice, n = 14 for WT poly(I:C)
treated mice and n = 23 for WT untreated mice). c B-ALL–specific survival of mice
treated with poly(I:C) (Pax5+/−, pink dashed line, n = 31; WT, blue dashed line, n = 14)
and nontreated mice (Pax5+/−, pink line, n = 35; WT, blue line, n = 23) following Fig. 7 | Suppression of innate immune signaling by immune stress triggers
leukemia development from Pax5+/−preleukemic precursors. Immune stress
(e.g. infection exposure, antibiotic treatment, etc.) can affect both healthy (a) and
Pax5+/−preleukemic carriers (b) alike, and in both of them it can elicit a partial
downregulation of Myd88 expression in individual B cell progenitors. However,
only in Pax5+/−preleukemic progenitors will this downregulation interfere with
innate immune signaling in a way that allows the appearance of secondary muta-
tions leading to the development of a full-blown leukemia.The incidence of B-ALL is
directly proportional to the percentage of Pax5+/−preleukemic cells having a
downregulation of Myd88 expression. Fig. 7 | Suppression of innate immune signaling by immune stress triggers
leukemia development from Pax5+/−preleukemic precursors. Immune stress
(e.g. infection exposure, antibiotic treatment, etc.) can affect both healthy (a) and
Pax5+/−preleukemic carriers (b) alike, and in both of them it can elicit a partial
downregulation of Myd88 expression in individual B cell progenitors. However, Fig. 7 | Suppression of innate immune signaling by immune stress triggers
leukemia development from Pax5+/−preleukemic precursors. https://doi.org/10.1038/s41467-023-40961-z Indeed, this in the clonal evolution of pre-malignant B-cell precursors toward
B-ALL as a result of immune stress. The molecular landscape char-
acterization of innate immune regulators that promote the initiation
of B-ALL has allowed us the identification of Myd88 as one key player
in this process. Indeed, a preleukemic B-cell-dependent role was
evidenced by an increase in B-ALL incidence upon Myd88 down-
regulation in the leukemia-prone Pax5+/−model (Fig. 7). The down-
regulation of Myd88 was shown to function in preleukemic B-cells by
upregulating the expression of genes involved in interferon-α
response, TNFα signaling via NFκB and interferon-γ response and
activation of signaling effectors (for example, Notch, apoptosis and
IL2-STAT5 signaling) associated with cell proliferation and survival. This inflammation-dependent role for Myd88 in triggering B-ALL
within preleukemic B-cells has also been shown for other TLRs in
cancer27. The characterization of the innate immunity signaling
within preleukemic precursor B cells showed that all Pax5+/−pre-
leukemic cells are not identical (Fig. 6). As proposed, this variability
in the expression of Myd88 under immune stress could provide a
means for preleukemic cells to adapt their behavior to the environ-
mental conditions and take advantage of potential fluctuations in the
immune proficiency of the organism under stress conditions2. We
speculate that such adaptation to new conditions might in turn lead
to a rewiring of preleukemic cells themselves, which might affect
immune molecules (such as TLRs and other immunomodulatory
molecules), favouring the acquisition of second hits. Indeed, this of all genetic preleukemic conditions tested. However, altering the
gut microbiome through bacterial depletion exclusively has a spe-
cific impact on B-ALL development in genetically predisposed
Pax5+/– mice,
therefore
highlighting
the complex
interaction
between genetic subtypes and environmental factors that drive
leukemogenesis in childhood B-ALL. Indeed, our study of variation
in susceptibility to B-ALL by genetic subtypes has allowed us to
identify for the first time an etiologic heterogeneity in the pro-
gression of the disease among subtypes of childhood B-ALL, and our
results suggest that the use of gut microbiota as a modifiable
therapeutic target to reduce or abolish the impact of immune stress
in predisposed carriers may only apply to specific genetic subtypes
of childhood B-ALL. Nature Communications| (2023) 14:5159 https://doi.org/10.1038/s41467-023-40961-z exposure to common infections. Log-rank (Mantel–Cox) test p = 0.7930 when
comparing Pax5+/−mice treated with poly(I:C) versus nontreated Pax5+/−mice,
p = 0.0034 when comparing Pax5+/−versus WT poly(I:C)-treated mice and
p = 0.0154 when comparing Pax5+/−versus WT mice without treatment. d Absence
of changes in B-ALL incidence in Pax5+/−mice treated with poly(I:C). The treatment
of Pax5+/−mice with poly(I:C) did not reduce nor increase the rate of B-ALL devel-
opment. Fisher exact test (two-tailed), p = 0.9999. 8 out 35 mice in the non-treated
group developed the disease (22.86%) and 8 out of 31 mice in the poly(I:C)-treated
group developed B-ALL (25.81%). e poly(I:C) treatment increases the latency of
B-ALL development in Pax5+/−mice. Pax5+/−untreated mice (n = 8) were diagnosed
with B-ALL between 6 and 15 months of age and the Pax5+/−mice treated with
poly(I:C) (n = 8) were diagnosed between 10.5 and 20.8 months of age. Error bars
represent the mean and SD. Statistic test Mann–Withney (two-tailed), p-value =
0.0019. Source data are provided as a Source Data file. Fig. 6 | Innate stimulation by poly(I:C) treatment delays B-ALL development in
Pax5+/−mice exposed to infections. a Study design. poly(I:C) treatment fully
described in methods section. SPF: specific pathogen free facility; CF: conventional
facility, where the mice area exposed to common infections. b Percentages of CD8+
T cells in peripheral blood of Pax5+/−and WT mice treated with poly(I:C) compared
to untreated mice. An increase in CD8+ T cells can be observed in the blood of
treated mice, only being statistically significant for the Pax5+/−mice (unpaired t test
(two-tailed), p-value = 0.0087) (for WT mice the unpaired t test has a nonsignificant
p-value = 0.5069). Each point represents the mean of the levels of the CD8+ T cells
in all mice in each group for each of the different time points (n = 31 for Pax5+/−
poly(I:C)-treated mice, n = 8 for Pax5+/−untreated mice, n = 14 for WT poly(I:C)
treated mice and n = 23 for WT untreated mice). c B-ALL–specific survival of mice
treated with poly(I:C) (Pax5+/−, pink dashed line, n = 31; WT, blue dashed line, n = 14)
and nontreated mice (Pax5+/−, pink line, n = 35; WT, blue line, n = 23) following Fig. 6 | Innate stimulation by poly(I:C) treatment delays B-ALL development in
Pax5+/−mice exposed to infections. a Study design. poly(I:C) treatment fully to untreated mice. https://doi.org/10.1038/s41467-023-40961-z Both male and female mice were used in all the studies with the
exception of the microbiome analysis in which only female mice were
included in the study in order to avoid gender biases in microbiome
results. Housing environmental conditions included a temperature of
21 °C ± 2°, humidity of 55% ±10%, and a 12 h∶12 h light: dark cycle. Mice
had access to food and water ad libitum. During housing, animals were
monitored daily for health status. When required, SPF-housed two
months old WT or Pax5+/−mice were i.p. injected with PBS or 35 mg LPS
corresponding to 35 EU (Escherichia coli 0111:B4, LPS-EB Ultrapure,
Invivogen). When the animals showed evidence of illness, they were
humanely killed, and the main organs were extracted by standard
dissection. The maximal tumor size/burden approved by the institu-
tional committees has not been exceeded in any mice included in the
experiments. All major organs were macroscopically inspected under
the stereo microscope, and then representative samples of tissue were
cut from the freshly dissected organs, and were immediately fixed. Differences in Kaplan–Meier survival plots of transgenic and WT mice
were analyzed using the log-rank (Mantel-Cox) test. Statistical analyses
were performed by using GraphPad Prism v5.01 (GraphPad Software). preleukemic heterogeneity could contribute to the variable disease
risks across susceptible children. The initial steps of B-ALL development in children pass usually
undetected and, when the disease becomes evident at diagnosis, the
delineation of the consecutive genetic events progressively leading to
the leukemia is complicated by the accumulation of many oncogenic
driver and passenger mutations. Therefore, key to the identification of
the driver role of innate immunity dysregulation is the study of leu-
kemia cells while they are appearing, in order to understand the
sequential events initiating infection-driven B-ALL. This approach has
allowed us to identify previously unknown molecular mechanisms
taking place in the still unnoticed stages of human B-cell leukemo-
genesis, thus opening new opportunities for the development of
effective strategies for the early detection and prevention of childhood
B-ALL. Collectively, these data raise the possibility that innate immu-
nity boosting might represent a viable strategy for risk reduction or
prevention in childhood-related B-ALL; early innate immune response
induction by Myd88-independent Toll-like receptor ligation during the
preleukemic phase in Pax5+/−mice resulted in significant delay of B-ALL
development under oncogenic environmental conditions, supporting
the view that immune modulation during the preleukemic stage can
significantly alter progression to B-ALL. Data reporting Sample sizes were determined on the basis of the literature describing
mouse modeling of natural infection-driven leukemia3–5,13,25 and were
justified by power calculations estimating 90% power to detect dif-
ferential leukemia incidence. The experiments were not randomized
and the investigators were not blinded to allocation during experi-
ments and outcome assessment. poly(I:C) treatment Pax5+/−and WT mice were born in SPF conditions and were maintained
there until they reached 1 month of age. At that time, 31 Pax5+/−and 14
WT mice were transferred to a conventional facility (CF) and admi-
nistered the first dose of poly(I:C) (intravenous injection of 200 μg/
mouse). As a control group, 35 Pax5+/−and 24 WT mice were trans-
ferred to CF and maintained under the same conditions without
poly(I:C) administration. 4 weeks later, the second dose of poly(I:C)
was administered to the previously treated animals. Mice were mon-
itored periodically. The endpoint was established at 2 years of age
(22–24 months) or when the mice showed signs of disease (clinical
signs or presence of blasts in peripheral blood). SPF: specific pathogen-
free facility; CF: conventional facility, where the mice area exposed to
common infections. Antibiotic treatment Mice were treated with an antibiotic regimen as described42. Groups of
mice were given a cocktail of antibiotics (ampicillin, 1 g/L, Ratiopharm;
vancomycin, 500 mg/L, Cell Pharm; ciprofloxacin, 200 mg/L, Bayer
Vital; imipenem, 250 mg/L, MSD; metronidazole, 1 g/L, Fresenius)
added to their drinking water ad libitum for a period of eight weeks. https://doi.org/10.1038/s41467-023-40961-z In agreement with this finding, our studies show
that, in Pax5+/−mice, immune training by early exposure to infection
does not have a protective effect against the conversion of the
preleukemic clone into B-ALL, therefore conflicting with the
“delayed infection” hypothesis for this specific molecular subtype
of B-ALL, and instead supporting Kinlen’s “population mixing”
explanation for the epidemiological findings. In this way, our results
reinforce the notion of etiologic variation in childhood B-ALL
development and the existence of a specific “gene/environment”
interaction (i.e., infection) triggering B-ALL development. of all genetic preleukemic conditions tested. However, altering the
gut microbiome through bacterial depletion exclusively has a spe-
cific impact on B-ALL development in genetically predisposed
Pax5+/– mice,
therefore
highlighting
the complex
interaction
between genetic subtypes and environmental factors that drive
leukemogenesis in childhood B-ALL. Indeed, our study of variation
in susceptibility to B-ALL by genetic subtypes has allowed us to
identify for the first time an etiologic heterogeneity in the pro-
gression of the disease among subtypes of childhood B-ALL, and our
results suggest that the use of gut microbiota as a modifiable
therapeutic target to reduce or abolish the impact of immune stress
in predisposed carriers may only apply to specific genetic subtypes
of childhood B-ALL. In agreement with this finding, our studies show
that, in Pax5+/−mice, immune training by early exposure to infection
does not have a protective effect against the conversion of the
preleukemic clone into B-ALL, therefore conflicting with the
“delayed infection” hypothesis for this specific molecular subtype
of B-ALL, and instead supporting Kinlen’s “population mixing”
explanation for the epidemiological findings. In this way, our results
reinforce the notion of etiologic variation in childhood B-ALL
development and the existence of a specific “gene/environment”
interaction (i.e., infection) triggering B-ALL development. Unexpectedly, our data also show that B-ALL development falls
into the category of cancers associated with a dysregulation of the
molecular mechanisms of innate immunity, which plays a central role Nature Communications| (2023) 14:5159 11 Article https://doi.org/10.1038/s41467-023-40961-z −;Myd88+/−mice (age of these mice range from 1 month-old until disease
development) of a mixed C57BL/6×CBA background were used in this
study. For the experiments, Pax5+/−, Sca1-ETV6-RUNX1, Sca1-Lmo2 and
Sca1-BCR-ABLp190, and Pax5+/−;Myd88+/−of the same litter were used. https://doi.org/10.1038/s41467-023-40961-z TLR signaling induction pro-
duces disparate effects depending on the genetic subtype of child-
hood B-ALL and the experimental approach used. While early innate
immune response induction by TLR ligation could reduce leukemia
penetrance in the Eμ-RET and E2A–PBX1 transgenic mouse models38, ex
vivo Toll-like receptor signaling induction by exposure to lipopoly-
saccharide triggers transformation of ETV6–RUNX1+ precursor B
cells39. Our results also suggest that the elimination of these rewired
preleukemic cells, or protection against this environmental adaption,
might help curtail or prevent leukemic development in genetically
predisposed carriers more effectively than strategies based on general
immune training. Likewise, immune training by early exposure to
infection does not prevent Pax5+/−preleukemic carriers against B-ALL. Overall, these findings have important implications for the under-
standing and potential therapeutic targeting of the preleukemic state
in children. V(D)J recombination V(D)J recombination analysis was carried out as previously described43. Immunoglobulin rearrangements were amplified by PCR using the
primers below. Cycling conditions consisted of an initial heat-
activation at 95 °C followed by 31–37 cycles of denaturation for 1 min
at 95 °C, annealing for 1 min at 65 °C, and elongation for 1 min 45 s at
72 °C. This was followed by a final elongation for 10 min at 72 °C. To
determine the DNA sequences of individual V(D)J rearrangements, the
PCR fragments were isolated from the agarose gel and cloned into the
pGEM-Teasy vector (Promega); the DNA inserts of at least ten clones
corresponding to the same PCR fragment were then sequenced. The
primer pairsused are listed in Table 1. Mouse model for natural immune stress-driven leukemia 100708), CD11b/Mac1 (M1/70, 1:200, cat. 553310), CD19
(1D3, cat. 152404), CD117/c-Kit (2B8, 1:200, cat. 105807), Ly-6G/Gr1
(RB6-8C5; cat. 108412), IgM (R6-60.2, cat. 406509) and CD25 (PC61,
cat. 553866) antibodies. All antibodies were purchased from BioLe-
gend and used at a 1:100 dilutions unless otherwise indicated. Mouse model for natural immune stress-driven leukemia Nucleated cells were obtained from total mouse bone marrow (flush-
ing from the long bones), peripheral blood, thymus, or spleen. Con-
taminating red blood cells were lysed with red cell lysis buffer (RCLB)
and the remaining cells were washed in PBS with 1% FCS. After staining,
all cells were washed once in PBS and were resuspended in PBS with 1%
FCS containing 10 µg/mL propidium iodide (PI) to excluded dead cells
from both analyses and sorting procedures. The samples and the data
were acquired in an AccuriC6 Flow Cytometer and analyzed using Flow
Jo software. Specific fluorescence of FITC, PE, PI, and APC excited at
488 nm (0.4 W) and 633 nm (30 mW), respectively, as well as known
forward and orthogonal light scattering properties of mouse cells were
used to establish gates. Nonspecific antibody binding was suppressed
by preincubation of cells with CD16/CD32 Fc-block solution (BD
Biosciences). For each analysis, a total of at least 50,000 viable (PI-)
cells were assessed. The gating strategy is exemplified in Supplemen-
tary Fig. S30. Pax5+/−, Sca1-ETV6-RUNX1, Sca1-Lmo2 and Sca1-BCR-ABLp190 mice have
been previously described4,14,15,40. The Sca1-ETV6-RUNX1 construct was
generated by placing the human ETV6-RUNX1 cDNA under the control
of the stem‐cell‐specific Sca1 promoter, as has been previously
described4. Pax5+/−
mice
were
crossed
with
Myd88+/−
mice
(B6.129P2(SJL)-Myd88<tm1.1Defr>/J)41 to generate Pax5+/−;Myd88+/−mice. These Pax5+/−;Myd88+/−mice were bred and maintained in the SPF area
of the animal house until the moment when they were exposed to
conventional pathogens present in non-SPF animal facilities, as pre-
viously described3. All mouse experiments were performed following
the applicable Spanish and European legal regulations, and had been
previously authorized by the pertinent institutional committees of
both University of Salamanca and Spanish Research Council (CSIC). Pax5+/−(age of these mice range from 1 month-old until disease devel-
opment), Sca1-ETV6-RUNX1 (2 month-old mice were used in the study),
Sca1-Lmo2 (2 month-old mice were used in the study), Sca1-BCR-
ABLp190 (2 month-old mice were used in the study), and Pax5+/ The following antibodies were used for flow cytometry: anti-B220
(RA3-6B2, cat. 103212), CD4 (RM4-5, 1:250, cat. 100516), CD8a (53-6.7, Nature Communications| (2023) 14:5159 12 Article https://doi.org/10.1038/s41467-023-40961-z Agilent Technologies 2100 Bioanalyzer using a DNA 1000 chip. V3-V4
paired-end sequencing was performed using an Illumina MiSeq (La
Jolla, CA) according to the manufacturer’s protocols. The base calls
(BCL) binary generated by the Illumina sequencer was converted into
FASTQ files utilizing the Illumina package cl2fastq. 1:250, cat. Histology Sequencing data processing and statistical analysis. Microbiome
bioinformatics were performed with QIIME 2 2019.1044. Raw sequence
data were demultiplexed and quality filtered using the q2‐demux
plugin followed by denoising with DADA245 (via q2‐dada2). All ampli-
con sequence variants (ASVs) were aligned with mafft46 (via q2‐align-
ment) and used to construct a phylogeny with fasttree247 (via q2‐
phylogeny). Alpha‐diversity metrics (observed features and Faith’s
Phylogenetic Diversity48), beta diversity metrics (weighted UniFrac49,
unweighted
UniFrac50,
Jaccard
distance,
and
Bray‐Curtis
dissimilarity51), and Principle Coordinate Analysis (PCoA) were esti-
mated using q2‐diversity after samples were rarefied (subsampled
without replacement) to 80992 sequences per sample. Taxonomy was
assigned to ASVs using the q2‐feature‐classifier52 classify‐sklearn naı̈ve
Bayes taxonomy classifier against the SILVA 132 99% OTUs reference
sequences53. For the comparative analysis of the taxonomy of the dif-
ferent groups, the feature table was filtered to keep features with a
minimum frequency of 10 and present in at least 10 samples. The
comparative analysis was determined using Linear Discriminant Ana-
lysis (LDA) Effect Size (LEfSe)54. Animals were humanely euthanized by cervical dislocation; tissue
samples were formalin-fixed and embedded in paraffin. Pathology
assessment was performed on hematoxylin-eosin stained sections
under the supervision of Dr. Oscar Blanco, an expert pathologist at the
Salamanca University Hospital. 16S rRNA sequencing and analysis Mouse toll-like receptor signaling pathway PCR array
RNA from sorted BM precursor B (B220low IgM−) cells of wild type,
and Pax5+/−was extracted with the TRIzol reagent (Invitrogen), and the
cDNAs prepared with the RT2 First Strand kit (Qiagen) were pre-
amplified using the RT² HT First Strand Kit cDNA cell cycle synthesis kit
(Qiagen). The expression of TLR genes and their signaling pathways
was analyzed in samples using the mouse RT² Profiler PCR Array
(Qiagen). The PCR array DataanalysisV4web (Qiagen) source was used
to calculate the threshold cycle (ΔCt) values and to obtain ΔCt values
normalized to those of housekeeping genes. To determine the differ-
ences in relative expression between B cell populations, comparative
analyses were performed (2–ΔΔCt) using the PCR Array Data Analysis
Software (Qiagen). DNA extraction. Fresh fecal samples were collected from each mouse
(WT or Pax5+/−mice) at defined time points. Fecal DNA was isolated
using QIAamp DNA Stool Mini Kit (Qiagen_Cat. No. 51604), following
the stool pathogen detection protocol. DNA was extracted following
the manufacturer’s instructions with a modification: heating step in
ASL buffer at 95 °C and eluting the DNA in 100 μl ATE buffer. Quantity
of DNA was done by Picogreen (Invitrogen, cat.#P7589) method using
Victor 3 fluorometry. After performing quality control, qualified sam-
ples proceed to library construction. Targeted 16S V4 region sequencing. We targeted and amplified the
V3-V4 region of the 16S rRNA gene by PCR using barcoded primers. Library construction was done by Macrogen (https://www.macrogen-
europe.com/) using the Library Kit Herculase II Fusion DNA Poly-
merase Nextera XT Index Kit V2. To verify the size of PCR-enriched
fragments, we checked the template size distribution by running on an Real-time PCR quantification (Q-PCR) of Myd88 Table 1 | Primers used for V(D)J recombination Nature Communications| (2023) 14:5159 Nature Communications| (2023) 14:5159 13 Article https://doi.org/10.1038/s41467-023-40961-z obtained from the total spleen of wild-type immunized mice. The fold
change in each group, calculated as 2-ΔΔCt sample, was compared. obtained from the total spleen of wild-type immunized mice. The fold
change in each group, calculated as 2-ΔΔCt sample, was compared. SV callers were implemented in the workflow for SV calling, including
Delly68, Lumpy69, Manta70, and Gridss71. The SV calls passing the default
quality filters of each caller were merged using SURVIVOR72 and gen-
otyped by SVtyper73. The intersected call sets were manually reviewed
for the supporting soft-clipped and discordant read counts at both
ends of a putative SV site using IGV. Microarray data analysis Total RNA was isolated in two steps using TRIzol (Life Technologies)
followed by RNeasy Mini-Kit (Qiagen) purification, following the
manufacturer’s RNA Clean-up Protocol with the optional On-Column
DNase treatment. The integrity and the quality of the RNA were verified
by electrophoresis, and its concentration was measured. Samples were
analyzed using Applied Biosystems Transcriptome Analysis Console
(TAC) software v4.0.1 with the following parameters: (i) Analysis Ver-
sion: version 1, (ii) Summarization Method: Gene Level—RMA, (iii)
Genome Version: mm10 (Mus musculus) and (iv) Annotation: MoGene-
1_0-st-v1.na36.mm10.transcript.csv. Differentially
expressed
genes
were evaluated by specific statistical criteria (p-value < 0.05 and
absolute fold change >2). Gene Set Enrichment Analyses were per-
formed using GSEAv4.3.255–57, hallmark collection of gene sets55, mur-
ine B cell developmental stages gene sets58, and Pax5-regulated gene
sets59,60. Gene expression signatures that are specifically upregulated
or downregulated in human B-ALL28–31 were also tested for enrichment
within tumor specimens using GSEA, the list of gene sets is included in
Supplementary Data 11. References 1. Cobaleda, C., Vicente-Duenas, C., Ramirez-Orellana, M. & Sanchez-
Garcia, I. Revisiting the concept of childhood preleukemia. Trends
Cancer 8, 887–889 (2022). 2. Cobaleda, C., Vicente-Duenas, C. & Sanchez-Garcia, I. Infectious
triggers and novel therapeutic opportunities in childhood B cell
leukaemia. Nat. Rev. Immunol. 21, 570–581 (2021). Quantification of serum cytokine levels Serum cytokine levels were analyzed using the Cytometric Bead Array
(CBA) immunoassay systems (BD Biosciences), which assesses simul-
taneously IL6, IL10, MCP1, IFNg, TNFa e IL12p70 (CBA Inflammation
Kit) and IL-2, IL-4, IL-6, IL-10, IL-17A, TNF alpha and IFN gamma in serum
from the mice (Mouse Th1 Th2 Th17 Cytokine Kit #560,485; BDB). Briefly, 50 μL of the serum was incubated for 2 h at room temperature
with 50 μL of anticytokine MAb-coated beads and 50 μL of the
appropriate PE-conjugated anticytokine antibody detector. After this
incubation period, samples were washed once (5 min at 200 g) to
remove the excess of detector antibodies. Immediately afterward, data
acquisition was performed on a FACSCanto II flow cytometer (BDB)
using the FACSDiva software program (BDB). During acquisition,
information was stored for 3,000 events corresponding to each bead
population analyzed per sample (total number of beads >9000). For
data analysis, FCAP Array Software v3.0 program (BDB) was used. Sanger sequencing
Mutations were validated using Sanger sequencing on a 3130 Genetic
Analyzer (Applied Biosystems). The following primer pairs were used:
mJak3, forward: CGGGATGTGGGGCTTTAACT, reverse: GCAGACA
CGGGGTATAGTGG; mJak1 forward: CCAGACAGCCAGGAGAACAG,
reverse: CGTCTGCATAGTACCCACCC; mPax5 forward: CTCGTACATG
CACGGAGACA, reverse: GGACCCTTCAGTACACCAGC. Preleukemic pro-B cell culture Pro-B cells were obtained from the BM of 2–3 weeks old WT, Pax5+/−,
and Pax5+/−;Myd88+/−mice, respectively. B cells were isolated from the
BM via positive MACS separation for B220. Cells were cultivated in
special pro-B-cell medium (IMDM medium + L-glutamine + HEPES, 2%
FCS, 1 mM penicillin–streptomycin, 0.03% Primaton, 50 µM beta-mer-
captoethanol, 2 µg/ml cyproxin) supplemented with 5 ng/ml murine
recombinant IL7 on ST2-feeder cells. Data availability Authors can confirm that all relevant data are included in the paper
and/or its supplementary information files. All data reported in this
article are deposited in the NCBI’s Gene Expression Omnibus (GEO)74,
available under the GEO Series accession number GSE221597 and to
NCBI’s Sequence Read Archive (SRA) under the BioProject accession
number PRJNA912234. The remaining data are available within the
Article, Supplementary Information or Source Data file. Source data
are provided with this paper. Statistical analysis Statistical analyses were performed using GraphPad Prism v5.01
(GraphPad Software). Statistical significance was calculated by
two-tailed unpaired t test, paired t test, Mann–Whitney test or
Kruskal–Wallis test. Data normality was tested using the D’Agos-
tino and Pearson test (alpha level = 0.05). Survival analyses were
performed using the log-rank/Mantel–Cox test. Two-side Fisher
exact test was used to compare leukemia incidence. P values are
depicted on the figures only for significant differences. According
to power analysis calculations, assuming the development of leu-
kemia as the experimental endpoint, and an expected proportion
of 0.23 B-ALL-positive untreated animals at 20 months of age, a
sample size of 34 in each group has a 73% power to detect a
decrease in the incidence of the disease to a proportion of 0.03 B-
ALL-positive mice in polyI:C-treated animals, with a significance
level (alpha) of 0.05. Real-time PCR quantification (Q-PCR) of Myd88 Real time PCR quantification (Q PCR) of Myd88
Expression of Myd88 was analyzed by Q-PCR in preleukemic samples
(sorted BM precursor B (B220low IgM-) cells of wild-type (n = 3), Pax5+/
−(n = 3) and Sca1-ETV6-RUNX1 (n = 5) and Pax5+/−(n = 3) and Sca1-ETV6-
RUNX1 (n = 5) mice treated with Abx for 8 weeks as well as in leukemic
samples (leukemic cells from Pax5+/−mice treated with Abx for 8 weeks
(n = 3) and leukemic cells from Myd88+/−;Pax5+/−mice (n = 3)). All of
them exposed to infections (in mice housed in conventional facility). cDNA for use in quantitative PCR studies was synthesized using reverse
transcriptase (SuperScript VILOTM cDNA Synthesis Kit; Invitrogen). Real-time PCR reactions were performed in an Eppendorf Mas-
terCycler Realplex machine. Assays used for quantitative PCR are
commercially available from IDT (Integrated DNA Technologies):
Myd88 (Assay ID: Mm.PT.58.8716051.gs) and GAPDH (Assay ID:
Mm.PT.39a.1). In addition, the probes were designed so that genomic
DNA would not be detected during the PCR. Measurement of GAPDH
gene product expression was used as an endogenous control. Total
bone marrow from Myd88−/−mice were used as negative control. All
samples were run in triplicate. The comparative CT Method (ΔΔCt) was
used to calculate relative expression of the transcript of interest and a
positive control. The change in threshold cycle (ΔCt) of each sample
was calculated as the Ct value of the tested gene (target) minus the Ct
value of GAPDH (endogenous control). The ΔΔCt of each sample was
obtained by subtracting the ΔCt value of the reference from the ΔCt
value of the sample. The ΔCt reference value used was the ΔCt Table 1 | Primers used for V(D)J recombination
VHJ558
forward
CGAGCTCTCCARCACAGCCTWCATGCARCTCARC
reverse
GTCTAGATTCTCACAAGAGTCCGATAGACCCTGG
VH7183
forward
CGGTACCAAGAASAMCCTGTWCCTGCAAATGASC
reverse
GTCTAGATTCTCACAAGAGTCCGATAGACCCTGG
VHQ52
forward
CGGTACCAGACTGARCATCASCAAGGACAAYTCC
reverse
GTCTAGATTCTCACAAGAGTCCGATAGACCCTGG
VHGam3.8
forward
CAAGGGACGGTTTGCCTTCTCTTTGGAA
reverse
GTCTAGATTCTCACAAGAGTCCGATAGACCCTGG
VH3609
forward
KCYYTGAAGAGCCRRCTCACAATCTCC
reverse
GTCTAGATTCTCACAAGAGTCCGATAGACCCTGG
DH
forward
TTCAAAGCACAATGCCTGGCT
reverse
GTCTAGATTCTCACAAGAGTCCGATAGACCCTGG
Cμ
forward
TGGCCATGGGCTGCCTAGCCCGGGACTT
reverse
GCCTGACTGAGCTCACACAAGGAGGA
M:A or C; S:C or G; R:A or G; W:A or T. Table 1 | Primers used for V(D)J recombination
VHJ558
forward
CGAGCTCTCCARCACAGCCTWCATGCARCTCARC
reverse
GTCTAGATTCTCACAAGAGTCCGATAGACCCTGG
VH7183
forward
CGGTACCAAGAASAMCCTGTWCCTGCAAATGASC
reverse
GTCTAGATTCTCACAAGAGTCCGATAGACCCTGG
VHQ52
forward
CGGTACCAGACTGARCATCASCAAGGACAAYTCC
reverse
GTCTAGATTCTCACAAGAGTCCGATAGACCCTGG
VHGam3.8
forward
CAAGGGACGGTTTGCCTTCTCTTTGGAA
reverse
GTCTAGATTCTCACAAGAGTCCGATAGACCCTGG
VH3609
forward
KCYYTGAAGAGCCRRCTCACAATCTCC
reverse
GTCTAGATTCTCACAAGAGTCCGATAGACCCTGG
DH
forward
TTCAAAGCACAATGCCTGGCT
reverse
GTCTAGATTCTCACAAGAGTCCGATAGACCCTGG
Cμ
forward
TGGCCATGGGCTGCCTAGCCCGGGACTT
reverse
GCCTGACTGAGCTCACACAAGGAGGA
M:A or C; S:C or G; R:A or G; W:A or T. Whole-genome sequencing 3. Martin-Lorenzo, A. et al. Infection exposure is a causal factor in
b-cell precursor acute lymphoblastic leukemia as a result of Pax5-
inherited susceptibility. Cancer Discov. 5, 1328–1343 (2015). Tumor DNA was derived from the BM where the percentage of blast
cells was higher than 80% in each B-ALL and germline control DNA was
obtained from the tail when the mice were 4-week old. Genomic DNA
libraries were prepared from sheared DNA with the HyperPrep Library
Preparation Kit (Roche PN 07962363001). Paired-end 150 cycle
sequencing was performed on a NovaSeq 6000 (Illumina). Illumina
paired-end reads were preprocessed and were mapped to the mouse
reference genome (mm10) with BWA61. We used an ensemble
approach to call somatic mutations (SNV/indels) with multiple pub-
lished tools, including Mutect262, SomaticSniper63, VarScan264, MuSE65,
and Strelka266. The consensus calls by at least two callers were con-
sidered as confident mutations. The consensus call sets were further
manually reviewed for the read depth, mapping quality, and strand
bias to remove additional artifacts. Somatic copy-number alternations
were determined by CNVkit67. For somatic structural variants (SV), four 4. Rodriguez-Hernandez, G. et al. Infection exposure promotes ETV6-
RUNX1 precursor B-cell leukemia via impaired H3K4 demethylases. Cancer Res. 77, 4365–4377 (2017). 5. Rodriguez-Hernandez, G. et al. Infectious stimuli promote malig-
nant B-cell acute lymphoblastic leukemia in the absence of AID. Nat. Commun. 10, 5563 (2019). 5. Rodriguez-Hernandez, G. et al. Infectious stimuli promote malig-
nant B-cell acute lymphoblastic leukemia in the absence of AID. Nat. Commun. 10, 5563 (2019). 6. Cazzaniga, G. et al. Possible role of pandemic AH1N1 swine flu
virus in a childhood leukemia cluster. Leukemia 31, 1819–1821
(2017). 6. Cazzaniga, G. et al. Possible role of pandemic AH1N1 swine flu
virus in a childhood leukemia cluster. Leukemia 31, 1819–1821
(2017). 7. Francis, S. S., Selvin, S., Yang, W., Buffler, P. A. & Wiemels, J. L. Unusual space-time patterning of the Fallon, Nevada leukemia
cluster: evidence of an infectious etiology. Chem. Biol. Interact. 196,
102–109 (2012). Nature Communications| (2023) 14:5159 14 Article https://doi.org/10.1038/s41467-023-40961-z 8. Hagan, T. et al. Antibiotics-driven gut microbiome perturbation
alters immunity to vaccines in humans. Cell 178, 1313–1328
e1313 (2019). 29. Juric, D. et al. Differential gene expression patterns and interaction
networks in BCR-ABL-positive and -negative adult acute lympho-
blastic leukemias. J. Clin. Oncol. 25, 1341–1349 (2007). 9. Kroll, M. E., Stiller, C. A., Murphy, M. F. & Carpenter, L. M. Whole-genome sequencing Today 19,
480–485 (1998). 17. Kamper-Jorgensen, M. et al. Childcare in the first 2 years of life
reduces the risk of childhood acute lymphoblastic leukemia. Leu-
kemia 22, 189–193 (2008). 38. Fidanza, M. et al. Inhibition of precursor B-cell malignancy pro-
gression by toll-like receptor ligand-induced immune responses. Leukemia 30, 2116–2119 (2016). 18. Ajrouche, R. et al. Childhood acute lymphoblastic leukaemia and
indicators of early immune stimulation: the Estelle study (SFCE). Br. J. Cancer 112, 1017–1026 (2015). 39. Swaminathan, S. et al. Mechanisms of clonal evolution in childhood
acute lymphoblastic leukemia. Nat. Immunol. 16, 766–774 (2015). 19. Amitay, E. L. & Keinan-Boker, L. Breastfeeding and childhood leu-
kemia incidence: a meta-analysis and systematic review. JAMA
Pediatr. 169, e151025 (2015). 40. Urbanek, P., Wang, Z. Q., Fetka, I., Wagner, E. F. & Busslinger, M. Complete block of early B cell differentiation and altered patterning
of the posterior midbrain in mice lacking Pax5/BSAP. Cell 79,
901–912 (1994). 20. Urayama, K. Y., Buffler, P. A., Gallagher, E. R., Ayoob, J. M. & Ma, X. A
meta-analysis of the association between day-care attendance and
childhood acute lymphoblastic leukaemia. Int J. Epidemiol. 39,
718–732 (2010). 41. Adachi, O. et al. Targeted disruption of the MyD88 gene results in
loss of IL-1- and IL-18-mediated function. Immunity 9, 143–150 (1998). 21. Greaves, M. A causal mechanism for childhood acute lympho-
blastic leukaemia. Nat. Rev. Cancer 18, 471–484 (2018). 42. Cording, S. et al. Commensal microbiota drive proliferation of
conventional and Foxp3(+) regulatory CD4(+) T cells in mesenteric
lymph nodes and Peyer’s patches Eur. J. Microbiol. Immunol. (Bp) 3,
1–10 (2013). 22. Auer, F. et al. Inherited susceptibility to pre B-ALL caused by
germline transmission of PAX5 c.547G>A. Leukemia 28,
1136–1138 (2014). 43. Cobaleda, C., Jochum, W. & Busslinger, M. Conversion of mature B
cells into T cells by dedifferentiation to uncommitted progenitors. Nature 449, 473–477 (2007). 23. Shah, S. et al. A recurrent germline PAX5 mutation confers sus-
ceptibility to pre-B cell acute lymphoblastic leukemia. Nat. Genet. 45, 1226–1231 (2013). 44. Bolyen, E. et al. Reproducible, interactive, scalable and extensible
microbiome data science using QIIME 2. Nat. Biotechnol. 37,
852–857 (2019). 24. Duployez, N. et al. Germline PAX5 mutation predisposes to familial
B-cell precursor acute lymphoblastic leukemia. Blood 137,
1424–1428 (2021). 45. Callahan, B. J. et al. DADA2: High-resolution sample inference from
Illumina amplicon data. Nat. Methods 13, 581–583 (2016). 25. Casado-Garcia, A. et al. Whole-genome sequencing Childhood
leukaemia and socioeconomic status in England and Wales 1976-
2005: evidence of higher incidence in relatively affluent commu-
nities persists over time. Br. J. Cancer 105, 1783–1787
(2011). 30. Chiaretti, S. et al. Gene expression profiles of B-lineage adult acute
lymphocytic leukemia reveal genetic patterns that identify lineage
derivation and distinct mechanisms of transformation. Clin. Cancer
Res. 11, 7209–7219 (2005). 31. Kohlmann, A. et al. Pediatric acute lymphoblastic leukemia (ALL)
gene expression signatures classify an independent cohort of adult
ALL patients. Leukemia 18, 63–71 (2004). 10. Greaves, M. F. Speculations on the cause of childhood acute lym-
phoblastic leukemia. Leukemia 2, 120–125 (1988). 32. Behera, A. K. et al. MyD88 deficiency results in tissue-specific
changes in cytokine induction and inflammation in interleukin-18-
independent mice infected with Borrelia burgdorferi. Infect. Immun. 74, 1462–1470 (2006). 11. Greaves, M., Cazzaniga, V. & Ford, A. Can we prevent childhood
Leukaemia? Leukemia 35, 1258–1264 (2021). 12. Kinlen, L. Evidence for an infective cause of childhood leukaemia:
comparison of a Scottish new town with nuclear reprocessing sites
in Britain. Lancet 2, 1323–1327 (1988). 33. Beneforti, L. et al. Pro-inflammatory cytokines favor the emergence
of ETV6-RUNX1-positive pre-leukemic cells in a model of
mesenchymal niche. Br. J. Haematol. 190, 262–273 (2020). 13. Vicente-Duenas, C. et al. An intact gut microbiome protects
genetically predisposed mice against leukemia. Blood 136,
2003–2017 (2020). 34. Ford, A. M. et al. The TEL-AML1 leukemia fusion gene dysregulates
the TGF-beta pathway in early B lineage progenitor cells. J. Clin. Invest. 119, 826–836 (2009). 14. Garcia-Ramirez, I. et al. Lmo2 expression defines tumor cell identity
during T-cell leukemogenesis EMBO J 37 (2018). 35. Portale, F. et al. Activin A contributes to the definition of a pro-
oncogenic bone marrow microenvironment in t(12;21) preleukemia. Exp. Hematol. 73, 7–12.e14 (2019). 15. Martin-Lorenzo, A. et al. Loss of Pax5 exploits Sca1-BCR-ABL(p190)
susceptibility to confer the metabolic shift essential for pB-ALL. Cancer Res 78, 2669–2679 (2018). 36. Alexopoulou, L., Holt, A. C., Medzhitov, R. & Flavell, R. A. Recogni-
tion of double-stranded RNA and activation of NF-kappaB by Toll-
like receptor 3. Nature 413, 732–738 (2001). 16. Rudant, J. et al. Childhood acute lymphoblastic leukemia and
indicators of early immune stimulation: a Childhood Leukemia
International Consortium study. Am. J. Epidemiol. 181,
549–562 (2015). 37. Ghia, P., ten Boekel, E., Rolink, A. G. & Melchers, F. B-cell develop-
ment: a comparison between mouse and man. Immunol. Acknowledgements The authors thank all the members of their laboratories for discussion. Research at C. Cobaleda’s laboratory was partially supported by
Ministerio de Ciencia e Innovación/AEI/FEDER (PID2021-122787OB-
I00), and a Research Contract with the “Fundación Síndrome de Wolf-
Hirschhorn o 4p-”. Institutional grants from the “Fundación Ramón
Areces” and “Banco de Santander” to the CBMSO are also acknowl-
edged. Research in C. Vicente-Dueñas group has been funded by
Instituto de Salud Carlos III through the project “ PI22/00379 and by a
“Miguel Servet Grant” [CPII19/00024-AES 2017-2020; co-funded by
European Regional Development Fund (ERDF)/European Social Fund
(ESF) “A way to make Europe”/“Investing in your future”]. Kim Nichols
receives funding from the American Lebanese Syrian Associated
Charities (ALSAC) and R01CA241452 from the National Cancer Insti-
tute Research in ISG group is partially supported by FEDER and by
SAF2015-64420-R MINECO/FEDER, UE; by RTI2018-093314-B-I00
MCIU/AEI/FEDER, UE; by PID2021-122185OB-I00 MCIU/AEI/FEDER, UE,
and by Junta de Castilla y León (UIC-017, CSI001U16, CSI234P18, and
CSI144P20). Research in BdA group is partially supported by RTI2019-
09114-B-I00 MCIU/AEI/FEDER, UE. M. Ramírez-Orellana and I. Sánchez-García have been supported by the Fundacion Unoen-
trecienmil (CUNINA project). C. Cobaleda, M. Ramírez-Orellana, and I. Sánchez-García have been supported by the Fundación Científica de
la Asociación Española contra el Cáncer (PRYCO211305SANC). A. Casado-García (CSI067-18) and M. Isidro-Hernández (CSI021-19) are
supported by FSE-Conserjería de Educación de la Junta de Castilla y
León 2019 and 2020 (ESF, European Social Fund) fellowship,
respectively. S. Alemán-Arteaga is supported by an Ayuda para
Contratos predoctorales para la formación de doctores (PRE2019-
088887). J. Martínez-Cano is supported by a predoctoral fellowship
FPI-UAM 2019. We also thank the Hartwell Center and Dr. Ti-Cheng
Chang from the Center for Bioinformatics at SJCRH for their support
of the whole genome sequencing and bioinformatic pipelines. 53. McDonald, D. et al. An improved Greengenes taxonomy with
explicit ranks for ecological and evolutionary analyses of bacteria
and archaea. ISME J. 6, 610–618 (2012). 54. Segata, N. et al. Metagenomic biomarker discovery and explana-
tion. Genome Biol. 12, R60 (2011). 55. Liberzon, A. et al. The Molecular Signatures Database (MSigDB)
hallmark gene set collection. Cell Syst. 1, 417–425 (2015). 56. Mootha, V. K. et al. PGC-1alpha-responsive genes involved in oxi-
dative phosphorylation are coordinately downregulated in human
diabetes. Nat. Genet. 34, 267–273 (2003). 57. Subramanian, A. et al. Gene set enrichment analysis: a knowledge-
based approach for interpreting genome-wide expression profiles. Proc. Natl Acad. Sci. Author contributions Initial conception of the project was designed by M.R.-O., C.V.-D., C.C.,
K.E.N., and I.S.-G. Development of methodology was performed by M.I.-
H., A.C.-G., N.O., S.A.-A., B.R.-C., J.M.-C., A.M., E.G.S., O.B., M.L.G., A.O.,
D.A.-L., J.D.L.R., S.R., P.P.-M., A.G.-M., F.J.G.C., M.B.G.C., M.R.-O., B.d.A.,
C.V.-D., C.C., K.E.N. and I.S.-G. O.B., M.B.G.C., F.J.G.C., and C.V.-D. performed pathology review. Management of patient information was
performed by O.B., M.B.G.C., F.J.G.C., S.R., P.P.-M., M.R.-O., C.V.-D.,
K.E.N., and I.S.-G. M.I.-H., A.C.-G., N.O., S.A.-A., B.R.-C., J.M.-C., A.M.,
A.O., D.A.-L., J.D.L.R, S.R., P.P.-M., A.G.-M., F.J.G.C., M.B.G.C., M.R.-O.,
B.d.A., C.V.-D., C.C., K.E.N. and I.S.-G. were responsible for analysis and
interpretation of the data (e.g., statistical analysis, biostatistics, com-
putational analysis). Paper preparation was performed by M.I.-H., A.C.-
G., N.O., S.A.-A., B.R.-C., J.M.-C., A.M., E.G.S., O.B., M.L.G., A.O., D.A.-L.,
J.D.L.R, S.R., P.P.-M., A.G.-M., F.J.G.C., M.B.G.C., M.R.-O., B.d.A., C.V.-D.,
C.C., K.E.N. and I.S.-G. Administrative, technical, or material support (i.e.,
reporting or organizing the data, constructing databases) was compiled
by M.I.-H., A.C.-G., N.O., S.A.-A., B.R.-C., D.A.-L., C.V.-D., C.C., K.E.N., and
I.S.-G. The study was supervised by M.R.-O., C.V.-D., C.C., K.E.N., and
I.S.-G. 65. Fan, Y. et al. MuSE: accounting for tumor heterogeneity using a
sample-specific error model improves sensitivity and specificity in
mutation calling from sequencing data. Genome Biol. 17, 178 (2016). 66. Kim, S. et al. Strelka2: fast and accurate calling of germline and
somatic variants. Nat. Methods 15, 591–594 (2018). 67. Talevich, E., Shain, A. H., Botton, T. & Bastian, B. C. CNVkit: genome-
wide copy number detection and visualization from targeted DNA
sequencing. PLoS Comput. Biol. 12, e1004873 (2016). 68. Rausch, T. et al. DELLY: structural variant discovery by integrated
paired-end and split-read analysis. Bioinformatics 28,
i333–i339 (2012). 69. Layer, R. M., Chiang, C., Quinlan, A. R. & Hall, I. M. LUMPY: a prob-
abilistic framework for structural variant discovery. Genome Biol. 15, R84 (2014). 70. Chen, X. et al. Manta: rapid detection of structural variants and
indels for germline and cancer sequencing applications. Bioinfor-
matics 32, 1220–1222 (2016). 71. Cameron, D. L. et al. GRIDSS: sensitive and specific genomic rear-
rangement detection using positional de Bruijn graph assembly. Genome Res. 27, 2050–2060 (2017). Whole-genome sequencing Transient inhibition of the JAK/STAT path-
way prevents B-ALL development in genetically predisposed mice. Cancer Res. 82, 1098–1109 (2022). 46. Katoh, K., Misawa, K., Kuma, K. & Miyata, T. MAFFT: a novel method
for rapid multiple sequence alignment based on fast Fourier
transform. Nucleic Acids Res. 30, 3059–3066 (2002). 26. Case, M. et al. Mutation of genes affecting the RAS pathway is
common in childhood acute lymphoblastic leukemia. Cancer Res. 68, 6803–6809 (2008). 47. Price, M. N., Dehal, P. S. & Arkin, A. P. FastTree 2-approximately
maximum-likelihood trees for large alignments. PLoS ONE 5,
e9490 (2010). 27. Man, S. M. & Jenkins, B. J. Context-dependent functions of pattern
recognition receptors in cancer. Nat. Rev. Cancer 22, 397–413
(2022). 48. Faith, D. P. Conservation evaluation and phylogenetic diversity. Biol. Conserv. 61, 1–10 (1992). 28. Linka, Y. et al. The impact of TEL-AML1 (ETV6-RUNX1) expression in
precursor B cells and implications for leukaemia using three dif-
ferent genome-wide screening methods. Blood Cancer J. 3,
e151 (2013). 49. Lozupone, C. A., Hamady, M., Kelley, S. T. & Knight, R. Quantitative
and qualitative beta diversity measures lead to different insights
into factors that structure microbial communities. Appl Environ. Microbiol 73, 1576–1585 (2007). Nature Communications| (2023) 14:5159 15 Article https://doi.org/10.1038/s41467-023-40961-z 50. Lozupone, C. & Knight, R. UniFrac: a new phylogenetic method for
comparing microbial communities. Appl Environ. Microbiol 71,
8228–8235 (2005). 73. Chiang, C. et al. SpeedSeq: ultra-fast personal genome analysis and
interpretation. Nat. Methods 12, 966–968 (2015). 73. Chiang, C. et al. SpeedSeq: ultra-fast personal genome analysis and
interpretation. Nat. Methods 12, 966–968 (2015). 73. Chiang, C. et al. SpeedSeq: ultra-fast personal genome analysis and
interpretation. Nat. Methods 12, 966–968 (2015). 74. Edgar, R., Domrachev, M. & Lash, A. E. Gene expression Omnibus:
NCBI gene expression and hybridization array data repository. Nucleic Acids Res. 30, 207–210 (2002). 51. Bray, J. R. & Curtis, J. T. An ordination of upland forest communities
of southern Wisconsin. Ecol. Monogr. 27, 325–349 (1957). 52. Bokulich, N. A. et al. Optimizing taxonomic classification of marker-
gene amplicon sequences with QIIME 2’s q2-feature-classifier plu-
gin. Microbiome 6, 90 (2018). Acknowledgements USA 102, 15545–15550 (2005). 58. Green, M. R. et al. Signatures of murine B-cell development impli-
cate Yy1 as a regulator of the germinal center-specific program. Proc. Natl Acad. Sci. USA 108, 2873–2878 (2011). 59. Revilla, I. D. R. et al. The B-cell identity factor Pax5 regulates distinct
transcriptional programmes in early and late B lymphopoiesis. EMBO J. 31, 3130–3146 (2012). 60. Schebesta, A. et al. Transcription factor Pax5 activates the chro-
matin of key genes involved in B cell signaling, adhesion, migration,
and immune function. Immunity 27, 49–63 (2007). 61. Li, H. & Durbin, R. Fast and accurate long-read alignment with
Burrows-Wheeler transform. Bioinformatics 26, 589–595
(2010). 62. Cibulskis, K. et al. Sensitive detection of somatic point mutations in
impure and heterogeneous cancer samples. Nat. Biotechnol. 31,
213–219 (2013). 63. Larson, D. E. et al. SomaticSniper: identification of somatic point
mutations in whole genome sequencing data. Bioinformatics 28,
311–317 (2012). 64. Koboldt, D. C. et al. VarScan 2: somatic mutation and copy number
alteration discovery in cancer by exome sequencing. Genome Res. 22, 568–576 (2012). Competing interests O.B. reports personal fees from Takeda and Clinigen outside the sub-
mitted work. K.N. reports grants from Incyte outside the submitted work. No disclosures were reported by the other authors. 72. Jeffares, D. C. et al. Transient structural variations have strong
effects on quantitative traits and reproductive isolation in fission
yeast. Nat. Commun. 8, 14061 (2017). Nature Communications| (2023) 14:5159 16 Article Additional information Additional information Open Access This article is licensed under a Creative Commons
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Carolina Vicente-Dueñas, César Cobaleda, Kim E. Nichols or Isidro
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isdictional claims in published maps and institutional affiliations. 1Experimental Therapeutics and Translational Oncology Program, Instituto de Biología Molecular y Celular del Cáncer, CSIC-USAL, Campus M. de Unamuno
s/n, Salamanca, Spain. 2Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain. 3Department of Oncology, St. Jude Children’s Research
Hospital, Memphis, TN, USA. 4Immune system development and function Unit, Centro de Biología Molecular Severo Ochoa (Consejo Superior de Investi-
gaciones Científicas -Universidad Autónoma de Madrid), Madrid, Spain. 5Servicio de Citometría, Departamento de Medicina, Biomedical Research Net-
working Centre on Cancer CIBER-CIBERONC (CB16/12/00400), Institute of Health Carlos III, and Instituto de Biología Molecular y Celular del Cáncer, CSIC/
Universidad de Salamanca, Salamanca, Spain. 6Department of Pediatric Hematology and Oncology, Hospital Infantil Universitario Niño Jesús, Universidad
Autónoma de Madrid, Madrid, Spain. 7Departamento de Anatomía Patológica, Universidad de Salamanca, Salamanca, Spain. 8Immunobiology Department,
Carlos III Health Institute, 28220 Majadahonda (Madrid), Spain. 9Bioinformatics Unit, Cancer Research Center (CSIC-USAL), Salamanca, Spain. 10Bioinfor-
matics and Functional Genomics Research Group, Cancer Research Center (CSIC-USAL), Salamanca, Spain. 11Department of Pediatrics, Hospital Universitario
de Salamanca, Paseo de San Vicente, 58-182, Salamanca 37007, Spain. 12Departamento de Cirugía, , Universidad de Salamanca, Salamanca, Spain. 13These
authors contributed equally: Marta Isidro-Hernández, Ana Casado-García, Ninad Oak, Silvia Alemán-Arteaga, Carolina Vicente-Dueñas, César Cobaleda, Kim
E. Nichols, Isidro Sánchez-García. e-mail: cvd@usal.es; cesar.cobaleda@csic.es; Kim.Nichols@STJUDE.ORG; isg@usal.es Nature Communications| (2023) 14:5159 17
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Recall of physical activity advice was associated with higher levels of physical activity in colorectal cancer patients
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BMJ open
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cc-by
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To cite: Fisher A, Williams K,
Beeken R, et al. Recall of
physical activity advice
was associated with higher
levels of physical activity in
colorectal cancer patients .
BMJ Open 2015;5:e006853.
doi:10.1136/bmjopen-2014-
006853 CRC survivors typically have lower levels of
PA than the general population4 and data from
the English
Longitudinal
Study of Ageing
showed a faster decline in activity over time in
participants who received a cancer diagnosis
than the rest of the cohort.5 Intervention
within the clinical care pathway to promote PA
could help CRC survivors achieve the health
gains associated with an active lifestyle. Primary and secondary outcome measures: Primary and secondary outcome measures:
Patients completed the ‘Living with and Beyond
Colorectal Cancer’ Patient-Reported Outcome Measures
(PROMS) survey in 2013. The survey included
questions on receiving exercise advice/information (‘PA
advice’), and the frequency of currently doing at least
30 min of brisk PA per day (‘PA level’: 0, 1–4 or
5–7 days, within the past week; with the top category
meeting UK guidelines). ▪Data on the context of the advice were not avail-
able and this should be a focus of future
research. ▪Data on the context of the advice were not avail-
able and this should be a focus of future
research. Results: A third of respondents (31%) recalled
receiving PA advice. Independent of demographics and
treatment, patients who recalled having PA advice were
more likely to be currently doing some brisk PA (51%
in the advice group vs 42% in the no advice group; OR
1.74, 95% CI 1.60 to 1.90; p<0.001), and more likely
to be meeting PA guidelines (25% vs 20%; OR 1.70,
CI 1.54 to 1.88; p<0.001). better
long-term
outcomes
following
a
diagnosis
of
colorectal
cancer
(CRC).1–3
A
meta-analysis
of
7
prospective
cohort
studies demonstrated that higher postdiagno-
sis physical activity (PA) was associated with
lower CRC-specific and all-cause mortality.1
A
report
from
the
Cancer
Prevention
Study-II (CPS-II) Nutrition Cohort showed
associations with all-cause mortality for both
postdiagnosis PA and sedentary time.2 PA has
also been associated with better quality of life
and lower levels of cancer-related fatigue.3 Conclusions: Recalling being given PA advice after a
diagnosis of CRC was associated with higher levels of
PA. However, less than a third of patients recalled
receiving advice. Future research should examine the
context in which advice is given and randomised trials
are required. However, encouraging clinicians working
with patients with CRC to give brief PA advice is
warranted and may help improve outcomes for CRC
survivors. Strengths and limitations of this study ABSTRACT
Objectives: The present study tested the hypothesis
that recall of receiving physical activity (PA) advice
would be associated with higher levels of PA in
patients with a diagnosis of colorectal cancer (CRC). Setting: Colorectal cancer patients who were
diagnosed in 2010 or 2011, and had been treated in
the English National Health Service (NHS). Participants: 17 753 respondents completed at least
one section of the survey relevant to the current study
and after exclusion of 171 with dementia (since results
relied on recall), 15 254 had complete data for the
current study. 60% were male, 67% were >65 years
and 96% were from a white ethnic group. Objectives: The present study tested the hypothesis
that recall of receiving physical activity (PA) advice
would be associated with higher levels of PA in
patients with a diagnosis of colorectal cancer (CRC). Setting: Colorectal cancer patients who were
diagnosed in 2010 or 2011, and had been treated in
the English National Health Service (NHS). ▪This is the first very large population-based
study to explore whether physical activity advice
is given to colorectal cancer patients; propor-
tions who recalled receiving advice were low. ▪Physical activity advice during the cancer care
pathway was associated with higher levels of
physical activity. Participants: 17 753 respondents completed at least
one section of the survey relevant to the current study
and after exclusion of 171 with dementia (since results
relied on recall), 15 254 had complete data for the
current study. 60% were male, 67% were >65 years
and 96% were from a white ethnic group. ▪The findings of this study provide strong impetus
to clinicians working with colorectal cancer to
recommend physical activity, a practice which
does not yet appear to be routine in the UK. ▸Prepublication history for
this paper is available online. To view these files please
visit the journal online
(http://dx.doi.org/10.1136/
bmjopen-2014-006853). ▪This study is limited in relying on patient report
of physical activity levels and recall of whether
they were given advice. However, the Patient-
Reported Outcome Measures data are designed
to collect data on patient experience on a large
scale. Recall of physical activity advice
was associated with higher levels of
physical activity in colorectal cancer
patients A Fisher, K Williams, R Beeken, J Wardle A Fisher, K Williams, R Beeken, J Wardle group.bmj.com
on July 23, 2015 - Published by
http://bmjopen.bmj.com/
Downloaded from group.bmj.com
on July 23, 2015 - Published by
http://bmjopen.bmj.com/
Downloaded from Open Access Open Access y
pp
Current PA was assessed by asking: ‘In the past week
how many days have you done 30 min or more of brisk
physical activity (This may include sport, exercise or
brisk walking or cycling for recreation or to get to and
from places, but should not include housework that or
physical activity that is part of your job)?’ Responses were
categorised as ‘none’ (0 days), ‘some’ (1–4 days) or
‘meeting guidelines’ (5–7 days), broadly in line with the
UK Government recommendations for the general popu-
lation19 and the American College of Sports Medicine
recommendations that cancer survivors should partici-
pate in at least 150 min per week of at least moderate
activity.20 There were also data on sex and age at diagno-
sis (obtained from registry data), and ethnicity (reported
by patients). Index of Multiple Deprivation (IMD) scores
were calculated for the home postcode (1–5 from least to
most deprived).21 IMD is an area level measure derived
from a composite of 38 indicators across seven domains
of deprivation, including income, employment, health,
education, housing and services, living environment and
crime.21 Brief PA advice in primary care has been shown to
increase PA in sedentary adults13 and a review con-
cluded that it was an effective intervention, at least in
the short-term.14 Studies in cancer survivors also indicate
that clinicians discussing PA with their patients might
have a positive effect on activity levels. A survey of 311
cancer survivors (38 with CRC), found that oncologist-
initiated discussion of PA was associated with higher
activity levels during treatment7 and an exploratory study
in 24 breast cancer survivors suggested that a clinician
recommendation was an important factor in exercise
adherence.15 In one of the few trials of brief advice in
cancer survivors, a simple PA recommendation doubled
the
percentage
of
breast
cancer
survivors
meeting
national exercise guidelines, with stronger effects among
those who correctly recalled the advice, although the
follow-up interval in this study was only 5 weeks.16 There were a variety of treatment questions, but for
these analyses we included: ‘How has your CRC responded
to treatment?’ (‘fully responded I am in remission’, ‘has
been treated but is still present’, ‘has not been treated’,
‘has come back after initial treatment’, ‘not certain what
is happening’). Length of time since treatment was also
reported. Open Access were 12–36 months postdiagnosis. Content and face val-
idity for the PROMS survey were identified through
expert reviews and consultations with patients, experts
and charity advisory groups.18 For the current study, a
formal request was made by the study authors to the
NCRS for health and lifestyle data for the secondary
analyses. Clinicians dealing with patients with cancer are in a
good position to offer activity advice, and they are the
patients’ preferred source of information.6 Patients also
specifically express a wish for those involved in their care
to initiate discussion about PA during consultation.7
However, evidence to date indicates that most patients
with cancer are not receiving PA advice. In a US survey,
conducted in 2008, 38% of oncologists and surgeons
said they did not enquire about patients’ activity levels.8
A similar study in the UK found that 56% of healthcare
professionals did not discuss PA with their patients with
breast cancer9 and a more recent survey suggested little
improvement in these figures.10 These findings are
broadly consistent with reports from cancer survivors
themselves, with only a third of patients with breast or
prostate cancer reporting being given any PA advice.11
Primary care physicians are another source of lifestyle
advice, but although US data indicate some increase in
provision of PA advice for patients with cancer in
primary care (25% in 2000, 36% in 2010), the rates are
still too low.12 The questionnaire section ‘Overall Support and Care’
included the question ‘Did you receive any advice or infor-
mation on physical activity and exercise (‘physical activity
advice’)?’ This was within a longer list of cancer-relevant
domains of information, including physical and psycho-
logical aspects of living with cancer, finance, employ-
ment, family and support services. Open Access Long-term health conditions (LTCs) were
assessed with ‘Do you have a long-standing health condition
other than cancer?’ (‘yes’, ‘no’, ‘can’t say’). As the focus of
this analysis was on recall of receiving PA advice, patients
reporting a diagnosis of dementia were excluded. The
full survey is available at cancerproms.ncr.nhs.uk. In
accordance with UCL Ethics Committee guidance, add-
itional ethical approval was not required for secondary
analyses of anonymous health surveillance survey data. p
y
y
The current study used data collected from a very
large sample of patients with CRC to test the hypothesis
that recalling being given PA advice would be related to
higher levels of physical activity. MATERIALS AND METHODS
Participants and measures Data were from the Living with and Beyond Colorectal
Cancer survey commissioned by the UK Department of
Health in 2013, as part of a programme of work
designed to ensure that the needs of patients with colo-
rectal cancer in the UK are met across a range of
health, psychosocial and lifestyle domains through the
recording of patient-reported outcomes (PROMS).17
The questionnaire was mailed by the National Cancer
Registration Service (NCRS), in 2013, to a sample of
34 467 adult patients with a recorded diagnosis of CRC
in 2010 or 2011, who were treated in the English
National Health Service, and who were >16 years and INTRODUCTION Correspondence to
Dr A Fisher;
abigail.fisher@ucl.ac.uk There is now a solid body of evidence that a
physically active lifestyle is associated with Fisher A, et al. BMJ Open 2015;5:e006853. doi:10.1136/bmjopen-2014-006853 1 group.bmj.com
on July 23, 2015 - Published by
http://bmjopen.bmj.com/
Downloaded from ANALYSES Where >75% of respondents fell into a single response
category, predictor variables were dichotomised for ana-
lyses. However, full descriptive data, including the pro-
portion of missing values, are presented in the results. Ethnic groups were categorised as ‘white’ versus ‘all 2 Fisher A, et al. BMJ Open 2015;5:e006853. doi:10.1136/bmjopen-2014-006853 group.bmj.com
on July 23, 2015 - Published by
http://bmjopen.bmj.com/
Downloaded from Open Access Men were more likely than women to recall being given
advice (35 vs 25%; OR 1.66, 95% CI 1.55 to 1.79;
p<0.001). Younger patients were more likely than older
patients to recall advice (37% in the <55-year-olds vs
20%
in
>85-year-olds
(OR
2.41,
CI
1.95
to
2.90;
p<0.001). Patients from higher SES groups were more
likely to recall advice than those from lower SES groups
(comparing highest to lowest SES, 32% vs 28%; OR 1.25,
CI 1.10 to 1.43; p<0.001), but there was no significant
association with ethnicity. Patients in remission were
more likely to recall being given PA advice (32% vs 27%;
OR 1.23, CI 1.12 to 1.30; p<0.001), and in the fully
adjusted model, patients with a LTC were statistically
more likely to recall advice, but the magnitude of this
difference was extremely small (32% vs 31%; p<0.001)
and unlikely to be clinically meaningful. Time since
treatment was not associated with advice in the adjusted
model. other’. Since health and treatment variables were sec-
ondary to the main research question, missing data were
recoded as ‘unknown’ for analyses to include as many
respondents as possible. Response to treatment was cate-
gorised as ‘in remission’ versus ‘all other’. Time since
treatment was coded as ‘Still having treatment’, ‘<1 year’
post-treatment, ‘>1 year’, or ‘unknown’. Presence of an
LTC was recorded as ‘yes’, ‘no’, or ‘unknown’. other’. Since health and treatment variables were sec-
ondary to the main research question, missing data were
recoded as ‘unknown’ for analyses to include as many
respondents as possible. Response to treatment was cate-
gorised as ‘in remission’ versus ‘all other’. Time since
treatment was coded as ‘Still having treatment’, ‘<1 year’
post-treatment, ‘>1 year’, or ‘unknown’. Presence of an
LTC was recorded as ‘yes’, ‘no’, or ‘unknown’. Descriptive statistics and percentages in each cat-
egory were calculated. Two types of logistic regression
were carried out. RESULTS Of the 34 467 questionnaires mailed out by NCRS,
21 802 (63.3%) were returned at least partially com-
pleted and further information on this sample are pro-
vided in a recent paper by Downing et al.22 Regarding
the data granted from NCRS for the current study,
17 753 patients had responded to at least 1 question in
the relevant parts of the survey. Compared to non-
responders, these participants were less likely to be from
deprived areas and fewer
were from the youngest
(<55 years)
and
oldest
(>85 years)
age
categories
(p<0.001). After exclusion of 171 (1.1%) patients with
dementia, complete data on the PA questions and demo-
graphics were available for 15 254 patients; this consti-
tuted the study sample. Characteristics of the 17 753
patients who completed at least 1 question in the rele-
vant parts of the survey and the study sample were very
similar, and are shown in table 1. There were associations between demographic vari-
ables and activity levels (men, younger patients and
those from higher SES backgrounds were more active;
table 2), but associations between PA advice and activity
levels remained significant after controlling for these
and the adjusted odds were similar to the unadjusted
values (OR 1.74, CI 1.60 to 1.90, p<0.001 for doing some
activity; OR 1.70, CI 1.54 to 1.88, p<0.001 for meeting
guidelines). Similarly, although some health and treatment factors
were related to activity levels (table 4), the association
between PA advice and current activity levels remained
after adjustment for treatment and presence of any LTC. 67% were >65 years old, 60% were male, and 96%
identified themselves as ‘white’. Most patients reported
that their cancer had fully responded to treatment and
they were in remission (79%), with smaller numbers
reporting that the cancer was still present (5%), had not
been treated (1%), had come back after initial treat-
ment (3%) or that they were not certain (9%); however,
4% did not respond to this question. The majority
(>80%) of patients were at least 1 year since treatment. Forty-seven per cent reported a long-standing health
condition other than cancer, 3% ‘couldn’t say’ and 11%
did not respond. Forty-five per cent of patients reported
doing at least some brisk activity and 22% met the guide-
lines for PA, but 33% reported doing none. ANALYSES First, to assess factors associated with
whether PA advice was given, a binary logistic regres-
sion with ‘advice’ or ‘no advice’ as the outcome was
carried out. Second, to assess whether advice related
to
activity levels, a multinomial
logistic regression
model
with
PA
level
(none/some/meeting
activity
guidelines) as the outcome was carried out. In each
case, simple associations and then models adjusted for
potential confounders were presented. Analyses were
carried out in SPSS V.18. Given the large sample size,
significance was set at p<0.01. Consistent with our hypothesis, recalling being given
PA advice was associated with higher levels of current
activity. Fifty-one per cent of patients who had been
given advice were doing at least some activity, with 25%
meeting the guidelines, compared with 42% and 20%,
respectively, in the ‘no advice’ group (see table 3 and
figure 1). Compared with the ‘no advice’ group, the
odds of doing some activity were 1.88 (CI 1.74 to 2.05;
p<0.001) and the odds of meeting guidelines were 1.90,
(CI 1.75 to 2.09; p<0.001) among those who were given
advice. DISCUSSION To the best of our knowledge, this was the largest
population-based study to explore the extent to which
patients with cancer are given PA advice, and the only
one to examine associations between recall of PA advice
and activity levels in patients with CRC. Consistent with
findings from other studies,8–11 a relatively small propor-
tion recalled being given PA advice. In support of our
hypothesis, recall of advice was associated with higher
activity levels, even after adjustment for sociodemo-
graphics and treatment factors. Overall, 31% of respondents recalled having received
any PA advice. The proportion receiving PA advice by
demographics, treatment and LTCs is shown in table 2. The finding that being given advice related to later
activity levels in patients with CRC is supported by evi-
dence from a small RCT in breast cancer survivors, 3 Fisher A, et al. BMJ Open 2015;5:e006853. DISCUSSION doi:10.1136/bmjopen-2014-006853 4 group.bmj.com
on July 23, 2015 - Published by
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Downloaded from Open Access Table 2
Associations between being given physical activity (PA) advice, demographic factors, treatment and activity levels
2–3 years later in 15 254 patients with colorectal cancer
PA advice
given n (%)
Model 1 (unadjusted)
OR (95% CI)
Model 2 (adjusted)
OR (95% CI)
Reference category No PA advice
Total
(n=15 254)
4734 (31.0)
–
–
Sex
Female (n=6091)
1506 (24.7)
1
1
Male (n=9163)
3228 (35.2)
1.66 (1.55 to 1.78)***
1.66 (1.55 to 1.79)***
Age
>85 (n=777)
156 (20.1)
1
1
75–84 (n=3797)
988 (26.0)
1.40 (1.16 to 1.69)**
1.35 (1.12 to 1.64)***
65–74 (n=5680)
1789 (31.5)
1.83 (1.52 to 2.20)***
1.73 (1.44 to 2.09)***
55–64 (n=3677)
1314 (35.7)
2.21 (1.83 to 2.67)***
2.15 (1.77 to 2.60)***
<55 (n=1323)
487 (36.8)
2.32 (1.88 to 2.86)***
2.41 (1.95 to 2.90)***
IMD
5 (n=1606)
455 (28.3)
1
1
4 (n=2441)
714 (29.3)
1.05 (0.91 to 1.20)
1.08 (0.94 to 1.24)
3 (n=3335)
1019 (30.6)
1.11 (0.98 to 1.27)
1.16 (1.02 to 1.33)*
2 (n=3844)
1245 (32.4)
1.21 (1.07 to 1.38)**
1.25 (1.10 to 1.43)***
1 (n=4028)
1301 (32.3)
1.21 (1.06 to 1.37)**
1.25 (1.10 to 1.43)***
Ethnicity
White (n=14 712)
4560 (31.0)
1
1
All other (n=542)
174 (32.1)
1.05 (0.88 to 1.27)
1.03 (0.85 to 1.24)
Time since treatment
Still having (n=289)
82 (28.4)
1
1
<1 year (n=1938)
597 (30.8)
1.36 (1.03 to 1.79)
1.06 (0.81 to 1.39)
>1 year (n=12 715)
3978 (31.3)
1.39 (1.07 to 1.80)
1.06 (0.80 to 1.41)
Unknown (n=312)
77 (24.7)
1.21 (0.41 to 1.74)
1.04 (0.72 to 1.50)
Treatment response
All other (n=3228)
868 (26.9)
1
1
In remission (n=12 026)
3866 (32.1)
1.29 (1.18 to 1.41)***
1.23 (1.12 to 1.34)***
Long-term condition
No (n=5995)
1855 (30.9)
1
1
Unknown (n=2099)
605 (28.8)
0.90 (0.81 to 1.00)
0.98 (0.89 to 1.10)
Yes (n=7160)
2274 (31.8)
1.04 (0.97 to 1.12)
1.13 (1.04 to 1.22)**
***p<0.001; **p≤0.01. IMD, index of multiple deprivation; 5, most deprived, 1, least deprived model 1 is a simple binary logistic regression with having received
physical activity (PA) advice (Y/N) as the dependent factor. Model 2 is a multivariable binary logistic regression model adjusting for all
sociodemographic and treatment factors. ‘All other’ treatment factors (been treated but still present/has not been treated/has come back/not certain). Fisher A, et al. BMJ Open 2015;5:e006853. doi:10.1136/bmjopen-2014-006853 DISCUSSION doi:10.1136/bmjopen-2014-006853 group.bmj.com
on July 23, 2015 - Published by
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Downloaded from group.bmj.com
on July 23, 2015 - Published by
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Downloaded from Table 1
Full demographics and treatment variables from responders and study sample
Responders
n=17 753
Study sample
n=15 254
Demographics
n
Per cent
n
Per cent
Sex
Female
7295
41.1
6091
39.9
Male
10 458
58.9
9163
60.1
Age
>85
969
5.5
777
5.1
75–84
4508
25.4
3797
24.9
65–74
6195
34.9
5680
37.2
55–64
3712
20.9
3677
24.1
<55
1585
8.9
1323
8.7
Missing
784
4.4
–
–
Ethnicity
White
17 070
96.2
14 712
96.4
Minority ethnic group
294
1.7
245
1.6
Unknown
389
2.2
297
1.9
IMD category
5 Most deprived
1947
11.0
1606
10.5
4
2935
16.5
2441
16.0
3
3919
22.1
3335
21.9
2
4419
24.9
3844
25.2
1 Least deprived
4533
25.5
4028
26.4
Physical activity (PA)
Received PA advice
Yes
5038
28.4
4734
31
No
11 489
64.7
10 520
69
Missing
1226
6.9
–
–
Activity levels
None
5803
32.7
5080
33.3
Some
7547
42.5
6877
45.1
Meeting guidelines
3626
20.4
3297
21.6
Missing
777
4.4
–
–
Treatment and health variables
Treatment response
In remission
13 804
77.8
12 026
78.8
Treated but still present
857
4.8
741
4.9
Has not been treated
159
0.9
123
0.8
Has come back
437
2.5
388
2.5
Not certain
1618
9.1
1341
8.8
Missing
878
4.9
635
4.2
Time since treatment
Still having
349
2.0
289
1.9
<3 months
173
1.0
139
0.9
3–12 months
2128
12.0
1799
11.8
1–5 years
14 501
81.7
12 598
82.6
>5 years
151
0.9
117
0.8
Do not know
48
0.3
32
0.2
Missing
403
2.3
280
1.8
Long-term condition
Yes
8225
46.3
7160
46.9
No
6733
37.9
5995
39.3
Do not know
552
3.1
444
2.9
Missing
2243
12.6
1655
10.8 Table 1
Full demographics and treatment variables from responders and study sample reported that the oncologist had initiated any discussion
around PA, but that this discussion was associated with
higher activity levels during treatment.7 Our results
strengthen the case for clinicians to recommend PA to which found a clear benefit from clinician advice over
usual care in the short term.16 Our results also concur
with an earlier survey which found that only 28% of
cancer survivors (predominantly breast and prostate) Fisher A, et al. BMJ Open 2015;5:e006853. DISCUSSION doi:10.1136/bmjopen-2014-006853 group.bmj.com
on July 23, 2015 - Published by
http://bmjopen.bmj.com/
Downloaded from Open Access Table 3
Associations between recall of being given physical activity advice, demographic factors and activity levels in
15 254 patients with colorectal cancer
PA level
(reference ‘None’)
Model 1 (unadjusted)
OR (95% CI)
Model 1 (unadjusted)
OR (95% CI)
Model 2 (adjusted)
OR (95% CI)
Model 2 (adjusted)
OR (95% CI)
Some PA
Meeting guidelines
Some PA
Meeting guidelines
PA advice given
No (n=10 520)
1
1
1
1
Yes (n=4734)
1.88 (1.74 to 2.05)***
1.90 (1.75 to 2.09)***
1.74 (1.60 to 1.90)***
1.70 (1.54 to 1.88)***
Sex
Female (n=6091)
1
1
1
1
Male (n=9163)
1.11 (1.03 to 1.20)***
1.75 (1.60 to 1.92)***
1.04 (0.96 to 1.12)
1.63 (1.49 to 1.80)***
Age
>85 (n=777)
1
1
1
1
75–84 (n=3797)
2.21 (1.85 to 2.66)***
2.20 (1.76 to 2.76)***
2.21 (1.84 to 2.65)***
2.15 (1.71 to 2.70)***
65–74 (n=5680)
4.11 (3.44 to 4.91)***
3.62 (2.90 to 4.52)***
4.05 (3.38 to 4.85)***
3.43 (2.74 to 4.29)***
55–64 (n=3677)
4.64 (3.86 to 5.57)***
3.64 (2.89 to 4.78)***
4.48 (3.72 to 5.40)***
3.42 (2.74 to 4.29)***
<55 (n=1323)
4.30 (3.49 to 5.30)***
3.06 (2.34 to 3.98)***
4.22 (3.41 to 5.21)***
3.00 (2.31 to 3.92)***
IMD
5 (n=1606)
1
1
1
1
4 (n=2441)
1.17 (1.01 to 1.34)
1.17 (0.99 to 1.39)
1.22 (1.06 to 1.41)**
1.23 (1.04 to 1.47)
3 (n=3335)
1.35 (1.18 to 1.54)***
1.24 (1.05 to 1.45)
1.41 (1.23 to 1.61)***
1.30 (1.10 to 1.53)**
2 (n=3844)
1.68 (1.47 to 1.92)***
1.46 (1.25 to 1.72)***
1.75 (1.53 to 2.00)***
1.53 (1.30 to 1.79)***
1 (n=4028)
1.85 (1.62 to 2.11)***
1.52 (1.30 to 1.79)***
1.95 (1.71 to 2.24)***
1.61 (1.37 to 1.89)***
Ethnicity
White (n=14 712)
1
1
1
1
All other (n=542)
1.01 (0.83 to 1.23)
1.04 (0.82 to 1.32)
1.00 (0.82 to 1.23)
1.02 (0.81 to 1.30)
***p<0.001; **p≤0.01. IMD, index of multiple deprivation, 1, least deprived, 5, most deprived. Model 1 simple multinomial logistic regression with PA none, some
(>30 min brisk activity on 1–4 days) and meeting guidelines (5–7 days) as the dependent variable. Model 2 adjusts for sex, age, IMD and
ethnicity. ‘All other’ treatment factors (been treated but still present/has not been treated/has come back/not certain). ethnicity. ‘All other’ treatment factors (been treated but still present/has not been treated/has come back/not certain). DISCUSSION Table 2
Associations between being given physical activity (PA) advice, demographic factors, treatment and activity levels
2–3 years later in 15 254 patients with colorectal cancer p<0.001;
p≤0.01. IMD, index of multiple deprivation; 5, most deprived, 1, least deprived model 1 is a simple binary logistic regression with having received
physical activity (PA) advice (Y/N) as the dependent factor. Model 2 is a multivariable binary logistic regression model adjusting for all
sociodemographic and treatment factors. ‘All other’ treatment factors (been treated but still present/has not been treated/has come back/not certain). Giving PA advice may not always be easy for healthcare
professionals. A recent qualitative study with cancer spe-
cialists identified lack of appropriate support as a barrier
to discussing lifestyle with patients with cancer.23 This
may be particularly applicable when discussing PA with
vulnerable groups, such as older patients and those
from lower SES groups. The same study also found that
some specialists did not believe that lifestyle change
would influence cancer risk and cited this as a reason
for not discussing it. In a recent survey completed by
323
CRC
clinicians
in
Scotland,
only
half
(52%)
reported being familiar with the lifestyle advice for
patients with CRC and many felt they lacked the skills,
confidence and knowledge to discuss it.24 In line with
this, a survey of 274 oncology nurses found being
‘unsure what to recommend’ was strongly related to
whether they gave advice or not.25 Insufficient time their patients with cancer. In the ‘advice’ group, the pro-
portion of participants doing at least some activity was
10% higher than in the ‘no advice’ group, and those
meeting the PA guidelines 5% higher. This difference is
potentially of real practical significance.14 Less than a third (31%) of our large sample of CRC
survivors recalled having received PA advice. Women,
older patients and those from lower SES backgrounds
were less likely to recall having been given advice. Older
patients were also less likely to have received advice in a
Canadian survey, which the authors speculated could be
due to them being less likely to initiate discussions about
PA,7 although nearly all patients expressed a wish for
their oncologist to initiate the discussion.7 Clinicians
may want to consider whether these populations need
more targeted advice to help make access to important
lifestyle advice more equitable. 5 Fisher A, et al. BMJ Open 2015;5:e006853. DISCUSSION in knowledge and skills while keeping in mind the time
constraints in consultations. Finally, the evidence that PA
reduces risk of recurrence and cancer-specific mortality
in CRC currently comes from observational cohorts;1
results of an RCT examining PA effects on survival of
CRC (CHALLENGE)26 are not yet available, so clini-
cians may not yet be convinced to recommend PA for
survival outcomes. However, in light of strong evi-
dence for a number of other important outcomes,
such
as
reductions
in
cancer-related
fatigue
and
improved quality of life,3 it is important for clinicians
to be advising their patients with CRC to be physically
active. during the consultation was identified as a barrier in
another study, along with unclear recommendations.8
These observations indicate a need to address the gaps Figure 1
Proportions of patients doing at least some activity,
or meeting the guidelines by advice. Values are from
n=15 254 colorectal cancer patients who reported the number
of days brisk physical activity (PA) was carried out in the past
week as none, some (1–4 days) or meeting guidelines
(5–7 days). χ2 value for difference s262.822; p<0.001. This study had limitations. Data were self-reported
using single item measures, so PA may have been under-
estimated or over-estimated. Additionally, no information
was gathered on who gave the advice and when in the
care pathway. This study was based on secondary analyses
of existing large-scale PROMS data and provides an
important
‘first
step’. Future
large-scale
surveys
in
cancer survivors should use validated measures of PA
and collect detailed information on the context of the
delivery of the PA advice. Information on receiving PA
advice depended on patient recall and therefore, could
also be under-estimated or over-estimated. Consistency
with
findings
from
other
studies,
both
from
the
patient
and
the
health
professional
perspective,
is Figure 1
Proportions of patients doing at least some activity,
or meeting the guidelines by advice. Values are from
n=15 254 colorectal cancer patients who reported the number
of days brisk physical activity (PA) was carried out in the past
week as none, some (1–4 days) or meeting guidelines
(5–7 days). χ2 value for difference s262.822; p<0.001. 6 Fisher A, et al. BMJ Open 2015;5:e006853. DISCUSSION doi:10.1136/bmjopen-2014-006853 group.bmj.com
on July 23, 2015 - Published by
http://bmjopen.bmj.com/
Downloaded from Table 4
Associations between being given physical activity (PA) advice, treatment and activity levels 2–3 years later in
15 254 patients with colorectal cancer
PA level (reference ‘none’)
Model 1 (unadjusted)
OR (95% CI)
Model 1 (unadjusted)
OR (95% CI)
Model 2 (adjusted)
OR (95% CI)
Model 2 (adjusted)
OR (95% CI)
Some PA
Meeting guidelines
Some PA
Meeting guidelines
PA advice given
No (n=10 520)
1
1
1
1
Yes (n=4734)
1.88 (1.74 to 2.05)***
1.90 (1.75 to 2.09)***
1.76 (1.61 to 1.91)***
1.79 (1.56 to 1.92)***
Time since treatment
Still having (n=289)
1
1
1
1
<1 year (n=1938)
1.34 (1.02 to 1.76)
1.26 (0.89 to 1.77)
1.06 (0.79 to 1.41)
0.96 (0.67 to 1.37)
≥1 year (n=12 715)
1.59 (1.22 to 2.05)***
1.57 (1.14 to 2.17)***
1.22 (0.93 to 1.60)
1.14 (0.82 to 1.60)
Unknown (n=312)
0.78 (0.50 to 1.22)
0.78 (0.49 to 1.22)
1.07 (0.73 to 1.54)
1.03 (0.64 to 1.63)
Treatment response
All other (n=3228)
1
1
1
1
In remission (n=12 026)
1.89 (1.73 to 2.06)***
2.15 (1.92 to 2.40)***
1.65 (1.50 to 1.81)***
1.90 (1.69 to 2.13)***
Long-term condition
Yes (n=7160)
1
1
1
1
Unknown (n=2099)
1.22 (1.10 to 1.37)***
1.42 (1.24 to 1.62)***
1.28 (13.14 to 1.43)***
1.49 (1.30 to 1.71)**
No (n=5995)
1.88 (1.74 to 2.04)***
2.24 (2.03 to 2.47)***
1.73 (1.59 to 1389)***
2.17 (1.97 to 2.40)***
***p<0.001. Model 1 simple multinomial logistic regression with PA none, some (>30 min brisk activity on 1–4 days) and meeting guidelines (>30 min brisk
activity on 1–4 days) as the dependent variable. Model 2 adjusts for treatment, long-term condition, sex, age and IMD. All other treatment factors (been treated but still present/has not been treated/has come back/not certain). p<0.001. Model 1 simple multinomial logistic regression with PA none, some (>30 min brisk activity on 1–4 days) and meeting guidelines (>30 min brisk
activity on 1–4 days) as the dependent variable. Model 2 adjusts for treatment, long-term condition, sex, age and IMD. All other treatment factors (been treated but still present/has not been treated/has come back/not certain). reassuring.7 9–11 It is also important to note that recall of
advice
is
an
important
outcome
in
itself
in
this
context.16 CRC patients to give brief PA advice is warranted and
may help improve outcomes for CRC survivors. Provenance and peer review Not commissioned; externally peer reviewed. Data sharing statement No additional data are available. Data sharing statement No additional data are available. Open Access This is an Open Access article distributed in accordance with
the terms of the Creative Commons Attribution (CC BY 4.0) license, which
permits others to distribute, remix, adapt and build upon this work, for
commercial use, provided the original work is properly cited. See: http://
creativecommons.org/licenses/by/4.0/ REFERENCES 1. Je Y, Jeon JY, Giovannucci EL, et al. Associations between
physical activity and mortality in colorectal cancer: a meta-analysis
of prospective cohort studies. Int J Cancer 2013;133:
1905–13. 2. Campbell PT, Patel AV, Newton CC, et al. Associations of
recreational physical activity and leisure time spent sitting with
colorectal cancer survival. J Clin Oncol 2013;31:876–85. DISCUSSION Acknowledgements The authors are extremely grateful to the patients who
completed the PROMS questionnaires and to the National Cancer Registration
Service in Public Health England for providing the data. The possibility that those recalling PA advice were
already more aware of the importance of PA or had
higher pretreatment levels cannot be ruled out, and lon-
gitudinal studies are warranted. However, this could also
suggest the need for research on how to make important
messages about PA more salient to those who are less
active and less engaged. One RCT in the field suggested
that clinician’s brief exercise advice increased subse-
quent activity levels of breast cancer survivors16 and
though this is very promising, trials in other cancers and
with long-term follow-ups are required. The use of
patient-reported disease and treatment variables should
also be viewed with caution. Additionally, the sample
were predominantly white, older and in remission,
which means that the findings cannot be generalised to
other populations. A further limitation is the lack of
data on cancer staging, because patients with more
advanced disease may be less likely to have been given
advice or to be doing any PA. In clinician surveys,
disease stage has not been raised as a major concern;
rather the lack of knowledge and time are cited as key
barriers.25 Contributors AF, KW, RB and JW contributed to the conception and design of
the work. AF carried out the analyses and drafted the paper. KW, RB and JW
contributed to interpretation of the analyses and revised the work critically for
important intellectual content. All authors approved the final version of
submission. Funding AF, RB and JW were funded by Cancer Research UK (grant number
C1418/A14133). KW was funded by a Cancer Research UK PhD studentship. Competing interests None declared. Provenance and peer review Not commissioned; externally peer reviewed. Provenance and peer review Not commissioned; externally peer reviewed. Open Access diagnosed breast cancer survivors: a single-blind randomized
controlled trial. Ann Behav Med 2004;28:105–13. 5. Williams K, Steptoe A, Wardle J. Is a cancer diagnosis a trigger for
health behaviour change? Findings from a prospective, population-
based study. Br J Cancer 2013;108:2407–12. ;
17. Glaser A, Fraser LK, Corner J, et al. Patient reported outcomes of
cancer survivors in England 1–5 years after diagnosis: a cross-
sectional survey. BMJ Open 2013;3:e002371. y
;
6. O’Leary KA, Estabrooks CA, Olson K, et al. Information acquisition
for woman facing surgical treatment for breast cancer: influencing
factors and selected outcomes. Patient Educ Couns 2007;69:5–19. 18. Hadi M, Gibbons E, Fitzpatrick R. A structured review of Patient
Reported Outcome Measures (PROMS) for colorectal cancer. Report to the Department of Health, 2010. http://phi.uhce.ox.ac.uk/
pdf/CancerReviews/PROMs_Oxford_Colorectal%20Cancer_012011. pdf/ (accessed Jun 2014). factors and selected outcomes. Patient Educ Couns 2007;69:5 19. 7. Jones LW, Courneya KS. Exercise discussions during cancer
treatment consultations. Cancer Pract 2002;10:66–74. 8. Karvinen KH, DuBose KD, Carney B, et al. Promotion of physical
activity amongst oncologists in the United States. J Support Oncol
2010;8:35–41. p
(
)
19. Department of Health. Start active, stay active: a report of physical
activity from the four home countries. Chief Medical Officers, 2011. file://ad.ucl.ac.uk/slms/home1/rmjdafi/Downloads/C–Documents%
20and%20Settings-sharona-Desktop-Start%20Active,%20Stay%
20Active%20-%20UK%20Guidelines%20for%20PA.pdf (accessed
Jun 2014). 9. Daley AJ, Bowden SJ, Rea DW, et al. What advice are oncologists
and surgeons in the United Kingdom giving to breast cancer
patients about physical activity? Int J Behav Nutr Physical Activity
2008;5:46. ;
10. MacMillan Move More, Physical activity the underrated wonder drug. 2011. http://www.macmillan.org.uk/Documents/AboutUs/Newsroom/
Physicalactivityreport.pdf (accessed Jun 2014). )
20. Schmitz KH, Courneya KS, Matthews C, et al. American College of
Sports Medicine round table on exercise guidelines for cancer
survivors. Med Sci Sports Excer 2010;42:1409–26. y
y
p
p
(
)
11. Stull VB, Snyder DC, Demark-Wahnefried W. Lifestyle interventions
in cancer survivors: designing programs that meet the needs of this
vulnerable and growing population. J Nutr 2013:243S–8. p
21. Department for Communities and Local Government: English Indices
of Multiple Deprivation. 2010. https://www.gov.uk/government/
statistics/english-indices-of-deprivation-2010 (accessed Feb 2015). g
g
12. Barnes MA, Schoenborn CA. Trends in adults recieving a
recommendation for exercise or other physical activity from a
physician or other health professional: NCHS data brief no 86. Hyattsville, MD: National Centre for Health Statistics, 2012. g
p
(
)
22. Downing A, Morris EJ, Richards M, et al. CONCLUSION Recalling being given PA advice after a diagnosis of CRC
was associated with higher levels of PA. However, less
than a third of patients recalled receiving advice. Future
research should examine the context in which advice is
given. However, encouraging clinicians working with 3. Peddle CJ, Au HS, Courneya KS. Associations between exercise,
quality of life, and fatigue in colorectal cancer survivors. Dis Colon
Rectum 2008;51:1242–8. 4. Hawkes A, Lynch BM, Owen N, et al. Health behaviours of
Australian colorectal cancer survivors compared to non-cancer
population controls. J Support Care Cancer 2008;16:1097–104. 4. Hawkes A, Lynch BM, Owen N, et al. Health behaviours of
Australian colorectal cancer survivors compared to non-cancer
population controls. J Support Care Cancer 2008;16:1097–104. 7 Fisher A, et al. BMJ Open 2015;5:e006853. doi:10.1136/bmjopen-2014-006853 group.bmj.com
on July 23, 2015 - Published by
http://bmjopen.bmj.com/
Downloaded from activity in colorectal cancer patients
associated with higher levels of physical
Recall of physical activity advice was
A Fi h
K Willi
R B
k
d J W
dl doi: 10.1136/bmjopen-2014-006853
2015 5:
BMJ Open doi: 10.1136/bmjopen-2014-006853
2015 5:
BMJ Open http://bmjopen.bmj.com/content/5/4/e006853
Updated information and services can be found at: Open Access Health-related quality after
colorectal cancer in England: a patient-reported outcomes study of
individuals 12 to 36 months after diagnosis. J Clin Oncol
2015;33:616–24. y
13. Calfas KJ, Long BJ, Sallis JF, et al. A controlled trial of physician
counseling to promote the adoption of physical activity. Prev Med
1996;25:225–33. 23. Miles A, Simon A, Wardle J. Answering patient questions about the
role lifestyle factors play in cancer onsent and reccurence: what do
health care professionals say? J Health Psychol 2010;15:291–8. 14. Hillsdon M, Foster C, Cavill N, et al. The effectiveness of public
health interventions of increasing physical activity among adults:
a review of reviews. NHS Health Development Agency, 2005. http://www.nice.org.uk/nicemedia/documents/physical_activity_
adults_ebsummary.pdf (accessed Jun 2014). p
y
y
24. Anderson AS, Caswell S, Wells M, et al. Obesity and lifestyle advice
in colorectal cancer survivors–how well are clinicians prepared? Colorectal Dis 2013;15:949–57. 25. Karvinen KH, McGourty S, Parent T, et al. Physical activity
promotion among oncology nurses. Cancer Nurs 2012;35:E41–8. 15. Segar ML, Katch VL, Roth RS, et al. The effect of aerobic exercise
on self-esteem and depressive and anxiety symptoms among breast
cancer survivors. Oncol Nurs Forum 1998;25:107–13. 26. Courneya KS, Booth CM, Gill S, et al. The Colon Health and
Life-Long Exercise Change (CHALLENGE) trial: a randomized trial
of the National Cancer Institute of Canada Clinical Trials Group. Curr
Oncol 2008;15:279–85. 16. Jones LW, Courneya KS, Fairey AS, et al. Effects of an oncologist’s
recommendation to exercise on self-reported behaviour in newly 8 Fisher A, et al. BMJ Open 2015;5:e006853. doi:10.1136/bmjopen-2014-006853 group.bmj.com
on July 23, 2015 - Published by
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https://openalex.org/W4381167583
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https://peerj.com/articles/15547v0.3/submission
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English
| null |
Peer Review #3 of "Expression, localization, and function of P4HB in the spermatogenesis of Chinese mitten crab (Eriocheir sinensis) (v0.1)"
| null | 2,023
|
cc-by
| 12,305
|
Expression, localization, and function of P4HB in the
spermatogenesis of Chinese mitten crab (Eriocheir sinensis)
Yulian Tang
Equal first author,
1 , Anni Ni
Equal first author,
1 , Shu Li
1 , Lishuang Sun
1 , Genliang Li
Corresp.
1 an Tang
Equal first author,
1 , Anni Ni
Equal first author,
1 , Shu Li
1 , Lishuang Sun
1 , Genliang Li
Corresp. 1 Shu Li
1 , Lishuang Sun
1 , Genliang Li
Corresp. 1 1 Youjiang Medical University for Nationalities, Baise, Guangxi, China
Corresponding Author: Genliang Li
Email address: ligenliang@163.com 1 Youjiang Medical University for Nationalities, Baise, Guangxi, China
Corresponding Author: Genliang Li 1 Youjiang Medical University for Nationalities, Baise, Guangxi, China Corresponding Author: Genliang Li
Email address: ligenliang@163.com Background The sperm of Chinese mitten crab (Eriocheir sinensis) have special
noncondensed nuclei. The formation and stability of the special nuclei are closely related
to the correct folding of proteins during spermatogenesis. P4HB plays a key role in protein
folding, but its expression and role in the spermatogenesis of E. sinensis are unclear. Objective To investigate the expression and distribution characteristics of P4HB in the
spermatogenesis of E. sinensis as well as its possible role. Methods The testis tissues of
adult and juvenile E. sinensis were used as materials. We utilized a variety of techniques,
including homology modeling, phylogenetic analysis, RT-qPCR, western blotting, and
immunofluorescence staining to predict the protein structure and sequence homology of
P4HB, analyze its expression in the testis tissues, and localize and semi-quantitatively
assess its expression in different male germ cells. Results The sequence of P4HB protein
in E. sinensis shared a high similarity of 58.09% with the human protein disulfide
isomerase, and the phylogenetic tree analysis indicated that the protein sequence was
highly conserved among crustaceans, arthropods, and other animals species. P4HB was
found to be expressed in both juvenile and adult E. sinensis testis tissues, with different
localization patterns observed all over the developmental stages of male germ cells. It was
higher expressed in the spermatogonia, spermatocytes, and stage I spermatids, followed
by the mature sperm than in the stage II and III spermatids. The subcellular localization
analysis revealed that P4HB was predominantly expressed in the cytoplasm, cell
membrane, and extracellular matrix in the spermatogonia, spermatocytes, stage I and
stage II spermatids, with some present in specific regions of the nuclei in the
spermatogonia. In contrast, P4HB was mainly localized in the nuclei of stage III spermatids
and sperm, with little expression observed in the cytoplasm. Conclusion P4HB was
expressed in the testis tissues of both adult and juvenile E. Manuscript to be reviewed Expression, localization, and function of P4HB in the
spermatogenesis of Chinese mitten crab (Eriocheir sinensis)
Yulian Tang
Equal first author,
1 , Anni Ni
Equal first author,
1 , Shu Li
1 , Lishuang Sun
1 , Genliang Li
Corresp.
1 sinensis, but the expression
and localization were different in male germ cells at various developmental stages. The
observed differences in the expression and localization of P4HB may be an essential factor
in maintaining the cell morphology and structure of diverse male germ cells in E. sinensis. PeerJ reviewing PDF | (2021:08:64840:2:0:NEW 17 May 2023) 6
△These authors contributed equally to this work. 6
△These authors contributed equally to this work. 7
[Abstract] Background The sperm of Chinese mitten crab (Eriocheir sinensis) have special noncondensed
8
nuclei. The formation and stability of the special nuclei are closely related to the correct folding of proteins
9
during spermatogenesis. P4HB plays a key role in protein folding, but its expression and role in the
10
spermatogenesis of E. sinensis are unclear. Objective To investigate the expression and distribution
11
characteristics of P4HB in the spermatogenesis of E. sinensis as well as its possible role. Methods The testis
12
tissues of adult and juvenile E. sinensis were used as materials. We utilized a variety of techniques, including
13
homology modeling, phylogenetic analysis, RT-qPCR, western blotting, and immunofluorescence staining to
14
predict the protein structure and sequence homology of P4HB, analyze its expression in the testis tissues, and
15
localize and semi-quantitatively assess its expression in different male germ cells. Results The sequence of
16
P4HB protein in E. sinensis shared a high similarity of 58.09% with the human protein disulfide isomerase,
17
and the phylogenetic tree analysis indicated that the protein sequence was highly conserved among crustaceans,
18
arthropods, and other animals species. P4HB was found to be expressed in both juvenile and adult E. sinensis
19
testis tissues, with different localization patterns observed all over the developmental stages of male germ cells. 20
It was higher expressed in the spermatogonia, spermatocytes, and stage I spermatids, followed by the mature
21
sperm than in the stage II and III spermatids. The subcellular localization analysis revealed that P4HB was
22
predominantly expressed in the cytoplasm, cell membrane, and extracellular matrix in the spermatogonia,
23
spermatocytes, stage I and stage II spermatids, with some present in specific regions of the nuclei in the
24
spermatogonia. In contrast, P4HB was mainly localized in the nuclei of stage III spermatids and sperm, with
25
little expression observed in the cytoplasm. Conclusion P4HB was expressed in the testis tissues of both adult
26
and juvenile E. sinensis, but the expression and localization were different in male germ cells at various
27
developmental stages. The observed differences in the expression and localization of P4HB may be an
28
essential factor in maintaining the cell morphology and structure of diverse male germ cells in E. sinensis. 6
△These authors contributed equally to this work. 29
Additionally, P4HB expressed in the nuclei of spermatogonia, late spermatids, and sperm may play an
30
indispensable role in maintaining the stability of the noncondensed spermatozoal nuclei in E. sinensis. 31 Manuscript to be reviewed Manuscript to be reviewed Additionally, P4HB expressed in the nuclei of spermatogonia, late spermatids, and sperm
may play an indispensable role in maintaining the stability of the noncondensed
spermatozoal nuclei in E. sinensis. PeerJ reviewing PDF | (2021:08:64840:2:0:NEW 17 May 2023) Manuscript to be reviewed 1
Expression, localization, and function of P4HB in the spermatogenesis of Chinese mitten crab
2
(Eriocheir sinensis) 1
Expression, localization, and function of P4HB in the spermatogenesis of Chinese mitten crab
2
(Eriocheir sinensis) 3
Yulian Tang1△, Anni Ni1△, Shu Li1, Lishuang Sun1 and Genliang Li1* 4
1.Youjiang Medical University for Nationalities, Baise, 533000, China 5
*Corresponding Author: Genliang Li. Email: ligenliang@ymun.edu.cn Manuscript to be reviewed 41
(Chen et al., 2020; Wu et al., 2016). The former appears in the sperm of many decapod crustaceans, such as E. 42
sinensis and is named noncondensed nucleus with genetic material in the form of fibrous chromatin filament
43
(Wei et al., 2019; Wu et al., 2015) while the latter presents in the sperm of most animals and is named
44
condensed nucleus. At present, the formation and stability mechanisms of these unique spermatozoal nuclei in
45
E. sinensis remain unclear. 41
(Chen et al., 2020; Wu et al., 2016). The former appears in the sperm of many decapod crustaceans, such as E. 42
sinensis and is named noncondensed nucleus with genetic material in the form of fibrous chromatin filament
43
(Wei et al., 2019; Wu et al., 2015) while the latter presents in the sperm of most animals and is named
44
condensed nucleus. At present, the formation and stability mechanisms of these unique spermatozoal nuclei in
45
E. sinensis remain unclear. 46
Cytoskeleton-related proteins are closely associated with the formation and stability of the noncondensed
47
spermatozoal nuclei in E. sinensis (Li et al., 2022; Wang et al., 2023). E. sinensis is able to maintain the
48
integrity of its unique spermatozoal nuclei and passes on genetic material to next generations, indicating the
49
pivotal role of cytoskeleton-related proteins which can stabilize the cellular or subcellular morphology and
50
structure. Prolyl 4-hydroxylase beta polypeptide (P4HB) is a cytoskeleton-related protein (Sobierajska et al.,
51
2014). This abundant multifunctional enzyme is known to be in the endoplasmic reticulum, acting as a subunit
52
of the microsomal triglyceride transporter protein. P4HB catalyzes the formation of 4-hydroxyproline in
53
collagen and other collagen-like proteins, and plays a key role in facilitating the folding of many newly
54
synthesized proteins as a chaperone-like multifunctional peptide (Kari & Johanna, 1998; Wu et al., 2021). 55
Protein disulfide isomerase (PDI) is a multifunctional protein located in the lumen of the rough endoplasmic
56
reticulum. It catalyzes the redox and isomerization of disulfide bonds, promoting the correct folding of proteins
57
(Li et al., 2018). P4HB gene in humans primarily encodes PDI, which is a chaperone protein responsible for
58
the folding of spermatocyte-specific protein present in the testis (Li et al., 2018; Wu et al., 2021). 32
Introduction 33
Spermatogenesis is a complex physiological process that includes spermatogonia proliferation and
34
differentiation into spermatocytes, spermatocyte meiosis resulting in the production of spermatids, and the
35
metamorphosis of spermatids into sperm (Nishimura & L'Hernault, 2017). Chinese mitten crab (Eriocheir
36
sinensis, E. sinensis) spermatogenesis differs significantly from that of other mammals. In E. sinensis, the
37
spherical-shaped spermatid nucleus is gradually depressed and squeezed into the one side of the cell and forms
38
a thin, cup-shaped spermatozoal nucleus that surrounds half of the acrosome as the proacrosomal vesicle
39
gradually enlarges during the later stage of spermatogenesis (Xu et al., 2018; Li et al., 2017). The cup-shaped
40
spermatozoal nucleus is susceptible to instability, compared to the stable, round or oval-shaped nuclei in cells PeerJ reviewing PDF | (2021:08:64840:2:0:NEW 17 May 2023) Manuscript to be reviewed Several
59
studies have reported PDI's presence in the testes of other mammals (e.g., rat, mouse, rabbit, sheep),
60
implicating its involvement in crucial processes such as spermatogenesis and sperm-egg fusion (Akama et al.,
61
2010; Lv et al., 2011; Zhao et al., 2013). 62
However, the role of P4HB in regulating spermatogenesis in decapod crustaceans has not been reported yet. 63
Therefore, we used testis tissues from adult and juvenile E. sinensis crabs to analyze the expression and
64
distribution characteristics of P4HB through RT-qPCR, western blotting, and immunofluorescence staining
65
techniques. Additionally, we also explored the possible functional roles of P4HB in the E. sinensis
66
spermatogenesis. Our study could provide a theoretical foundation for comprehending the molecular
67
mechanisms of spermatogenesis and reproductive regulation in E. sinensis. These results can also serve as a
68
valuable reference for species conservation and aquaculture management of E. sinensis. 69
2 M t
i l
d M th d Manuscript to be reviewed Hoffmann-La Roche Co., Ltd. (Basel,
84
Switzerland); The ultra-sensitive multifunctional imager was purchased from Cytiva Co., Ltd. (Marlborough,
85
America), and the laser confocal microscope was purchased from Olympus Co., Ltd. (Tokyo, Japan). 86
P4HB and β-Actin primers were purchased from Shenggong Bioengineering Co., Ltd. (Shanghai, China). Anti-
87
P4HB antibody [EPR949] (ab137110), Anti-beta Actin antibody [mAbcam 8226] (ab8226), Anti-DDX4/MVH
88
antibody [mAbcam27591] (ab27591), Goat Anti-Mouse IgG H&L (HRP) (ab6789), Goat Anti-Mouse IgG
89
H&L (Alexa Fluor® 594) (ab150116), Goat Anti-Rabbit IgG H&L (HRP) (ab6721), and Goat Anti-Rabbit IgG
90
H&L (Alexa Fluor® 488) (ab150077) were all purchased from the same company, Abcam Trading Co., Ltd. 91
(Shanghai, China). 82
Blaze Taq SYBR® Green qPCR mix was purchased from iGine Biotechnology Co., Ltd. (Guangzhou, China). 83
The Roche LightCycler 96 PCR instrument was purchased from F. Hoffmann-La Roche Co., Ltd. (Basel,
84
Switzerland); The ultra-sensitive multifunctional imager was purchased from Cytiva Co., Ltd. (Marlborough,
85
America), and the laser confocal microscope was purchased from Olympus Co., Ltd. (Tokyo, Japan). 86
P4HB and β-Actin primers were purchased from Shenggong Bioengineering Co., Ltd. (Shanghai, China). Anti-
87
P4HB antibody [EPR949] (ab137110), Anti-beta Actin antibody [mAbcam 8226] (ab8226), Anti-DDX4/MVH
88
antibody [mAbcam27591] (ab27591), Goat Anti-Mouse IgG H&L (HRP) (ab6789), Goat Anti-Mouse IgG
89
H&L (Alexa Fluor® 594) (ab150116), Goat Anti-Rabbit IgG H&L (HRP) (ab6721), and Goat Anti-Rabbit IgG
90
H&L (Alexa Fluor® 488) (ab150077) were all purchased from the same company, Abcam Trading Co., Ltd. 91
(Shanghai, China). 70
2.1 Materials 72
Chinese mitten crabs were purchased from Ice King Aquatic Products Sales Co., Ltd. (Suqian, China). Thirty
73
adult crabs weighing between 100-150 g and thirty juvenile crabs weighing between 20–30 g, with sound
74
limbs and uniform size and vitality were selected, respectively. After acquisition, they were temporarily
75
housed for 1 day under fully aerated tap water (water temperature 24 °C, pH 6.8, and dissolved oxygen levels
76
above 5.0 mg/L) and subjected to testis tissue isolation and extraction. All animal procedures were performed
77
at the Experimental Animal Center of Youjiang Medical University for Nationalities, and the study was
78
conducted in accordance with national animal welfare regulations. The Total RNA Extraction Kit was purchased from Solabo Technology Co., Ltd. (Beijing, 80
The Total RNA Extraction Kit was purchased from Solabo Technology Co., Ltd. (Beijing, China). The First-
81
Strand cDNA Synthesis Kit was purchased from Thermo Fisher Technology Co., Ltd. (Shanghai, China). 80
The Total RNA Extraction Kit was purchased from Solabo Technology Co., Ltd. (Beijing, China). The First-
81
Strand cDNA Synthesis Kit was purchased from Thermo Fisher Technology Co., Ltd. (Shanghai, China). PeerJ reviewing PDF | (2021:08:64840:2:0:NEW 17 May 2023) 93
2.2.1 RNA Extraction and Sequencing 94
Approximately 25 mg of testis tissue was collected was taken from each adult and juvenile crab, and every 4–5
95
crab samples from the same group were pooled to obtain a sample of approximately 100 mg. Each group,
96
including adult and juvenile crabs, had three biologically replicates (n = 3). The samples were immediately
97
flash-frozen in liquid nitrogen and ground to a fine powder. Total RNA extraction was performed on each
98
sample using the Total RNA Extraction Kit, and the resulting RNA solution was considered qualified when the
99
260/280 ratio was between 1.8–2.0 and the 260/230 ratio was greater than 2.0. The purified RNA samples
100
were sent to Shenzhen BGI Gene Co., Ltd. for RNA-seq. Poly(A) mRNA was isolated using the NEBNext
101
Poly(A) mRNA Magnetic Isolation Module (NEB), the mRNA fragmentation and priming were performed
102
using the NEBNext First Strand Synthesis Reaction Buffer and NEBNext Random Primers. The libraries were
103
then multiplexed with different indices and loaded onto an Illumina HiSeq instrument (Illumina, San Diego,
104
CA, USA). Sequencing was carried out using a 2 x 150 bp paired-end (PE) configuration, followed by image
105
analysis and base calling by the HiSeq instrument. Lastly, the P4HB mRNA sequence was obtained by
106
comparing the genome sequence of E. sinensis. 107
2.2.2 P4HB Protein Tertiary Structure Prediction and Phylogenetic Tree Construction
108
The P4HB mRNA sequence was analyzed using the Expasy Online website (https://web.expasy.org/translate/)
109
to obtain the corresponding amino acid sequence. Subsequently, the tertiary structure model of this protein was
110
constructed
using
the
SWISS-MODEL
homology
modeling
website
111
(https://swissmodel.expasy.org/interactive). Additionally, sequence comparison was performed using the
112
NCBI-BLAST online tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi), and the MAGA 7.0 software was used for
113
sequence analysis. Finally, the evolutionary history of P4HB was investigated using neighbor-joining (NJ)
114
analysis, and a phylogenetic tree was generated. Manuscript to be reviewed 82
Blaze Taq SYBR® Green qPCR mix was purchased from iGine Biotechno
83
The Roche LightCycler 96 PCR instrument was purchased from F. Ho
84
Switzerland); The ultra-sensitive multifunctional imager was purchased fr
85
America), and the laser confocal microscope was purchased from Olympus
86
P4HB and β-Actin primers were purchased from Shenggong Bioengineerin
87
P4HB antibody [EPR949] (ab137110), Anti-beta Actin antibody [mAbcam
88
antibody [mAbcam27591] (ab27591), Goat Anti-Mouse IgG H&L (HRP
89
H&L (Alexa Fluor® 594) (ab150116), Goat Anti-Rabbit IgG H&L (HRP)
90
H&L (Alexa Fluor® 488) (ab150077) were all purchased from the same
91
(Shanghai, China). 92
2.2 Methods
93
2.2.1 RNA Extraction and Sequencing
94
Approximately 25 mg of testis tissue was collected was taken from each ad
95
crab samples from the same group were pooled to obtain a sample of a
96
including adult and juvenile crabs, had three biologically replicates (n =
97
flash-frozen in liquid nitrogen and ground to a fine powder. Total RNA
98
sample using the Total RNA Extraction Kit, and the resulting RNA solutio
99
260/280 ratio was between 1.8–2.0 and the 260/230 ratio was greater th
100
were sent to Shenzhen BGI Gene Co., Ltd. for RNA-seq. Poly(A) mRN
101
Poly(A) mRNA Magnetic Isolation Module (NEB), the mRNA fragmen
102
using the NEBNext First Strand Synthesis Reaction Buffer and NEBNext
103
then multiplexed with different indices and loaded onto an Illumina HiSe
104
CA, USA). Sequencing was carried out using a 2 x 150 bp paired-end (PE
105
analysis and base calling by the HiSeq instrument. Lastly, the P4HB
106
comparing the genome sequence of E. sinensis. 107
2.2.2 P4HB Protein Tertiary Structure Prediction and Phylogenetic Tr
108
The P4HB mRNA sequence was analyzed using the Expasy Online websi
109
to obtain the corresponding amino acid sequence. Subsequently, the tertiar
110
constructed
using
the
SWISS-MODEL
homol
111
(https://swissmodel.expasy.org/interactive). Additionally, sequence com
112
NCBI-BLAST online tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi), and th
113
sequence analysis. Finally, the evolutionary history of P4HB was inves
114
analysis, and a phylogenetic tree was generated. 115
2.2.3 P4HB mRNA Expression Analysis Using RT-qPCR
116
Total RNA from testis tissues of both adult and juvenile crab samples,
117
crabs in the same group, was reverse transcribed into cDNA. Real-time qu 82
Blaze Taq SYBR® Green qPCR mix was purchased from iGine Biotechnology Co., Ltd. (Guangzhou, China). 83
The Roche LightCycler 96 PCR instrument was purchased from F. 5 P4HB Protein Expression Analysis Using Immunofluorescence Localization 134
2.2.5 P4HB Protein Expression Analysis Using Immunofluorescence Localization 135
Fresh testis tissues from both adult and juvenile crabs were immediately fixed using formaldehyde and
136
subsequently made into paraffin sections. The sections were then dewaxed, rehydrated, and subjected to
137
antigen retrieval. Primary antibodies were added to the slices at a 1:100 dilution and incubated overnight at
138
4°C. Subsequently, fluorescent secondary antibodies were added at a 1:5000 dilution and incubated in the dark
139
for 1 hour. The slices were then stained with a fluorescent cell nuclear dye and observed and photographed
140
using a confocal microscope. The average fluorescence intensity in different cells was analyzed using ImageJ
141
software. The formula used for calculating the average fluorescence intensity was: average fluorescence
142
intensity = integrated density in the region / area of the region. 144
A statistical analysis of the data was performed using SPSS 13.0 statistical software. The results were
145
expressed as mean ± standard deviation. After testing for normality and the chi-square test, a two-samples
146
independent t-test was used to compare the two types of samples that conformed to a normal distribution. For
147
multiple samples that conformed to a normal distribution, a completely randomized ANOVA was used for
148
comparison. Differences were considered statistically significant at P < 0.05. 149
3 Results 150
3.1 P4HB Protein Structure Model and Sequence Analysis
151
The mRNA and protein sequences of E. sinensis P4HB were obtained through RNA sequencing and
152
annotation (Fig. 1). The P4HB mRNA sequence was 1584 bp in length, and the protein had a molecular weight
153
of 58.61 kDa. The protein consisted of 527 amino acids with an isoelectric point of 4.61. To predict the tertiary
154
structure of the P4HB protein, we used the homology modeling method with the human protein disulfide
155
isomerase (PDI) sequence as the template. The predicted tertiary structure model of P4HB is shown in Fig. 2. 156
The similarity between this model and the tertiary structure model of the human PDI protein was 58.09%,
157
which exceeded the threshold of 30% and even 50%. These results indicate that the predicted model is highly
158
reliable and meets the basic criteria of homology modeling. 159
The position of Fig. 1
160
The position of Fig. 2
161
Phylogenetic tree analysis was performed to determine the evolutionary relationships of P4HB in E. 115
2.2.3 P4HB mRNA Expression Analysis Using RT-qPCR 116
Total RNA from testis tissues of both adult and juvenile crab samples, which were pooled from every 4–5
117
crabs in the same group, was reverse transcribed into cDNA. Real-time quantitative PCR was then performed
118
using a fluorescent probe. The reaction conditions were set as follows: pre-denaturation at 95℃ for 30 seconds,
119
followed by 40 cycles of denaturation at 95℃ for 5 seconds, and annealing/extension at 60℃ for 30 seconds. 120
The dissolution curve reaction was conducted as per the recommendations of the Roche LightCycler96
121
instrument. The relative expression of P4HB mRNA between adult and juvenile crab samples was calculated
122
using the 2-ΔΔCt method. 116
Total RNA from testis tissues of both adult and juvenile crab samples, which were pooled from every 4–5
117
crabs in the same group, was reverse transcribed into cDNA. Real-time quantitative PCR was then performed
118
using a fluorescent probe. The reaction conditions were set as follows: pre-denaturation at 95℃ for 30 seconds,
119
followed by 40 cycles of denaturation at 95℃ for 5 seconds, and annealing/extension at 60℃ for 30 seconds. 120
The dissolution curve reaction was conducted as per the recommendations of the Roche LightCycler96
121
instrument. The relative expression of P4HB mRNA between adult and juvenile crab samples was calculated
122
using the 2-ΔΔCt method. PeerJ reviewing PDF | (2021:08:64840:2:0:NEW 17 May 2023) Manuscript to be reviewed 2.2.4 P4HB Protein Expression Analysis Using Western Blotting 123
2.2.4 P4HB Protein Expression Analysis Using Western Blotting
124
Testis tissues were obtained from both adult and juvenile crab samples (200 mg each) and rapidly ground into
125
a fine powder using liquid nitrogen. The tissue lysate was then added to each sample (1 ml of lysate per 50-100
126
mg of powder), mixed thoroughly, and placed on ice for 5 minutes before being ground using a tissue grinder. 127
The total protein solution was then obtained by centrifugation at 12000 r/min for 5 minutes at 4℃. The
128
concentration of total protein was determined using the BCA method, and 5× loading buffer was added to
129
adjust the protein solution to the same concentration. The mixture was then heated and boiled for 5 minutes to
130
obtain a denatured protein. SDS-PAGE and Western blotting were conducted according to the method
131
proposed by Pang et al (Pang et al., 2021). The primary antibody was used at a 1:100 dilution, while the
132
second antibody was used at a 1:5000 dilution. The membranes were then imaged using an ultra-sensitive
133
multifunctional imager and quantitatively analyzed using Image J software. Manuscript to be reviewed 175
The position of Table 1
176
The position of Fig. 4
177
3.3 Expression of P4HB Protein in Different Male Germ Cells
178
Immunofluorescence localization was conducted to investigate the expression pattern and subcellular
179
localization of P4HB in male germ cells at different developmental stages in E. sinensis testis tissues. The
180
results showed that P4HB was expressed in both adult and juvenile testis tissues, but the expression level in
181
male germ cells at different developmental stages in E
i
i
testis tissues was different (Fig 5) The 171
The expression of P4HB mRNA and protein in the testis tissues of both adult and juvenile E. sinensis was
172
detected using RT-qPCR and Western blotting, respectively. β-Actin was used as the internal reference gene
173
and reference protein. The primer sequences are shown in Table 1. The results demonstrated that P4HB was
174
expressed in both adult and juvenile E. sinensis testis tissues, as showed in Fig. 4. 175
The position of Table 1 Manuscript to be reviewed 164
costatus, Ctenocephalides felis, Anopheles stephensi, Ctenocephalides felis, Anoplophora glabripennis, Thrips
165
palmi, and other arthropods. Meanwhile, Vargula hilgendorfii, Limulus polyphemus, Tetranychus truncatus,
166
and Araneus ventricosus were clustered into another group (Fig. 3). It can be observed that P4HB has some
167
degree of species conservation, and the evolutionary relationships of P4HB among different species are
168
relatively conserved. 164
costatus, Ctenocephalides felis, Anopheles stephensi, Ctenocephalides felis, Anoplophora glabripennis, Thrips
165
palmi, and other arthropods. Meanwhile, Vargula hilgendorfii, Limulus polyphemus, Tetranychus truncatus,
166
and Araneus ventricosus were clustered into another group (Fig. 3). It can be observed that P4HB has some
167
degree of species conservation, and the evolutionary relationships of P4HB among different species are
168
relatively conserved. 164
costatus, Ctenocephalides felis, Anopheles stephensi, Ctenocephalides felis, Anoplophora glabripennis, Thri
165
palmi, and other arthropods. Meanwhile, Vargula hilgendorfii, Limulus polyphemus, Tetranychus truncatu
166
and Araneus ventricosus were clustered into another group (Fig. 3). It can be observed that P4HB has som
167
degree of species conservation, and the evolutionary relationships of P4HB among different species a
168
relatively conserved. 169
The position of Fig. 3
170
3.2 Expression of P4HB mRNA and Protein in Testis Tissues
171
The expression of P4HB mRNA and protein in the testis tissues of both adult and juvenile E. sinensis w
172
detected using RT-qPCR and Western blotting, respectively. β-Actin was used as the internal reference gen
173
and reference protein. The primer sequences are shown in Table 1. The results demonstrated that P4HB w
174
expressed in both adult and juvenile E. sinensis testis tissues, as showed in Fig. 4. 175
The position of Table 1
176
The position of Fig. 4
177
3.3 Expression of P4HB Protein in Different Male Germ Cells
178
Immunofluorescence localization was conducted to investigate the expression pattern and subcellul
179
localization of P4HB in male germ cells at different developmental stages in E. sinensis testis tissues. Th
180
results showed that P4HB was expressed in both adult and juvenile testis tissues, but the expression level
181
male germ cells at different developmental stages in E. sinensis testis tissues was different (Fig. 5). Th
182
fluorescence intensity of each cell type, from strong to weak, was spermatogonia, spermatocytes, stage
183
spermatids, mature sperm, stage III spermatids, and stage II spermatids. 5 P4HB Protein Expression Analysis Using Immunofluorescence Localization sinensis
162
with other arthropods. The results showed that E. sinensis P4HB protein was clustered with Portunus
163
trituberculatus, Penaeus vannamei, and Armadillidium vulgare, forming one large branch with Cyphomyrmex 161
Phylogenetic tree analysis was performed to determine the evolutionary relationships of P4HB in E. sinensis
162
with other arthropods. The results showed that E. sinensis P4HB protein was clustered with Portunus
163
trituberculatus, Penaeus vannamei, and Armadillidium vulgare, forming one large branch with Cyphomyrmex PeerJ reviewing PDF | (2021:08:64840:2:0:NEW 17 May 2023) 177
3.3 Expression of P4HB Protein in Different Male Germ Cells 177
3.3 Expression of P4HB Protein in Different Male Germ Cells
178
Immunofluorescence localization was conducted to investigate the expression pattern and subcellular
179
localization of P4HB in male germ cells at different developmental stages in E. sinensis testis tissues. The
180
results showed that P4HB was expressed in both adult and juvenile testis tissues, but the expression level in
181
male germ cells at different developmental stages in E. sinensis testis tissues was different (Fig. 5). The
182
fluorescence intensity of each cell type, from strong to weak, was spermatogonia, spermatocytes, stage I
183
spermatids, mature sperm, stage III spermatids, and stage II spermatids. There were no significant differences
184
in the fluorescence intensity between the stage I spermatids with mature sperm, stage II spermatids with
185
mature sperm, stage III spermatids with mature sperm, and stage II spermatids with stage III spermatids (n = 3,
186
P > 0.05). However, there were significant differences between other cell types (n = 3, P < 0.05). This result
187
showed that P4HB was mainly expressed in spermatogonia, spermatocytes, and stage I spermatids, followed
188
by mature sperm, and was less expressed in stage II and stage III spermatids. Furthermore, P4HB was mainly
189
expressed in the cytoplasm, cell membrane, intercellular substance, and extracellular matrix of spermatogonia,
190
spermatocytes, stage I spermatids, and stage II spermatids. Additionally, P4HB was partially expressed in
191
specific regions of the nuclei of spermatogonia, and it was mainly located in the nuclei and a small amount in
192
the cytoplasm of stage III spermatids and mature sperm. Manuscript to be reviewed There were no significant differenc
184
in the fluorescence intensity between the stage I spermatids with mature sperm, stage II spermatids wi
185
mature sperm, stage III spermatids with mature sperm, and stage II spermatids with stage III spermatids (n =
186
P > 0.05). However, there were significant differences between other cell types (n = 3, P < 0.05). This resu
187
showed that P4HB was mainly expressed in spermatogonia, spermatocytes, and stage I spermatids, followe
188
by mature sperm, and was less expressed in stage II and stage III spermatids. Furthermore, P4HB was main
189
expressed in the cytoplasm, cell membrane, intercellular substance, and extracellular matrix of spermatogoni
190
spermatocytes, stage I spermatids, and stage II spermatids. Additionally, P4HB was partially expressed
191
specific regions of the nuclei of spermatogonia, and it was mainly located in the nuclei and a small amount
192
the cytoplasm of stage III spermatids and mature sperm. 193
The position of Fig. 5
194
4 Discussion
195
In most animals, the spermatozoal nuclei are highly compacted and reduced in size to protect the genet
196
material from external factors before fertilization. However, unlike mammals, the decapod crustacean
197
sinensis possesses noncondensed chromatin that does not damage DNA (Wu et al., 2016; Sela et al., 201
198
Hime et al., 1996). The noncondensed state of the spermatozoal nuclei in E. sinensis makes the genet
199
material loose. Therefore, we conducted a study on the expression and localization of P4HB in testis tissues 164
costatus, Ctenocephalides felis, Anopheles stephensi, Ctenocephalides felis, Anoplophora glabripennis, Thrips
165
palmi, and other arthropods. Meanwhile, Vargula hilgendorfii, Limulus polyphemus, Tetranychus truncatus
166
and Araneus ventricosus were clustered into another group (Fig. 3). It can be observed that P4HB has some
167
degree of species conservation, and the evolutionary relationships of P4HB among different species are
168
relatively conserved. 169
The position of Fig. 3
170
3.2 Expression of P4HB mRNA and Protein in Testis Tissues
171
The expression of P4HB mRNA and protein in the testis tissues of both adult and juvenile E. sinensis was
172
detected using RT-qPCR and Western blotting, respectively. β-Actin was used as the internal reference gene
173
and reference protein. The primer sequences are shown in Table 1. The results demonstrated that P4HB was
174
expressed in both adult and juvenile E. sinensis testis tissues, as showed in Fig. 4. Manuscript to be reviewed Spermatogonia
215
and spermatocytes are in the early stages of spermatogenesis and actively engage in cell proliferation and
216
differentiation. Consequently, coordinated cytoskeletal reorganization is crucial in these stages (Carlton et al.,
217
2020; Frappaolo et al., 2022). Our study discovered elevated P4HB levels in the spermatogonia and
218
spermatocytes, indicating its importance in maintaining cytoskeletal reorganization. 219
P4HB has been widely demonstrated to play a crucial role in the endoplasmic reticulum, mitochondria, and
220
cytoplasm in various studies (Zhu et al., 2022). However, our study showed that P4HB is expressed in specific
221
regions of spermatogonia nuclei, in addition to the cytoplasm, cell membrane, and extracellular matrix. 222
Furthermore, in E. sinensis stage III spermatids and mature sperm, it is mainly expressed in the nuclei and to a
223
lesser extent in the cytoplasm. These findings suggest that P4HB's spatial distribution in the male germ cells in
224
E. sinensis differs from the common site of P4HB expression in the somatic cells in other species (Zhu et al.,
225
2022). So, we speculate that this unusual expression pattern may be related to the mechanism of noncondensed
226
nuclei formation in E. sinensis. Unlike mammals, the spermatozoal nuclei in E. sinensis undergoes a complex,
227
decondensed process during spermatogenesis (Chen et al., 2020), culminating in the unique morphology of its
228
sperm, with flagella-free, microtubule sheaths, and cup-shaped nuclei with radial arms on the surface of sperm
229
(Sela et al., 2012; Wang et al., 2015). This intricate process is completed in an orderly manner, which requires
230
appropriate protein folding (Wang et al., 2015) and the chromatin remodeling (Wu et al., 2016) to maintain the
231
structural integrity of the sperm. Therefore, the unique expression pattern of P4HB in E. sinensis suggests its
232
potential involvement in ensuring proper protein folding and subsequent sperm development. 219
P4HB has been widely demonstrated to play a crucial role in the endoplasmic reticulum, mitochondria, and
220
cytoplasm in various studies (Zhu et al., 2022). However, our study showed that P4HB is expressed in specific
221
regions of spermatogonia nuclei, in addition to the cytoplasm, cell membrane, and extracellular matrix. 222
Furthermore, in E. sinensis stage III spermatids and mature sperm, it is mainly expressed in the nuclei and to a
223
lesser extent in the cytoplasm. These findings suggest that P4HB's spatial distribution in the male germ cells in
224
E. 194
4 Discussion 195
In most animals, the spermatozoal nuclei are highly compacted and reduced in size to protect the genetic
196
material from external factors before fertilization. However, unlike mammals, the decapod crustacean E. 197
sinensis possesses noncondensed chromatin that does not damage DNA (Wu et al., 2016; Sela et al., 2012;
198
Hime et al., 1996). The noncondensed state of the spermatozoal nuclei in E. sinensis makes the genetic
199
material loose. Therefore, we conducted a study on the expression and localization of P4HB in testis tissues of
200
adult and juvenile E. sinensis. Our study found that P4HB was expressed in both adult and juvenile testis
201
tissues of E. sinensis. However, the expression localization varied among male germ cells at different
202
developmental stages. 203
Previous studies have highlighted the multifaceted roles of P4HB, including catalyzing the formation,
204
breakage, and rearrangement of disulfide bonds, acting as a reductase on the cell membrane surface, and PeerJ reviewing PDF | (2021:08:64840:2:0:NEW 17 May 2023) Manuscript to be reviewed 205
connecting the extracellular matrix to the intracellular skeletal network (Wan et al., 2020; Bi et al., 2011;
206
Mezghrani et al., 2000). Furthermore, P4HB can act as a chaperone protein that inhibits the aggregation of
207
misfolded proteins and facilitates the folding of cytoplasmic proteins (Yu et al., 2020; Bi et al., 2011; Lumb &
208
Bulleid, 2002; Zhao et al., 2005). These findings suggest that P4HB plays a crucial role in the synthesis and
209
structural modification of proteins in the cell membrane and cytoplasm. In our study, we observed that P4HB
210
is abundantly expressed in the cell membrane and cytoplasm, particularly on the outer side of the cell
211
membrane and cytoplasm of spermatogonia and spermatocytes. P4HB in these regions can interact with
212
proteins such as integrins, which are closely associated with the actin cytoskeleton (Kong et al., 2020). 213
Therefore, P4HB in the cytoplasm can convey extracellular signals and trigger subsequent cascade reactions
214
such as protein phosphorylation and cytoskeletal reorganization (Ponamarczuk et al., 2018). Spermatogonia
215
and spermatocytes are in the early stages of spermatogenesis and actively engage in cell proliferation and
216
differentiation. Consequently, coordinated cytoskeletal reorganization is crucial in these stages (Carlton et al.,
217
2020; Frappaolo et al., 2022). Our study discovered elevated P4HB levels in the spermatogonia and
218
spermatocytes, indicating its importance in maintaining cytoskeletal reorganization. 206
Mezghrani et al., 2000). Furthermore, P4HB can act as a chaperone protein that inhibits the aggregation of
207
misfolded proteins and facilitates the folding of cytoplasmic proteins (Yu et al., 2020; Bi et al., 2011; Lumb &
208
Bulleid, 2002; Zhao et al., 2005). These findings suggest that P4HB plays a crucial role in the synthesis and
209
structural modification of proteins in the cell membrane and cytoplasm. In our study, we observed that P4HB
210
is abundantly expressed in the cell membrane and cytoplasm, particularly on the outer side of the cell
211
membrane and cytoplasm of spermatogonia and spermatocytes. P4HB in these regions can interact with
212
proteins such as integrins, which are closely associated with the actin cytoskeleton (Kong et al., 2020). 213
Therefore, P4HB in the cytoplasm can convey extracellular signals and trigger subsequent cascade reactions
214
such as protein phosphorylation and cytoskeletal reorganization (Ponamarczuk et al., 2018). Manuscript to be reviewed 246
In addition, in our study, we observed differential expressed of P4HB in male germ cells at different
247
developmental stages in E. chinensis. Previous studies have proved that P4HB can interact with various other
248
proteins in different cellular contexts. For instance, P4HB retained on the surface of Th2 cells can increase
249
disulfide bond reductase activity, altering plasma membrane redox state and enhancing cell migration (Bi et al.,
250
2011). P4HB interactions with basic fibroblast growth factor and transforming growth factor in pediatric aortic
251
cells can promote the synthesis of DNA, collagen and matrix proteins (Fu et al., 2004). P4HB binding to
252
insulinogen has been found to influence disulfide bonds maturation (Insook et al., 2019). Additionally, binding
253
of P4HB to the thyroid hormone T3 can inhibit disulfide bond formation in nascent and misfolded proteins
254
(Hashimoto et al., 2012). These findings show that P4HB's diverse functions can be determined by certain
255
factors. Our immunofluorescence localization results showed that the subcellular location and expression of
256
the P4HB protein varied in male germ cells at different developmental stages, which suggests that it is
257
influenced by various factors in its extensive functional activity during spermatogenesis. 258
P4HB is distributed at high levels in the extracellular matrix or intracellular regions of spermatogonia,
259
spermatocytes, and stage I spermatids and may be involved in spermatogenesis mainly through its
260
oxidoreductase, chaperone, and isoenzyme effects (Zhu et al., 2022), which promote the maintenance of the
261
cell's morphology and structure. As spermatids progress from the stage II to the stage III and eventually
262
specialize into the sperm, they discard most of the organelles and excess substances in the cells, further
263
simplifying the cell structure (Dalton, 2013). Therefore, the low expression of P4HB in these stages of cells
264
may be due to the reduced requirement of P4HB for its structural maintenance function during this phase of
265
spermatogenesis. Nevertheless, expression of P4HB at appropriate levels during these stages is vital to ensure
266
the proper formation of protamine and the appropriate compaction of sperms, which is critical for successful
267
fertilization. Hence, understanding the role of P4HB during different stages of spermatogenesis is essential for
268
a comprehensive understanding of how this protein regulates spermatogenesis. 269
Furthermore, our study provided a preliminary analysis of the mRNA and protein sequences of P4HB in E. Manuscript to be reviewed sinensis differs from the common site of P4HB expression in the somatic cells in other species (Zhu et al.,
225
2022). So, we speculate that this unusual expression pattern may be related to the mechanism of noncondensed
226
nuclei formation in E. sinensis. Unlike mammals, the spermatozoal nuclei in E. sinensis undergoes a complex,
227
decondensed process during spermatogenesis (Chen et al., 2020), culminating in the unique morphology of its
228
sperm, with flagella-free, microtubule sheaths, and cup-shaped nuclei with radial arms on the surface of sperm
229
(Sela et al., 2012; Wang et al., 2015). This intricate process is completed in an orderly manner, which requires
230
appropriate protein folding (Wang et al., 2015) and the chromatin remodeling (Wu et al., 2016) to maintain the
231
structural integrity of the sperm. Therefore, the unique expression pattern of P4HB in E. sinensis suggests its
232
potential involvement in ensuring proper protein folding and subsequent sperm development. 233
A decrease in P4HB can lead to the production of unfolded proteins, reduce cell survival, and result in
234
genomic instability by down-regulating genes involved in DNA repair and cell cycle regulation (Liu et al.,
235
2019). The chromatin structure of DNA plays a vital role in genome compaction (Ou et al., 2017). Therefore,
236
the high expression of P4HB in the nuclei of E. sinensis spermatogonia is likely involved in the formation of
237
its noncondensed chromatin during spermatogenesis. As spermatogonia differentiate, cytoplasmic proteins
238
undergo folding and structural changes, while the nucleus undergoes simultaneous condensation and plasticity
239
changes. Both the later stage of spermatid development and the spermatozoal maturation involve gradual loss
240
of cytoplasm, replacement of histones with alkaline protamine, and repackaging of genetic material in the
241
nuclei (Wu et al., 2016). The cysteine sulfhydryl group (-SH) in protamine undergoes oxidation, forming
242
disulfide bonds (-S-S) within and between alkaline protamine molecules in the process, ultimately leading to
243
sperm maturation (Sharma et al., 2019). Therefore, the expression of P4HB in the nuclei of the later stages of
244
spermatids and mature sperm may play a critical role in maintaining homeostasis in the noncondensed
245
spermatozoal nuclei of E. chinensis. PeerJ reviewing PDF | (2021:08:64840:2:0:NEW 17 May 2023) Manuscript to be reviewed 270
sinensis and found that the protein sequence of P4HB was similar to that of human PDI protein, indicating a
271
similar spatial binding and biological functions (Kapli et al., 2020). Phylogenetic tree analysis further revealed
272
that the P4HB sequence in E. sinensis shared some similarities and affinities with other crustaceans and
273
arthropods, indicating some degree of species conservation. This conservation of functional domains suggests
274
that different species perform similar functions in the process of evolution. However, different species exhibit
275
diversity in function and phenotype due to the unique environmental pressures and evolutionary adaptations
276
they face (Sidi et al., 2013). This is valuable in the evolutionary analysis of species affinity and can be used as
277
a molecular clock to analyze the homology and evolutionary status of arthropods to a certain extent. This
278
diversity in function and phenotype is also reflected in the sequence characteristics of P4HB in different
279
species. Therefore, the analysis of P4HB sequence characteristics and functional domains in different species
280
can help to understand the evolution of species and how it contributes to the diversity of function and
281
phenotype in animals. 282
Overall, our study deepens our understanding of the role of P4HB in E. sinensis spermatogenesis and
283
highlights its potential contribution to the maintenance of cell morphology, stability of spermatozoal nuclei,
284
and genetic stability. This study also provides a foundation for future research on the functional and
285
evolutionary analysis of P4HB in other crustaceans and arthropods. 286
5 Conclusion PeerJ reviewing PDF | (2021:08:64840:2:0:NEW 17 May 2023) Manuscript to be reviewed 287
P4HB was expressed in both the adult and juvenile testis tissues of E. sinensis, but the expression level and
288
localization varied among the male germ cells at the different developmental stages (as illustrated in Fig. 6). 289
As P4HB plays an important role in the formation, folding, and stabilization of disulfide bonds in proteins, the
290
differences in its expression and localization are likely to be a significant factor in maintaining the cell
291
morphology and structure of different male germ cells of E. sinensis. In particular, P4HB expressed in the
292
nuclei of spermatogonia, late spermatids, and mature sperm of E. sinensis may play a crucial role in the
293
stabilization of the noncondensed spermatozoal nuclei. 287
P4HB was expressed in both the adult and juvenile testis tissues of E. sinensis, but the expression level a
288
localization varied among the male germ cells at the different developmental stages (as illustrated in Fig. 6
289
As P4HB plays an important role in the formation, folding, and stabilization of disulfide bonds in proteins, t
290
differences in its expression and localization are likely to be a significant factor in maintaining the c
291
morphology and structure of different male germ cells of E. sinensis. In particular, P4HB expressed in t
292
nuclei of spermatogonia, late spermatids, and mature sperm of E. sinensis may play a crucial role in t
293
stabilization of the noncondensed spermatozoal nuclei. 294
The position of Fig. 6
295
296
Data Deposition
297
The following information was supplied regarding data availability:
298
The raw data are available Supplemental Files. 299
300
Supplemental Information
301
Supplemental information for this article can be found online at
302
https://www.ncbi.nlm.nih.gov/sra?linkname=bioproject_sra_all&from_uid=354440. 303
304
References:
305
Akama, K., Horikoshi, T., Sugiyama, A., Nakahata, S., Akitsu, A., Niwa, N., Intoh, A., Kakui, Y., Sugaya, M
306
Takei, K., Imaizumi, N., Sato, T., Matsumoto, R., Iwahashi, H., Kashiwabara, S., Baba, T., Nakamura, M
307
and Toda, T. 2010. Protein disulfide isomerase-P5, down-regulated in the final stage of boar epididymal sper
308
maturation, catalyzes disulfide formation to inhibit protein function in oxidative refolding of reduced denatur
309
lysozyme. Biochim Biophys Acta 1804:1272-1284. 10.1016/j.bbapap.2010.02.004
310
Bi, S., Hong, P.W., Lee, B., and Baum, L.G. 2011. Galectin-9 binding to cell surface protein disulfide isomera
311
regulates the redox environment to enhance T-cell migration and HIV entry. Manuscript to be reviewed Proc Natl Acad Sci U S A 108:1065
312
10655. 10.1073/pnas.1017954108
313
Carlton, J.G., Jones, H., and Eggert, U.S. 2020. Membrane and organelle dynamics during cell division. Nat R
314
Mol Cell Biol 21:151-166. 10.1038/s41580-019-0208-1
315
Chen, T., Mu, S., Sun, Z., Zhang, H., Li, C., Guo, M., Li, Y., Kang, X., and Wang, Z. 2020. Spermiogen
316
histone
transitions
and
chromatin
decondensation
in
Decapoda. THERIOGENOLOGY
156:242-25
317
10.1016/j.theriogenology.2020.07.003
318
Dalton, S. 2013. G1 compartmentalization and cell fate coordination. CELL 155:13-14. 10.1016/j.cell.2013.09.015
319
Frappaolo, A., Piergentili, R., and Giansanti, M.G. 2022. Microtubule and Actin Cytoskeletal Dynamics in Ma
320
Meiotic Cells of Drosophila melanogaster. Cells 11. 10.3390/cells11040695
321
Fu, P., Sodian, R., Luders, C., Lemke, T., Kraemer, L., Hubler, M., Weng, Y., Hoerstrup, S.P., Meyer, R
322
and Hetzer, R. 2004. Effects of basic fibroblast growth factor and transforming growth factor-beta on maturation
323
human pediatric aortic cell culture for tissue engineering of cardiovascular structures. ASAIO JOURNAL 50:9-1
324
10 1097/01 mat 0000104815 21056 08 297
The following information was supplied regarding data availability:
298
The raw data are available Supplemental Files. 300
Supplemental Information 301
Supplemental information for this article can be found online at 301
Supplemental information for this article can be found online at 302
https://www.ncbi.nlm.nih.gov/sra?linkname=bioproject_sra_all&from_uid=354440. 303 Manuscript to be reviewed 328
communications 68. 328
communications 68. 329
Hime, G.R., Brill, J.A., and Fuller, M.T. 1996. Assembly of ring canals in the male germ line from structural
330
components of the contractile ring. JOURNAL OF CELL SCIENCE 109 ( Pt 12):2779-2788. 331
Insook, J., Anita, P., Juthakorn, P., Jing, Y., Jacqueline, L., Adriana, C., Zhouji, C., 331
Insook, J., Anita, P., Juthakorn, P., Jing, Y., Jacqueline, L., Adriana, C., Zhouji, C., Donalyn, L.S., Ming, L.,
332
Pamela, I., Peter, A., and Randal, J.K. 2019. PDIA1/P4HB is required for efficient proinsulin maturation and ? 333
cell health in response to diet induced obesity. eLife 8. 332
Pamela, I., Peter, A., and Randal, J.K. 2019. PDIA1/P4HB is required for efficient proinsulin maturation and ? 333
cell health in response to diet induced obesity. eLife 8. 334
Kapli, P., Yang, Z., and Telford, M.J. 2020. Phylogenetic tree building in the genomic age. NATURE REVIEWS
335
GENETICS 21:428-444. 10.1038/s41576-020-0233-0 336
Kari, I.K., and Johanna, M. 1998. Prolyl 4-hydroxylases and their protein disulfide isomerase subunit. MATRIX
337
BIOLOGY 16. 338
Kong, L., Wang, B., Yang, X., He, B., Hao, D., and Yan, L. 2020. Integrin-associated molecules and signalling
339
cross talking in osteoclast cytoskeleton regulation. JOURNAL OF CELLULAR AND MOLECULAR MEDICINE
340
24:3271-3281. 10.1111/jcmm.15052 338
Kong, L., Wang, B., Yang, X., He, B., Hao, D., and Yan, L. 2020. Integrin-associated molecules and signalling
339
cross talking in osteoclast cytoskeleton regulation. JOURNAL OF CELLULAR AND MOLECULAR MEDICINE 340
24:3271-3281. 10.1111/jcmm.15052 341
Li, G., Kang, X., Mu, S., Guo, M., Huang, S., Chen, Q., Nong, S., Huang, X., Hu, H., and Sun, K. 2017. 342
H3K9ac involved in the decondensation of spermatozoal nuclei during spermatogenesis in Chinese mitten crab
343
Eriocheir sinensis. CYTOTECHNOLOGY 69:75-87. 10.1007/s10616-016-0038-3 344
Li, H., Yang, K., Wang, W., Niu, Y., Li, J., Dong, Y., Liu, Y., Wang, C., Wang, L., and Liang, H. 2018. Crystal
345
and solution structures of human protein-disulfide isomerase-like protein of the testis (PDILT) provide insight into
346
its chaperone activity. The Journal of biological chemistry 293. 347
Li, Z.F., Hao, S.L., Wang, L.M., Qi, H.Y., Wang, J.M., Tan, F.Q., and Yang, W.X. 2022. mTORC1/C2 regulate
348
spermatogenesis in Eriocheir sinensis via alterations in the actin filament network and cell junctions. CELL AND
349
TISSUE RESEARCH 390:293-313. 304
References: 305
Akama, K., Horikoshi, T., Sugiyama, A., Nakahata, S., Akitsu, A., Niwa, N., Intoh, A., Kakui, Y., Sugaya, M.,
306
Takei, K., Imaizumi, N., Sato, T., Matsumoto, R., Iwahashi, H., Kashiwabara, S., Baba, T., Nakamura, M.,
307
and Toda, T. 2010. Protein disulfide isomerase-P5, down-regulated in the final stage of boar epididymal sperm
308
maturation, catalyzes disulfide formation to inhibit protein function in oxidative refolding of reduced denatured
309
lysozyme. Biochim Biophys Acta 1804:1272-1284. 10.1016/j.bbapap.2010.02.004 310
Bi, S., Hong, P.W., Lee, B., and Baum, L.G. 2011. Galectin-9 binding to cell surface protein disulfide isomerase
311
regulates the redox environment to enhance T-cell migration and HIV entry. Proc Natl Acad Sci U S A 108:10650-
312
10655. 10.1073/pnas.1017954108 313
Carlton, J.G., Jones, H., and Eggert, U.S. 2020. Membrane and organelle dynamics during cell division. Nat Rev
314
Mol Cell Biol 21:151-166. 10.1038/s41580-019-0208-1 315
Chen, T., Mu, S., Sun, Z., Zhang, H., Li, C., Guo, M., Li, Y., Kang, X., and Wang, Z. 2020. Spermiogenic
316
histone
transitions
and
chromatin
decondensation
in
Decapoda. THERIOGENOLOGY
156:242-252. 317
10.1016/j.theriogenology.2020.07.003 318
Dalton, S. 2013. G1 compartmentalization and cell fate coordination. CELL 155:13-14. 10.1016/j.cell.2013.09.015 319
Frappaolo, A., Piergentili, R., and Giansanti, M.G. 2022. Microtubule and Actin Cytoskeletal Dynamics in Male
320
Meiotic Cells of Drosophila melanogaster. Cells 11. 10.3390/cells11040695 321
Fu, P., Sodian, R., Luders, C., Lemke, T., Kraemer, L., Hubler, M., Weng, Y., Hoerstrup, S.P., Meyer, R.,
322
and Hetzer, R. 2004. Effects of basic fibroblast growth factor and transforming growth factor-beta on maturation of
323
human pediatric aortic cell culture for tissue engineering of cardiovascular structures. ASAIO JOURNAL 50:9-14. 324
10.1097/01.mat.0000104815.21056.08 325
Hashimoto, S., Ito, L., Okumura, M., Shibano, T., Nawata, M., Kumasaka, T., Yamaguchi, H., and Imaoka, S. 326
2012. Crystallization and preliminary crystallographic analysis of the complex between triiodothyronine and the bb' 325
Hashimoto, S., Ito, L., Okumura, M., Shibano, T., Nawata, M., Kumasaka, T., Yamaguchi, H., and Imaoka, S. 326
2012. Crystallization and preliminary crystallographic analysis of the complex between triiodothyronine and the bb'
327
fragment of rat protein disulfide isomerase. Acta crystallographica. Section F, Structural biology and crystallization 327
fragment of rat protein disulfide isomerase. Acta crystallographica. Section F, Structural biology and crystallization PeerJ reviewing PDF | (2021:08:64840:2:0:NEW 17 May 2023) Manuscript to be reviewed 369
mitten crab (Eriocheir sinensis). Sci Rep 11:4492. 10.1038/s41598-021-83984-6 370
Ponamarczuk, H., Popielarski, M., Stasiak, M., Bednarek, R., Studzian, M., Pulaski, L., Babinska, A., and
371
Swiatkowska, M. 2018. Contribution of activated beta3 integrin in the PDI release from endothelial cells. Front
372
Biosci (Landmark Ed) 23:1612-1627. 10.2741/4663 373
Sela, Y., Molotski, N., Golan, S., Itskovitz-Eldor, J., and Soen, Y. 2012. Human embryonic stem cells exhibit
374
increased propensity to differentiate during the G1 phase prior to phosphorylation of retinoblastoma protein. STEM
375
CELLS 30:1097-1108. 10.1002/stem.1078 375
CELLS 30:1097-1108. 10.1002/stem.1078 376
Sharma, P., Ghanghas, P., Kaushal, N., Kaur, J., and Kaur, P. 2019. Epigenetics and oxidative stress: A twin-
377
edged sword in spermatogenesis. ANDROLOGIA 51:e13432. 10.1111/and.13432 378
Sidi, C., Benjamin, H.K., and Manyuan, L. 2013. New genes as drivers of phenotypic evolution. NATURE
379
REVIEWS GENETICS 14. 380
Sobierajska, K., Skurzynski, S., Stasiak, M., Kryczka, J., Cierniewski, C.S., and Swiatkowska, M. 2014. 380
Sobierajska, K., Skurzynski, S., Stasiak, M., Kryczka, J., Cierniewski, C.S., and Swiatkowska, M. 2014. 381
Protein disulfide isomerase directly interacts with beta-actin Cys374 and regulates cytoskeleton reorganization. 382
JOURNAL OF BIOLOGICAL CHEMISTRY 289:5758-5773. 10.1074/jbc.M113.479477 381
Protein disulfide isomerase directly interacts with beta-actin Cys374 and regulates cy y
y
g
y
g
382
JOURNAL OF BIOLOGICAL CHEMISTRY 289:5758-5773. 10.1074/jbc.M113.479477 382
JOURNAL OF BIOLOGICAL CHEMISTRY 289:5758-5773. 10.1074/jbc.M113.479477 383
Wan, X.L., Zhou, Z.L., Wang, P., Zhou, X.M., Xie, M.Y., Mei, J., Weng, J., Xi, H.T., Chen, C., Wang, Z.Y.,
384
and Wang, Z.B. 2020. Small molecule proteomics quantifies differences between normal and fibrotic pulmonary
385
extracellular matrices. Chin Med J (Engl) 133:1192-1202. 10.1097/CM9.0000000000000754 386
Wang, J.M., Li, Z.F., Qi, H.Y., Zhao, Z., and Yang, W.X. 2023. es-Arp3 and es-Eps8 regulate spermatogenesis
387
via microfilaments in the seminiferous tubule of Eriocheir sinensis. TISSUE & CELL 81:102028. 388
10.1016/j.tice.2023.102028 389
Wang, Y.L., Sun, W.J., He, L., Li, Q., and Wang, Q. 2015. Morphological alterations of all stages of
390
spermatogenesis and acrosome reaction in Chinese mitten crab Eriocheir sinensis. CELL AND TISSUE RESEARCH
391
360:401-412. 10.1007/s00441-014-2092-5 391
360:401-412. 10.1007/s00441-014-2092-5 392
Wei, Y.L., Yang, T., Kovacs, T., and Yang, W.X. 2019. C-terminal kinesin motor es-KIFC1 regulates nuclear
393
formation
during
spermiogenesis
in
Chinese
mitten
crab
Eriocheir
sinensis. GENE
719:144074. 394
10.1016/j.gene.2019.144074 395
Wu, J., Mu, S., Guo, M., Chen, T., Zhang, Z., Li, Z., Li, Y., and Kang, X. 2016. Manuscript to be reviewed 10.1007/s00441-022-03680-3 350
Liu, Y., Ji, W., Shergalis, A., Xu, J., Delaney, A.M., Calcaterra, A., Pal, A., Ljungman, M., Neamati, N., and
351
Rehemtulla, A. 2019. Activation of the Unfolded Protein Response via Inhibition of Protein Disulfide Isomerase 350
Liu, Y., Ji, W., Shergalis, A., Xu, J., Delaney, A.M., Calcaterra, A., Pal, A., Ljungman, M., Neamati, N., and
351
Rehemtulla, A. 2019. Activation of the Unfolded Protein Response via Inhibition of Protein Disulfide Isomerase 352
Decreases the Capacity for DNA Repair to Sensitize Glioblastoma to
Radiotherapy. CANCER RESEARCH
353
79:2923-2932. 10.1158/0008-5472.CAN-18-2540 354
Lumb, R.A., and Bulleid, N.J. 2002. Is protein disulfide isomerase a redox-dependent molecular chaperone? The
355
EMBO journal 21. 356
Lv, L.X., Ujisguleng, B., Orhontana, B., Lian, W.B., and Xing, W.J. 2011. Molecular cloning of sheep and
357
cashmere goat Pdia3 and localization in sheep testis. REPRODUCTION IN DOMESTIC ANIMALS 46:980-989. 358
10.1111/j.1439-0531.2011.01771.x 358
10.1111/j.1439-0531.2011.01771.x 359
Mezghrani, A., Courageot, J., Mani, J.C., Pugniere, M., Bastiani, P., and Miquelis, R. 2000. Protein-disulfide
360
isomerase (PDI) in FRTL5 cells. pH-dependent thyroglobulin/PDI interactions determine a novel PDI function in
361
the post-endoplasmic reticulum of
thyrocytes. JOURNAL OF BIOLOGICAL CHEMISTRY 275:1920-1929. 362
10.1074/jbc.275.3.1920 362
10.1074/jbc.275.3.1920 363
Nishimura, H., and L'Hernault, S.W. 2017. Spermatogenesis. CURRENT BIOLOGY 27:R988-R994. 364
10.1016/j.cub.2017.07.067 365
Ou, H.D., Phan, S., Deerinck, T.J., Thor, A., Ellisman, M.H., and O'Shea, C.C. 2017. ChromEMT: Visualizing
366
3D chromatin structure and compaction in interphase and mitotic cells. SCIENCE 357. 10.1126/science.aag0025 367
Pang, Y.Y., Huang, G.Y., Song, Y.M., Song, X.Z., Lv, J.H., He, L., Niu, C., Shi, A.Y., Shi, X.L., Cheng, Y.X.,
368
and Yang, X.Z. 2021. Effects of miR-143 and its target receptor 5-HT2B on agonistic behavior in the Chinese 367
Pang, Y.Y., Huang, G.Y., Song, Y.M., Song, X.Z., Lv, J.H., He, L., Niu, C., Shi, A.Y 368
and Yang, X.Z. 2021. Effects of miR-143 and its target receptor 5-HT2B on agonistic behavior in the Chinese 368
and Yang, X.Z. 2021. Effects of miR-143 and its target receptor 5-HT2B on agonisti PeerJ reviewing PDF | (2021:08:64840:2:0:NEW 17 May 2023) Manuscript to be reviewed Manuscript to be reviewed 410
protein disulfide isomerase involves disulfide cross-link and dimer to tetramer switch. JOURNAL OF
411
BIOLOGICAL CHEMISTRY 280:13470-13476. 10.1074/jbc.M413882200
412
Zhao, X.J., Tang, R.Z., Wang, M.L., Guo, W.L., Liu, J., Li, L., and Xing, W.J. 2013. Distribution of PDIA3
413
transcript and protein in rat testis and sperm cells. REPRODUCTION IN DOMESTIC ANIMALS 48:59-63. 414
10.1111/j.1439-0531.2012.02024.x
415
Zhu, J., Ma, X., Jing, Y., Zhang, G., Zhang, D., Mao, Z., Ma, X., Liu, H., and Chen, F. 2022. P4HB
416
UFMylation regulates mitochondrial function and oxidative stress. Free Radic Biol Med 188:277-286. 417
10.1016/j.freeradbiomed.2022.06.237
418
419
Figure legends
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Fig. 1 The P4HB mRNA sequence and its protein sequence in Eriocheir sinensis
421
Note: This figure shows the translation of the P4HB mRNA sequence in E. sinensis into the corresponding
422
protein sequence. The P4HB mRNA sequence has a length of 1584 bp, and its translated has a molecular
423
weight of 58.61 kDa. The protein contains 527 amino acids and has an isoelectric point of 4.61. Open reading
424
frames in the mRNA sequence are highlighted in red. 425
426
Fig. 2 The prediction of the tertiary structure model of P4HB protein in Eriocheir sinensis
427
Note: This figure shows the predicted tertiary structure of the P4HB protein in E. sinensis using homology
428
modeling techniques. (A) displays the sequence alignment between the P4HB protein in E. sinensis and the
429
human disulfide isomerase (PDI), where "6i7s. 2. A" denotes a protein sequence of human PDI protein with a
430
known spatial structure from the SWISS-MODEL template library (SMTL) which was used as a template for
431
prediction of the tertiary structure model of the P4HB protein in E. sinensis. "Model_06" represents the amino
432
acid sequence corresponding to the predicted tertiary structure model of the P4HB protein in E. sinensis.. The
433
arrows in the figure show the direction of alignment between the target and template sequence. (B) shows a
434
model of the protein tertiary structure of P4HB in E. sinensis displayed in the interactive viewer. 435
436
Fig. 3 Phylogenetic tree analysis of amino acid sequence of P4HB protein in Eriocheir sinensis
437
Note: Phylogenic tree was constructed using the neighbor-joining method in the MEGA 7.0 software, based on
438
twenty species' amino acid sequences. Manuscript to be reviewed Histone H2B gene cloning, with
396
implication for its function during nuclear shaping in the Chinese mitten crab, Eriocheir sinensis. GENE 575:276-
397
284. 10.1016/j.gene.2015.09.005 397
284. 10.1016/j.gene.2015.09.005 398
Wu, J.L., Kang, X.J., Guo, M.S., Mu, S.M., and Zhang, Z.H. 2015. Cloning and Functional Analysis of Histones
399
H3 and H4 in Nuclear Shaping during Spermatogenesis of the Chinese Mitten Crab, Eriocheir sinensis. PLoS One
400
10:e126623. 10.1371/journal.pone.0126623 400
10:e126623. 10.1371/journal.pone.0126623 401
Wu, Y., Peng, Y., Guan, B., He, A., Yang, K., He, S., Gong, Y., Li, X., and Zhou, L. 2021. P4HB: A novel
402
diagnostic and prognostic biomarker for bladder carcinoma. Oncology Letters 21:95. 10.3892/ol.2020.12356 403
Xu, Y.R., Wang, G.Y., Zhou, Y.C., and Yang, W.X. 2018. The characterization and potential roles of bone
404
morphogenetic protein 7 during spermatogenesis in Chinese mitten crab Eriocheir sinensis. GENE 673:119-129. 405
10.1016/j.gene.2018.06.020 406
Yu, J., Li, T., Liu, Y., Wang, X., Zhang, J., Wang, X., Shi, G., Lou, J., Wang, L., Wang, C.C., and Wang, L. 407
2020. Phosphorylation switches protein disulfide isomerase activity to maintain
proteostasis and attenuate ER
408
stress. EMBO JOURNAL 39:e103841. 10.15252/embj.2019103841 409
Zhao, T.J., Ou, W.B., Xie, Q., Liu, Y., Yan, Y.B., and Zhou, H.M. 2005. Catalysis of creatine kinase refolding by PeerJ reviewing PDF | (2021:08:64840:2:0:NEW 17 May 2023) Manuscript to be reviewed Numbers in the phylogram nodes indicate the percent bootstrap, and the
439
bar at the bottom indicates a 5% amino acid divergence in sequence. 440
441
Table 1 The primers of genes used in the study for RT-qPCR
442
443
Fig. 4 The expression of P4HB mRNA and protein in testis tissues of adult and juvenile Eriocheir sinensis
444
Note: (A) shows the expression of P4HB mRNA in the testis tissues of adult and juvenile E. sinensis. The
445
amplified products of RT-qPCR were identified using agarose electrophoresis. (B) shows the expression of
446
P4HB protein in the testis tissues of adult and juvenile E. sinensis using western blotting. The results indicate
447
that P4HB is expressed in the testis tissues of both adult and juvenile E. sinensis. 448
449
Fig 5 The expression of P4HB protein in the different male germ cells of Eriocheir sinensis (n = 3) 410
protein disulfide isomerase involves disulfide
411
BIOLOGICAL CHEMISTRY 280:13470-13476. 10
412
Zhao, X.J., Tang, R.Z., Wang, M.L., Guo, W.L
413
transcript and protein in rat testis and sperm c
414
10.1111/j.1439-0531.2012.02024.x
415
Zhu, J., Ma, X., Jing, Y., Zhang, G., Zhang,
416
UFMylation regulates mitochondrial function
417
10.1016/j.freeradbiomed.2022.06.237
418
419
Figure legends
420
Fig. 1 The P4HB mRNA sequence and its protein
421
Note: This figure shows the translation of the P
422
protein sequence. The P4HB mRNA sequence
423
weight of 58.61 kDa. The protein contains 527 a
424
frames in the mRNA sequence are highlighted in
425
426
Fig. 2 The prediction of the tertiary structure mod
427
Note: This figure shows the predicted tertiary s
428
modeling techniques. (A) displays the sequence
429
human disulfide isomerase (PDI), where "6i7s. 2
430
known spatial structure from the SWISS-MODE
431
prediction of the tertiary structure model of the P
432
acid sequence corresponding to the predicted tert
433
arrows in the figure show the direction of alignm
434
model of the protein tertiary structure of P4HB in
435
436
Fig. 3 Phylogenetic tree analysis of amino acid se
437
Note: Phylogenic tree was constructed using the n
438
twenty species' amino acid sequences. Numbers i
439
bar at the bottom indicates a 5% amino acid diver
440
441
Table 1 The primers of genes used in the study fo
442
443
Fig. Manuscript to be reviewed 4 The expression of P4HB mRNA and protei
444
Note: (A) shows the expression of P4HB mRN
445
amplified products of RT-qPCR were identified
446
P4HB protein in the testis tissues of adult and ju
447
that P4HB is expressed in the testis tissues of both
448 415
Zhu, J., Ma, X., Jing, Y., Zhang, G., Zhang, D., Mao, Z., Ma, X., Liu, H., and Chen, F. 2022. P4HB
416
UFMylation regulates mitochondrial function and oxidative stress. Free Radic Biol Med 188:277-286. 417
10.1016/j.freeradbiomed.2022.06.237 Manuscript to be reviewed 450
Note: (A) shows the expression of P4HB protein in the male germ cells at different developmental stages of E. 451
chinensis. (B) presents the comparison of the average fluorescence intensity of P4HB protein in the male germ
452
cells at different developmental stages of E. chinensis. The results indicate that P4HB was prominently
453
expressed in spermatogonia, spermatocytes, and stage I spermatids, followed by mature sperm, and less so in
454
stage II and stage III spermatids. In spermatogonia, spermatocytes, stage I spermatids, and stage II spermatids,
455
P4HB was mainly expressed in the cytoplasm, cell membrane, intercellular matrix, and extracellular matrix,
456
while in stage III spermatids and mature sperm, P4HB was mainly expressed in the nuclei and to a lesser
457
degree in the cytoplasm, in addition to being partially expressed in specific regions of the nuclei of
458
spermatogonia. DDX4 is a marker protein of male germ cells, primarily distributed in the cytoplasm. The scale
459
of immunofluorescence was 5 μm. * Compared with spermatogonia: *P < 0.05; # Compared with
460
spermatocytes: #P < 0.05; & Compared with stage I spermatids: &P < 0.05. 461
462
Fig. 6 Expression pattern diagram of P4HB in male germ cells at different developmental stages of Eriocheir
463
sinensis
464
Note: The red dot in the figure represents P4HB, and each alphabet refers to different developmental stages,
465
i.e., a: spermatogonia; b: spermatocytes; c: stage I spermatids; d: stage II spermatids; e: stage III spermatids; f:
466
sperm. N denotes nuclei, PV represents proacrosomal vesicle, AT represents acrosomal tubule, and AC stands
467
for apical cap. The expression and localization of P4HB protein in male germ cells is shown in the figure as
468
follows: (1) spermatogonia: extracellular, cytoplasm, nuclear (some); (2) spermatocytes: extracellular,
469
cytoplasm, nuclei (less); (3) stage I spermatids: extracellular, cytoplasm, nuclei (less); (4) stage II spermatids:
470
cytoplasm, nuclei (more), proacrosomal vesicle (less); (5) stage III spermatids: nuclei (abundant),
471
proacrosomal vesicle (very little); (6) sperm: nuclei (very abundant), acrosome (very little). 472 450
Note: (A) shows the expression of P4HB protein in the male germ cells at different developmental stages of E. 451
chinensis. (B) presents the comparison of the average fluorescence intensity of P4HB protein in the male germ
452
cells at different developmental stages of E. chinensis. 419
Figure legends 421
Note: This figure shows the translation of the P4HB mRNA sequence in E. sinensis into the corresponding
422
protein sequence. The P4HB mRNA sequence has a length of 1584 bp, and its translated has a molecular
423
weight of 58.61 kDa. The protein contains 527 amino acids and has an isoelectric point of 4.61. Open reading
424
frames in the mRNA sequence are highlighted in red. 37
Note: Phylogenic tree was constructed using the neighbor-joining method in the MEGA 7.0 software, based on
38
twenty species' amino acid sequences. Numbers in the phylogram nodes indicate the percent bootstrap, and the
39
bar at the bottom indicates a 5% amino acid divergence in sequence. 437
Note: Phylogenic tree was constructed using the neighbor-joining method in the MEGA 7.0 software, based on
438
twenty species' amino acid sequences. Numbers in the phylogram nodes indicate the percent bootstrap, and the
439
bar at the bottom indicates a 5% amino acid divergence in sequence. PeerJ reviewing PDF | (2021:08:64840:2:0:NEW 17 May 2023) Manuscript to be reviewed The results indicate that P4HB was prominently
453
expressed in spermatogonia, spermatocytes, and stage I spermatids, followed by mature sperm, and less so in
454
stage II and stage III spermatids. In spermatogonia, spermatocytes, stage I spermatids, and stage II spermatids,
455
P4HB was mainly expressed in the cytoplasm, cell membrane, intercellular matrix, and extracellular matrix,
456
while in stage III spermatids and mature sperm, P4HB was mainly expressed in the nuclei and to a lesser
457
degree in the cytoplasm, in addition to being partially expressed in specific regions of the nuclei of
458
spermatogonia. DDX4 is a marker protein of male germ cells, primarily distributed in the cytoplasm. The scale
459
of immunofluorescence was 5 μm. * Compared with spermatogonia: *P < 0.05; # Compared with
460
spermatocytes: #P < 0.05; & Compared with stage I spermatids: &P < 0.05. 461 462
Fig. 6 Expression pattern diagram of P4HB in male germ cells at different developmental stages of Eriocheir
463
sinensis
464
Note: The red dot in the figure represents P4HB, and each alphabet refers to different developmental stages,
465
i.e., a: spermatogonia; b: spermatocytes; c: stage I spermatids; d: stage II spermatids; e: stage III spermatids; f:
466
sperm. N denotes nuclei, PV represents proacrosomal vesicle, AT represents acrosomal tubule, and AC stands
467
for apical cap. The expression and localization of P4HB protein in male germ cells is shown in the figure as
468
follows: (1) spermatogonia: extracellular, cytoplasm, nuclear (some); (2) spermatocytes: extracellular,
469
cytoplasm, nuclei (less); (3) stage I spermatids: extracellular, cytoplasm, nuclei (less); (4) stage II spermatids:
470
cytoplasm, nuclei (more), proacrosomal vesicle (less); (5) stage III spermatids: nuclei (abundant),
471
proacrosomal vesicle (very little); (6) sperm: nuclei (very abundant), acrosome (very little). 472 462
Fig. 6 Expression pattern diagram of P4HB in male germ cells at different developmental stages of Eriocheir
463
sinensis
464
Note: The red dot in the figure represents P4HB, and each alphabet refers to different developmental stages,
465
i.e., a: spermatogonia; b: spermatocytes; c: stage I spermatids; d: stage II spermatids; e: stage III spermatids; f:
466
sperm. N denotes nuclei, PV represents proacrosomal vesicle, AT represents acrosomal tubule, and AC stands
467
for apical cap. Figure 1 The P4HB mRNA sequence and its protein sequence in Eriocheir sinensis Manuscript to be reviewed The expression and localization of P4HB protein in male germ cells is shown in the figure as
468
follows: (1) spermatogonia: extracellular, cytoplasm, nuclear (some); (2) spermatocytes: extracellular,
469
cytoplasm, nuclei (less); (3) stage I spermatids: extracellular, cytoplasm, nuclei (less); (4) stage II spermatids:
470
cytoplasm, nuclei (more), proacrosomal vesicle (less); (5) stage III spermatids: nuclei (abundant),
471
proacrosomal vesicle (very little); (6) sperm: nuclei (very abundant), acrosome (very little). 472 PeerJ reviewing PDF | (2021:08:64840:2:0:NEW 17 May 2023) Manuscript to be reviewed Manuscript to be reviewed The P4HB mRNA sequence and its protein sequence in Eriocheir sinensis Note: This figure shows the translation of the P4HB mRNA sequence in E. sinensis into the
protein sequence. The length of the P4HB mRNA sequence was 1584 bp, and its protein
molecular weight was 58.61 KD. This protein contains 527 amino acids with an isoelectric
point of 4.61. Open reading frames are highlighted in red in the figure. PeerJ reviewing PDF | (2021:08:64840:2:0:NEW 17 May 2023) Manuscript to be reviewed Manuscript to be reviewed PeerJ reviewing PDF | (2021:08:64840:2:0:NEW 17 May 2023) Figure 2 The prediction of the tertiary structure model of P4HB protein in Eriocheir sinensis Note: This figure shows the modeling of the tertiary structure of the P4HB protein based on
the amino acid sequence of the protein using homology modeling techniques. (A) is a
sequence alignment between the P4HB protein in E. sinensis and the human disulfide
isomerase (PDI). The "6i7s. 2. A" represents a protein sequence of human PDI protein with a
known spatial structure from the SWISS-MODEL template library (SMTL), which was used as a
template for the prediction of the tertiary structure model of the P4HB protein in E. sinensis. The "Model_06" represents a tertiary structure model of the P4HB protein in E. sinensis
predicted based on the template. The arrows in the figure show the direction of the
alignment between the target sequence and the template sequence and the coverage of the
target sequence on the template sequence, and the boxes show the coverage of a target
sequence on the template sequence. If an amino acid on the target sequence is not covered
on the template sequence, the corresponding amino acid on the template sequence will be
shown in gray. (B) shows a model of the protein tertiary structure of P4HB in E. sinensis
displayed in the interactive viewer. PeerJ reviewing PDF | (2021:08:64840:2:0:NEW 17 May 2023) PeerJ reviewing PDF | (2021:08:64840:2:0:NEW 17 May 2023) Manuscript to be reviewed Manuscript to be reviewed PeerJ reviewing PDF | (2021:08:64840:2:0:NEW 17 May 2023) Manuscript to be reviewed Figure 3 Phylogenetic tree analysis of amino acid sequence of P4HB protein in Eriocheir sinensis
Note: Phylogenic tree constructed based on the sequences of amino acid from twenty species
using the neighbor-joining method in the MEGA 7.0 software. Numbers in the phylogram Phylogenetic tree analysis of amino acid sequence of P4HB protein in Eriocheir sinensis Note: Phylogenic tree constructed based on the sequences of amino acid from twenty species
using the neighbor-joining method in the MEGA 7.0 software. Numbers in the phylogram
nodes indicate the percent bootstrap, and the bar at the bottom indicates a 5% amino acid
divergence in sequence. PeerJ reviewing PDF | (2021:08:64840:2:0:NEW 17 May 2023) Manuscript to be reviewed Figure 4 The expression of P4HB mRNA and protein in testis tissues of adult and juvenile
Eriocheir sinensis Note: (A) shows the expression of P4HB mRNA in the testis tissues of adult and juvenile E. sinensis. The amplified products of RT-qPCR were identified using agarose electrophoresis. (B) shows the expression of P4HB protein in the testis tissues of adult and juvenile E. sinensis
using western blotting. The above results show that P4HB is both expressed in the testis
tissues of adult and juvenile E. sinensis. PeerJ reviewing PDF | (2021:08:64840:2:0:NEW 17 May 2023) PeerJ reviewing PDF | (2021:08:64840:2:0:NEW 17 May 2023) PeerJ reviewing PDF | (2021:08:64840:2:0:NEW 17 May 2023) Manuscript to be reviewed Figure 5 The expression of P4HB protein in the different male germ cells of Eriocheir sinensis (n
= 3) Note: (A) shows the expression of P4HB protein in the male germ cells at different
developmental stages of E. chinensis. (B) shows the comparison of the average fluorescence
intensity of P4HB protein in the male germ cells at different developmental stages of E. chinensis. The results showed that P4HB was mainly expressed in the spermatogonia,
spermatocytes, and stage I spermatids, followed by the mature sperm, and was less
expressed in the stage II and stage III spermatids. In spermatogonia, spermatocytes, stage I
spermatids, and stage II spermatids, P4HB was mainly expressed in the cytoplasm, cell
membrane, intercellular matrix, and extracellular matrix; in stage III spermatids and mature
sperm, P4HB was mainly expressed in the nuclei and to a lesser extent in the cytoplasm, in
addition to being partially expressed in specific regions of the nuclei of spermatogonia. DDX4
is a marker protein of male germ cells, mainly distributed in the cytoplasm. The scale of
immunofluorescence was 5 μm. * Compared with spermatogonia: *P < 0.05; # Compared
with spermatocytes:
#P < 0.05; & Compared with stage I spermatids:
&P < 0.05. PeerJ reviewing PDF | (2021:08:64840:2:0:NEW 17 May 2023) Manuscript to be reviewed PeerJ reviewing PDF | (2021:08:64840:2:0:NEW 17 May 2023) PeerJ reviewing PDF | (2021:08:64840:2:0:NEW 17 May 2023) Figure 6 Expression pattern diagram of P4HB in male germ cells at different developmental
stages of Eriocheir sinensis Note: The red dot in the figure represents P4HB; a: spermatogonia; b: spermatocytes; c:
stage I spermatids; d: stage II spermatids; e: stage III spermatids; f: sperm; N: nuclei; PV:
proacrosomal vesicle; AT: acrosomal tubule; AC: apical cap. As seen in the figure, the
expression and localization of P4HB protein is: (1) spermatogonia: extracellular, cytoplasm,
nuclear (some) (2) spermatocytes: extracellular, cytoplasm, nuclei (less) (3) stage I
spermatids: extracellular, cytoplasm, nuclei (less) (4) stage II spermatids: cytoplasm, nuclei
(more), proacrosomal vesicle (less) (5) stage III spermatids: nuclei (abundant), proacrosomal
vesicle (very little) (6) sperm: nuclei (very abundant), acrosome (very little). PeerJ reviewing PDF | (2021:08:64840:2:0:NEW 17 May 2023) Manuscript to be reviewed Manuscript to be reviewed PeerJ reviewing PDF | (2021:08:64840:2:0:NEW 17 May 2023) Manuscript to be reviewed Manuscript to be reviewed Table 1(on next page) Table 1(on next page) Table 1(on next page) The primers of genes used in the study for RT-qPCR PeerJ reviewing PDF | (2021:08:64840:2:0:NEW 17 May 2023) Manuscript to be reviewed 1
Table 1 The primers of genes used in the study for RT-qPCR
Gene
forward primer（5'-3'）
reverse primer （5'-3'）
P4HB
ACAGGCGGTGTCTATGGTCAGG
CCTCGTCCTCCTCTTCCTCATCC
β-Actin
ACCTCGGTTCTATTTTGTCGG
ATGCTTTCGCAGTAGTTCGTC 2 PeerJ reviewing PDF | (2021:08:64840:2:0:NEW 17 May 2023)
|
https://openalex.org/W4293207240
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https://chemrxiv.org/engage/api-gateway/chemrxiv/assets/orp/resource/item/630661bf90802dd2bd68c2ec/original/analytical-evaluation-of-lead-iodide-precursor-impurities-affecting-halide-perovskite-device-performance.pdf
|
English
| null |
Analytical Evaluation of Lead Iodide Precursor Impurities Affecting Halide Perovskite Device Performance
| null | 2,022
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cc-by
| 4,837
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Performance Ross A. Kerner,a Earl D. Christensen,a Steven P. Harvey,a Jonah Messinger,b Severin N. Habisreutinger,a Fei Zhang,a,c Giles E. Eperon,d Laura T. Schelhas,a Kai Zhu,a Joseph J. Berry,a,e,f and David T. Moore a Corresponding Author Corresponding Author * David T. Moore: David.Moore@nrel.gov * David T. Moore: David.Moore@nrel.gov 1 Abstract Mirroring established semiconductor technologies, halide perovskite materials synthesized from
higher quality reagents display improved optoelectronic performance. In this study, we performed
a semiquantitative analytical characterization of five different commercial PbI2 sources to
determine the identity and concentration of impurities that affect perovskite devices. It was
possible to single out acetate (OAc) and potassium (K) as key species in as-received materials,
both plausibly remnant from synthesis or purification. We removed these impurities through
aqueous recrystallization revealing contrasting impacts on device performance: removal of OAc
was beneficial but reducing K could be detrimental. This observation indicates that the highest
purity PbI2 does not guarantee the highest performing perovskite material, since certain extrinsic
impurities, such as KI, can improve device performance. Fundamental and applied studies will
both benefit from improved purification procedures coupled with analytical studies to better
understand and control the effects of individual impurities in halide perovskite materials. TOC GRAPHIC 2 2 Halide perovskite materials are touted as tolerant to the presence of native structural defects;
native refers to “intrinsic” lattice point defects, including vacancies and interstitials, which may or
may not be induced by an extrinsic impurity/defect.1-3 That is to say, with the possible exception
of positively charged iodine interstitials4, native defects are optoelectronically inert and do not
prevent these materials from performing their primary photovoltaic or electroluminescent
function.1 This limited tolerance to crystallographic imperfections may mean native defects in
perovskites are superficially benign, but their interactions with extrinsic species are certainly
consequential. Reactions between native halide vacancies and molecular oxygen influence doping5
and facilitate degradation.6,7 Moreover, it is understood that concentrations of native defects and
extrinsic impurities are directly linked. Ionic conductivity measurements of undoped and doped
PbBr2 crystals illustrate how, for example, Na+ impurities occupying Pb2+ sites induce halide
vacancies for charge neutrality, which are available to participate in ionic conduction.8 We
speculate that halide perovskite defect populations are similarly dominated by impurities present
within the reagents, a result of using processing additives, and released by in situ reactions during
synthesis. Further, a growing body of literature9-18 indicates that the full efficiency and stability
potential of halide perovskite materials will, to some degree, be unlocked by improving precursor
quality, motivating us to scrutinize the impurity content of PbI2 reagents for perovskite
applications. From this study, we present results from a semiquantitative analytical characterization of five
commercially available PbI2 materials. Three main techniques were used: inductively coupled
plasma optical emission spectroscopy (ICP-OES) to determine I:Pb stoichiometry; 1H and 13C
nuclear magnetic resonance (NMR) spectroscopy to reveal hydrogen-containing organic
impurities; and time-of-flight secondary ion mass spectrometry (TOF-SIMS) to compare relative 3 3 intensities of lightweight atomic and molecular impurities. These techniques can be used
separately or in combination to collect different information. For example, ICP-OES is ideal to
quantify many elements, but molecular species such as organics, water, hydroxide, carbonate, or
nitrate may need to be measured by other methods like NMR, ion chromatography, or titrations. A rigorous study on PbI2 should evaluate purity along several axes for a comprehensive
understanding of the impact of trace impurities on the resulting perovskite material quality and
device performance. Our analysis details two specific impurities in different source materials that were relatively
abundant, positively identifiable, and successfully removed by aqueous recrystallization to
measure an effect on devices. TOC GRAPHIC We found that iodide substoichiometry (I:Pb ratio < 2) was largely
due to substitution of iodide by oxygen-containing anions including hydroxide (OH-), proposed
previously11, as well as acetate (OAc) unambiguously identified in the lowest quality PbI2
examined here. Oxidic impurities are notoriously difficult to remove from Pb halides,19 but
reducing their concentration has been shown to have a positive effect on perovskite devices.11 In
contrast, the high quality PbI2 sources investigated displayed relatively low concentrations of
oxidic anion impurities. One PbI2 reagent, in particular, simultaneously indicated elevated levels
of potassium (K) and superstoichiometry (I:Pb > 2) suggesting KI as a plausible impurity. When
oxidic anion concentrations are low, the beneficial effect of even small amounts of alkali iodide
impurities like KI20-25 may become visible, helping to explain this PbI2’s high material quality and
device performance. Simply put, the beneficial effects of K can be easily overshadowed by other
impurities. This was substantiated by purifying the as-received PbI2 sources via aqueous
recrystallization which reduced both OAc and adventitious K, among other impurities, and
amended stoichiometries nearer to 2. Finally, we compared devices fabricated from all ten 4 as-received and recrystallized PbI2 sources which, in combination with the analytical
characterization, provides insight regarding dominant impurities and rationalizes expected
outcomes in devices. Our work demonstrates that a competition can and does exist between
beneficial (e.g. KI) and detrimental (e.g. oxidic anions) impurities which influence the overall
performance of the materials at the device level. While we suspect some impurities remain unidentified and/or unquantified, analytical
verification of key species such as OAc and K allowed us to better explain observed differences
in precursor solubility, deviation from nominal stoichiometry, and correlations to device
performance. This work additionally demonstrates that common analytical techniques provide
accessible means to evaluate purity levels of perovskite precursors and that practical purification
methods can effectively raise the purity of most PbI2. These techniques are relevant to fundamental
scientists for understanding the impact of synthetic conditions and intrinsic versus extrinsic
material properties, as well as to applied scientists for tactics to improve quality control,
performance, and reproducibility. Abbreviations used throughout the manuscript for the five PbI2 products, arranged according to
measured stoichiometry, are shown in Table 1, along with their nominal purity, physical form,
I:Pb ratio, and impurity information inferred from our data. TOC GRAPHIC The nomenclature is based on the
supplier name and the number of “9s” in the quoted purity using metals basis or trace metals basis
when reported. 5 Table 1. Abbreviation (Abbr.), product information, stoichiometry, solubility, and highlighted
impurities suggested by SIMS and NMR data for the five commercial PbI2 sources evaluated in
this study, listed in order of I:Pb ratio measured by ICP-OES. Abbr. Supplier
Purity
Form
I:Pb
ratio*
Solubility
Confirmed
Suspected
Impurities
Impurities
SA2
Sigma Aldrich
99%
Powder
1.84
Good
Na, K, OAc
OH-, O2-
SA5
Sigma Aldrich
99.999%
Powder
1.92
Poor**
Na, K
OH-, O2-, OAc
AA4
Alfa Aesar
99.9985%
Powder
1.97
Poor
Na, K, organics
NO3-
TCI4
TCI
99.99%
Powder
2.03
Good
organics
AA5
Alfa Aesar
99.999%
Beads
2.10
Moderate
KI
* ICP-OES data has an average error of +/- 5.8 % with a maximum error of +/- 6.7%
**Would not dissolve in perovskite inks precluding device fabrication. **Would not dissolve in perovskite inks precluding device fabrication. **Would not dissolve in perovskite inks precluding device fabrication. Perfectly pure, stoichiometric PbI2 should display an I:Pb ratio equal to two; therefore,
stoichiometry is suggestive of overall purity or quality,15 but might not be predictively reliable. Table 1 and Fig. 1a show I:Pb ratios of the as-received PbI2 materials (measured by ICP-OES)
varied from 8% deficient to 5% excess in iodide. Despite statistical considerations, the trend in as-
received I:Pb ratio appears reflected in the corresponding device efficiency in Fig. 1b reported as
stabilized power output (SPO). A similar trend in as-received PbI2 efficiency is observed in devices
with differing perovskite compositions, processing, and architectures made by different
researchers (Supplementary Figures S1 and S2) confirming that the noted effects are not limited
to a specific composition, architecture, nor processing route. Note that SA5 is omitted from as-
received device sets because its solubility was too low to produce films adequate for devices. We subsequently performed an aqueous recrystallization of all five PbI2 sources to remove
impurities that might alter the measured stoichiometry. As shown in Fig. 1a, the variation in I:Pb
ratio was successfully reduced among the newly recrystallized (RC) PbI2 reagents. Most 6 6 significant, the I:Pb ratio of the two outliers, SA2 and AA5, became statistically equivalent and
much closer to two. Devices were fabricated from the recrystallized PbI2, and the measured SPO
in Fig. 1b shows efficiency no longer correlates to the new stoichiometry. TOC GRAPHIC However, though it is
significantly less obvious, the performance trend resembles that observed for the as-received
materials. Note the recrystallized device data includes SA5 that became soluble after
recrystallization, and the solubility of AA4 also improved slightly, which is a consistent
improvement observed by several groups.11,13 This experiment illustrates that I:Pb ratio of the PbI2
reagent has some influence on the perovskite quality, but it is not a reliable indicator of device
efficiency. Device level metrics are likely dependent on the specific impurities that alter I:Pb ratio,
so we turned to more in-depth characterization techniques to reveal the identity of the dominant
impurity species. significant, the I:Pb ratio of the two outliers, SA2 and AA5, became statistically equivalent and
much closer to two. Devices were fabricated from the recrystallized PbI2, and the measured SPO
in Fig. 1b shows efficiency no longer correlates to the new stoichiometry. However, though it is
significantly less obvious, the performance trend resembles that observed for the as-received
materials. Note the recrystallized device data includes SA5 that became soluble after
recrystallization, and the solubility of AA4 also improved slightly, which is a consistent
improvement observed by several groups.11,13 This experiment illustrates that I:Pb ratio of the PbI2
reagent has some influence on the perovskite quality, but it is not a reliable indicator of device
efficiency. Device level metrics are likely dependent on the specific impurities that alter I:Pb ratio,
so we turned to more in-depth characterization techniques to reveal the identity of the dominant
impurity species. 7 7 Figure 1. (a) I:Pb ratio measured by ICP-OES for as-received PbI2 reagents compared to after
aqueous recrystallization. (b) Stabilized power output (SPO) of devices fabricated with as-received
and recrystallized PbI2 precursors. (c) 1H-NMR of Sigma Aldrich 99% PbI2 as-received (SA2), the
SA2 sample spiked with a ~3 mg particle of lead acetate trihydrate (Pb[OAc]2•3H2O), and a sample
of recrystallized SA2 (SA2-RC). (d) Integrated SIMS K+ ion intensity (summation of K counts
normalized to total ion counts over 43 data points) of the PbI2 powders as-received and after
recrystallization (denoted by -RC suffix). Figure 1. (a) I:Pb ratio measured by ICP-OES for as-received PbI2 reagents compared to afte
lli
i
(b) S bili
d
(SPO) f d
i
f b i
d
i h
i
d Figure 1. (a) I:Pb ratio measured by ICP-OES for as-received PbI2 reagents compared to after
aqueous recrystallization. TOC GRAPHIC (b) Stabilized power output (SPO) of devices fabricated with as-received
and recrystallized PbI2 precursors. (c) 1H-NMR of Sigma Aldrich 99% PbI2 as-received (SA2), the
SA2 sample spiked with a ~3 mg particle of lead acetate trihydrate (Pb[OAc]2•3H2O), and a sample
of recrystallized SA2 (SA2-RC). (d) Integrated SIMS K+ ion intensity (summation of K counts
normalized to total ion counts over 43 data points) of the PbI2 powders as-received and after
recrystallization (denoted by -RC suffix). Figure 1. (a) I:Pb ratio measured by ICP-OES for as-received PbI2 reagents compared to after
aqueous recrystallization. (b) Stabilized power output (SPO) of devices fabricated with as-received
and recrystallized PbI2 precursors. (c) 1H-NMR of Sigma Aldrich 99% PbI2 as-received (SA2), the
SA2 sample spiked with a ~3 mg particle of lead acetate trihydrate (Pb[OAc]2•3H2O), and a sample
of recrystallized SA2 (SA2-RC). (d) Integrated SIMS K+ ion intensity (summation of K counts
normalized to total ion counts over 43 data points) of the PbI2 powders as-received and after
recrystallization (denoted by -RC suffix). Using 1H-NMR, we identified OAc as a major anionic impurity in as-received SA2 (Fig. 1c)
which we confirmed by spiking with lead acetate trihydrate and additional 13C-NMR (inset). 8 8 Acetate is likely remnant from PbI2 synthesis which commonly involves mixing water soluble
Pb(OAc)2 or Pb(NO3)2 salts with KI to precipitate PbI2.12-14,26,27 Spiking experiments quantified
the OAc concentration as ~2.2 ± 0.3 mol% with respect to Pb (Supplementary Figure S3). It is
particularly interesting to note the agreement between our inferred synthetic origin of SA2 and
reports by Eckstein et al. that the Pb(OAc)2 route to PbI2 typically results in lower stoichiometry
than via Pb(NO3)2.27 The measured amount of OAc accounts for only one fourth of the full 8%
iodide deficiency in SA2, implying other extrinsic impurities are present in similar percentages. Figure 1c additionally shows that OAc was largely removed by the aqueous recrystallization to
which we partially attribute the increase in stoichiometry and improved device performance for
SA2 (Fig. 1a and 1b). Since the OAc concentration, by itself, does not fully explain the device
performance trends, either before or after recrystallization, we performed TOF-SIMS to identify
species not detectable by NMR. TOC GRAPHIC Acetate is likely remnant from PbI2 synthesis which commonly involves mixing water soluble
Pb(OAc)2 or Pb(NO3)2 salts with KI to precipitate PbI2.12-14,26,27 Spiking experiments quantified The most notable impurity we identified with TOF-SIMS was K, which also likely originates
from synthesis. Figure 1d shows the integrated K+ intensity of the PbI2 materials as-received and
after recrystallization (denoted by -RC suffix) highlighting large variation in relative K
concentration by 3-4 orders of magnitude among the 10 different samples. The relative SIMS
intensities for other alkali cations such as Li, Na, and Rb only varied by ~1 order of magnitude
among the PbI2 (Supplementary Figure S4), which justifies our focus on K in relation to changes
in device performance. As-received, AA5 displays K+ intensity 2 orders of magnitude higher than
TCI4, the second-highest quality and most pure reagent (see below). Hydrocarbon/carbohydrate
(Fig. 2a), oxide/hydroxide (Fig. 2b), and nitrogen oxide (Fig. 2c) SIMS fragment intensities
suggest TCI4 and AA5 contain the lowest concentrations of oxygen-containing anion impurities
(Fig. 2a fragment intensities further reflect significant OAc impurity in SA228). Together, these 9 9 two measurements on AA5 – low oxidic anion concentrations and I:Pb ratio greater than 2 – imply
that KI is the main impurity in this commercial source. For all reagents except AA5, due to I:Pb ≤
2, the K impurities can be regarded as KOH, KOAc, or KNO3. Potassium iodide is known as a
beneficial processing additive for perovskite devices20-25, and residual KI in AA5 plausibly
explains the observation that AA5-derived perovskites slightly outperform those processed from
TCI4 in all our device data sets (Fig. 1b and Supplementary Fig. S1 and S2). Finally,
recrystallization reduced K+ intensity in AA5 by nearly 3 orders of magnitude (Fig. 1d). The
relative decrease in other alkali cations was a maximum of ~ 1 order of magnitude, again
suggesting the large drop in K for AA5 is significant. Removing KI from a PbI2 reagent free of
anionic impurities is consistent with the observed decrease in ICP-OES stoichiometry (Fig. 1a)
and lower device performance (Fig. 1b) after recrystallization. two measurements on AA5 – low oxidic anion concentrations and I:Pb ratio greater than 2 – imply
that KI is the main impurity in this commercial source. For all reagents except AA5, due to I:Pb ≤
2, the K impurities can be regarded as KOH, KOAc, or KNO3. TOC GRAPHIC Potassium iodide is known as a
beneficial processing additive for perovskite devices20-25, and residual KI in AA5 plausibly
explains the observation that AA5-derived perovskites slightly outperform those processed from
TCI4 in all our device data sets (Fig. 1b and Supplementary Fig. S1 and S2). Finally,
recrystallization reduced K+ intensity in AA5 by nearly 3 orders of magnitude (Fig. 1d). The
relative decrease in other alkali cations was a maximum of ~ 1 order of magnitude, again
suggesting the large drop in K for AA5 is significant. Removing KI from a PbI2 reagent free of
anionic impurities is consistent with the observed decrease in ICP-OES stoichiometry (Fig. 1a)
and lower device performance (Fig. 1b) after recrystallization. 10 Figure 2. Integrated ion fragment SIMS intensities of (a) hydrocarbons and carbohydrates Figure 2. Integrated ion fragment SIMS intensities of (a) hydrocarbons and carbohydrates, (b)
id
d h d
id
d ( )
it
id
i
th
i
d PbI
Th
f
t Figure 2. Integrated ion fragment SIMS intensities of (a) hydrocarbons and carbohydrates, (b)
oxide and hydroxide, and (c) nitrogen oxides in the as-received PbI2 sources. The fragments
associated with acetate are indicated by * in (a).28 Either TCI4 or AA5 displays the lowest intensity
for each fragment. Figure 2. Integrated ion fragment SIMS intensities of (a) hydrocarbons and carbohydrates, (b)
oxide and hydroxide, and (c) nitrogen oxides in the as-received PbI2 sources. The fragments
associated with acetate are indicated by * in (a).28 Either TCI4 or AA5 displays the lowest intensity
for each fragment. The stoichiometry and device results are only partially explained by OAc and KI impurities,
suggesting perovskite material quality is sensitive to other trace impurities that remain difficult to
identify. For example, increased nitrogen oxide SIMS intensities for AA4 (Fig. 2c) suggest
elevated nitrate impurities in this reagent that was removed by purification (Supplementary Figure
S5). Indeed, several additional molecular impurities that might impact perovskite synthesis are 11 visible by 1H-NMR of the as-received PbI2s (Fig. 3a). There are unassigned, hydrogen-containing
impurities present in all as-received PbI2 sources except AA5 (SA2 additionally showed a small
impurity peak downfield at 10.7 ppm). Recrystallized materials in Fig. 3b display reduced
concentrations of original 1H-NMR impurities (compare, for example, the quartet at 3.54 ppm in
AA4 and AA4-RC in Fig. 3a and Fig. TOC GRAPHIC 3b, respectively), yet contain small signals from new
impurities, one of which was identified as isopropanol (IPA, doublet at 0.99 ppm) that we used
during filtration and collection. The NMR and additional SIMS data (Supplementary Fig. S4-S6)
all indicate the recrystallization decreases impurity concentrations in most categories. However,
OAc and K removal by recrystallization exemplify a competition between detrimental and
beneficial impacts of impurities. Therefore, while recrystallizing PbI2 may increase the purity, the
degree to which the purified reagent improves the perovskite active layer and resulting devices
may vary due to a given process’s sensitivity to the trace beneficial impurities removed, as well as
new trace species introduced, by the purification. 12 Figure 3. Upfield region 1H-NMR spectra of PbI2 sources as-received (a) and after aqueous
lli
i
(b) d
d b
RC
ffi
(i
d
d di
h l
lf
id
[DMSO d6]) Th Figure 3. Upfield region 1H-NMR spectra of PbI2 sources as-received (a) and after aqueous
recrystallization (b) denoted by -RC suffix (in deuterated dimethylsulfoxide [DMSO-d6]). The
DMSO-d5 solvent peak is omitted by the x-axis break and several impurity peak positions are
indicated for clarity. Figure 3. Upfield region 1H-NMR spectra of PbI2 sources as-received (a) and after aqueous
recrystallization (b) denoted by -RC suffix (in deuterated dimethylsulfoxide [DMSO-d6]). The
DMSO-d5 solvent peak is omitted by the x-axis break and several impurity peak positions are
indicated for clarity. Consequently, researchers may leverage this information about the different major impurities in
as-received PbI2 reagents or incorporate a purification protocol to best suit their goals. Our
observations help rationalize difficulties reproducing results with PbI2 from different suppliers or
even batch-to-batch reproducibility from a single supplier. Improvements to purification methods
and analytical characterization are necessary efforts for fundamental halide perovskite studies;
accurate quantification of the impact of impurities and additives requires first reducing their levels
to negligible concentrations before titrating back into the reagents. Ultimately, aqueous
recrystallization methods may not achieve a desirable purity level nor optimal perovskite quality
in devices, but alternative methods such as dissolution and precipitation from dimethylsulfoxide 13 (DMSO)11 or sublimation in the dark (to avoid photolysis26,29.30) under vacuum remain viable and
should be simultaneously explored. Although the ideal purification technique relevant to
perovskite semiconductors remains to be identified, fundamental- and device-oriented research
will benefit from deeper analytical insight into perovskite precursors and the resulting materials. TOC GRAPHIC In summary, we studied a set of five commercial PbI2 precursor sources often used to fabricate
halide perovskite devices. Cumulative information gathered from three analytical chemistry
techniques elucidate relationships between synthesis, I:Pb stoichiometry, impurities, properties,
and performance. We substantiated our conclusions by performing purifications via aqueous
recrystallization and evaluated its effectiveness. Our semiquantitative analysis provides significant
insight into specific impurities such as acetate and K that influence the functional performance of
the resulting halide perovskite material. Identifying the remaining impurities, and optimizing
purification methods, will help to clarify subtle yet impactful compositional variations that
influence lab-to-lab or process-specific differences, and contribute to perovskites reaching their
full potential. Like all semiconductor materials preceding it, the halide perovskite technology will
undoubtedly benefit from rigorous scrutiny of precursor composition and purification methods. Notes The authors declare no competing financial interest. ASSOCIATED CONTENT Supporting Information. Detailed materials lists, device fabrication and testing, and
characterization methods, performance results of two additional device composition-architectures
using the commercial PbI2 reagents, 1H-NMR quantification of acetate figure, three figures with
additional TOF-SIMS data. 14 ACKNOWLEDGMENT This work was authored by the National Renewable Energy Laboratory, operated by Alliance for
Sustainable Energy, LLC, for the U.S. Department of Energy (DOE) under Contract No. DE-
AC36-08GO28308. Funding provided by U.S. Department of Energy Office of Energy Efficiency
and Renewable Energy, Solar Energy Technologies Office (SETO) project “De-risking Halide
Perovskite Solar Cells” program (DE-FOA-0000990). R.A.K. acknowledges support from the
Laboratory Directed Research and Development (LDRD) Program at NREL. The views expressed
in the article do not necessarily represent the views of the DOE or the U.S. Government. The U.S. Government retains and the publisher, by accepting the article for publication, acknowledges that
the U.S. Government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or
reproduce the published form of this work, or allow others to do so, for U.S. Government purposes. 1. Steirer, K. X.; Schulz, P.; Teeter, G.; Stevanovic, V.; Yang, M.; Zhu, K.; Berry, J. J. Defect
Tolerance in Methylammonium Lead Triiodide Perovskite. ACS Energy Lett. 2016, 1, 360–
366. AUTHOR INFORMATION Corresponding author contact: David.Moore@nrel.gov
Notes 8. Schoonman, J. The Ionic Conductivity of Pure and Doped Lead Bromide Single Crystals.
J. Solid State Chem., 1972, 4, 466–474. REFERENCES 1. Steirer, K. X.; Schulz, P.; Teeter, G.; Stevanovic, V.; Yang, M.; Zhu, K.; Berry, J. J. Defect
Tolerance in Methylammonium Lead Triiodide Perovskite. ACS Energy Lett. 2016, 1, 360–
366. 15 2. Walsh, A.; Scanlon, D. O.; Chen, S.; Gong, X. G.; Wei, S.-H. Self-Regulation Mechanism
for Charged Point Defects in Hybrid Halide Perovskites. Angew. Chem. Int. Ed., 2015, 54,
1791–1794. 3. Yin, W.-J.; Shi, T.; Yan, Y. Unusual defect physics in CH3NH3PbI3 Perovskite Solar Cell
Absorber. Appl. Phys. Lett., 2014, 104, 063903. 4. Zhang, X.; Turiansky, M. E.; Shen, J.-X.; Van de Walle, C. G. Iodine Interstitials as a
Cause of Nonradiative Recombination in Hybrid Perovskites. Phys. Rev. B, 2020, 101,
140101(R). 5. Shin, D.; Zu, F.; Cohen, A. V.; Yi, Y.; Kronik, L.; Koch, N. Mechanism and Timescales
of Reversible p-Doping of Methylammonium Lead Triiodide by Oxygen. Adv. Mater.,
2021, 33, 2100211. 6. Aristidou, N.; Eames, C.; Sanchez-Molina, I.; Bu, X.; Kosco, J.; Islam, M. S.; Haque, S. A. Fast Oxygen Diffusion and Iodide Defects Mediate Oxygen-Induced Degradation of
Perovskite Solar Cells. Nat. Comm., 2017, 8, 15218. 7. Bryant, D.; Aristidou, N.; Pont, S.; Sanchez-Molina, I.; T. Chotchuangchutchaval, T.;
Wheeler, S.; Durrant, J. R.; Haque, S. A. Light and Oxygen Induced Degradation Limits
the Operational Stability of Methylammonium Lead Triiodide Perovskite Solar Cells. Energy Environ. Sci., 2016, 9, 1655–1660. 8. Schoonman, J. The Ionic Conductivity of Pure and Doped Lead Bromide Single Crystals. J. Solid State Chem., 1972, 4, 466–474. 16 9. Yao, J.; Yang, L.; Cai, F.; Yan, Y.; Gurney, R. S.; Liu, D.; Wang, T. The Impacts of PbI2
Purity on the Morphology and Device Performance of One-step Spray-coated Planar
Heterojunction Perovskite Solar Cells. Sustain. Energy Fuels, 2018, 2, 436–443. 10. Chang, J.; Zhu, H.; Li, B.; Isikgor, F. H.; Hao, Y.; Xu, Q.; Ouyang, J. Boosting the
Performance of Planar Heterojunction Perovskite Solar Cell by Controlling the Precursor
Purity of Perovskite Materials. J. Mater. Chem. A, 2016, 4, 887–893. 11. Senevirathna, D. C.; Yu, J. C.; Peiris, T. A. N.; Li, B.; Michalska, M.; Li, H.; Jasieniak, J. J. Impact of Anion Impurities in Commercial PbI2 on Lead Halide Perovskite Films and
Solar Cells. ACS Materials Lett. 2021, 3, 351–355. 12. Chen, B.; Fei, C.; Chen, S.; Gu, H.; Xiao, X.; Huang, J. Recycling Lead and Transparent
Conductors from Perovskite Solar Modules. Nat. Comm. 2021, 12, 5859. 13. Lee, C.-H.; Shin, Y.-J.; Jeon, G. 15. Tsevas, K.; Smith, J. A.; Kumar, V.; Rodenburg, C.; Fakis, M.; Yusoff, A. R. b. M.;
Vasilopoulou, M.; Lidzey, D. G.; Nazeeruddin, M. K.; Dunbar, A. D. F. Controlling PbI2 21. Tang, Z.; Bessho, T.; Awai, F.; Kinoshita, T.; Maitani, M. M.; Jono, R.; Murakami, T. N.;
Wang, H.; Kubo, T.; Uchida, S.; Segawa, H. Hysteresis-free Perovskite Solar Cells Made
of Potassium-doped Organometal Halide Perovskite. Sci. Rep., 2017, 7, 12183. REFERENCES G.; Kang, D.; Jung, J.; Jeon, B.; Park, J.; Kim, J.; Yoon,
S. J. Cost-efficient, Effect of Low-Quality PbI2 Purification to Enhance Performances of
Perovskite Quantum Dots and Perovskite Solar Cells. Energies, 2021, 14, 201. 14. Lee, C.-H.; Shin, Y.-J.; Villanueva-Antolí, A.; Adhikari, S. D.; Rodriguez-Pereira, J.;
Macak, J. M.; Mesa, C. A.; Giménez, S.; Yoon, S. J.; Gualdrón-Reyes, A. F.; Mora-Seró,
I. Efficient and Stable Blue- and Red-Emitting Perovskite Nanocrystals through Defect
Engineering: PbX2 Purification. Chem. Mater. 2021, 33, 8745–8757. 15. Tsevas, K.; Smith, J. A.; Kumar, V.; Rodenburg, C.; Fakis, M.; Yusoff, A. R. b. M.;
Vasilopoulou, M.; Lidzey, D. G.; Nazeeruddin, M. K.; Dunbar, A. D. F. Controlling PbI2 17 Stoichiometry during Synthesis to Improve the Performance of Perovskite Photovoltaics. Chem. Mater. 2021, 33, 554–566. Stoichiometry during Synthesis to Improve the Performance of Perovskite Photovoltaics. Chem. Mater. 2021, 33, 554–566. 16. Min, H.; Kim, M.; Lee, S.-U.; Kim, H.; Choi, K.; Lee, J. H.; Seok, S. I. Efficient, Stable
Solar Cells by Using Inherent Bandgap of α-phase Formamidinium Lead Iodide. Science,
2019, 366, 749–753. 17. Zhang, Y.; Seo, S.; Lim, S. Y.; Kim, Y.; Kim, S.-G.; Lee, D.-K.; Lee, S.-H.; Shin, H.;
Cheong, H.; Park, N.-G. Achieving Reproducible and High-Efficiency (>21%) Perovskite
Solar Cells with a Presynthesized FAPbI3 Powder. ACS Energy Lett. 2020, 5, 360–366. 18. Tong, G.; Son, D.-Y.; Ono, L, K.; Kang, H.-B.; He, S.; Qiu, L.; Zhang, H.; Liu, Y.; Hieulle,
J.; Qi, Y. Removal of Residual Compositions by Powder Engineering for High Efficiency
Formamidinium-based Perovskite Solar Cells with Operation Lifetime Over 2000 h. Nano
Energy, 2021, 87, 106152. 19. Tonna, J.; Matuchovab, M.; Danilewsky, A. N.; Cröll, A. Removal of Oxidic Impurities
for the Growth of High Purity Lead Iodide Single Crystals. J. Cryst. Growth, 2015, 416,
82–89. 20. Zhao, W.;Yao, Z.; Yu, F.; Yang, D.; Liu, S. (F.). Alkali Metal Doping for Improved
CH3NH3PbI3 Perovskite Solar Cells. Adv. Sci., 2018, 5, 1700131. 21. Tang, Z.; Bessho, T.; Awai, F.; Kinoshita, T.; Maitani, M. M.; Jono, R.; Murakami, T. N.;
Wang, H.; Kubo, T.; Uchida, S.; Segawa, H. Hysteresis-free Perovskite Solar Cells Made
of Potassium-doped Organometal Halide Perovskite. Sci. Rep., 2017, 7, 12183. 18 22. Abdi-Jalebi, M.; Andaji-Garmaroudi, Z.; Cacovich, S.; Stavrakas, C.; Philippe, B.; Richter,
J. M.; Alsari, M.; Booker, E. P.; Hutter, E. M.; Pearson, A. J.; Lilliu, S.; Savenije, T. J.;
Rensmo, H.; Divitini, G.; Ducati, C.; Friend, R. 30. Fornaro, L.; Saucedo, E.; Mussio, L.; Yerman, L.; X. Ma, X.; Burger, A. Lead iodide film
deposition and characterization. Nucl. Instrum. Methods., 2001, 458, 406–412. 29. Saucedo, E.; Fornaro, L.; Mussio, L.; Gancharov, A. New Ways for Purifying Lead Iodide
Appropriate as Spectrometric Grade Material. IEEE Trans. Nucl. Sci., 2002, 49, 1974–
1977. REFERENCES H.; Stranks, S. D. Maximizing and
stabilizing luminescence from halide perovskites with potassium passivation. Nature,
2018, 555, 497–501. 23. Yang, Y.; Wu, L.; Hao, X.; Tang, Z.; Lai, H.; Zhang, J.; Wang, W.; Feng, L. Beneficial
effects of potassium iodide incorporation on grain boundaries and interfaces of perovskite
solar cells. RSC Adv., 2019, 9, 28561–28568. 24. Son, D.-Y.; Kim, S.-G.; Seo, J.-Y.; Lee, S.-H.; Hyunjung Shin, H.; Lee, D.; Park, N.-G. Universal Approach toward Hysteresis-Free Perovskite Solar Cell via Defect Engineering. J. Am. Chem. Soc., 2018, 140, 1358–1364. 25. Su, R.; Xu, Z.; Wu, J.; Luo, D.; Hu, Q.; Yang, W.; Yang, X.; Zhang, R.; Yu, H.; Russell,
T. P.; Gong, Q.; Zhang, W.; Zhu, R. Dielectric screening in perovskite photovoltaics. Nat. Comm. 2021, 12, 2479. 26. Albrecht, M.G. (1975). The photolysis of lead iodide. PhD Thesis (University of London,
Imperial College). 27. Eckstein, J.; Erler, B.; Benz, K. W. High purity lead iodide for crystal growth and its
characterization. Mater. Res. Bull., 1992, 27, 537–544. 28. NIST Standard Reference Database 69: NIST Chemistry WebBook Acetic acid (nist.gov),
https://webbook.nist.gov/cgi/cbook.cgi?ID=C64197&Units=SI&Mask=200#Mass-Spec,
date accessed 11/26/2021 19 29. Saucedo, E.; Fornaro, L.; Mussio, L.; Gancharov, A. New Ways for Purifying Lead Iodide
Appropriate as Spectrometric Grade Material. IEEE Trans. Nucl. Sci., 2002, 49, 1974–
1977. 30. Fornaro, L.; Saucedo, E.; Mussio, L.; Yerman, L.; X. Ma, X.; Burger, A. Lead iodide film
deposition and characterization. Nucl. Instrum. Methods., 2001, 458, 406–412. 20
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Ultrastructural Analysis of<i>Leishmania infantum chagasi</i>Promastigotes Forms Treated<i>In Vitro</i>with Usnic Acid
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Correspondence should be addressed to Jo˜ao S. B. da Luz; j.soaresbrito704@gmail Received 31 October 2014; Revised 12 January 2015; Accepted 16 January 2015 Academic Editor: Thomas Efferth Copyright © 2015 Jo˜ao S. B. da Luz et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited. Leishmaniasis is considered by the World Health Organization as one of the infectious parasitic diseases endemic of great relevance
and a global public health problem. Pentavalent antimonials used for treatment of this disease are limited and new phytochemicals
emerge as an alternative to existing treatments, due to the low toxicity and cost reduction. Usnic acid is uniquely found in lichens
and is especially abundant in genera such as Alectoria, Cladonia, Evernia, Lecanora, Ramalina, and Usnea. Usnic acid has been
shown to exhibit antiviral, antiprotozoal, antiproliferative, anti-inflammatory, and analgesic activity. The aim of this study was
to evaluate the antileishmanial activity of usnic acid on Leishmania infantum chagasi promastigotes and the occurrence of drug-
induced ultrastructural damage in the parasite. Usnic acid was effective against the promastigote forms (IC50 = 18.30 ± 2.00 𝜇g/mL). Structural and ultrastructural aspects of parasite were analyzed. Morphological alterations were observed as blebs in cell membrane
and shapes given off, increasing the number of cytoplasmic vacuoles, and cellular and mitochondrial swelling, with loss of cell
polarity. We concluded that the usnic acid presented antileishmanial activity against promastigote forms of Leishmania infantum
chagasi and structural and ultrastructural analysis reinforces its cytotoxicity. Further, in vitro studies are warranted to further
evaluate this potential. Hindawi Publishing Corporation
e Scientific World Journal
Volume 2015, Article ID 617401, 7 pages
http://dx.doi.org/10.1155/2015/617401 Hindawi Publishing Corporation
e Scientific World Journal
Volume 2015, Article ID 617401, 7 pages
http://dx.doi.org/10.1155/2015/617401 Hindawi Publishing Corporation
e Scientific World Journal
Volume 2015, Article ID 617401, 7 pages
http://dx.doi.org/10.1155/2015/617401 1Pos-Graduation Program in Pathology, Federal University of Pernambuco, Avenida Prof. Moraes Rego 1235,
50670-901 Recife, PE, Brazil f
2Pos-Graduation Program in Therapeutic Innovation, Federal University of Pernambuco, Avenida Prof. Moraes Rego 1235,
50670-901 Recife, PE, Brazil 2Pos-Graduation Program in Therapeutic Innovation, Federal University of Pernambuco, Avenida Prof. Moraes Rego 1235,
50670-901 Recife, PE, Brazil f
3Department of Histology and Embryology, Federal University of Pernambuco, Avenida Prof. Moraes Rego 1235,
50670-901 Recife, PE, Brazil 3Department of Histology and Embryology, Federal University of Pernambuco, Avenida Prof. Moraes Rego 1235,
50670-901 Recife, PE, Brazil f ,
,
4Department of Biochemistry, Federal University of Pernambuco, Avenida Prof. Moraes Rego 1235, 50670-901 Recife, PE, Brazil
5Laboratory of Immunopathology Keizo Asami (LIKA) and Research Center Aggeu Magalh˜aes, CPqAM/FIOCRUZ, Federal University
of Pernambuco, Avenida Prof. Moraes Rego, 1235, 50670-901 Recife, PE, Brazil f
4Department of Biochemistry, Federal University of Pernambuco, Avenida Prof. Moraes Rego 1235, 50670-901 Recife, PE, Brazil
5Laboratory of Immunopathology Keizo Asami (LIKA) and Research Center Aggeu Magalh˜aes, CPqAM/FIOCRUZ, Federal University
f P
b
A
d P
f M
R
R
f
PE B
l 4Department of Biochemistry, Federal University of Pernambuco, Avenida Prof. Moraes Rego 1235, 50670-901 Rec 5Laboratory of Immunopathology Keizo Asami (LIKA) and Research Center Aggeu Magalh˜aes, CPqAM/FIOCRUZ
of Pernambuco, Avenida Prof. Moraes Rego, 1235, 50670-901 Recife, PE, Brazil Correspondence should be addressed to Jo˜ao S. B. da Luz; j.soaresbrito704@gmail.com Correspondence should be addressed to Jo˜ao S. B. da Luz; j.soaresbrito704@gmail.com Leishmaniasis is endemic in areas of tropics and sub-
tropics, including southern Europe, Asia, Africa, and the
Americas [3–5].
Leishmania infantum chagasi, a trypanosomatid parasite,
is an etiological agent of visceral leishmaniasis (VL) in the
American continent and it is now admitted to be the same
species causing visceral leishmaniasis in Europe and certain
parts of Africa [6]. João S. B. da Luz,1,2,3 Erwelly B. de Oliveira,3 Monica C. B. Martins,4
Nicácio H. da Silva,4 Luiz C. Alves,5 Fábio A. B. dos Santos,5 Luiz L. S. da Silva,1,3
Eliete C. Silva,1,2,3 and Paloma L. de Medeiros1,2,3 João S. B. da Luz,1,2,3 Erwelly B. de Oliveira,3 Monica C. B. Martins,4
Nicácio H. da Silva,4 Luiz C. Alves,5 Fábio A. B. dos Santos,5 Luiz L. S. da Silva,1,3
Eliete C. Silva,1,2,3 and Paloma L. de Medeiros1,2,3 1. Introduction After 72 h of incubation
with usnic acid and amphotericin B 25 𝜇L of MTT solution
was added (5 mg/mL). After 3 h of incubation at 37∘C, MTT
solution was aspirated and 100 𝜇L DMSO was added for the
solubilization of the formazan crystals. After solubilization,
the absorbance was determined in a spectrophotometer at
585 nm wavelength. The results were expressed as percentage
of relative viability of cells to the negative control group [32]. All the experiments were performed in triplicate. Several natural products with antileishmanial activity
have been recently reported, including naphthoquinones,
lignans, neolignans, alkaloids, flavanol, and terpenoids [13,
14], but none has reached clinical use. Lichens synthesize over eight hundred types of metabo-
lites [15] and depsides, depsidones, dibenzofurans, xanthenes,
anthraquinones, and usnic acids are amongst the more
extensively studied lichen metabolites [16]. The search for
new phytochemicals with antiparasitic action emerges as an
alternative to existing treatments, due to the low toxicity and
cost reduction. Some studies showed that usnic acid, secondary metabo-
lite of the lichen Cladonia substellata Vainio, was initially
used in the treatment of pulmonary tuberculosis [17], and
there are data regarding its biological activities as antibiotic
[18], antiproliferative [19], analgesic and antipyretic [20], anti-
inflammatory [21], antiviral [22, 23], antifungal [24], against
the parasite Trypanosoma cruzi [25], and an immunologic
modulator [26]. In addition, its mutagenic and cytotoxic
activities have been determined against normal and malig-
nant human cells lines [27–30].f 2.4. Scanning Electron Microscopy. To evaluate parasite ultra-
structural alterations by scanning electron microscopy, Leish-
mania infantum chagasi promastigotes were grown for 72 h
as described in LIT-DMSO or the same medium containing
25 𝜇g/mL usnic acid; they were subsequently collected by
centrifugation at 1500 g, washed twice in 0.1 M Phosphate
buffer (pH 7.2), and fixed in a solution containing 2.5% glu-
taraldehyde, 4% paraformaldehyde, and 0.1 M T phosphate
buffer. After washing twice in the same buffer, the parasites
were adhered to glass slides previously coated with 0.1%
poly-I-lysine for 30 min at 37∘C. Subsequently, the slides
were washed twice with 0.1 M phosphate buffer, postfixed in
solution of OsO4 for 1 h at room temperature, and washed
twice again with 0.1 M phosphate buffer. 1. Introduction All samples were
dehydrated gradually increasing the ethanol concentrations
(30–100%) and were critical point dried using CO2, mounted
on metal stubs, and coated with gold (5–30 nm) for observa-
tion in a scanning electron microscope (JEOL T-200). Taking into account the side effects and the resistance that
pathogenic protozoan parasites develop against drugs cur-
rently used in the treatment of leishmaniasis, more atten-
tion should be given to extracts and biologically active
compounds isolated from plant species commonly used in
herbal medicine. Thus, in this work, we have analyzed the
in vitro effect of usnic acid on growth and ultrastructure of
Leishmania infantum chagasi promastigote forms. 1. Introduction Leishmaniasis is endemic in areas of tropics and sub-
tropics, including southern Europe, Asia, Africa, and the
Americas [3–5]. Leishmaniasis is one of the most neglected diseases that
remain a public health problem worldwide, affecting approxi-
mately 12 million people in more than 98 countries, with more
than 350 million people at risk and when not treated, death
is expected to occur after a period of four months to one year
[1, 2]. Leishmania infantum chagasi, a trypanosomatid parasite,
is an etiological agent of visceral leishmaniasis (VL) in the
American continent and it is now admitted to be the same
species causing visceral leishmaniasis in Europe and certain
parts of Africa [6]. 2 2 The Scientific World Journal by Dr. Osvaldo Pomp´ılio de Melo Neto at Department
of Microbiology of Research Center Aggeu Magalh˜aes,
FIOCRUZ. The promastigotes were routinely grown in Liver
Infusion Tryptose medium (LIT) at 26∘C, supplemented
with 10% heat-inactivated fetal bovine serum (FBS) (LGC
Biotechnology), 0.1% penicillin and streptomycin, and 0.2%
hemin (Sigma). Pentavalent antimonials such as sodium stibogluconate
and meglumine antimoniate have been used in the treatment
of all forms of leishmaniasis for more than half a century;
although the mechanism of action of pentavalent antimonial
is not fully understood, it is generally accepted that the active
form of the metal is the reduced form Sb (III), which can
lead to serious limitations due to high toxicity and resistance
to drugs [2, 7]. In addition, a major problem in antimonial
chemotherapy is the emergence of clinical resistance against
pentavalent antimonial drugs that has reached epidemic
proportions in parts of India [8–10]. Newer drugs such as mil-
tefosine and amphotericin B have shown promising results,
but they also cause some side effects that may limit their use. In this context, the discovery of new active and promising
compounds with antileishmanial potential remains essential
for control and prevention of leishmaniasis [11, 12]. 2.3. In Vitro Antileishmanial Activity. The antileishmanial ac-
tivity was assessed by the colorimetric method MTT [3-{4.5-
dimethylthiazol-2-yl}-2.5-diphenyl-tetrazolium]
(SIGMA)
that is based on the conversion of the tetrazolium salt into-
the colored formazan product, the concentration of which
can be determined spectrophotometrically. The promasti-
gotes were seeded (2 × 106 cells/mL) with LIT medium in
96-well microplates and incubated with 50 𝜇L of different
concentrations of usnic acid (100 to 0.195 𝜇g/mL) and
amphotericin B (positive control). LIT medium (50 𝜇L)
(negative control) was also added. 2.2. Parasites Culture. The strain of Leishmania infantum
chagasi (MHOM/BR2000/Merivaldo2) was kindly provided 3. Results Usnic acid
Amphotericin B Amphotericin B The antileishmanial activity was directly proportional to
usnic acid and amphotericin B concentrations. Usnic acid
antileishmanial activity was estimated by the IC50 concentra-
tion at 72 h after incubation. Usnic acid was found to exhibit a
lower inhibitory activity against Leishmania infantum chagasi
(IC50 = 18.30 ± 2.00 𝜇g/mL) than the reference drug ampho-
tericin B (1.36 ± 0.067 𝜇g/mL) (Figure 1).h Figure 1: In vitro effects of different concentrations of usnic acid
and amphotericin B on the growth of Leishmania infantum chagasi
promastigote forms. Means followed by different letters indicating
statistically significant differences according to the Anova-Tukey test
(𝑃< 0.05, compared to controls). The analysis of scanning electron micrographs of treated
parasites demonstrated that usnic acid affected the parasite
surface. Morphological alterations were observed as the
presence of blebs in the cell membrane and of shapes given
off. No ultrastructural change was observed in promastigotes
grown with LIT-DMSO for 72 h, showing the elongated
normal morphology (Figures 2(a)–2(f)). (atranorin and norstictic acid), IC50 values for Leishmania
infantum chagasi at concentrations of 30 and 40 𝜇g/mL,
respectively. p
y
In a literature review conducted until October 2006 few
references regarding the leishmanicidal activity of com-
pounds isolated from lichens were found. Fournet et al. [35]
revealed that among the lichen substances tested in vitro
against promastigote forms of L. braziliensis, L. amazonensis
and L. donovani, the usnic acid presented total lysis of
parasites in the concentration of 25 𝜇g/mL. The promastigote
forms of L. amazonensis and L. braziliensis under the effect of
usnic acid exhibited lysis of parasites at concentration of
10 𝜇g/mL (80–90%) and against L. donovani the activity
was total. Already, depsidones (panarin and 1-cloropanarin)
were active only in concentrations equal to or greater than
50 𝜇g/mL. gy
g
Morphological alterations of Leishmania infantum cha-
gasi promastigotes treated with 25 𝜇g/mL usnic acid were
observed by transmission electron microscopy. The com-
pound caused changes in the cytoplasm density, cell swelling,
and loss of cell polarity. Changes in mitochondrial mor-
phology were characterized by marked swelling. We also
noticed an increased number of intracellular vacuoles. Blebs
were observed in the plasma membrane and detached from
the membrane. It was also observed that the membrane
was separated from the cytoplasm and there was a higher
accumulation of fat compared to controls. 2. Materials and Methods 2.1. Extraction of Usnic Acid. Usnic acid was isolated from
the crude extract of the lichen Cladonia substellata Vainio,
and was obtained from Dr. Nic´acio Henrique da Silva at
Department of Biochemistry, Federal University of Pernam-
buco. Fractionation and purification of this compound were
performed as previously described [31]. A stock solution was
prepared at 10 mg/mL in DMSO and stored at 4∘C until use. 2.5. Transmission Electron Microscopy. Control and treated
culture promastigotes with 25 𝜇g/mL were harvested, at room
temperature by centrifugation at 1500 g, washed twice in PBS,
and fixed for 2 h at 4∘C in a solution containing 2.5%
glutaraldehyde, 4% paraformaldehyde, and 0.1 M cacodylate
buffer, pH 7.2. After washing in this similar buffer, cells were
postfixed for 1 h in a solution containing 1% OsO4, 0.8%
potassium ferricyanide, and 5 mM CaCl2 in 0.1 M cacodylate
buffer, pH 7.2. The cells were then dehydrated in acetone and
embedded in epoxy resin. Ultrathin sections were stained 2.5. Transmission Electron Microscopy. Control and treated
culture promastigotes with 25 𝜇g/mL were harvested, at room
temperature by centrifugation at 1500 g, washed twice in PBS,
and fixed for 2 h at 4∘C in a solution containing 2.5%
glutaraldehyde, 4% paraformaldehyde, and 0.1 M cacodylate
buffer, pH 7.2. After washing in this similar buffer, cells were
postfixed for 1 h in a solution containing 1% OsO4, 0.8%
potassium ferricyanide, and 5 mM CaCl2 in 0.1 M cacodylate
buffer, pH 7.2. The cells were then dehydrated in acetone and
embedded in epoxy resin. Ultrathin sections were stained 2.2. Parasites Culture. The strain of Leishmania infantum
chagasi (MHOM/BR2000/Merivaldo2) was kindly provided 3 The Scientific World Journal with uranyl acetate and lead citrate and examined in a Zeiss
EM 109 transmission electron microscope. Viability (%) 2.6. Statistical Analysis. Statistical analysis of growth dif-
ferences between treated and control cultures (IC50) was
performed using the Anova with Tukey test, with 𝑃
<
0.05. The results were expressed as mean values ± standard
deviation (S.D.). 2.6. Statistical Analysis. Statistical analysis of growth dif-
ferences between treated and control cultures (IC50) was
performed using the Anova with Tukey test, with 𝑃
<
0.05. The results were expressed as mean values ± standard
deviation (S.D.). 3. Results Nucleus was
observed with presence of an electron-lucent space, when
compared to the control (Figures 3(a)–3(f)). Scanning electron micrographs reveal the formation of
blebs in the plasma membrane, membrane rupture, and
loss of polarity. da Silva et al. [6] when using warifteine
drug against promastigotes of Leishmania infantum chagasi
found similar results in scanning microscopy, such as blebs,
suggesting that usnic acid metabolism operates through a
change in intracellular calcium concentration.f 4. Discussion Usnic acid is the most abundant constituent of various
species of lichens, including those belonging to the genus
Cladonia and Usnea [33]. This compound is known to present
several biological activities, acting as an antiprotozoan [34,
35] and antiparasitic [25]. Research with usnic acid in vitro
against promastigote forms of Leishmania sp. demonstrated
the effectiveness of usnic acid in their antileishmanial activity
[35].h Comparing the ultrastructural effects of usnic acid with
those produced by other drug (amiodarone), against the
promastigotes and amastigotes forms of L. amazonensis
evaluated by da Silva et al. [6], was observed similar effects on
the membranes of parasites, such as lipid accumulation and
loss of integrity of nuclear envelope and plasma membrane,
leading to cell death by necrosis. The concentration which inhibited 50% growth of a
culture of Leishmania infantum chagasi promastigote forms
obtained in our experiments (18.30 𝜇g/mL) was close to that
obtained by de Carvalho et al. [25] who detected an IC50
around 20 𝜇g/mL for another trypanosomatid parasite (T. cruzi). Our results demonstrated a promising leishmanicidal
activity of usnic acid against the Leishmania infantum chagasi
promastigote forms in vitro when confronted with the study
of Jota et al. [36] who obtained for other depsides from lichens According to Abo-Khatwa et al. [37] usnic acid would
be similar to the classical uncoupler 2, 4-dinitrophenol
(DNP) oxidative phosphorylation, and thereby promote ATP
depletion. Furthermore, usnic acid has lipophilic character-
istics and ionizing could alter the mitochondrial membrane,
causing damage of this organelle [15]. Einarsd´ottir et al. [38] showed that mitochondrial mem-
brane potential loss appears to be dose-dependent with The Scientific World Journal (a)
(b)
(c)
(d)
∗
(e)
(f)
anning electron micrographs of Leishmania infantum chagasi promastigotes exposed to usnic acid. (a) C
, c, d, e, and f) treated group usnic acid at a concentration of 25 𝜇g/mL for 72 hours. Blebs on the surface
membrane rupture (full arrow), swelling (asterisk), and the formation pore (arrowhead). Bars: 1 𝜇m (a, d);
s: (a) 11.000x, (b) 5.000x, (c) 8.000x, (d) 13.000x, (e) 5.500x, and (f) 8.500x. (a) (b) (b) (a) (d) (c) (d) (c) ∗
(e) (f) (f) (e) Figure 2: Scanning electron micrographs of Leishmania infantum chagasi promastigotes exposed to usnic acid. (a) Control group (no
treatment); (b, c, d, e, and f) treated group usnic acid at a concentration of 25 𝜇g/mL for 72 hours. 4. Discussion Blebs on the surface of promastigotes
(thin arrows), membrane rupture (full arrow), swelling (asterisk), and the formation pore (arrowhead). Bars: 1 𝜇m (a, d); 2 𝜇m (c, e and f). Magnifications: (a) 11.000x, (b) 5.000x, (c) 8.000x, (d) 13.000x, (e) 5.500x, and (f) 8.500x. Adade and Souto-Padr´on [39] studying the ultrastruc-
tures of the genera Trypanosoma and Leishmania regarding
the antiparasitic drugs observed that the ultrastructural
effects are similar among the various drugs used as well as
between herbal medicines, including usnic acid.hf respect to the effect of usnic acid in the two cell lines such as
breast cancer T-47D and pancreatic cancer Capan-2. These
data demonstrate and confirm one of the mechanisms of
action of usnic acid previously cited by M¨uller [15], where
changes in membrane lipid composition alter the physical
properties with loss of mitochondrial function. We suggest,
based on prior observations, that the usnic acid destabilized
membranes allowing the leishmanicidal effect. The study in question was relevant to effectiveness of
usnic acid in the face of Leishmania infantum chagasi pro-
mastigote forms, which leads us to suggest it as a possible 5 The Scientific World Journal 10𝜇m
N
Ct
V
V
V
L
L
L L
(a)
20𝜇m
Bl
Bl
dM
(b)
10𝜇m
V
V
V V
V
L
L
Bd
N
Bf
Mt
(c)
10𝜇m
V
V
dM
dM
Bd
Bd
Bd
(d)
10𝜇m
Mt
Ct
(e)
10𝜇m
V
dM
Ep
(f)
Figure 3: Transmission electron micrographs promastigote forms of Leishmania infantum chagasi. (a) Control group; (b), (c), (d), (e), and (f)
treated groups (AU) at a concentration of 25 𝜇g/mL for 72 hours. Vacuoles (V), acidocalcisomes (arrows), nucleus (N), lipid (L), kinetoplast
(Ct), flagellar pocket (Bf), mitochondrial swelling (Mt), detachment of the plasma membrane (dM), blebs (Bl), detached blebs (Bd), and
electron-lucent space (Ep). Bars 10 𝜇m (a, c, d, e, and f), Bars 20 𝜇m (b). 20𝜇m
Bl
Bl
dM
(b) 10𝜇m
N
Ct
V
V
V
L
L
L L
(a) (a) (b) 10𝜇m
V
V
V V
V
L
L
Bd
N
Bf
Mt
(c) 10𝜇m
V
V
dM
dM
Bd
Bd
Bd
(d) (d) (c) 10𝜇m
V
dM
Ep
(f) 10𝜇m
Mt
Ct
(e) (f) (e) Figure 3: Transmission electron micrographs promastigote forms of Leishmania infantum chagasi. (a) Control group; (b), (c), (d), (e), and (f)
treated groups (AU) at a concentration of 25 𝜇g/mL for 72 hours. References phytotherapic agent in the treatment of visceral leishmani-
asis. In this context, further studies are needed to elucidate
the mechanisms of action of usnic acid and its physiological
effects in vivo. [1] World Health Organization, “Technical report series: control of
leishmaniases,” Report of a Meeting of the WHO Expert Com-
mittee on the Control of Leishmaniases, WHO, Geneva, Switz-
erland, 2010, http://whqlibdoc.who.int/ trs/WHO TRS 949 eng
.pdf. [2] C. Dos Santos Ferreira, P. Silveira Martins, C. Demicheli, C. Brochu, M. Ouellette, and F. Fr´ezard, “Thiol-induced reduction
of antimony(V) into antimony(III): a comparative study with
trypanothione, cysteinyl-glycine, cysteine and glutathione,”
BioMetals, vol. 16, no. 3, pp. 441–446, 2003. Conflict of Interests The authors declare that there is no conflict of interests that
could affect the impartiality of the research reported. [3] J.-C. Dujardin, L. Campino, C. Ca˜navate et al., “Spread of
vector-borne diseases and neglect of leishmaniasis, Europe,”
Emerging Infectious Diseases, vol. 14, no. 7, pp. 1013–1018, 2008. 4. Discussion Vacuoles (V), acidocalcisomes (arrows), nucleus (N), lipid (L), kinetoplast
(Ct), flagellar pocket (Bf), mitochondrial swelling (Mt), detachment of the plasma membrane (dM), blebs (Bl), detached blebs (Bd), and
electron-lucent space (Ep). Bars 10 𝜇m (a, c, d, e, and f), Bars 20 𝜇m (b). Acknowledgments [4] C. M. Machado, T. C. Martins, I. Colturato et al., “Epidemiology
of neglected tropical diseases in transplant recipients. Review
of the literature and experience of a Brazilian HSCT center,”
Revista do Instituto de Medicina Tropical de S˜ao Paulo, vol. 51,
no. 6, pp. 309–324, 2009. The authors like to thank the PROPESQ/UFPE, the Coor-
dination for the Graduated Personnel (CAPES), and Aggeu
Magalh˜aes Institute/Oswaldo Cruz Foundation (CPqAM-
FIOCRUZ). The Scientific World Journal 6 and whose main target is RNA transcription,” Biochimie, vol. 84,
no. 4, pp. 329–334, 2002. and whose main target is RNA transcription,” Biochimie, vol. 84,
no. 4, pp. 329–334, 2002. [5] A. Pavli and H. C. Maltezou, “Leishmaniasis, an emerging infec-
tion in travelers,” International Journal of Infectious Diseases,
vol. 14, no. 12, pp. e1032–e1039, 2010. [23] [23] P. Scirpa, G. Scambia, V. Masciullo et al., “Adjuvant treatment
of HPV infection. A perspective and controlled study on the
efficacy and safety of intravaginal administration of usnic acid
and zinc sulphate compound,” Minerva Ginecologica, vol. 51, no. 6, pp. 255–260, 1999. [6] E. C. da Silva, C. D. Rayol, P. L. Medeiros et al., “Antileishmanial
activity of warifteine: a bisbenzylisoquinoline alkaloid isolated
from Cissampelos sympodialis Eichl. (Menispermaceae),” The
Scientific World Journal, vol. 2012, Article ID 516408, 5 pages,
2012. [24] P. Halama and C. van Haluwin, “Antifungal activity of lichen
extracts and lichenic acids,” BioControl, vol. 49, no. 1, pp. 95–
107, 2004. [7] J. Chakravarty and S. Sundar, “Drug resistance in leishmaniasis,”
Journal of Global Infectious Diseases, vol. 2, no. 2, pp. 167–176,
2010. [25] E. A. B. de Carvalho, P. P. Andrade, N. H. Silva, E. C. Pereira,
and R. C. B. Q. Figueiredo, “Effect of usnic acid from the
lichen Cladonia substellata on Trypanosoma cruzi in vitro: an
ultrastructural study,” Micron, vol. 36, no. 2, pp. 155–161, 2005. [8] P. L. Olliaro, P. J. Guerin, S. Gerstl, A. A. Haaskjold, J. A. Rottingen, and S. Sundar, “Treatment options for visceral
leishmaniasis: a systematic review of clinical studies done in
India, 1980-2004,” The Lancet Infectious Diseases, vol. 5, no. 12,
pp. 763–774, 2005. [26] L. C. Santos, N. K. Honda, I. Z. Carlos, and W. Vilegas,
“Intermediate reactive oxygen and nitrogen from macrophages
induced by Brazilian lichens,” Fitoterapia, vol. 75, no. 5, pp. 473–
479, 2004. [9] S. L. Croft, K. Seifert, and V. Acknowledgments Yardley, “Current scenario of
drug development for leishmaniasis,” Indian Journal of Medical
Research, vol. 123, no. 3, pp. 399–410, 2006. [27] F. Bucar, I. Schneider, H. ¨Ogmundsd´ottir, and K. Ing´olfsd´ottir,
“Anti-proliferative lichen compounds with inhibitory activity
on 12(S)-HETE production in human platelets,” Phytomedicine,
vol. 11, no. 7-8, pp. 602–606, 2004. [10] S. Sundar, D. K. More, M. K. Singh et al., “Failure of pentavalent
antimony in visceral leishmaniasis in India: report from the
center of the Indian epidemic,” Clinical Infectious Diseases, vol. 31, no. 4, pp. 1104–1107, 2000. [28] M. Mayer, M. A. O’Neill, K. E. Murray et al., “Usnic acid: a non-
genotoxic compound with anti-cancer properties,” Anti-Cancer
Drugs, vol. 16, no. 8, pp. 805–809, 2005. [11] F. Chappuis, S. Sundar, A. Hailu et al., “Visceral leishmaniasis:
what are the needs for diagnosis, treatment and control?”
Nature Reviews Microbiology, vol. 5, no. 11, pp. 873–882, 2007. [29] N. P. da Silva Santos, S. C. Nascimento, M. S. O. Wanderley
et al., “Nanoencapsulation of usnic acid: improvement on the
antitumour activity and reduction of hepatotoxicity,” European
Journal of Pharmaceutics and Biopharmaceutics, vol. 64, no. 2,
pp. 154–160, 2006. [12] T. P. C. Dorlo, M. Balasegaram, J. H. Beijnen, and P. J. de vries,
“Miltefosine: a review of its pharmacology and therapeutic effi-
cacy in the treatment of leishmaniasis,” Journal of Antimicrobial
Chemotherapy, vol. 67, no. 11, pp. 2576–2597, 2012. [13] A. Fournet and V. Mu˜noz, “Natural products as trypanocidal,
antileishmanial and antimalarial drugs,” Current Topics in
Medicinal Chemistry, vol. 2, no. 11, pp. 1215–1237, 2002. [30] M.-A. Bazin, A.-C. Le-Lamer, J.-G. Delcros et al., “Synthesis
and cytotoxic activities of usnic acid derivatives,” Bioorganic &
Medicinal Chemistry, vol. 16, no. 14, pp. 6860–6866, 2008. [14] J. D. F. Inacio, M. M. Canto-Cavalheiro, and E. E. Almeida-
Amaral, “In vitro and in vivo effects of (−)-epigallocatechin
3-𝑂-gallate on Leishmania amazonensis,” Journal of Natural
Products, vol. 76, no. 10, pp. 1993–1996, 2013. [31] F. Odabasoglu, A. Cakir, H. Suleyman et al., “Gastroprotective
and antioxidant effects of usnic acid on indomethacin-induced
gastric ulcer in rats,” Journal of Ethnopharmacology, vol. 103, no. 1, pp. 59–65, 2006. [15] K. M¨uller,
“Pharmaceutically
relevant
metabolites
from
lichens,” Applied Microbiology and Biotechnology, vol. 56, no. 1-2, pp. 9–16, 2001. [32] T. Mosmann, “Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays,”
Journal of Immunological Methods, vol. 65, no. 1-2, pp. 55–63,
1983. [16] N. Acknowledgments K. Honda and W. Vilegas, “A qu´ımica dos liquens,” Qu´ımica
Nova, vol. 6, no. 21, pp. 110–125, 1998. [33] Y. Nishitoba, I. Nishimura, T. Nishiyama, and J. Mizutani,
“Lichen acids, plant growth inhibitors from Usnea longissima,”
Phytochemistry, vol. 26, no. 12, pp. 3181–3185, 1987. [17] A. Plichet, “Usnic acid from lichens in therapy of tuberculosis,”
La Presse M´edicale, vol. 63, no. 17, article 350, 1955. [18] M. Cocchietto, N. Skert, P. Nimis, and G. Sava, “A review
on usnic acid, an interesting natural compound,” Naturwis-
senschaften, vol. 89, no. 4, pp. 137–146, 2002. [34] J. Wu, M. Zhang, D. Ding, T. Tan, and B. Yan, “Effect of Cladonia
alpestris on Trichomonas vaginalis in vitro,” Chinese Journal of
Parasitology & Parasitic Disease, vol. 13, no. 2, pp. 126–129, 1995. [19] M. Cardarelli, G. Serino, L. Campanella et al., “Antimitotic
effects of usnic acid on different biological systems,” Cellular and
Molecular Life Sciences, vol. 53, no. 8, pp. 667–672, 1997. [35] A. Fournet, M. E. Ferreira, A. R. de Arias et al., “Activity of
compounds isolated from Chilean lichens against experimental
cutaneous leishmaniasis,” Comparative Biochemistry and Phys-
iology C: Pharmacology, Toxicology and Endocrinology, vol. 116,
no. 1, pp. 51–54, 1997. [20] E. Okuyama, K. Umeyama, M. Yamazaki, Y. Kinoshita, and Y. Yamamoto, “Usnic acid and diffractaic acid as analgesic and
antipyretic components of Usnea diffracta,” Planta Medica, vol. 61, no. 2, pp. 113–115, 1995. [36] V. C. S. Jota, P. P. Andrade, N. H. Silva, E. C. Pereira, R. Figueiredo, and N. K. Honda, “An´alise in vitro de substˆancias
liquˆenicas sobre Leishmania chagasi,” in II Encontro do Grupo
Brasileiro de Liquen´ologos (EGBL-2), Livro de Resumo, Campo
Grande, Brazil, 1998. [21] C. S. Vijayakumar, S. Viswanathan, M. Kannappa Reddy,
S. Parvathavarthini, A. B. Kundu, and E. Sukumar, “Anti-
inflammatory activity of (+)-usnic acid,” Fitoterapia, vol. 71, no. 5, pp. 564–566, 2000. [37] A. N. Abo-Khatwa, A. A. Al-Robai, and D. A. Al-Jawhari,
“Lichen acids as uncouplers of oxidative phosphorylation of
mouse-liver mitochondria,” Natural Toxins, vol. 4, no. 2, pp. 96–
102, 1996. [22] L. Campanella, M. Delfini, P. Ercole, A. Iacoangeli, and G. Risuleo, “Molecular characterization and action of usnic acid: a
drug that inhibits proliferation of mouse polyomavirus in vitro The Scientific World Journal 7 7 [38] E. Einarsd´ottir, J. Groeneweg, G. G. Bj¨ornsd´ottir et al., “Cellular
mechanisms of the anticancer effects of the lichen compound
usnic acid,” Planta Medica, vol. Acknowledgments 76, no. 10, pp. 969–974, 2010. [39] C. M. Adade and T. Souto-Padr´on, “Contributions of ultrastruc-
tural studies to the cell biology of trypanosmatids: targets for
anti-parasitic drugs,” The Open Parasitology Journal, vol. 4, no. 1, pp. 178–187, 2010.
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Experimental Investigation into Closed-Loop Control for HTPB-Based Hybrid Rocket Motors
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Aerospace
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cc-by
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aerospace aerospace aerospace Citation: Tan, G.; Tian, H.; Wang, Z.;
Guo, Z.; Gao, J.; Zhang, Y.; Cai, G. Experimental Investigation into
Closed-Loop Control for HTPB-Based
Hybrid Rocket Motors. Aerospace
2023, 10, 421. https://doi.org/
10.3390/aerospace10050421 Article
Experimental Investigation into Closed-Loop Control for
HTPB-Based Hybrid Rocket Motors Guang Tan 1,2, Hui Tian 1,2,*, Zhongshuo Wang 1,2, Zihao Guo 1,2, Jingfei Gao 1,2, Yuanjun Zhang 3
and Guobiao Cai 1,2 Guang Tan 1,2, Hui Tian 1,2,*, Zhongshuo Wang 1,2, Zihao Guo 1,2, Jingfei Gao 1,2, Yuanjun Zhang 3
and Guobiao Cai 1,2 1
School of Astronautics, Beihang University, Beijing 100191, China 1
School of Astronautics, Beihang University, Beijing 100191, China
2
Key Laboratory of Spacecraft Design Optimization & Dynamic Simulation Technologies,
Ministry of Education, Beijing 100816, China
3
Research Institute of Aero-Engine, Beihang University, Beijing 100191, China
*
Correspondence: tianhui@buaa.edu.cn 1
School of Astronautics, Beihang University, Beijing 100191, China
2
Key Laboratory of Spacecraft Design Optimization & Dynamic Simulation Technologies,
Ministry of Education, Beijing 100816, China
3
Research Institute of Aero-Engine, Beihang University, Beijing 100191, China
*
Correspondence: tianhui@buaa.edu.cn 1
School of Astronautics, Beihang University, Beijing 100191, China
2
Key Laboratory of Spacecraft Design Optimization & Dynamic Simulation Technologies,
Ministry of Education, Beijing 100816, China
3
Research Institute of Aero-Engine, Beihang University, Beijing 100191, China
*
Correspondence: tianhui@buaa.edu.cn g
y
j
g
2
Key Laboratory of Spacecraft Design Optimization & Dynamic Simulation Technologies
Ministry of Education, Beijing 100816, China *
Correspondence: tianhui@buaa.edu.cn *
Correspondence: tianhui@buaa.edu.cn Abstract: Space exploration greatly facilitates the development of advanced propulsion systems. Extensive research has shown that hybrid rocket motors have bright prospects for use in variable-
thrust propulsion systems. However, the variable-thrust precision control of a hybrid rocket motor
with a high-mass fraction of aluminum has not been adequately explored. In this paper, we propose
a closed-loop control system for a high-performance laboratory-scale hybrid rocket motor, and verify
its performance through tests on a hybrid rocket motor containing 98% hydrogen peroxide and
hydroxyl-terminated polybutadiene with 58% of an aluminum additive. The results show that, first,
the average value of thrust in the stable sections in the three stages were 400.7 N, 599.1 N, and 400.1 N
when the target values were 400 N, 600 N, and 400 N, respectively. Second, the average thrust was
stable, and the control error of the average value was better than 0.5%. Third, the real-time error
in thrust was controlled to within ± 20 N with a steady-state error smaller than 5%. These results
indicate that the proposed closed-loop control strategy for hybrid rocket motors with a high-mass
fraction of aluminum can maintain a constant thrust and smooth transitions in case of variable thrust. Article
Experimental Investigation into Closed-Loop Control for
HTPB-Based Hybrid Rocket Motors Keywords: experimental investigation; closed-loop control system; hybrid rocket motors; thrust regulation aerospace aerospace 1. Introduction The use of hybrid rocket motors in a variety of fields has promoted research on effective
and accurate motor control. They have received considerable attention in studies on space
missions due to their safety, easily controlled thrust, on-demand restart capability, and low
cost [1,2]. They also play a supporting role in missions involving space transportation [3,4],
manned lunar landings [5,6], and Mars vehicles [7,8]. The control of hybrid rocket motors
thus warrants further research, especially from the perspectives of their specific impulse
performance and accuracy of thrust control. Academic Editor: Stephen Whitmore Received: 30 March 2023
Revised: 24 April 2023
Accepted: 27 April 2023
Published: 29 April 2023 Received: 30 March 2023
Revised: 24 April 2023
Accepted: 27 April 2023
Published: 29 April 2023 Research on high-performance hybrid rocket motors has focused on their regression
rate and specific impulse [9–11] as well as the disadvantages incurred by their poor me-
chanical strength [12–14]. Our team has added metals to fuels of hydroxyl-terminated
polybutadiene (HTPB) to improve their performance. Adding 58% aluminum to HTPB-
based fuels improves the specific impulse and regression rate [15,16], but large amounts
of aluminum additives lead to nozzle ablation, uncertainty in regression rate, and the
instability of combustion pressure [15,17,18] that directly yield unpredictable and unstable
thrust and indirectly lead to debris emissions [5,19,20]. Therefore, we seek to compensate
for nozzle ablation and oscillations in thrust by regulating the flow rate of the oxidizer from
the perspective of the control system. In this way, we can ensure the stability of the thrust
and improve the accuracy of thrust control during the operation of a high-performance
hybrid rocket motor. Copyright:
© 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article
distributed
under
the
terms
and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). https://www.mdpi.com/journal/aerospace Aerospace 2023, 10, 421. https://doi.org/10.3390/aerospace10050421 Aerospace 2023, 10, 421 2 of 21
le thro
b id Thrust control has long been a vital and challenging issue in research on rocket motors. The thrust control of liquid rocket motors is mainly achieved by adjusting the flow rates of
the oxidizer and the fuel. This is complicated because it requires simultaneously regulating
the mass flow rates of both based on their mixing ratio [21,22]. 1. Introduction Thrust control is realized
in solid rocket motors by changing the area of the nozzle throat by using a pintle valve
or secondary-flow injection [23,24]. Thrust control in hybrid rocket motors is achieved by
adjusting the flow rate of the oxidizer by using a control valve [25]. This suggests that it is
easy to control the thrust of hybrid rocket motors. A turndown ratio of 67:1 in the thrust
was reported in one study by using an inexpensive off-the-shelf throttle valve in a hybrid
rocket motor containing nitrous oxide and HTPB [26]. Closed-loop throttle control is easier
to implement in a hybrid rocket motor due to the controllability of its thrust [27]. Past
research has reported the closed-loop throttle control of a laboratory-scale hybrid rocket
motor containing nitrous oxide and HTPB [28]. The results showed that closed-loop throttle
control significantly reduced the mean run-to-run variations in thrust from ±9.1% to lower
than ±3.9%. The variation in thrust was reduced to ±1.5% when the effects of erosion of
the nozzle were considered [28]. A closed-loop thrust control scheme for a laboratory-scale
paraffin wax/nitrous oxide hybrid rocket motor was implemented by using a low-cost ball
valve as the controlling hardware. The results of the experiments showed that the motor
remained throttled within ± 2.4% of the maximum thrust [29]. y
j
g
y
g
gests that it is easy to control the thrust of hybrid rocket motors. A turndow
in the thrust was reported in one study by using an inexpensive off-the-shelf
in a hybrid rocket motor containing nitrous oxide and HTPB [26]. Closed
control is easier to implement in a hybrid rocket motor due to the contro
thrust [27]. Past research has reported the closed-loop throĴle control of a lab
hybrid rocket motor containing nitrous oxide and HTPB [28]. The results
closed-loop throĴle control significantly reduced the mean run-to-run varia
from ±9.1% to lower than ±3.9%. The variation in thrust was reduced to ±1
effects of erosion of the nozzle were considered [28]. A closed-loop thrust co
for a laboratory-scale paraffin wax/nitrous oxide hybrid rocket motor was
by using a low-cost ball valve as the controlling hardware. The results of th
showed that the motor remained throĴled within ± 2.4% of the maximum th
Adding 58% of an aluminum additive to HTPB can significantly impro
sion rate and specific impulse of the hybrid rocket motor [15]. 1. Introduction However, osci
thrust of the propellant containing an aluminum additive are severer than
propellant without an aluminum additive [30] The pressure of the combus Adding 58% of an aluminum additive to HTPB can significantly improve the regression
rate and specific impulse of the hybrid rocket motor [15]. However, oscillations in the
thrust of the propellant containing an aluminum additive are severer than those in the
propellant without an aluminum additive [30]. The pressure of the combustion chamber
and thrust of hybrid rocket motors with a high mass fraction of aluminum are shown
in Figure 1. Unstable combustion is generated with the addition of a large number of
aluminum particles. Moreover, the attendant oscillations do not occur as the motor starts
operating but instead emerge after a certain duration of its operation. As combustion
proceeds, the oscillations in thrust gradually increase and the average thrust increasingly
varies. Adding a large number of aluminum particles to the fuel can increase the regression
rate and specific impulse of the motor, but leads to the new problem of wide fluctuations in
thrust that limit its use. Moreover, few closed-loop control systems use linear regulating
valves for a high-performance hybrid rocket motor with HTPB-based fuel with aluminum
additives. To fill this gap in research, we propose a closed-loop control system for a high-
performance hybrid motor in this study. We design a closed-loop throttle control strategy
to ensure constant thrust in the range of operation of the motor and a smooth transition
during variable thrust. propellant without an aluminum additive [30]. The pressure of the combus
and thrust of hybrid rocket motors with a high mass fraction of aluminum
Figure 1. Unstable combustion is generated with the addition of a large num
num particles. Moreover, the aĴendant oscillations do not occur as the mot
ating but instead emerge after a certain duration of its operation. As combus
the oscillations in thrust gradually increase and the average thrust increa
Adding a large number of aluminum particles to the fuel can increase the r
and specific impulse of the motor, but leads to the new problem of wide fl
thrust that limit its use. Moreover, few closed-loop control systems use line
valves for a high-performance hybrid rocket motor with HTPB-based fuel w
additives. To fill this gap in research, we propose a closed-loop control syste
performance hybrid motor in this study. 2.1. Incremental PID Control Algorithm We use the variable-area cavitating venturi as an actuator in this study. The input
to the actuator in each control cycle is the increase in the pintle of the valve. We use the
incremental PID control algorithm to solve for this. The formula of recurrence for the
incremental PID is obtained from the literature [18]: u(k) = kperror(k) + ki
k
∑
i=1
error(i) + kd[error(k) −error(k −1)]. (1) (1) The deviation in the control signal error(k) in the k-th control cycle is the difference between
the set value yd(k) and the measured value y(k) in the current cycle. error(k) = yd(k) −y(k)
(2) (2) The output of the k-th control cycle is the increment ∆u(k), which is the difference
between the total output of the current control cycle u(k) and the total output of the previous
control cycle u(k −-1). ∆u(k) = u(k) −u(k −1)
(3) (3) The increment ∆u(k) can be obtained by combining Equation (1) with Equation (3): (k) −error(k −1)) + kierror(k) + kd(error(k) −2error(k −1) + error(k −2)),
(4) (4) where ∆u(k) depends only on the control parameters (kp, ki, and kd) and deviations in the
control signal (u(k), u(k −1), and u(k −2)). Therefore, incorrect movements of the control
valve have a weak impact on the control system such that it maintains its stability. 1. Introduction We design a closed-loop throĴle co
to ensure constant thrust in the range of operation of the motor and a smo
during variable thrust. Figure 1. Pressure in the combustion chamber and thrust of the hybrid rocket motor [30]. Figure 1. Pressure in the combustion chamber and thrust of the hybrid rocket motor [30]. Aerospace 2023, 10, 421 3 of 21 3 of 21 Based on the above considerations, the major objective of this study is to improve
the applicability of the variable-thrust hybrid rocket motor with a high mass fraction of
aluminum through a method of closed-loop control. We use the design of a segmented
proportional–integral–derivative (PID) controller to control the average thrust and achieve
a smooth transition in variable thrust. We verified the design through one hot firing test
on a hybrid rocket motor containing 98% hydrogen peroxide and HTPB with 58% of an
aluminum additive. 2. Control System 2.1. Incremental PID Control Algorithm 2.2. Segmented PID Algorithm The above algorithm can ensure both the rapidity of transitions of the thrust and the
accuracy of its steady states. 2.3. Quasi-Steady-State Assumptions
The object of control in this paper is a laboratory sized hybrid rocket motor Because 2.3. Quasi-Steady-State Assumptions The object of control in this paper is a laboratory-sized hybrid rocket motor. Because
many disturbing factors related to dynamic changes are in play during actual ignition
tests of hybrid rocket motors, it is difficult to determine them one by one. For example,
the hybrid rocket motor has different characteristics of thrust response to the flow rate of
the oxidizer in different stages of its operation, where the diameter of the grain port and
the area of combustion are variable. Therefore, we make quasi-steady-state assumptions
regarding the hybrid rocket motor to simplify the object of control: 1. The first half of the control cycle of the motor is dynamic and the flow rate is variable. The displacement of the pintle valve is fixed in the second half of the control cycle
and the flow rate is constant. 2. The dynamic characteristics of the motor during the first half of the control cycle
are neglected. 3. The motor reaches a quasi-steady state before the end of the second half of the
control cycle. 4. A quasi-steady state means that the motor reaches a steady state at the end of the control
cycle, and its performance-related parameters satisfy the characteristic static equations The purpose of the quasi-steady-state assumptions is to simplify the equations of
dynamic control into characteristic static equations of control, at the cost of waiting for
half a control cycle so that the motor reaches a quasi-steady state. A certain opening of
the valve corresponds to a certain flow rate in the characteristic static equations, and this
corresponds to a certain thrust. There is no dynamic term in the relationship. 2.2. Segmented PID Algorithm Because the hybrid rocket engine considered in this study uses 98% hydrogen peroxide
and HTPB fuel containing 58% Al as a propellant, the thrust signal oscillates severely, and
deviations in the control signal error(k) are uncertain and random. To ensure the stability
and accuracy of control of the thrust, we limit the calculated control parameters and the
increment in control ∆u(k) to within a narrow range such that the signal response of actual
thrust is slow. When the set target thrust changes sharply, the deviation in the control
signal error(k) is large within a short period, and a long response time is required to reach
the target thrust in this case. We propose the segmented PID algorithm to appropriately
balance the response time and the accuracy of control of the variations in thrust. The idea underlying the segmented PID algorithm is as follows: When the actual
thrust deviates significantly from the set thrust, large values of the control parameters (kp0,
ki0, and kd0) are used. The output increment in control ∆u(k) is thus larger and the thrust
response is fast. When the deviation in thrust is reduced to within a certain range, small
control parameters (kp1, ki1, and kd1) are chosen to avoid overshooting, and to ensure the
stability and accuracy of the thrust response of the motor. The use of segmented control
can simultaneously ensure the rapidity, accuracy, and stability of the thrust response of the
motor. The implementation of the segmented PID algorithm consists of three steps. First,
we set the threshold value ε > 0. Second, we select the control parameters. Larger control
parameters (kp0, ki0, and kd0) are used when |error(k)| > ε, and smaller control parameters Aerospace 2023, 10, 421 4 of 21 (kp1, ki1, and kd1) obtained through calculations are used when |error(k)| < ε. Finally, the
segmented incremental PID control algorithm can be expressed as: ∆u(k) =
kp0(error(k) −error(k −1)) + ki0error(k) + kd0(error(k) −2error(k −1) + error(k −2))
|error(k)>ε|
kp1(error(k) −error(k −1)) + ki1error(k) + kd1(error(k) −2error(k −1) + error(k −2))
|error(k)<ε|. (5) ∆u(k) =
kp0(error(k) −error(k −1)) + ki0error(k) + kd0(error(k) −2error(k −1) + error(k −2))
|error(k)>ε|
kp1(error(k) −error(k −1)) + ki1error(k) + kd1(error(k) −2error(k −1) + error(k −2))
|error(k)<ε|. (5) (5) The above algorithm can ensure both the rapidity of transitions of the thrust and the
accuracy of its steady states. rol Valve
2.5. Flow Control Valve
thrust of the motor b o
a ve
ator of the control system is a flow control valve that indirectly regulates the
motor by adjusting the flow rate of the oxidizer. The flow control valve is
ntrol loop because its linearity and response characteristics significantly in-
The actuator of the control system is a flow control valve that indirectly regulates the
thrust of the motor by adjusting the flow rate of the oxidizer. The flow control valve is vital
in the control loop because its linearity and response characteristics significantly influence
the accuracy and response time of the control system. vital in the control loop because its linearity and response characteristics significa
fluence the accuracy and response time of the control system. We used a variable-area cavitating venturi as the flow control valve due to
vantages of high linearity, fast response, and isolation from fluctuations in the curacy and response time of the control system. a variable-area cavitating venturi as the flow control valve due to its ad-
igh linearity, fast response, and isolation from fluctuations in the down-
We used a variable-area cavitating venturi as the flow control valve due to its advan-
tages of high linearity, fast response, and isolation from fluctuations in the downstream
pressure. A schematic diagram of the variable-area cavitating venturi is shown in Figure 3. vantages of high linearity, fast response, and isolation from fluctuations in the
stream pressure. A schematic diagram of the variable-area cavitating venturi is sh
Figure 3. re. A schematic diagram of the variable-area cavitatin
Figure 3. Schematic of variable-area cavitating venturi [31
Figure 3. Schematic of variable-area cavitating venturi [31]. A schematic d Figure 3. Schematic of variable-area cavitating venturi
Figure 3. Schematic of variable-area cavitating venturi [31]. matic of variable-area cavitating venturi [31]. able-area cavitating venturi mainly consists of a servo motor, a transmission
intle, and a venturi tube. The servo motor converts the rotational motion of
aft into the linear motion of the pintle through the transmission assembly
The variable-area cavitating venturi mainly consists of a servo motor, a transm
assembly, a pintle, and a venturi tube. The servo motor converts the rotational mo
the output shaft into the linear motion of the pintle through the transmission ass
and the area of throttling changes with the linear motion of the pintle [32–34]. 2.4. Components of the Control System The PLC then converts these digital signals into real-time thrust values, and then
differentiates the latter from the set target thrust to obtain the deviation in control error(k)
in the current control cycle. converts the 4 20 mA analog signals into digital signals in the range of 0
LC then converts these digital signals into real-time thrust values, and then
the laĴer from the set target thrust to obtain the deviation in control error(k)
the
current
control
cycle
in
the
current
control in
the
current
control
cycle. Figure 2. Functional block diagram of the control system. Figure 2. Functional block diagram of the control system. Figure 2. Functional block diagram of the control system. 2.5. Flow Control Valve
The actuator of the control system is a flow control valve that indirectly reg ional block diagram of the control system. Figure 2. Functional block diagram of the control system. 2.5. Flow Control Valve
The actuator of the control system is a flow 2.4. Components of the Control System The object of control in this study is a laboratory-sized hybrid rocket motor and the
control variable is the thrust of the motor. The measuring element for collecting actual
thrust signals is an s-type compression force transducer. A variable-area cavitating venturi
is used as the actuator, and a programmable logic controller (PLC) is the value of the set
thrust-providing element. p
g
A functional block diagram of the control system is shown in Figure 2. The PLC serves
as the controller, provides the value of the target thrust, and implements the segmented
incremental PID algorithm. The output of the PLC is the control increment ∆u(k), which is
a certain number of high-speed pulses. The input signal to the servo motor driver consists
of high-speed pulses and the output is a PWM signal (pulse width modulation). The
servo motor driver actuates the servo motor to rotate at a certain angle depending on
the number of high-speed pulses. The servo motor converts rotational motion into linear
motion through couplings, ball screws, and other transmission devices to drive the pintle
of the variable-area cavitating venturi to move and change its throttling area, where this
changes the flow rate of the oxidizer. The hybrid rocket motor accepts the oxidizer from
the feed system of the propellant, and generates thrust through combustion and expansion. The thrust of the motor is transferred to the compression force transducer through the
thrust test bench. The compression force transducer converts the real-time thrust into an
output signal of 4–20 mA. This signal is subjected to low-pass filtering (100 Hz) through
an isolator to eliminate high-frequency interference from it. The analog-to-digital (AD)
conversion module converts the 4–20 mA analog signals into digital signals in the range of Aerospace 2023, 10, 421
test b
signa 5 of 21
he rang
lues an 0–32000. The PLC then converts these digital signals into real-time thrust values, and then
differentiates the latter from the set target thrust to obtain the deviation in control error(k)
in the current control cycle. converts the 4 20 mA analog signals into digital signals in the range of 0
LC then converts these digital signals into real-time thrust values, and then
the laĴer from the set target thrust to obtain the deviation in control error(k)
the
current
control
cycle
in
the
current
control 0–32000. rol Valve
2.5. Flow Control Valve
thrust of the motor b Th
tionship between the throttling area of the variable-area cavitating venturi and the
The variable-area cavitating venturi mainly consists of a servo motor, a transmission
assembly, a pintle, and a venturi tube. The servo motor converts the rotational motion of
the output shaft into the linear motion of the pintle through the transmission assembly, and
the area of throttling changes with the linear motion of the pintle [32–34]. The relationship
between the throttling area of the variable-area cavitating venturi and the pintle stroke is
shown in Figure 4. EW
6 haft into the linear motion of the pintle through the transmission assembly,
of throttling changes with the linear motion of the pintle [32–34]. The rela-
ween the throttling area of the variable-area cavitating venturi and the pintle
wn in Figure 4. stroke is shown in Figure 4. Figure 4. Design throĴling area of the variable-area cavitating venturi corresponding to the
stroke. 0
2
4
6
8
10
12
14
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Design results of throttling area
A (mm2)
x (mm)
Figure 4. Design throttling area of the variable-area cavitating venturi corresponding to the pintle stroke. Figure 4. Design throĴling area of the variable-area cavitating venturi corresponding to the
t
k
Figure 4. Design throttling area of the variable-area cavitating venturi corresponding to the pintle stroke. Aerospace 2023, 10, 421 6 of 21
uri wa When the pressure at the inlet of the variable-area cavitating venturi was almost
constant and it was in a state of cavitation, the flow rate of the oxidizer was determined by
the pintle stroke. The latter and the flow rate of the variable-area cavitating venturi obeyed
the following relationship [32–34]:
the pintle stroke. The latter and the flow rate of the variable-area cavitating ve
the following relationship [32–34]:
2
1
1
2 (
)
(
) 2 (
)
d
s
d
s
Q
C A
p
p
C ax
bx
c
p
p
. Q = CdA
q
2ρ(p1 −ps) ≈Cd(ax −bx2 + c)
q
2ρ(p1 −ps). rol Valve
2.5. Flow Control Valve
thrust of the motor b (6)
(6), Q is the mass flow rate (unit: kg/s), Cd is the coefficient of f
a (unit: m2), ρ is the density of the liquid (unit: m2), p1 is the tota (6)
of f
tota In Equation (6), Q is the mass flow rate (unit: kg/s), Cd is the coefficient of flow, A is the
throttling area (unit: m2), ρ is the density of the liquid (unit: m2), p1 is the total pressure
at the inlet of the variable-area cavitating venturi (unit: MPa), ps is the saturated vapor
pressure of the fluid at a given temperature (unit: MPa), x is the pintle stroke (unit: mm),
and a and b are constant coefficients measured by the cold flow test. In the test, the inlet
pressure of the venturi was 6 MPa; the outlet pressure of the venturi was atmospheric
pressure. The temperature of the ultrapure water was room temperature, about 15 ◦C. The
results of the cold flow test of the variable-area cavitating venturi are shown in Figure 5. g
ρ
y
q
p
the inlet of the variable-area cavitating venturi (unit: MPa), ps is the saturated
sure of the fluid at a given temperature (unit: MPa), x is the pintle stroke (un
a and b are constant coefficients measured by the cold flow test. In the test, th
sure of the venturi was 6 MPa; the outlet pressure of the venturi was atmos
sure. The temperature of the ultrapure water was room temperature, abou
results of the cold flow test of the variable-area cavitating venturi are shown Figure 5. Results of the cold flow test of the variable-area cavitating
0
2
4
6
8
10
12
14
0
50
100
150
200
250
300
350
400
Test data points (P1 = 6MPa)
Fitted curve of test data (Q=35.1346x−0.6740x2−1.7423)
Q (g/s)
x (mm)
Figure 5. Results of the cold flow test of the variable-area cavitating venturi. igure 5. Results of the cold flow test of the variable-area cavitatin
Figure 5. Results of the cold flow test of the variable-area cavitating venturi. rol Valve
2.5. Flow Control Valve
thrust of the motor b (8)
ox
ox
I
m
. mpulse of the oxidizer F is the thrust and 𝑚̇ ௫ (8) In Equation (8), Iox is the specific impulse of the oxidizer, F is the thrust, and
.mox is the flow
rate of the oxidizer. The specific impulse of the oxidizer represents the thrust produced
per unit mass of the oxidizer. This is different from the specific impulse that indicates the
thrust produced per unit mass of the oxidizer and the fuel. q
p
p
flow rate of the oxidizer. The specific impulse of the oxidizer represents the thru
duced per unit mass of the oxidizer. This is different from the specific impulse tha
cates the thrust produced per unit mass of the oxidizer and the fuel. Thus the thrust of the motor can be expressed as the product of the flow rate In Equation (8), Iox is the specific impulse of the oxidizer, F is the thrust, and
.mox is the flow
rate of the oxidizer. The specific impulse of the oxidizer represents the thrust produced
per unit mass of the oxidizer. This is different from the specific impulse that indicates the
thrust produced per unit mass of the oxidizer and the fuel. q
p
p
flow rate of the oxidizer. The specific impulse of the oxidizer represents the thrus
duced per unit mass of the oxidizer. This is different from the specific impulse tha
cates the thrust produced per unit mass of the oxidizer and the fuel. Thus the thrust of the motor can be expressed as the product of the flow rate Thus, the thrust of the motor can be expressed as the product of the flow rate of the
oxidizer
.mox and its specific impulse Iox:
Thus, the thrust of the motor can be expressed as the product of the flow rate
oxidizer 𝑚̇ ௫ and its specific impulse Iox: F = (
.mox +
.m f )Isp =
.mox(1 +
.m f
.mox
)Isp =
.moxIox. (9)
(
)
(1
)
f
ox
f
sp
ox
sp
ox
ox
ox
F
m
m
I
m
I
m I
m
. p is the specific impulse and 𝑚̇ is the mass flow rate of fuel The (9) In Equation (9), Isp is the specific impulse and
.m f is the mass flow rate of fuel. rol Valve
2.5. Flow Control Valve
thrust of the motor b The fluid used for the cold flow test was ultrapure water, and the cor
b
fl
d h
k
f h
l
b
d
f ll
The fluid used for the cold flow test was ultrapure water, and the correspondence
between its flow rate and the strokes of the pintle can be represented as follows: and the strokes of the pintle can be represented as follo
2
35 1346
0 6740
1 7423
Q
x
x
QH2O = 35.1346x −0.6740x2 −1.7423. (7) and the strokes of the pintle can b
2
35 1346
0 6740
Q
x
x
QH2O = 35.1346x −0.6740x2 −1.7423. follo
(7) 2
H O
Q
In Equation (7), QH2O is the mass flow rate of ultrapure water (unit: g/s) and x
In Equation (7), QH2O is the mass flow rate of ultrapure water (unit: g/s) and x is the pintle
stroke (unit: mm). stroke (unit: mm). 2.6. Static Characteristics of Hybrid Rocket Motors The process of thrust control of hybrid rocket motors has time-varying and time-lag
characteristics. The time-varying characteristics occur because the regression rate, area
of combustion, and diameter of the throat of the nozzle are variable, and this results in
a correspondence between the flow rate of the oxidizer and the thrust with respect to the
duration of operation of the motor. The time-lag characteristic occurs because the injection
of the oxidizer, pyrolysis of fuel, combustion of the propellant, and energy conversion in
the nozzle do not occur instantaneously, but require a certain response time. The control system simplifies the process of thrust response of the motor into a quasi-
steady-state process. The controller needs to use static characteristics to solve for the
magnitude of displacement of the pintle corresponding to the error in the thrust in each
control cycle. The change in the stroke of the pintle is obtained from that in the flow rate of
the oxidizer, which is in turn obtained from the error in the thrust. Aerospace 2023, 10, 421 7 of 21
w rate Before introducing the static characteristics of the hybrid rocket motor, we introduce
the concept of the specific impulse of the oxidizer Iox to calculate the control parameters. Before introducing the static characteristics of the hybrid rocket motor, we intr
the concept of the specific impulse of the oxidizer Iox to calculate the control parame
F Iox =
F
.mox
. rol Valve
2.5. Flow Control Valve
thrust of the motor b The relation-
ship between the specific impulse and the specific impulse of the oxidizer is as follows:
q
( ),
p
p
p
tionship between the specific impulse and the specific impulse of the oxidizer is as fol
m Iox = (1 +
.m f
.mox
)Isp. (10)
(1
)
ox
sp
ox
I
I
m
. as calculated by using rocket propulsion analysis ( (10) The above relationship was calculated by using rocket propulsion analysis (RPA)
software, as shown in Figure 6. The oxidizer consisted of 98% hydrogen peroxide, the fuel
consisted of 42% HTPB with 58% of an aluminum additive, and the ratio of expansion of
the nozzle was three. p
y
g
p
p
y
software, as shown in Figure 6. The oxidizer consisted of 98% hydrogen peroxide, th
consisted of 42% HTPB with 58% of an aluminum additive, and the ratio of expans
the nozzle was three. Figure 6. Specific impulses of the oxidizer under different oxidizer-to-fuel mass r
0
5
10
15
20
0
1000
2000
3000
4000
5000
6000
Iox (m / s)
mox / mf
Figure 6. Specific impulses of the oxidizer under different oxidizer-to-fuel mass ratios. Figure 6. Specific impulses of the oxidizer under different oxidizer-to-fuel mass
Figure 6. Specific impulses of the oxidizer under different oxidizer-to-fuel mass ratios. As shown in Figure 6, the specific impulse of the oxidizer decreased with an increase
in the oxidizer-to-fuel mass ratio by internal ballistic calculation. The initial conditions
were as follows: The nozzle expansion ratio was 3; the combustion chamber pressure was
3 MPa. The impact of combustion pressure on the specific pulse was slight reflecting the
feature of hybrid rocket motors [31]. Therefore, the impact was not considered. During
the operation of the motor, the mass flow rate of the oxidizer remained constant while the
ratio of the mixture varied in response to changes in the mass flux. This indicates that the
specific impulse of the oxidizer changed during the operation of the motor. p
p
g
g
p
For example, when the oxidizer-to-fuel mass ratio was one, the specific impulse of the
oxidizer was 4249 N·s/kg. That is, assuming an oxidizer-to-fuel ratio of one, a flow rate
of the oxidizer of 1 kg/s resulted in a motor thrust of 4249 N. rol Valve
2.5. Flow Control Valve
thrust of the motor b When the oxidizer-to-fuel Aerospace 2023, 10, 421 8 of 21 mass ratio was three, the same flow rate of the oxidizer (1 kg/s) generated a thrust of only
2923 N. The time-varying nature of hybrid rocket motors can thus be explained by the fact
that the same flow rate of the oxidizer resulted in varying levels of thrust. We needed to carry out internal ballistic calculations on the hybrid rocket motor to
obtain an accurate range of variations in the oxidizer-to-fuel mass ratio during the operation
of the motor. We set the inner diameter of the tube grain to 25 mm, its outer diameter
to 100 mm, and its length to 375 mm. We used measurements made by our team in the
equation of the regression rate [15]. .r = 0.014Gox0.874. (11) (11) In Equation (11),
.r is the regression rate (unit: mm/s) and Gox is the mass flux of the oxidizer
(unit: kg/m2·s). Gox =
.mox
Ap
= 4
.mox
πd2 . (12) (12) In Equation (12), AP is the cross-sectional area of flow of the oxidizer (unit: m2) and d is the
inner diameter of the tube grain (unit: m). The mass flow rate of fuel
.m f can be obtained by the equation for the regression rate. .m f = ρ f Ab
.r × 10−3,
(13) (13) In Equation (13), ρf is the density of the fuel (unit: kg/m3)—we used 42% HTPB and 58%
Al
ith
d
it
f 1634 k /
3
d A i th
f
b
ti
(
it
2) In Equation (13), ρf is the density of the fuel (unit: kg/m3)—we used 42% HTPB and 58%
Al, with a density of 1634 kg/m3—and Ab is the area of combustion (unit: m2). In Equation (13), ρf is the density of the fuel (unit: kg/m3)—we used 42% HTPB and 58%
Al, with a density of 1634 kg/m3—and Ab is the area of combustion (unit: m2). W
l
l
d h
f
b
i
f h
b
i
f ll Al, with a density of 1634 kg/m3—and Ab is the area of combustion (unit: m2). We calculated the area of combustion of the tube grain as follows: y
g
b
(
)
We calculated the area of combustion of the tube grain as follows: g
area of combustion of the tube grain as follows: Ab = πdL. rol Valve
2.5. Flow Control Valve
thrust of the motor b (14) (14) In Equation (14), L is the length of the tube grain (unit: m). In Equation (14), L is the length of the tube grain (unit: m). eburned =
Z t
0
.r(t)dt. (15) (15) In Equation (15), eburned is the web thickness of the burned grain (unit: mm), t is the working
time (unit: s), and
.r(t) is the regression rate (unit: mm/s) corresponding to the moment t. .r
is assumed to be constant along the axes of the fuel rod. The steady-state pressure of the combustion chamber of the hybrid rocket motor was
obtained as follows: Pc = (
.mox +
.m f )C∗
At
. (16) (16) In Equation (16), Pc is the pressure of the combustion chamber (unit: Pa), c* is the charac-
teristic velocity (unit: m/s) obtained by thermal calculation with RPA (rocket propulsion
analysis) software, and At is the throttling area of the nozzle throat (unit: m2). In detail,
c* depends on the oxidizer-fuel ratio and the combustion chamber pressure. When the
oxidizer-fuel ratio is constant, c* and Pc are interacting and coupled. The exact Pc and c* are
calculated in four iterations. Specifically, the first step is to set an initial Pc. The second step
is to obtain c* by interpolating Pc and the oxidizer-fuel ratio. Next, we use c* to obtain the
iterated Pc. Finally, Pc is updated and the program returns to the second step. When the
number of iterations is greater than four, the calculation is terminated and Pc is considered
to be converged. Internal ballistic calculations were performed for the tube grain with a flow rate of the
oxidizer of 160 g/s of hydrogen peroxide. This yielded a curve of the oxidizer-to-fuel mass
ratio corresponding to different web thicknesses of the burned grain. Curves of the specific impulses of the oxidizer for different web thicknesses of the
burned grain are shown in Figure 7. The variation of Iox was due to the premises that the
oxidizer flow rate was 160 g/s and that both the oxidizer-fuel ratio and pressure varied. As Aerospace 2023, 10, 421 9 of 21
mises th
ssure v
ssure v
ff
t
f 9 of 21
mises th
ssure v
ssure v
ff
t
f shown in the conclusion in reference [31], in hybrid rocket motors, the effect of combustion
chamber pressure on the specific impulse was slight. rol Valve
2.5. Flow Control Valve
thrust of the motor b Therefore, the effect of combustion
chamber pressure on Isp and Iox was not considered in this paper. As the combustion of
the tube grain proceeded, the oxidizer-to-fuel mass ratio gradually increased from 1.5 at
the outset to 4.2. However, the specific impulse of the oxidizer gradually decreased from
3698 m/s to 2648 m/s. This indicates that an initial flow rate of the oxidizer of 160 g/s
generated a thrust of 592 N, whereas at the end of the process, the same flow rate generated
a thrust of 423.6 N: a 20.6% decrease in thrust. The correspondence between the thrust
and the pressure of the combustion chamber, and the web thickness of the burned grain is
shown in Figure 8. bustion chamber pressure on the specific impulse was slight. Therefore, the effect o
bustion chamber pressure on Isp and Iox was not considered in this paper. As the com
tion of the tube grain proceeded, the oxidizer-to-fuel mass ratio gradually increased
1.5 at the outset to 4.2. However, the specific impulse of the oxidizer gradually decr
from 3698 m/s to 2648 m/s. This indicates that an initial flow rate of the oxidizer of 1
generated a thrust of 592 N, whereas at the end of the process, the same flow rate g
ated a thrust of 423.6 N: a 20.6% decrease in thrust. The correspondence between the
and the pressure of the combustion chamber, and the web thickness of the burned
is shown in Figure 8. bustion chamber pressure on Isp and Iox was not considered in this paper. As the co
tion of the tube grain proceeded, the oxidizer-to-fuel mass ratio gradually increased
1.5 at the outset to 4.2. However, the specific impulse of the oxidizer gradually decr
from 3698 m/s to 2648 m/s. This indicates that an initial flow rate of the oxidizer of 1
generated a thrust of 592 N, whereas at the end of the process, the same flow rate
ated a thrust of 423.6 N: a 20.6% decrease in thrust. The correspondence between the
and the pressure of the combustion chamber, and the web thickness of the burned
is shown in Figure 8. Figure 7. Variations in the oxidizer-to-fuel mass ratio and a specific impulse of the oxidize
different web thicknesses of the burned grain. rol Valve
2.5. Flow Control Valve
thrust of the motor b 0
5
10
15
20
25
30
35
40
0
1
2
3
4
5
6
7
mox / mf
mox / mf
eburned (mm)
0
1000
2000
3000
4000
Iox (m / s)
Iox (m / s)
Figure 7. Variations in the oxidizer-to-fuel mass ratio and a specific impulse of the oxidizer with
different web thicknesses of the burned grain. Figure 7. Variations in the oxidizer-to-fuel mass ratio and a specific impulse of the oxidize
different web thicknesses of the burned grain. 0
5
10
15
20
25
30
35
40
0
1
2
3
4
5
6
7
mox / mf
mox / mf
eburned (mm)
0
1000
2000
3000
4000
Iox (m / s)
Iox (m / s) Figure 7. Variations in the oxidizer-to-fuel mass ratio and a specific impulse of the oxidize
different web thicknesses of the burned grain. Figure 7. Variations in the oxidizer-to-fuel mass ratio and a specific impulse of the oxidizer with
different web thicknesses of the burned grain. Figure 7. Variations in the oxidizer-to-fuel mass ratio and a specific impulse of the oxidize
different web thicknesses of the burned grain. Figure 8. Variations in the thrust and the pressure of the combustion chamber with differen
thicknesses of the burned grain
0
5
10
15
20
25
30
35
40
0
100
200
300
400
500
600
700
F (N)
F (N)
eburned (mm)
0
1
2
3
4
5
6
7
Pc (MPa)
Pc (MPa)
Figure 8. Variations in the thrust and the pressure of the combustion chamber with differen
thicknesses of the burned grain. 0
5
10
15
20
25
30
35
40
0
100
200
300
400
500
600
700
F (N)
F (N)
eburned (mm)
0
1
2
3
4
5
6
7
Pc (MPa)
Pc (MPa)
Figure 8. Variations in the thrust and the pressure of the combustion chamber with different web
thicknesses of the burned grain. Figure 8. Variations in the thrust and the pressure of the combustion chamber with differe
h k
f h b
d
burned (
)
Figure 8. Variations in the thrust and the pressure of the combustion chamber with differe
thicknesses of the burned grain. Figure 8. Variations in the thrust and the pressure of the combustion chamber with different web
thicknesses of the burned grain. The error signal error(k) in the k-th control cycle is as follows: The error signal error(k) in the k-th control cycle is as follows: error(k) = ∆F(k) = Fd(k) −F(k). (17) (17) Fd(k) and F(k) are the set thrust and the actual output thrust in the k-th control cycle,
respectively, and ∆F(k) is an error in the thrust that is equal to error(k). First, we set the control period to 20 ms based on the quasi-steady-state assumption. During the first half of the control cycle (0–10 ms), the pintle of the valve was actuated, the
flow rate changed, and the thrust was adjusted. However, in the second half of the control
cycle (10–20 ms), the pintle of the valve was fixed, the flow tended to stabilize, and the
thrust and the pressure in the chamber were steady. We assumed that the hybrid rocket
motor had reached the quasi-steady state by the end of the control cycle (20 ms). Second, we used the segmented PID algorithm to set the threshold ε > 0, and divided
the process of transition into three stages according to the error signal. Stage 1: Saturation output. When the error in the thrust signal was too large
(|error(k)| > 100 N), the control output was saturated, and the speed of adjustment of
the pintle of the valve reached its limit of saturation. This limit was the maximum permit-
ted speed of adjustment of the pintle, 4.3 mm/s. Equivalently, the rate of change of flow
was limited to 133 g/s2, where this was approximately equal to a rate of change in thrust of
567 N/s. Stage 2:
Rapid transition. When the error in the thrust signal was large
(100 N > |error(k)| > 20 N), large control parameters (kp0, ki0, and kd0) were used. The stage
of rapid transition was designed to improve the transition-related performance and reduce
the response time. p
Stage 3:
Slow convergence. When the error in the thrust signal was small
(20 N > |error(k)|), smaller control parameters (kp1, ki1, and kd1) were used. The stage of
slow convergence was designed to improve the accuracy and stability of control. Finally, the control parameters were calculated by using static characteristics. Figure 7
shows that when the flow rate of hydrogen peroxide was maintained at 160 g/s, the specific
impulse of the oxidizer decreased from 3698 m/s to 2648 m/s over the period of operation
of the motor. (18) F =
.moxIox
Iox ∈[2648 ∼3698 m/s]. thicknesses of the burned grain.
2.7. Solving for the Control Parameters The control parameters in this paper needed to be provided on a case-by-case basis. Open-loop control was used during the periods of ignition and shutdown of the motor,
and closed-loop control was ineffective. Closed-loop control was switched on when the
motor was operating normally. Different values of the control parameters were used in the
closed-loop control stage by determining whether the absolute value of the error signal
|error(k)| was greater than the set threshold ε. Aerospace 2023, 10, 421 10 of 21 The error signal error(k) in the k-th control cycle is as follows: (21) (21) The units of
.mox and x in Equation (21) are the same as in Equation (19). Similarly, the
quasi-steady state equation for the (k −1)-th control cycle, i.e., t = (k −1)T, is as below. F(k −1) =
.mox(k −1)Iox(k −1),
(22) F(k −1) =
.mox(k −1)Iox(k −1), (22) F(k −1) =
.mox(k −1)Iox(k −1),
(22)
.mox(k −1) = 10−3 × (42.1615x(k −1) −0.8088x2(k −1) −2.0907). (23)
sing Equations (20) and (22), we obtain: .mox(k −1) = 10−3 × (42.1615x(k −1) −0.8088x2(k −1) −2.0907). (23)
sing Equations (20) and (22), we obtain: (23) ∆F(k) = F(k) −F(k −1) =
.mox(k)Iox(k) −
.mox(k −1)Iox(k −1). (24) (24) The thrust F(k), mass flow rate of oxidizer
.mox(k), and displacement of the pintle x(k)
at the end of the k-th control cycle can be calculated by adding F(k −1),
.mox(k −1), x(k −1),
and ∆F(k) to ∆
.mox(k) and ∆x(k). The thrust F(k), mass flow rate of oxidizer
.mox(k), and displacement of the pintle x(k)
at the end of the k-th control cycle can be calculated by adding F(k −1),
.mox(k −1), x(k −1),
and ∆F(k) to ∆
.mox(k) and ∆x(k).
F(k) = F(k −1) + ∆F(k)
.mox(k) =
.mox(k −1) + ∆
.mox(k)
x(k) = x(k −1) + ∆x(k)
. (25) (25) The specific impulse of the oxidizer ranged from 2955 m/s to 3689 m/s because the
duration of operation of the hybrid rocket motor in this hot test was 21 s. To be specific,
during the internal ballistic calculation at 21 s, Iox and Isp were 2955 m/s and 2195 m/s,
respectively; oxidizer flow rate was 160 g/s; the oxidizer–fuel ratio was 2.9; Pc was 2.4 MPa;
F was 472.8 N, and the thickness of burned flesh was 17.3 mm. When the time changed
from 0 s to 21 s, the specific impulse of the oxidizer decreased from 3698 m/s to 2955 m/s. p
p
In this paper, the same set of control parameters was used for the whole ignition period
for the sake of realizability and reliability. Iox was set as the average value of 2955 m/s
and 3698 m/s (3326.5 m/s). We assumed that the specific impulse of the oxidizer was
constant throughout the period of operation, and this enabled us to obtain constant control
parameters. The unit of 3326.5 below is m/s. The error signal error(k) in the k-th control cycle is as follows: F =
.moxIox
Iox ∈[2648 ∼3698 m/s]. (18) In Equation (18), F is the thrust (unit: N),
.mox is the mass flow rate of the oxidizer (unit:
kg/s), and Iox is the specific impulse of the oxidizer (unit: m/s). Its range of values was
obtained from internal ballistic calculations. The tube grain used in this paper had a range
of specific impulses of the oxidizer of 3698 to 2648 m/s from the initial surface of the
combustion until the end of combustion. Since the density of 98% hydrogen dioxide is 1.4 times larger than ultra water, the
mass flow rate of ultra water is multiplied by
√
1.4 when inlet pressures are the same
(P1 = 6 MPa) to obtain the mass flow rate of hydrogen dioxide. Here, we set the inlet
pressure and the saturation vapor pressure to 6 MPa and 0, respectively. The reason for this
value is twofold. First, the saturation vapor pressure of water at 15 ◦C was 1705.6 Pa. The
saturation vapor pressure was so smaller than the inlet pressure, around 6 MPa that it was
regarded as 0. Second, the upstream pressure did not change violently in the test process
and the error caused by the minor change could thus be omitted. Accordingly, we set the
inlet pressure to a fixed value of 6 MPa. .mox = QH2O2 =
√
1.4QH2O = 10−3 × (42.1615x −0.8088x2 −2.0907)
x ∈[0 ∼13 mm] 2 =
√
1.4QH2O = 10−3 × (42.1615x −0.8088x2 −2.0907)
x ∈[0 ∼13 mm],
(19) (19) where x is the displacement of the pintle (unit: mm) in the range of 0 to 13 mm, obtained
from the cold test of the variable-area cavitating venturi. In Equation (19), the unit of
.mox is
kg/s; the unit of x is mm. g
Each variable was discretized to calculate the discrete state of the output of the controller. Aerospace 2023, 10, 421 11 of 21 11 of 21 The values of the thrust, flow rate, and displacement of the pintle in the k-th control
cycle (t = kT) were F(k),
.mox(k), and x(k), respectively. When the hybrid rocket motor reached
the quasi-steady state, it satisfied the following quasi-steady state equations: F(k) =
.mox(k)Iox(k),
(20) (20) .mox(k) = 10−3 × (42.1615x(k) −0.8088x2(k) −2.0907). (21) .mox(k) = 10−3 × (42.1615x(k) −0.8088x2(k) −2.0907). The error signal error(k) in the k-th control cycle is as follows: Experimental Setup
The experimental system consisted of three subsystems: a throttleable hybrid 3. Experimental Setup
dizer feeding system, a
motor, an oxidizer fe The experimental system consisted of three subsystems: a throttleable hybrid rocket
motor, an oxidizer feeding system, and a measurement and control system. e Hybrid Rocket Motor
3.1. ThroĴleable Hybrid Rocket Motor
Figure 9 shows a drawing of the assembly of the throttleable hybrid rocket m The error signal error(k) in the k-th control cycle is as follows: ∆F(k) = [
.mox(k) −
.mox(k −1)]Iox = ∆
.mox(k)Iox = 3326.5∆
.mox(k). (26) (26) By using Equations (22) and (24), we obtain: ∆
.mox(k) =
.mox(k) −
.mox(k −1) = 10−3 ×
h
42.1615[x(k) −x(k −1)] −0.8088[x2(k) −x2(k −1)] ∆
.mox(k) =
.mox(k) −
.mox(k −1) = 10−3 ×
h
42.1615[x(k) −x(k −1)] −0.8088[x2(k) −x2(k −1)]
i
. (27) (k) −
.mox(k −1) = 10−3 ×
h
42.1615[x(k) −x(k −1)] −0.8088[x2(k) −x2(k −1)]
i
. (27) (27) A variable-area cavitating venturi is regarded as a linear component owing to its
igh linearity. A variable-area cavitating venturi is regarded as a linear component owing to its
high linearity. ∆
.mox(k) = 10−3 × [42.1615[x(k) −x(k −1)]] = 10−3 × 42.1615∆x(k). (28) (28) By using Equations (25) and (27), we obtain: ∆F(k) = 3326.5∆
.mox(k) = 3326.5∆
.mox(k) = 140.25∆x(k). (29) (29) The units of ∆F(k), ∆
.mox, and ∆x(k) are N, kg/s, and mm, respectively. Thus, ∆F(k), the
error in thrust in the current control cycle, can be calculated. ∆x(k) is then solved for: The units of ∆F(k), ∆
.mox, and ∆x(k) are N, kg/s, and mm, respectively. Thus, ∆F(k), the
error in thrust in the current control cycle, can be calculated. ∆x(k) is then solved for: ∆x(k) = 0.007130∆F(k). (30) (30) Aerospace 2023, 10, 421
By us 12 of 21 12 of 21 By using Equations (4) and (30), we obtain:
w that kp0 = 0, ki0 = 0.007130, and kd0 = 0. M
Then, we know that kp0 = 0, ki0 = 0.00713 By using Equations (4) and (30), we obtain:
w that kp0 = 0, ki0 = 0.007130, and kd0 = 0. M
Then, we know that kp0 = 0, ki0 = 0.00713 By using Equations (4) and (30), we obtain:
w that kp0 = 0, ki0 = 0.007130, and kd0 = 0. Meanwhile, we set kp1 = 0, ki1 = 500,
Then, we know that kp0 = 0, ki0 = 0.007130, and kd0 = 0. Meanwhile, we set kp1 = 0, k ∆u(k) = 0.007130 · error(k). (31)
us tests. d on the previous tests. (31) Then, we know that kp0 = 0, ki0 = 0.007130, and kd0 = 0. Meanwhile, we set kp1 = 0, ki1 = 500,
ki0 = 3.565, and kd0 = 0 based on the previous tests. tal Setup
rimental system consisted of three subsystems: a throttleable hybrid rocket
3. 3.1. Throttleable Hybrid Rocket Motor
shows a drawing of the assembly o
catalyst bed, a pre-combustion c
g
g
consisted of a catalyst bed, a pr Figure 9 shows a drawing of the assembly of the throttleable hybrid rocket motor. It
consisted of a catalyst bed, a pre-combustion chamber, a fuel grain, a post-combustion
chamber, and a nozzle (the physical photograph is shown in Figure 10). The material of the
nozzle is tungsten-infiltrated copper which can be cooled down by regenerative cooling. y
,
p
,
g
,
p
a nozzle (the physical photograph is shown in Figure 10). The material of
ungsten-infiltrated copper which can be cooled down by regenerative cool-
chamber, and a nozzle (the physical photograph is shown in Figure 10). The ma
the nozzle is tungsten-infiltrated copper which can be cooled down by regenerati
ing. Figure 9. Drawing of the assembly of the throĴleable hybrid rocket motor. Figure 9. Drawing of the assembly of the throttleable hybrid rocket motor. Figure 9. Drawing of the assembly of the throĴleable hybrid rocket motor. Figure 9. Drawing of the assembly of the throĴleable hybrid rocket motor. Figure 9. Drawing of the assembly of the throttleable hybrid rocket motor. Figure 9. Drawing of the assembly of the throĴleable hybrid rocket motor. ing of the assembly of the throĴleable hybrid rocket motor. Figure 9. Drawing of the assembly of the throttleable hybrid rocket motor. Figure 9. Drawing of the assembly of the throĴleable hybrid rocket ng of the assembly of the throĴleable hybrid rocket motor. Figure 9. Drawing of the assembly of the throttleable hybrid rocket motor. Figure 9. Drawing of the assembly of the throĴleable hybrid rocket tograph of the components of the hybrid rocket motor. Figure 10. Photograph of the components of the hybrid rocket mo
Figure 10. Photograph of the components of the hybrid rocket motor. ograph of the components of the hybrid rocket motor. Figure 10. Photograph of the components of the hybrid rocket m
Figure 10. Photograph of the components of the hybrid rocket motor. The catalyst bed is the igniter of the motor. Hydrogen peroxide was decomposed into
oxygen and water vapor by the catalyst bed. The temperature of the catalytic products,
oxygen, and water vapor, exceeded 1100 K. The high-temperature oxygen and water vapor
thus ignited the motor. g
Table 1 lists the main design parameters of the throttleable hybrid rocket motor. Aerospace 2023, 10, 421 13 of 21 Table 1. Main design parameters of the throttleable hybrid rocket motor. g
ngth of fuel grain
375 Table 1. 3.2. Oxidizer Feeding System
as tank, a pressure regul Figure 11 shows a schematic of the oxidizer feeding system. It was composed of a high-
pressure gas tank, a pressure regulator, hand valves, solenoid valves, a buffer tank, pressure
transducers, a mass flowmeter, a variable-area cavitating venturi, and other components. The high-pressure gas tank contains nitrogen gas to exert pressure on the oxidizer tank. ucers, a mass flowmeter, a variable-area cavitating venturi, and other com-
gh-pressure gas tank contains nitrogen gas to exert pressure on the oxi- Figure 11. Schematic of the oxidizer feeding system. Figure 11. Schematic of the oxidizer feeding system. tic of the oxidizer feeding system. Figure 11. Schematic of the oxidizer feeding system. 3.1. Throttleable Hybrid Rocket Motor
shows a drawing of the assembly o
catalyst bed, a pre-combustion c
g
g
consisted of a catalyst bed, a pr Main design parameters of the throttleable hybrid rocket motor. Parameter
Value
Unit
Mass flow rate of oxidizer
0.16–0.24
kg/s
Inner diameter of fuel grain
25
mm
Outer diameter of fuel grain
100
mm
Length of fuel grain
375
mm
Inner diameter of pre-chamber
80
mm
Length of pre-chamber
35
mm
Inner diameter of post-chamber
80
mm
Length of post-chamber
50
mm
Diameter of nozzle throat
15
mm
Expansion ratio of nozzle
3
–
g
ngth of fuel grain
375
mm
ameter of pre-chamber
80
mm
gth of pre-chamber
35
mm
ameter of post-chamber
80
mm
gth of post-chamber
50
mm
eter of nozzle throat
15
mm
nsion ratio of nozzle
3
–
ding System
hows a schematic of the oxidizer feeding system It was composed of a 4. Experimental Results and Discussion
p
p
mented PID control system. The measur We performed a closed-loop throttle test to evaluate the performance of the segmented
PID control system. The measured thrust was used as the feedback measurement for the
hot test, and 98% hydrogen peroxide and HTPB with 58% of aluminum additive were used
as the propellant for the closed-loop throttle test. for the hot test, and 98% hydrogen peroxide and HTPB with 58% of aluminum ad
were used as the propellant for the closed-loop throttle test. 4.1. Time Sequence of Test Table 2. Error range and basic information of measurement components.
Technology
Beijing Weisitezhonghang Table 2. Error range and basic information of measurement components. Component
Company
Product Number
Error
Mass flowmeter
Emerson Technology
CMF025M172N2BPMKZZ
<0.1%
Thrust sensor
Beijing Weisitezhonghang Technology
CYB-602S
<0.2%
Pressure sensor
Beijing Weisitezhonghang Technology
Cyb-20S-HX
<1.5%
Technology
Pressure sensor
Beijing Weisitezhonghang
Technology
Cyb-20S-HX
1
4. Experimental Results and Discussion
W
f
d
l
d l
th
ttl
t
t t
l
t
th
f
f th 4.1. Time Sequence of Test
Figure 12 shows th
l
d l
t
l
it Figure 12 shows the temporal sequence of the closed-loop throttle test. Before the
closed-loop control switch was opened at 4 s, the main valve was opened at 1 s and the
oxidizer had thus been available for 3 s. This was intended to pre-heat the catalytic bed and
prepare for grain ignition. The core component of the 45 mm-diameter catalytic bed was
composed of nickel-based silver meshes catalyzing and decomposing hydrogen peroxide
efficiently due to the good catalytic property of silver monomers. Before the main value was
turned off at 26 s, the closed-loop control switch was turned off at 25 s to avoid the incorrect
action of the pintle of the control valve under the command of the feedback control system
during the shutdown process. closed-loop control switch was opened at 4 s, the main valve was opened at 1 s an
oxidizer had thus been available for 3 s. This was intended to pre-heat the catalyti
and prepare for grain ignition. The core component of the 45 mm-diameter catalyt
was composed of nickel-based silver meshes catalyzing and decomposing hydroge
oxide efficiently due to the good catalytic property of silver monomers. Before the
value was turned off at 26 s, the closed-loop control switch was turned off at 25 s to
the incorrect action of the pintle of the control valve under the command of the fee
control system during the shutdown process. Figure 12. Time sequence of the closed-loop throĴle test. 4.2. Closed-Loop ThroĴle Test
Figure 13 shows the time history of the response profile of the thrust of the m
The prescribed profile of thrust was divided into three steady stages. The target valu
thrust in the three stages were 400 N, 600 N, and 400 N, and the average thrust valu
the stable section in each stage were 400.7 N, 599.1 N, and 400.1 N, respectively. Th
erage thrust was thus stable and the control error of the average value of the thrus
44
43
27.5
27
26.5
26
25
4
1
0.5
Tank relief valve
Blow-off valve
Closed loop control switch
Main valve
Tank pressurization valve
Tank pressurization valve
Main valve
Closed loop control switch
Blow-off valve
Tank relief valve
Time (s)
Figure 12. Time sequence of the closed-loop throttle test. 4.2. Closed-Loop Throttle Test
Figure 13 shows the time history of the response profile of the thrust of the motor. 4.1. Time Sequence of Test
Figure 12 shows th
l
d l
t
l
it The
prescribed profile of thrust was divided into three steady stages. The target values of thrust
in the three stages were 400 N, 600 N, and 400 N, and the average thrust values of the stable
section in each stage were 400.7 N, 599.1 N, and 400.1 N, respectively. The average thrust
was thus stable and the control error of the average value of the thrust was better than 0.5%. Although the average thrust was very close to the target value, the instantaneous values
of thrust were not stable in the three steady stages, and steady-state error was obtained. Figure 14 illustrates the snapshots of plume exhausting from the nozzle for the hot test
photographed by an infrared camera. Figure 12. Time sequence of the closed-loop throĴle test. 44
43
27.5
27
26.5
26
25
4
1
0.5
Tank relief valve
Blow-off valve
Closed loop control switch
Main valve
Tank pressurization valve
Tank pressurization valve
Main valve
Closed loop control switch
Blow-off valve
Tank relief valve
Time (s)
Figure 12. Time sequence of the closed-loop throttle test. Tank pressurization valve
Main valve
Closed loop control switch
Blow-off valve
Tank relief valve Figure 12. Time sequence of the closed-loop throĴle te
Figure 12. Time sequence of the closed-loop throttle test. t and Control System
3.3. Measurement and Control System t and Control System
stem used a programmable logic controller (PLC) to control the variable-
venturi. A general automatic test system based on the PXI bus was used to
imental data on the temperature, pressure, mass flow rate, and thrust, and
ling during the test was 2000 Hz. The repeated accuracy of positioning of
e of the variable-area cavitating venturi was 0.02 mm and the accuracy of
The test system used a programmable logic controller (PLC) to control the variable-
area cavitating venturi. A general automatic test system based on the PXI bus was used
to collect the experimental data on the temperature, pressure, mass flow rate, and thrust,
and the rate of sampling during the test was 2000 Hz. The repeated accuracy of positioning
of the pintle stroke of the variable-area cavitating venturi was 0.02 mm and the accuracy
of the displacement sensor was 10 microns. An Emerson mass flowmeter with a range of
0–360 g/s, a measurement accuracy at the level of 0.1, and a measurement error of less than
0.1% was used to measure the mass flow rate. The S-type tensile pressure sensor was used
to measure the thrust. The range of the sensor used to measure thrust was 0–1 kN, and
the error in the measured thrust was smaller than 0.2%. Error in the measurement of each Aerospace 2023, 10, 421 14 of 21 14 of 21 pressure sensor was smaller than 1.5%. Table 2 lists the error range and basic information
of measurement components. p
p
y
Mass flowmeter
Emerson Technology
CMF025M172N2BPMKZZ 0
Thrust sensor
Beijing Weisitezhonghang
CYB 602S
0 pressure sensor was smaller than 1.5%. Table 2 lists the error range and basic information
of measurement components. Mass flowmeter
Emerson Technology
CMF025M172N2BPMKZZ 0
Thrust sensor
Beijing Weisitezhonghang
CYB 602S
0 Table 2. Error range and basic information of measurement components. Technology
Beijing Weisitezhonghang Table 2. Error range and basic information of measurement components. Technology
Beijing Weisitezhonghang 4 2 Closed Loop ThroĴle Test
4.2. Closed-Loop Throttle Test 4.2. Closed-Loop ThroĴle Test
Figure 13 shows the time history of the response profile of the thrust of the m
The prescribed profile of thrust was divided into three steady stages. The target valu
thrust in the three stages were 400 N, 600 N, and 400 N, and the average thrust valu
the stable section in each stage were 400.7 N, 599.1 N, and 400.1 N, respectively. Th
erage thrust was thus stable and the control error of the average value of the thrus
Figure 13 shows the time history of the response profile of the thrust of the motor. The
prescribed profile of thrust was divided into three steady stages. The target values of thrust
in the three stages were 400 N, 600 N, and 400 N, and the average thrust values of the stable
section in each stage were 400.7 N, 599.1 N, and 400.1 N, respectively. The average thrust
was thus stable and the control error of the average value of the thrust was better than 0.5%. Although the average thrust was very close to the target value, the instantaneous values
of thrust were not stable in the three steady stages, and steady-state error was obtained. Figure 14 illustrates the snapshots of plume exhausting from the nozzle for the hot test
photographed by an infrared camera. Aerospace 2023, 10, 421
error w
zle for t 15 of 21
from the
he in-
-state Figure 13. Response profile of the thrust in the closed-loop throĴle test. (a)
(b)
(c)
Figure 14. Snapshots of plume exhausting from the nozzle for the hot test at (a) 8 s, (b) 15 s, a
23 s. 0
5
10
15
20
25
30
0
200
400
600
800
Raw thrust signal
Commanded thrust profile
Filtered thrust signal
Thrust (N)
Time (s)
Figure 13. Response profile of the thrust in the closed-loop throttle test. Figure 13. Response profile of the thrust in the closed-loop throĴle test. (a)
(b)
(c)
Figure 14. Snapshots of plume exhausting from the nozzle for the hot test at (a) 8 s, (b) 15 s, and (c)
23 s. 0
5
10
15
20
25
30
0
200
400
600
800
Raw thrust signal
Commanded thrust profile
Filtered thrust signal
Thrust (N)
Time (s)
Figure 14. Snapshots of plume exhausting from the nozzle for the hot test at (a) 8 s, (b) 15 s, and
(c) 23 s. 4 2 Closed Loop ThroĴle Test
4.2. Closed-Loop Throttle Test g
p
p
g
( )
, ( )
,
( )
23 s. Ablation of the nozzle occurred due to the large amount of aluminum particles
fuel. The nozzle throat diameters before and after the test were 14.787 mm and 15.132
respectively, with a difference of 0.345 mm. The fuel consumption was obtained as
the nozzle occurred due to the large amount of aluminum particles in the
throat diameters before and after the test were 14.787 mm and 15.132 mm,
th a difference of 0.345 mm. The fuel consumption was obtained as 1.715
weighing method
Ablation of the nozzle occurred due to the large amount of aluminum particles in the
fuel. The nozzle throat diameters before and after the test were 14.787 mm and 15.132 mm,
respectively, with a difference of 0.345 mm. The fuel consumption was obtained as 1.715 kg
by the mass weighing method. Ablation of the nozzle occurred due to the large amount of aluminum particles in the
fuel. The nozzle throat diameters before and after the test were 14.787 mm and 15.132 mm,
respectively, with a difference of 0.345 mm. The fuel consumption was obtained as 1.715
kg by the mass weighing method. Because the period of control in this study was set to 20 ms the cut-off frequency of kg by the mass weighing method. Because the period of control in this study was set to 20 ms, the cut-off frequen
the filter was set to 50 Hz. The filtered curves of the thrust over time were then obt
as shown in Figure 15. The real-time error in the thrust was controlled to within ±
weighing method. e period of control in this study was set to 20 ms, the cut-off frequency of
t to 50 Hz. The filtered curves of the thrust over time were then obtained
ure 15. The real-time error in the thrust was controlled to within ± 20 N
Because the period of control in this study was set to 20 ms, the cut-off frequency of
the filter was set to 50 Hz. The filtered curves of the thrust over time were then obtained as
shown in Figure 15. The real-time error in the thrust was controlled to within ± 20 N with
a steady-state error smaller than 5%. 4 2 Closed Loop ThroĴle Test
4.2. Closed-Loop Throttle Test zle for the hot test photographed by an infrared camera. Figure 13. Response profile of the thrust in the closed-loop throĴle test. (a)
(b)
(c)
Figure 14. Snapshots of plume exhausting from the nozzle for the hot test at (a) 8 s, (b) 15 s, and (c)
23 s. 0
5
10
15
20
25
30
0
200
400
600
800
Raw thrust signal
Commanded thrust profile
Filtered thrust signal
Thrust (N)
Time (s) Figure 13 Response profile of the thrust in the closed loop throĴle test
0
5
10
15
20
25
30
0
200
400
600
800
Raw thrust signal
Commanded thrust profile
Filtered thrust signal
Thrust (N)
Time (s)
Figure 13 Response profile of the thrust in the closed loop throttle test
Figure 13. Response profile of the thrust in the closed-loop throĴle test. 0
5
10
15
20
25
30
0
200
400
600
800
Raw thrust signal
Commanded thrust profile
Filtered thrust signal
Thrust (N)
Time (s)
zle for the hot test photographed by an infrared camera. Fi
13 R
fil
f th th
t i
th
l
d l
th
Ĵl t
t
0
5
10
15
20
25
30
0
200
400
600
800
Raw thrust signal
Commanded thrust profile
Filtered thrust signal
Thrust (N)
Time (s) Raw thrust signal
Commanded thrust profile
Filtered thrust signal
rofile
Raw thrust signal
Commanded thrust profile Figure 13. Response profile of the thrust in the closed-loop throĴle
Figure 13. Response profile of the thrust in the closed-loop throttle test. e profile of the thrust in the closed-loop throĴle test. igure 13. Response profile of the thrust in the closed-loop throĴle test. osed-loop throĴle test. b)
(c
d loop throttle test. (c)
loop throĴle test. (c)
zzle for the hot test at (a) 8 s (b) 15 s (a)
(
Fi
14 S a)
(b)
)
(b)
hots of plume exhausting from the n (a) (b)
h (c) Figure 14. Snapshots of plume exhausting from the nozzle for the hot test at (a) 8 s, (b) 15 s,
23 s. ots of plume exhausting from the nozzle for the hot test at (a) 8 s, (b) 15 s, and (c)
Figure 14. Snapshots of plume exhausting from the nozzle for the hot test at (a) 8 s, (b) 15 s, and
(c) 23 s. 4 2 Closed Loop ThroĴle Test
4.2. Closed-Loop Throttle Test Because the period of control in this study was set to 20 ms, the cut off frequency of
the filter was set to 50 Hz. The filtered curves of the thrust over time were then obtained
as shown in Figure 15. The real-time error in the thrust was controlled to within ± 20 N
with a steady-state error smaller than 5%. with a steady-state error smaller than 5%. 0
5
10
15
20
25
0
200
400
600
800
400N-20N
400N+20N
600N-20N
Filtered thrust signal
Thrust (N)
600N+20N
ate error smaller than 5%. 5
10
15
20
25
30
400N-20N
400N+20N
600N-20N
tered thrust signal
600N+20N
Figure 15. Filtered curves of the thrust over time with an error band. 0
5
10
15
20
25
30
0
200
400
600
800
400N-20N
400N+20N
600N-20N
Filtered thrust signal
Thrust (N)
Time (s)
600N+20N
Figure 15. Filtered curves of the thrust over time with an error band. 0
5
10
15
20
05
10
15
20
25
Figure 15. Filtered curves of the thrust over time with an error band. Figure 15. Filtered curves of the thrust over time with an error band. 0
4.3. Discussion riations in the oxidizer-to-fuel mass ratio and the specific impulse of the
n in Figure 17. The oxidizer-to-fuel ratio and the specific impulse of the
slightly in the first two stages of steady thrust. The oxidizer-to-fuel ratio
Curves of variations in the oxidizer-to-fuel mass ratio and the specific impulse of the
oxidizer are shown in Figure 17. The oxidizer-to-fuel ratio and the specific impulse of the
oxidizer changed slightly in the first two stages of steady thrust. The oxidizer-to-fuel ratio
gradually increased in the third section of steady thrust, and this led to a gradual decrease
in the specific impulse of the oxidizer, mainly because of the low mass flux and the reduced
regression rate in the late stage of combustion. i
igu e
e o idi e to ue
atio a d t e speci ic i
pu se o t e
d slightly in the first two stages of steady thrust. The oxidizer-to-fuel ratio
sed in the third section of steady thrust, and this led to a gradual decrease
mpulse of the oxidizer, mainly because of the low mass flux and the re-
n rate in the late stage of combustion. dually increased in the third section of steady thrust, and this led to a gradual decrease
he specific impulse of the oxidizer, mainly because of the low mass flux and the re-
ed regression rate in the late stage of combustion. ure 17 Curves of variations in the oxidizer-to-fuel mass ratio and the specific impulse of the
6
8
10
12
14
16
18
20
22
24
26
0
1
2
3
4
5
6
7
Oxidizer-to-fuel mass ratio
Oxidizer specific impulse (m/s)
Time(s)
ox
f
0
500
1000
1500
2000
2500
3000
3500
4000
Iox (m/s)
Figure 17. Curves of variations in the oxidizer-to-fuel mass ratio and the specific impulse of the
oxidizer. When calculating the control parameters, we assumed that the average specific im-
pulse of the oxidizer was 3326.5 m/s during the entire process of operation. The actual
6
8
10
12
14
16
18
20
22
24
26
0
1
2
3
4
5
6
7
Oxidizer-to-fuel mass ratio
Oxidizer specific impulse (m/s)
Time(s)
mox / mf
0
500
1000
1500
2000
2500
3000
3500
4000
Iox (m/s)
Figure 17. Curves of variations in the oxidizer-to-fuel mass ratio and the specific impulse of the oxidizer. 0
4.3. Discussion 3
6
9
12
15
18
21
24
27
0.5
1.0
1.5
2.0
2.5
Regression rate (IRRM)
Fuel mass flow rate (IRRM)
Regression rate (Reconstruction Technique)
Fuel mass flow rate (Reconstruction Technique)
Time(s)
r (mm/s)
0
50
100
150
mf (g/s)
Figure 16. Curves of variation in the regression rate and mass flow rate of the fuel. Figure 16. Curves of variation in the regression rate and mass flow rate of the fuel. In consideration of the thrust throĴling operation of the motor, the reconstruction
technique proposed in previous articles [35,36] was referred to in order to obtain the real-
ti
*
d
t
i
t
d f
l fl
t
hi h i
h
i
Fi
3
6
9
12
15
18
21
24
27
0.5
1.0
1.5
2.0
Fuel mass flow rate (Reconstruction Technique)
Time(s)
r (mm/s)
0
50
100
mf (g/s) Regression rate (IRRM)
Fuel mass flow rate (IRRM)
Regression rate (Reconstruction Technique)
Fuel mass flow rate (Reconstruction Technique)
100 variation in the regression rate and mass flow rate of the fuel. Figure 16. Curves of variation in the regression rate and mass flow rate of the fuel. g
p
sed in previous articles [35,36] was referred to in order to obtain ion of the thrust throĴling operation of the motor, the reconstruction
ed in previous articles [35,36] was referred to in order to obtain the real
e accurate regression rate and fuel flow rate, which is shown in Figure
In consideration of the thrust throttling operation of the motor, the reconstruction
technique proposed in previous articles [35,36] was referred to in order to obtain the real-
time c* and a more accurate regression rate and fuel flow rate, which is shown in Figure 16. Compared with the IRRM method, the OPTMA-FE-L method added real-time information
about the thrust and took into account the real-time variation of c*. ore accurate regression rate and fuel flow rate, which is shown in Figure
with the IRRM method, the OPTMA-FE-L method added real-time infor-
e thrust and took into account the real-time variation of c*. variations in the oxidizer-to-fuel mass ratio and the specific impulse of the
wn in Figure 17. The oxidizer-to-fuel ratio and the specific impulse of the h the IRRM method, the OPTMA-FE-L method added real-time infor-
thrust and took into account the real-time variation of c*. 0
4.3. Discussion Figure 15. Filtered curves of the thrust over time with an error band. d curves of the thrust over time with an error band. We used the instantaneous regression rate-based method (IRRM) previously developed
by our team to obtain the real-time regression rate of the motor and the real-time mass flow
rate of the fuel [30]. The curves of variations in the regression rates and mass flow rate
of fuel are shown in Figure 16. As combustion proceeded, the regression rate gradually Aerospace 2023, 10, 421
gradually d
mass flu
rate of th 16 of 21
er and
ects 16 of 21
er and
ects decreased, but when the thrust was 600 N, the flow rates of the oxidizer and mass flux
increased first, followed by an increase in the regression rate. The mass flow rate of the fuel
did not vary greatly in each section of stable thrust due to the joint effects of the gradually
decreasing regression rate and the gradually increasing area of combustion. d not vary greatly in each section of stable thrust due to the joint effect
ecreasing regression rate and the gradually increasing area of combus
Regression rate (IRRM)
Fuel mass flow rate (IRRM)
Regression rate (Reconstruction Technique)
150 Figure 16. Curves of variation in the regression rate and mass flow rate of the fuel. 3
6
9
12
15
18
21
24
27
0.5
1.0
1.5
2.0
2.5
Regression rate (IRRM)
Fuel mass flow rate (IRRM)
Regression rate (Reconstruction Technique)
Fuel mass flow rate (Reconstruction Technique)
Time(s)
r (mm/s)
0
50
100
150
mf (g/s)
Figure 16. Curves of variation in the regression rate and mass flow rate of the fuel. Figure 16. Curves of variation in the regression rate and mass flow rate of the fuel. In consideration of the thrust throĴling operation of the motor, the reconstruction
technique proposed in previous articles [35,36] was referred to in order to obtain the real
*
d
d f
l fl
h h
h
F
3
6
9
12
15
18
21
24
27
0.5
1.0
1.5
2.0
Fuel mass flow rate (Reconstruction Technique)
Time(s)
r (mm/s)
0
50
100
mf (g/s) Figure 16. Curves of variation in the regression rate and mass flow rate of the fuel. 0
4.3. Discussion ly increased whereas the specific impulse of the oxidizer decreased
mpulse of the oxidizer in the third stage was lower than that in the firs As the combustion progressed, the nozzle ablation occurred. The diameter of the
nozzle throat gradually increased whereas the specific impulse of the oxidizer decreased. Thus, the specific impulse of the oxidizer in the third stage was lower than that in the
first stage. ws curves of the variations in the thrust, mass flow rate of the oxidizer
e pintle over time during the closed-loop throttle test. It also illustrates
collected. The thrust generated by 0 s was the initial compression. The
i l
i
t
t th
t h
k d
i
th i
iti
f th Figure 18 shows curves of the variations in the thrust, mass flow rate of the oxidizer,
and position of the pintle over time during the closed-loop throttle test. It also illustrates
the thrust signals collected. The thrust generated by 0 s was the initial compression. The
purpose of the initial compression was to prevent thrust shock during the ignition of the
motor in order to better collect thrust signals. The initial compression had been subtracted
in the subsequent data processing. According to Figure 18, the peak thrust at the beginning
of ignition occurred due to the delay in ignition. Because the catalytic bed had not been
fully preheated and the motor had not completed ignition, the real-time thrust was much
lower than the design thrust. Error in the thrust signals received by the controller of the
control system was too large, and led to a significant increase in the position of the pintle. This in turn increased the mass flow rate of the oxidizer and the thrust. ial compression was to prevent thrust shock during the ignition of the
etter collect thrust signals. The initial compression had been subtracted
data processing. According to Figure 18, the peak thrust at the begin-
ccurred due to the delay in ignition. Because the catalytic bed had not
ed and the motor had not completed ignition, the real-time thrust was
the design thrust. Error in the thrust signals received by the controller
em was too large, and led to a significant increase in the position of the
n increased the mass flow rate of the oxidizer and the thrust. igure 18. 0
4.3. Discussion When calculating the control parameters, we assumed that the average specific impulse
of the oxidizer was 3326.5 m/s during the entire process of operation. The actual average
specific impulses of the oxidizer at three stages were 2864.9 m/s, 2930.8 m/s, and 2663 m/s,
respectively, and the errors were 13.8%, 11.9%, and 19.9% all of which are lower than 20%. The experimental results show that the actual specific impulse of the oxidizer is consistent ually increased in the third section of steady thrust, and this led to a gradual decrease
he specific impulse of the oxidizer, mainly because of the low mass flux and the re-
ed regression rate in the late stage of combustion. 3
4
5
6
7
Oxidizer-to-fuel mass ratio
Oxidizer specific impulse (m/s)
1500
2000
2500
3000
3500
4000
Iox (m/s)
6
8
10
12
14
16
18
20
22
24
26
0
1
2
3
4
5
6
7
Oxidizer-to-fuel mass ratio
Oxidizer specific impulse (m/s)
Time(s)
mox / mf
0
500
1000
1500
2000
2500
3000
3500
4000
Iox (m/s) 1000
of variations in the oxidizer-to-fuel mass ratio and the specific impulse of the
Figure 17. Curves of variations in the oxidizer-to-fuel mass ratio and the specific impulse of the oxidizer. of variations in the oxidizer to fuel mass ratio and the specific impulse of the
10
12
14
16
18
20
22
24
26
Time(s)
0
500
ulating the control parameters, we assumed that the average specific im-
dizer was 3326.5 m/s during the entire process of operation. The actual
When calculating the control parameters, we assumed that the average specific impulse
of the oxidizer was 3326.5 m/s during the entire process of operation. The actual average
specific impulses of the oxidizer at three stages were 2864.9 m/s, 2930.8 m/s, and 2663 m/s,
respectively, and the errors were 13.8%, 11.9%, and 19.9% all of which are lower than 20%. The experimental results show that the actual specific impulse of the oxidizer is consistent Aerospace 2023, 10, 421
impulse be
difference b 17 of 21
efore, a 17 of 21
efore, a with its pre-set specific impulse. At the same time, in the internal ballistic calculation, the
actual specific impulse of the oxidizer was lower than the pre-set specific impulse because
the efficiency of the specific impulse was not considered. Therefore, a difference between
them was obtained. 0
4.3. Discussion Curves of variations in the thrust, mass flow rate of the oxidizer, and position of the pintle
ver time in the closed-loop throĴle test. 0
5
10
15
20
25
30
0
200
400
600
x (mm)
mox (g/s)
Thrust
Oxidizer mass flow rate
Pintle position
Time (s)
Thrust (N)
0
50
100
150
200
250
300
30
40
50
60
70
80
90
100
Figure 18. Curves of variations in the thrust, mass flow rate of the oxidizer, and position of the pintle
over time in the closed-loop throttle test. variations in the thrust, mass flow rate of the oxidizer, and position of the pintle
ed-loop throĴle test. Figure 18. Curves of variations in the thrust, mass flow rate of the oxidizer, and position of the pintle
over time in the closed-loop throttle test. control period in this paper was set to 20 ms, the control system could
ow-frequency oscillations in thrust. The main frequency of oscillations
4 Hz, based on the Fourier transform of signals of the thrust sensor. To
variations in the control parameters, signals of the sensor were discre-
increases in the thrust, mass flow rate of oxidizer, and the pintle stroke
dure was as follows: The measurements of the sensor were filtered by a
Although the control period in this paper was set to 20 ms, the control system could not
suppress the low-frequency oscillations in thrust. The main frequency of oscillations in the
thrust was 24 Hz, based on the Fourier transform of signals of the thrust sensor. To easily
analyze the variations in the control parameters, signals of the sensor were discretized to
obtain the increases in the thrust, mass flow rate of oxidizer, and the pintle stroke. The specific
procedure was as follows: The measurements of the sensor were filtered by a third-order
Butterworth filter with a cut-off frequency of 30 Hz. We then selected one data point every
12 Hz from the filtered data; Finally, pairs of adjacent data points were subtracted from each
other, that is, the increase in each physical quantity was obtained every 83.33 ms. worth filter with a cut-off frequency of 30 Hz. 0
4.3. Discussion We then selected one data
from the filtered data; Finally, pairs of adjacent data points were sub-
other, that is, the increase in each physical quantity was obtained every
ws the curves of variations in the increases in the thrust, mass flow rate
d the position of the pintle over time. The four green lines in the figure
ning on feedback control, instantaneously increasing the target thrust to
usly reducing the target thrust to 400 N and turning off feedback con-
Figure 19 shows the curves of variations in the increases in the thrust, mass flow rate
of the oxidizer, and the position of the pintle over time. The four green lines in the figure
correspond to turning on feedback control, instantaneously increasing the target thrust
to 600 N, instantaneously reducing the target thrust to 400 N, and turning off feedback
control. Different from Figure 18, the change at 2 s in Figure 19 stems from the thrust
generated by the catalytic bed in its initial working stage. The catalytic bed catalyzed
and decomposed hydrogen peroxide into high-temperature gas, which expanded through
the nozzle to generate thrust. At this point, the hybrid rocket motor was regarded as
a hydrogen peroxide monopropellant rocket motor. At 4 s, feedback control was turned on
and the control system increased the flow rate to speed up the ignition of the motor. This Aerospace 2023, 10, 421 18 of 21
t 18 s, t
f declin 18 of 21
t 18 s, t
f declin led to a large overshoot in thrust. This was not a bug in the control system, but occurred
because the delay in ignition was too long. At 11 s, the target thrust was modified to
600 N. The position of the pintle and the flow rate increased rapidly, yet the thrust did not
respond immediately to these changes. The delay in the period of increase in the thrust was
approximately 160 ms. The stepwise response time was 0.83 s. At 18 s, the target thrust was
modified to 400 N. The thrust response was fast in the stage of decline and the delay was
only 15 ms. The response time in the stepwise descent phase was 0.78 s. At 25 s, feedback
control was turned off and the position of the pintle no longer changed. y
p
p
g
ed to a large overshoot in thrust. 0
4.3. Discussion This was not a bug in the control system, but occurred
because the delay in ignition was too long. At 11 s, the target thrust was modified to 600
N. The position of the pintle and the flow rate increased rapidly, yet the thrust did not
respond immediately to these changes. The delay in the period of increase in the thrust
was approximately 160 ms. The stepwise response time was 0.83 s. At 18 s, the target
thrust was modified to 400 N. The thrust response was fast in the stage of decline and the
delay was only 15 ms. The response time in the stepwise descent phase was 0.78 s. At 25
s, feedback control was turned off and the position of the pintle no longer changed. s, feedback control was turned off and the position of the pintle no longer chang
−50
0
50
△F
△Q
200
400
6
8
10 Figure 19. Curves of variations in the increases in the thrust (ΔF), mass flow rate of the oxidizer
(Δ𝑚̇ ௫), and position of the pintle (Δx) over time in the closed-loop throĴle test. 0
5
10
15
20
25
30
−300
−250
−200
−150
−100
−50
0
50
Δx (mm)
ΔF (N)
Δmox (g/s)
△F
△Q
△x
Time (s)
−400
−200
0
200
400
0
2
4
6
8
10
−2
Figure 19. Curves of variations in the increases in the thrust (∆F), mass flow rate of the oxidizer
(∆
.mox), and position of the pintle (∆x) over time in the closed-loop throttle test. Figure 19. Curves of variations in the increases in the thrust (ΔF), mass flow rate of th
(Δ𝑚̇ ௫), and position of the pintle (Δx) over time in the closed-loop throĴle test. Figure 20 shows the curves of pressure obtained from each pressure senso
0
5
10
15
20
25
30
−300
−250
−200
−150
−100
ΔF (N)
Δmox (g/s)
△x
Time (s)
−400
−200
0
0
2
4
−2 igure 19. Curves of variations in the increases in the thrust (ΔF), mass flow rate of the oxidizer
Δ𝑚̇ ௫), and position of the pintle (Δx) over time in the closed-loop throĴle test. Figure 19. Curves of variations in the increases in the thrust (∆F), mass flow rate of the oxidizer
(∆
.mox), and position of the pintle (∆x) over time in the closed-loop throttle test. 0
4.3. Discussion Δ𝑚௫), and position of the pintle (Δx) over time in the closed loop throĴle test. Figure 20 shows the curves of pressure obtained from each pressure sens Figure 20 shows the curves of pressure obtained from each pressure sensor during
the closed-loop throttle test. Although the downstream pressure of the venturi and that in
the combustion chamber changed, the upstream pressure of the venturi did not change
significantly, indicating that the variable-area cavitating venturi had effectively isolated
fluctuations in the downstream pressure. Figure 20 shows the curves of pressure obtained from each pressure sensor during
the closed-loop throttle test. Although the downstream pressure of the venturi and that
in the combustion chamber changed, the upstream pressure of the venturi did not change
significantly, indicating that the variable-area cavitating venturi had effectively isolated
fluctuations in the downstream pressure. the closed-loop throttle test. Although the downstream pressure of the venturi an
the combustion chamber changed, the upstream pressure of the venturi did no
significantly, indicating that the variable-area cavitating venturi had effectively
fluctuations in the downstream pressure. Figure 20. Curves of pressure obtained from each pressure sensor in the closed-loop throĴle test. 0
5
10
15
20
25
30
0
1
2
3
4
5
6
7
Pressure (MPa)
Time (s)
Oxidant tank pressure
Venturi upstream pressure
Venturi downstream pressure
Combustion chamber pressure
Figure 20. Curves of pressure obtained from each pressure sensor in the closed-loop thr
0
5
10
15
20
25
30
0
1
2
3
4
5
6
7
Pressure (MPa)
Time (s)
Oxidant tank pressure
Venturi upstream pressure
Venturi downstream pressure
Combustion chamber pressure
Figure 20. Curves of pressure obtained from each pressure sensor in the closed-loop throttle test. 5 Conclusions Figure 20 Curves of pressure obtained from each pressure sensor in the closed-loop th
Figure 20. Curves of pressure obtained from each pressure sensor in the closed-loop throttle test. Figure 20. Curves of pressure obtained from each pressure sensor in the closed-loop th
Figure 20. Curves of pressure obtained from each pressure sensor in the closed-loop throttle test. 5. Conclusions g
5. Conclusions In this study, the authors conducted a closed-loop thrust throttle test to maintain
a constant average thrust in a hybrid rocket motor composed of 98% hydrogen peroxide Aerospace 2023, 10, 421 19 of 21 19 of 21 and HTPB with 58% of an aluminum additive. A segmented PID controller was applied to
ensure the smooth transition of the variable thrust. The findings of this paper are as follows:
First, the segmented PID controller was used to realize a high-performance hybrid rocket
motor. The closed-loop throttle control strategy was effective in ensuring a constant thrust
during the operation of the motor. The results show that the average values of thrust in the
stable section in each stage were 400.7 N, 599.1 N, and 400.1 N when the target values of
thrust were 400 N, 600 N, and 400 N, respectively. The average thrust was thus stable and
the average accuracy of thrust control was better than 0.5%. The real-time error in thrust
was controlled to within ± 20 N with a steady-state error smaller than 5%. y
Second, the results here experimentally verify the effectiveness of closed-loop throttle
control for a high-performance hybrid rocket motor with a high mass fraction of aluminum. Thrust throttle control was achieved in the proposed scheme by using a variable-area
cavitating venturi to isolate fluctuations in the downstream pressure. The measured thrust
was used as the feedback measurement. The parameters of closed-loop throttle control
were derived from the specific impulse of the oxidizer, which was obtained in turn from
the internal ballistics of the hybrid rocket motor and did not require multiple tests to tune
the parameters. p
Third, closed-loop control can solve the problem of oscillations in the thrust of the
hybrid rocket motor containing 58% of an aluminum additive. The average thrust was
stable during the entire period of operation of the motor, and no large fluctuation in it
was observed. During the closed-loop throttle test with a duration of 21 s, the average
thrust remained close to the target value without any significant fluctuation. This is
an important difference between the tests reported here and the hot test conducted under
open-loop control. Author Contributions: Conceptualization, G.T. and H.T; methodology, G.T. and H.T.; software, G.T. and Y.Z.; validation, Z.W. and Z.G.; formal analysis, G.T. and Y.Z.; investigation, G.T. and Y.Z.;
resources, G.T. and J.G.; data curation, G.T. and J.G.; writing—original draft preparation, G.T. g
5. Conclusions and
H.T.; writing—review and editing, G.T., Z.W. and Z.G.; visualization, G.T. and J.G.; supervision, H.T. and G.C.; project administration, H.T. and G.C.; funding acquisition, H.T. and G.C. All authors have
read and agreed to the published version of the manuscript. Funding: This research was funded by the National Natural Science Foundation of China, grant
number U20B2034. Data Availability Statement: Data is contained within the article. Variables
QH2O
mass flow rate of ultrapure water, g/s
Isp
specific impulse, m/s
u(k)
controller output of kth control cycle
F
thrust, N
u(k −1)
controller output of (k −1)th control cycle
.mox
mass flow rate of oxidizer, kg/s
error(k)
deviation in the control signal of kth control cycle
.m f
mass flow rate of fuel, kg/s
kp, ki, and kd
PID control parameters
Iox
specific impulse of oxidizer, m/s
yd(k)
set value
.r
regression rate, mm/s
y(k)
measured value
Gox
mass flux of oxidizer, kg·s−1·m2
∆u(k)
increment in controller output
AP
cross-sectional area of flow of oxidizer, m2
ε
threshold value
d
inner diameter of tube grain, m
kp0, ki0, and kd0
large PID control parameters
ρf
fuel density, kg/m3
kp1, ki1, and kd1
small PID control parameters
Ab
combustion area, m2
Q
mass flow rate, kg/s
L
length of tube grain, m
Cd
flow coefficient
eburned
web thickness of burned grain, mm
A
throttling area, m2
Pc
pressure of combustion chamber, MPa
ρ
liquid density, kg/m3
c*
characteristic velocity, m/s
p1
total inlet pressure of the variable-area cavitating venturi, MPa
At
throttling area of the nozzle throat, m2
ps
saturated vapor pressure of the fluid at a given temperature, MPa
Fd(k)
set thrust value, N
x
pintle stroke, mm
F(k)
actual output thrust value, N
a, b
constant coefficients
∆F(k)
error in thrust value, N 20 of 21 Aerospace 2023, 10, 421 References Thomas, J.C.; Paravan, C.; Stahl, J.M.; Tykol, A.J.; Rodriguez, F.A.; Galfetti, L.; Petersen, E.L. Experimental evaluation of
HTPB/paraffin fuel blends for hybrid rocket applications. Combust. Flame 2021, 229, 111386. [CrossRef] , J
;
,
;
, J
;
y
,
J ;
g
,
;
,
;
,
p
PB/paraffin fuel blends for hybrid rocket applications. Combust. Flame 2021, 229, 111386. [CrossRef] 15. Tian, H.; Wang, Z.; Guo, Z.; Yu, R.; Cai, G.; Zhang, Y. Effect of Metal and Metalloid Solid-Fuel Additives on Performance and
Nozzle Ablation in a Hydroxy-terminated Polybutadiene based Hybrid Rocket Motor. Aerosp. Sci. Technol. 2022, 123, 107493. [CrossRef] [
]
16. Guo, Z.; Tian, H.; Wang, Z.; Meng, X.; Cai, G. Numerical and Experimental Study on 95% Hydrogen Peroxide Catalytic Ignition
of Hybrid Rocket Motors with HTPB-based Aluminum Additive Fuel. Acta Astronaut. 2022, 195, 98–108. [CrossRef] 17. Liu, Y.; Guan, Y.; Li, J.; Liu, P.; Zhang, X. Insulator Ablation Modes in Different Impact Conditions of Alumina Droplets onto Wall
Surfaces. Acta Astronaut. 2018, 153, 138–145. [CrossRef] 18. Karakas, H.; Kahraman, B.; Ozkol, I.; Karabeyoglu, A. Hybrid Rocket Nozzle Erosion with Microaluminum-Added Fuel. J. Propuls. Power 2022, 38, 901–910. [CrossRef] a, T. Performance of Mixture-Ratio-Controlled Hybrid Rockets Under Uncertainties in Fuel Regression
1, 37, 86–99. [CrossRef] 19. Ozawa, K.; Shimada, T. Performance of Mixture-Ratio-Controlled Hybrid Rockets Under Unce
J. Propuls. Power 2021, 37, 86–99. [CrossRef] 20. Casalino, L.; Masseni, F.; Pastrone, D. Robust Design Approaches for Hybrid Rocket Upper Stage. J. Aerosp. Eng. 2019, 32,
04019087. [CrossRef] 21. Cui, P.; Li, Q.; Cheng, P.; Chen, L. System Scheme Design for LOX/LCH4 Variable Thrust Liquid Rocket Engines Using Motor
Pump. Acta Astronaut. 2020, 171, 139–150. [CrossRef] 22. Yue, C.; Li, J.; Hou, X.; Feng, X.; Yang, S. Summarization on Variable Liquid Thrust Rocket Engines. Sci. China Ser. E Technol. Sci. 2009, 52, 2918–2923. [CrossRef] 23. Song, A.; Wang, N.; Li, J.; Ma, B.; Chen, X. Transient Flow Characteristics and Performance of a Solid Rocket Motor with a Pintle
Valve. Chin. J. Aeronaut. 2020, 33, 3189–3205. [CrossRef] 24. Guo, C.; Wei, Z.; Xie, K.; Wang, N. Thrust Control by Fluidic Injection in Solid Rocket Motors. J. Propuls
[CrossRef] 25. Zhao, S.; Cai, G.; Tian, H.; Yu, N.; Zeng, P. Experimental Tests of Throttleable H2O2/PE Hybrid Rocket Motors. In Proceedings of
the 51st AIAA/SAE/ASEE Joint Propulsion Conference, Orlando, FL, USA, 27–29 July 2015. J
p
J
y
26. References 1. Okninski, A.; Kopacz, W.; Kaniewski, D.; Sobczak, K. Hybrid Rocket Propulsion Technology for Space Transportation Revisited–
Propellant Solutions and Challenges. FirePhysChem 2021, 1, 260–271. [CrossRef] 2. Casalino, L.; Masseni, F.; Pastrone, D. Hybrid Rocket Engine Design Optimization at Politecnico di Torino: A Review. Aerospace
2021, 8, 226. [CrossRef] ]
o, L.; Pinarello, G. Paraffin-Based Hybrid Rocket Engines Applications: A Review and a Market Perspective
, 126, 286–297. [CrossRef] 3. Mazzetti, A.; Merotto, L.; Pinarello, G. Paraffin-Based Hybrid Rocket Engines Applications: A Revie
Acta Astronaut. 2016, 126, 286–297. [CrossRef] 4. Austin, B.; Heister, S.; Dambach, E.; Meyer, S.; Wernimont, E. Variable Thrust, Multiple Start Hybrid Motor Solutions for Missile
and Space Applications. In Proceedings of the 46th AIAA/ASME/SAE/ASEE Joint Propulsion Conference & Exhibit, Nashville,
TN, USA, 25–28 July 2010. y
5. Lee, D.; Han, S.; Moon, H. Development of 200 N-class Throttleable Hybrid Rocket Motor for Lun
FirePhysChem 2021, 1, 251–259. [CrossRef] y
oon, H. Development of 200 N-class Throttleable Hybrid Rocket Motor for Lunar Module Application
1, 251–259. [CrossRef] 6. Stoll, E.; Härke, P.; Linke, S.; Heeg, F.; May, S. The Regolith Rocket—A Hybrid Rocket Using Lunar Resources. Acta Astronaut. 2021, 179, 509–518. [CrossRef] 7. Casalino, L.; Masseni, F.; Pastrone, D. Optimal Design of Electrically Fed Hybrid Mars Ascent Vehicle. Aerospace 2021, 8, 181. [CrossRef] [
]
8. Kara, O.; Karaka¸s, H.; Karabeyo˘glu, M.A. Hybrid Rockets with Mixed N2O/CO2 Oxidizers for Mars Ascent Vehicles. Acta Astro-
naut. 2020, 175, 254–267. [CrossRef] 9. Liu, L.-L.; He, X.; Wang, Y.; Chen, Z.-B.; Guo, Q. Regression Rate of Paraffin-based Fuels in Hybrid Rocket Motor. Aerosp. Sci. Technol. 2020, 107, 106269. [CrossRef] ; Ravikumar, V. Mechanical Characterization of Paraffin-Based Hybrid Rocket Fuels. Mater. Today Proc. 201
sRef] 10. Pal, Y.; Ravikumar, V. Mechanical Characterization of Paraffin-Based Hybrid Rocket Fuels. Mater. Today Proc. 2019, 16, 939–948. [CrossRef] 11. Paccagnella, E.; Santi, M.; Ruffin, A.; Barato, F.; Pavarin, D.; Misté, G.A.; Venturelli, G.; Bellomo, N. Testin
Paraffin-Based Hybrid Rocket Motor. J. Propuls. Power 2019, 35, 432–442. [CrossRef] 12. Wu, Y.; Zhang, Z.; Wang, Q.; Wang, N. Combustion Characteristics of Skeleton Polymer Reinforced Paraffin-Wax Fuel Grain for
Applications in Hybrid Rocket Motors. Combust. Flame 2022, 241, 112055. [CrossRef] 13. Wang, Y.; Hu, S.-Q.; Liu, X.-L.; Liu, L.-L. Regression Rate Modeling of HTPB/Paraffin Fuels in Hybrid Rocket Motor. Aerosp. Sci. Technol. 2022, 121, 107324. [CrossRef] 14. Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content. References Whitmore, S.A.; Peterson, Z.W.; Eilers, S.D. Deep Throttle of a Nitrous Oxide and Hydroxyl-Terminated Polybutadiene Hybrid
Rocket Motor. J. Propuls. Power 2014, 30, 78–86. [CrossRef] 27. Messineo, J.; Shimada, T. Theoretical Investigation on Feedback Control of Hybrid Rocket Engines. Aerospace 2019, 6, 65. [CrossRef] 28. Whitmore, S.A.; Peterson, Z.W.; Eilers, S.D. Closed-Loop Precision Throttling of a Hybrid Rocket Motor. J. Propuls. Power 2014, 30,
325–336. [CrossRef] 21 of 21 21 of 21 Aerospace 2023, 10, 421 29. Velthuysen, T.; Brooks, M.; Pitot, J. Closed Loop Throttle Control of a Liquefying Fuel Hybrid Rocket Motor. J. Aerosp. Technol. Manag. 2021, 13, e2421. [CrossRef] g
30. Tian, H.; Wang, Z.; Zhu, H.; Lu, Y.; Gao, J.; Cai, G. Effect of High-Mass Fraction of Aluminum on Catalytic Hybrid Rocket Motor. Appl. Sci. 2022, 12, 13023. [CrossRef] 31. Guang, T.; Hui, T.; Xianzhu, J.; Ruikai, C.; Yudong, L.; Yuanjun, Z. Experimental Study on Precise Thrust Control of Hydrogen
Peroxide and Polyethylene Hybrid Rocket Motors. Acta Astronaut. 2022, 196, 303–313. [CrossRef] 32. Tian, H.; Zeng, P.; Yu, N.; Cai, G. Application of Variable Area Cavitating Venturi as a Dynamic Flow Controller. Flow Meas. Instrum. 2014, 38, 21–26. [CrossRef] 33. Zeng, P.; Tian, H.; Yu, N.; Cai, G. Numerical Investigation on Flow in the Variable Area Cavitating Vent
49th AIAA/ASME/SAE/ASEE Joint Propulsion Conference, San Jose, CA, USA, 14–17 July 2013. 33. Zeng, P.; Tian, H.; Yu, N.; Cai, G. Numerical Investigation on Flow in the Variable Area Cavitating Venturi. In Proceedings of the
49th AIAA/ASME/SAE/ASEE Joint Propulsion Conference, San Jose, CA, USA, 14–17 July 2013. 4
Zh
X W
D L
R Zh
H Sh B S
d
f M
h
l Ch k d V
N
l
U
d f
L
d Fl
C
ll ao, R.; Zhao, H.; Shi, B. Study of Mechanical Choked Venturi Nozzles Used for Liquid Flow Controlling. 9, 65, 158–165. [CrossRef] 34. Zhang, X.; Wang, D.; Liao, R.; Zhao, H.; Shi, B. Study of Mechanical Choked Venturi Nozzles Used
Flow Meas. Instrum. 2019, 65, 158–165. [CrossRef] 35. Carmicino, C.; Sorge, A.R. Influence of a Conical Axial Injector on Hybrid Rocket Performance. J. Propuls. Power 2006, 22, 984–995. [CrossRef] [
]
36. Saito, Y.; Kamps, L.T.; Tsuji, A.; Nagata, H. Reconstruction techniques for determining O/F in hybrid rockets. In Proceedings of
the AIAA Propulsion and Energy 2021 Forum, Virtual Event, 9–11 August 2021. 36. 36.
Saito, Y.; Kamps, L.T.; Tsuji, A.; Nagata, H. Reconstruction techniques for determining O/F in hybrid r
the AIAA Propulsion and Energy 2021 Forum, Virtual Event, 9–11 August 2021. References Saito, Y.; Kamps, L.T.; Tsuji, A.; Nagata, H. Reconstruction techniques for determining O/F in hybrid rockets. In Proceedings of
the AIAA Propulsion and Energy 2021 Forum, Virtual Event, 9–11 August 2021. Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content. Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.
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https://openalex.org/W3043051316
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http://ojs.uho.ac.id/index.php/JPM/article/download/9902/pdf
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Indonesian
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Pembelajaran Kooperatif Think Talk Write (TTW) Untuk Meningkatkan Komunikasi Matematis
|
Jurnal pendidikan matematika/Jurnal Pendidikan Matematika
| 2,020
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cc-by
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(Think Talk Write (TTW) Cooperative Learning to Improve Mathematical Communication) Keywords: think talk write (TTW), algebraic function, mathematical communication * Korespondensi Penulis. E-mail: abd.qohar.fmipa@um.ac.id (Think Talk Write (TTW) Cooperative Learning to Improve Mathematical Communication) Anis Rochma Pratiwi 1), Abd.Qohar 1)*
1 Jurusan Matematika, Universitas Negeri Malang, Kota Malang, Indonesia. Abstrak: Penelitian ini bertujuan untuk menjelaskan langkah-langkah pembelajaran Think Talk Write (TTW)
yang dapat meningkatkan komunikasi matematis tertulis siswa pada materi fungsi aljabar bagi siswa kelas XI
E-4 SMA Negeri 5 Malang. Jenis peneltian ini adalah Penelitian Tindakan Kelas yang terdiri dari 2 siklus. Masing-masing siklus terdiri dari tiga pertemuan dan satu kali tes akhir siklus. Subjek penelitian ini adalah
siswa kelas XI E-4 SMA Negeri 5 Malang yang berjumlah 35 siswa. Data penelitian ini berupa hasil
observasi, hasil catatan lapangan, dan hasil tes akhir siklus. Proses pembelajaran TTW terdiri dari tiga tahap
yaitu (1) tahap think, (2) tahap talk, (3) tahap write. Hasil penelitian menunjukkan bahwa pembelajaran Think
Talk Write (TTW) dapat meningkatkan komunikasi matematis tertulis siswa pada materi fungsi aljabar. Kriteria keberhasilan yang ditetapkan peneliti tercapai pada siklus II yaitu: 85,71% siswa memperoleh nilai
tes kemampuan komunikasi matematis tertulis dengan kategori baik, aktivitas guru dan siswa juga masuk
dalam kategori baik. Oleh karena itu disarankan pada para guru agar menerapkan pembelajaran TTW untuk
meningkatkan komunikasi matematis tertulis siswa. Kata kunci: fungsi aljabar, think talk write (TTW), komunikasi matematis. Kata kunci: fungsi aljabar, think talk write (TTW), komunikasi matematis. Abstract: This study aims to describe the steps of the Think Talk Write (TTW) learning model that can
improve students' mathematical written communication on algebraic function material for students of class
XI E-4 SMA Negeri 5 Malang. This type of research is Classroom Action Research with two cycles. Each
cycle consists of three learning meetings and one final test cycle. The subjects of this study were students of
class XI E-4 SMA Negeri 5 Malang, totaling 35 students. This research data in the form of observations, field
notes and final cycle results. The learning process of TTW consists of three stages, namely (1) the think
stage, (2) the talk stage, (3) the write stage. The results of this study indicate that the Think Talk Write (TTW)
learning can improve students' written mathematical communication on the algebraic function material. The
success criteria determined by researchers were achieved in the second cycle, namely: 85.71% of students
received a score of the written mathematical communication skills test in the good category, teacher and
student activities also included in the good category. It is therefore recommended that teachers apply TTW
learning to improve students' written mathematical communication. Penerbit: Jurusan Pendidikan Matematika FKIP Universitas Halu Oleo PENDAHULUAN & Lubis (2018), komunikasi antara satu
dengan yang lainya adalah kunci utama
untuk membangun kehidupan yang lebih
baik, tanpa komunikasi tidak akan mungkin
terjadinya pertukaran pola pikir untuk
kemajuan bangsa ini Komunikasi
matematis
merupakan
proses mengkonstruksi pemahaman ide-ide
matematika dan membuat lebih mudah
untuk dimengerti (Hasibuan & Amry, 2017). (Senjayawati, 2015) mengemukakan bahwa
kemampuan komunikasi matematis menjadi
modal
penting
dalam
menyelesaikan,
mengekplorasi,
dan
menginvestigasi
matematikadan mempertajam ide untuk
meyakinkan orang lain. Menurut Pane, Jaya, Devlin (2009) mengungkapkan bahwa
pembelajaran
yang
efektif
setidaknya
memenuhi
beberapa
hal
berikut:
(1)
mengembangkan siswa pada kemampuan Penerbit: Jurusan Pendidikan Matematika FKIP Universitas Halu Oleo 99 Jurnal Pendidikan Matematika, 11 (1) (2020): 99-109
Pembelajaran Kooperatif Think Talk Write (TTW) Untuk Meningkatkan Komunikasi Matematis Jurnal Pendidikan Matematika, 11 (1) (2020): 99-109
Pembelajaran Kooperatif Think Talk Write (TTW) Untuk Meningkatkan Komunikasi Matematis Jurnal Pendidikan Matematika, 11 (1) (2020): 99-109
Pembelajaran Kooperatif Think Talk Write (TTW) Untuk Meningkatkan Komunikasi Matematis berpikir kritis, komunikasi, kolaborasi dan
berpikir
kreatif,
(2)
mengembangkan
pemahaman
konsep
dasar
siswa,
(3)
pembelajaran
terhubung
dengan
permasalahan sehari-hari. Selain itu menurut
Qohar
(2011)
komunikasi
matematis
digunakan
untuk
memahami
ide-ide
matematika
secara
benar. Lemahnya
kemampuan
komunikasi
akan
mengakibatkan
lemahnya
kemampuan
memahami
konsep-konsep
matematika
lainnya. Sehingga kemampuan komunikasi
matematis
merupakan
salah
satu
kemampuan yang perlu dimiliki siswa
dalam pembelajaran matematika. guru dan siswa. NCTM (2000:29) tentang
Standar Proses pembelajaran matematika
adalah
problem
solving
(pemecahan
masalah), reasoning and proof (memberikan
alasan
dan
bukti),
communication
(komunikasi),
connections
(menghubungkan),
and
representations
(menggambarkan). Sehingga dari kurikulum
ini guru wajib mengembangkan kemampuan
komunikasi siswa menjadi lebih baik. Berdasarkan hasil studi pendahuluan
yang dilakukan peneliti di kelas XI E-4
SMAN
5
Malang
ditemukan
bahwa
komunikasi matematis tertulis siswa masih
rendah. Pada saat itu di kelas XI E-4 sedang
berlangsung pembahasan soal tentang materi
limit. Pada saat diskusi kelompok, banyak
diantara siswa mengalami kesulitan dalam
menyelesaikan soal limit. Sehingga banyak
siswa yang melakukan kesalahan dalam
menyelesaikan soal limit. Oleh karena
banyaknya kesalahan siswa ini, maka materi
limit dipilih untuk penelitian tindakan kelas
ini. Contoh
kesalahan
komunikasi
matematis dalam penyelesaian soal limit
dapat dilihat pada Gambar 1 berikut Mengembangkan
kemampuan
komunikasi
matematis
sejalan
dengan
paradigm baru pembelajaran matematika. Menurut Qohar (2011), pada pendekatan
lama,
guru
lebih
dominan
dalam
pembelajaran
dan
hanya
bersifat
memberikan
ilmu
pengetahuan
kepada
siswa, sedangkan para siswa bersikap diam
dan pasif menerima ilmu pengetahuan dari
guru tersebut. Guru membantu siswa dalam
meluruskan pemahaman siswa yang kurang
tepat melalui komunikasi yang dilakukan Gambar 1. PENDAHULUAN Kesalahan Komunikasi Matematis Siswa KR Pada Penyelesaian Soal Limit
Fungsi Gambar 1. Kesalahan Komunikasi Matematis Siswa KR Pada Penyelesaian Soal Limit
Fungsi Pada Gambar 1, dapat dilihat bahwa
siswa tidak menggambarkan grafik fungsi
dan menentukan nilai limit fungsi. Padahal
disebutkan pada soal bahwa siswa diminta
untuk
menggambarkan
grafik
dan
menentukan nilai limit dari suatu fungsi. Ia
hanya mensubstitusikan nilai 𝑥 pada fungsi
sesuai dengan syarat yang ditentukan. Berdasarkan
wawancara
yang
telah
dilakukan peneliti dengan siswa KR, ia mengaku
bahwa
belum
mampu
menggambarkan grafik fungsi. Sehingga KR
tidak bisa menentukan nilai limit fungsi
tersebut. Kesalahan yang dilakukan KR
dapat
disebabkan
oleh
kurangnya
kemampuan komunikasi matematis tertulis. Kurangnya kemampuan komunikasi siswa
dalam menggambar grafik tidak hanya
dialami oleh KR. Banyak juga siswa lain
yang melakukan kesalahan serupa. Beberapa mengaku
bahwa
belum
mampu
menggambarkan grafik fungsi. Sehingga KR
tidak bisa menentukan nilai limit fungsi
tersebut. Kesalahan yang dilakukan KR
dapat
disebabkan
oleh
kurangnya
kemampuan komunikasi matematis tertulis. Kurangnya kemampuan komunikasi siswa
dalam menggambar grafik tidak hanya
dialami oleh KR. Banyak juga siswa lain
yang melakukan kesalahan serupa. Beberapa 100 Jurnal Pendidikan Matematika, 11 (1) (2020): 99-109
Anis Rochma Pratiwi, Abd.Qohar matematis,
yaitu
kesalahan
dalam
menuliskan notasi limit, penulisan tanda
yang menyebabkan kesalahan pada jawaban
akhir. Hal tersebut dapat dilihat pada
Gambar 2 berikut. siswa mampu menggambarkan grafik fungsi
namun tidak lengkap, bisa menggambarkan
grafik tetapi tidak bisa menentukan nilai
limit fungsi. Selain KR, ada juga siswa yang
melakukan
kesalahan
komunikasi Selain KR, ada juga siswa yang
melakukan
kesalahan
komunikasi Gambar 2. Kesalahan Komunikasi Matematis Siswa RZA Pada Penyelesaian Soal Nilai
Limit Fungsi Gambar 2. Kesalahan Komunikasi Matematis Siswa RZA Pada Penyelesaian Soal Nilai
Limit Fungsi Berdasarkan Gambar 2, dapat dilihat
bahwa RZA tidak menuliskan notasi limit,
ia hanya menuliskan fungsinya saja. Padahal
sebelumnya sudah diberi petunjuk agar
menuliskan notasi limit saat masih ada
variabel dan tidak perlu menuliskan
notasi limit saat mensubstitusikan nilai x
yang didekati. Kesalahan lainnya yaitu,
tidak memberikan tanda kurung pada (𝑥 +
2), sehingga menyebabkan kesalahan pada
jawaban akhir. Seharusnya jawabannya
berupa bilangan yang terdefinisi yaitu
3
3
2
. Dari dua contoh pekerjaan siswa,
yaitu pekerjaan KR dan RZA menunjukkan
bahwa komunikasi matematis siswa masih
rendah. Lebih lanjut mereka mengatakan
bahwa dalam belajar sering menghafal suatu
materi
dari
buku
atau
catatan
yang
dibuatnya sendiri. Sehingga materi yang
didapat menjadi tidak bermakna. peningkatan motivasi belajar matematika
siswa
yang
menggunakan
model
pembelajaran
TTW. PENDAHULUAN Maisyarah
(2017)
dalam penelitiannya menerapkan model
pembelajaran kooperatif tipe Student Teams
Achievement
Division
(STAD)
dapat
meningkatkan komunikasi matematis siswa
SMA. Penelitian-penelitian
tersebut
berfokus terhadap penggunaan Think, Talk,
Write (TTW) pada hasil belajar dan motivasi
belajar. Selain
itu,
penelitian
terkait
komunikasi matematis menggunakan model
STAD. Dari beberapa penelitian tersebut,
masih diperlukan penelitian terkait dengan
komunikasi matematis menggunakan model
pembelajaran TTW. TTW merupakan model pembelajaran
yang dapat memfasilitasi komunikasi lisan
dan tulis (Huda, 2013). Model pembelajaran
ini
diperkenalkan
oleh
Huinker
dan
Laughlin (1996). Menurut Laughlin dalam
Huda (2013) alur kemajuan komunikasi
dimulai dari keterlibatan siswa dalam
berpikir atau berdialog dengan dirinya
sendiri, kemudian berbicara dan berbagi ide
satu sama lain, dan selanjutnya menulis. Sesuai namanya, model pembelajaran ini
memiliki tiga tahapan. Tahap pertama
adalah think, di mana siswa akan diberikan
teks berupa masalah dan dalam tahap ini Beberapa
penelitian
terdahulu
mengenai
pembelajaran
yang
telah
dilakukan, diantaranya Riski, Rijal, &
Linawati (2017) meneliti tentang hasil
belajar siswa di kelas VIII C SMP Negeri 9
Palu dengan hasil adanya peningkatan
dengan menggunakan pembelajaran TTW. Penelitian
Jariswandana,
Yerizon,
&
Nilawasti
(2012)
menemukan
adanya 101 Jurnal Pendidikan Matematika, 11 (1) (2020): 99-109
Pembelajaran Kooperatif Think Talk Write (TTW) Untuk Meningkatkan Komunikasi Matematis Jurnal Pendidikan Matematika, 11 (1) (2020): 99-109
Pembelajaran Kooperatif Think Talk Write (TTW) Untuk Meningkatkan Komunikasi Matematis Jurnal Pendidikan Matematika, 11 (1) (2020): 99-109
Pembelajaran Kooperatif Think Talk Write (TTW) Untuk Meningkatkan Komunikasi Matematis pembelajaran lain seperti STAD, Teams
Games
Tournament
(TGT),
jigsaw,
Numbered Head Together (NHT), dan
investigasi kelompok tidak ada tahapan
seperti
itu
di
awal
pembelajaran
(Ratumanan, 2015). Pada strategi TTW
terdapat tes awal yang dilakukan pada awal
pembelajaran, tetapi tes ini bertujuan untuk
menempatkan
siswa
pada
program
individual (Ratumanan, 2015). Sehingga,
model pembelajaran TTW dirasa menjadi
model pembelajaran yang lebih mendukung
untuk
meningkatkan
kemampuan
komunikasi matematis siswa. Tujuan yang
ingin dicapai dalam penelitian ini adalah
untuk
mendeskripsikan
langkah-langkah
pembelajaran kooperatif tipe TTW yang
dapat meningkatkan komunikasi matematis
siswa kelas XI E-4 SMA Negeri 5 Malang
pada materi turunan fungsi aljabar. siswa memikirkan kemungkinan jawaban
serta membuat catatan kecil tentang ide-ide
dari bacaan yang telah diberikan. Tahap
kedua adalah talk, dimana siswa diberikan
kesempatan untuk mengungkapkan hasil
penyelidikan dari tahap pertama. Dengan
kata
lain
siswa
diberi
kesempatan
mengomunikasikan ide-ide matematis untuk
masalah yang diberikan. Tahap terakhir
adalah
write,
dimana
peserta
didik
menuliskan
ide-ide
matematis
yang
diperolehnya
secara
jelas. Cara
menuliskannya pun secara matematis. METODE PENELITIAN Penelitian
ini
menggunakan
pendekatan kualitatif dan kuantitatif. Jenis
penelitian ini adalah Penelitian Tindakan
Kelas (PTK). Penelitian dilaksanakan pada
semester genap 2018/2019 di Kelas XI E-4
SMA Negeri 5 Malang yang terdiri dari 35
siswa dengan 21 siswa perempuan dan 14
siswa laki-laki. siklus. Indikator komunikasi matematis
yang digunakan pada penelitian ini adalah
(1) menyatakan permasalahan ke dalam
model matematika (gambar, tabel, diagram,
grafik dan ekspresi aljabar) yang tepat, (2)
menjelaskan ide dan model matematika
(gambar, tabel, diagram, grafik dan ekspresi
aljabar) secara tertulis dengan tepat, (3)
menggunakan simbol dan notasi matematika
dengan benar. Instrumen yang digunakan dalam
penelitian ini adalah lembar tes, lembar
observasi kegiatan guru dan observasi
kegiatan siswa, lembar catatan lapangan
serta dokumentasi. Penelitian ini terdiri dari
dua siklus dengan masing-masing terdiri
dari
tiga
pertemuan. Kemudian
pada
pertemuan keempat diberikan tes akhir Penelitian ini dikatakan berhasil jika (a)
hasil
pengamatan
aktivitas
guru
dalam
pembelajaran minimal baik, (b) minimal 75%
dari semua siswa minimal berada pada kategori
baik. PENDAHULUAN Model pembelajaran TTW ini dipilih
karena
dalam
tahapannya,
model
pembelajaran ini memberikan kesempatan
kepada
siswa
untuk
memikirkan
penyelesaian dari suatu permasalahan yang
diberikan secara individu sebagai bentuk
pemahaman awal atau pemikiran awal
(tahap
think),
sedangkan
pada
model HASIL PENELITIAN Pada penerapan pembelajaran TTW,
guru
merencanakan
pembelajaran
tiap
siklusnya mulai dari siklus pertama. Setiap
siklus terdiri dari tiga pertemuan dan satu
kali tes akhir siklus. Pada siklus I
pertemuan pertama, kegiatan pembelajaran
diawali dengan salam pembuka, do’a dan
mengecek kehadiran siswa. Selanjutnya dilakukan pemberian apersepsi terkait nilai
fungsi, grafik fungsi, limit fungsi dan
gradien garis lurus. Pada tahap Think, siswa mengamati
permasalahan dalam kehidupan sehari-hari
tentang gradien garis dan menuliskan
pertanyaan terkait yang diamati. Tahap Talk,
siswa berdiskusi untuk menemukan definisi 102 Jurnal Pendidikan Matematika, 11 (1) (2020): 99-109
Anis Rochma Pratiwi, Abd.Qohar turunan sebagai suatu limit fungsi. Tahap
Write,
siswa
menuliskan
kesimpulan
pembelajaran dan mengerjakan soal kuis
secara individu. Kemudian, pada kegiatan
penutup, siswa merefleksi pembelajaran
dengan mengisi lembar self assessment. Pada pertemuan kedua dan ketiga, siswa
diberi apersepsi tentang definisi turunan. Pada tahap think, siswa mengamati beberapa
fungsi beserta turunannya. Pada tahap talk,
siswa berdiskusi untuk menemukan sifat-
sifat turunan fungsi aljabar menggunakan
definisi turunan. Pada tahap write, siswa
menuliskan
hasil
diskusi
berdasarkan
pemahaman
kelompoknya,
menuliskan
kesimpulan pembelajaran dan mengerjakan soal tentang sifat-sifat turunan fungsi
aljabar. Pada kegiatan penutup, siswa
mengisi lembar self assessment untuk
merefleksi pembelajaran pada pertemuan
tersebut. Pada saat tes akhir siklus I, ditemukan
beberapa
kesalahan
siswa
dalam
menyelesaikan soal, salah satunya dapat
dilihat pada Gambar 3. Siswa masih
melakukan
kesalahan
pada
penulisan
pangkat, langkah penyelesaian yang kurang
tepat, dan siswa tidak menuliskan sifat
turunan fungsi apa yang digunakan untuk
menentukan turunan dari fungsi yang
diberikan. Gambar 3 Pekerjaan Siklus I Salah Satu Siswa Gambar 3. Pekerjaan Siklus I Salah Satu Siswa Gambar 3. Pekerjaan Siklus I Salah Satu Siswa
Gambar 4. Hasil Observasi dan Hasil Tes Akhir Siklus Gambar 4. Hasil Observasi dan Hasil Tes Akhir Siklus I menunjukkan bahwa dari 35 siswa kelas E-
4 terdapat 13 siswa yang mendapatkan
kategori sangat baik, 11 siswa mendapatkan
kategori baik, 6 siswa mendapatkan kategori
cukup dan 5 siswa mendapatkan kategori
kurang, sehingga persentase secara klasikal Berdasarkan Gambar 4 dapat dilihat
hasil observasi aktivitas guru dan siswa dari
dua observer diperoleh rata-rata nilai dengan
kategori sangat baik untuk aktivitas guru
dan kategori baik untuk aktivitas siswa. HASIL PENELITIAN Sedangkan berdasarkan hasil tes akhir siklus 103 Jurnal Pendidikan Matematika, 11 (1) (2020): 99-109
Pembelajaran Kooperatif Think Talk Write (TTW) Untuk Meningkatkan Komunikasi Matematis Jurnal Pendidikan Matematika, 11 (1) (2020): 99-109
operatif Think Talk Write (TTW) Untuk Meningkatkan Komunikasi Matematis siswa
yang
memiliki
kemampuan
komunikasi matematis tertulis minimal baik
adalah 68,57%. dilakukan berdasarkan beberapa kelemahan
dan kekurangan pembelajaran yang terjadi
selama siklus I. Tabel 1 berikut, merupakan
beberapa kelemahan tersebut dan tindakan
perbaikan pembelajaran yang dilakukan
pada siklus II. Berdasarkan hasil tersebut, penelitian
dilanjutkan pada siklus II. Siklus ini juga
dilaksanakan dalam tiga pembelajaran dan
satu kali tes akhir siklus. Perbaikan Tabel 1. Refleksi Siklus I dan Tindakan Perbaikan Siklus II Solusi Perbaikan
Guru lebih tegas dan disiplin terhadap waktu,
bila perlu menegur siswa yang berbicara hal-hal
di luar materi Kekurangan
Waktu yang dibutuhkan pada beberapa
tahapan kegiatan lebih lama dari yang
telah direncanakan
Perhatian guru kurang merata Guru berkeliling ke setiap kelompok dan
berusaha memberikan perhatian berupa
penjelasan dan pengarahan secara merata
Pengawasan terhadap siswa ditambah lagi
dengan cara guru lebih aktif berkeliling ke tiap-
tiap kelompok diskusi Masih banyak siswa yang ramai dan
tidak aktif saat diskusi kelompok
Ada beberapa kelompok yang bekerja
secara individu Masih banyak siswa yang ramai dan
tidak aktif saat diskusi kelompok Masih banyak siswa yang ramai dan
tidak aktif saat diskusi kelompok Ada beberapa kelompok yang bekerja
secara individu Ada beberapa kelompok yang bekerja
secara individu Guru menekankan bahwa jawaban dalam satu
kelompok merupakan hasil diskusi/pemikiran
bersama-sama, maka dari itu siswa harus
berdiskusi dalam menyelesaikannya. Guru juga
mengingatkan
bahwa
boleh
bergantian
menuliskan jawaban atau dibagi-bagi, tetapi
jawaban
tersebut
adalah
hasil
pemikiran
bersama Ada kelompok yang tidak
mendengarkan presentasi teman dari
kelompok lainnya Ada kelompok yang tidak
mendengarkan presentasi teman dari
kelompok lainnya Guru memberikan pertanyaan dan mengambil
perhatian siswa yang tidak memperhatikan
presentasi teman dari kelompok lainnya dengan
cara menanyakan apa yang telah disampaikan
teman sebelumnya dan mengulangi jawaban
temannya Ada beberapa siswa yang masih
bingung/belum paham dengan materi Guru harus berusaha lebih peka terhadap
seluruh
siswa,
guru
dapat
memastikan
pemahaman siswa pada saat berkeliling dengan
tanya jawab dan memperhatikan jawaban yang
ditulis siswa di LKS Tulisan di papan tulis kurang teratur Tulisan di papan tulis kurang teratur Jika memang membutuhkan papan tulis, Jika memang membutuhkan papan tulis,
sebaiknya papan tulis dibagi menjadi beberapa
bagian dan memberinya nomor agar lebih teratur
penulisannya Pada siklus II fokus guru memperbaiki
komunikasi matematis tulis siswa seperti
menuliskan
notasi/simbol
matematika,
memodelkan
permasalahan
ke
dalam
matematika melalui pembelajaran aplikasi
turunan. Sebelum
memasuki
tahapan
pembelajaran TTW, siswa diberi apersepsi terkait turunan fungsi aljabar dan gradien
garis
singgung. Selain
itu,
guru
menginformasikan
tujuan
pembelajaran
yaitu menemukan bahwa gradien suatu garis
singgung suatu fungsi dapat ditentuan
dengan menggunakan turunan pertama,
kemudian
guru
memberikan
motivasi 104 Jurnal Pendidikan Matematika, 11 (1) (2020): 99-109
Anis Rochma Pratiwi, Abd.Qohar ketika fungsi naik dan turun dengan
menggunakan turunan pertama. Kelompok
yang digunakan untuk pertemuan kedua
merupakan kelompok yang sama dengan
pertemuan sebelumnya. Pada tahap write,
guru
mengarahkan
siswa
untuk
menyimpulkan hasil diskusi, dilanjutkan
dengan pemberian soal terkait penentuan
interval
naik
dan
turun. PEMBAHASAN berdialog dengan dirinya sendiri melalui
pemikirannya,
tanpa
harus
berdiskusi
dengan siswa lain (Huinker & Laughlin,
1996). Berdasarkan Gambar 4 dapat dilihat
hasil observasi aktivitas guru dan siswa
menunjukkan bahwa telah mencapai nilai
pada kategori sangat baik. Hal tersebut
berarti
bahwa
hasil
observasi
telah
memenuhi kriteria keberhasilan penelitian. Sedangkan berdasarkan hasil tes akhir siklus
II, siswa yang mendapatkan nilai dengan
kategori minimal baik sebanyak 85,71%. Hal tersebut berarti pemahaman siswa
terkait materi turunan juga meningkat. Hasil
ini
menunjukkan
bahwa
pembelajaran
dengan menggunakan model TTW dapat
meningkatkan
komunikasi
matematika
tertulis siswa pada materi turunan fungsi
aljabar. Pada tahap talk, guru membagi siswa
menjadi
beberapa
kelompok
secara
heterogen. Pengelompokan
tersebut
dilakukan sesuai dengan pendapat Lie
(2010) yaitu kelompok dengan anggota yang
heterogen akan memberikan peluang untuk
saling
mengajar
(peer
tutorial)
dan
memudahkan
pengelolaan
kelas. Selanjutnya
siswa
diberi
kesempatan
mendiskusikan LKS yang diberikan oleh
guru untuk menguatkan atau mengonfirmasi
hipotesis jawaban dan pertanyaan siswa
yang telah ditulis pada tahap think (Huda,
2013). Oleh karena itu, siswa tidak hanya
menggali
informasi
untuk
menemukan
konsep matematika pada tahap ini, namun
juga
mengaitkan/menganalisis
informasi
yang sudah didapat agar menjadi lebih
kompleks dan bermakna bagi siswa. Pembelajaran
dimulai
dengan
pemberian
apersepsi,
siswa
diberi
kesempatan
untuk
mengaitkan
pemahamannya
dengan
pemahaman
sebelumnya. Menurut
Ausubel
dalam
Subanji (2013), kegiatan tersebut dapat
menimbulkan proses belajar bermakna. Hal
tersebut dikarenakan siswa dapat membuat
suatu
skema
yang
menyambungkan
pemahaman yang telah dimiliki siswa
dengan pemahaman yang
akan
diperolehnya (Croker & Buchanan, 2011). Selanjutnya
pemberian
motivasi
pembelajaran berupa manfaat yang didapat
dari permasalahan dalam kehidupan sehari-
hari maupun fakta-fakta tentang materi yang
akan dipelajari. Menurut Lestari (2017),
untuk membangkitkan motivasi belajar
siswa maka perlu diberikan pembelajarna
yang baik sesuia dengan keinginan peserta
didik yang pada akhirnya berpengaruh pada
hasil belajarnya. Pada tahap write, siswa menuliskan
hasil diskusinya pada tempat yang telah
disediakan pada LKS. Ide-ide matematis
yang didiskusikan dan ditulis siswa pada
tahap talk dan write masih memiliki
kemungkinan belum lengkap atau bahkan
miskonsepsi (Huinker dan Laughlin, 1996). Oleh karena itu, guru memberi kesempatan
kepada beberapa siswa untuk menuliskan
jawabannya pada papan tulis untuk dibahas
secara klasikal. Kemampuan matematis
siswa dalam proses pembelajaran dapat
meningkat melalui kegiatan cara menulis
(Kosko & Gao, 2017) dan pembelajaran
kooperatif (Kosko & Gao, 2017; Munir,
Baroutian, & Young, 2018; Qohar &
Sumarmo, 2013). Tabel 1. Refleksi Siklus I dan Tindakan Perbaikan Siklus II Pada
akhir
pembelajaran, siswa mengisi lembar self
assessment untuk merefleksi pembelajaran. pembelajaran. Pada tahap think pertemuan
pertama siklus II, siswa diberi bahan
pengamatan berupa masalah tentang gradien
garis singgung dan menuliskan pertanyaan
berkaitan dengan hasil pengamatannya. Pada tahap talk, siswa berkelompok dengan
kelompok yang sama pada pembelajaran
siklus
1
untuk
mencapai
tujuan
pembelajaran. Pada kegiatan diskusi, guru
lebih
memperhatikan
siswa
yang
membutuhkan
motivasi
untuk
belajar. Selanjutnya, setelah setiap kelompok telah
mencapai
tujuan
pembelajaran,
siswa
diminta untuk mendiskusikan penyelesaian
soal pada kegiatan Ayo Menalar. Pada tahap
write, siswa menuliskan kesimpulan dari
pembelajaran dan mengerjakan soal kuis
yang memuat soal untuk menentukan
persamaan garis singgung suatu fungsi
dengan
menggunakan
turunan
pertama
fungsi. Pada pertemuan ketiga, siswa diberi
apersepsi dan motivasi terkait materi nilai
maksimum dan minimum suatu fungsi. Pada
tahap think, siswa mengamati grafik fungsi
yang
memuat
nilai
maksimum
dan
minimum, kemudian siswa menuliskan
informasi yang diperolehh dan membuat
pertanyaan terkait pengamatannya. Pada
tahap
talk,
siswa
berdiskusi
untuk
menemukan
cara
menentukan
nilai
maksimum
dan
minimu
suatu
fungsi
menggunakan turunan pertama fungsi. Pada
tahap write, guru mengarahkan siswa untuk
menyimpulkan hasil diskusi, dilanjutkan
dengan pemberian soal terkait penentuan
nilai maksimum dan minimum. Pada akhir
pembelajaran, siswa mengisi lembar self
assessment untuk merefleksi pembelajaran. Pada tes akhir siklus II, terlihat pada
Gambar 5 bahwa siswa sudah menuliskan
langkah-langkah
penyelesaian
dengan
lengkap. Selanjutnya pada pertemuan kedua,
guru
memperbaiki cara
menyimpulkan
melalui pembelajaran kemonotonan fungsi. Pada awal pembelajaran, siswa mengingat
kembali gradien garis singgung fungsi
menggunakan turunan pertama. Selanjutnya
guru
menginformasikan
tujuan
pembelajaran dan memberikan motivasi
Pada tahap think, siswa mengamati grafik
fungsi kemudian meminta siswa untuk
menyebutkan interval suatu fungsi saat naik
atau
turun. Pada
tahap
talk,
secara
berkelompok siswa menemukan interval Gambar 5 Pekerjaan Siklus II Salah Satu Siswa Gambar 5 Pekerjaan Siklus II Salah Satu Siswa 105 105 Jurnal Pendidikan Matematika, 11 (1) (2020): 99-109
Pembelajaran Kooperatif Think Talk Write (TTW) Untuk Meningkatkan Komunikasi Matematis PEMBAHASAN Masingila & Wisniowska
(1996) dalam tulisannya menyatakan bahwa
kegiatan menulis bisa membantu siswa
dalam
menyatakan
pengetahuan
dan
gagasan yang masih belum terungkap agar
lebih
terlihat
dan
merefleksikan
pengetahuan dan ide-ide mereka. Selain itu
aktivitas
menulis
dalam
pembelajaran
matematika
dapat
menunjukkan Pada tahap pertama yaitu tahap think
Siswa mengamati sajian yang ditampilkan
guru
berupa
gambar,
ilustrasi,
atau
permasalahan kontekstual yang berkaitan
dengan
materi
yang
akan
dipelajari. Kemudian siswa memikirkan informasi
penting apa saja yang ada pada sajian atau
solusi dari permasalahan yang ditampilkan
dan menuliskannya pada kotak catatan. Pada
aktivitas ini siswa berpikir secara invidual, 106 Jurnal Pendidikan Matematika, 11 (1) (2020): 99-109
Anis Rochma Pratiwi, Abd.Qohar menggunakan bahasanya sendiri (Huda,
2013:). keberhasilan
tujuan
utama
dalam
pembelajaran, yaitu siswa dapat memahami
materi yang telah diajarkan (Shield &
Swinson, 1996). Selanjutnya siswa juga
menuliskan hasil pembelajaran yang mereka
dapatkan mulai dari proses diawal hingga
diakhir berupa kesimpulan. Guru meminta
siswa untuk menuliskan kesimpulan agar
siswa bisa merumuskan serta merangkum
pengetahuan yang mereka dapatkan dengan Pada
akhir
pembelajaran
siswa
mengerjakan soal kuis untuk mengecek
pemahaman siswa pada satu pertemuan. Setelah itu siswa mengisi lembar self
assessment untuk merefleksi pembelajaran
yang sudah dilakukan pada pertemuan
tersebut. KESIMPULAN DAN SARAN siswa atau 68,57% yang memperoleh nilai
dengan kategori minimal baik. Penelitian
dinyatakan belum berhasil karena belum
mencapai
75%
siswa
yang
mencapai
kategori minimal baik. Oleh karena itu,
dilakukan
refleksi
dan
perbaikan
pembelajaran pada siklus II. Pada tes akhir
siklus II terdapat 30 siswa yang memenuhi
kriteria
atau
85,
71%. Selain
itu,
berdasarkan hasil observasi kegiatan guru
dan siswa diperoleh bahwa telah memenuhi
kriteria minimal baik pada pembelajaran
siklus I maupun II. Oleh karena itu,
penelitian
telah
memenuhi
indikator
keberhasilan. Berdasarkan hasil dan pembahasan
pada penelitian ini, pembelajaran dengan
model Think Talk Write (TTW) dapat
meningkatkan
komunikasi
matematis
tertulis siswa kelas XI E-4 SMAN 5 Malang
pada materi turunan dan aplikasi turunan
fungsi aljabar. Pembelajaran tersebut terdiri
dari 2 siklus yang masing-masing terdiri dari
tiga pertemuan dan 1 pertemuan tes akhir
siklus. Pembelajaran
kooperatif
TTW
meliputi tahap think, tahap talk dan tahap
write. Tahap think memberikan kesempatan
kepada
siswa
untuk
memikirkan
penyelesaian, mencatat informasi penting
dan menuliskan pertanyaan terkait masalah
yang diamati. Tahap talk, siswa berdiskusi
secara berkelompok yang terdiri delapan
kelompok untuk menemukan definisi, sifat,
maupun aplikasi turunan fungsi aljabar. Tahap write, siswa menuliskan hasil diskusi
di
LKS
yang
diberikan
berdasarkan
pemahaman kelompok pada saat diskusi,
menuliskan hasil diskusi di papan tulis
kemudian
dibahas
secara
klasikal,
meyimpulkan kesimpulan dari pembelajaran
dan mengerjakan tugas individu/kuis secara
individu. Berdasarkan hasil tes akhir siklus I
menunjukkan bahwa terdapat 24 dari 35 Terdapat beberapa saran bagi peneliti
lain yang ingin menggunakan pembelajaran
dengan
model
TTW
meningkatkan
kemampuan komunikasi matematis tulis
terhadap
suatu
materi,
yaitu:
(a)
pembelajaran sebaiknya dikemas dengan
menarik untuk mengatasi kejenuhan siswa
karena melakukan pembelajaran dengan
model yang sama; (b) manajemen waktu
harus
diperhatikan,
agar
pembelajaran
berjalan sesuai dengan RPP yang sudah
dibuat DAFTAR PUSTAKA https://doi.org/10.1348/026151010X
496906. Croker, S., & Buchanan, H. (2011). Scientific Reasoning in A Real‐
World Context: the Effect of Prior
Belief and Outcome on Children's
Hypothesis‐Testing
Strategies. British Journal of Developmental
Psychology,
29(3),
409-424. Devlin, M. (2010). The Criteria of Effective
Teaching in a Changing Higher
Education
Context. Higher
Education
Research
and Devlin, M. (2010). The Criteria of Effective
Teaching in a Changing Higher
Education
Context. Higher
Education
Research
and 107 Jurnal Pendidikan Matematika, 11 (1) (2020): 99-109
operatif Think Talk Write (TTW) Untuk Meningkatkan Komunikasi Matematis Pembelajaran
Kooperatif
Tipe
STAD di Kelas XII IPS. EDU-MAT
Jurnal Pendidikan Matematika, 5(1),
76-85. http://dx.doi.org/10.20527/edumat.v
5i1.3824 Development,
29(2),
111-124. https://doi.org/10.1080/07294360903
244398. Hasibuan, I.S., & Amry, Z. (2017). Differences
Of
Students
Mathematical
Communication
Ability Between Problems Based
Learning, Realistic Mathematical
Education And Inquiri Learning In
SMP Negeri 1 Labuhan Deli. IOSR
Journal of Research & Method in
Education (IOSR-JRME), 7(6), 54-
60. Masingila, J. O., & Wisniowska, E.P. (1996). Developing and Assessing
Mathematical
Understanding
in
Calculus Through Writing. In P.C
Elliott, and M. J Kenney (Eds).1996
Yearbook. Communication
in
Mathematics. K-12 and Beyond. USA: NCTM. Huda, M. (2013). Model-model Pengajaran
dan
Pembelajaran. Yogyakarta:
Pustaka Pelajar. Munir, M.T., Baroutian, S., Young, B.R., &
Carter, S. (2018). Flipped classroom
with cooperative learning as a
cornerstone. Educational Chemical
Engineering,
23,
25–33. https://doi.org/10.1016/j.ece.2018.05
.001. Huinker, D. & Laughlin, C. (1996). Talk
Your
Way
into
Writing. Communication in Mathematics, K-
12 and Beyond. 1996 Yearbook. Reston, VA: NCTM. NCTM. (2000). Principles and Standards
for School Mathematics. Reston:
NCTM Inc. Jariswandana, L., Yerizon, & Nilawasti, Z. A. (2012). Meningkatkan Motivasi
Belajar Matematika Siswa Dengan
Penerapan
Model
Pembelajaran
Kooperatif Tipe Think Talk Write. Jurnal Pendidikan Matematika, 1
(1), 81-86. Pane, N.S., Jaya, I., & Lubis, M.S. (2018). Analisis Kemampuan Komunikasi
Matematis
Siswa
Pada
Materi
Penyajian Data di Kelas VII MTs
Islamiyah Medan T.P 2017/2018. Axiom: Jurnal
Pendidikan
Dan
Matematika,
8(2),
97-109. tp://dx.doi.org/10.3082/axiom.v7i1.1
779 Kosko,
K.W.,
&
Gao,
Y. (2017). Mathematical
Communication
in
State Standards Before the Common
Core. Educational Policy, 31, 275–
302. https://doi.org/10.1177/08959048155
95723. Qohar, A. (2011). Pengembangan Instrumen
Komunikasi Matematis Untuk Siswa
SMP. Makalah
disajikan
pada
Lomba dan Seminar Matematika ke
XIX,
Yogyakarta. Retrived
1
September
2018,
from
https://core.ac.uk/download/pdf/110
64561.pdf Lestari, W. (2017). Pengaruh Kemampuan
Awal Matematika dan Motivasi
Belajar
Terhadap
Hasil
Belajar
Matematika. Jurnal Analisa, 3(1),
76-84. https://doi.org/10.15575/ja.v3i1.1499 Qohar, A., & Sumarmo, U. (2013). DAFTAR PUSTAKA Improving
Mathematical
Communication Ability and Self
Regulation Learning Of Yunior High
Students
by
Using
Reciprocal
Teaching. Journal on Mathematics
Education,
4,
59–74. Lie, A. (2010). Cooperative Learning:
Mempraktikkan
Cooperative
Learning di Ruang-Ruang Kelas. Jakarta: Grasindo Maisyarah. (2017). Meningkatkan
Kemampuan Komunikasi Matematis
Peserta Didik Menggunakan Model 108 Jurnal Pendidikan Matematika, 11 (1) (2020): 99-109
Anis Rochma Pratiwi, Abd.Qohar https://doi.org/10.22342/jme.4.1.562. 59-74. https://doi.org/10.22342/jme.4.1.562. 59-74. Ratumanan,
T. G. (2015). Inovasi
Pembelajaran. Yogyakarta: Penerbit
Ombak. Riski, R., Rijal, M., & Linawati. (2017). Penerapan
Model
Pembelajaran
Kooperatif Tipe Think Talk Write
(TTW) Untuk Meningkatkan Hasil
Belajar
Siswa
Pada
Materi
Hubungan Sudut Pusat, Panjang
Busur, Dan Luas Juring Di Kelas
VIII C SMP Negeri 9 Palu. Jurnal
elektronik Pendidikan Matematika
Tadulako, 5 (2), 175-189. Senjayawati,
E. (2015). Penerapan
Pendekatan
Kontekstual
untuk
Meningkatkan
Kemampuan
Komunikasi
Matematik
Siswa
SMkKdi Kota Cimahi. Didaktik,
9(1), 33–39. Shield, M. & Swinson, K. (1996). The Link
Shet: A Comunication Aid for
Clarifying
and
Developing
Mathematical Ideas and Processes. In. P. C. Elliot and M. J Kenney
(Eds). Year
Book
1996. Comunication in Mathematics K-12
and Beyond. USA: NCTM. Subanji. (2013). Pembelajaran Matematika
Kreatif dan Inovatif. Malang: UM
Press 109
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English
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Feasibility of a physical activity intervention during and shortly after chemotherapy for testicular cancer
|
BMC research notes
| 2,017
|
cc-by
| 6,300
|
© The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/
publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Given the risk of developing acute and long-term adverse effects in patients receiving cisplatin-based
chemotherapy for testicular cancer (TC), risk-reducing interventions, such as physical activity (PA), may be relevant. Limited knowledge is available on the challenges met when conducting PA intervention trials in patients with TC
during and shortly after chemotherapy. The aims of the present feasibility study are therefore to determine patient
recruitment, compliance and adherence to a PA intervention. Results: Patients with metastatic TC referred to cisplatin-based chemotherapy were eligible. They followed an indi-
vidual low-threshold PA intervention, including counseling from a personal coach during and 3 months after chemo-
therapy. Outcomes were recruitment rate, compliance rate and adherence to the intervention including preferences
for type of PA and barriers for PA. During 8 months 12 of 18 eligible patients were invited, all consented, but three
dropped out. Walking and low intensity activities were preferred and nausea and feeling unwell were the most often
reported barriers towards PA. Discussion: In order to achieve adequate recruitment, compliance and complete data in future PA intervention trials,
close cooperation with treating physicians, individual PA plans and availability of personalized coaching are required. Trial registration NCT01749774, November 2012, ClinicalTrials.gov Keywords: Testicular cancer, Chemotherapy, Physical activity, Feasibility Feasibility of a physical activity
intervention during and shortly
after chemotherapy for testicular cancer Lene Thorsen1*, Camilla Kirkegaard2, Jon Håvard Loge3, Cecilie E. Kiserud1, Merethe Lia Johansen4,
Gunhild M. Gjerset1, Elisabeth Edvardsen5, Hanne Hamre6, Tone Ikdahl7 and Sophie D. Fosså1 Thorsen et al. BMC Res Notes (2017) 10:214
DOI 10.1186/s13104-017-2531-y Thorsen et al. BMC Res Notes (2017) 10:214
DOI 10.1186/s13104-017-2531-y BMC Research Notes Background After chemotherapy, long-term TC survivors are at risk
of several late effects and display a higher risk of diabe-
tes and myocardial infarction compared to age-matched
men from the general population [4, 5]. Interventions
that can reduce the risk of acute and long-term adverse
effects are therefore warranted. g
Testicular cancer (TC) is the most common cancer
among men between 18–40 years of age [1]. Standard
treatment for patients with low- or intermediate risk
metastatic disease is chemotherapy including three or
four cycles with etoposide and cisplatin with or without
bleomycin (EP or BEP) [2]. This treatment often leads
to acute toxic effects such as bone marrow suppression,
nausea, thromboembolic events, reduced kidney func-
tion, and peripheral neuropathy, resulting in reduced
well-being and physical capacity during treatment [3]. f
Several meta-analyses have concluded that physical
exercise is a feasible and beneficial intervention for sev-
eral health outcomes in cancer patients, however most
studies have been conducted in breast cancer patients
and after cancer treatment [6–9]. In a recent review, only
17 studies were identified assessing the effects of physical
exercise during adjuvant cancer treatment, of which 14
studies included breast cancer patients, two studies lung
cancer patients and one study patients with several diag-
noses [10]. Studies examining the feasibility and effects of *Correspondence: LKA@ous‑hf.no
1 National Advisory Unit on Late Effects After Cancer Treatment, Oslo
University Hospital, Box 4950, Nydalen, 0424 OSLO Oslo, Norway
Full list of author information is available at the end of the article Thorsen et al. BMC Res Notes (2017) 10:214 Page 2 of 9 for three or four cycles with BEP/EP, without physi-
cal and psychological co-morbidities contraindicating
the assessments and/or intervention as assessed by the
responsible oncologist, age ≥18 years and no prior can-
cer (except non-melanoma skin cancer). physical activity (PA) during chemotherapy treatment in
patients with other diagnoses than breast cancer, such as
cisplatin-based chemotherapy in patients with metastatic
TC are needed [11]. One rationale for initiating PA during chemotherapy
is to maintain physical fitness, both in terms of aerobic
capacity and muscle strength, and thereby possibly reduce
troublesome side-effects and increase well-being. On the
other side acute side-effects might be barriers towards
PA, and more knowledge on the optimal content of a PA
intervention during such treatment and how these inter-
ventions should be conducted in practice is needed. Background Before
planning and implementing randomized clinical trials
(RCTs) including PA in patients with metastatic TC dur-
ing chemotherapy, patient recruitment, compliance and
patients’ adherence to such intervention are important to
ensure that a RCT can be successfully performed. A review
by Oldervoll et al. [12] focused on recruitment, compli-
ance and adherence to PA interventions during and after
cancer treatment. In 12 RCTs, the average recruitment
rate was 43%, the average compliance rate was 86% and
the adherence to the interventions ranged from 72–100%. The majority of the participants (62%) were breast cancer
patients during and after chemotherapy [12].h Intervention Due to lack of literature regarding PA in patients with
metastatic TC, we conducted upfront semi-structured
interviews among 11 men who previously had received
BEP/EP chemotherapy for TC. These interviews revealed
reduced wellbeing, fatigue and weakness especially on
the days 4–7 in each cycle. Wellbeing gradually decreased
throughout the treatment period. After completion of
chemotherapy, the interviewed patients reported that
recovery to pre-treatment physical capacity could take
several months. Most interviewed patients expressed
skepticism towards performing high intensity (HI) physi-
cal exercise during chemotherapy and preferred PAs with
low intensity (LI) or moderate intensity (MI). On this background the intervention in the present fea-
sibility study was designed as an individual low-thresh-
old PA intervention during (Phase I) and shortly after
(Phase II) chemotherapy (Fig. 1). The intervention aimed
to avoid inactivity and maintenance of physical capacity. All patients got a personal coach to motivate, counsel and
encourage them to follow the public health PA guidelines
(≥150 min MI activity per week or ≥75 min HI activity)
[13]. During the first week of treatment an individualized
PA plan was developed. The patient could choose differ-
ent types of PAs and the intensity should be adapted to
the patient’s day-to-day wellbeing. The coach contacted
the patient in person at least once during each hospitali-
zation, and if desirable, counseled the patient during PA
sessions at the training centre at the hospital. Between
the hospitalizations the coach phoned the patient at least
once per week, but no face-to-face counseled PA sessions
were performed outside the hospital. The young age, male gender and the BEP/EP regimen,
with its specific toxic effects, might interfere differently
on the feasibility in PA studies in patients with metastatic
TC than other cancer populations. Therefore, the aims
of the present feasibility study are to determine patient
recruitment, compliance and adherence to a PA interven-
tion during and shortly after cisplatin-based chemother-
apy, in patients with newly diagnosed metastatic TC. Patients and treatmenth This feasibility study was performed as a single institutional
prospective one-armed intervention study at Oslo Uni-
versity Hospital (OUH) from December 2012 to July 2013
(8 months). OUH is the only institution treating patients
with newly diagnosed TC within a population of approxi-
mately 2.8 million individuals, living in the south eastern
part of Norway. Most patients undergo orchiectomy at their
local hospital, but are thereafter referred to OUH for staging
and eventually further treatment. Risk-adapted treatment of
patients with metastatic TC comprises three or four cycles
of BEP or four EP [2]. Each cycle of 21 days includes 5 days
at OUH receiving chemotherapy infusions (days 1–5), and
16 days at home (days 6–21). At day 15, the patients receive
bleomycin intravenously at their local hospital. Post-chem-
otherapy residual tumor often requires surgery, most fre-
quently retroperitoneal lymph node dissection.i Phase II started after the last BEP/EP cycle and lasted
for 12 weeks. The coach and the patient worked out an
individual PA plan depending on the patient’s prefer-
ences. All PAs were to be performed near by the patients’
home. The coach phoned the patient every second week,
encouraging him to increase the frequency, duration and
intensity of PAs, at least to a level corresponding to the
public health PA guidelines. Patient recruitment, refusal and compliance Patient recruitment, refusal and compliance y
Patient recruitment, refusal and compliance Eligible patients were recruited by the responsible oncolo-
gists 1–2 weeks before the first BEP/EP cycle (Fig. 1). After short oral information by the treating oncolo-
gist, the patients received written information and more Patients eligible for the present study fulfilled the fol-
lowing criteria: newly diagnosed metastatic TC, planned Thorsen et al. BMC Res Notes (2017) 10:214 Page 3 of 9 Fig. 1 Design of the feasibility study and timeline for each patient Fig. 1 Design of the feasibility study and timeline for each patient Fig. 1 Design of the feasibility study and timeline for each patient 12 to 14 were categorized as MI sessions, and activities
above 14 as HI sessions [14]. detailed oral information from the study coordinator if
they expressed an interest in participating. The recruit-
ment rate was defined as the proportion of invited patients
among eligible patients. Reasons for eligible patients
not being invited were identified retrospectively. Refusal
rate was defined as the proportion of patients among the
invited that did not consent to participate. Completion
rate was defined as the proportion of consenting patients
who completed any measurement at the final assessment. At T2 the patients went through a semi-structured
interview, including questions related to PA preferences
(Table 4) and barriers asking; “On a scale from 0 to10,
how did the listed adverse effects (Table 5) hamper your
level of PA during chemotherapy?” (1 = not all to 10 = to
a very high degree). Ethicsh i
The patients were evaluated during the week before the
first BEP/EP cycle (T0), the week after the last BEP/EP
cycle (after phase I) (T1) and 12 weeks after the last BEP/
EP cycle (after Phase II) (T2) (Fig. 1). The assessments
included fasting blood sampling, questionnaires, a cardio-
pulmonary exercise test (CPET), dual-energy X-ray absorp-
tiometry (DXA) and muscle strength tests. Assessment
completion rate was defined as the proportion of completed
assessments, at T0, T1 and T2 separately and summed up. The study was approved by the Regional Committee for
Medical and Health Research Ethics, South-East Region
(2011/2008/REK South-East A) and registered in Clini-
calTrial.gov (NCT01749774). Eligible patients willing
to participate signed an informed consent form prior to
testing at T0. Adherence to the interventionh 2 Patient flow through the study Excluded (n = 7)
- another cancer diagnosis (n = 2)
- back pain that contraindicated physical tests (n = 1)
- advanced disease that required immediate treatment (n = 4) Excluded (n = 7)
- another cancer diagnosis (n = 2)
- back pain that contraindicated physica
- advanced disease that required immed Dropped out (n = 3)
- educational obligations in other parts of Norway (n = 2)
- hospitalization due to another medical condition that
contraindicated further participation (n = 1) Table 1 Baseline characteristics of included patients (n = 12)
a According to Royal Marsden Hospital Stage system [15]
b Patients with stage IIIa/b had recidiv from their seminoma
Mean (SD)
Years of age
36 (7.7)
N
Married/cohabitant
9
Education
Primary/secondary school
10
College/university
2
Histology
Seminoma
7
Non-seminoma
5
Stagea
IIa/b
10
IIIa/bb
2
Treatment
3BEP
12 Table 1 Baseline characteristics of included patients (n = 12) T1 (but before phase II of the intervention), because of
educational obligations outside the region (n = 2) and
hospitalization due to a medical condition (n = 1), giving
a compliance rate of 75%. At T0, T1 and T2 the assessment completion rates
were 92, 89 and 62%, respectively. Of 216 possible assess-
ments 175 (81%) were completed. The lowest completion
rate was observed for DXA-scans, due to lack of staff at
the lab (Table 2). Adherence to the interventionh From December 2012 to July 2013, 18 of 25 patients with
stage II–IV TC were eligible for the study, of which 12
patients (mean age 36 years) were invited, all of them
receiving three BEP cycles (Fig. 2; Table 1).h The patients were asked to report all types of PA includ-
ing the intensity and duration in a PA log during each
chemotherapy cycle. Intensity was reported according to
the Borg scale [14]. PAs rated below 12 at the Borg scale
were categorized as LI sessions, activities rated from The study achieved a recruitment rate of 66%, logistics
were the main reason participants did not enter the study, Thorsen et al. BMC Res Notes (2017) 10:214 Page 4 of 9 Referred to chemotherapy (n=25)
Excluded (n = 7)
- another cancer diagnosis (n = 2)
- back pain that contraindicated physical tests (n = 1)
- advanced disease that required immediate treatment (n = 4)
)
8
1
=
n
(
elbigil
E
Not invited (n = 6)
- not invited due to holiday (n = 1)
- treating oncologist not aware of the protocol (n = 5)
)
2
1
=
n
(
d
etiv
n
I
Consenting (n=12)
)
2
1
=
n
(
0
T
T1 (after phase I)
(n=12)
Dropped out (n = 3)
- educational obligations in other parts of Norway (n = 2)
- hospitalization due to another medical condition that
contraindicated further participation (n = 1)
T2 (after phase II)
(n=9)
Fig. Adherence to the intervention Eleven patients completed their PA log during chemo-
therapy (Table 3). During the first cycle (week 1–3),
three patients met the PA guidelines each week (only
one patient all 3 weeks). During the second cycle (week
4–6), two patients met the PA guidelines in week 4 and
6, whereas three patients met the PA guidelines in week
5. During the third cycle (week 7–9) one patient met the
PA guidelines each week (different patient each week)
(Table 3) and (Additional file 1). i
During nine weeks of chemotherapy the number of HI, MI
and LI sessions decreased gradually paralleled by an increas-
ing proportion of patients reporting no PA at all (Table 3). particularly summer holiday and oncologists not being
aware of the study. None of the invited patients refused
to participate (refusal rate: 0%). No patients dropped out
between T0 and T1, but three patients dropped out after Nine of 12 underwent a semi-structured interview on
their preferences and experienced barriers related to PA at Thorsen et al. BMC Res Notes (2017) 10:214 Page 5 of 9 Page 5 of 9 Table 2 Assessment
completion
among
consenting
patients (n = 12)
Reasons for missing data: CPET: T0, 1 due to lack of personnel at the lab; T1,
1 due to lack of personnel at the lab; T2, 1 due to lack of personnel at the lab. DXA: T0, 3 due to lack of personnel at the lab; T1, 3 due to lack of personnel at
the lab; T2, 2 due to lack of personnel at the lab. Leg press: T0, 1 due to pain
in the back and 1 due to surgery; T1, 1 due to pain in the back and 1 due to
thrombosis in the leg; T2, 1 due to pain in the back and 1 due to thrombosis in
the leg. Chest press: T1, 1 due to pain in the arm; T2, 1 due to pain in the arm and
2 due to broken chest press instrument. Adherence to the intervention Leg press: T0, 1 due to pain
in the back and 1 due to surgery; T1, 1 due to pain in the back and 1 due to
thrombosis in the leg; T2, 1 due to pain in the back and 1 due to thrombosis in
the leg. Chest press: T1, 1 due to pain in the arm; T2, 1 due to pain in the arm and
2 due to broken chest press instrument. Pull down: T1, 1 due to pain in the arm;
T2, 1 due to pain in the arm T2. Walking and LI activities were most preferred (Table 4). The most often reported barriers towards PA during chem-
otherapy were nausea, feeling unwell, reduced general con-
dition and being exhausted/tired (Table 5). Eight patients
said that they preferred a flexible program and five would
have preferred a personal trainer during the exercise ses-
sions. Six patients said that they could have been more
active than they actually had been and four said they could
have been pushed more by their personal coach. All patients invited to participate in the study agreed. In
the study of Christensen et al. [16] 73% eligible patients
were willing to participate and included. The reasons
for refusal were lack of time or interest or long traveling
distance. The flexible and individually adjusted interven-
tion in our feasibility study might have favored a positive
attitude towards participation and been an important
reason for no drop-outs during chemotherapy. On the
other hand the flexible individualized approach and lack
of standardized training dose for each patients, limit the
possibility to conclude on the exact dose of PA that are
feasible and necessary to maintain physical capacity in
future intervention trials. Standardized supervised exer-
cise sessions at the hospital might increase the effects of
a PA intervention, but the inconvenience with traveling
to the hospital several times per week might be a threat
to the overall adherence to the intervention and external
validity. Adherence to the intervention Pull down: T1, 1 due to pain in the arm;
T2, 1 due to pain in the arm
T0
T1
T2
Total
Questionnaire
12
12
9
33
CPET
11
11
8
30
DXA
9
9
7
25
Leg press
10
10
7
27
Chest press
12
11
6
29
Pull down
12
11
8
31
Total possible assessments
72
72
72
216
Total completed assessments
66
64
45
175
Assessment completion rate (%)
92
89
62
81 Table 2 Assessment
completion
among
consenting
patients (n = 12) between the involved researchers and clinicians. Our fea-
sibility study was lead by researchers within the field of
PA, and highly involved clinicians are also necessary to
increase the recruitment rate in future PA intervention
trials. Two patients withdrew during follow-up due to
studies at universities in other parts of the country, and
this is probably a more common reason for drop-out in a
young cancer population than in older patients.f To examine the effects of PA interventions, compre-
hensive and long-lasting pre-post intervention assess-
ments involving staff from several labs are often required. The interval from detection of the metastasis to initiation
of chemotherapy is only a few days. A challenge is there-
fore to coordinate the assessments at the labs within the
same day, preferable within half a day. Close cooperation
between the researchers and the labs is required. Warn-
ing the test-leaders and allocating time for testing, with
the risk that it might be cancelled at last minute might
be necessary. A possible solution might be to start the
first BEP/EP cycle on Monday afternoon giving the time
before lunch for testing. In a hectic daily hospital life
adjustments related to such a study might claim extra
work for the doctors and nurses and their goodwill and
cooperation is needed. The number of necessary assess-
ments before and during the study should carefully be
considered in future intervention trials. Reasons for missing data: CPET: T0, 1 due to lack of personnel at the lab; T1,
1 due to lack of personnel at the lab; T2, 1 due to lack of personnel at the lab. DXA: T0, 3 due to lack of personnel at the lab; T1, 3 due to lack of personnel at
the lab; T2, 2 due to lack of personnel at the lab. Discussion
Th
f The present feasibility study illustrates several factors that
need to be considered before conducting PA intervention
trials in patients with metastatic TC; possible loss of eli-
gible patients, logistic challenges related to assessments
and need for face-to-face PA counseling to increase the
adherence to the intervention. On the other hand, the
interest to participate in PA intervention studies seems
to be high among these patients during and shortly after
chemotherapy. Among all the patients referred to three or four BEP/EP
during the study period, more than one fourth (7 of 25)
was excluded. Four of seven patients (57%) were ineligi-
ble due to their need for immediate treatment. In future
PA intervention trials this type of ineligibility should
be taken into account when estimating the number of
patients to be available. To avoid a high refusal rate in future PA intervention
trials in patients with TC, we suggest that the interven-
tion, when the patients are not hospitalized, should be
conducted at fitness centres near the patients’ homes
under supervision of qualified personal trainers or physi-
otherapists. PA near by the patients’ home might be of
special importance in rural areas, and makes it easier to
continue the program after the intervention period. It has
been shown that patients with lung cancer performing Even in large cancer centres the number of patients
refereed to chemotherapy for metastatic TC is relatively
low, and performing multi-centre studies in future tri-
als will reduce the recruitment period. Optimal patient
recruitment also requires a well established cooperation Thorsen et al. BMC Res Notes (2017) 10:214 Page 6 of 9 Table 3 Physical activity log during each BEP cycle (n = 11) Table 3 Physical activity log during each BEP cycle (n = 11) Table 3 Physical activity log during each BEP cycle (n = 11)
N
HI sessions (≥15 BS)
MI sessions (12–14 BS)
LI sessions (≤11 BS)
BEP 1
Week 1
Meeting guidelines (n)
3
No activities reported (n)
2
Patients (n)/no of sessions performed
3/9
6/9
6/25
Mean duration of the sessions [min. (range)]
47 (15–120)
35 (15–60)
36 (15–90)
Types of activities
ST, bicycling, running
Bicycling, ST, running, walking,
SAG
Walking, HK, SAG, jogging
Week 2
Meeting guidelines (n)
3
No activities reported (n)
0
Patients (n)/no of sessions performed
5/10
6/14
9/37
Mean duration of the sessions [min. Discussion
Th
f (range)]
46 (25–120)
39 (20–90)
28 (10–95)
Types of activities
TT, CCS, bicycling,
walking, ST
TT, walking, bicycling, ST, Foot-
ball, SAG
Walking, ST
Week 3
Meeting guidelines (n)
3
No activities reported (n)
2
Patients (n)/no of sessions performed
6/9
5/10
6/27
Mean duration of the sessions [min. (range)]
43 (20–90)
42 (20–90)
25 (10–68)
Types of activities
CCS, ST, running, TT,
jogging
TT, CCS, walking, ST, jogging,
SAG
Walking, ST, gardening
BEP 2
Week 4
Meeting guidelines (n)
2
No activities reported (n)
3
Patients (n)/no of sessions performed
3/6
2/2
6/23
Mean duration of the sessions [min. (range)]
87 (20–160)
58 (40–75)
40 (10–240)
Types of activities
Jogging, CCS, walking,
ST
Walking, SAG
Walking
Week 5
Meeting guidelines (n)
3
No activities reported (n)
2
Patients (n)/no of sessions performed
2/4
4/5
6/19
Mean duration of the sessions [min. (range)]
36 (10–60)
141(30–360)
33 (10–60)
Types of activities
CCS, walking
Curling, hiking, HK, ST, walking
Walking, gardening,
swimming, HK, SAG
Week 6
Meeting guidelines (n)
2
No activities reported (n)
3
Patients (n)/no of sessions performed
3/4
3/6
5/31
Mean duration of the sessions [min. (range)]
28 (10–58)
94 (30–270)
41 (20–240)
Types of activities
CCS, walking, cross-fit
Paddling, football, SAG, walking
Walking, SAG, gardening,
HK, SAG
BEP 3
Week 7
Meeting guidelines (n)
1
No activities reported (n)
5
Patients (n)/no of sessions performed
2/4
0
4/10
Mean duration of the sessions [min. (range)]
74 (30–180)
27 (10–45)
Types of activities
Running, walking
Walking, gardening N
HI sessions (≥15 BS)
MI sessions (12–14 BS)
LI sessions (≤11 BS) Thorsen et al. BMC Res Notes (2017) 10:214 Page 7 of 9 HI, high intensity; BS, Borg Scale; MI, moderate intensity; LI, low intensity; ST, strength training; SAG, session at the gym; HK, house keeping; CCS, cross-country skiing;
TT, table tennis
Table 3 continued
N
HI sessions (≥15 BS)
MI sessions (12–14 BS)
LI sessions (≤11 BS)
Week 8
Meeting guidelines (n)
1
No activities reported (n)
7
Patients (n)/no of sessions performed
1/1
2/4
1/2
Mean duration of the sessions [min. (range)]
30
81 (40–160)
75 (60–90)
Types of activities
ST
Walking, ST, SAG
Gardening, SAG
Week 9
Meeting guidelines (n)
1
No activities reported (n)
6
Patients (n)/no of sessions performed
1/1
2/6
1/2
Mean duration of the sessions [min. Discussion
Th
f (range)]
20
43 (20–90)
120 (120)
Types of activities
ST
Walking, SAG
Gardening Table 3 continued N
HI sessions (≥15 BS)
MI sessions (12–14 BS)
LI sessions (≤11 BS) HI, high intensity; BS, Borg Scale; MI, moderate intensity; LI, low intensity; ST, strength training; SAG, session at the gym; HK, house keeping; CCS, cross-country skiing;
TT, table tennis HI, high intensity; BS, Borg Scale; MI, moderate intensity; LI, low intensity; ST, strength training; SAG, session at the gym; HK, house keeping; CCS, cross-country skiing;
TT table tennis PA after surgery had positive effects on cardiorespira-
tory fitness, muscle strength, and quality of life of a
supervised aerobic and strength training program, sup-
ported by a personal trainer at a fitness centre near the
patients’ home [17]. In the present feasibility study, the intervention was individualised and flexible according to
the patient’s daily well-being, with regular counselling by
the coach. Nevertheless, the level of PA during chemo-
therapy was lower than expected and only a few patients
met the PA guidelines. Treatment related adverse effects
have shown to account for more than 50% of the bar-
riers to perform PA in cancer patients treated with
chemotherapy [18]. The patients in our study reported
well-known chemotherapy-related acute adverse effects
as barriers to perform PA. Interestingly, after the inter-
vention, more than half of the patients indicated that they
could have been able to be more active than they actually
were, and that they could have been pushed more by the Table 4 Physical activity preferences during chemother-
apy (n = 9)
N
What kind of activity did you prefer during chemotherapy
(open question)
Walking
6
Walking/jogging/stair climbing
1
Outdoor activities
1
Pleasurable activities
1
Which intensity did you prefer? Low
5
Low/moderate
2
Moderate/hard
1
Adjusted to the condition
1
What do you think is most suitable during BEP treatment? A strict exercise program
1
A flexible program
8
Would you have preferred a personal trainer during the PA sessions
outside the hospital? Yes
5
No
4
Would you have been able to be more active than you actually were? Yes
6
No
3
Could your coach have pushed you more? Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub-
lished maps and institutional affiliations. Received: 1 June 2016 Accepted: 5 June 2017 Received: 1 June 2016 Accepted: 5 June 2017 Ethics Several aspects should be addressed before planning
and implementing PA intervention trials in patients with
metastatic TC during chemotherapy. A close cooperation
between researchers, the clinicians’ and staff at the labs
is important in order to increase the recruitment- and
assessment compliance rate. The experiences from this
feasibility study suggest that the interest for PA during
chemotherapy in patients with metastatic TC is high, but
face-to-face counselling by a personal trainer or physi-
otherapist might be important to increase the level of PA
during chemotherapy. The challenges regarding logistics
related to inclusion and assessments procedures should
seriously be taken into account when planning and per-
forming PA intervention trial in patients with metastatic
TC. The study was approved by the Regional Committee for Medical and Health
Research Ethics, South-East Region (2011/2008/REK South-East A) and reg-
istered in ClinicalTrial.gov (NCT01749774). The participants provided written
informed consent. Funding The study was funded by Gjensidige Foundation, Active against Cancer, The
Research Council of Norway (NFR 218312, entitled ‘Late effects—early detec-
tion, follow-up and treatment‘) and South-Eastern Norway Regional Health
Authority (2010063—Cancer rehabilitation—content and effects). Abbreviations
TC
i
l TC: testicular cancer; PA: physical activity; EP: etoposide and cisplatin; BEP:
etoposide, cisplatin and bleomycin; RCT: randomized clinical trials; OUH: Oslo
University Hospital; HI: high intensity; LI: low intensity; MI: moderate intensity;
T0: the week before the first BEP/EP cycle; T1: the week after the last BEP/EP
cycle; T2: 12 weeks after the last BEP/EP cycle; CPET: cardiopulmonary exercise
test; DXA: dual-energy X-ray absorptiometry. 3. Fossa SD, de Wit R, Roberts JT, Wilkinson PM, de Mulder PH, Mead GM,
et al. Quality of life in good prognosis patients with metastatic germ cell
cancer: a prospective study of the European Organization for Research
and Treatment of Cancer Genitourinary Group/Medical Research
Council Testicular Cancer Study Group (30941/TE20). J Clin Oncol. 2003;21(6):1107–18. Consent to publish
The participants provided written informed consent to publish. The participants provided written informed consent to publish. References 1. Cancer Reistry of Norway. Cancer in Norway 2015 - Cancer incidence,
mortality, survival and prevalence in Norway. Oslo: Cancer Reistry of
Norway; 2016. 1. Cancer Reistry of Norway. Cancer in Norway 2015 - Cancer incidence,
mortality, survival and prevalence in Norway. Oslo: Cancer Reistry of
Norway; 2016. Additional file 1. Physical activity log during each BEP cycle. Additional file 1. Physical activity log during each BEP cycle. 2. Oldenburg J, Fossa SD, Nuver J, Heidenreich A, Schmoll HJ, Bokemeyer
C, et al. Testicular seminoma and non-seminoma: ESMO Clinical Practice
Guidelines for diagnosis, treatment and follow-up. Ann Oncol Off J Eur
Soc Med Oncol ESMO. 2013;24(Suppl 6):vi125–32. Author details
1 1 National Advisory Unit on Late Effects After Cancer Treatment, Oslo
University Hospital, Box 4950, Nydalen, 0424 OSLO Oslo, Norway. 2 Clinical
Manager, Medical Affairs, Coloplast A/S, Humlebæk, Denmark. 3 Department
of Behavioral Sciences in Medicine, University of Oslo/Regional Advisory
Unit in Palliative Care, Oslo University Hospital, Oslo, Norway. 4 Department
of Cancer Rehabilitation, Oslo University Hospital, Oslo, Norway. 5 Department
of Pulmonary Medicine, Oslo University Hospital, Oslo, Norway. 6 Department
of Oncology, Akershus University Hospital, Lorenskog, Norway. 7 Akershus
University Hospital, Lorenskog, Norway. 8. Mishra SI, Scherer RW, Snyder C, Geigle P, Gotay C. Are exercise programs
effective for improving health-related quality of life among cancer
survivors? A systematic review and meta-analysis. Oncol Nurs Forum. 2014;41(6):E326–42. 9. Stene GB, Helbostad JL, Balstad TR, Riphagen II, Kaasa S, Oldervoll LM. Effect of physical exercise on muscle mass and strength in cancer
patients during treatment-a systematic review. Crit Rev oncol hematol. 2013;88(3):573–93. 10. Loughney L, West MA, Kemp GJ, Grocott MP, Jack S. Exercise intervention
in people with cancer undergoing neoadjuvant cancer treatment and
surgery: a systematic review. Eur J Surg oncol J Eur Soc Surg Oncol Br
Assoc Surg Oncol. 2016;42(1):28–38. Authors’ contributions 4. Haugnes HS, Bosl GJ, Boer H, Gietema JA, Brydoy M, Oldenburg J, et al. Long-term and late effects of germ cell testicular cancer treatment and
implications for follow-up. J Clin Oncol. 2012;30(30):3752–63. LT and CK were involved in the conception and design of the study, acquisi-
tion of data, analysis and interpretation of data and drafting and preparation
of the manuscript. JHL, SDF, CEK and TI were involved in the conception and
design of the study and preparation of the manuscript. MLJ, GMG and EE
were involved in the conception and design of the study, acquisition of data
and preparation of the manuscript. HH was involved in acquisition of data
and preparation of the manuscript. All authors read and approved the final
manuscript. 5. Haugnes HS, Wethal T, Aass N, Dahl O, Klepp O, Langberg CW, et al. Car-
diovascular risk factors and morbidity in long-term survivors of testicular
cancer: a 20-year follow-up study. J Clin Oncol. 2010;28(30):4649–57. 6. Speck RM, Courneya KS, Masse LC, Duval S, Schmitz KH. An update of
controlled physical activity trials in cancer survivors: a systematic review
and meta-analysis. J Cancer Surviv. 2010;4(2):87–100. 6. Speck RM, Courneya KS, Masse LC, Duval S, Schmitz KH. An update of
controlled physical activity trials in cancer survivors: a systematic review
and meta-analysis. J Cancer Surviv. 2010;4(2):87–100. y
7. Meneses-Echavez JF, Gonzalez-Jimenez E, Ramirez-Velez R. Effects of
supervised exercise on cancer-related fatigue in breast cancer survivors: a
systematic review and meta-analysis. BMC cancer. 2015;15:77. 7. Meneses-Echavez JF, Gonzalez-Jimenez E, Ramirez-Velez R. Effects of
supervised exercise on cancer-related fatigue in breast cancer survivors: a
systematic review and meta-analysis. BMC cancer. 2015;15:77. Discussion
Th
f Yes
4
No
5 Table 4 Physical activity preferences during chemother-
apy (n = 9) N Table 5 Physical activity barriers during chemotherapy
(n = 9)
“On a scale from 0 to 10, how did the following adverse effects hampered your
level of physical activity during chemotherapy?” (1 = not at all to 10 = to a very
high degree). Mean value for the nine patients that were interviewed, for each
adverse effect
Mean value
Nausea
5.7
Feeling unwell
5.7
Reduced general condition
4.8
Exhausted/tired
4.6
Headache
3.6
Breathlessness
3.6
Diarrhea
2.6
Reduced muscle strength
2.6
Dyspnea
2.4
Constipation
2.1
Increased heart rate
1.6
Neuropathy
1.4 Table 5 Physical activity barriers during chemotherapy
(n = 9)
“On a scale from 0 to 10, how did the following adverse effects hampered your
level of physical activity during chemotherapy?” (1 = not at all to 10 = to a very
high degree). Mean value for the nine patients that were interviewed, for each
adverse effect
Mean value
Nausea
5.7
Feeling unwell
5.7
Reduced general condition
4.8
Exhausted/tired
4.6
Headache
3.6
Breathlessness
3.6
Diarrhea
2.6
Reduced muscle strength
2.6
Dyspnea
2.4
Constipation
2.1
Increased heart rate
1.6
Neuropathy
1.4 Table 5 Physical activity barriers during chemotherapy
(n = 9) Page 8 of 9 Thorsen et al. BMC Res Notes (2017) 10:214 Competing interests
The authors declare that they have no competing interests. Competing interests coach. Christensen et al. showed that patients with TC in
average performed 70% of the planned resistance training
sessions during BEP supervised by a trainer. In further
intervention trials more frequent face-to-face counseling
by a personal trainer during the exercise sessions might
be necessary to increase the PA level during BEP/EP. Availability of data and materials
The dataset supporting the conclusion of this manuscript cannot be shared
due to protection of the patients’ confidentiality. Consent to publish
h Consent to publish
The participants provided written informed consent to publish. Thorsen et al. BMC Res Notes (2017) 10:214 11. Christensen JF, Bandak M, Campbell A, Jones LW, Hojman P. Treatment-
related cardiovascular late effects and exercise training countermeasures
in testicular germ cell cancer survivorship. Acta Oncol. 2015;54(5):592–9.
12. Oldervoll LM, Kaasa S, Hjermstad MJ, Lund JÅ, Loge JH. Physical exercise
results in the improved subjective well-being of a few or is effective
rehabilitation for all cancer patients? Eur J Cancer. 2004;40:951–62.
13. Rock CL, Doyle C, Demark-Wahnefried W, Meyerhardt J, Courneya KS,
Schwartz AL, et al. Nutrition and physical activity guidelines for cancer
survivors. CA Cancer J Clin. 2012;62(4):243–74.
14. Borg GA. Perceived exertion. Exerc Sport Sci Rev. 1974;2:131–53.
15. Horwich A. Testicular cancer. In: Horwich A, editor. Oncology—a multidis-
ciplinary textbook. London: Chapman and Hall; 1995. p. 485–98. 16. Christensen JF, Jones LW, Tolver A, Jorgensen LW, Andersen JL, Adamsen
L, et al. Safety and efficacy of resistance training in germ cell cancer
patients undergoing chemotherapy: a randomized controlled trial. Br J
Cancer. 2014;111(1):8–16.
17. Edvardsen E, Skjonsberg OH, Holme I, Nordsletten L, Borchsenius F,
Anderssen SA. High-intensity training following lung cancer surgery: a
randomised controlled trial. Thorax. 2015;70(3):244–50.
18. Courneya KS, McKenzie DC, Reid RD, Mackey JR, Gelmon K, Friedenreich
CM, et al. Barriers to supervised exercise training in a randomized con-
trolled trial of breast cancer patients receiving chemotherapy. Ann Behav
Med Publ Soc Behav Med. 2008;35(1):116–22. 16. Christensen JF, Jones LW, Tolver A, Jorgensen LW, Andersen JL, Adamsen
L, et al. Safety and efficacy of resistance training in germ cell cancer
patients undergoing chemotherapy: a randomized controlled trial. Br J
Cancer. 2014;111(1):8–16. 17. Edvardsen E, Skjonsberg OH, Holme I, Nordsletten L, Borchsenius F,
Anderssen SA. High-intensity training following lung cancer surgery: a
randomised controlled trial. Thorax. 2015;70(3):244–50. 15. Horwich A. Testicular cancer. In: Horwich A, editor. Oncology—a multidis-
ciplinary textbook. London: Chapman and Hall; 1995. p. 485–98. Acknowledgements
h
k h We thank the patients for participating and the clinicians that identified and
included the patients. Page 9 of 9 • We accept pre-submission inquiries
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CM, et al. Barriers to supervised exercise training in a randomized con-
trolled trial of breast cancer patients receiving chemotherapy. Ann Behav
Med Publ Soc Behav Med. 2008;35(1):116–22. 13. Rock CL, Doyle C, Demark-Wahnefried W, Meyerhardt J, Courneya KS,
Schwartz AL, et al. Nutrition and physical activity guidelines for cancer
survivors. CA Cancer J Clin. 2012;62(4):243–74. g
p
12. Oldervoll LM, Kaasa S, Hjermstad MJ, Lund JÅ, Loge JH. Physical exercise
results in the improved subjective well-being of a few or is effective
rehabilitation for all cancer patients? Eur J Cancer. 2004;40:951–62. 11. Christensen JF, Bandak M, Campbell A, Jones LW, Hojman P. Treatment-
related cardiovascular late effects and exercise training countermeasures
in testicular germ cell cancer survivorship. Acta Oncol. 2015;54(5):592–9.
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Infinitely many solutions for Schrödinger equations with Hardy potential and Berestycki-Lions conditions
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Open Mathematics 2024; 22: 20230175
Research Article
Shan Zhou*
Infinitely many solutions for Schrödinger
equations with Hardy potential
and Berestycki-Lions conditions
https://doi.org/10.1515/math-2023-0175
received August 30, 2023; accepted January 2, 2024
Abstract: In this article, we investigate the following Schrödinger equation:
− Δu −
μ
u = g (u )
∣x∣2
in N ,
(N − 2)2
μ
where N ≥ 3, ∣ x ∣2 is called the Hardy potential and g satisfies Berestycki-Lions conditions. If 0 < μ < 4 ,
we will take symmetric mountain pass approaches to prove the existence of infinitely many solutions of this
problem.
Keywords: Berestycki-Lions conditions, Hardy potential, infinitely many solutions, variation method, symmetric mountain pass
MSC 2020: 35Jxx, 46Txx, 49J35, 49J40
1 Introduction
In [1], Berestycki and Lions considered the following equation:
− Δu = g (u) ,
u ∈ Hr1(N ) ,
(1)
where g satisfies
(g1) g ∈ C ( , ) is odd,
(g2) −∞ < liminfs → 0+
(g3)
g (s )
limsups →+∞ s 2 *−1
g (s )
s
≤ limsups → 0+
≤ 0, where 2* =
g (s )
=
s
2N
,
N−2
ζ
(g4) there exists ζ > 0 such that G (ζ ) ≔
− M < 0,
∫0 g (t )dt > 0,
then, by using the constrained minimization method, they obtained the existence of infinitely many solutions
of problem (1). Moreover, they proved that problem (1) has at least one ground state solution in [2]. Here, we
should point out that ( g1 )–( g4 ) is referred to as the Berestycki-Lions condition, which is an almost necessary
and sufficient condition for the existence of ground state solutions. Subsequently, Hirata et al. used mountain
pass and symmetric mountain pass approaches to prove the existence of infinitely many solutions of problem
(1) in [3].
At the same time, many researchers studied the perturbation of problem (1), that is, the following Schrödinger equation:
* Corresponding author: Shan Zhou, Global Graduate School, Anyang University in Korea, Gyeonggi-do 430714, South Korea,
e-mail: zhoushan0812@163.com
Open Access. © 2024 the author(s), published by De Gruyter.
International License.
This work is licensed under the Creative Commons Attribution 4.0
2 Shan Zhou
x ∈ N , N ≥ 3,
− Δu + V (x )u = g (u) ,
(2)
with V suitable potential function. Azzollini and Pomponio [4] considered a potential V satisfying
(V1) V ∈ C 1(N , [0, +∞)) , V (x ) ≢ 0, and lim∣ x ∣→∞V (x ) = 0,
(V2) ∣⟨∇V , x ⟩+∣N2 < 2S , where ⟨∇V , x ⟩+ = max{⟨∇V , x ⟩, 0} and
⎫
⎧
S ≔ inf ⎨ ∣∇u∣2 dx : u ∈ D 1,2(N ) , ∣u∣2* dx = 1⎬,
⎭
⎩ N
N
∫
∫
(V3) V is radially symmetric.
In the radial space Hr1(N ), by using the Jeanjean theorem [5], they obtained that problem (2) has a ground
state solution. Liu et al. [6] also investigated the existence of ground state solutions of problem (2). In [6],
V is no longer a radially symmetric function but V satisfies
(V4) V ∈ C 1(N , ( − M , 0]) and lim∣ x ∣→+∞V (x ) = 0;
(V5) ∣⟨∇V , x ⟩−∣N2 < (N − 2)S and ∣⟨∇V , x ⟩+∣N2 < 2S , where
⟨∇V , x ⟩± = max{ ± ⟨∇V , x ⟩, 0} .
Sato and Shibata [7] proved the existence of infinitely many solutions of problem (2). They assumed that
the potential function V satisfies the following:
(V6) V ∈ C 1(N , (0, ∞)), lim∣ x ∣→∞V (x ) = V∞ > 0.
(V7) There exists η ∈ L2 (N ) ∩ W 1, ∞(N ) such that
(x ⋅∇V (x )) ≤ η(x )2
for all x ∈ N .
(V8) There exists ρ ∈ (0, 1) such that, for any α > 0,
lim
inf
(x ⋅∇V ( y ))e α ∣ x ∣ = ∞.
∣ x ∣→∞ y ∈ B(x , ρ ∣ x ∣)
and the nonlinearity g satisfies the following:
(g1′) g ∈ C 1( , ) is odd,
(g2′) g ′ (0) = 0,
g ′ (s )
(g3′) There exists p ∈ (1, (N + 2)∕(N − 2)) such that lims →∞ s p − 1 = 0,
(g4′) lims →∞
g (s )
s
= ∞.
Cerami et al. [8,9] supposed that the potential V satisfies V (x ) → V∞ > 0 as ∣x∣ → ∞ and slow decay assumptions
but does not need to fulfill any symmetry property. Then the existence of infinitely many positive solutions, by
purely variational methods, is proved. Further results for related problems can be found in [10–13]. One can
easily see that −Δ may be replaced by the more general operator through appropriate modifications on g . We
highlight the results on ( p , q )-Laplacian equations [14], the quasi-linear Schrödinger equation [15], and
p-Laplace equation [16] in this regard. In [14,15], multiplicity results were obtained by employing the symmetric mountainpass theorem and fountain theorem. In particular, [14] falls in the symmetric setting and [16]
treats critical p-Laplacian equations with Hardy terms. Nevertheless, we do not want to go further in that
direction.
μ
In this article, we focus on the Hardy potential − ∣ x ∣2 , which is also called the localized inverse-square
potential and arises in many other areas such as quantum mechanics, nuclear physics, molecular physics, and
quantum cosmology [17,18]. Motivated by the aforementioned works, we consider the existence of infinitely
many solutions for problem (2) with the Hardy potential in this article, that is,
Infinitely many solutions for Schrödinger equations
⎧⎪− Δu − μ u = g (u) ,
∣x∣2
⎨
⎪
1
⎩u ∈ Hr (N ) ,
for x ∈ N ,
3
( ) .
Exactly we give our main result.
Theorem 1.1. Assume that N ≥ 3, 0 < μ <
sequence of distinct solutions.
(N − 2)2
,
4
and ( g1 )–( g4 ) hold. Then problem ( ) possesses an infinite
Remark 1.2. We point out, as far as we are concerned, that this article seems to be the first attempt to obtain
the existence of infinitely many solutions to problem ( ). About Schrödinger equations with the Hardy
potential, most of the authors consider critical nonlinearities, see [19–23]. Our aim here is to illustrate the
existence of solutions for the nonlinearity under Berestycki-Lions conditions, which is different from those
results in the literature involving a potential having a different shape. For subcritical nonlinearities, there are
many similar research studies, please refer to the literature [24], where the assumptions on the nonlinear term
are stronger than Berestycki-Lions conditions.
Compared with [4,6], we want to obtain the existence of infinitely many solutions when V (x ) is a Hardy
potential, which clearly does not satisfy (V2 ) or (V5). Of course, the methods dealing with our problem
are different from ones in [4,6]. Our work extend and complete some results in [4,6].
The organization of this article is as follows. In the next section, we introduce our framework
and we establish some preliminary results. Theorem 1.1 is proved in Section 3.
2 Preliminaries
From now on, we will use the following notations.
• H 1(N ) is the usual Sobolev space endowed with the usual norm
‖u‖2 =
∫ (∣∇u∣2 + u2)dx .
N
• L p (N ) is the usual Lebesgue space endowed with the norm
∣u∣pp =
∣u(x )∣
∫ ∣u∣p dx and ∣u∣∞ = essxsup
∈
•
•
•
•
for all p ∈ [1, +∞) .
N
N
meas Ω denotes the Lebesgue measure of the set Ω .
⟨ ⋅ , ⋅ ⟩ denotes action of dual.
Br ( y ) ≔ {x ∈ N : ∣x − y∣ ≤ r } and Br ≔ {x ∈ N : ∣x∣ ≤ r } .
C denotes a positive constant, which changes value from line to line.
We reclaim the Hardy inequality
∣u∣2
2
2
∫ ∣x∣2 dx ≤ ⎛⎝ N − 2 ⎞⎠ ∫ ∣∇u∣2 dx .
N
(3)
N
Following [2], define
s0 ≔ min{s ∈ [ζ , +∞) : g (s ) = 0}(s0 = +∞ if g (s ) ≠ 0
and set g͠ : → the function such that
for any s ≥ ζ )
4 Shan Zhou
⎧ g (s ) ,
g͠ (s ) = ⎨0,
⎩
s ∈ [0, s0],
s ∈ (s0, +∞) .
For s ≦ 0, g͠ is defined (as g ) by g͠ (s ) = − g ( − s ) . Observe that g͠ satisfies the same conditions as g . Hence,
there is no loss in generality in replacing g by g͠ in the following discussion. With this modification, g satisfies
the stronger condition
(g3′ ) lim
s →∞
g (s )
= 0.
∣s∣2*− 1
In this article, we will work on the space Hr1(N ) and define the energy functional : Hr1(N ) → given by
(u) =
1
2
μ
∫ ⎛⎝∣∇u∣2 − ∣x∣2 u2⎞⎠dx − ∫ G(u)dx .
⎜
⎟
N
N
By a standard argument, we can say that is of class C 1 whose derivative is given by
⟨′(u) , v⟩ =
μ
∫ ⎛⎝∇u ⋅∇v − ∣x∣2 uv⎞⎠dx − ∫ g (u)vdx ,
⎜
⎟
N
v ∈ Hr1(N ) .
N
Formally, critical points of are solutions of problem ( ). Following from [3], we introduce h ∈ C ( , ) by
⎧ max{Mt + g (t ) , 0}
h (t ) = ⎨
⎩− h( − t )
for t ≥ 0,
for t < 0.
Furthermore, we choose p0 ∈ (1, (N + 2)∕(N − 2)) and set
h (τ )
⎧ p
t 0 sup
⎪ 0 < τ ≤ t τ p0
f (t ) = ⎨
0
⎪
⎩− f (−t )
for t > 0,
for t = 0,
for t < 0.
We also set
t
t
H (t ) = h(s )ds ,
∫
F (t ) = f (s )ds .
0
0
∫
From the definition of h(ξ ) , f (ξ ), and m 0, we have
Lemma 2.1. Assume that N ≥ 3 and (g1) − (g4 ) hold. Then
(a) Mt + g (t ) ≤ h(t ) ≤ f (t ) for all t ≥ 0;
(b) h(t ) ≥ 0, f (t ) ≥ 0 for all t ≥ 0;
(c) There exists δ0 > 0 such that h(t ) = f (t ) = 0 for t ∈ [0, δ0];
(d) There exists t0 > 0 such that 0 < h(t0) ≤ f (t0);
(e) t ↦ f (t )∕t p0 is non-decreasing on (0, ∞);
(f) h(t ) , f (t ) satisfies (g3′ ) .
Proof. The proof is similar to the proof of [3, Lemma 2.1]. We omit the details.
We define the functional (u) : Hr1(N ) → by
(u) =
1
2
μ
∫ ⎛⎝∣∇u∣2 + Mu2 − ∣x∣2 u2⎞⎠dx − ∫ F (u)dx .
⎜
N
⎟
N
□
Infinitely many solutions for Schrödinger equations
5
Critical points of (u) are solutions of the following problem:
⎧⎪− Δu + Mu − μ u = f (u) ,
∣x∣2
⎨
⎪
1
⎩u ∈ Hr (N ) .
for x ∈ N ,
(P∞)
Lemma 2.2. Assume that N ≥ 3 and ( g1 )–( g3 ) hold. Then possesses the mountain pass geometry, namely
(i) there exists ρ > 0 such that inf‖u ‖= ρ(u) ≥ inf‖u ‖= ρ(u) > 0,
(ii) For any n ∈ , there exists an odd continuous mapping
γ0, n : S n − 1 = {σ = (σ1, …, σn ) ∈ n; ∣σ ∣ = 1} → Hr1(N )
such that
I (γ0n(σ )) < 0, J (γ0n(σ )) < 0 for all σ ∈ S n − 1.
Proof. (i): Obviously, (u) ≥ (u) for all u ∈ Hr1(N ). It follows from (e)–(f) in Lemma 2.1 that there exist C such
that for any s ∈
∣F (s )∣ ≤ C (s p0 + 1 + ∣s∣2* ) .
Thus, one has
(u) ≥
u2
1
2
∫ ⎛⎝∣∇u∣2 − ∣x∣2
⎜
N
⎞
+ Mu2 dx − C
⎠
⎟
∫ ∣u∣p +1 + ∣u∣2* dx
0
N
1
2 ⎞2 ⎫ 2
⎧
≥ min ⎨M , 1 − μ⎛
⎬‖u‖ − C (‖u‖p0 + 1 + ‖u‖2* ) .
⎝N − 2⎠ ⎭
2
⎩
Since p0 ∈ (1, (N + 2)∕(N − 2)) , 2* > 2 , there exists ρ > 0 such that (u) ≥ (u) ≥
1
4
2
⎫
⎧
2
min ⎨m , 1 − μ⎛ N − 2 ⎞ ⎬ρ2 > 0
⎝
⎠
⎭
⎩
for ‖u‖ = ρ > 0.
(ii): We can argue as in [2, Theorem 10] and find for any n ∈ an odd continuous mapping
πn : S n − 1 → Hr1(N ) such that
∫ G(πn(σ ))dx ≥ 1
0 ∉ πn(S n − 1) ,
for all σ ∈ S n − 1.
N
For t ≥ 1, set
γ0, n(σ )(x ) = πn(σ )(x ∕t ) : S n − 1 → Hr1(N ) .
Then
(γ0, n(σ )(x )) =
t N −2
2
μ
∫ ⎛⎝∣∇πn(σ )∣2 dx − ∣x∣2 πn2(σ )⎞⎠dx − t N ∫ G(πn(σ ))dx .
⎜
⎟
N
N
Thus for sufficiently large t = tn ≥ 1, γ0, n(σ ) has the desired property for . Moreover, γ0, n(σ ) also has
□
the desired property for . This completes the proof.
We consider the level
bn = inf max (γ(σ )) ,
γ ∈ Γn σ ∈ Dn
cn = inf max (γ(σ )) .
γ ∈ Γn σ ∈ Dn
Here Bn = {σ = (σ1, …, σn ) ∈ n : ∣σ ∣ ≤ 1} and a family of mappings Γn is defined by
Γn = {γ ∈ C (Bn , Hr1(N )) : γ( − σ ) = − γ(σ ) , γ(σ ) = γ0, n(σ )
for all σ ∈ ∂Bn} .
(4)
6 Shan Zhou
We remark that
⎧
⎛σ ⎞
∣σ ∣γ0, n
,
γ (σ ) = ⎨
⎝ ∣σ ∣ ⎠
⎪
⎩0,
⎪
⎜
for σ ∈ Bn \{0} ,
⎟
(5)
for σ = 0,
belongs to Γn and Γn ≠ ∅ for all n . We can easily see that
γ(Bn ) ∩ {u ∈ Hr1(N ) : ‖u‖ = ρ} ≠ ∅
for all γ ∈ Γn .
Thus, it follows from Lemma 2.2 that
bn ≥ cn ≥
1
2 ⎞2 ⎫ 2
⎧
min ⎨m , 1 − μ⎛
⎬ρ > 0.
⎝N − 2⎠ ⎭
4
⎩
(6)
Lemma 2.3. Assume that N ≥ 3 and ( g1 )–( g4 ) hold. Then there exists {um, n} satisfying
(um, n ) → bn ,
(um, n ) → 0,
′(um, n ) → 0,
and
(7)
where
(u) =
N−2
2
∫ ∣∇u∣2 dx −
N
(N − 2)μ
2
u2
∫ ∣x∣2 dx − N ∫ G(u)dx .
N
N
Proof. Following Jeanjean [5,11] (see also [3,25]), we define the map Φ : × Hr1(N ) → Hr1(N )
for t ∈ , v ∈ Hr1(N ), and x ∈ N by
Φ(t , v)(x ) = v(e −tx ) .
For every t ∈ and v ∈ Hr1(N ) , the functional ∘ Φ is computed as
(Φ(t , v)) =
e (N − 2)t
2
∫ ∣∇v∣2 dx −
N
μe (N − 2)t
2
v2
∫ ∣x∣2 dx − e Nt ∫ G(v)dx .
3
N
In view of (g1)–( g4 ), ∘ Φ is continuously Fréchet-differentiable on × Hr1(N ) . We equip a standard product
norm ‖(θ , u)‖ × Hr1 = (∣θ∣2 + ‖u‖2 )1 ∕ 2 to × Hr1(N ) . We define minimax values b̃n for ∘ Φ by
∼
bn = inf max( ∘ Φ)(γ˜(σ )) ,
γ˜ ∈ Γ̃n σ ∈ Bn
Γn = {γ∼(σ ) ∈ C (Bn , × Hr1(N )): γ∼(σ ) = (θ (σ ) , η(σ )) satisfies (θ ( − σ ) , η( − σ )) = (θ (σ ) , − η(σ )) for all
where ∼
σ ∈ Bn and (θ (σ ) , η(σ )) = (0, γ0, n(σ )) for all σ ∈ ∂Bn} .
Γn and we may
Then we have b˜n = bn for all n ∈ . Indeed, for any γ ∈ Γn we can see that (0, γ(σ )) ∈ ∼
∼
∼
regard Γn ⊂ ∼
Γn . Thus by the definitions of bn and bn , we have bn ≤ bn . On the other hand, for any given
γ∼(σ ) = (θ (σ ) , η(σ )) ∈ ∼
Γn , we set γ(σ ) = η(σ )(e −θ(σ )x ) . We can verify that γ(σ ) ∈ Γn , (γ(σ )) = ∘ Φ(γ∼(σ ))
∼
for all σ ∈ Bn . Thus, we also have bn ≥ bn .
As in [3, Proposition 4.2], there exists a sequence {(tm, n , vm, n )} ⊂ × Hr1(N ) such that as m → ∞
tm, n → 0,
( ∘ Φ)(tm, n , vm, n ) → bn ,
( ∘ Φ)′ (tm, n , vm, n ) → 0.
Since for every (h, w ) ∈ × Hr1(N )
( ∘ Φ)′ (tm, n , vn )[h, w] = ′(Φ(tm, n , vn ))[Φ(tm, n , w )] + (Φ(tm, n , vn ))h.
Then, by taking um, n = Φ(tm, n , vm, n ) we have
(um, n ) → bn ,
This completes the proof.
(um, n ) → 0,
and ′(um, n ) → 0.
□
Infinitely many solutions for Schrödinger equations
7
3 Proof of Theorem 1.1
Now we are in a position to prove Theorem 1.1. We adopt some techniques in the proof of Theorem 1.1 in [3]
(see also [26]). We divide our proof into two steps.
Proof. Step 1. We show that for all n there exists a nontrivial solution un such that
(un ) = bn .
In Lemma 2.3, we recall that {um, n} satisfies as m → ∞,
(um, n ) → bn ,
(um, n ) → 0,
and ′(um, n ) → 0.
Indeed, we know
N
2
2 ⎞⎫
⎧
≥ ⎨1 − μ⎛
⎬
⎝
N − 2⎠ ⎭
⎩
∣um, n∣2
dx
∣x∣2
N
∫ ∣∇um,n∣2 dx − μ ∫
Nbn + o(1) = N (um, n ) − (um, n ) =
∫ ∣∇um,n∣2 dx .
N
This implies that {um, n} is bounded in D 1,2(N ). On the other hand, we have
2
⎧
⎛ 2 ⎞⎫
⎨1 − μ
⎬
⎝N − 2⎠ ⎭
⎩
∫ ∣∇un∣2 dx ≤
N
=
N−2
2N
∫ ∣∇um,n∣2 dx −
N
(N − 2)μ
2N
∣um, n∣2
dx
∣x∣2
N
∫
∫ G(um,n)dx + o(1)
N
≤−
mN
2
∫ ∣um,n∣2 dx + C ∫ ∣um,n∣2* dx + o(1).
N
N
Then {um, n} is bounded in L2 (N ) from the Sobolev embedding. So that {um, n} is bounded in Hr1(N ).
2
( N ) , u m , n (x ) → u n (x )
Furthermore, there exists un ∈ Hr1(N ) such that um, n ⇀ un in Hr1(N ), um, n → u in Lloc
∞
N
N
a.e. in . For any φ ∈ C0 ( ), one sees
0 = ⟨′(um, n ) , φ⟩ + o(1) = ⟨′(un ) , φ⟩.
Namely, un is a solution of problem ( ). We claim that um, n → un in Hr1(N ). Indeed,
o(1) = ⟨′(um, n ) − (un ) , um, n − un⟩
=
μ
∫ ∣∇(um,n − u)∣2 + ⎛⎝M − ∣x∣2 ⎞⎠(um,n − un)2dx
⎜
⎟
(8)
N
+
∫ [g (un) − g (um,n)](um,n − un) − M (um,n − un)2dx .
N
We observed that
∫ h(um,n)um,ndx → ∫ h(un)undx
N
N
and
h(um, n(x ))um, n(x ) − Mum2 , n(x ) − g (um, n(x ))um, n(x ) ≥ 0
for all n ∈ ,
x ∈ N .
8 Shan Zhou
Therefore, by Fatou’s lemma, as n large enough we have
∫ g (un)un − g (um,n)um,n + M (un2 − um2 ,n)dx
N
=
∫ h(um,n)um,n − h(un)undx − ∫ h(um,n)um,n − h(un) − [g (un)un − g (um,n)um,n + M (un2 − um2 ,n)]dx ≤ 0.
N
N
Combining with the Hardy inequality, from (8) we have
0 = liminf
n →∞
μ
∫ ∣∇(un − u)∣2 + ⎛⎝M − ∣x∣2 ⎞⎠(un − u)2dx ≤ 0.
⎜
⎟
N
Hr1(N )
This implies um, n → un strongly in
as m → ∞.
Step 2. Finally, we shall prove that bn → ∞ as n → ∞. This implies that has infinitely many critical points.
We can also see that cn(n ∈ ) are critical values of (see [3,26]). We apply an argument in [26, Chapter 9].
We set
Γ n = {h(Dm \Y ) : h ∈ Γm , m ≥ n, Y ∈ m and genus (Y ) ≤ m − n} .
Here m is the family of closed sets A ⊂ m\{0} such that − A = A and genus (A) is the Krasnoselskii’s genus
of A. We define another sequence of minimax values by
dn = inf max (u) .
A ∈ Γn u ∈ A
Then we have cn ≥ dn for all n ∈ , d1 ≤ d2≤ … ≤ dn ≤ dn + 1≤ … . Note that satisfies the Palais-Smale condition.
By modifying the argument in [26, Chapter 9] slightly, we have dn → ∞ as n → ∞. Thus, cn → ∞ as n → ∞. By (6),
the minimax values bn satisfy bn → ∞ as n → ∞.
Therefore, up to a subsequence, problem ( ) possesses an infinite sequence of distinct solutions {un} such
that (un ) = bn , where bn → ∞.
□
Acknowledgements: The author is very grateful to the anonymous referees for their knowledgeable reports,
which helped him to improve the manuscript.
Conflict of interest: The author states no conflicts of interest.
References
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
H. Berestycki and P.-L. Lions, Nonlinear scalar field equations, II, Existence of infinitely many solutions, Arch. Ration. Mech. Anal. 82
(1983), 347–375.
H. Berestycki and P.-L. Lions, Nonlinear scalar field equations, I, Existence of a ground state, Arch. Ration. Mech. Anal. 82 (1983),
313–345.
J. Hirata, N. Ikoma, and K. Tanaka, Nonlinear scalar field equations in N : mountain pass and symmetric mountain pass approaches,
Topol. Methods Nonlinear Anal. 35 (2010), 253–276.
A. Azzollini and A. Pomponio, On the Schrödinger equation in N under the effect of a general nonlinear term, Indiana Univ. Math. J. 58
(2009), 1361–1378.
L. Jeanjean, On the existence of bounded Palais-Smale sequences and application to a Landesman-Lazer-type problem set on N , Proc.
Roy. Soc. Edinburgh Sect. A 129 (1999), 787–809.
J. Liu, T. Liu, and J.-F. Liao, A perturbation of nonlinear scalar field equations, Nonlinear Anal. Real World Appl. 45 (2019), 531–541.
Y. Sato and M. Shibata, Infinitely many solutions for a nonlinear Schrödinger equation with general nonlinearity, Calc. Var. Partial
Differential Equations 57 (2018), no. 5, 137.
G. Cerami, G. Devillanova, and S. Solimini, Infinitely many bound states for some nonlinear scalar field equations, Calc. Var. Partial
Differential Equations 23 (2005), 139–168.
Infinitely many solutions for Schrödinger equations
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
9
G. Cerami, D. Passaseo, and S. Solimini, Infinitely many positive solutions to some scalar field equations with nonsymmetric coefficients,
Comm. Pure Appl. Math. 66 (2013), 372–413.
G. Cerami, R. Molle, and D. Passaseo, Multiplicity of positive and nodal solutions for scalar field equations, J. Differential Equations 257
(2014), no. 10, 3554–3606.
L. Jeanjean and K. Tanaka, A remark on least energy solutions in N , Proc. Amer. Math. Soc. 131 (2003), 2399–2408.
G.-D. Li, Y.-Y. Li, and C.-L. Tang, Existence and asymptotic behavior of ground state solutions for Schrödinger equations with Hardy
potential and Berestycki-Lions type conditions, J. Differential Equations 275 (2021), 77–115.
G.-D. Li, Y.-Y. Li, and C.-L. Tang, Ground state solutions for critical Schrödinger equations with Hardy potential, Nonlinearity 35 (2022),
5076–5108.
L. Baldelli, Y. Brizi, and R. Filippucci, On symmetric solutions for (p,q)-Laplacian equations in N with critical terms, J. Geom. Anal. 32
(2022), 120.
L. Baldelli and R. Filippucci, Singular quasilinear critical Schrödinger equations in N , Commun. Pure Appl. Anal. 21 (2022), 2561–2586.
R. Filippucci, P. Pucci, and F. Robert, On a p-Laplace equation with multiple critical nonlinearities, J. Math. Pures Appl. 91 (2009), no. 2,
156–177.
V. Felli, E. M. Marchini, and S. Terracini, On Schrödinger operators with multipolar inverse-square potentials, J. Funct. Anal. 250 (2007),
265–316.
W. M. Frank, D. J. Land, and R. M. Spector, Singular potentials, Rev. Modern Phys. 43 (1971), 36–98.
E. N. Dancer, F. Gladiali, and M. Grossi, On the Hardy-Sobolev equation, Proc. Roy. Soc. Edinburgh Sect. A 147 (2017), 299–336.
Y. Deng, L. Jin, and S. Peng, Solutions of Schrödinger equations with inverse square potential and critical nonlinearity, J. Differential
Equations 253 (2012), 1376–1398.
N. Ghoussoub and F. Robert, The Hardy-Schrödinger operator with interior singularity: the remaining cases, Calc. Var. Partial
Differential Equations 56 (2017), 149.
N. Ghoussoub and F. Robert, Hardy-singular boundary mass and Sobolev-critical variational problems, Anal. PDE 10 (2017), 1017–1079.
C. Wang and Y.-Y. Shang, Existence and multiplicity of solutions for Schrödinger equation with inverse square potential and Hardy-Sobolev
critical exponent, Nonlinear Anal. Real World Appl. 46 (2019), 525–544.
Q. Guo and J. a. Mederski, Ground states of nonlinear Schrödinger equations with sum of periodic and inverse-square potentials,
J. Differential Equations 260 (2016), 4180–4202.
V. Moroz and J. Van Schaftingen, Existence of groundstates for a class of nonlinear Choquard equations, Trans. Amer. Math. Soc. 367
(2015), 6557–6579.
P. H. Rabinowitz, Minimax Methods in Critical Point Theory with Applications to Differential Equations, CBMS Regional Conference Series
in Mathematics, Vol. 65, American Mathematical Society, Providence, 1986.
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Leverage dan Market Share sebagai Faktor Penentu Posisi Persaingan Pasar Perusahaan Manufaktur di Indonesia
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Suggested Citation: Mitani, Hidetaka (2014). Capital Structure and Competitive Position in Product Market. International Review of
Economic and Finance, 29 (2014) 358-371. Leverage dan Market Share sebagai Faktor Penentu Posisi Persaingan Pasar
Perusahaan Manufaktur di Indonesia Leverage dan Market Share sebagai Faktor Penentu Posisi Persaingan Pasar
Perusahaan Manufaktur di Indonesia Juwita Sari*
Sekolah Tinggi Ilmu Ekonomi Indonesia Surabaya
juwitasari@stiesia.ac.id
Puput Tri Komalasari
Universitas Airlangga, Surabaya
puput.tri@feb.unair.ac.id Juwita Sari*
Sekolah Tinggi Ilmu Ekonomi Indonesia Surabaya
juwitasari@stiesia.ac.id
Puput Tri Komalasari
Universitas Airlangga, Surabaya
puput.tri@feb.unair.ac.id Submitted Date :
9 September 2021
Accepted Date :
22 November 2021 Abstract: This study aims to examine the simultaneous relationship between leverage and market share as a determinant of market
competition for manufacturing companies in Indonesia. The model used in this study is the TSLS (Two Stage Least Square)
and testing with OLS (Ordinary Least Square), the testing tool used is SPSS. The object of research used in this study is a
Manufacturing Company listed on the Indonesia Stock Exchange from 2015-2020 with a total of 122 companies that have fit
with criteria for the research sample. The results of the study show that the two endogenous variables: leverage and market
share, have a significant positive correlation with each other, which means that there is a simultaneous relationship between
leverage policy and market share as an indicator of the competitive position of the product market. Keywords: Capital Structure; Competitive Position; Leverage; Market Share Keywords: Pangsa Pasar; Posisi Kompetitif; Rasio Hutang; Struktur Modal Volume 8 No 2
2021
Hlm. 380 - 391 Volume 8 No 2
2021
Hlm. 380 - 391 Jurnal Bisnis dan Manajemen
http://jurnal.unmer.ac.id/index.php/jbm Latar Belakang Salah satu aspek penting Perusahaan dalam upaya mencapai tujuan Perusahaan adalah optimalisasi rencana
pendanaan (Khairov et al., 2016; Soboleva & Parshutina, 2016). Aktifitas operasional Perusahaan menjadi lebih
efektif dan efisien dengan pengaturan rencana pendanaan yang tepat. Sumber pendanaan menurut Sudana
(2009, 2011) ada tiga yaitu penggunaan hutang, modal sendiri dan menerbitkan sekuritas. Perusahaan di
Indonesia yang didominasi Perusahaan keluarga, umumnya lebih memilih menggunakan kebijakan untuk
berhutang (Bambang & Hermawan, 2013; Santos et al., 2020). Selain sebagai upaya mengurangi biaya pajak,
dengan berhutang manajemen kepemilikan pada suatu Perusahaan tidak terpecah oleh pemilik ekternal atau
pemilik yang bukan anggota keluarga di Perusahaan tersebut (Mukhibad et al., 2020). Menentukan sumber dana
dari alternatif diatas, Perusahaan sebaiknya mampu menentukan komposisi yang optimal dengan biaya yang
minimal dalam membiayai operasional Perusahaan. Kebijakan pendanaan yang baik disebut juga dengan
struktur modal atau struktur modal optimal (Naomi et al., 2018; Schorr & Lips, 2019). (
)
Definisi dari struktur modal sendiri merupakan pembelanjaan jangka panjang suatu Perusahaan yang
diukur dengan perbandingan utang jangka panjang dengan modal sendiri (Sudana, 2011). Struktur modal yang
optimal adalah jumlah prosentase utang optimum yang dapat digunakan Perusahaan dalam membiayai
aktifitasnya, sehingga menjadi penting untuk memahami permasalahan terkait penentuan struktur modal yang
optimal dan upaya meminimkan biaya-biaya yang timbul pada operasional Perusahaan (Das & Swain, 2018). y
y
y y
g
(
)
Struktur modal yang merupakan bagian dari financial leverage akan berbeda-beda pada tiap Perusahaan
(Wati et al., 2020). Perbedaan ini didasari oleh kondisi keuangan Perusahaan dan pola pengambilan keputusan
tiap manajer dalam pemilihan dana Perusahaannya (Bas et al., 2009; Kayo et al., 2018). Penggunaan leverage
seringkali digunakan pada Perusahaan Manufaktur yang memiliki bermacam-macam Industri (Amedi & Mustafa,
2020; Shaferi et al., 2020) sehingga penggunaan struktur modal menjadi lebih kompleks (Gobenvy, 2014),
ditambah lagi dengan persaingan pada dunia bisnis yang semakin tajam yang mengharuskan Perusahaan untuk
terus mempertimbangkan faktor-faktor persaingan pasar yang terus berkembang (Boubaker et al., 2017). Dalam persaingan tersebut, mayoritas Industri Manufaktur mampu memproduksi barang-barang yang
jumlahnya jauh lebih banyak dari jumlah yang dapat dibeli oleh konsumen di pasar (Mitani, 2014). Menurut
Kotler (2004) kelebihan kapasitas merupakan hasil dari proyeksi yang dilakukan oleh masing-masing pesaing
bahwa market share tumbuh lebih besar dari kemungkinan yang sebenarnya. Kesalahan fatal terjadi ketika
Perusahaan memproyeksikan pertumbuhan Perusahaan sebesar 10 persen dan pasar total ternyata tumbuh
hanya 3 persen, hasilnya adalah kelebihan dalam kapasitas barang. Abstrak: Penelitian ini bertujuan untuk melihat hubungan simultan antara leverage dan market share sebagai penentu persaingan
pasar perusahaan manufaktur di Indonesia. Model pengujian yang digunakan dalam penelitian ini yaitu model TSLS (Two
Stage Least Square) dan pengujian diperkuat dengan OLS, alat pengujian yang digunakan adalah SPSS. Objek penelitian
yang digunakan dalam penelitian adalah Perusahaan Manufaktur yang terdaftar di BEI dari tahun 2015-2020 dengan jumlah
122 perusahaan yang sudah sesuai dengan kriteria sampel penelitian. Hasil penelitian menunjukkan kedua variabel
endogen yaitu leverage dan market share berkorelasi positif signifikan satu sama lain yang artinya terdapat pengaruh
simultan antara leverage perusahaan dan market share sebagai indikator posisi persaingan pasar produk. Keywords: Pangsa Pasar; Posisi Kompetitif; Rasio Hutang; Struktur Modal Keywords: Pangsa Pasar; Posisi Kompetitif; Rasio Hutang; Struktur Modal JEL Classification: G32
*Corresponding Author JEL Classification: G32
*Corresponding Author *Corresponding Author 380 Volume 8, No 2 (380-391), 2021 Latar Belakang Jurnal Bisnis dan Manajemen
http://jurnal.unmer.ac.id/index.php/jbm bentuk persaingan tertentu, merupakan kebaruan dari artikel ini. Sehingga, tujuan dari penelitian ini adalah
untuk menguji pengaruh simultan yang terjadi antara leverage dan market share perusahaan manufaktur di
Indonesia bentuk persaingan tertentu, merupakan kebaruan dari artikel ini. Sehingga, tujuan dari penelitian ini adalah
untuk menguji pengaruh simultan yang terjadi antara leverage dan market share perusahaan manufaktur di
Indonesia Porter (1998) mendefinisikan keunggulan bersaing sebagai strategi dari Perusahaan yang melakukan
kerjasama untuk berkompetisi lebih efektif di pasar. Strategi harus didesain untuk mewujudkan keunggulan
bersaing yang terus menerus, sehingga Perusahaan dapat mendominasi pasar lama maupun pasar baru
(Moeinaddin et al., 2013). Dalam hal ini, prosentase market share pada Perusahaan dapat dijadikan indikator
untuk mengetahui kedudukan Perusahaan tersebut dan posisi para pesaingnya di pasar. Prosentase market
share Perusahaan dapat digunakan untuk menyusun rencana-rencana strategis dalam upaya bersaing di pasar
(Blundell et al., 1999). Kotler (2005); Kotler & Keller (2016) menjelaskan posisi Perusahaan dapat dibedakan
sebagai market leader, market challenger, market follower, dan market nicher. Pemimpin pasar (market leader)
menguasai empat puluh persen atas pasar keseluruhan, penantang pasar (market challenger) menguasai tiga
puluh persen atas pasar keseluruhan, penurut/pengikut pasar (market follower) yaitu Perusahaan yang
cenderung stabil dan berupaya mempertahankan market sharenya menguasai dua puluh persen atas
keseluruhan pasar. Relung pasar (market nicher) yaitu Perusahaan yang melayani segmen pasar kecil/pasar
yang tidak terlayani oleh Perusahaan besar. Perusaahan dengan posisi market nicher hanya mengasai sepuluh
persen atas market share secara keseluruhan (Porter, 1998). Mitani (2014) mengatakan bahwa Perusahaan dengan market share yang tinggi cenderung menurunkan
rasio leverage-nya. Market share yang tinggi mengindikasikan Perusahaan tersebut berada pada posisi
pemimpin pasar (Michael Adiwijaya, 2007). Strategi utama yang dapat dilakukan Perusahaan adalah bertahan
pada jumlah market share yang ada, namun tetap berusaha memperluas prosentase market share (Ande,
2018). Perusahaan dengan posisi sebagai pemimpin pasar pada umumnya mengambil kebijakan hutang yang
rendah (Liu et al., 2019), karena selain memiliki cash flow yang tinggi serta aset yang besar untuk mendanai
operasionalnya Perusahaan juga mengantisipasi risiko kebangkrutan di masa datang (Mu et al., 2017; Uzliawati
et al., 2018). Gambar 1. Hubungan simultan antara leverage dan market share Gambar 1. Hubungan simultan antara leverage dan market share
Sumber: Kerangka Hipotesis Peneliti (2021)
Mitani (2014) berpendapat bahwa terdapat hubungan simultan antara leverage dan market share
Perusahaan. Latar Belakang Selanjutnya, Perusahaan akan menerapkan
strategi diskon besar-besaran agar barang yang berlebih dapat segera terjual dan tidak memenuhi gudang, yang
pada akhirnya Perusahaan akan merugi. Menghindari kerugian tersebut, Perusahaan sebaiknya harus
berproduksi secara efisien dengan memahami posisi Perusahaan dalam persaingan pasar (Kotler, 2005; Kotler
& Keller, 2016). Salah satu komponen keuangan dari sudut pandang persaingan yang dapat dijadikan penentu
posisi persaingan Perusahaan di pasar produk adalah market share (Sumarwan et al., 2010). p
p
g
p
p
(
)
Selain itu, Perusahaan juga dapat mengetahui kedudukannya dan posisi pesaing-pesaingnya di pasar
melalui prosentase market share (Ferrier et al., 1999; Varadarajan & Dillon, 1981). Perusahaan diharapkan
dapat lebih adaptif dalam mengambil strategi yang tepat atas penggunaan hutangnya (Boubaker et al., 2017). Perusahaan yang bertujuan untuk berekspansi dan menjadi pemimpin pasar harus berani mengambil risiko lebih
besar dengan penggunaan hutang yang juga besar (Buzzell et al., 1975; Varadarajan & Dillon, 1981) agar dapat
meningkatkan market share-nya (Kartikasari & Merianti, 2016; Lumapow, 2018). Misalnya, Perusahaan dapat
berinvestasi pada proyek baru yang bonafit atau profitable di masa mendatang seperti menciptakan produk
inovatif baru yang belum pernah ada di pasar. Sehingga, hubungan simultan akan terjadi ketika Perusahaan
menggunakan market share sebagai indikator untuk memilih seberapa besar hutang yang dibutuhkan
Perusahaan, begitu pula sebaliknya. Perusahaan dapat menggunakan rasio hutangnya untuk kebijakan strategi
meningkatkan market sharenya (Mitani, 2014). Pada penelitian Guney et al., (2011) dengan objek penelitian Perusahaan Manufaktur di Cina,
menggunakan tobinsQ sebagai variabel indikator atas persaingan. Menggunakan model analisis regresi linear
berganda yang menunjukan hubungan satu arah antara variabel persaingan pasar terhadap leverage. Sedangkan Mitani (2014) menggunakan market share sebagai bentuk lain persaingan pasar menguji
Perusahaan di Jepang dengan membagi posisi persaingan menjadi 2 yaitu Cournot Competition dan Bertrand
Competition. Perbedaan riset pada penelitian terdahulu menjadikan penelitian ini menarik untuk diulas. Pada
jurnal ini data yang diolah merupakan data keuangan dari Perusahaan Manufaktur yang listing di BEI pada
tahun 2015-2020. Menggunakan metode penelitian simultan dan tidak membagi posisi persaingan pasar dalam 381 Volume 8 No 2
2021
Hlm. 380 - 391 Volume 8 No 2
2021
Hlm. 380 - 391 Jurnal Bisnis dan Manajemen
http://jurnal.unmer.ac.id/index.php/jbm p
p
gg
g
j
(
g
)
Perusahaan yang mempertimbangkan peningkatan biaya agensi hutang akan mengantipasi dengan
meminimalkan biaya agensi hutang yang timbul (Bolton & Scharfstein, 1990; S. Ross et al., 2009). Perusahaan
dengan market share yang lebih rendah akan berusaha memperluas market share mereka dengan
mengoptimalkan pengaruh strategis atas keuntungan dari penggunaan hutang yang lebih besar (Das & Swain,
2018; Uzliawati et al., 2018), daripada pembiayaan biaya agensi hutang yang timbul (Bolton & Scharfstein,
1990). Perusahaan dengan market share yang tinggi menandakan bahwa Perusahaan tersebut mampu
menurunkan rasio hutangnya (leverage) (Mitani, 2014). Dari penjelasan diatas, maka hipotesis yang dirumuskan
adalah terdapat hubungan simultan, atau hubungan bersama-sama antara market share dengan leverage. Terdapat 6 variabel yang mempengaruhi Leverage sebagai variabel endogen: market share, tangibility,
size, GO, risiko bisnis, profitability. Market share, diukur melalui penjualan tahunan dibagi total penjualan
industri. Prosentase market share Perusahaan dapat dijadikan indikator dalam menganalisis posisi persaingan
perusahaan dalam industri (Moeinaddin et al., 2013), sehingga Perusahaan dapat mengetahui seberapa besar
hutang yang dibutuhkan Perusahaan (Mu et al., 2017). Mitani (2014) mengatakan bahwa perusahaan dengan
market share yang tinggi cenderung menurunkan rasio leveragenya. Perusahaan dengan posisi sebagai
pemimpin pasar pada umumnya mengambil kebijakan hutang yang rendah, karena selain memiliki cash flow
yang tinggi serta aset yang besar untuk mendanai operasionalnya (Frésard, 2008) perusahaan juga
mengantisipasi risiko kebangkrutan di masa datang (Schorr & Lips, 2019). Growth Opportunity. Perusahaan dalam industri yang mempunyai laju mendanai pembelanjaan
perusahaan (Rushpaka, 2020; Wati et al., 2020). Perusahaan dapat memanfaatkan dana eksternal atau hutang
untuk pendanaan investasi baru, untuk itu terdapat hubungan positif signifikan antara growth opportunity dan
leverage (Ibhagui & Okoloyo, 2018). Tangibility atau aset berwujud, merupakan aset yang dimiliki perusahaan, berumur lebih dari satu tahun,
dan dalam kegiatan operasional perusahaan selalu digunakan (S. A. Ross, 1977; Varadarajan & Dillon, 1981). Perusahaan dengan aset berwujud yang tinggi dapat menggunakan hutang sebagai pendanaan karena aktiva
tersebut bisa dijadikan sebagai dapat memperoleh kemudahan dalam melakukan aktivitas (Kartikasari &
Merianti, 2016; Moeinaddin et al., 2013). Perusahaan yang sebagian besar operasionalnya berasal dari aset
berwujud diharapkan memiliki lebih banyak hutang (Bambang & Hermawan, 2013; Niu, 2009). j
p
y
g (
g
)
Size, perusahaan adalah variabel yang digunakan dalam menjelaskan ukuran besar/kecilnya perusahaan
yang diukur melalui Ln of sales (Sundas, 2019). Jurnal Bisnis dan Manajemen
http://jurnal.unmer.ac.id/index.php/jbm Perusahaan berhutang untuk menerapkan strategi baru agar dapat meningkatkan market share
(Singh & Faircloth, 2005), begitupula market share digunakan sebagai faktor dalam menentukan posisi
persaingan Perusahaan yang nantinya digunakan untuk memutuskan seberapa besar hutang yang dibutuhkan
oleh Perusahaan (Edeling & Himme, 2018). Hubungan timbal simultan yang terjadi antara leverage dan market
share juga menimbulkan risiko kebangkrutan bagi masa depan Perusahaan (Y. Chen & Hammes, 2003; Mu et
al., 2017). Peningkatan rasio leverage juga akan meningkatkan debt agency cost (biaya agensi utang) (Bolton &
Scharfstein, 1990; Kayo et al., 2018; Niu, 2009; Wati et al., 2020). Biaya agensi hutang merupakan biaya yang Sumber: Kerangka Hipotesis Peneliti (2021) Sumber: Kerangka Hipotesis Peneliti (2021) Mitani (2014) berpendapat bahwa terdapat hubungan simultan antara leverage dan market share
Perusahaan. Perusahaan berhutang untuk menerapkan strategi baru agar dapat meningkatkan market share
(Singh & Faircloth, 2005), begitupula market share digunakan sebagai faktor dalam menentukan posisi
persaingan Perusahaan yang nantinya digunakan untuk memutuskan seberapa besar hutang yang dibutuhkan
oleh Perusahaan (Edeling & Himme, 2018). Hubungan timbal simultan yang terjadi antara leverage dan market
share juga menimbulkan risiko kebangkrutan bagi masa depan Perusahaan (Y. Chen & Hammes, 2003; Mu et
al., 2017). Peningkatan rasio leverage juga akan meningkatkan debt agency cost (biaya agensi utang) (Bolton &
Scharfstein, 1990; Kayo et al., 2018; Niu, 2009; Wati et al., 2020). Biaya agensi hutang merupakan biaya yang Mitani (2014) berpendapat bahwa terdapat hubungan simultan antara leverage dan market share
Perusahaan. Perusahaan berhutang untuk menerapkan strategi baru agar dapat meningkatkan market share
(Singh & Faircloth, 2005), begitupula market share digunakan sebagai faktor dalam menentukan posisi
persaingan Perusahaan yang nantinya digunakan untuk memutuskan seberapa besar hutang yang dibutuhkan
oleh Perusahaan (Edeling & Himme, 2018). Hubungan timbal simultan yang terjadi antara leverage dan market
share juga menimbulkan risiko kebangkrutan bagi masa depan Perusahaan (Y. Chen & Hammes, 2003; Mu et
al., 2017). Peningkatan rasio leverage juga akan meningkatkan debt agency cost (biaya agensi utang) (Bolton &
Scharfstein, 1990; Kayo et al., 2018; Niu, 2009; Wati et al., 2020). Biaya agensi hutang merupakan biaya yang 382 Volume 8, No 2 (380-391), 2021 timbul karena kreditur mengantisipasi kerugian atas dana yang dipinjamkan kepada Perusahaan dalam bentuk
pembatasan penggunaan hutang oleh Manajer (M. C. Jensen & Smith, Jr., 2005; M. Jensen & Meckling, 1976). Jurnal Bisnis dan Manajemen
http://jurnal.unmer.ac.id/index.php/jbm Perusahaan size besar diharapkan memiliki rasio leverage yang
lebih tinggi karena perusahaan besar lebih mudah dalam mendapatkan modal eksternal daripada perusahaan
dengan size kecil (Kartikasari & Merianti, 2016). Kemudahan akses tersebut menunjukkan bahwa perusahaan
size besar memiliki kesempatan melakukan pinjaman dalam jumlah yang besar pula dibandingkan perusahaan
kecil (Ibhagui & Okoloyo, 2018). (
g
y
)
Risiko bisnis, pada jurnal ini risiko bisnis menggunakan DOL (Degree of Operating Leverage) yang diukur
melalui delta ebit dibagi dengan delta penjualan perusahaan (S. Ross et al., 2009; Sarkar, 2018; Sudana, 2011). Perusahaan dengan risiko bisnis yang tinggi umumnya mempertahankan rasio operating leverage yang rendah
untuk mempertahankan fleksibilitas keuangan (Kale et al., 1991; Moeinaddin et al., 2013; Utami, 2019). Fleksibilitas keuangan/financial flexibility merupakan kemampuan perusahaan untuk mengakses dan
merestrukturisasi keuangannya dengan biaya transaksi yang rendah. Terdapat hubungan negatif signifikan
antara risiko bisnis dan kebijakan hutang (leverage) (Moeinaddin et al., 2013; Utami, 2019). ROA. Perusahaan dengan nilai ROA yang tinggi mengartikan bahwa perusahaan tersebut berada pada
posisi profitable (menguntungkan) yang berarti perusahaan mampu membiayai operasional perusahaan tanpa
menarik dana dari luar (Ross, 1977). Selanjutnya, terdapat 5 variabel yang mempengaruhi market share sebagai variabel endogen: Leverage,
Likuiditas, Specificity, HHI, GO. Leverage. Leverage diharapkan memiliki hubungan positif signifikan terhadap
market share karena perusahaan mengambil kebijakan berhutang untuk meningkatkan market share (Boubaker
et al., 2017). Rasio leverage yang meningkat akan membantu manajer perusahaan dalam mengambil langkah-
langkah strategis untuk menaikkan posisi persaingan di pasar (Buzzell et al., 1975; Harris & Raviv, 1990). g
g
g
(
)
Likuiditas. Likuiditas yang tinggi menunjukan perusahaan mampu mengembalikan hutang lancar
sehingga menumbuhkan kepercayaan bagi investor untuk mengalirkan dananya pada perusahaan tersebut
(Kuan et al., 2012). Likuiditas dihitung dengan membandingkan modal kerja (aktiva lancar dikurangi kewajiban
lancar) terhadap nilai buku aset (Ross et al., 2009). Likuiditas yang besar diharapkan dapat meningkatkan
market share perusahaan (Frésard, 2008; Kuan et al., 2012). 383 Volume 8 No 2
2021
Hlm. 380 - 391 Metode Penelitian Metode Penelitian Jenis penelitian ini merupakan penelitian kuantitatif eksplanatif. Populasi yang digunakan pada jurnal ini
merupakan seluruh Perusahaan Manufaktur yang go public dan terdaftar di Bursa Efek Indonesia (BEI) periode
tahun 2015-2020. Terdapat 537 sampel dari 122 perusahaan observasi yang memenuhi kriteria penelitian. Dalam penelitian ini menggunakan 13 (tiga belas) variabel dimana dua sebagai variabel endogen/dependen
sedangkan sembilan lainnya bersifat sebagai variabel eksogen/prediktor dan instrumental. Variabel endogen
tersebut yaitu leverage it dan market share it sedangkan variabel eksogennya adalah leverage t-1, likuiditas,
specificity, konsentrasi Industri, growth opportunity, market share t-1, aset berwujud, size, risiko bisnis, dan
profitability. Teknik analisis data yang digunakan pada riset ini adalah Two Stage Least Squares (TSLS/2SLS),
lalu diperkuat dengan analisis sensitivitas menggunakan OLS (Ordinary Least Square) (Ghozali, 2009). Gambar 2. Kerangka Pemikiran
Sumber: Data Diolah (2021)
Hasil
Data yang diperoleh peneliti selanjutnya diolah dan dilakukan analisis deskriptif. Teknik analisis data yang
digunakan pada riset ini adalah Two Stage Least Squares (TSLS/2SLS). Hasil yang didapat diuraikan pada
Tabel 1 dan 2 dibawah ini. Gambar 2. Kerangka Pemikiran Sumber: Data Diolah (2021) Sumber: Data Diolah (2021) Jurnal Bisnis dan Manajemen
http://jurnal.unmer.ac.id/index.php/jbm Specificity. Pada dunia pemasaran specificity berkaitan dengan karakteristik tertentu dan kinerja tertentu
yang diperlukan untuk membuat suatu produk ataupun menjual produk (Gopalakrishnan et al., 2015). Mitani
(2014) mengukur spesifitas perusahaan melalui beban penjualan ditambah beban administrasi lalu dibagi
dengan total aset. Hasil yang dihasilkan pada pengukuran tersebut menunjukan besarnya nilai beban penjualan
dan beban administrasi dalam membiayai operasional perusahaan. Diharapkan perusahaan memiliki market
share meningkat dengan menaikkan nilai specificity (Lyandres, 2006). Growth Opportunity. Perusahaan yang memiliki banyak peluang investasi adalah perusahaan yang
berada dalam industri yang mempunyai laju pertumbuhan yang tinggi (Rushpaka, 2020). Perusahaan yang
memiliki peluang investasi dan mampu mengelola investasinya dengan baik dapat memanfaatkan proyek-proyek
baru untuk membuat produk yang lebih berinovasi dan memiliki daya saing di pasar (Frésard, 2008; Kalhor &
Rooker, 2017; Soboleva & Parshutina, 2016). Manajer pemasaran juga dapat memanfaatkan situasi tersebut
untuk menyerang kompetitior dengan strategi-strategi yang tepat sehingga dapat meningkatkan market share
perusahaan, untuk itu diharapkan terjadi hubungan positif signifikan antara growth opportunity dan market share. Konsentrasi Industri/HHI. Menurut Davies & Geroski (1997) hubungan positif terjadi antara konsentrasi industri
dan market share perusahaan. Perusahaan dengan industri yang terkonsentrasi menghadapi persaingan yang
kurang kompetitif karena mereka cenderung memiliki lebih banyak kesempatan untuk memperluas market share
mereka. Hasil Data yang diperoleh peneliti selanjutnya diolah dan dilakukan analisis deskriptif. Teknik analisis data yang
digunakan pada riset ini adalah Two Stage Least Squares (TSLS/2SLS). Hasil yang didapat diuraikan pada
Tabel 1 dan 2 dibawah ini. Data yang diperoleh peneliti selanjutnya diolah dan dilakukan analisis deskriptif. Teknik analisis data yang
digunakan pada riset ini adalah Two Stage Least Squares (TSLS/2SLS). Hasil yang didapat diuraikan pada
Tabel 1 dan 2 dibawah ini. Data yang diperoleh peneliti selanjutnya diolah dan dilakukan analisis deskriptif. Teknik analisis data yang
digunakan pada riset ini adalah Two Stage Least Squares (TSLS/2SLS). Hasil yang didapat diuraikan pada
Tabel 1 dan 2 dibawah ini. 384 Volume 8, No 2 (380-391), 2021 Tabel 1. Hasil TSLS: Leverage Sebagai Variabel Endogen
Variabel
Unstandardized
Coefficients
Beta
t
Sig. B
Std. Error
(Constant)
,059
,167
,351
,726
Market Share
,100
,049
,095
2,066
,039
Tangibility
-,021
,047
-,019
-,453
,651
Size
,015
,006
,110
2,382
,018
Resiko Bisnis
-,016
,007
-,091
-2,333
,020
Growth Opportunity
,016
,006
,116
2,464
,014
Profitability
-1,033
,107
-,477
-9,672
,000
Sumber: Data Diolah (2021) Tabel 1. Hasil TSLS: Leverage Sebagai Variabel Endogen Tabel 1. Hasil TSLS: Leverage Sebagai Variabel Endogen Pada Tabel 1 diatas, menunjukkan adanya pengaruh positif signifikan antara market share terhadap
leverage. Hal ini ditunjukan dari tingkat signifikansi market share sebesar 0.039 dimana nilai ini dibawah
signifikansi 5%. Hasil ini tidak sejalan dengan teori yang dikembangkan dimana market share mampu
mempengaruhi leverage secara negatif, tetapi hasil ini menunjukkan hubungan positif signifikan. Selanjutnya
variabel size, risiko bisnis, growth opportunity dan profitability juga berpengaruh terhadap leverage karena
memiliki probabilitas signifikansi dibawah taraf signifikansi 5%. Variabel size berpengaruh positif signifikan
terhadap leverage, yang artinya semakin besar ukuran perusahaan maka rasio hutang/penggunaan hutang juga
akan meningkat. Berbeda dengan resiko bisnis yang memiliki pengaruh negatif signifikan terhadap leverage,
yang artinya ketika perusahaan dengan risiko bisnis kecil maka perusahaan lebih mudah dalam mendapatkan
pendanaan hutang. Variabel growth opportunity menunjukkan hubungan positif signifikan terhadap tingkat
leverage. Sedangkan variabel profitability berpengaruh negatif signifikan terhadap leverage dibawah nilai
statistik 0.000. Variabel Tangibility tidak berpengaruh terhadap leverage karena memiliki nilai signifikansi 0.651
dimana nilai tersebut diatas batas signifikansi 5%. Selanjutnya, pada Tabel 2 dibawah ini merupakan hasil uji
TSLS dengan market share sebagai variabel endogen. Tabel 2. Hasil TSLS: Market Share Sebagai Variabel Endogen
Variabel
Unstandardized
Coefficients
Beta
t
Sig. B
Std. Hasil Error
(Constant)
,031
,020
1,508
,132
Leverage
,073
,033
,076
2,195
,029
Likuiditas
-,007
,028
-,008
-,262
,793
Specificity
,001
,007
,002
,109
,913
Growth Opportunity
,007
,003
,053
2,507
,012
HHI
1,190
,027
,875
43,349
,000
Sumber: Data Diolah (2021) Tabel 2. Hasil TSLS: Market Share Sebagai Variabel Endogen Tabel 2. Hasil TSLS: Market Share Sebagai Variabel Endogen Sumber: Data Diolah (2021) Tabel 2 menunjukkan korelasi antar variabel yang diteliti dengan market share sebagai variabel endogen. Leverage mempengaruhi market share dengan dibuktikan pada nilai signifikansi sebesar 0.029 dan nilai tersebut
dibawah taraf signifikansi 5%. Sedangkan likuiditas tidak memiliki pengaruh terhadap market share karena
bernilai 0.793 dimana nilai tersebut lebih besar dari 5%. Specificity memiliki nilai probabilitas signifikansi sebesar
0.913 yang berada diatas taraf signifikansi 5% yang artinya variabel specificity tidak memiliki pengaruh terhadap
market share. Selanjutnya variabel growth opportunity dan HHI berpengaruh positif signifikan terhadap market
share karena nilai signifikansi yang dihasilkan sebesar 0.012 dan 0.000 dibawah taraf signifikansi 5%. Uji Sensitivitas Menggunakan OLS (Ordinary Least Square) Pada penelitian ini, setelah dilakukan uji menggunakan TSLS selanjutnya data diuji menggunakan OLS. Pengujian dengan OLS digunakan untuk memastikan adanya hubungan simultan antar variabel dependen yang
diuji. Menurut Ghozali (2009) jika tidak terdapat hubungan interdependen antara stochastic error dan variabel
endogen penjelas maka sebaiknya menggunakan metode OLS dalam mengestimasi persamaan simultan. 385 Jurnal Bisnis dan Manajemen
http://jurnal.unmer.ac.id/index.php/jbm
Volume 8 No 2
2021
Hlm. 380 - 391 Tabel 3. Hasil Uji OLS:Leverage Sebagai Variabel Dependen
Model
Unstandardized
Coefficients
Standardized
Coefficients
t
Sig. B
Std. Error
Beta
(Constant)
,078
,166
,472
,637
Market Share
,113
,046
,108
2,453
,014
Tangibility
-,021
,047
-,019
-,452
,652
Size
,014
,006
,105
2,283
,023
Resiko Bisnis
-,016
,007
-,091
-2,333
,020
Growth Opportunity
,016
,006
,116
2,469
,014
Profitability
-1,034
,107
-,477
-9,685
,000
Sumber: Data Diolah (2021) Tabel 3. Hasil Uji OLS:Leverage Sebagai Variabel Dependen Tabel 3. Hasil Uji OLS:Leverage Sebagai Variabel Dependen Sumber: Data Diolah (2021) Dari Tabel 3 diatas, dapat diketahui bahwa market share berpengaruh positif signifikan terhadap leverage
dengan ditunjukkan oleh nilai probabilitas signifikan sebesar 0.014. Hasil ini sama dengan ketika dilakukan uji
TSLS (Tabel 1). Selanjutnya, tangibility menunjukkan tidak ada pengaruh terhadap leverage. Size berpengaruh
positif signifikan terhadap leverage dengan nilai 0.023 nilai ini dibawah tingkat signifikansi 5%. Variabel resiko
bisnis berpengaruh negatif signifikan terhadap leverage dengan nilai 0.020. Growth opportunity memiliki nilai
probabilitas signifikansi sebesar 0.014 yang berarti positif signifikan dibawah tingkat signifikansi 5%. Variabel
profitability juga menunjukkan adanya pengaruh negatif signifikan terhadap leverage. Tabel 4. Hasil Uji OLS:Market Share Sebagai Variabel Dependen Tabel 4. Hasil Uji OLS:Market Share Sebagai Variabel Dependen Tabel 4. Hasil Uji OLS:Market Share Sebagai Variabel Dependen
Sumber: Data Diolah (2021)
Model
Unstandardized
Coefficients
Standardized
Coefficients
t
Sig. B
Std. Error
Beta
(Constant)
,030
,017
1,758
,079
Leverage
,074
,026
,078
2,863
,004
Likuiditas
-,006
,025
-,007
-,250
,802
Specificity
,001
,007
,002
,102
,919
Growth Opportunity
,007
,003
,053
2,512
,012
HHI
1,190
,027
,875
43,355
,000 Tabel 4. Hasil Uji OLS:Market Share Sebagai Variabel Dependen Tabel 4 menunjukkan leverage berpengaruh positif signifikan terhadap market share. Hasil ini mengkonfirmasi
hasil pada Tabel 2 sebelumnya. Selanjutnya, variabel likuiditas tidak berpengaruh terhadap market share. Specificity juga tiddak berpengaruh terhadap market share karena memiliki nilai signifikansi diatas 5%. Uji Sensitivitas Menggunakan OLS (Ordinary Least Square) Variabel
growth opportunity berpengaruh positif signifikan terhadap market share, begiupula HHI/Konsentrasi industri
yang juga berpengaruh positif signifikan terhadap market share dengan dibuktikan oleh besarnya nilai
probabilitas signifikansi yang lebih kecil dari batas signifikansi 5%. Dari hasil uji OLS pada Tabel 3 dan 4 dapat
dipahami bahwa hasil dari penelitian ini adalah mengkonfirmasi hasil pada Tabel 1 dan 2 yang artinya, temuan
ini menerima dugaan awal adanya pengaruh simultan (pengaruh bersama) antar 2 variabel dependen yaitu
market share dan leverage. Pembahasan Hasil penelitian ini sesuai dengan penelitian sebelumnya oleh Guney et al., (2011); (Kalhor & Rooker, 2017);
dan sedikit berbeda dengan penelitian Mitani, (2014). Jika pada penelitian sebelumnya ditemukan bahwa market
share berpengaruh negatif terhadap leverage, pada penelitian ini market share berpengaruh positif signifikan
terhadap leverage Perusahaan. Perusahaan di Indonesia yang memiliki posisi persaingan yang competitive
memiliki pangsa pasar terbesar dalam Industri cenderung meningkatkan rasio leveragenya sehingga terjadi
pengaruh hubungan positif signifikan antara market share dan leverage. Hal tersebut terjadi karena Perusahaan
dengan posisi market leader cenderung memiliki size besar, sehingga memudahkan Perusahaan mendapatkan
dana dari kreditor (Rushpaka, 2020; Sundas, 2019). 386 Volume 8, No 2 (380-391), 2021 Variabel lainnya yaitu tangibility/asset berwujud pada penelitian ini menunjukkan bahwa variabel aset
berwujud tidak berpengaruh terhadap leverage, karena signifikasi diatas 0.05. Hal tersebut menunjukan
bahwasannya Perusahaan manufaktur di Indonesia yang memiliki aset yang besar mungkin saja tidak terlalu
memerlukan hutang yang tinggi dalam menjalankan Perusahaannya. Aset berwujud yang besar dapat
menghasilkan laba dan cash flow yang cukup untuk operasional Perusahaan. Begitu pula Perusahaan dengan
aset berwujud yang kecil, tetap dapat meminjam kepada kreditur. Kreditur mungkin mempertimbangkan banyak
faktor seperti capacity, collateral, credit, character dan condition. ROA berpengaruh negatif terhadap leverage (Santoso, 2018). Hal tersebut berarti ketika ROA
Perusahaan tersebut mengalami kenaikan maka leveragenya mengalami penurunan, sebaliknya ketika ROA
Perusahaan mengalami penurunan maka akan terjadi peningkatan pada nilai leverage. Perusahaan yang
memiliki ROA kecil akan meningkatkan hutangnya untuk pengembangan strategi-strategi baru, namun
Perusahaan dengan tingkat keuntungan yang tinggi akan berusaha menurunkan nilai leveragenya karena
Perusahaan menggunakan dana internal untuk membiayai strategi-strateginya. Likuiditas pada penelitian ini tidak berpengaruh terhadap pangsa pasar karena uji statistik memaparkan
hasil signifikasi diatas 0.05. Perusahaan manufaktur di Indonesia pada umumnya meningkatkan pangsa pasar
tanpa terpengaruh kemampuan mereka dalam memenuhi kewajibannya. Perusahaan akan tetap meningkatkan
posisi persaingan produknya di pasar tanpa terbebani rasio kewajiban hutang jangka pendeknya (likuiditas). Size berpengaruh positif terhadap leverage. Hasil tersebut memiliki kesamaan pada riset oleh Mitani,
(2014); Santoso (2018) yang berargumentasi bahwasannya Perusahaan dengan size besar lebih cenderung
untuk menggunakan dana eksternal dalam komposisi struktur modalnya. Hal ini karena kebutuhan dana
Perusahaan yang semakin meningkat. Terlebih lagi bagi Perusahaan yang memiliki rencana untuk melakukan
ekspansi/pengembangan Perusahaan, kebutuhan dana Perusahaan akan melonjak serta akan memerlukan
dana tambahan selain dari dana internal. Pada penelitian ini risiko bisnis berpengaruh negatif signifikan. Pembahasan Kemungkinan hal tersebut terjadi karena
masih banyak Perusahaan yang berhutang karena dipengaruhi oleh kebutuhan akan investasi ataupun
operasional Perusahan, sehingga tingkat risiko bisnis kurang berpengaruh terhadap kebijkan berhutang. Risiko
bisnis mungkin saja kurang berpengaruh terhadap leverage karena risiko bisnis sendiri tidak hanya dipengaruhi
oleh operating leverage yang tinggi tetapi bisa banyak faktor yang mempengaruhi seperti risiko penjualan, risiko
investasi, risiko manajemen, risiko kredit, dan risiko pasar (S. Ross et al., 2009; Sudana, 2011). Pada penelitian ini growth opportunity berkorelasi positif signifikan pada leverage Perusahaan (Ibrahim,
2017). Hasil yang sama dengan penelitian Mitani (2014) menunjukan bahwa Perusahaan di Indonesia umumnya
meningkatkan leverage ketika Perusahaan memiliki peluang pertumbuhan yang tinggi. Perusahaan dengan
peluang pertumbuhan yang tinggi akan berupaya meningkatkan hutangnya dengan berinvestasi pada proyek
yang menguntungkan bagi Perusahaan dimasa depan. Selanjutnya, variabel Specificity pada penelitian ini tidak berpengaruh terhadap pangsa pasar dengan
dibuktikan pada Tabel 4 dimana signifikasi diatas 0.05. Hasil yang berbeda dari penelitian Mitani (2014) ini, bisa
saja dikarenakan investasi atau pendanaan khusus yang besar belum tentu mempengaruhi pangsa pasar. Misalnya pendanaan khusus yang dilakukan Perusahaan yaitu Perusahaan meningkatkan biaya promosi
penjualan. Promosi penjualan yang besar belum tentu membuat konsumen membeli produk tersebut. Banyak
faktor-faktor lain yang mempengaruhi konsumen dalam keputusan membeli sebuah produk, ditambah lagi
konsumen pada era globalisasi mulai cerdas dalam memilih produk karena banyak informasi yang didapat
konsumen sebelum membeli produk. Promosi penjualan yang bagus namun memiliki produk yang kurang
dibutuhkan konsumen tetap saja tidak akan mempengaruhi pangsa pasar Perusahaan. Growth opportunity berpengaruh positif terhadap pangsa pasar. Artinya Perusahaan yang memiliki
peluang investasi dan mampu mengelola investasinya dengan baik dapat memanfaatkan proyek-proyek baru
untuk membuat produk yang lebih berinovasi dan memiliki daya saing di pasar. Manajer pemasaran juga dapat
memanfaatkan situasi tersebut untuk menyerang kompetitior dengan strategi-strategi yang tepat sehingga dapat
meningkatkan pangsa pasar Perusahaan. Pada penelitian ini juga, HHI berpengaruh positif signifikan terhadap market share. Hal ini berarti
karakteristik Perusahaan di Indonesia mungkin saja dalam upaya peningkatan pangsa pasar dipengaruhi oleh
tingkat konsentrasi Industri. Terkonsentrasi atau tidaknya Perusahaan dalam Industri tersebut mempengaruhi
secara positif upaya menaikkan pangsa pasar karena memperluas pangsa pasar tergantung pada kemampuan
masing-masing Perusahaan dalam mendapatkan konsumen pada Industri. 387 Kesimpulan Volume 8 No 2
2021
Hlm. 380 - 391 Jurnal Bisnis dan Manajemen
http://jurnal.unmer.ac.id/index.php/jbm Kesimpulan Sesuai hasil analisis dan pembahasan pada pemaparan diatas, dapat ditarik kesimpulan bahwasannya terdapat
pengaruh simultan antara leverage dan market share. Leverage mencerminkan struktur modal atau besaran
hutang yang digunakan perusahaan untuk menjalankan bisnisnya. Sedangkan pangsa pasar/market share
dalam penelitian ini menunjukkan tingkat posisi persaingan pasar. Hal ini dibuktikan oleh uji statistik pengaruh
leverage terhadap market share sebesar 0.029 yang menunjukkan tingkat signifikasi dibawah 5%. Begitu pula
pengaruh market share terhadap leverage yang menunjukkan nilai sebesar 0.039 positif signifikan dibawah
tingkat signifikansi 5%. Dengan penelitian ini menunjukkan bahwa Perusahaan manufaktur di Indonesia
menggunakan kebijakan berhutang untuk meningkatkan posisi persaingan pasar. Begitu pula sebaliknya,
melalui pemahaman posisi persaingan pasar perusahaan dapat dengan mudah meningkatkan kebijakan
hutangnya. Kedepannya hasil penelitian ini tidak hanya sebagai kontribusi akademis bagi peneliti selanjutnya,
tetapi hasil temuan ini bisa digunakan saran untuk manajer keuangan sebagai bahan pertimbangan dalam
mengelola kebijakan hutang Perusahaan. g
j
g
Sesuai riset yang telah dilakukan, terdapat keterbatasan yang ditemui, yaitu penelitian ini terbatas data
yang hanya 5 tahun dan tidak membagi posisi persaingan produk pada teori persaingan pasar tertentu. Sehingga saran pada penelitian selanjutnya diharapkan dapat memberikan kontribusi pengetahuan yang
berbeda dengan dukungan data yang lebih panjang dan membagi persaingan produk kedalam kategori tertentu. Penambahan jumlah variabel, sampel atau periode tahun sehingga mempunyai tingkat akurasi hasil statistik
yang lebih baik kedepannya, atau dapat menambahkan sampel Perusahaan dari sektor lain yang terdaftar di
BEI y
g
BEI Daftar Pustaka Daftar Pustaka Amedi, A. M. R., & Mustafa, A. S. (2020). Board Characteristics and Firm Performance: Evidence from
Manufacture
Sector
of
Jordan. Accounting
Analysis
Journal,
9(3),
146–151. https://doi.org/10.15294/aaj.v9i3.39577 p
g
j
Ande, D. F. (2018). PT. HM. Sampoerna Tbk and Its Strategies Becoming a Market Leader Since 2006. Jurnal
Ekonomika Dan Manajemen, 7(1), 121. Bambang, M., & Hermawan, M. S. (2013). Founding Family Ownership and Firm Performance in Consumer
Goods
Industry:
Evidence
from
Indonesia. SSRN
Electronic
Journal,
4(2),
112–131. https://doi.org/10.2139/ssrn.2292375 p
g
Bas, T., Muradoglu, G., & Phylaktis, K. (2009). Determinants of Capital Structure in Developing Countries. Business, February 2016, 1–38. Blundell, R., Griffith, R., & Van Reenen, J. (1999). Market share, market value and innovation in a panel of
British manufacturing firms. Review of Economic Studies, 66(3), 529–554. https://doi.org/10.1111/1467-
937X.00097 Bolton, P., & Scharfstein, D. S. (1990). A theory of predation based on agency problems in financial contracting. The American Economic Review, 80(1), 93–106. http://www.jstor.org/stable/10.2307/2006736 Boubaker, S., Saffar, W., & Sassi, S. (2017). Product Market Competition and Debt Choice. Journal Of
Corporate Finance, 2017, 1–28. Buzzell, R. D., Gale, B. T., & Sultan, R. G. M. (1975). Market Share - A Key to Profitibility. Harvard Business
Review, 53(1), 1–9. Chen, J., Jiang, C., & Lin, Y. (2014). What determine firms’ capital structure in China? Managerial Finance,
40(10), 1024–1039. https://doi.org/10.1108/MF-06-2013-0163 Chen, Y., & Hammes, K. (2003). Capital structure. Theories and Empirical Result - A Panel Data Analysis. Journal of Economic Perspectives, 15(2), 81–102. https://doi.org/10.1257/jep.15.2.81 Das, C. P., & Swain, R. K. (2018). Influence of Capital Structure on Financial Performance. Parikalpana, KIIT
Journal of Management, 14(1), 161–171. es, S. W., & Geroski, P. A. (1997). Changes in concentration, turbulence, and the dynamics of mark
shares. Review of Economics and Statistics, 79(3), 383–389. https://doi.org/10.1162/003465300556977 Davies, S. W., & Geroski, P. A. (1997). Changes in concentration, turbulence, and the dynamics of market
shares. Review of Economics and Statistics, 79(3), 383–389. https://doi.org/10.1162/003465300556977
Edeling, A., & Himme, A. (2018). When does market share matter? New empirical generalizations from a meta-
analysis of the market share-performance relationship. Journal of Marketing, 82(3), 1–24. ( )
p
g
Edeling, A., & Himme, A. (2018). When does market share matter? New empirical generalizations from a meta-
analysis of the market share-performance relationship. Journal of Marketing, 82(3), 1–24. 388 Volume 8, No 2 (380-391), 2021 https://doi.org/10.1509/jm.16.0250 Ferrier, W. J., Smith, K. G., & Grimm, C. M. (1999). Daftar Pustaka The role of competitive action in market share erosion and
industry dethronement: A study of industry leaders and challengers. Academy of Management Journal,
42(4), 372–388. https://doi.org/10.2307/257009 ( )
p
g
Frésard, L. (2008). Financial Strength and Product Market Behaviors : The Real Effects of Corporate Cash
Holdings. May, 1–46. g
y
Ghozali, I. (2009). Analisis Multivariate Lanjutan dengan Program SPSS. Badan Penerbit Universitas
Diponegoro. Gobenvy, O. (2014). Pengaruh Profitabilitas, Financial Leverage dan Ukuran Perusahaan Terhadap Financial
Distress pada Perusahaan Manufaktur yang Terdaftar di Bursa Efek Indonesia Tahun 2009-2011. Jurnal
Akuntansi Universitas Negeri Padang, 2(1), 1–24. ( )
Gopalakrishnan, M., Libby, T., Samuels, J. A., & Swenson, D. (2015). The effect of cost goal specificity and new
product development process on cost reduction performance. Accounting, Organizations and Society, 42,
1–11. https://doi.org/10.1016/j.aos.2015.01.003 p
g
j
Goyal, K., & Kumar, S. (2021). Financial literacy: A systematic review and bibliometric analysis. International
Journal of Consumer Studies, 45(1), 80–105. https://doi.org/10.1111/ijcs.12605 Guney, Y., Li, L., & Fairchild, R. (2011). The relationship between product market competition and capital
structure in Chinese listed firms. International Review of Financial Analysis, 20(1), 41–51. https://doi.org/10.1016/j.irfa.2010.10.003 p
g
j
Harris, M., & Raviv, A. (1990). Capital Structure and the Informational Role of Debt. The Journal of Finance,
45(2), 321–349. https://doi.org/10.1111/j.1540-6261.1990.tb03693.x Hendra, S. T. N., & Hartomo, D. D. (2018). Pengaruh Konsentrasi Dan Pangsa Pasar Terhadap Pengambilan
Resiko Bank. Jurnal Bisnis Dan Manajemen, 17(2), 35. https://doi.org/10.20961/jbm.v17i2.17176 Ibhagui, O., & Okoloyo, F. O. (2018). Leverage and Firm Performance: New Evidence on The Role of Firm Size. The
North
American
Journal
of
Economics
and
Finance,
40(2),
366–374. https://doi.org/doi.org/10.1016/j.najef.2018.02.002 Ibrahim, M. (2017). Capital Structure and Firm Value in Nigerian Listed Manufacturing Companies: an Empirical
Investigation Using Tobin’s Q Model. International Journal of Innovative Research in Social Sciences &
Strategic
Management
Techniques,
4(2),
112–125. http://www.internationalpolicybrief.org/images/2017/SEPT-JOURNALS/IRSSSMT/ARTICLE9.pdf g
g
q
( )
p://www.internationalpolicybrief.org/images/2017/SEPT-JOURNALS/IRSSSMT/ARTICLE9.pdf Jensen, M. C., & Smith, Jr., C. W. (2005). Stockholder, Manager, and Creditor Interests: Applications of Agency
Theory. SSRN Electronic Journal, July. https://doi.org/10.2139/ssrn.173461 Jensen, M., & Meckling, W. (1976). Theory of the firm: Managerial behavior, agency costs, and ownership
structure. The
Economic
Nature
of
the
Firm:
A
Reader,
Third
Edition,
283–303. https://doi.org/10.1017/CBO9780511817410.023 p
g
Kale, J. R., Noe, T. H., & Ramirez, G. G. (1991). The Effect of Business Risk on Corporate Capital Structure:
Theory and Evidence. The Journal of Finance, 46(5), 1693. https://doi.org/10.2307/2328569 Kalhor, C., & Rooker, H. (2017). Daftar Pustaka The relationship between capital structure and product market competition in
US listed company [LUND University]. In International Review of Financial Analysis (Vol. 20, Issue 1). https://doi.org/10.1016/j.irfa.2010.10.003 g
j
Kartikasari, D., & Merianti, M. (2016). The effect of leverage and firm size to profitability of public manu
companies in Indonesia. International Journal of Economics and Financial Issues, 6(2), 409–413. Kayo, E. K., Brunaldi, E. O., & Aldrighi, D. M. (2018). Capital Structure Adjustment in Brazilian Family Firms. Revista
de
Administração
Contemporânea,
22(1),
92–114. https://doi.org/10.1590/1982-
7849rac2018170004 Khairov, B. G., Khairova, S. M., Biryukov, V. V., & Romanenko, E. V. (2016). Optimization of financial flows of
the enterprise based on logistical approach. Indian Journal of Science and Technology, 9(14). https://doi.org/10.17485/ijst/2016/v9i14/91513 Kotler, P. (2004). Marketing Insights From A to Z. Erlangga. Kotler, P. (2005). Manajemen Pemasaran. jilid 1. (11th ed.). Intan Sejati Klaten. (
)
j
j
(
)
j
Kotler, P., & Keller, K. L. (2016). Marketing Management (5th ed.). Pearson Education Limitted. (
)
g
g
(
)
Kuan, T. H., Li, C. S., & Liu, C. C. (2012). Corporate governance and cash holdings: A quantile regression
approach. International Review of Economics and Finance, 24(February 2019), 303–314. https://doi.org/10.1016/j.iref.2012.04.006 g
j
apow, L. S. (2018). The Influence of Managerial Ownership and Firm Size On Debt Policy. Internation Lumapow, L. S. (2018). The Influence of Managerial Ownership and Firm Size On Debt Policy. International 389 Jurnal Bisnis dan Manajemen
http://jurnal.unmer.ac.id/index.php/jbm
Volume 8 No 2
2021
Hlm. 380 - 391 Volume 8 No 2
2021
Hlm. 380 - 391 Volume 8 No 2
2021
Hlm. 380 - 391 Journal
of
Applied
Business
and
International
Management,
3(1),
47–55. https://doi.org/10.32535/ijabim.v3i1.76 Lyandres, E. (2006). Capital structure and interaction among firms in output markets: Theory and evidence. Journal of Business, 79(5), 2381–2421. https://doi.org/10.1086/505239 Michael Adiwijaya. (2007). Analisa Strategi Reposisi Merek Dalam Persaingan Pasar. Jurnal Manajemen
Pemasaran, 2(2). http://puslit2.petra.ac.id/ejournal/index.php/mar/article/view/17004 Mitani, H. (2014). Capital structure and competitive position in product market. International Review of
Economics and Finance, 29, 358–371. https://doi.org/10.1016/j.iref.2013.06.009 Moeinaddin, M., Nayebzadeh, S., & Ghasemi, M. (2013). The Relationship between Product Market Competition
and Capital Structure of the Selected Industries of the Tehran Stock Exchange. International Journal of
Academic Research in Accounting, Finance and Management Sciences, 3(3), 221–233. https://doi.org/10.6007/ijarafms/v3-i3/132 Mu, C., Wang, A., & Yang, J. (2017). Optimal capital structure with moral hazard. International R
Economics and Finance, 48, 326–338. https://doi.org/10.1016/j.iref.2016.12.006 Mukhibad, H., Subowo, S., Maharin, D. Daftar Pustaka O., & Mukhtar, S. (2020). Determinants of debt policy for public
companies in Indonesia. Journal of Asian Finance, Economics and Business, 7(6), 29–37. https://doi.org/10.13106/JAFEB.2020.VOL7.NO6.029 p
g
Naomi, P., Ekaputra, I. A., & Wibowo, B. (2018). The role of Industrial factors on optimal capital structure proxy:
An empirical study on Indonesia manufacture companies. Serbian Journal of Management, 13(1), 105–
113. https://doi.org/10.5937/sjm13-16216 p
g
j
Niu, X. (2009). Theoretical and Practical Review of Capital Structure and its Determinants. International Journal
of Business and Management, 3(3), 133–139. https://doi.org/10.5539/ijbm.v3n3p133 Panda, B., & Leepsa, N. M. (2017). Agency theory: Review of theory and evidence on problems and
perspectives. Indian
Journal
of
Corporate
Governance,
10(1),
74–95. https://doi.org/10.1177/0974686217701467 g
Porter, M. E. (1998). Competitive Strategy. Techniques Fors Analyzing Industries and Competitors (Vol. 2, Issue
1). The Free Press. https://doi.org/10.1002/smj.4250020110 )
p
g
j
Ross, S. A. (1977). Determination of Financial Structure: the Incentive-Signalling Approach. Bell J Econ, 8(1),
23–40. https://doi.org/10.2307/3003485 p
g
Ross, S., Westerfield, R. W., & Jordan, B. D. (2009). Pengantar Keuangan Perusahaan. Buku 1. Terjemahan
Oleh Ali Akbar Yulianto. Salemba Empat. p
Rushpaka, B. N. V. (2020). Pengaruh Struktur Modal Terhadap Daya Saing Pasar Produk. Jurnal Ilmiah Bisnis
Dan Ekonomi Asia, 14(1), 65–73. https://doi.org/10.32812/jibeka.v14i1.157 Santos, A., Rindra, E., Hidayat, A. A., & Adelina, Y. E. (2020). Dividend and leverage in Indonesian
intergenerational
family
firms. Jurnal
Siasat
Bisnis,
24(1),
43–58. https://doi.org/10.20885/jsb.vol24.iss1.art4 Santoso, A. (2018). Pengaruh Profitabilitas, Ukuran Perusahaan dan Tingkat Pertumbuhan terhadap Nilai
Perusahaan Manufaktur di Indonesia dengan Struktur Modal sebagai Variabel Moderating. Petra Business
& Management Review, 4(1), 130–141. http://publication.petra.ac.id/index.php/breview/article/view/9160 Sarkar, S. (2018). Optimal DOL (degree of operating leverage) with investment and production flexibility. International Journal of Production Economics, 202, 172–181. https://doi.org/10.1016/j.ijpe.2018.05.022 Sarkar, S. (2018). Optimal DOL (degree of operating leverage) with investment and production flexibility. International Journal of Production Economics, 202, 172–181. https://doi.org/10.1016/j.ijpe.2018.05.022
Schorr, A., & Lips, M. (2019). The optimal capital structure of Swiss dairy farms. Agricultural Finance Review,
79(3) 323–337 https://doi org/10 1108/AFR-05-2018-0034 p
g
j jp
Schorr, A., & Lips, M. (2019). The optimal capital structure of Swiss dairy farms. Agricultural Finance
79(3), 323–337. https://doi.org/10.1108/AFR-05-2018-0034 Shaferi, I., Wahyudi, S., Mawardi, W., Hidayat, R., & Puspitasari, I. (2020). The manufacture and service
companies differ leverage impact to financial performance. International Journal of Financial Research,
11(2), 281–286. https://doi.org/10.5430/ijfr.v11n2p281 ( )
p
g
j
p
Singh, M., & Faircloth, S. (2005). The impact of corporate debt on long term investment and firm performance. Daftar Pustaka Applied Economics, 37(8), 875–883. https://doi.org/10.1080/00036840500076762 ( )
p
g
j
p
Singh, M., & Faircloth, S. (2005). The impact of corporate debt on long term investment and firm performance. Applied Economics, 37(8), 875–883. https://doi.org/10.1080/00036840500076762 Soboleva, Y. P., & Parshutina, I. G. (2016). Management of investment attractiveness of the region by improving
company
strategic
planning. Indian
Journal
of
Science
and
Technology,
9(14). https://doi.org/10.17485/ijst/2016/v9i14/91522 Soboleva, Y. P., & Parshutina, I. G. (2016). Management of investment attractiveness of the region by improving
company
strategic
planning. Indian
Journal
of
Science
and
Technology,
9(14). https://doi.org/10.17485/ijst/2016/v9i14/91522 390 Volume 8, No 2 (380-391), 2021 Volume 8, No 2 (380-391), 2021 Volume 8, No 2 (380-391), 2021 Sudana, I. M. (2009). Manajemen Keuangan. Teori Dan Praktik. Airlangga University Press. Sudana, I. M. (2011). Manajemen Keuangan Perusahaan. Teori&Praktik. Penerbit Erlangga. (
)
j
g
gg
Sumarwan, U., Fachrodji, A., Nursal, A., Nugroho, A., Nurzal, E. R., Setiadi, I. A., Suharyono, & Alamsyah, Z. (2010). Pemasaran Strategik. Perspektif Value-Based Marketing&Pengukuran Kinerja. IPB Press. Sundas, S. (2019). Firm Size And Financial-Leverage Choice Evidence From An Emerging Economy. International Journal of Commerce and Finance, 5(1), 25–33. ( )
Utami, E. S. (2019). Analysis Capital Structure on Indonesia Stock Exchange. 100(Icoi), 686–690. https://doi.org/10.2991/icoi-19.2019.120 Uzliawati, L., Yuliana, A., Januarsi, Y., & Santoso, M. I. (2018). Optimisation of capital structure and firm v
European Research Studies Journal, 21(2), 705–713. https://doi.org/10.35808/ersj/1034 Varadarajan, P., & Dillon, W. R. (1981). Competitive position effects and market share: An exploratory
investigation. Journal of Business Research, 9(1), 49–64. https://doi.org/10.1016/0148-2963(81)90031-X Wati, M., Wijayanti, A., & Siddi, P. (2020). Faktor-Faktor Yang Mempengaruhi Struktur Modal. Accounting Global
Journal, 4(1), 97–113. https://doi.org/10.24176/agj.v4i1.4318 ( )
g
gj
Yerdavletova, F., Ermekbaeva, B., Zhunissova, G., & Mukhametzhanova, Z. (2020). Overview of Actions to
Strengthen Financial Stability of Companies. E3S Web of Conferences 159, 04016. Zingales, L. (1998). Survival of the fittest or the fattest? Exit and financing in the trucking industry. In Journal of
Finance (Vol. 53, Issue 3, pp. 905–938). https://doi.org/10.1111/0022-1082.00039 391
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Purification and characterization of a thermostable cyclodextrin glycosyltransferase from Thermoanaerobacter sp. P4
|
African journal of biotechnology
| 2,012
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cc-by
| 5,872
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African Journal of Biotechnology Vol.11(45), pp. 10407-10415, 5 June, 2012
Available online at http://www.academicjournals.org/AJB
DOI:10.5897/AJB11.3513
ISSN 1684-5315 ©2012 Academic Journals African Journal of Biotechnology Vol.11(45), pp. 10407-10415, 5 June, 2012
Available online at http://www.academicjournals.org/AJB
DOI:10.5897/AJB11.3513
ISSN 1684-5315 ©2012 Academic Journals African Journal of Biotechnology Vol.11(45), pp. 10407-10415, 5 June, 2012
Available online at http://www.academicjournals.org/AJB
DOI:10.5897/AJB11.3513
ISSN 1684-5315 ©2012 Academic Journals Ayse Avci1* and Sedat Dönmez2 1Department of Food Engineering, Faculty of Engineering, Sakarya University, 54040, Sakarya, Turkey. 2Department of Food Engineering, Faculty of Engineering, Ankara University, 06110 Dışkapı, Ankara, Turkey. Accepted 23 May, 2012 Accepted 23 May, 2012 Cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) from a thermophilic anaerobic bacterium,
Thermoanaerobacter sp. P4, was purified by ammonium sulfate precipitation followed by α-cyclodextrin
epoxy activated-sepharose 6B column chromatography. Enzyme was purified 141 fold and had the
specific activity of 143.8 U/mg proteins. Purification yields after ammonium sulfate precipitation and
affinity chromatography were 25.8 and 17.8%, respectively. SDS-PAGE analysis showed that enzyme
was purified successfully and had a single band. Molecular weight of the enzyme was determined as
68.7 kDa. The enzyme had optimum cyclization activity at 80 to 90°C and hydrolyzing activity at 90°C
and maintained 87 and 95% of these activities at 95°C, respectively. Optimal pH was found as 7.0. It
retained full activity at 80°C for 4 h. Enzyme was strongly inhibited by HgSO4 and AgNO3. Addition of 1
mM CaCl2 increased the enzymatic activity up to 7%. This novel enzyme could be a good candidate for
industrial applications according to its characteristic found in the current study. Key words: Cyclodextrin glycosyltransferase, cyclodextrin production, Thermoanaerobacter sp. P4,
thermophilic, enzyme purification, enzyme characterization. *Corresponding author. E-mail: Ayse.Avci@ars.usda.gov. Tel:
1-309-681-6029. Fax: 1-309-681-6427. Key words: Cyclodextrin glycosyltransferase, cyclodextrin production, Thermoanaerobacter sp. P4,
thermophilic, enzyme purification, enzyme characterization. ull Length Research Paper
Purification and characterization of a thermostable
cyclodextrin glycosyltransferase from
Thermoanaerobacter sp. P4 Full Length Research Paper Ammonium sulfate precipitation and dialysis Solid ammonium sulfate was slowly added to the crude enzyme
solution until 85% of final concentration with continuous agitation for
8 h at 4°C and it was allowed to stand one night at 4°C to improve
the precipitation efficiency. The precipitate was collected by
centrifugation at 12000 x g for 15 min. It was dissolved in 10 mM
Na acetate buffer pH 5.5 and dialyzed against the same buffer by
using 10000 MWCO dialysis tube (Sigma, USA). Interests in the usage of thermophilic enzymes are
increasing due to their various advantages over
mesophilic enzymes (Haki and Rakshit, 2003). Working
with thermophilic enzymes at high temperatures reduces
risk of contamination, improves reaction rate, lowers
viscosity and increases solubility of substrates (Biwer et
al., 2002). Researches are focused on the improvement
of activity, selectivity, stability of these enzyme by site
directed
mutagenesis,
chemical
modification
and
immobilization of these enzymes as well as isolation and
characterization of new enzymes (Alcalde et al., 2001;
Haki and Rakshit, 2003; Cowan and Lafuente, 2011). Enzyme assay Cyclization activity of CGTase was determined by the phenol-
phthalein method as described by Shim et al. (2004). The method is
based on the decrease in colour intense due to inclusion complex
formation of β-CD with phenolphthalein (Alcalde et al., 1999). 2%
(w/v) soluble starch was used as substrate in 50 mM sodium
acetate buffer pH 6.0. 0.1 ml of enzyme and 1 ml of soluble starch
solution were incubated at 80°C for 10 min. The concentration of β-
CD was determined by measuring the decrease in absorbance at
550 nm using a standard curve. One unit of enzyme activity was
defined as the amount of enzyme producing 1 µmol of β-CD per
minute. Unless otherwise indicated, enzyme activity or CGTase
activity refers to cyclization activity in the text. INTRODUCTION pneumoniae AS-22 (Gawande and Patkar, 2001) and
Brevibacterium sp 9605 (Mori et al., 1994). In general, B. macerans is used in the commercial productions (Biwer
et al., 2002). However industrial production of CDs
require higher temperatures for the liquefaction of starch
at which CGTases from mesophilic bacteria are not
stable. It is performed by using thermostable α-amylases
or by jet cooking before the addition of CGTases (Biwer
et al., 2002; Jorgensen et al., 1997; Wind et al., 1995). CGTase production CGTase was produced under anaerobic conditions in 1 l serum
bottles containing 750 ml medium. Medium contained soluble
potato starch, 30 g/l; yeast extract, 5 g/L; casein, 5 g/l;
MgSO4.7H2O, 1 g/l, K2HPO4 1.5 g/l with an initial pH of 7.5 to 8.0. After 30 h of growth at 65°C, bacterial cells were removed from the
growth medium by centrifugation at 12000 x g for 15 min. Cell-free
supernatant was used as the crude enzyme. Some
extremophilic
CGTases
from
thermophilic
anaerobic bacteria Thermoanaerobacter sp (Norman and
Jorgensen, 1992) and Thermoanaerobacterium EM1
(Wind et al., 1995), archea Thermococcus sp. (Tachibana
et al., 1999), Thermocoocus kodakaraensis (Rashid et
al., 2002) and Pyrococcus furiosus (Lee et al., 2007)
were isolated and characterized in the last two decades. These enzymes have optimal activities between 80 and
100°C and they also have higher hydrolyzing activities
than the CGTases of mesophilic bacteria. Therefore
production of CDs can be realized in one step with a
single enzyme (Alcalde et al., 1999). MATERIALS AND METHODS CD standards were purchased from Sigma (USA) and soluble
potato starch, yeast extract, peptone, ammonium sulfate and
sodium dodecyl-sulphate were from Merck (Germany). Sepharose
6B was purchased from Amersham Biosciences (GE Healtcare,
Sweden). Molecular weight kit for electrophoresis was purchased
from Biorad (USA). 3,5-Dinitrosalicylic acid (DNS) was purchased
from Sigma. Hydrolytic activity was assayed by incubating 0.1 mL of enzyme
solution with 0.3% (w/v) soluble starch in 50 mM sodium acetate
buffer pH 6.0 for 20 min at 80°C. Then, the increase in reducing
sugar content was measured by DNS method (Forouchi and Gunn,
1983). One unit of activity was defined as the amount of enzyme
releasing 1 µmol of glucose per min under assay conditions. Affinity chromatography Freeze dried epoxy-activated Sepharose 6B was used in the
column. Affinity matrix was prepared using the method described by
Sian et al. (2005). The gel was loaded into a 15 × 200 mm column
(Pharmacia). Dialyzed enzyme solution was loaded onto column,
and then it was flushed with 10 mM sodium acetate buffer pH 5.5
for 5 h at a flow rate of 36 ml/h. Elution of the bound CGTase was
accomplished by using same buffer containing 1% α-CD. 3 ml
fractions were collected by the help of the fraction collector
(Spectra/Chrom CF-1) and each fraction was analyzed for CGTase
activity and protein content. Although there are number of mesophilic CGTase
producing strains characterized, CGTases from thermo-
philic bacteria are restricted. Thermoanaerobacter sp. P4
is thermophilic anaerobic bacterium which produces
CGTase. It was identified and CGTase production
conditions were determined in our previous study (Avci
and Donmez, 2009). In this work, purification results and
some
properties
of
the
purified
CGTase
from
Thermoanaerobacter sp. P4 are demonstrated. INTRODUCTION Cyclodextrin glycosyltransferase (CGTase, EC2.4.1.19)
is a starch converting extracellular enzyme which belongs
to α-amylase family (family 13) of glycoside hydrolases
(Bertoldo and Atranikian, 2002; van der Maarel et al.,
2002). It is a unique enzyme capable of producing cyclo-
dextrins (CDs) through intramolecular transglycosylation
reaction called cyclization (Ibrahim et al., 2005; Tonkova,
1998). Beside the cyclization reaction, this enzyme also
catalyzes intermolecular transglycosylation reactions,
namely, coupling reaction (opening of the CD ring and
transferring the resulting linear oligosaccharides to
acceptor molecules) and disproportionation reaction
(transfer
of
linear
oligosaccharides
to
acceptors)
(Nakamura et al., 1994). CGTase also possesses weak
hydrolyzing activity (Alcalde et al., 2003). CDs are cyclic oligosaccharides consisting of mainly six,
seven and eight glucose units named as α, β and γ-CD,
respectively, linked via α(1-4) glucosidic bonds (Biwer et
al., 2002; van der Ween et al., 2000). Due to their
hydrophobic cavity and hydrophilic outer surface, they
are able to form inclusion (host-guest) complexes with a
variety of organic and inorganic molecules, thus changing
the chemical and physical properties of the guest
molecules (Del Valle, 2004; Singh et al., 2002). Inclusion
complex formation makes CDs attractive for the use in
wide industrial applications including food, pharmacy,
agriculture, cosmetics, analytical chemistry (Brewster and
Loftsson, 2007; Del Valle, 2004; Loftsson and Duchene,
2007; Song et al., 2009; Szente and Szejtli, 2004). CGTases are produced by various bacteria, mainly by
mesophilic bacteria such as Bacillus macerans (Stavn
and Granum, 1979), Bacillus circulans (Szerman et al.,
2007), Bacillus firmus (Saverage et al., 2008), Bacillus
agaradhaerens LS-3C (Martins and Hatti Kaul, 2003),
Paenibacillus pabuli US132 (Jemli et al., 2007) Klebsiella Afr. J. Biotechnol. 10408 pneumoniae AS-22 (Gawande and Patkar, 2001) and
Brevibacterium sp 9605 (Mori et al., 1994). In general, B. macerans is used in the commercial productions (Biwer
et al., 2002). However industrial production of CDs
require higher temperatures for the liquefaction of starch
at which CGTases from mesophilic bacteria are not
stable. It is performed by using thermostable α-amylases
or by jet cooking before the addition of CGTases (Biwer
et al., 2002; Jorgensen et al., 1997; Wind et al., 1995). (Avci and Donmez, 2009). DSMZ medium no 144 was used for the
growth of bacteria (www.dsmz.de/mediumlist). The medium was
prepared under anaerobic conditions and dispensed to Hungate
tubes or serum bottles with aluminum flacon under nitrogen
atmosphere. Growth of the culture was carried out at 65°C for 24 h. Kinetic parameters Protein content was determined according to Lowry et al. (1951)
using Bovine serum albumin (Sigma, USA) as standard. Km and Vmax values of the enzyme were determined by incubating
0.35 µg of purified CGTase in 1 ml 50 mM sodium acetate buffer at
pH 6.0 containing various concentrations of soluble starch, ranging
from 0.4 to 20 mg/ml at 80°C for 10 min. The Km and Vmax of the
enzyme were determined from Lineweaver-Burk plot. Purification of CGTase The crude enzyme having a specific activity of 1.02 U/mg
was purified in two steps including ammonium sulfate
precipitation and affinity chromatography. Purification
results of the enzyme are given in Table 1. After the
purification with α-CD bound–epoxy activated Sepharose
6B affinity chromatography, the CGTase was purified 141
fold with a yield of 17.8%. The specific activity of the
purified enzyme was found as 143.8 U/mg. Other than
cyclization, hydrolysis and coupling activities of the
enzyme were determined and they are given in Table 2 in
comparison with some reported CGTases. Optimum temperature and pH of the enzyme Optimum pH for the activity of purified CGTase was determined by
measuring the cyclization and hydrolyzing activities using soluble
starch with designated buffers of sodium acetate buffer (50 mM, pH
4.0 to 6.0), tris HCl buffer (100 mM, pH 7.0 to 8.0) and glycine-
NaOH buffer (100 mM, pH 9.0 to 11.0). Incubations were carried
out at 80°C for 10 min by using 0.1 ml enzyme. Optimum
temperature was determined by measuring the hydrolyzing and
cyclization activities at temperatures between 40 to 95°C in the
presence of soluble starch dissolved in 50 mM sodium acetate
buffer at pH 6.0 and 0.1 ml of enzyme. Single band was obtained on SDS-PAGE which
confirms the purity of the enzyme. Molecular weight of
the enzyme was estimated as 68.7 kDa on SDS-PAGE
gel (Figure 1). Effects of metals on the activity as donors and methyl α-D-glucopyranoside (10 mM) as acceptor
(Alcalde et al., 1999). The incubation was carried out at 80°C for 10
min. Then, amyloglucosidase (from Aspergillus niger, Sigma) was
used to convert the linear oligosaccharides formed during the
reaction to glucose units at 37°C for 30 min. The amount of glucose
formed was determined by DNS method (Forouchi and Gunn,
1983). One unit of enzyme activity was defined as the amount of
enzyme converting 1 µmol of CD per minute under the assay
conditions. Effects of metal ions on cyclization activity were investigated by
incubating 0.1 ml purified enzyme with 0.2 ml of 50 mM sodium
acetate buffer pH 6.0 containing 1 and 10 mM of AlCl3.6H2O
AgNO3,
CoCl3,
CuSO4.5H2O,
MgSO4.7H2O,
MnCl.4H2O,
FeCl2.4H2O, BaCl2.2H2O, KCl, ZnSO4.7H2O, and HgSO4, CaCl2. After the incubation for 30 min at 25°C, the residual enzyme activity
for cyclization was measured. SDS-PAGE analysis Molecular weight of CGTase was estimated by sodium dodecyl-
sulphate
polyacrylamide
gel
(SDS-PAGE)
electrophoresis
according to Laemmli (1970) on a vertical slab gel containing 10%
polyacrylamide gel using Mini Protean electrophoresis apparatus
(Biorad
Laboratories). Standard
molecular
weight
markers
(Fermantas) ranging from 25 to 200 kDa were used for the esti-
mation of molecular weight. The gel was stained with Coomassie
Brilliant Blue R-20 (Merck, Germany). Molecular weight was
calculated by using KODAK Molecular Imaging Software V.4.5.1
(Japan). Bacterial strain Thermophilic anaerobic bacterium was isolated from an oil field
(Raman/Turkey) and identified as Thermoanaerobacter sp. P4
according to 16S rRNA studies and biochemical characteristics Coupling activity was performed according to the method
described by Nakamura et al. (1993) using α-, and β-CD (2.5 mM) 10409 Avci and Dönmez 1 Table 1. Summary of the purification results of CGTase from Thermoanaerobacter sp.P4. Purification step
Volume (ml)
Total protein
(mg)
Total activity
(U)
Specific activity
(U/mg)
Yield (%)
Purification
fold
Supernatant
1000
168
172
1.02
100
1
Ammonium sulfate precipitate
15
2.04
44.5
21.8
25.8
21.37
Affinity chromatography
156
0.226
32.5
143.8
17.75
141 Effects of metals on the activity Effect of pH and temperature on CGTase activity Determination of molecular weight of purified CGTase on SDS-
PAGE. 1,Molecular weight standard; 2, purified CGTase. (2)
(1)
68.7 kDa
30 kDa
40 kDa
50 kDa
60 kDa
70 kDa
85 kDa
100 kDa
120 kDa
150 kDa
200 kDa
Figure 1. Determination of molecular weight of purified CGTase on SDS-
PAGE. 1,Molecular weight standard; 2, purified CGTase. (1) Figure 1. Determination of molecular weight of purified CGTase on SDS-
PAGE. 1,Molecular weight standard; 2, purified CGTase. were determined as 157.3 and 55.3 U/mg at this pH,
respectively. It retained over 90% of its activities between
pH 6.0 and pH 8.0. There was no complete inhibition at
extreme pH values like pH 4.0 and pH 11.0 at which over
65 and 40% of both activities were retained, respectively
(Figure 2). and hydrolyzing activity at 90°C. The enzyme requires
higher
temperatures
for
hydrolyzing
activity
than
cyclization which increased significantly with increasing
temperatures and it was still high even at 95°C. Effect of pH and temperature on CGTase activity In order to determine pH stability of CGTase, the purified enzyme
was incubated with varying buffers indicated above for 1 h at 80°C
without substrate, and the remaining activity was determined using
the cyclization method mentioned above. Temperature stability was
determined by incubating purified enzyme in 50 mM sodium acetate
buffer (pH 6.0) at 80, 90 and 100°C for 4 h without substrate, and
residual cyclization activity was measured as mentioned in the
enzyme assay section. Optimum pH and temperature of the enzyme was
determined for both hydrolyzing and cyclization activities
(Figures 2 and 3). Enzyme was active at a wide range of
pH values having the maximum at pH 7.0 for both
activities. Specific cyclization and hydrolyzing activities 10410 Afr. J. Biotechnol. 10410 Table 2. Specific enzymatic activities of CGTases from different sources Table 2. Specific enzymatic activities of CGTases from different sources
Strain
Cyclization
(U/mg)
Hydrolyzing
(U/mg)
Coupling (U/mg)
Reference
-CD
-CD
Thermoanaerobacter sp. P4*
157.3
55.3
399
238
This work
T. thermosulfurigenesEM1
86
23
-
74
(Wind et al., 1995)
Thermoanaerobacter sp.501
252
82
270
48
(Alcade et al., 1999)
Klebsiella pneumonia AS-22
523
0
498
-
(Gawande and Patkar 2001)
B. agaradhaerens LS-3C
14.8
7.75
7.37
3.9
(Martins and Hatti-Kaul, 2003)
Activities were determined at 80°C and pH 6.0 with soluble starch. p
y
Strain
Cyclization
(U/mg)
Hydrolyzing
(U/mg)
Coupling (U/mg)
Reference
-CD
-CD
Thermoanaerobacter sp. P4*
157.3
55.3
399
238
This work
T. thermosulfurigenesEM1
86
23
-
74
(Wind et al., 1995)
Thermoanaerobacter sp.501
252
82
270
48
(Alcade et al., 1999)
Klebsiella pneumonia AS-22
523
0
498
-
(Gawande and Patkar 2001)
B. agaradhaerens LS-3C
14.8
7.75
7.37
3.9
(Martins and Hatti-Kaul, 2003)
Activities were determined at 80°C and pH 6.0 with soluble starch. (2)
(1)
68.7 kDa
30 kDa
40 kDa
50 kDa
60 kDa
70 kDa
85 kDa
100 kDa
120 kDa
150 kDa
200 kDa
Figure 1. Determination of molecular weight of purified CGTase on SDS-
PAGE. 1,Molecular weight standard; 2, purified CGTase. (2)
(1)
68.7 kDa
30 kDa
40 kDa
50 kDa
60 kDa
70 kDa
85 kDa
100 kDa
120 kDa
150 kDa
200 kDa
Figure 1. Determination of molecular weight of purified CGTase on SDS-
PAGE. 1,Molecular weight standard; 2, purified CGTase. (2)
(1)
68.7 kDa
30 kDa
40 kDa
50 kDa
60 kDa
70 kDa
85 kDa
100 kDa
120 kDa
150 kDa
200 kDa
Figure 1. DISCUSSION It is known that CGTases catalyze four different reactions
named
as
cyclization,
hydrolyzing,
coupling
and
disproportionation. These activities vary depending on
the source of organisms (Martins and Hatti-Kaul, 2003). In our study, cyclization, hydrolyzing and coupling
activities of the purified enzyme were determined and it
was demonstrated that the enzyme had all the activities
measured (Table 2). Hence, it is a true CGTase. It was
reported that CGTases from thermophilic anaerobic
bacteria
generally
have
higher
hydrolysis
activity
compared with mesophilic strains such as Bacillus and
Klebsiella (Alcalde et al., 1999; Gawande and Patkar,
2001). CGTase from Thermoanaerobacter sp P4 had It is known that CGTases catalyze four different reactions
named
as
cyclization,
hydrolyzing,
coupling
and
disproportionation. These activities vary depending on
the source of organisms (Martins and Hatti-Kaul, 2003). In our study, cyclization, hydrolyzing and coupling
activities of the purified enzyme were determined and it
was demonstrated that the enzyme had all the activities
measured (Table 2). Hence, it is a true CGTase. It was
reported that CGTases from thermophilic anaerobic
bacteria
generally
have
higher
hydrolysis
activity
compared with mesophilic strains such as Bacillus and
Klebsiella (Alcalde et al., 1999; Gawande and Patkar,
2001). CGTase from Thermoanaerobacter sp P4 had Kinetic parameters of the enzyme 80% of its activity at pH levels between 4.0 and 10.0. Lowest stability was observed at pH 11.0, at which 60%
of the activity was retained. The kinetic parameters for the CGTase were determined
using soluble starch at 80°C. The values of Km, Vmax
and kcat were calculated as 1.96 ± 0.36 mg/ml, 161.3 ±
5.4 µmol/min/mg, and 184.7 ± 6.2 s-1, respectively from
Lineweaver-Burk plot (data not shown). Thermal stability of the enzyme was tested by
incubating the enzyme at 80, 90 and 100°C for 4 h and
residual activities were analyzed every 30 min during this
period (Figure 5). Enzyme was fully stable at 80°C and
no loss activity was observed at the end of 4 h of
incubation. When the temperature was increased to
90°C, only 9% of loss in the activity was detected in 1 h. It lost 72% of its activity in 4 h at this temperature. The
half life of the enzyme was 35 min at 100°C. Effects of pH and temperature on CGTase stability (
g
)
The effect of temperature on the cyclization and
hydrolyzing activities is illustrated in Figure 3. At
temperatures below 70°C, both activities were quite low. The higher temperatures promoted both activities and
maximum cyclization activity was obtained at 80 to 90°C Figure 4 shows the stability of CGTase at varying pH
values measured in terms of cyclization activity. Enzyme
was stable within a broad pH range. It maintained over 10411 Avci and Dönmez Figure 2. Effect of pH on the cyclization (▲) and hydrolyzing (□) activities of purified
CGTase from Thermoanaerobacter sp. P4. 100% corresponds to 159.4 U/mg for
cyclization activity and 58.1 U/mg for hydrolyzation activity. Incubations were carried out at
80oC with soluble starch. Figure 2. Effect of pH on the cyclization (▲) and hydrolyzing (□) activities of purified
CGTase from Thermoanaerobacter sp. P4. 100% corresponds to 159.4 U/mg for
cyclization activity and 58.1 U/mg for hydrolyzation activity. Incubations were carried out at
80oC with soluble starch. Figure 3. Effect of temperature on the cyclization (▲) and hydrolyzing (□) activities of purified
CGTase from Thermoanaerobacter sp. P4. Activities were measured at pH 6.0 with soluble
starch as substrate. 100% corresponds to 157.3 U/mg for cyclization activity and 55.3 U/mg for
hydrolyzation activity. Figure 3. Effect of temperature on the cyclization (▲) and hydrolyzing (□) activities of purified
CGTase from Thermoanaerobacter sp. P4. Activities were measured at pH 6.0 with soluble
starch as substrate. 100% corresponds to 157.3 U/mg for cyclization activity and 55.3 U/mg for
hydrolyzation activity. 10412 Afr. J. Biotechnol. Figure 4. Effect of pH on the stability of purified CGTase from Thermoanaerobacter sp. P4 determined in
terms of cyclization activity. The enzyme was incubated at 80oC in buffers having varying pH values for 1 h
without substrate and cyclization assay was performed to determine residual activity. 100% corresponds to
157.3 U/mg specific activity. Figure 4. Effect of pH on the stability of purified CGTase from Thermoanaerobacter sp. P4 determined in
terms of cyclization activity. The enzyme was incubated at 80oC in buffers having varying pH values for 1 h
without substrate and cyclization assay was performed to determine residual activity. 100% corresponds to
157.3 U/mg specific activity. Effects of various metals on CGTase activity Effects of some metals on CGTase activity were tested at
1 and 10 mM final concentrations of these metals, and
the results are given in Table 3. CGTase was almost
stable (over 84%) at 1 mM concentrations of the metals
except for Hg and Ag, with which enzyme was inhibited
41 and 75%, respectively. As the concentration was
increased to 10 mM, complete inhibition by HgSO4 and
AgNO3 was detected. 10413 Avci and Dönmez Figure 5. Effect
of temperature
on the stability
of purified CGTase from
Thermoanaerobacter sp. P4 determined in terms of cyclization activity. The enzyme was
incubated at varying temperatures in sodium acetate buffer pH 6.0. 100 % corresponds to
157.3 U/mg specific activity Figure 5. Effect
of temperature
on the stability
of purified CGTase from
Thermoanaerobacter sp. P4 determined in terms of cyclization activity. The enzyme was
incubated at varying temperatures in sodium acetate buffer pH 6.0. 100 % corresponds to
157.3 U/mg specific activity Figure 5. Effect
of temperature
on the stability
of purified CGTase from
Thermoanaerobacter sp. P4 determined in terms of cyclization activity. The enzyme was
incubated at varying temperatures in sodium acetate buffer pH 6.0. 100 % corresponds to
157.3 U/mg specific activity Table 3. Effects of 1 and 10 mM concentrations of various metals
on the cyclization activity of Thermoanaerobacter sp. P4 CGTase. Table 3. Effects of 1 and 10 mM concentrations of various metals
on the cyclization activity of Thermoanaerobacter sp. P4 CGTase. PAGE (Figure 1), which is in accord with the molecular
weights of the reported CGTases. Even though there are
some exceptions, most of the CGTases have molecular
weights of around 70 kDa. Molecular weights of CGTases
from Thermoanaerobacterium thermosulfurigenes EM1
(Wind et al., 1995), Thermoanaerobacter sp. 53627
(Norman
and
Jorgensen,
1992),
alkalophilic
Anaerobranca gottschalkii (Theimann et al., 2004),
archeae Thermococcus sp B1001 (Tachibana et al.,
1999), Thermococcus kodakarensis (Rashid et al., 2002)
and Pyrococcus furiosus (Lee et al., 2007) were reported
as 68, 75.5, 78, 83, 67 and 65 kDa, respectively. 55.3 U/mg which is as high as the other reported
CGTases from thermophilic bacteria.
Molecular weight was estimated as 68.7 kDa on SDS- REFERENCES Alcalde M, Plo FJ, Andersen C, Martin MT, Pedersen S, Ballesteros A
(1999). Chemical modification of lysine side chains of cyclodextrin
glycosyltransferase to α-amylase specificity. FEBS Lett., 445: 333-
337. Alcalde M, Plou FJ, Martin MT, Valdes I, Mendez E, Ballesteros A
(2001). Succinylation of cyclodextrin glycosyltransferase from
Thermoanaerobacter sp. 501 enhances its transferase activity using
starch as donor. J. Biotechnol., 86:71-80. Alcalde M, Plou FJ, Boada MP, Arellano HG, Valdes I, Mendez E,
Ballesteros A (2003). Chemical modification of carboxylic residues in
a cyclodextrin glucanotransferase and its implication in the
hydrolysis/transglycosylation ratio of the α-amylase family. J. Mol. Catal., 26: 57-67. Avci A, Donmez S (2009). A novel thermophilic anaerobic bacteria
producing cyclodextrin glycosyltransferase. Process Biochem., 44:
36-42. Bertoldo C, Atranikian G (2002). Starch hydrolyzing enzymes from
thermophilic archaea and bacteria. Curr. Opin. Chem. Biol., 6: 151-
160. (
)
The inhibition of the CGTase activity by most of the
metals except CaCl2, which increases the activity and
stability, is known. The inhibitory effect is attributed to the
metal catalyzed oxidation of amino acid residues
essential for the enzyme activity (Jemli et al., 2007;
Martins and Hatti-Kaul, 2003; Sian et al., 2005). CGTase
activity of the enzyme was also enhanced by the addition
of 1 mM and 10 mM concentrations of CaCl2 which led to
7 and 2% increases in the activity, respectively. Effect of
CaCl2 on the activity of CGTase was studied by various
researchers (Rashid et al., 2002; Theimann et al., 2004;
Wind et al., 1995) and it has been reported that
stimulation of CGTase activity in the presence of CaCl2 is
a common phenomenon among CGTases (Martins and
Hatti-Kaul, 2003). Biwer A, Antranikian G, Heinzle E (2002). Enzymatic production of
cyclodextrins. Appl. Microbiol. Biotechnol., 59: 609-617. y
pp
Brewster ME, Loftsson T (2007). Cyclodextrins as pharmaceutical
solubilizers. Adv. Drug. Deliv. Rev., 59: 645-666. Cowan DA, Lafuente RF (2011). Enhancing the functional properties of
thermophilic enzymes by chemical modification and immobilization. Enzyme Microbial. Technol., 49: 326-346. y
Del Valle EMM (2004). Cyclodextrins and their uses: a review. Process
Biochem., 39:1033-1046. Forouchi E, Gunn DJ (1983). Some effects of metal ions on the
estimation of reducing sugars in biological media. Biotechnol. Bioeng., 25: 1905-1911. g
Gawande BN, Patkar AY (2001). Purification and properties of a novel
raw starch degrading-cyclodextrin glycosyltransferase from Klebsiella
pneumoniae AS-22. Enzyme Microbial. Technol., 28:735-743. Haki GD, Rakshit SK (2003). Conclusion resistance of the enzyme to the high temperature
applications. When temperature was raised from 90 to
95°C, thermal inactivation was only 13% (Figure 3). Enzyme
exhibited
hydrolyzing
activity
at
higher
temperatures than that of the cyclization activity, which is
maximal at 90 to 95°C. It seems that hydrolyzing
activities at higher temperatures than cyclization activities
is a common property of CGTases. Similarly, CGTase
from T. thermosulfurigenes EM1 had same characteristic
(Wind et al., 1995). A new CGTase from Thermoanaerobacter sp. P4 was
purified and characterized in terms of molecular weight,
pH, temperature, effect of metals and kinetic parameters. The enzyme shows high activities at 80 to 95°C which is
desirable in the industrial productions of CDs. Another
promising property of the enzyme is the higher
hydrolyzing activities at elevated temperatures. Thus,
considering
the
limited
amounts
of
thermostable
CGTases, this enzyme can be a potential for industrial
application. (
,
)
CGTase from Thermoanaerobacter sp. P4 is highly
thermostable possessing no activity loss at 80°C during
24 h incubation period (data not shown). The thermal
stabilities of the CGTases from other thermophilic
anaerobic bacteria were reported by some researchers. Norman and Jorgensen (1992) found that CGTase from
Thermoanaerobacter sp ATCC 53627 retained 95 to
100% of its activity at 75°C for 60 min. Wind et al. (1995)
tested the thermal stability of T. thermosulfurigenes EM1
in the presence of Paselli SA2, and found 10% loss at
80°C and 35% at 90°C after 5 h. It must be pointed out
that thermal stability was measured without addition of
any substrate into the medium in this study. It was stated
that substrate addition enhances the stability of CGTase
(Martins and Hatti-Kaul, 2003; Theimann et al., 2004;
Wind et al., 1995). Unlike our enzyme that can retain 63
and
35%
of
its
activity
at
90°C
without
any
supplementation like starch and calcium, CGTase from T. thermosulfurigenes EM1 has been reported to lose
completely its activity in 1 h at the same conditions (Wind
et al., 1995). CGTase from Thermoanaerobacter sp. P4
possessed similar thermal characteristics to other
thermostable CGTases except CGTase from hyper-
thermophilic archaeon Thermococcus sp. that has the
highest CGTase activity (100°C) among all the reported
CGTases (Tachibana et al., 1999). ACKNOWLEGMENT This study was supported by the Scientific and
Technological Research Council of Turkey (TUBITAK)
grant no. 106 O 319. Effects of various metals on CGTase activity Compound
Relative activity (%)
1 mM
10 mM
Control
100
100
AlCl.6H2O
89
76
AgNO3
75
0
CoCl3
86
75
CuSO4.5H2O
90
67
MgSO4.7H2O
96
83
MnCl.4H2O
96
67
FeCl2.4H2O
94
64
BaCl2.2H2O
96
78
KCl
95
75
ZnSO4.7H2O
98
75
HgSO4
41
10
CaCl2
107
102
Enzyme was incubated in 50 mM sodium buffer pH 6.0 in the
presence of 1 and 10 mM of the metals at 25oC for 30 min and
residual activities were measured. p
y
The optimum pH and the temperature of the CGTase
were determined in the supernatant of the bacterium and
reported elsewhere (Avci and Dönmez, 2009). In this
study, those from the purified CGTase have been also
determined in order to confirm the properties of the
enzyme before and after purification. Almost the same
results were acquired in both studies. Purified CGTase
displayed an optimum cyclization activity at 80 to 90°C
which is similar to the optimum temperatures of
thermophilic
anaerobic
Thermoanaerobacterium
thermosulfurigenes EM1 (Wind et al., 1995) and
Thermoanaerobacter sp. (Jorgensen et al., 1997). In
general, extracellular enzymes may be stable at
temperatures considerably higher than the growth
temperatures (Cowan and Lafuente, 2011). Activity at
95°C was also high enough as an indication of the Enzyme was incubated in 50 mM sodium buffer pH 6.0 in the
presence of 1 and 10 mM of the metals at 25oC for 30 min and
residual activities were measured. 55.3 U/mg which is as high as the other reported
CGTases from thermophilic bacteria. Molecular weight was estimated as 68.7 kDa on SDS- Afr. J. Biotechnol. 10414 resistance of the enzyme to the high temperature
applications. When temperature was raised from 90 to
95°C, thermal inactivation was only 13% (Figure 3). Enzyme
exhibited
hydrolyzing
activity
at
higher
temperatures than that of the cyclization activity, which is
maximal at 90 to 95°C. It seems that hydrolyzing
activities at higher temperatures than cyclization activities
is a common property of CGTases. Similarly, CGTase
from T. thermosulfurigenes EM1 had same characteristic
(Wind et al., 1995). Avci and Dönmez 10415 Avci and Dönmez 10415 (1997). Cloning
and nucleotide sequence
of
a thermostable
cyclodextrin glycosyltransferase gene from Thermoanaerobacter
sp.ATCC 53627 and its expression in Escherichia coli. Biotechnol. Lett., 19: 1027-1031. Song LX, Bai L, Xu XM, He, J, Pan SZ (2009). Inclusion complexation,
encapsulation interaction and inclusion number in cyclodextrin
chemistry. Coord. Chem. Rev., 253: 1276-1284. y
Stavn A, Granum PE (1979). Purification and physicochemical
properties
of
an
extracellular
cycloamylose
(cyclodextrin)
glucanotransferase from Bacillus macerans. Carbohydr. Res., 75:
243-250. Laemmli UK (1970). Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature, 277: 680-685. Lee MH, Yang SJ, Woo Kim J, Lee HS, Waan Kim J, Park KH (2007). Characterization of a thermostable cyclodextrin glucanotransferase
from Pyrococcus furiosus DSM3638. Extremophiles, 11: 537-541. Szente L, Szejtli J (2004). Cyclodextrins as food ingredients. Food Sci. Technol., 15: 137-142. y
p
Loftsson T, Duchene D (2007). Cyclodextrins and their pharmaceutical
application. Int. J. Pharm., 329(1-2): 1-11. Szerman N, Schroh I, Rossi AL, Rosso AM, Krymkiewicz N, Ferrarotti
SA
(2007). Cyclodextrin
production
by
cyclodextrin
glycosyltransferase from Bacillus circulans DF 9R. Bioresour. Technol., 98: 2886-2891. pp
Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ (1951). Protein
measurement with folin phenol reagent. J. Biol. Chem., 193: 265-275. Martins RF, Hatti-Kaul R (2002). A new cyclodextrin glycosyltransferase
from an alkaliphilic Bacillus agaradhaerens isolate: purification and
characterisation, Enzyme Microb. Technol., 30: 116-124. Tachibana Y, Kuramura A, Shirasaka N, Suzuki Y, Yamamoto T,
Fujiwara S, Takagi M, Imanaka T (1999). Purification and
characterization of an extremely thermostable cyclomaltodextrin
glucanotransferase
from
a
newly
isolated
hyperthermophilic
archaeon, a Thermococcus sp. Appl. Environ. Microbiol., 65: 1991-
1997. y
Martins RF, Hatti-Kaul R (2003). Bacillus agaradhaerens LS-3C
cyclodextrin glycosyltransferase: activity and stability features. Enzyme Microb. Technol., 33: 819-827. Theimann V, Dönges C, Prowe SG, Sterner R, Atranikian G (2004). Characterisation
of
a
thermoalkali-stable
cyclodextrin
glycosyltransferase from the anaerobic thermoalkaliphilic bacterium
Anaerobranca gottschalkii. Arch. Microbiol., 182: 226-235. y
,
Mori S, Hirose S, Oya T, Kitahata S (1994). Purification and properties
of cyclodextrin glucanotransferase from Brevibacterium sp. 9605. Biosci. Biotechnol. Biochem., 56: 207-210. Nakamura A, Haga K, Yamane K (1993). Three histidine residues in the
active center of cyclodextrin glucanotransferase from alkalophilic
Bacillus sp. 1011. Effects of the replacement on pH dependence and
transition-state stabilization. Biochemistry, 32: 6624-6631. Tonkova A (1998). Bacterial cyclodextrin glucanotransferase. Enzyme
Microbial Technol., 22: 678-686. REFERENCES Developments in industrially important
thermostable enzymes: a review. Bioresour. Technol., 89: 17-34. Ibrahim HM, Yusoff WMW, Hamid AA, Illias RM, Hassan O, Omar O
(2005). Optimization of medium for the production of β-cyclodextrin
glucanotransferase using Central Composite Design (CCD). Process
Biochem., 40:753-758. )
The CGTases from various sources exhibit different Km
values for soluble starch ranging from 0.52 to 21.2 mg/ml
(Gawande and Patkar, 2001; Martins and Hatti-Kaul,
2002; Rahman et al., 2006; Stavn and Granum, 1979). Km value of CGTase from Thermoanaerobacter sp. P4
was calculated as 1.95 mg/ml indicating that the enzyme
has high affinity for soluble starch. Jemli S, Messaoud B, Zouari DA, Naili B, Khemakhem B, Bejar SA
(2007). A β-cyclodextrin glycosyltransferase from a newly isolated
Paenibacillus pabuli US132 strain: Purification, properties and
potential use in bread-making. Biochem. Eng. J., 34: 44-50. p
g
g
,
Jorgensen ST, Tangney M, Starnes RL, Amemiya K, Jorgensen PL Avci and Dönmez 10415 van der Maarel MJEC, van de Ween B, Uitdehaag JCM, Leemhuis H,
Dijkhuizen L (2002). Properties and applications of starch-converting
enzymes of the α-amylase family. J. Biotech., 94: 137-155. Nakamura A, Haga K, Yamane K (1994). The transglycosylation
reaction of cyclodextrin glucano transferase is operated by a ping
pong mechanism. FEBS Lett., 337: 66-70. van der Ween BA, Uitdehaag JCM, Dijkstra BW, Dijkhuizen L (2000)
Engineering of cyclodextrin glycosyltransferase reaction and product
specificity. Biochim. Biophys. Acta, 1543: 336-360. p
g
Norman BE, Jorgensen ST (1992). Thermoanaerobacter sp. CGTase its
properties and application. Denpun Kagaku, 39: 101-108. Wind RD, Liebl W, Buitelaar RM, Penningra D, Spreinat A, Dijkhuizen
L, Bahl H (1995). Cyclodextrin formation by the thermostable α-
amylase of Thermoanaerobacterium thermosulfurigenes EM1 and
reclassification of the enzyme as a cyclodextrin glycosyltransferase. Appl. Environ. Microbiol., 61: 1257-1265. Rahman K, Illias RM, Hassan O, Mahmood, NAN, Rashid NAA (2006)
Molecular cloning of a cyclodextrin glucanotransferase gene from
alkolophilic
Bacillus
sp. TS1-1
and
characterization
of
the
recombinant enzyme, Enzyme Microb. Technol., 39: 74-84. Rashid N, Cornista J, Ezaki S, Fukui T, Atomi H, Imanaka T (2002). Characterization of an archaeal cyclodextrin glucanotransferase with
a novel C-terminal domain. J. Bacteriol., 184: 777-784. Saverage LS, Dhule SS, Jogdand VV, Nene SN, Gadre RV (2008). Production
and
single
step
purification
of
cyclodextrin
glycosyltransferase from alkalophilic Bacillus firmus by ion extchange
chromatography. Biochem. Eng. J., 39: 510-515. Shim JH, Kim YW, Kim TJ, Chae HY, Park JH, Cha H, Kim JW, Kim YR,
Schaefer T, Spendler T, Moon TW, Park KH (2004). Improvement of
cyclodextrin glucanotransferase as an antistaling enzyme by error-
prone PCR. Protein Eng. Des. Sel., 17: 205-211. Sian HK, Said M, Hassan O, Kamaruddin K, Ismail AF, Rahman RA,
Mahmood NAN, Illias RM (2005). Purification and characterization of
cyclodextrin glucanotransferase from alkalophilic Bacillus sp. G1. Process Biochem., 40: 1101-1111. Singh M, Sharma R, Banerjee UC (2002). Biotechnological applications
of cyclodextrins. Biotech. Adv., 20: 341-359.
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https://openalex.org/W4386824835
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https://www.researchsquare.com/article/rs-2915312/latest.pdf
|
English
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Investigating the effect of edible pineapple on the ripening of the cervix and the onset of labor.
|
Research Square (Research Square)
| 2,023
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cc-by
| 7,498
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Results According to T-test, two groups were homogeneous in terms of age (p = 0.439). The average Bishop score
was the same at the beginning of the study and according to the analysis of variance test with repeated
values, the changes in the first 48 hours, 72 hours after the start of the study, and hospitalization time
were significantly different between the two groups (p < 0.001). There was a significant difference (p <
0.001) in the time of labor and the amount of oxytocin used in the intervention and control groups. The
two intervention and control groups had no significant differences between the Apgar scores of the first
and fifth minutes of the newborns, the contamination of the amniotic fluid with meconium, the type of
delivery, and the condition of the fetal heart during hospitalization. Research Article Keywords: ripening of cervix, pineapple, delivery, women
Posted Date: September 18th, 2023
DOI: https://doi.org/10.21203/rs.3.rs-2915312/v1
License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Keywords: ripening of cervix, pineapple, delivery, women Additional Declarations: No competing interests reported. Additional Declarations: No competing interests reported. Page 1/15 Page 1/15 Methods In this randomized controlled clinical trial, 66 pregnant women with a gestational age of 40–41 weeks,
referring to the maternity of Omo Albanin Hospital in Mashhad, were selected and randomly divided into
two intervention and control groups, and the Bishop Cervix score at the time of study entry. It is registered. Women in the intervention group consumed a 250-gram packet of edible pineapple every 24 hours for 3
consecutive days. In the control group, routine care was performed. Research units were followed up in
terms of Bishop's Cervix score and labor onset symptoms 48 and 72 hours later and during
hospitalization. Data collection tools were personal and midwifery information questionnaires,
observation and examination forms, daily records, and follow-ups, which were used for content validity
and simultaneous observation to check the tool’s reliability. The data was analyzed using SPSS 18
repeated measures comparison test. A significance level of 0.05 was considered. Background Labor induction in post-term pregnancies is still one of the most common midwifery interventions, and
preparation of the cervix before labor induction is one of the most important reasons for the success of
labor. Therefore, this research was conducted to investigate the effect of edible pineapple on ripening the
cervix and the onset of labor. Background Childbirth is a part of the natural physiological process and it happens spontaneously when the
pregnancy period is completed without complications, and at this time, the cervix is often ready for
childbirth (1, 2). Also, natural childbirth requires many changes and transformations in the function of the
uterus and cervix. Before contractions begin, the cervix must undergo extensive deformation. Changing
the shape of the cervix includes changes in the soft tissue, which is called ripening of the cervix, and is
one of the main steps at the beginning of labor (3). Preparation of the cervix before induction of labor is
necessary for the success of labor and is one of the most common measures in the termination of
pregnancy (4, 5). The researchers of "Safe Labor Consortium" state that elective induction has resulted in vaginal delivery
in 97% of multiparous women and 76% of nulliparous women, but induction of labor has been more
successful if the cervix is ready, and the possibility of cesarean delivery is related to Induction is strongly
influenced by the time spent for induction (especially in cases of the unfavorable cervix) (3). Women undergoing elective induction undergo more interventions such as amniotomy, electronic fetal
monitoring, and epidural anesthesia compared to women who have spontaneous labor (6). Also,
cesarean delivery, Chorioamnionitis, uterine scar rupture, and postpartum hemorrhage due to uterine
atony, uterine tachysystole, and fetal heart rate changes are maternal and fetal complications related to
Liberia induction (3). For this reason, there is an effort to prepare the cervix by various methods before
induction of labor (7). Various medical methods for preparing the cervix include prostaglandin E1 and E2 (in the form of gels,
suppositories, and oral tablets) and mechanical methods including (intracervical Foley catheter with or
without extra amniotic injection of normal saline, cervical hygroscopic dilators) and tearing of the
curtains (3) and other methods include nipple stimulation, acupuncture, sexual intercourse, enema and
medicinal plants (8). Medical and surgical methods have significant side effects such as bleeding during
and after delivery, prolonged delivery, suffering and fetal injuries and uterine rupture(9), and tearing of the
membranes with pain, vaginal bleeding, and irregular contractions without accompanying labor(3). Therefore, finding a method that helps prepare the cervix with fewer complications has always been of
interest. Conclusion Daily consumption of edible pineapple positively affects the progress of labor to prepare the cervix and
induce labor and does not harm the mother and the fetus. It is also suggested to use a larger sample size
and time to start consumption at a gestational age of fewer than 40 weeks and a longer duration of
pineapple consumption in future research. Page 2/15 Background So, with the process of preparation of the cervix,
which includes the change in the shape of the connective tissue, the decrease in the concentration of
collagen and proteoglycan, and the increase in water content compared to the non-pregnant uterus (3), it
can cause the preparation of the cervix for childbirth. Oral bromelain dose-dependently decreases plasma bradykinin and prostaglandin E2 levels and
increases prostacyclin in inflammatory areas (23). Considering that prostaglandin E2 keeps the uterus
relaxed (3), reducing the level of prostaglandin PGE2 can cause uterine contractions. Also, an increase in
prostacyclin causes an increase in collagenase enzyme and muscle relaxation. The effect of bromelain
plant protease by inhibiting the effect of collagen in the process of angiogenesis has also been observed
in the laboratory study of Mushtaq et al(2019) (24). So, with the process of preparation of the cervix,
which includes the change in the shape of the connective tissue, the decrease in the concentration of
collagen and proteoglycan, and the increase in water content compared to the non-pregnant uterus (3), it
can cause the preparation of the cervix for childbirth. Although no research shows the effect of bromelain on the initiation of uterine contractions in humans, in
many cases it is generally recommended for pregnant women to initiate labor, and some researchers also
believe that the fiber and sugar in pineapple are due to intestinal-digestive movements. It affects creating
freedom (2). It may be possible to justify the effectiveness of pineapple on the cervix with the following
mechanisms: Firstly, the main factor in the structural arrangement of the cervix is collagen (3) and on the other hand,
bromelain enzyme increases the hydrolytic activity in the connective tissue, especially on collagen (18). It
has been mentioned that the hydrolysis of collagen by bromelain increases the elasticity and flexibility of
the uterine muscle (18). And since collagen is responsible for the main arrangement of the connective
tissue of the cervix, this effect can also justify the effectiveness of pineapple in the preparation of the
cervix. Secondly, the bromelain enzyme is an external proteolytic enzyme in the cysteine proteinase group,
which is widely used for the regression of connective tissue (25), which may be one of the reasons for the
effect of bromelain enzyme on cervical connective tissue to soften and prepare the cervix, its proteolytic
effect. Background Among these solutions is the use of medicinal plants that are usually used to prepare the cervix, including
castor oil (10, 11), red raspberry leaf (12), primrose oil (13), sodas and (9) and evening primrose oil (14)
and dates (15–17). Pineapple is one of the fruits mentioned in various cultures to start childbirth. Pineapple is a plant from the bromelainase family and is one of the foods that stimulate labor (2). Pineapple (Ananas Comosus) contains phytonutrients, vitamin C, and a compound called bromelain. Bromelain is a proteolytic or protease enzyme that is found only in the pineapple plant and its highest
amount is in the stem and fruit of pineapple (18). Most of the medicinal effects of bromelain are related
to its proteolytic properties (19) and it has various therapeutic properties such as anti-asthma, anti-
inflammatory, fibrinolytic, anti-cancer, antibiotic, anti-thrombotic and anticoagulant (18). Page 3/15 In the traditions of old medicine, pineapple is known to induce abortion in pregnant women and as a pain
reliever, and in other words, because it has a contraction effect on the muscles of the uterus, it can be
used in prolonged pregnancy to prevent medical interventions (20). In the study of Faezeh Manji et al. (2016) at the Gynecology and Obstetrics Center of Singapore, the effect of pineapple extract on the
contractile activity of rat and human uterine myometrium samples was shown (20), and in another study
by Nwankudu et al (2015) the oxytocin effect of pineapple juice on muscle contractions. The uterus of
mice has been investigated and confirmed (21). It is also stated in the study of Padma (2012) that
pineapple bromelain enzyme is effective in uterine contractions by stimulating prostaglandin secretion
(22). Oral bromelain dose-dependently decreases plasma bradykinin and prostaglandin E2 levels and
increases prostacyclin in inflammatory areas (23). Considering that prostaglandin E2 keeps the uterus
relaxed (3), reducing the level of prostaglandin PGE2 can cause uterine contractions. Also, an increase in
prostacyclin causes an increase in collagenase enzyme and muscle relaxation. The effect of bromelain
plant protease by inhibiting the effect of collagen in the process of angiogenesis has also been observed
in the laboratory study of Mushtaq et al(2019) (24). Method In this randomized controlled clinical trial study, 64 pregnant mothers with a gestational age of 40–41
weeks were referred to the maternity ward of Omo Albanin Hospital in Mashhad. The subjects were
selected by the access method and were divided into two groups (intervention, control) by simple random
assignment method. Due to the lack of a completely similar study, the sample size was determined based
on the effect size formula and considering the error of 5% and the test power of 80%, and the average
effect size (0.5) of 30 people in each group. The researcher presented an official letter of introduction from the Mashhad Nursing and Midwifery
College and went to Omo Albanin Hospital in Mashhad City after obtaining approval from the relevant
authorities, she settled in the hospital to collect information and take samples. The criteria for mothers to
enter the study include maternal age of 18–35 years, gestational age of 40–41 weeks based on the first
day of the last menstrual period or ultrasound under the first trimester of pregnancy, singleton fetus with
the cephalic presentation, presence of normal AFI according to the ultrasound of the third trimester of
pregnancy, absence Allergy to pineapple, no known chronic disease, no history of cesarean section or
childbirth complications (hydramnios, oligohydramnios, preeclampsia, eclampsia), Bishap’s score less
than or equal to 4, healthy amnion sac, normal non-stress test, no sexual intercourse from 24 hours
before From the beginning of the study to the end of it, no use of any herbal medicine three days before
and during the study. Background Thirdly, the significant changes that occur in the extracellular matrix during the ripening of the
cervix in the second stage of pregnancy are associated with the invasion of inflammatory cells into the
stroma. In this model, the chemical absorbents present in the cervix attract inflammatory cells to the site,
and these cells also release proteases that may facilitate the breakdown of collagen and other matrix
components (3). Also, by stimulating leukocytes, bromelain increases the level of interleukin 6, 1, 8 and
tumor necrosis factor-alpha in the inflamed area. The increase of cytokines, especially interleukin 8, leads
to the recruitment of granulocytes and monocytes to the inflammatory area and increases the activity of Page 4/15 Page 4/15 phagocytosis and chemotaxis (19). Therefore, according to the above mechanisms, probably pineapple
with bromelain enzyme can prepare the cervix Therefore, considering that the goal of midwifery is to emphasize natural and safe childbirth by
maintaining the health of the mother and fetus and using non-pharmacological approaches, performing
vaginal delivery without medical intervention (induction of labor), and preventing prolonged labor for the
mother and the fetus is vitally important (9). Therefore, the current study was designed and implemented
to determine the effect of pineapple consumption on the readiness of the cervix and the onset of labor. Informed consent was signed by each patient regarding the intervention application, its possible complications, and
outcomes. This study protocol was approved by the Medical ethics committee of Mashhad University of
Medical Sciences with the ethical code IR.MUMS.NURSE.REC.1397.055 and all methods in this study
were carried out in accordance with relevant guidelines and regulations of RCT studies. At the beginning of the study, the researcher identified the research units in three shifts, morning, evening,
or night, among the mothers who went to the midwifery clinic of the hospital without labor symptoms to
determine the time of delivery or pregnancy control, or with a complaint of passing the due date of
delivery, and after describing The purpose and method of the research was included in the study after
obtaining written consent (pregnancy age was calculated according to LMP or based on ultrasound of Page 5/15 the first trimester). The selection form of the research unit was completed through study, interview, taking
a complete history, and viewing ultrasounds and pregnancy control records. First, a preliminary
examination was performed to determine the Bishop score of the cervix, and the research units were
included in the intervention if they had a Bishop score of less than or equal to 4. An equal number of “a”
and “b” cards were placed in a box, and the mothers were assigned to one of the study groups by
removing the “a” (intervention) and b (control) cards. To avoid storing more than 72 hours divided and packaged pineapples, fresh pineapples were purchased
from the fruit shop immediately after the selection of the research unit. Then its skin was separated and
three 250g packs for each mother were packed in disposable containers and delivered to the research
unit. The mother was advised to keep the fruit on the middle shelf of the refrigerator and to consume a
whole pack every 24 hours alternately or all at once according to her wish (for 3 consecutive days). In
addition, both intervention and control groups received routine measures. Both groups were asked to
return to the maternity hospital 48 and 72 hours after the start of the intervention in case of no onset of
labor symptoms, so that a vaginal examination can be performed to determine the Bishop's score, as well
as auscultation of the fetal heart and, if necessary, an echocardiogram of the fetus. Results In this study, 66 pregnant women were examined, 36 in the intervention group and 30 in the control group. In the control group, we had 6 people dropped out of the study. 2 people were excluded from the study
due to the mother's visit to the doctor's office and vaginal examination and the possibility of cervical
stripping, 3 people were excluded from the study because they did not visit and 1 person was excluded
from the study because of having sex. The average age of the intervention and control groups was 26.4 ±
4.47 and 27.3 ± 4.41 respectively. There was no statistically significant difference between the ages of the
two groups (p-value = 0.439). The majority of the participants had primary education (18 people (27.3
percent)) and were housewives (48 people (72.7 percent)). The demographic information of the
participants according to the treatment groups is presented in (Table No. 1). The two groups were similar
in terms of age, education level, occupation of the woman and her husband, and economic status of the
family. The mean gestational age and birth weight of the baby were not significantly different between the two
groups. Also, in terms of the type of pregnancy and the number of pregnancies, the heart rate of the fetus
at the time of admission, no significant difference was observed between the two study groups (Table No. 2). In the intervention group, more cervical dilatation and amnion sac rupture were observed than in the
control group. But more blood secretions were recorded in the control group. There was a significant
difference between the two study groups in terms of labor onset symptoms. In the pineapple consumer
group, only 3 people (8.3 percent) used cervical preparation methods, while in the control group, 23
people (76.7 percent) used these methods, and the two groups had a significant difference in They used
the methods of preparing the cervix. In mothers consuming pineapple, oxytocin was used for 6 people
(16.7%) during labor, while in the control group, oxytocin was used for 17 people (56.7%) for
strengthening. Contractions were used (P = 0.001). (Table No. 3) The mean score of Bishop’s score before the intervention was not significantly different between the two
groups (p-value = 0.566), but over time, changes in addiction were observed between the Bishop scores of
the two groups. Informed consent The time of the onset of labor was investigated in three time periods within 48 and 72 hours from the
start of the intervention and the hospitalization time of the mothers. It should be noted that the examinations of the subjects in subsequent visits were performed by the
assistant researcher, who was unaware of the type of groups. During the study, the research units were
asked to contact the maternity hospital in case of labor pains, spotting or bleeding, discharge, reduced
fetal movements, or any other problem while calling the researcher. With the onset of labor symptoms
and hospitalization of the patient (Bishop Cervix score was also determined at the time of p
p
(
p
hospitalization), her follow-up was done by the researcher and assistant researcher from the time of
hospitalization until the end of delivery. Demographic and midwifery information questionnaires and
research unit follow-up form and research unit information from the beginning of hospitalization to
delivery, which includes the date and time of hospitalization, the reason for visiting the maternity hospital,
initial examinations at the time of admission (vital signs, fetal heart rate, and vaginal examination)
Childbirth, use of cervical preparation methods and its type, use of oxytocin, type of delivery, newborn's
Apgar score, degree of amniotic fluid contamination with meconium were recorded by the researcher and
the researcher's assistant using records and observation. Also, the daily registration form for fetal
movements (with training) was given to the mother to be completed by the research unit during the
intervention, and the fetal heart rate was taken from the mothers every other day until hospitalization. Finally, the average cervical preparation score in the two groups was determined and compared during
the three time periods mentioned. Also, the time of the onset of labor was recorded and compared in both
groups (during the first 48–72 hours and hospitalization time) from the beginning of the intervention. The
economic status of the family according to the family income was divided into 3 levels (below the
sufficiency level, above the sufficiency level, and more than the sufficiency level), and the level of
education was also considered into 5 levels (illiterate, elementary, secondary, diploma and university). Informed consent Page 6/15 Page 6/15 After collecting and recording data in SPSS software version 18, the data were analyzed in two
descriptive and analytical formats by a statistician who was unaware of the division of the intervention
and control groups at a significance level of α = 0.05. Became In the analysis section, the normality of
data distribution was checked and t-test, chi-square, and repeated measures analysis of variance were
used to answer the hypotheses of this study. Discussion Outpatient use of herbal and oral medicines as one of the non-pharmacological solutions with fewer side
effects to prepare the cervix and induce labor has always been the focus of researchers. As some
examples, Razali et al. (2017), Kariman (2015), and Kordi (2013) investigated the effect of dates on the
onset of labor (26). Okoro et al. (2019), Gilad (2018), Kohnmoi Aghdam (2014), and Sabri (2008)
investigated the effect of edible castor oil on the onset of labor (11). Kelati et al. (2018) investigated the
effect of evening primrose oral capsule on cervical readiness, and Najafi (2019) and Hashminejad (2019)
also investigated the effect of vaginal evening primrose on cervical readiness and labor initiation (26). Pineapple is one of the fruits mentioned in various cultures to start childbirth. In the study of Faze Manji
et al. (2016), pineapple extract caused contractile activity in mouse and human uterine myometrium
samples (20), and in the study of Nwankudz (2015), ripe pineapple fruit juice caused contraction of the
uterine muscles of non-pregnant mice (21). Pineapple bromelain enzyme is effective in uterine
contractions by stimulating prostaglandin secretion (13). Prostaglandin also improves the preparation of
the cervix by increasing the water under the cervical mucosa and causing changes in the collagen bands
and causing an increase in the sensitivity of the uterine myometrium to oxytocin (3). In the study of
Harianja (2017), which examined the effect of pineapple juice on the fundal height of primiparous
mothers in the postpartum period, in addition to the effect of bromelain in stimulating prostaglandin
secretion and its contractile effect on the uterine myometrium, the proteolytic property of pineapple
bromelain enzyme on connective tissue and collagen Uterine myometrium is effective in uterine
involution (18). Considering the effects of pineapple in the above articles and the review of various articles, no study was
found that investigated the effect of pineapple consumption on the onset of labor in human samples. This study was conducted to investigate the effect of pineapple consumption on the preparation of the
cervix and the onset of labor in primiparous women. The findings of the research showed that in the pineapple-consuming group, 63.9% of the mothers had
labor symptoms within 72 hours after the intervention, while in the intervention group, most of the
mothers had these symptoms at the time of hospitalization and more than 3 days had passed. Results To compare the changes in the Bishop score in the two study groups, the repeated
measures test was used with the control of baseline values. Based on the obtained results, in addition to
the increase in Bishop's score over time, significant changes in Bishop's score were seen as a result of the
intervention used (p-value < 0.001). That is, the consumption of pineapple has caused a greater increase
in Bishop's score. (Table No. 4 and Fig. 1) Page 7/15 Page 7/15 Finally, we compared the consequences and possible complications of childbirth of participants between
study groups. As the results show only induction duration was statistically significant between control
and intervention groups. (Table No. 5) Finally, we compared the consequences and possible complications of childbirth of participants between
study groups. As the results show only induction duration was statistically significant between control
and intervention groups. (Table No. 5) Discussion However, in the old study of Aghdam (31), castor oil did not have a significant effect on the
onset of labor, and in the study of Gilad (32), castor oil did not have a significant effect on the
spontaneous onset of labor in primiparous mothers, and it is not consistent with the present study, which
could be due to the sample size. Higher in these two studies and the possibility of using a placebo and
reducing the possibility of bias compared to similar studies and the present study. Pineapple bromelain enzyme is effective in uterine contractions by stimulating prostaglandin secretion
(22), which is comparable to the present study due to the similar mechanism of the date effect, which can
act on prostaglandin receptors and cause early stimulation of uterine contractions (27). In the study of
Kurdi (16) and Kariman (17) entitled "Effect of Dates on the Beginning of Labor", a significant increase in
the spontaneous onset of labor was reported, which was in line with the present study. In the present
study, 63.9% of mothers were hospitalized with cervical dilatation, which is one of the signs of labor. However, in Razali et al.'s study (2017), dates did not affect the spontaneous onset of labor (15), which
was not consistent with the results of our study. Razali's study started with a larger sample size and only
in primiparous mothers with a gestational age of 36 weeks, which can be one of the reasons for not being
consistent with the present study. According to the results of some studies, which show that castor oil
Ricinoleic acid increases the readiness of the cervix and uterine contractions with the effect of EP3
prostaglandin receptors on the uterine and intestinal smooth muscles (26), the results of the relevant
studies are comparable with the present study. In Pirdade Biranvand's (28) and Azarkish's (29) study titled
the effect of oral castor oil on the Onset of Labor, a significant increase in the spontaneous onset of labor
symptoms was reported, which was consistent with the present study. In Saberi's study (30), with the
effect of castor oil on labor induction, only the onset of labor was considered, which was in line with the
present study. Discussion the In the
pineapple-consuming group, only 8.4% of cases used one of the methods of preparing the cervix,
including extra-amnion tube and misoprostol, while in the control group, 76.7% of the mothers used one
of these two methods, which indicates that Better Bishop's score and better preparation of the cervix in
the intervention group. Also, the percentage of oxytocin use and the duration of oxytocin use to
strengthen labor contractions in the control group were significantly higher than the intervention group (P
= 0.001) and (P < 0.001). Page 8/15 Pineapple bromelain enzyme is effective in uterine contractions by stimulating prostaglandin secretion
(22), which is comparable to the present study due to the similar mechanism of the date effect, which can
act on prostaglandin receptors and cause early stimulation of uterine contractions (27). In the study of
Kurdi (16) and Kariman (17) entitled "Effect of Dates on the Beginning of Labor", a significant increase in
the spontaneous onset of labor was reported, which was in line with the present study. In the present
study, 63.9% of mothers were hospitalized with cervical dilatation, which is one of the signs of labor. However, in Razali et al.'s study (2017), dates did not affect the spontaneous onset of labor (15), which
was not consistent with the results of our study. Razali's study started with a larger sample size and only
in primiparous mothers with a gestational age of 36 weeks, which can be one of the reasons for not being
consistent with the present study. According to the results of some studies, which show that castor oil
Ricinoleic acid increases the readiness of the cervix and uterine contractions with the effect of EP3
prostaglandin receptors on the uterine and intestinal smooth muscles (26), the results of the relevant
studies are comparable with the present study. In Pirdade Biranvand's (28) and Azarkish's (29) study titled
the effect of oral castor oil on the Onset of Labor, a significant increase in the spontaneous onset of labor
symptoms was reported, which was consistent with the present study. In Saberi's study (30), with the
effect of castor oil on labor induction, only the onset of labor was considered, which was in line with the
present study. Discussion In Hasheminejad's study (34), despite taking twice the dose of vaginal evening primrose capsules
compared to Najafi's study, evening primrose was not effective on the onset of labor, which is not Page 9/15 Page 9/15 consistent with our study, and the reason for this could be the use of capsules in the 37th week of
pregnancy and only by mothers. be a virgin However, the present study was conducted in primiparous
mothers of 40 weeks and more, which can be justified due to the greater increase in oxytocin receptors
and greater preparation of the cervix with increasing gestational age, in addition to this, elective induction
before the 39th week of pregnancy has a significant amount of adverse and severe side effects. The
period of infancy will be accompanied (3). In the study of Harianja (2017) entitled "Investigation of the effect of pineapple juice on the height of the
uterus after delivery", pineapple has been effective in increasing the contraction of the uterus and the
return of the uterus in the postpartum period due to the presence of bromelain enzyme and its effect on
the collagen level and with its hydrolytic properties. 18). Although the results of this study are not the
same as the purpose of the present study, they are in line with the study done in justifying the effect of
pineapple on uterine contractions and helping to start labor as a result of uterine contractions. In the
Haryana study, 250 grams of pineapple juice containing fruit puree was given to the research units daily
for 7 days. While the mothers in the present study were pregnant and the time of the intervention started
from the 40th week of pregnancy and the probability of the mothers being hospitalized at 41 weeks due
to various reasons by the doctors or the insistence of the mothers was high, consuming 250 grams of
pineapple for 3 days It was recommended. In the present study, there were no significant differences between the two groups in amniotic fluid color,
Apgar scores of 1 and 5 minutes, and type of delivery. In this study, mothers' willingness to consume
pineapple fruit was very high, which is considered one of the strengths of the study. Due to the pleasant
taste of pineapple, the researcher did not face any problems in consuming the fruit. Discussion However, in the old study of Aghdam (31), castor oil did not have a significant effect on the
onset of labor, and in the study of Gilad (32), castor oil did not have a significant effect on the
spontaneous onset of labor in primiparous mothers, and it is not consistent with the present study, which
could be due to the sample size. Higher in these two studies and the possibility of using a placebo and
reducing the possibility of bias compared to similar studies and the present study. According to the findings of the present study, the average score of Bishop in the pineapple group
compared to the control group increased significantly within 48 hours from the start of the intervention (p
= 0.001) and 72 hours after the start of the intervention (p < 0.001), as well as the average score Bishop at
the time of hospitalization, was 8.08 in the intervention group and 4.30 in the control group, which had a
significant difference (p < 0.001). In the study of Kurdi and Kariman, a significant increase in Bishop's
score was reported, which was consistent with the present study. Bishop's score was not evaluated in
Razali's intervention. A significant increase in Bishop's scores was also reported in the study of Pirdade
Biranvand(28) and Azarkish(29), which was in line with the present study. In Saberi's study (30) with the
effect of castor oil on induction of labor, Bishop Cervix scores were not considered. In the ancient study of
Aghdam (31), castor oil did not have a significant effect on Bishop's score. In Najafi's study (33), the effect of taking evening primrose capsules from week 38 to delivery was
investigated, which was effective on Bishop's score and was consistent with the present study. Evening
primrose contains large amounts of essential unsaturated fatty acids that facilitate the synthesis of
prostaglandins. He does. Prostaglandins play an essential role in the preparation process of the cervix by
affecting pro-inflammatory factors such as cytokines and leukocytes (27). Therefore, considering the
similar mechanism mentioned in the effect of pineapple on the preparation of the cervix, it is comparable. Discussion In addition, due to the
possibility of using it at home, it is possible to avoid the problems related to visiting medical centers and
related costs that are seen in other methods such as acupressure or acupuncture. Among the limitations of this study, the following points can be mentioned: the accuracy of the research
units in consuming the specified amounts of fruit completely by the individual himself, the inability to
blind the patients as the main blinding part of the clinical trial studies, the physical characteristics of the
subjects in the labor process. And childbirth, psychological characteristics, and personality characteristics
of people in learning the training provided were effective in how to answer the questions of the
questionnaire, which were not fully controllable, and the researcher made the accuracy of the statements
of the research units a criterion. Also, another limitation was the high price of pineapple fruit in Iran,
which in addition to the high cost of the study, it may be difficult to obtain this fruit for poor socio-
economic classes. Funding This clinical trial is a pilot study of a project grant by Mashhad University of Medical Sciences. The
funding organizations had no role in the preparation of this manuscript for submission. RCT RCT
Randomized controlled trial Randomized controlled trial Consent for publication It is not applicable. Availability of data and materials le. All relevant data are within the paper and its Supporting Information files It is available. All relevant data are within the paper and its Supporting Info Conclusion Based on the findings of this research, it seems that the consumption of pineapple fruit orally is effective
in the preparation of the cervix and the beginning of labor in pregnant mothers, and the consumption of
this fruit can be recommended at the end of pregnancy. The amount of fruit consumed was considered to Page 10/15 Page 10/15 be within the range of safe doses for the mother and fetus and can be consumed daily in this study. Finally, according to the positive effects of using vaginal methods in labor induction and the positive
effect of pineapple bromelain in cervical preparation, research entitled Investigating the effect of vaginal
bromelain in the onset of labor and cervical preparation with more samples is suggested. be within the range of safe doses for the mother and fetus and can be consumed daily in this study. Finally, according to the positive effects of using vaginal methods in labor induction and the positive
effect of pineapple bromelain in cervical preparation, research entitled Investigating the effect of vaginal
bromelain in the onset of labor and cervical preparation with more samples is suggested. Ethics approval and consent to participate The study was approved by The Medical ethics committee of Mashhad University of Medical
Sciences. Ethical Lot Number: IR.MUMS.NURSE.REC.1397.055 and registered in the clinical trial database
of Iran with the code IRCT20181007041266N2 the hold on (15/01/ 2019). We confirm that all methods
were carried out in accordance with relevant guidelines and regulations. For participants who are illiterate, we provided informed consent from their legal guardian/ a legally
authorized representative. It is not applicable under Consent for publication our study. Competing interests The authors declare that they have no competing interests. Authors’ contributions All authors read and approved the final manuscript. SSM participated in the conception and design of the
study, coordinated the study, collected data, participated in the data analysis and interpretation of data,
and wrote the manuscript. RM participated in data collection, conducted the data analysis, assisted with Page 11/15 Page 11/15 the interpretation of the data, and commented on the manuscript. MR participated in the data collection,
the interpretation of data, and co-wrote the manuscript. ES & RM participated in the conception and
design of the study, assisted in the interpretation of the data, and commented on the different versions of
the manuscript. YR & SSM participated in the conception and design of the study, data collection, the
interpretation of data, and co-wrote the manuscript. All authors read and approved the final manuscript. Acknowledgment: This article is the result of a student project at the Mashhad Nursing and Midwifery College, which was
approved by the Medical ethics committee of Mashhad University of Medical Sciences. The authors
thank the mothers who helped us in conducting this research. References 1. Koh LM, Percival B, Pauley T, Pathak S. Complementary therapy and alternative medicine: effects on
induction of labor and pregnancy outcome in low-risk post-date women. Heliyon. 2019;5(11):e02787. 1. Koh LM, Percival B, Pauley T, Pathak S. Complementary therapy and alternative medicine: effects on
induction of labor and pregnancy outcome in low-risk post-date women. Heliyon. 2019;5(11):e02787. 2. Evans M. Postdates pregnancy and complementary therapies. Complement Ther Clin Pract. 2009;15(4):220–4. 3. Cunningham FGLK, Bloom SL, Haut JC, Rouse D, Sponger CY. Williams Obstetrics. New York:
McGraw-Hill; 2018. p. 799. 3. Cunningham FGLK, Bloom SL, Haut JC, Rouse D, Sponger CY. Williams Obstetrics. New York:
McGraw-Hill; 2018. p. 799. 4. Simpson KR. Cervical ripening and labor induction and augmentation. Nurs Women's Health. 2020;24(4):1–S41. 4. Simpson KR. Cervical ripening and labor induction and augmentation. Nurs Women's Health. 2020;24(4):1–S41. 5. Saunders SJ, Grisamore JL, Wong T, Torrejon Torres R, Saunders R, Einerson B. Moving pre-induction
cervical ripening to a lower acuity inpatient setting using the synthetic hygroscopic cervical dilator: a
cost-consequence analysis for the United States. J Med Econ. 2022;25(1):1185–98. 6. Fareed P, Bashir S, Lone SA. Hazards and benefits of elective induction of labor at term. Int J Reprod
Contracept Obstet Gynecol. 2020;9(9):3626–31. 7. Wheeler V, Hoffman A, Bybel M. Cervical ripening and induction of labor. Am Family Phys. 2022 105(2) 177 86 7. Wheeler V, Hoffman A, Bybel M. Cervical ripening and induction of labor. Am Family Phys. 2022;105(2):177–86. 8. Yazdizadeh H, Apedi P, Najar S, Ahmadi K, Zaker Hoseiyni V. The effect of vaginal administration
isosorbide mononitrate on cervical ripening and induction of labor in post-term pregnancy. J
Jundishapur Univ Med Sci. 2012;11(6):675–84. 8. Yazdizadeh H, Apedi P, Najar S, Ahmadi K, Zaker Hoseiyni V. The effect of vaginal administration
isosorbide mononitrate on cervical ripening and induction of labor in post-term pregnancy. J
Jundishapur Univ Med Sci. 2012;11(6):675–84. 9. Zahrani ST, Dasumi S, Tansaz M, Akbarzadeh A. The effects of paganism Harmala Smoke cervical
ripening, initiation, and outcome of labor. Med History J. 2016;8(27):151–77. 9. Zahrani ST, Dasumi S, Tansaz M, Akbarzadeh A. The effects of paganism Harmala Smoke cervical
ripening, initiation, and outcome of labor. Med History J. 2016;8(27):151–77. 10. Moradi M, Niazi A, Mazloumi E, Lopez V. Effect of Castor Oil on Cervical Ripening and Labor
Induction: a systematic review and meta-analysis. J Pharmacopunct. 2022;25(2):71. 10. Moradi M, Niazi A, Mazloumi E, Lopez V. References Effect of Castor Oil on Cervical Ripening and Labor
Induction: a systematic review and meta-analysis. J Pharmacopunct. 2022;25(2):71. 11. Amerizadeh A, Farajzadegan Z, Asgary S. Effect and safety of castor oil on labor induction and
prevalence of vaginal delivery: A systematic review and meta-analysis. Iran J Nurs Midwifery Res. 2022;27(4):251. Page 12/15 Page 12/15 12. Simpson M, Parsons M, Greenwood J, Wade K. Raspberry leaf in pregnancy: its safety and efficacy in
labor. J Midwifery Women’s Health. 2001;46(2):51–9. 13. Tenore JL. Methods for cervical ripening and induction of labor. Am Family Phys. 2003;67(10):2123–
8. 13. Tenore JL. Methods for cervical ripening and induction of labor. Am Family Phys. 2003;67(10):2123–
8. 14. Moradi M, Niazi A, Heydarian Miri H, Lopez V. The effect of evening primrose oil on labor induction
and cervical ripening: A systematic review and meta-analysis. Phytother Res. 2021;35(10):5374–83. 14. Moradi M, Niazi A, Heydarian Miri H, Lopez V. The effect of evening primrose oil on labor induction
and cervical ripening: A systematic review and meta-analysis. Phytother Res. 2021;35(10):5374–83. 15. Razali N, Mohd Nahwari SH, Sulaiman S, Hassan J. Date fruit consumption at term: Effect on length
of gestation, labour and delivery. J Obstet Gynaecol. 2017;37(5):595–600. 15. Razali N, Mohd Nahwari SH, Sulaiman S, Hassan J. Date fruit consumption at term: Effect on length
of gestation, labour and delivery. J Obstet Gynaecol. 2017;37(5):595–600. 16. Kordi M, Aghaei Meybodi F, Tara F, Nematy M, Shakeri MT. The effect of date consumption in late
pregnancy on the onset of labor in nulliparous women. Iran J Obstet Gynecol Infertility. 2013;16(77):9–15. 17. Kariman N, Yousefy Jadidi M, Jam Bar Sang S, Rahbar N, Afrakhteh M, Lary H. The effect of
consumption date fruit on cervical ripening and delivery outcomes. Pajoohandeh J. 2015;20(2):72–
7. 17. Kariman N, Yousefy Jadidi M, Jam Bar Sang S, Rahbar N, Afrakhteh M, Lary H. The effect of
consumption date fruit on cervical ripening and delivery outcomes. Pajoohandeh J. 2015;20(2):72–
7. 18. Harianja WY, Setiani O, Umaroh U, Widyawati MN, Mashoedi ID, Pujiastuti SE. THE IMPACT OF
PINEAPPLE (ANANAS COMOSUS (L.) MERR.) JUICE ON FUNDAL HEIGHT IN PRIMIGRAVIDA
MOTHERS DURING POSTPARTUM PERIOD. Belitung Nurs J. 2017;3(2):134–41. 19. Golozar S, Namjouian F, Latifi SM, Mirahi A. Evaluating the effect of oral bromelain (pineapple) on
episiotomy wound healing in primiparus women. Feyz Journals of Kashan University of Medical
Sciences. 2011;15(2). 20. References Monji F, Adaikan PG, Lau LC, Said BB, Gong Y, Tan HM, et al. Investigation of uterotonic properties of
Ananas comosus extracts. J Ethnopharmacol. 2016;193:21–9. 20. Monji F, Adaikan PG, Lau LC, Said BB, Gong Y, Tan HM, et al. Investigation of uterotonic properties of
Ananas comosus extracts. J Ethnopharmacol. 2016;193:21–9. 21. Nwankudu N, Ndibe N, Ijioma S. Oxytocic effect of Ananas comosus fruit juice on isolated pregnant
rats uteri. Nigerian Veterinary Journal. 2015;36(4):1318–26. 21. Nwankudu N, Ndibe N, Ijioma S. Oxytocic effect of Ananas comosus fruit juice on isolated pregnant
rats uteri. Nigerian Veterinary Journal. 2015;36(4):1318–26. 22. Padma P, Jayakumar K, Mathai V, Chintu S, Sarath B. Immobilization and kinetic studies of
bromelain: a plant cysteine protease from pineapple (Ananas comosus) plant parts. Int J Med Health
Sci. 2012;3:10–4. 23. Mirlashari BM, Abedian Z, Rakhshandeh H, Esmaily H. Comparison of the effects of bromelain and
mefenamic acid on menstrual bleeding in students with primary dysmenorrhea: A double-blind
randomized clinical trial. Iran J Obstet Gynecol Infertility. 2019;21(11):61–70. 24. PARIVAR MOSHTAGHIS, BAHARARA K, HAYATI J. N, KERACHIAN M. Synergic Application Effects of
Bromelain and Low-Frequency Electromagnetic Field on the Aortic Ring on Angiogenesis. 2019. 24. PARIVAR MOSHTAGHIS, BAHARARA K, HAYATI J. N, KERACHIAN M. Synergic Application Effects of
Bromelain and Low-Frequency Electromagnetic Field on the Aortic Ring on Angiogenesis. 2019. 25. Ionescu A, Aprodu I, Pascaru G, EFFECT OF PAPAIN AND BROMELIN ON MUSCLE, AND COLLAGEN
PROTEINS IN BEEF MEAT. Annals of the University Dunarea de Jos of Galati Fascicle VI–Food
Technol. 2008;1(31). 25. Ionescu A, Aprodu I, Pascaru G, EFFECT OF PAPAIN AND BROMELIN ON MUSCLE, AND COLLAGEN
PROTEINS IN BEEF MEAT. Annals of the University Dunarea de Jos of Galati Fascicle VI–Food
Technol. 2008;1(31). 26. Ghasemi M, Ebrahimzadeh-Zagami S, Ghavami V. The Effect of Outpatient Herbal Drugs on Cervical
Ripening and Onset of Labor: A Systematic Review and Meta-Analysis. J Isfahan Med School. 2022;39(655):999–1009. 26. Ghasemi M, Ebrahimzadeh-Zagami S, Ghavami V. The Effect of Outpatient Herbal Drugs on Cervical
Ripening and Onset of Labor: A Systematic Review and Meta-Analysis. J Isfahan Med School. 2022;39(655):999–1009. Page 13/15 Page 13/15 27. Bagherzadeh Karimi A, Elmi A, Mirghafourvand M, Baghervand Navid R. Effects of date fruit (Phoenix
dactylifera L.) on labor and delivery outcomes: a systematic review and meta-analysis. BMC
Pregnancy Childbirth. 2020;20:1–14. 28. Pirdadeh Beiranvand S, Azhari S, Lotfalizadeh M, Shakeri M. References The effect of castor oil on initiating
labor in 40–42 weeks pregnancy. J Babol Univ Med Sci. 2007;9(4):33–8. 29. Azarkish F, Absalan N, Roudbari M, Barahooie F, Mirlashari S, Bameri M. Effect of oral castor oil on
labor pain in post-term pregnancy. Sci J Kurdistan Univ Med Sci. 2008;13(3):1–6. 30. Saberi F, Abedzadeh M, Sadat Z, Eslami A. Effect of castor oil on induction of labor. KAUMS J
(FEYZ). 2008;11(4):19–23. 31. KahnamoyiAghdam F, Aghamohammadi M, Rostamnejad M. Effect of castor oil on induction of
labor. J Health Care. 2013;15(3):63–58. 32. Gilad R, Hochner H, Savitsky B, Porat S, Hochner-Celnikier D. Castor oil for induction of labor in post-
date pregnancies: A randomized controlled trial. Women Birth. 2018;31(1):e26–e31. 33. Najafi M, Loripoor M, Saghafi Z, Kazemi M. The effect of vaginal evening primrose on the Bishop
score of term nulliparous women. Nurs Pract Today. 2019;6(4):202–11. 33. Najafi M, Loripoor M, Saghafi Z, Kazemi M. The effect of vaginal evening primrose on the Bishop
score of term nulliparous women. Nurs Pract Today. 2019;6(4):202–11. 34. Hashemnejad M, Ataei M, Modarresi M, Forutan F. Investigating the effect of Primrose Capsule
(Primula Flower Oil) on cervix preparation and commencement of child delivery pains. Revista
Latinoam de Hipertensión. 2019;14(1):119. 34. Hashemnejad M, Ataei M, Modarresi M, Forutan F. Investigating the effect of Primrose Capsule
(Primula Flower Oil) on cervix preparation and commencement of child delivery pains. Revista
Latinoam de Hipertensión. 2019;14(1):119. Tables Table 1 to 5 are available in the Supplementary Files section. Comparison of bishop scores in two groups during time Comparison of bishop scores in two groups during time Figures Page 14/15
Figures
Figure 1 Page 14/15
Figures
Figure 1 Figure 1 Page 14/15 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. tables.docx tables.docx Page 15/15
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Knowledge and attitude of the first year student at Faculty of Medical and Applicable Sciences at Yanbu governorate about some aspects of reproductive health.
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Tanta Scientific Nursing Journal
Knowledge and attitude of the first year student at Faculty of Medical and Applicable Sciences
at Yanbu governorate about some aspects of reproductive health.
Wafaa Ahmed Ibrahim:
Assistant professor of obstetrics and gynecological nursing
Faculty of nursing , Alexandria University, Egypt
Abstract
The aim of this study was to identify the knowledge and attitude of the first year students at Faculty
of Medical and Applicable Sciences at yanbu governorate about some aspects of reproductive health
.Subjects and method ; An exploratory study and a Convenience sample ware used (all the students
in first year of the faculty ). Data collection tools: A structured / questionnaire was used to collect
students Knowledge about reproductive health and a likert - like scale was adopted to assess their
attitudes towards reproductive health. Scores of knowledge and attitude were done. The result:
reveled most of the study subjects shocked and cried as reaction during menarche. Also said that
menstruation is spoiled blood the body get mid of it. There is a need for more improving student
knowledge and attitude regarding RH through the following Recommendation Strengthen the RH
component in the school curricula, the role of the families ,mass media ,and school accurate
information about RH must be enhanced, a program to educate parents on RH issues should be
carried out, teachers should be well informed about RH issues , so they can prevent some of the
problems by means of carful and open education.
Key wards: Applicable Sciences ,reproductive health
Introduction
Reproductive health (RH) is one of the center
onset
stone's of an individual's health and well-
organization defines adolescence as the period
being, and an important component of
of life between age 11 and 21 years
country's human social development, limited
adolescence is characterized by physical
access to RH information among female can
psychological ,and social change. It represents
increase their vulnerability to health problem.
a window of opportunity to prepare for a
There for, it is important to provide them with
healthy adult. The health adult population
accurate and age appropriate in formation"1,
1200 million persons or about 19% of the total
adolescence is a period of transition from
population -faces a series challenges not only
childhood to adulthood ,which status with the
Vol. 13 No. 2 November, 2017
90
of
puberty.
The
world
health
Tanta Scientific Nursing Journal
affecting their growth and development but
, natural part of her life ". Issues associated
also their life hood as adults (1).
with menstruation are never discussed openly
In many parts of the world adolescents are
and this burdens young girls by keeping them
poorly informed about their health , bodies
ignorant of this biological function
and physical well-being . Adolescent girls in
Even after the attachment of menarche ,very
particular are often kept away from learning
little information is given to young girl about
about reproductive health issues because of a
physiological processes involved and the
cultural
is
hygienic practices to followed. It is obvious
particularly in most of the Arab Countries ,
that the young youth receive inadequate
where adolescents are often reluctant to ask
information about puberty from parents and
for RH information from adult in their
health professionals, so they resort to other
families, communities ,or in professional
sources such as fiends and media , which may
settings. Furthermore social prohibitions and
be interpreted differently.10
negative attitude of parents in discussing the
As regarded premarital counseling is a type of
related issue openly which has blocked the
therapy that
access
marriage .Premarital counseling can help
and
of
religious
adolescent
taboos.
girls
This
to
connect
helps
couples prepare for
information (6).
ensure that you and your partner have a strong
Adolescence and puberty can be difficult
healthy relationship giving you a better chance
times for all young people, but for girls in
for a stable and satisfying marriage. Premarital
many countries, puberty, ,especially the onset
counseling
of menstruation, poses particular challenges.
weaknesses that could become problems
Menarche ,or the onset of menstruation is a
during marriage Premarital counseling is often
landmark feature of female puberty and
provided by licensed therapists known about
signals reproductive maturity. The menarche
marriage
is often horrifying and traumatic to an
therapists have graduate or postgraduate
adolescent girl because it usually occurs
degrees.( 12,14,15)
without her awareness about it. Anxiety ,fear
Also premarital counseling can help couples
,confusion ,and even depression are frequently
improve their relationships before marriage
reportedas experiences of menarche.(7)
.Through premarital counseling ,couples are
As menstruation play an important role in the
encouraged to discuss topics related to
health of female. It is crucial that the female
marriage, such as Finances, Communication,
obtain accurate knowledge about menstruation
Beliefs and values ,Roles in marriage,
and learns to accept menstruation as a positive
Affection and sex ,Children and parenting
Vol. 13 No. 2 November, 2017
91
can
and
also
family
help
you
therapists.
identify
These
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,Family
relationships
,
Decision-making,
Dealing with anger, Time spent together
More
ever
Premarital
counseling
governorate
about
some
aspects
of
reproductive health.
helps
Research questions:
partners improve their ability to communicate,
What are the knowledge and attitude of the
set realistic expectations for marriage and
student about reproductive health.
develop conflict-resolution skills. In addition,
Subjects and method
premarital
Research design : Descriptive research design
counseling
can
help
couples
establish a positive attitude about seeking help
was used in this study
down the road (16,17) .
Setting : The study was conducted in the
Acquiring knowledge and developing attitude
faculty of medical and applicable sciences at
takes place during adolescence , which can
Yanbu governorate.
have lifelong effects on the individual ; family
Subject: convenience sampling of all student
and society , and also changes in the pattern of
in the first year participate in the study , their
thinking , attitude , relationships , moral
number equal 200 student.
standards and abilities take place in this period
Tools of data collection
. Therefore knowledge of reproductive health
designed and used by the research to collect
and the means to protect oneself against
the necessary data :
reproductive problems diseases should be
Tool I : Structure
available to adolescents . Better knowledge
which entailed two parts :
and education and counseling on human
Part (a) : Socio -demographic data.
reproductive
responsible
Part (b): knowledge about menstrual period
parenthood among young adults will lead to
and premarital education as an aspect of a
correct attitudes and responsible reproductive
reproductive health.
health behavior . On the other hand Inadequate
Scoring
Knowledge in this area may lead to serious
knowledge included:
consequences in the reproductive health1 3. For
Correct and complete answer was scored (2)
all of these causes a prefer ,to search on this
Correct and incomplete answer was scored (1)
topic and also this is the first time for
Incorrect or no answer was scored
application at Yanbu City(9).
Total scores of students ' knowledge were
Aim of the study
calculated and classified as follow:
This study aimed to identify the knowledge
More than 70% were considered good.
and attitude of the first year student at Faculty
60-70 % were considered fair.
of Medical and Applicable Sciences at Yanbu
Less than 60% were considered poor.
health
and
Vol. 13 No. 2 November, 2017
92
system
: Two tools was
questionnaire schedule
for
studied
students
(0)
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Tool II: (reproductive health attitude scale)
Each student was individual interviewed and
likert like scale form of there continuum
informed about the aim of the study in order to
(agree, not sure, disagree) was adopted to
obtain her consent to participate in the study.
assess
The tools of the study were introduced to ten
the
student,
attitudes
towards
reproductive health (13)
juries' expert in the field of obstetric nursing
- The total score of attitudes was classified as
to test its validity. Modification was done
follow :
accordingly.
Positive attitude (75% or more), Neutral
Pilot study was carried out on10%of study
attitudes (50% : < 75%) and negative attitude
sample to test tool reliability the necessary
(<50%).
modification was carried and not excluded
Method
from the study.
A approval of the directors of the faculty was
The questionnaire sheet and the attitude scale
obtained after explanation of the aims of the
ware disturbed to the student during their free
study.
time between classes.
Ethical and legal consideration:
It look 10-15 minutes to voluntarily complete
An informed consent for participation in the
the tool
study was obtained.
Statistical analysis :
Nature of the study was not caused any harm
Statistical analysis was done by the researcher.
and/or pain for the entire sample.
A scoring system for students' knowledge and
Confidentiality and privacy was taken into
attitudes regarding reproductive health was
consideration regarding data collection.
adopted. The correct answers was determined
according to literature and the question were
coded.
Results
It showed(table1) distribution of the study
subjects where agree and about 20.5% (one
subjects according to their knowledge about
fifth) where disagree . Concerning this
menstrual period as regard the first reaction
awareness regarding menstruation process
during menses 66% (less than two third) of
43% (less than one half) said it is normal
study subjects shocked and cried and 34%
physical process and 57% (more than one half)
(more than one third) where normal reaction .
said it is spoiled blood the body get rid of it .
Regarding to information about menstruation
Among the knowledge about the normal
onset 79.5% (the majority) of the study
duration of menstruation 47.5% (less than one
Vol. 13 No. 2 November, 2017
93
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half) responded correctly and about 52.5%
study subjects were illiterate / read & write
(more than one half)of the study subjects
while 36.50% (about more than one third)
where responded wrongly or they don’t know .
were less than university also the same
Regarding to the knowledge about the normal
percentage were university . Regarding to
interval between menstruation 69.5% (less
father occupation 79% (three quarter) were
than three quarter) of the study subjects where
work but ,only 21% (nearly to one quarter)
correct answer and 30.5% (more than one
were not work . Concerning mother education
quarter) said wrong answer or don’t know.
63% (three fifth ) of the study subjects were
Concerning the frequency of changing pad
less than university and 38% (two fifth) were
/day of the study subjects 4.5% (little of study
illiterate/
subjects) one time , 8.5% (little of study
percentage were university . Regarding to
subject) changing bad tow day and 87% (the
mother occupation 71.5% (three quarter) were
majority of the study subject) change bad three
house wife but ,only 28.5% (more than one
or more day . In addition 86% (the majority of
quarter) were worker . Among residence
study subjects) agree with bathing during
75.5% (three qual)
read
&write
also
the
same
menstruation is necessary while 14%(one
tenth) disagree about bathing during
It shows the knowledge about premarital
menstruation . In addition to causes about the
counseling 88.5% of the majority of the study
bathing necessary during menstruation 4.5%
subjects mentioned that 20 or more is the
(little of study subject) said correct answer
suitable age for marriage while 11.5% (more
61% (three fifth) said correct but incomplete
than one tenth) of the study subjects
answer and 34.5% (more than one third )of
mentioned less than 20 is the suitable age for
study subjects said wrong answer or don’t
marriage. Concerning hearing about premarital
know . Regarding to causing about the bathing
counseling all of the study subjects 100%
is unnecessary during menstruation all of them
mention yes . Regarding to important of
responded wrongly and don’t known.
premarital counseling 73% (less than three
quarter) responded were correctly and
It showed table (2):distribution of the study
incomplete answer or don't know , neither of
subjects according to their sociodemographic
them were written a correct answer. Regarding
characteristic as regard age 67% (less than tow
to contents of premarital counseling 77% the
third) of the study subjects were in the age
majority of the study subjects were show the
group 20 or more . Concerning father
wrong answer or don’t know and 18.5% (less
education 33% (more than one third) of the
than one fifth) were correct but incomplete
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94
Tanta Scientific Nursing Journal
answer, little of the them were written correct
answer.
Table (1) : Distribution of the study subjects according to their socio demographic data
Social demographic data
N =200
%
- Less than 20
134
67%
- 20 or more
66
33%
- illiterate /read &write
73
36.5%
- Less than university
66
33%
- University
61
30.5%
- Work
158
79%
- Not work
42
21%
- Illiterate/read &write
38
19%
- Less than university
126
63%
- University
36
18%
57
28.5%
Age:
Father education :
Father occupation :
Mother education :
Mother occupation :
- Work
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Table
(2):distribution
of the study subjects according to their
menstrual
- House
wife
143 Knowledge about
71%
Period
Knowledge
Residence :about menstrual period
- Urban when the menarche occur :
Reaction
- Shocked and cried
Rural reaction
- -Normal
N =200
151
%
75.5%
132
6
66%
68 24.5% 34%
Information about menarche before its onset:
79.5%
rter)-Yes
of the study subjects were from urban area ,while 24.5% (nearly to one 159
quarter) were
from
- No
41
20.5%
rural
area.
Awareness regarding menstruation process :
- Normal physical process
86
43%
- Spoiled blood the body get mid of it
114
57%
- Correct answer
95
47.5%
- Wrong answer or don’t know
105
52.5%
- Correct answer
139
69.5%
- Wrong answer or don’t know
61
30.5%
- One
9
4.5%
- Two
17
8.5%
- Three or more
174
87%
- Yes
172
86%
- No
28
14%
- Correct answer
9
4.5%
- Correct but incomplete answer
122
61%
- Wrong answer or don’t know
69
34.5%
Know the normal duration of menstruation :
Know the normal interval between menstruation:
Know frequency of changing pad /day:
Is bathing during menstruation necessary :
Why bathing during menstruation necessary :
Why bathing during menstruation is unnecessary :
- Correct
answer of the study subjects according to their knowledge about6 premarital3%
Table
3 : Distribution
counseling
-Knowledge
Correct butabout
incomplete
answer
premarital
counseling
- Wrong answer or don’t know
Vol. 13 No. 2 November, 2017
N=200
17
177
96
%
8.5%
88.5%
Tanta Scientific Nursing Journal
Know suitable age for marriage :
- Less than 20
23
11.5%
- 20 Or more
177
88.5%
-yes
200
100%
-no
0
0%
- correct answer
2
1%
- correct but incomplete answer
146
73%
- wrong answer or don't know
52
26%
- correct answer
9
4.5%
- correct but incomplete answer
37
18.5%
- wrong answer or don’t know
154
77%
Hearing about premarital counseling:
Important of premarital counseling :
Contents of premarital counseling :
Figure (1) : study subjects total score of
knowlegde about reproductive health
0
20.9
38.5
GOOD
SATISFACTORY
POOR
40.6
Figure(1) Study subjects Total score of Knowledge about reproductive health
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Figure 2 : Study subjects sources of knowledge about reproductive health
40%
35%
30%
25%
20%
15%
10%
5%
0%
35%
30%
15%
10%
Mother,
Sister
10%
Relative,
friends
School,
Culture
Mass media
Books,
Magazines
Source of knowledge
Figure(2) Study subjects sources of knowledge about reproductive health
It show that about one third 35% & 31 % of the stud subjects obtained their knowledge from mass
media and relative or friend respectively and 15% from school classes while 10% from mothers
&sisters and also 10% from books and magazines .
ATTITUDE
0
10.09%
Positive
24%
Natural
65.91%
Negative
Figure (3) : Total score of attitude of study subject about reproductive health
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98
Tanta Scientific Nursing Journal
It show total score of attitudes of study subjected about reproductive health, about tow third of the
study subjects 65.91 % obtained positive score , while 24% (one quarter) obtained natural score and
only 10.09% (one tenth) obtained negative score.
Discussion
A doles cannot represent one of the main
NagrandAimol (2011) also found that in
pillars of any society , they form a large
Tribal Areas of India , slightly more than half
significant and growing population group in
of the study subjects know that menstruation is
many countries. Despite that they do not
unclean/dirty / bad blood .(22) In addition ,
resave much attention and suffer from poor
Adinma (2008) found in his study that slightly
knowledge about reproductive health. This
less than two - fifth of study subjects
knowledge about reproductive health issue and
perceived menstruation as a physiological
health seeking behavior in the course of their
process , while slightly more than one half
future life is very important(18,19).
viewed menstruation as release of bad blood
This study revealed general lack of knowledge
(23)
about reproductive health among almost three
by Kuwar and Kundon (2011) in Urban
fifth of the study subject this finding is more
Indian revealed that three - fifth of adolescent
than to that of EL Sadek et at (2008) who
girls thought menarche to be a natural process
found in Cairo , Egypt that more than one -
24
half of the subject had poor knowledge about
awareness and knowledge about the subject.
RH.(20) Nezam (2010) formed in Syria that
The manner in which a girl learns about
slightly more than two third of the study
menstruation and its associated changes may
subject
about
have an impact on her response to the event of
reproductive health(21), In particular the study
menarche. Although menstruation is a natural
subjects
process
had
good
knowledge
knowledge
about
menstruation
. On the other hand , the study conducted
. The reaction to menstruation depends upon
,
it
is
liked
misconceptions
normal psychological process while more than
sometimes
one half said it is spoiled blood that the body
outcome(25). Regarding
gets rid of this finding is relatively similar to
menarche occur. The present study revealed
that of Eswited (2013) who found that more
the two third of the study subject were
than half of the participants reported that
shocked and cries while one third had normal
menstruation is an event that happens to the
reaction this may arise from an unexpected
girl during puberty, it occurs monthly and
appearance of blood pre vagina at menarche
Vol. 13 No. 2 November, 2017
99
(9)
.
result
practice
several
revealed that more than two fifth said it is
spoiled blood the body get rid of it
and
with
into
,
which
adverse
health
reaction when the
and this finding is similar to that of
Tanta Scientific Nursing Journal
AbdelTwaab et al ( 2012 ) who found in
(2011) and as well as Dhingra at (2007) in
survey of young people in Egypt had two third
India.(29-31) Which revealed that 58%, 30% and
were shocked and cries as well as being scared
43.5% of girls respectively had no - prior
when the menarche occurred
(11)
also the
knowledge
finding is relatively similar to that of Dube
menarche.
and Sharina(2012) who found in India that
Hygiene is one important aspect especially
fear and panic was the dominant reaction
during menstruation proper understanding of
among the majority of the study subject when
menarche
they had their first menses(26)Bobhate (2011)
menstruation
also found slightly less than two thirds agreed
reproductive health of adolescents.
to being scared on their first menstruation
The majority of the present study subject
while slightly less than one third felt irritated
reported that sanitary pads should be used
(27)
and
during the menstruation and changed three
Elsabagh(2013)found that one half of the girls
times/ day, while 4% , 8% respectively change
were scared from first menstruation, and the
the bad one and twice /day . This finding is in
remaining were anxious and get syncope ( 28 ).
line with that of MalleshAppaetale (2012)
As regard awareness the majority of the study
who found in India that almost all the study
subject were informal about menarche and
subject (97.5%) reported that sanitary pad
menstruation before its onset, the current
should be used during menses , and also it
finding is in agreement with that of Eewietal
should be changed regularly
(2012), Juyal et al (2012) and Omidvar ,25
bathing during menstruation the majority of
(2010), whose found that 14%, 66% and 64.5
present
of girls respectively had been informed about
bathing menstruation is important and their
menarche before it's onset.( 9,10,24) The current
reasons are for personal hygiene and reform
study in different with that of bob hate (2011)
,to stimulate blood circulation ,help for sleep
who found of that 80% of the participant were
and relax ,and prevent the bad odor , and about
not aware about menstruation before menarche
more than one tenth of the study subject
27
. Also with that of Abdullah E ( 2013) in
mentioned bathing is unnecessary and did not
Zagazig City , Egypt which revealed that 65%
reported any reasons this finding is in line
of
for
with Nager and Aimal who found that tow
i n addition the present
third of the responding considered having bath
.
In
girls
addition,
did
not
menarche.(28)
have
AbdAllah
preparation
about
and
study
hygiene
significantly
subject
should
during
influences
(32)
. Regarding
menstruation
be
used
that
twice
Aparaqita (2012)1 west Bengal , Thakre et al
menstruation' . Also the present study show
100
day
personal
before
study on the convers with Sudehan and
Vol. 13 No. 2 November, 2017
a
menstruation
during
Tanta Scientific Nursing Journal
that majority of the study subject mention this
reproductive health . the main sources of
result 20 or more is the suitable age for
students' knowledge about RH were mass
marriage. This result is online with agreement
media and relatives and friends.
with that of ELSadek et al (2008) who found
Recommendation :
that (93.6%) of the study girls reported that
Strengthen the RH component in the school
they heard about pre-marital examination
curricula.
counseling
study
(20)
. Although the majority of the
subject
heard
about
pre
marital
The role of the families , mass media ,and
school accurate information about RH must be
counseling yet almost mean to three quarter of
enhanced.
the study subject and more than one quarter
A program to educate parents on RH issues
responded incomplete and wrong or did not
should be carried out.
know respectively regarded important of pre-
Teaches should be well informed about RH
marital counseling and the majority of them
issues ,so they can prevent some of the
mentioned wrong answer did not know as
problems by means of carful and open
regard contact of pre-marital counseling. This
education .
finding not on line with Lamada (2004) who
Further studies :
found that knowledge of adolescent girt about
Assess male versus female secondary school
premarital counseling and examination was
students knowledge and attitudes regarding
insufficient and a sizeable proportion of them
reproductive health .
did not know the important of premarital
References
(33)
counseling
students
. In the current study the
got
their
knowledge
Population
Reference
Bureau
:
World
about
population data sheet. Minding the gap
reproductive health from many sources ,the
Alexandria , talking to girls in school about
main sources ware mass media ,relatives and
reproductive health ,2012.
friend. This finding is in line with Farag
World health organization. Report on violence
(2012) who found that the main sources of
and health.WOH, Geneva, 2010.
knowledge about RH were mass media and
Tegegn A, Gelaus Y. Reproductive health
internet (34) .
knowledge and Attitude among adolescents ;
Conclusion
Jimma Town, southwest Ethiopia. 2008
Based on the finding of the present study ,it
;22(3):243..51.
can be concluded that only less than tow fifth
EL - Zanaty F, way A. Demographic and
of the students had good knowledge while tow
health survey, 2009.
third of the had positive attitude about
Vol. 13 No. 2 November, 2017
101
Tanta Scientific Nursing Journal
Population
Reference
population
sheet.
Bureau
Mother
-
:
world
daughter
:
health
.
Kathmandu
university
Medical
Journal 2005:3(4):380-3
communication about sexual and reproductive
Geaft N , Likert scales, levels of measurement
health in rural areas of Alexandria,(2011).
and the (laws) of statistics. Advances in health
Mudey A , Goyal R Agross - sectional study
science Education 2010: 15(5) :625-32.
on awareness regarding safe and hygienic
Marriage preparation. American Association
practices among school adolescent girls in
for
rural area of Warha District, India.Glo . J.
Therapy.http://www.aamft.org/iMIS15/Conten
Health2010,2(2) :22 5-31.
t/Consumer_Updates/Marr
Marriage
and
Rao R, Lena H, and Kamalh A. Effectiveness
iage_Preparation.aspx.
of reproductive health education among
2014.
Family
Accessed,
natural adolescent girls in Udupitaluk, Indian J
Marriage and family therapists: The friendly
Med Sci 2008; 62(11):439-93.
mental
Amiebue U, Aniebue P, Nwankwo O. The
Association
inpact of pre -menarchealTrining on menstrual
15/AAMFT/Content/Consumer_Updates/Marr
practicesnand hygiene of Nigerian school
iage_and_Family_Therapists.aspx. Accessed,
girls, the par African Medical goinrnal
2014.
2009;(9)2:l-9.
Nine psychological tasks for a good marriage.
Eswi A, Helal H and Efarousy W. Menstrual
American
A Attitude and knowledge among Egyptian
http://www.apa.org/helpcenter/mamage.aspx.
adolescents female . journal of American
Accessed, 2014.
science 2013, 8161: 555-65.
Murray CE, et al. Solution-focused premarital
Juyal R and Negi K . practices of menstrual
counseling: Helping couples build a vision for
hygiene among adolescent girls in a district of
their marriage. Journal of Marital and Family
UttarKhand . Indian Journal of community
Therapy. 2004;30:349 .
health 2012; 24 (2): 124-8.
Hilal T, Soliman C, and Oraby D. Meeting
Abdel Twab N, Roushely N, and sieeverding
Adolescent Reproductive health Needs in
M. survey of young
Egypt: Quantitative Assessment of youth -
people in Egypt policy brief series No.l :2012
friendly clinics. Family Health international
Dhital
2007.
A
,
paudel
Rand
uprety
D.
health
for
professionals.
Marriage
American
and
Psychological
Family
Association.
Effectiveness of structured teaching program
Muzammil K and semwal J. study of
in impresving knowledge and attitude of
knowledge
school going adolescent on reproductive
Vol. 13 No. 2 November, 2017
102
and
attitudes
of
adolescent
Tanta Scientific Nursing Journal
regarding Reproductive health. Indian. Prev.
Dube S and Sharma K. Knowledge,
Soc. Mad. 2009;40(2):89-95.
Attitude
El- Sadek A, Kasim K, abd and-Elhady A
Reproductive Health among Urban
.knowledge and Attitude of Adolescent Girl
and Rural Girls:A comparative study.
towards Reproductive health in cairo ,Egypt,
Ethno Med 2012;6(2):85-94.
the Egyptian journal of community Medicine
2008:26(1): 1-15.
practice
Regarding
Bobhate P and Shrivastava S . Accross
sectional study of knowledge and
21 - Nezarna A . knowledge and
attitude of female Tichreen university
students about reproductive health Master thesis , Alexandria: Faculty of
Nursing Alexandria university ,2008.
Adolescent
Girls
Menstruation
Tribal
Stud
Regarding
areas
Tribes
of
TRIBALS
2010;8(l)27-30.
among Fomale Adolescents in amurb
slum of Mumbai. Journal of Family and
reproductive health 2011;5(4): 117 -
Abd Allah E and Elsabagh E. Impact of
health
education
Knowledge
and
Menstruation
Intervention
paretic
among
on
about
female
secondary school students in zigzag
Adima E and A dinma J. perception
and practices on Menstruation among
Nigerian secondary school Girls. Afr
Report Health 2008; 12( 1 ):74-83.
kumor G and Kundan M. Psycho-social
behavior of urban Indian adolescent
girls during menstruation. AMT 201
l;44t48-52.
city. Journal of American science
2011;7(9):737-47.
Omidvarps ,and Begum K . Factors
influencing hygienic practice during
menses among girls from south India .
International journal of collaborative
Research on Internal Medicine and
public Health
Abd EL- Hamed N, and Ahmed E .
Assessment of Dysmenorrheal and
Menstrual Hygiene practices among
Adolescent Girls in some nursing
schools at EL - Minia Governorate .
Egypt. Journal of American science
2011;7(9):216 — 23.
Vol. 13 No. 2 November, 2017
practices about reproductive health
24 .
Nagar S and Aimol K .Knowledge of
Meghalaya.
and
103
2010;2(12):411-23.
Thaker SB ,Reddy M and Pathok K.
Menstrual Hygiene : knowledge and
practice among adolescent school
Girls of sooner Nagpur
Tanta Scientific Nursing Journal
District.
Journal
of
clinical
and
Diagnostic research 2011;5(5) 102733.
Dhingra
R
and
Mushtaq
A.
A
comparative study of understand of
menstrual process among Married
Women Belonging to two Ecological
setting. J. Hum. Eeol 2007;22(3):261-6.
Malleshappa
K
.Knowledge
and
and
Reproductive
Nandinic
attitude
health
about
among
adolescent girls in kuppammandal: An
intervention
study
.
Biomedical
Research 2011;22(3):305-10.
Lamada S. Knowledge , attitudes and
practices
of
regarding
RH
Alexandria
:
adolescent
females
.Doctorate
Faculty
thesis,
Alexandria
university,2004.
Farag
W.
university
students
perspective about reproductive health
needs. Master
faculty
of
thesis, Alexandria:
Nursing,
Alexandria
university,2012
Vol. 13 No. 2 November, 2017
104
Tanta Scientific Nursing Journal
رقــم االيــــداع
2012 ) لسنة207 (
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https://openalex.org/W2988943128
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https://www.biorxiv.org/content/biorxiv/early/2019/07/02/684910.full.pdf
|
English
| null |
Loss of adaptive capacity in asthmatic patients revealed by biomarker fluctuation dynamics after rhinovirus challenge
|
eLife
| 2,019
|
cc-by
| 28,130
|
.
CC-BY 4.0 International license
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certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was not
this version posted July 2, 2019.
;
https://doi.org/10.1101/684910
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bioRxiv preprint . CC-BY 4.0 International license
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bioRxiv preprint . CC-BY 4.0 International license
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The copyright holder for this preprint (which was not
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bioRxiv preprint Loss of adaptive capacity in asthmatics revealed by biomarker fluctuation
dynamics upon experimental rhinovirus challenge Anirban Sinhaa,b,c,1, René Luttera,b, Binbin Xud, Tamara Dekkerb, Barbara Dierdorpb, Peter J. Sterka, Urs Freyc,
Edgar Delgado-Eckertc a Amsterdam UMC, University of Amsterdam, Department of Respiratory Medicine, Meibergdreef 9, 1105 AZ,
Amsterdam, The Netherlands. b Amsterdam UMC, University of Amsterdam, Department of Experimental Immunology, Meibergdreef 9, 1105
AZ, Amsterdam, The Netherlands. b Amsterdam UMC, University of Amsterdam, Department of Experimental Immunology, Meibergdreef 9, 1105
AZ, Amsterdam, The Netherlands. c University of Basel, University Children’s Hospital (UKBB), Spitalstrasse 33, Postfach, 4031 Basel, Switzerland. d University of Bordeaux, Inserm, Bordeaux Population Health Research Center, UMR 1219, F-33000 Bordeaux,
France. 1 Corresponding author. Email: a.sinha@amc.uva.nl, anirban.sinha@ukbb.ch, Phone: +31 20 566 4356 Abstract: Asthma is a dynamic disease, in which lung mechanical and inflammatory processes often
interact in a complex, unpredictable manner. We hypothesize that this may be explained by
respiratory disease-related systems instability and loss of adaptability to changing
environmental conditions, resulting in highly fluctuating biomarkers and symptoms. Using time
series of inflammatory (eosinophils, neutrophils, FeNO), clinical and lung function biomarkers
(PEF, FVC, and FEV1), we estimated this loss of adaptive capacity (AC) during an experimental
perturbation with a rhinovirus in 24 healthy and asthmatic volunteers. Loss of AC was estimated
by comparing similarities between pre- and post-challenge time series. Unlike healthy
participants, the asthmatic’s post-viral-challenge state resembled significantly more other
rhinovirus-infected asthmatics than their own pre-viral-challenge state (hypergeometric-test:
p=0.029). This reveals loss of AC, and supports the novel concept that not only single
physiological mechanisms, but interacting dynamic disease properties are altered in asthma and
contribute to a more vulnerable phenotype. . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was not
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bioRxiv preprint . CC-BY 4.0 International license
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The copyright holder for this preprint (which was not
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bioRxiv preprint Introduction CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was not
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bioRxiv preprint . CC-BY 4.0 International license
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The copyright holder for this preprint (which was not
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bioRxiv preprint adaptive capacity, i.e., its ability to cope with external perturbations (10), is more difficult and
dependent on the physiological context. For instance, researchers have directly linked the
capacity of rats to adapt to environmental heat stresses to the ability of the animal’s liver cells
to rapidly express the heat shock protein HSP70 in high quantities (18). Other scientists have
suggested "the capacity of a physiological system to bring itself autonomously back to the
normal homeostatic range after a challenge" as a more workable definition of adaptive capacity
(19). We found evidence for a loss of adaptive capacity by quantitatively comparing the
similarities of means and fluctuations in the pre- and post-viral-challenge time series of
biomarkers in an unassuming data driven manner, using hierarchical clustering (20) and
statistically assessing the clusters found by means of enrichment analysis (21-23). The aim of this study is to test the hypothesis, whether in asthmatics the adaptive capacity to a
standardized environmental perturbation, such as an experimental viral challenge, is altered in
comparison to healthy subjects. As a proof of concept, such changes in the homeokinetic system
properties in disease would support evidence that not only single factors, such as inflammation,
but also system properties contribute to disease dynamics and phenotype stability. In a prospective, longitudinally designed study comprising healthy and asthmatic subjects, we
measured time series of a set of standard lung functional and inflammatory/immune biomarkers
two months prior to and one month following an experimental rhinovirus 16 (RV16) infection
induced by controlled and deliberate inoculation of healthy and asthmatic volunteers. This
choice was driven by the fact that rhinovirus (RV) infections in asthmatics have been found to
be among the most prominent external triggers of acute worsening of asthma symptoms, and of
asthma exacerbations and loss of control (24, 25). Introduction Adaptive capacity to the rhinovirus infection
was compared between asthmatic and healthy subjects for the abovementioned biomarkers. Introduction The dynamics of (patho)physiology are particularly relevant in chronic diseases. In
uncontrolled conditions, the patients are almost always clinically unstable, exhibiting a
fluctuation pattern of symptoms, including unexpected loss of control or even sudden,
potentially life-threatening exacerbations. Asthma, a chronic airway inflammation with variable
airway obstruction is an archetypical example of such a dynamic disease. It is well known that
the relationship between the strength of the environmental trigger, and the resulting degree of
airway inflammation, airway obstruction, and symptoms is highly nonlinear, thus asthma is
often insufficiently controlled with anti-inflammatory drugs (www.ginasthma.org) (1). The temporal behavior of disease biomarkers in asthma is related to the inherently complex,
interacting pathophysiology of the respiratory system, involving environmental, inflammatory
and airway mechanical components (2). We and others have hypothesized that in health, the
respiratory system exhibits a so-called ‘homeokinetic’ stable equilibrium state, enabling the
respiratory system to easily adapt to an external perturbation (3). However, the homeokinesis
of the respiratory system in asthma may be altered and adaptability to external stimuli (adaptive
capacity) may be decreased, leading to a less stable system with more temporal fluctuations and
exacerbations (3). Thus, asthma instability and fluctuation dynamics may not just relate to the
degree of airway inflammation or the strength of the environmental trigger, but to the innate
properties of the homeokinetic system itself. Homeokinesis is the ability of a physiological system to return to a dynamic equilibrium after
a perturbation (4-7); The remarkable complexity of such systems originates from non-linear
interactions between their constitutive parts (8-10). The healthy homeokinetic respiratory
system is characterized by a stable equilibrium and sufficient adaptability in the face of
changing environmental conditions such as pollutants, pathogens and allergens (10). A
concomitant phenomenon of homeokinesis is the fluctuation behavior of physiological
processes and related physiological parameters and biomarkers around a stable medium value
when measured longitudinally (11, 12). Over the past decades, a considerable research effort
has been invested into mathematically analyzing and characterizing physiologic time series (8,
13-16). Moreover, it has been shown that time series properties, such as unpredictability, long-
range correlations, fractality, and information content are associated with states of health and
disease (10, 17). Such changes in dynamic behavior are potentially related to loss of adaptive
capacity of the complex respiratory system. However, quantifying such loss of the system’s . Experimental rhinovirus challenge while monitoring cohort participants Table 1: Biomarkers/parameters measured in each cohort participant during two months before, and during one
month immediately after deliberate experimental inoculation with rhinovirus. The corresponding sampling
frequencies can be found in columns 2 and 3. See the Methods section below for more details on the study
design, and on the measurement procedures and laboratory assays used. FEV1: forced expiratory volume in one
second. FVC: forced vital capacity. PEF: peak expiratory flow. FeNO: fractional expired concentration of nitric
oxide. Table 1: Biomarkers/parameters measured in each cohort participant during two months before, and during one
month immediately after deliberate experimental inoculation with rhinovirus. The corresponding sampling
frequencies can be found in columns 2 and 3. See the Methods section below for more details on the study
design, and on the measurement procedures and laboratory assays used. FEV1: forced expiratory volume in one
second. FVC: forced vital capacity. PEF: peak expiratory flow. FeNO: fractional expired concentration of nitric
oxide. Hierarchical clustering of biomarker time series In order to quantitatively establish the degree of similarity or “proximity” between two time
series of a given biomarker, we used the Earth Mover’s Distance (EMD), which regards each
of the time series as a univariate empirical distribution of the biomarker at hand (see Methods
and SI for more details). The pre- and post-challenge time series of individual biomarker time
series from all participants (both healthy and asthmatics) were clustered using the EMD as the
distance metric between the time series. The outcomes for the levels of exhaled nitric oxide
(FeNO), and the percentage of eosinophils in nasal lavage fluid are presented here, whereas the
results for the other biomarkers are presented in the SI. Experimental rhinovirus challenge while monitoring cohort participants Experimental rhinovirus challenge while monitoring cohort participants
In all cohort participants (12 healthy and 12 asthmatic volunteers), the biomarkers/parameters
listed in Table 1 below were measured during two months before, and during one month
immediately after deliberate experimental inoculation with rhinovirus, resulting in pre- and
post-viral-challenge time series of each biomarker/parameter. Plots of the time series of each
biomarker can be found in Supplementary Figures File 2. For the healthy and the asthmatics
groups separately, summary statistics of the average before the viral challenge (average over 2 . CC-BY 4.0 International license
under a
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bioRxiv preprint months) and after the viral challenge (average over 1 month) of each of these
biomarkers/parameters can be found in the SI. Biomarker or parameter
Sampling frequency before
rhinovirus challenge
Sampling frequency after
rhinovirus challenge
Lung function (FEV1, FVC,
FEV1/FVC, PEF)
2x daily
2x daily
Exhaled
Nitric
Oxide
(FeNO)
3x weekly
3x weekly
Eosinophil and neutrophil
cell density in nasal lavage
fluid
1x weekly
3x weekly
Table 1: Biomarkers/parameters measured in each cohort participant during two months before, and during one
month immediately after deliberate experimental inoculation with rhinovirus. The corresponding sampling
frequencies can be found in columns 2 and 3. See the Methods section below for more details on the study
design, and on the measurement procedures and laboratory assays used. FEV1: forced expiratory volume in one
second. FVC: forced vital capacity. PEF: peak expiratory flow. FeNO: fractional expired concentration of nitric
oxide. i) Time series of exhaled Nitric Oxide (FeNO) Findings are summarized in Table 2. The corresponding dendrogram is depicted in Figure 1. In
brief, we found three clusters. Cluster 1 consists of four time series stemming from 2 asthmatics. As can be read off of the dendrogram in Fig. 1 below, and of the distance matrix depicted in
Panel C of Fig. 2 (see Methods section below), these two participants are prominently different
from the rest (regarding their FeNO time series), and might be regarded as outliers. Cluster 2
contains more healthy participants than expected by chance. In other words, Cluster 2 is
enriched in healthy participants. Conversely, due to the balanced design of the cohort (equal
numbers of healthy and of asthmatic participants), Cluster 2 is also depleted of asthmatic
participants, i.e., it contains fewer asthmatic participants than expected by chance. And finally,
Cluster 3, which is enriched in asthmatic participants. The marked difference between the . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
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bioRxiv preprint members of Cluster 1 and Cluster 3 regarding FeNO time series revealed by the hierarchical
clustering evidences a higher heterogeneity among the asthmatics, as compared to the healthy
participants. In Cluster 2, the tendency for infected participants to be clustered together with
their corresponding uninfected counterpart is statistically significant (p-value = 0.038, see Table
2 below). This is not the case for Cluster 3. The difference in this regard between Cluster 2
(mainly healthy participants) and Cluster 3 (mainly asthmatic participants) is further
underpinned by the fact that the distributions of cophenetic distances between the infected
cluster members and their uninfected counterparts are statistically significantly different
between these two clusters (p-value=0.033, see also Fig. 3 in the SI). More specifically, an
average of 34.5 in Cluster 3, as opposed to 12.0 in Cluster 2. i) Time series of exhaled Nitric Oxide (FeNO) In Table 3, the sub-clusters found within Clusters 2 and 3, respectively (marked with orange
and blue rectangles in Figure 1), are analyzed in terms of enrichment in or depletion of pre- and
post-challenge time series. This analysis provides evidence for a statistically significant
separation of pre- and post-challenge time series within Cluster 3. Indeed, the union of
subclusters 3.1 and 3.2 is enriched in pre-challenge time series (p-value=0.029, see Table 3
below), whereas subcluster 3.3 is enriched in post-challenge time series (p-value=0.029, see
Table 3 below). Such a separation cannot be observed within Cluster 2. A bootstrap based sensitivity analysis of these findings can be found in the SI. . CC-BY 4.0 International license
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bioRxiv preprint Characteristic \ Cluster number
Cluster 1
Cluster 2
Cluster 3
Size (%)
4 (8.33 %)
26 (54.17 %)
18 (37.5 %)
Fully represented healthy participants
0
11
0
Partially represented healthy participants
0
1
1
Fully represented asthmatic participants
2
1
8
Partially represented asthmatic participants
0
1
1
Number of time series from healthy participants
(%)
0 (0%)
23 (88.46 %)
1 (5.56 %)
p-value
0.055
1.78E-09
1.15E-06
Number of neighboring pre- and post-pairs (%)
2 (100 %)
4 (28.57 %)
2 (20 %)
Empirical p-value
0.003
0.007
0.097
Table 2: Composition, enrichment analysis, and grouping characteristics of the clusters found by comparison of
each participant’s pre- and post-challenge time series of FeNO. Enrichment is marked in bold letters, depletion in
italics; the corresponding p-values were calculated using the hypergeometric test. The empirical p-values for the
proportion of pre- and post-pairs were calculated using simulated permutations (see Methods section). A
participant is fully represented in a given cluster if both their pre- and post-challenge time series of measurements
are contained in the cluster. i) Time series of exhaled Nitric Oxide (FeNO) ;
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bioRxiv preprint Figure 1: Cluster dendrogram obtained via hierarchical clustering of the participants’ pre- and post-challenge time
series of FeNO. The distance between any two-time series was calculated using the EMD. Rectangles mark the
clusters and sub-clusters identified. From top to bottom: Cluster 1, Cluster 2 (subdivided into Clusters 2.1, 2.2,
and 2.3), and Cluster 3 (subdivided into Clusters 3.1 and 3.2, and 3.3). Patient IDs are indicated by Pxy, their
health status using H/A, denoting Healthy or Asthmatic, and their RV infection status by Uninf/Inf, which stands
for Uninfected/Infected. Cluster 1 consists of asthmatics which are prominently different from other asthmatic
subjects in Cluster 3 and also from healthy subjects in Cluster 2. These might be regarded as outliers. Figure 1: Cluster dendrogram obtained via hierarchical clustering of the participants’ pre- and post-challenge time
series of FeNO. The distance between any two-time series was calculated using the EMD. Rectangles mark the
clusters and sub-clusters identified. From top to bottom: Cluster 1, Cluster 2 (subdivided into Clusters 2.1, 2.2,
and 2.3), and Cluster 3 (subdivided into Clusters 3.1 and 3.2, and 3.3). Patient IDs are indicated by Pxy, their
health status using H/A, denoting Healthy or Asthmatic, and their RV infection status by Uninf/Inf, which stands
for Uninfected/Infected. Cluster 1 consists of asthmatics which are prominently different from other asthmatic
subjects in Cluster 3 and also from healthy subjects in Cluster 2. These might be regarded as outliers. Figure 1: Cluster dendrogram obtained via hierarchical clustering of the participants’ pre- and post-challenge time
series of FeNO. The distance between any two-time series was calculated using the EMD. Rectangles mark the
clusters and sub-clusters identified. From top to bottom: Cluster 1, Cluster 2 (subdivided into Clusters 2.1, 2.2,
and 2.3), and Cluster 3 (subdivided into Clusters 3.1 and 3.2, and 3.3). Patient IDs are indicated by Pxy, their
health status using H/A, denoting Healthy or Asthmatic, and their RV infection status by Uninf/Inf, which stands
for Uninfected/Infected. Cluster 1 consists of asthmatics which are prominently different from other asthmatic
subjects in Cluster 3 and also from healthy subjects in Cluster 2. These might be regarded as outliers. i) Time series of exhaled Nitric Oxide (FeNO) For example, the healthy participant "P08H" is fully represented in Cluster 2, as both
their pre- and post-challenge time series of FeNO measurements are members of Cluster 2 (see Fig. 1 below). Partial representation corresponds to the scenario in which only one of the two time series (pre- and post-challenge)
is a member of the cluster. For instance, the asthmatic participant " P07A" is only partially represented in Cluster
2, because their pre-challenge time series of FeNO measurements is part of Cluster 2, whereas their post-challenge
time series of FeNO belongs to Cluster 3 (see Fig. 1 below). See also the Methods section for the definition of
neighbors. Sub-cluster number \ Characteristic Size (%)
Number of pre-challenge series (%)
p-value
Cluster 2.1
7 (26.92 %)
3 (42.86 %)
0.404
Cluster 2.2
11 (42.31 %) 8 (72.73 %)
0.104
Cluster 2.3
8 (30.77 %)
3 (37.5 %)
0.246
Cluster 3.1 & 3.2
13 (72.22 %) 8 (61.54 %)
0.029
Cluster 3.3
5 (27.78 %)
0 (0.00 %)
0.029
Table 3: Enrichment analysis of the sub-clusters found within the clusters described in Table 2 above by
comparison of each participant’s pre- and post-challenge time series of FeNO. Enrichment is marked in bold
letters, depletion in italics; the corresponding p-values were calculated using the hypergeometric test. Table 3: Enrichment analysis of the sub-clusters found within the clusters described in Table 2 above by
comparison of each participant’s pre- and post-challenge time series of FeNO. Enrichment is marked in bold
letters, depletion in italics; the corresponding p-values were calculated using the hypergeometric test. Table 3: Enrichment analysis of the sub-clusters found within the clusters described in Table 2 above by
comparison of each participant’s pre- and post-challenge time series of FeNO. Enrichment is marked in bold
letters, depletion in italics; the corresponding p-values were calculated using the hypergeometric test. . CC-BY 4.0 International license
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this version posted July 2, 2019. ii) Time series of percentage of eosinophils in nasal lavage fluid Findings are summarized in Table 4. The corresponding dendrogram is depicted in Fig.1 in the
SI. In brief, three clusters were identified. Cluster 1 consists of four time series stemming from
3 asthmatics; As can be read off of the dendrogram depicted in Fig. 1 in the SI, these time series . CC-BY 4.0 International license
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bioRxiv preprint are prominently different from all the other time series, and might be regarded as outliers. Cluster 2 is enriched in healthy participants. And finally, Cluster 3, which is enriched in
asthmatic participants. As seen in the analysis of FeNO, the marked difference between the
members of Cluster 1 and Cluster 3 evidences a higher heterogeneity among the asthmatics, as
compared to the healthy participants. However, Cluster 1 in the eosinophil analysis and Cluster
1 in the FeNO analysis only have one asthmatic patient in common. Again, in Cluster 2, the
tendency for infected participants to be clustered together with their corresponding uninfected
counterpart is statistically significant (p-value = 0.001, see Table 4 below). This is not the case
for clusters 1 and 3. The difference in this regard between Cluster 2 (mainly healthy
participants) and Cluster 3 (mainly asthmatic participants) is further substantiated by the fact
that the distributions of cophenetic distances between the infected cluster members and their
uninfected counterparts are statistically significantly different between these two clusters (p-
value= 8.96e-05, one-tailed Mann-Whitney-U-test, see also Fig. 2 in the SI). More specifically,
an average of 36.0 in Cluster 3, as opposed to 0.5 in Cluster 2. ii) Time series of percentage of eosinophils in nasal lavage fluid Characteristic \ Cluster number
Cluster 1
Cluster 2
Cluster 3
Size (%)
4 (8.33 %)
26 (54.17 %)
18 (37.50 %)
Fully represented healthy participants
0
11
1
Partially represented healthy participants
0
0
0
Fully represented asthmatic participants
1
2
7
Partially represented asthmatic participants
2
0
2
Number of time series from healthy participants
(%)
0 (0%)
22 (84.62 %)
2 (11.11 %)
p-value
0.055
1.09E-07
2.89E-05
Number of neighboring pre- and post-pairs (%)
1 (33.33 %)
5 (38.46 %)
1 (10 %)
Empirical p-value
0.123
0.001
0.424
Table 4: Composition, enrichment analysis, and grouping characteristics of the clusters found by comparison of
each participant’s pre- and post-challenge time series of percentage of eosinophils in nasal lavage fluid. Enrichment is marked in bold letters, depletion in italics; the corresponding p-values were calculated using the
hypergeometric test. The empirical p-values for the proportion of pre- and post-pairs were calculated using
simulated permutations (see Methods section). A participant is fully represented in a given cluster if both their pre-
and post-challenge time series of measurements are contained in the cluster. Partial representation corresponds to
the scenario in which only one of the two time series (pre- and post-challenge) is a member of the cluster. See also
the Methods section for the definition of neighbors. Individual response to the viral challenge with respect to the biomarkers measured In order to test the effectiveness of the virus challenge, we measured the individual patient’s
response with respect to each predefined biomarker. The results of blood antibody tests (RV16
seroconversion) along with RV Polymerase Chain Reaction (PCR) conducted on nasal lavage
fluid taken from every participant after the inoculation indicated that 11 out of 12 healthy
participants and 12 out of 12 asthmatics were effectively infected with the RV16 after . CC-BY 4.0 International license
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bioRxiv preprint inoculation (Table 2 in the SI). According to these laboratory tests, one healthy participant did
not become infected. However, this participant did develop cold symptoms within a few days
after the virus inoculation, suggesting that the laboratory tests failed to detect the ongoing
infection although the participant was positively infected. Consequently, this participant was
included in the analyses. After having established the efficacy of the inoculation with RV16, we then explored, for each
of the biomarkers measured (listed in the first column of Table 5), for how many participants a
statistically significant within-subject change upon infection can be observed (“responders”,
see Table 5). To this end, two criteria for “responders” were implemented. The first criterion,
which regards time series as univariate empirical distributions of the biomarker at hand, aimed
at detecting distributional changes in a given biomarker induced by the viral challenge: Here,
each participant’s pre- and post-challenge time series of each biomarker were compared using
the Kolmogorov-Smirnov test. The second criterion aimed at detecting short-term and transient
relative changes induced by the viral challenge in the context of the relative changes observed
prior to the challenge. Here, throughout the entire period of observation, we assessed the
relative change of each biomarker taking place within time intervals of 10 days. (see Subsection
5.2 and Fig. 3 in the Methods section below). Biomarker name
% Healthy
responders
(distributional
changes)
% Asthmatic
responders
(distributional
changes)
% Healthy
responders (relative
change within 10
days)
% Asthmatic
responders
(relative change
within 10 days)
PEF (% of predicted)
50.0%
75.0%
0.0%
0.0%
Normalized FEV1
75.0%
66.7%
0.0%
0.0%
Normalized FVC
83.3%
100.0%
0.0%
0.0%
Normalized FEV1/FVC
75.0%
66.7%
16.7%
0.0%
FeNO
8.3%
0.0%
41.7%
8.3%
Cell density in nasal
lavage fluid
0.0%
0.0%
66.7%
41.7%
Neutrophils in nasal lavage
fluid (%)
0.0%
0.0%
25.0%
16.7%
Eosinophils in nasal lavage
fluid (%)
8.3%
0.0%
0.0%
16.7%
Table 5: Proportions of responders within the groups of healthy and asthmatic participants, respectively. Two different
criteria were used in order to establish a statistically significant response. According to the first criterion, a participant is
considered a responder with respect to a given biomarker if the outcome of comparing the pre-challenge time series and the
post-challenge time series of the same biomarker by means of the Kolmogorov-Smirnov test results in a p-value <= 0.05
(columns 2 and 3). ii) Time series of percentage of eosinophils in nasal lavage fluid According to the second criterion, a participant is considered a responder with respect to a given biomarker
if the outcome of comparing, by means of a Mann-Whitney-U-test, the magnitude of relative changes observed during 10-
day time intervals prior to the challenge with the magnitude of relative changes that took place during 10-day time intervals
that contained the day of the challenge results in a p-value <= 0.05 (columns 4 and 5). For calculating the proportion of
responders within each group the p-values were corrected for multiple testing using the false discovery rate (FDR) method
of Benjamini and Hochberg. FEV1: forced expiratory volume in one second. FVC: forced vital capacity. PEF: peak expiratory
flow. FeNO: fractional expired concentration of nitric oxide. The lung function parameters FEV1 and FVC, and thereby their Table 5: Proportions of responders within the groups of healthy and asthmatic participants, respectively. Two different
criteria were used in order to establish a statistically significant response. According to the first criterion, a participant is
considered a responder with respect to a given biomarker if the outcome of comparing the pre-challenge time series and the
post-challenge time series of the same biomarker by means of the Kolmogorov-Smirnov test results in a p-value <= 0.05
(columns 2 and 3). According to the second criterion, a participant is considered a responder with respect to a given biomarker
if the outcome of comparing, by means of a Mann-Whitney-U-test, the magnitude of relative changes observed during 10-
day time intervals prior to the challenge with the magnitude of relative changes that took place during 10-day time intervals
that contained the day of the challenge results in a p-value <= 0.05 (columns 4 and 5). For calculating the proportion of
responders within each group the p-values were corrected for multiple testing using the false discovery rate (FDR) method
of Benjamini and Hochberg. FEV1: forced expiratory volume in one second. FVC: forced vital capacity. PEF: peak expiratory
flow. FeNO: fractional expired concentration of nitric oxide. The lung function parameters FEV1 and FVC, and thereby their Table 5: Proportions of responders within the groups of healthy and asthmatic participants, respectively. Two different
criteria were used in order to establish a statistically significant response. ii) Time series of percentage of eosinophils in nasal lavage fluid CC-BY 4.0 International license
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bioRxiv preprint ratio FEV1/FVC, were normalized using the standardized reference equations recommended by Global Lung Function
Initiative (GLI) Task Force for comparisons across different populations. ratio FEV1/FVC, were normalized using the standardized reference equations recommended by Global Lung Function
Initiative (GLI) Task Force for comparisons across different populations. The notion of adaptive capacity in a clinical and epidemiological context The notion of adaptive capacity in a clinical and epidemiological context f
p
p
y
p
g
Epidemiological asthma research has provided indirect clues about a loss of adaptive capacity
in the human respiratory system due to asthma. For instance, respiratory comorbidities have
been found to be remarkably more prevalent in asthma than in non-asthma patients (26). Furthermore, provocation tests in asthma diagnostics (recently reviewed in (27)) implicitly rely
on the assumption of loss of adaptive capacity in asthmatics. Loss of adaptive capacity
manifests itself in increased vulnerability to external perturbations and e.g. the well-known fact
that the common cold is a primary driver of asthma exacerbations (24, 28-38). Discussion In this proof of concept study, we provided experimental evidence for the loss of adaptive
capacity in the human respiratory system due to asthma. To this end, we hypothesized that a
loss of adaptive capacity could be experimentally demonstrated by means of a detection of a
similarity diminution between the pre- and post-perturbation dynamics of the system. Using a
data-driven clustering approach, we have shown that, in particular, FeNO and eosinophil time
series were similar prior to and following the challenge in healthy subjects, suggesting stable
homeokinetic behavior. In asthmatics, however, this similarity was predominantly reduced,
suggesting a marked impact of the asthmatic condition on dynamic properties of the respiratory
system, consistent with more unstable behavior and loss of adaptive capacity following the
perturbation with viral infection. Adaptive capacity is a system characteristic of a homekinetic
system. Our data on the respiratory system in asthma and health support the hypothesis that
these homeokinetic system characteristics of the lung likely contribute to the stability and
dynamic phenotype of asthmatic disease. This is particularly astonishing, since the average
FeNO, lung function values, and nasal lavage fluid eosinophil counts are, in general, not
different following a viral challenge. ii) Time series of percentage of eosinophils in nasal lavage fluid According to the first criterion, a participant is
considered a responder with respect to a given biomarker if the outcome of comparing the pre-challenge time series and the
post-challenge time series of the same biomarker by means of the Kolmogorov-Smirnov test results in a p-value <= 0.05
(columns 2 and 3). According to the second criterion, a participant is considered a responder with respect to a given biomarker
if the outcome of comparing, by means of a Mann-Whitney-U-test, the magnitude of relative changes observed during 10-
day time intervals prior to the challenge with the magnitude of relative changes that took place during 10-day time intervals
that contained the day of the challenge results in a p-value <= 0.05 (columns 4 and 5). For calculating the proportion of
responders within each group the p-values were corrected for multiple testing using the false discovery rate (FDR) method
of Benjamini and Hochberg. FEV1: forced expiratory volume in one second. FVC: forced vital capacity. PEF: peak expiratory
flow. FeNO: fractional expired concentration of nitric oxide. The lung function parameters FEV1 and FVC, and thereby their Table 5: Proportions of responders within the groups of healthy and asthmatic participants, respectively. Two different
criteria were used in order to establish a statistically significant response. According to the first criterion, a participant is
considered a responder with respect to a given biomarker if the outcome of comparing the pre-challenge time series and the
post-challenge time series of the same biomarker by means of the Kolmogorov-Smirnov test results in a p-value <= 0.05
(columns 2 and 3). According to the second criterion, a participant is considered a responder with respect to a given biomarker
if the outcome of comparing, by means of a Mann-Whitney-U-test, the magnitude of relative changes observed during 10-
day time intervals prior to the challenge with the magnitude of relative changes that took place during 10-day time intervals
that contained the day of the challenge results in a p-value <= 0.05 (columns 4 and 5). For calculating the proportion of
responders within each group the p-values were corrected for multiple testing using the false discovery rate (FDR) method
of Benjamini and Hochberg. FEV1: forced expiratory volume in one second. FVC: forced vital capacity. PEF: peak expiratory
flow. FeNO: fractional expired concentration of nitric oxide. The lung function parameters FEV1 and FVC, and thereby their . Interpretation of our findings in a physiological context Our study and findings for the first time demonstrate this at the system level using respiratory
biomarker fluctuation dynamics. Loss of adaptive capacity cannot be determined by single-time
point observations of biomarkers, it was, however, possible in a time series experiment
involving asthma biomarkers during a standardized perturbation of the respiratory system. . CC-BY 4.0 International license
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bioRxiv preprint Nevertheless, the following considerations have to be discussed. We started off with the
question: For which type of participant, healthy or asthmatic, and for which biomarker is the
disruption introduced by the viral challenge strong enough to render infected individuals more
similar among themselves than to their uninfected counterparts? Our results indicate that, with
respect to cell density in nasal lavage fluid, there is a statistically significant separation of pre-
and post-challenge time series. However, this effect does not seem to be cluster specific, even
though we found a cluster of size 7 enriched in asthmatic participants within the subgroup of
mainly post-challenge time series (see SI). A more distinctive scenario results from our cluster
analysis of the pre- and post-challenge time series of the percentage of eosinophils in nasal
lavage fluid: In Cluster 2, which is statistically significantly enriched in healthy participants,
the tendency for infected participants to be clustered together with their corresponding
uninfected counterpart is statistically significant and clearly higher than in Cluster 3, which is
mainly composed of asthmatic participants. Finally, in the clustering of the pre- and post-
challenge time series of FeNO we found Cluster 2, mainly composed of healthy participants,
and Cluster 3, made of nearly 95% asthmatics. Furthermore, within Cluster 3 we found a
statistically significant separation of pre- and post-challenge time series. No such separation
was found within Cluster 2. Effectiveness of the perturbation (viral challenge) Our analyses were systematically carried out with the general goal of testing the hypothesis of
a loss of adaptive capacity of the respiratory system in asthmatic cohort participants. However,
towards this goal, it was pertinent to determine whether and in which individuals the viral
inoculation was effective. The presence of cold symptoms and/or positive blood antibody tests
along with RV PCR provide strong evidence of all participants being effectively challenged
shortly after RV16 inoculation. Indeed, RV PCR was performed on day 3 post inoculation and
in all but one participant the presence of RV was detected. This is in concordance with other
studies showing that RV concentrations in the nasal wash could be detected 2-3 days after viral
inoculation, with a signal peak on the 5th/6th day (39, 40). ariable and heterogeneous effect on lung function and inflammatory/immune biomarkers This is in line with the results
of previous studies (41-44), which concluded that after RV challenge lung function in asthmatic
subjects did change, but did not decline dramatically in comparison to the changes observed in
healthy controls. In contrast, our analyses indicate that changes in the inflammatory or
immunological biomarkers at the cellular or molecular level are short-term and transient in
nature (see rows 5-8 in Table 5 above, and Figures in Supplementary Figures File 1). Nevertheless, for these parameters fewer responders were found, when compared to the lung
function parameters. However, our results also hint at a relatively short time scale of response
of these inflammatory/immunological biomarkers. Thus, the sampling frequency used in this
study may not entirely capture the rapidly changing magnitudes. The observed differences in
the type of response between the lung function and the inflammatory/immunological
biomarkers may be a manifestation of the interplay of different temporal and spatial scales. There is a clear macroscopic/functional manifestation of the kindling RV infection, as reflected
at the level of distributional changes induced by the viral challenge on lung function parameters. Indeed, with respect to this criterion, 50% or more statistically significant responders in each
of the two groups (healthy and asthmatics) were found (see rows 1-4 in Table 5 above). Notably,
significant differences found between pre- and post-challenge time series were, in general, not
attributable to changes in the variance, as verified using Levene’s test (results not shown). Nevertheless, for most participants, the lung function parameters did not show short-
term/transient relative changes induced by the viral challenge that were statistically
significantly different in magnitude from short-term changes observed during the pre-challenge
phase (see columns 4 and 5, and rows 1-4 in Table 5 above, and Figures in Supplementary
Figures File 1). Taken together, these results suggest that the changes in lung function elicited
by the viral challenge are, both for healthy and asthmatic participants, subtle, spread over
comparatively longer time periods, and unlike a transient decline. This is in line with the results
of previous studies (41-44), which concluded that after RV challenge lung function in asthmatic
subjects did change, but did not decline dramatically in comparison to the changes observed in
healthy controls. ariable and heterogeneous effect on lung function and inflammatory/immune biomarkers In contrast, our analyses indicate that changes in the inflammatory or
immunological biomarkers at the cellular or molecular level are short-term and transient in
nature (see rows 5-8 in Table 5 above, and Figures in Supplementary Figures File 1). Nevertheless, for these parameters fewer responders were found, when compared to the lung
function parameters. However, our results also hint at a relatively short time scale of response
of these inflammatory/immunological biomarkers. Thus, the sampling frequency used in this
study may not entirely capture the rapidly changing magnitudes. The observed differences in
the type of response between the lung function and the inflammatory/immunological
biomarkers may be a manifestation of the interplay of different temporal and spatial scales. ariable and heterogeneous effect on lung function and inflammatory/immune biomarkers Variable and heterogeneous effect on lung function and inflammatory/immune biomarkers
We carried out a quantitative characterization of individual response to the viral perturbation. This was done using two computational/statistical approaches. One approach aimed to capture
the changes elicited by the viral challenge taking place over longer time periods (comparison
of the per- and post-challenge time series, viewed as empirical distributions), whereas the other . CC-BY 4.0 International license
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bioRxiv preprint assessed relative short-term changes occurring at shorter time scales (comparison of the
magnitude of relative changes observed during 10-day time intervals). g
g
g
y
)
There is a clear macroscopic/functional manifestation of the kindling RV infection, as reflected
at the level of distributional changes induced by the viral challenge on lung function parameters. Indeed, with respect to this criterion, 50% or more statistically significant responders in each
of the two groups (healthy and asthmatics) were found (see rows 1-4 in Table 5 above). Notably,
significant differences found between pre- and post-challenge time series were, in general, not
attributable to changes in the variance, as verified using Levene’s test (results not shown). Nevertheless, for most participants, the lung function parameters did not show short-
term/transient relative changes induced by the viral challenge that were statistically
significantly different in magnitude from short-term changes observed during the pre-challenge
phase (see columns 4 and 5, and rows 1-4 in Table 5 above, and Figures in Supplementary
Figures File 1). Taken together, these results suggest that the changes in lung function elicited
by the viral challenge are, both for healthy and asthmatic participants, subtle, spread over
comparatively longer time periods, and unlike a transient decline. Conclusion and implications Conclusion and implications
We have found evidence supporting the notion that a chronic disease such as asthma may alter
the properties of a homeokinetic physiologic system in a way that compromises its capacity to
appropriately react to a possibly harmful environmental stimulus. This loss of adaptive capacity
in the asthmatic lung may be understood as changes that render the system overly unstable (3). As a proof of concept, such changes in homeokinetic system properties would support evidence
that not only single factors, such as inflammation, but also system properties contribute to
disease dynamics and phenotype stability. This systems-level understanding of chronic asthma may open up new avenues for the
understanding of asthma and other chronic dynamic diseases. Already in this small sample size,
it is obvious that there is remarkable individual temporal variability in inflammatory and
physiological biomarkers, not only in disease but also in health. Thus, dynamic fluctuations of
physiological processes around an equilibrium state, including their related biomarkers, are an
intrinsic feature of the respiratory system. Moreover, even in health there are strong inter-
individual differences in these dynamic characteristics. Nevertheless, within a given healthy
subject fluctuations remain similar following the virus challenge, indicating that, these dynamic
fluctuations seem to be in a stable equilibrium state in health. This characteristic is lost in
asthma. Future studies involving time series of biomarker measurements may help us understand this
system instability in chronic asthma, even in the absence of severe airway inflammation or
obstruction. Furthermore, future therapeutic approaches may want to focus on maintaining a
stable homeokinetic equilibrium of the respiratory system, rather than just normalizing single
physiological or inflammatory biomarkers. Materials and Methods This study was approved by the medical ethical committee from the Amsterdam University
Medical Center and registered at the Netherlands Trial Register (NTR5426). Limitations of the study Our findings need to be judged in light of the limitations of our study. One of the limitations is
that we only included mild asthmatics for ethical reasons. However, more severe asthmatics are
likely to be on corticosteroid treatment, which may potentially influence the inflammatory
response, therefore introducing a confounding factor. Another potential shortcoming of this
study is the relatively small sample size. However, this drawback is compensated for by the
unprecedented high sampling frequency at which the participants were screened in our study. . CC-BY 4.0 International license
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bioRxiv preprint In fact, the sample size was a compromise between the number of subjects and the number of
assessment visits. Nevertheless, as discussed above, the sampling frequency for some of the
inflammatory/immunological biomarkers may still not have been high enough in order to
capture their temporal dynamics. In fact, the sample size was a compromise between the number of subjects and the number of
assessment visits. Nevertheless, as discussed above, the sampling frequency for some of the
inflammatory/immunological biomarkers may still not have been high enough in order to
capture their temporal dynamics. 1. Participant cohort . CC-BY 4.0 International license
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bioRxiv preprint Twelve non-smoking, atopic (as determined by positive skin prick test to common aero
allergens), mild to moderate asthmatic subjects (based on ATS/ERS criteria), not using steroids
were chosen for inclusion. Similarly, 12 non-smoking, non-atopic healthy subjects were also
included in the study as controls. All participants provided written informed consent. The
demographics of the study population are summarized in Table 7. All the participants were
required to have their serum antibody titer of RV16 < 1:8 during screening. The age group for
the study population was 18-30 years. Individuals with concomitant disease and pregnant
women were excluded. The basic inclusion criteria for the study populations followed standard recommendations and
were as shown in Table 6 below. Healthy
Asthmatics
No history of episodic chest symptoms
History of episodic chest symptoms
Baseline FEV1 ≥ 80 % predicted
Baseline FEV1 ≥ 70% predicted
AHR to methacholine (PC20) ≥ 19.6 mg/ml
AHR to methacholine (PC20) £ 9.8 mg/ml
SPT negative for all 12 common
Aeroallergens
SPT positive for at least 1 out of 12 common
Aeroallergens
Table 6: Basic characteristics of the study population. FEV1: forced expiratory volume in one second, AHR:
Airway Hyper Responsiveness, PC20: Provocative Concentration causing a 20% fall in FEV1, SPT: Skin Prick basic inclusion criteria for the study populations followed standard recommendations an
as shown in Table 6 below. 1. Participant cohort Demographic features
Healthy
Asthmatic
Total number, n
12
12
Female gender, n (%)
7 (58.3%)
8 (66.7%)
Age (years), mean (SD)
21 ± 1.5
22.2 ± 2.2
Ethnicity (Caucasian), n (non-Caucasian, n)
11
9
BMI, mean (SD)
22.2 ± 1.6
22.8 ± 3.1
Smoking (pack years), n
1 (0.17 PY)
--
Height (centimeters)
177.7 ± 8.6
172.5 ± 13.0
Weight (KG)
70.4 ± 10.1
67.8 ± 12.4
Baseline spirometry
FEV1 %predicted
105.7 ± 11.6
101.0 ± 10.0
FVC %predicted
104.2 ± 10.5
104.2 ± 10.2
PEF %predicted
108.4 ± 14.0
104.7 ± 12.2
mean ± standard deviation
Table 7: The demographics of the study population. BMI is Body Mass Index. Only 1 healthy subject smoked 2
pack years or less 2 years before recruitment to our study, which is considered an insignificant smoking history. FEV1: forced expiratory volume in one second. PEF: peak expiratory flow. 2 S
d D
i Table 7: The demographics of the study population. BMI is Body Mass Index. Only 1 healthy subject smoked 2
pack years or less 2 years before recruitment to our study, which is considered an insignificant smoking history. FEV1: forced expiratory volume in one second. PEF: peak expiratory flow. Table 7: The demographics of the study population. BMI is Body Mass Index. Only 1 healthy subject smoked 2
pack years or less 2 years before recruitment to our study, which is considered an insignificant smoking history. FEV1: forced expiratory volume in one second. PEF: peak expiratory flow. Table 7: The demographics of the study population. BMI is Body Mass Index. Only 1 healthy subject smoked 2
pack years or less 2 years before recruitment to our study, which is considered an insignificant smoking history. FEV1: forced expiratory volume in one second. PEF: peak expiratory flow. 2. Study Design The project represents a prospective observational, follow-up study including patients with
asthma and healthy controls with an experimental RV intervention. . CC-BY 4.0 International license
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bioRxiv preprint The study participants were recruited after meticulous screening of volunteers (as mentioned in
SI). The study was mainly divided into 2 phases. Phase 1 (stable phase) consisted of 2 months
where these subjects were followed up and sampled diligently every alternate day (3 times a
week for most of the measurements) with constant frequency at the hospital clinic. After that
they were subjected to a standardized nasal dose of RV inoculation in the laboratory and
followed up at the same frequency for one additional month (also called Phase 2 or unstable
phase). In total the study consisted of 3 months of sampling period with a minimum of 180
measurements of lung function, 33 FeNO data points, and 20 cytokine and cell count
measurements per subject. The schematic work flow of the phases mentioned, is provided in the Fig 10 of the SI. 3.1 Lung function assessment Spirometry was performed only once on the screening visit at the clinic to include participants
based on inclusion criteria using a daily calibrated spirometer according to European
Respiratory Society (ERS) recommendations (45). Home monitoring of morning and evening lung function was done by hand held devices (Micro
Diary, CareFusion, yielding the FEV1, FVC, FEV1/FVC and PEF values analyzed in this study. Moreover, the Asthma Control Questionnaire was administered. 3.2 Exhaled Nitric Oxide (FeNO) Measurement of fractionated exhaled nitric oxide (FENO) was performed using the NIOX
MINO® (Aerocrine AB, Sweden). Single measurements per person were recorded at the clinic,
thrice weekly, according to recommendations by the ATS (46). Refer to SI for further details. Refer to SI for further details. 3.3 Nasal lavage Measurements of biomarkers
Frequency before
rhinovirus
challenge
Frequency after rhinovirus challenge
Lung function with pocket-
size
spirometers
(FEV1,
FVC, FEV1/FVC, PEF)
2x daily
2x daily
Exhaled
Nitric
Oxide
(FeNO)
3x weekly
3x weekly
Differential cell counts
1x weekly
3x weekly
Asthma Control
Questionnaire
2x daily
2x daily
Spirometry
Performed once during screening to include subjects in the
study
Methacholine challenge
Performed once during screening to include subjects in the
study
Rhinovirus challenge
Performed after 2 months into the study Table 8: The overview of different measurements performed in the study along with the frequency of sampling
before and after rhino-virus challenge. Measures 1-4 include repeated measurements and 5,6 represent one-time
measurement to screen the subjects for the study. 8 refers to the experimental intervention in the study. FEV1:
forced expiratory volume in one second. FVC: forced vital capacity. PEF: peak expiratory flow. FeNO: fractional
expired concentration of nitric oxide. Table 8: The overview of different measurements performed in the study along with the frequency of sampling
before and after rhino-virus challenge. Measures 1-4 include repeated measurements and 5,6 represent one-time
measurement to screen the subjects for the study. 8 refers to the experimental intervention in the study. FEV1:
forced expiratory volume in one second. FVC: forced vital capacity. PEF: peak expiratory flow. FeNO: fractional
expired concentration of nitric oxide. Table 8: The overview of different measurements performed in the study along with the frequency of sampling
before and after rhino-virus challenge. Measures 1-4 include repeated measurements and 5,6 represent one-time
measurement to screen the subjects for the study. 8 refers to the experimental intervention in the study. FEV1:
forced expiratory volume in one second. FVC: forced vital capacity. PEF: peak expiratory flow. FeNO: fractional
expired concentration of nitric oxide. 3.3 Nasal lavage Nasal lavage was collected from the study participants once weekly before RV challenge and
was up scaled to thrice weekly after the challenge at the clinic as previously described (42)
[Refer to SI for details]. Table 8 provides an overview of the different sample measurements along with their frequency
before and after rhinovirus challenge. Table 8 provides an overview of the different sample measurements along with their frequency
before and after rhinovirus challenge. . CC-BY 4.0 International license
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certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
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bioRxiv preprint Measurements of biomarkers
Frequency before
rhinovirus
challenge
Frequency after rhinovirus challenge
Lung function with pocket-
size
spirometers
(FEV1,
FVC, FEV1/FVC, PEF)
2x daily
2x daily
Exhaled
Nitric
Oxide
(FeNO)
3x weekly
3x weekly
Differential cell counts
1x weekly
3x weekly
Asthma Control
Questionnaire
2x daily
2x daily
Spirometry
Performed once during screening to include subjects in the
study
Methacholine challenge
Performed once during screening to include subjects in the
study
Rhinovirus challenge
Performed after 2 months into the study
Table 8: The overview of different measurements performed in the study along with the frequency of sampling
before and after rhino-virus challenge. Measures 1-4 include repeated measurements and 5,6 represent one-time
measurement to screen the subjects for the study. 8 refers to the experimental intervention in the study. FEV1:
forced expiratory volume in one second. FVC: forced vital capacity. PEF: peak expiratory flow. FeNO: fractional
expired concentration of nitric oxide. 4. Rhinovirus challenge The study participants were exposed to rhinovirus 16 (RV16) using a standardized and validated
challenge approach, based on previous studies by ourselves and other groups (47-52). All
participants were screened for the presence of respiratory viruses just before the challenge, to
rule out a concomitant infection resulting in a cold (see SI for more details). Those participants
with a positive outcome of this test were excluded from the study. An experimental RV16
infection was induced by using a relatively low-dose inoculum of 100 TCID50 (Tissue Culture
Infective Dose determining the amount of virus required to cause cytopathy in 50% of the cells)
to mimic a natural exposure. The study protocol along with the viral dose used and its safety
have been approved by the institutional Medical Ethics Committee in Amsterdam University
Medical Centre, the details of which have been included in SI. Data from our previous study
show that a low dose is sufficient to induce mild cold-symptoms (52, 53). Furthermore, this
low-dose inoculum previously resulted in a slight decrease of FEV1 (loss of asthma control) in
asthmatic patients between day 4 and 6 after RV16 exposure, whereas no decrease has been
observed in healthy controls (50). 5. Statistical and computational Analysis Statistical tests resulting in a p-value less or equal to 0.05 were regarded as significant. . CC-BY 4.0 International license
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bioRxiv preprint 5.1 Assessment of differences: Pre- vs. post-viral-challenge 5.1 Assessment of differences: Pre- vs. post-viral-challenge CC-BY 4.0 International license
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bioRxiv preprint Figure 2: Panel A depicts two pre-challenge time series of FeNO obtained from a healthy (blue curve), and from
an asthmatic (red curve) participant, respectively. In Panel B, each of the time series is represented as empirical
distribution. This representation of the two-time series allows for the calculation of a distance or “dissimilarity”
between the two by means of the Earth Mover’s Distance (EMD). The EMD-comparison of all possible pairs of
time series (both pre- and post-challenge) results in a symmetric matrix of pair-wise distances, as shown in Panel
C using a color-coded (violet to green) heat-map. Each row in this matrix corresponds to one time series. The color
bar on the left hand side of the matrix encodes the “type” of time series: Cyan marks a pre-challenge time series
originating from a healthy participant; Blue marks a post-challenge time series originating from a healthy
participant; Pink marks a pre-challenge time series originating from an asthmatic participant; Red marks a post-
challenge time series originating from an asthmatic participant. The information stored in the matrix of pair-wise
distances is then used within an agglomerative clustering algorithm in order to group the time series in different
clusters. The outcome of this procedure is represented using a dendrogram as depicted in Figure 1 above. 5 2 C l
l ti
f h
t t
/t
i
t h Figure 2: Panel A depicts two pre-challenge time series of FeNO obtained from a healthy (blue curve), and from
an asthmatic (red curve) participant, respectively. In Panel B, each of the time series is represented as empirical
distribution. This representation of the two-time series allows for the calculation of a distance or “dissimilarity”
between the two by means of the Earth Mover’s Distance (EMD). The EMD-comparison of all possible pairs of
time series (both pre- and post-challenge) results in a symmetric matrix of pair-wise distances, as shown in Panel
C using a color-coded (violet to green) heat-map. Each row in this matrix corresponds to one time series. 5.1 Assessment of differences: Pre- vs. post-viral-challenge For each participant, their time series of a given biomarker prior to and after the viral challenge
were compared. This comparison was based on the Kolmogorov-Smirnov test, whereby the
time series were treated as empirical distributions, thus disregarding the chronological order of
the measurements. Differences in the variance between the pre- and post-challenge distributions were assessed
using Levene’s test (54). Differences in the variance between the pre- and post-challenge distributions were assessed
using Levene’s test (54). Multiple comparison correction was performed where required, using the false discovery rate
(FDR) method of Benjamini and Hochberg (55), setting the expected proportion of falsely
rejected null hypotheses to 0.05. The time series of a given biomarker, prior to and after the viral challenge, were regarded as
empirical distributions and compared to each other using the Earth Mover’s Distance (EMD)
(56). The resulting pair-wise distances between distributions were then used for hierarchical
clustering of pre- and post-challenge distributions. See Fig. 2 below and the SI for more details. Our clustering approach makes use of the entire time series (distributions) of values measured
before and after the challenge, respectively, and does not amalgamate the information into a
single magnitude (e.g., the mean value). This method unveils subtle differences and similarities
between the participants’ measurements that are less likely to be captured by conventional
methods based on averages. The time series of a given biomarker, prior to and after the viral challenge, were regarded as
empirical distributions and compared to each other using the Earth Mover’s Distance (EMD)
(56). The resulting pair-wise distances between distributions were then used for hierarchical
clustering of pre- and post-challenge distributions. See Fig. 2 below and the SI for more details. Our clustering approach makes use of the entire time series (distributions) of values measured
before and after the challenge, respectively, and does not amalgamate the information into a
single magnitude (e.g., the mean value). This method unveils subtle differences and similarities
between the participants’ measurements that are less likely to be captured by conventional
methods based on averages. . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
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bioRxiv preprint . 5.1 Assessment of differences: Pre- vs. post-viral-challenge The color
bar on the left hand side of the matrix encodes the “type” of time series: Cyan marks a pre-challenge time series
originating from a healthy participant; Blue marks a post-challenge time series originating from a healthy
participant; Pink marks a pre-challenge time series originating from an asthmatic participant; Red marks a post-
challenge time series originating from an asthmatic participant. The information stored in the matrix of pair-wise
distances is then used within an agglomerative clustering algorithm in order to group the time series in different
clusters. The outcome of this procedure is represented using a dendrogram as depicted in Figure 1 above. 5.2 Calculation of short-term/transient changes 5.2 Calculation of short-term/transient changes For each participant individually, and for each biomarker, throughout the entire period of
observation, the biomarker’s relative change in value taking place within time intervals of 10
days was calculated. This choice of time interval length was made based on published literature
whereby 5 days post exposure to respiratory viruses was shown to be critical. Hence a 10-day
window for comparison would include 5 days before challenge to contrast with 5 days after
challenge (57). This was done throughout the entire period of observation considering all . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
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bioRxiv preprint possible time intervals consisting of 10 consecutive days. In order to assess the statistical
significance of the short-term relative changes possibly elicited by the viral challenge, the
magnitude of relative changes observed during 10-day time intervals starting at least 10 days
prior to the challenge were compared, by means of a Mann-Whitney-U-test, to the magnitude
of relative changes that took place during 10-day time intervals that contained the day of the
challenge. See Fig. 3 below and the SI for more details. Figure 3: Panel A: Graphical representation of a biomarker time series ti. For the calculation of short-term/transient
changes, a gliding interval or window is moved, one day at a time, along the time series. The relative change
between the first and last entry of the gliding window is calculated, resulting in a new time series of short-term
relative changes ri. In Panel B a healthy participant’s time series of short-term relative changes in FeNO is depicted. A gliding interval of size 10 days was used to calculate it from the participant’s time series of FeNO measurements. The start position of the gliding window is expressed relative to the day of the viral challenge, which is marked as
day 0. 5.2 Calculation of short-term/transient changes When the position of the gliding window was such that the day of the viral challenge was contained within
the gliding window, the corresponding value of the relative change is marked in red. In order to assess the statistical
significance of the short-term relative changes possibly elicited by the viral challenge, the relative change values
located to the left of those marked in red were compared to the values marked in red by means of a Mann-Whitney-
U-test. Visual inspection of the time series in Panel B correctly suggests that the outcome of this test is not
significant. The reason being that the relative changes within time intervals of 10 days observed prior to the viral
challenge are comparable to changes observed within intervals of the same length containing the day of the viral
challenge. In Panel C, depicting data from a different healthy participant, the situation is clearly different, as
verified by a significant outcome of the corresponding Mann-Whitney-U-test. In such cases, the participant is
called a “responder” with respect to the “relative change within 10 days criterion”. Figure 3: Panel A: Graphical representation of a biomarker time series ti. For the calculation of short-term/transient
changes, a gliding interval or window is moved, one day at a time, along the time series. The relative change
between the first and last entry of the gliding window is calculated, resulting in a new time series of short-term
relative changes ri. In Panel B a healthy participant’s time series of short-term relative changes in FeNO is depicted. A gliding interval of size 10 days was used to calculate it from the participant’s time series of FeNO measurements. The start position of the gliding window is expressed relative to the day of the viral challenge, which is marked as
day 0. When the position of the gliding window was such that the day of the viral challenge was contained within
the gliding window, the corresponding value of the relative change is marked in red. In order to assess the statistical
significance of the short-term relative changes possibly elicited by the viral challenge, the relative change values
located to the left of those marked in red were compared to the values marked in red by means of a Mann-Whitney-
U-test. Visual inspection of the time series in Panel B correctly suggests that the outcome of this test is not
significant. 5.2 Calculation of short-term/transient changes The reason being that the relative changes within time intervals of 10 days observed prior to the viral
challenge are comparable to changes observed within intervals of the same length containing the day of the viral
challenge. In Panel C, depicting data from a different healthy participant, the situation is clearly different, as
verified by a significant outcome of the corresponding Mann-Whitney-U-test. In such cases, the participant is
called a “responder” with respect to the “relative change within 10 days criterion”. 5.3 Characterization of the dendrogram clusters 5.3 Characterization of the dendrogram clusters In order to evaluate the discriminatory power of a given biomarker, the clusters found in the
clustering dendrogram were tested for enrichment in or depletion of healthy or asthmatic . CC-BY 4.0 International license
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bioRxiv preprint participants, and/or for enrichment in or depletion of pre- or post-challenge distributions. Statistically significant enrichment or depletion were established using the hypergeometric test
(21-23). participants, and/or for enrichment in or depletion of pre- or post-challenge distributions. Statistically significant enrichment or depletion were established using the hypergeometric test
(21-23). The relative location of leaves in the clustering dendrogram was quantitatively evaluated using
the cophenetic distance (58). The cophenetic distance between two leaves of a dendrogram is
defined as the height of the dendrogram at which the two largest branches that individually
contain the two leaves merge into a single branch. For every cohort participant and any given biomarker there is a pre-challenge and a post-
challenge time series, which we call the participant’s pre- and post-pair. If the disruption caused
by the viral challenge is not strong enough, the pre- and post-challenge distributions of a given
participant will tend to cluster together. Therefore, a cluster in which pre- and post-pairs are
closely located in terms of the cophenetic distance within the dendrogram, represents a
subgroup of participants for which the viral challenge caused a relatively weaker disruption, at
least with respect to the biomarker under scrutiny. Two dendrogram leaves are called neighbors if their mutual cophenetic distance is equal to the
minimum of all cophenetic distances from one of the leaves to all the other leaves in the
dendrogram. If this condition is fulfilled for both leaves simultaneously, then the two leaves
form a two-element cluster in the dendrogram. Competing interests None of the authors have any competing interest/s related to this manuscript. 5.3 Characterization of the dendrogram clusters If the condition is only fulfilled for one of the
leaves, the two are still considered neighbors, even if this is not always visually obvious from
inspecting the dendrogram (see Figure 11 in the SI). Under the null-hypothesis that the branching in the dendrogram is the result of a purely random
process, the number of neighboring pre- and post-pairs to be expected just by chance within a
given cluster can be estimated by simply permuting the labels of the leaves in the dendrogram
and counting the number of neighboring pre- and post-pairs. This permutation test is used for
calculating the empirical p-values displayed in Tables 2 and 4 above. A participant is fully represented in a given cluster if both their pre- and post-challenge time
series of measurements are contained in the cluster. For example, the healthy participant
"P08H" is fully represented in Cluster 2, as both their pre- and post-challenge time series of
FeNO measurements are members of Cluster 2 (see Fig. 1 above). Partial representation
corresponds to the scenario in which only one of the two time series (pre- and post-challenge)
is a member of the cluster. For instance, the asthmatic participant " P07A" is only partially
represented in Cluster 2, because their pre-challenge time series of FeNO measurements is part . CC-BY 4.0 International license
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bioRxiv preprint of Cluster 2, whereas their post-challenge time series of FeNO belongs to Cluster 3 (see Fig. 1
above). Acknowledgments The salary of AS was sponsored from the European Respiratory Society-Marie Sklodowska
Curie actions COFUND RESPIRE 2 fellowships (MCF-7077-2014) and also from a grant
supported by Lungenliga Schweiz (2017_14). The work was supported by an unrestricted grant from Chiesi Pharmaceuticals, institutional
funding from the Academic Medical Centre, Amsterdam UMC, University of Amsterdam
(IA601011). References 1. Chung KF. Clinical management of severe therapy-resistant asthma. Expert Review of Respiratory
Medicine. 2017;11(5):395-402. ( )
2. Frey U, Suki B. Complexity of chronic asthma and chronic obstructive pulmonary disease: implication
for risk assessment, and disease progression and control. The Lancet. 2008;372(9643):1088-99. 2. Frey U, Suki B. Complexity of chronic asthma and chronic obstructive pulmonary disease: implications
for risk assessment, and disease progression and control. The Lancet. 2008;372(9643):1088-99. 3. Frey U, Maksym G, Suki B. Temporal complexity in clinical manifestations of lung disease. Journal of
Applied Physiology. 2011;110(6):1723-31. 3. Frey U, Maksym G, Suki B. Temporal complexity in clinical manifestations of lung disease. Journal of
Applied Physiology. 2011;110(6):1723-31. 4. Macklem PT. Emergent phenomena and the secrets of life. Journal of Applied Physiology. 2008;104(6):1844-6. 4. Macklem PT. Emergent phenomena and the secrets of life. Journal of Applied Physiology. 2008;104(6):1844-6. ( )
5. Macklem PT, Seely A. Towards a definition of life. Perspect Biol Med. 2010;53(3):330-40. Macklem PT, Seely A. Towards a definition of life. Perspect Biol Med. 2010;53(3):330-40. ,
y
p
;
( )
6. Que C-L, Kenyon CM, Olivenstein R, Macklem PT, Maksym GN. Homeokinesis and short-term
variability of human airway caliber. Journal of Applied Physiology. 2001;91(3):1131-41. 6. Que C-L, Kenyon CM, Olivenstein R, Macklem PT, Maksym GN. Homeokinesis and short-term
variability of human airway caliber. Journal of Applied Physiology. 2001;91(3):1131-41. y
y
pp
y
gy
( )
7. Yates FE. The 10th JAF Stevenson Memorial Lecture Outline of a physical theory of physiological
systems. Canadian journal of physiology and pharmacology. 1982;60(3):217-48. 7. Yates FE. The 10th JAF Stevenson Memorial Lecture Outline of a physical theory of physiological
systems. Canadian journal of physiology and pharmacology. 1982;60(3):217-48. 8. Garfinkel A. A mathematics for physiology. The American journal of physiology. 1983;245(4)
66. 9. Goldberger AL. Non-linear dynamics for clinicians: chaos theory, fractals, and complexity at the
bedside. Lancet. 1996;347(9011):1312-4. 9. Goldberger AL. Non-linear dynamics for clinicians: chaos theory, fractals, and complexity at the
bedside. Lancet. 1996;347(9011):1312-4. (
)
10. Goldberger AL, Amaral LA, Hausdorff JM, Ivanov P, Peng CK, Stanley HE. Fractal dynamics in
physiology: alterations with disease and aging. Proceedings of the National Academy of Sciences of the United
States of America. 2002;99 Suppl 1:2466-72. 11. Glass L. Synchronization and rhythmic processes in physiology. Nature. 2001;410:277. k
i
k
l h
i
i
f
l
i
i 12. Eke A, Herman P, Kocsis L, Kozak LR. References A survey of exact inference for contingency tables. Statistical Science. 1992:131-5 22. Fleiss JL, Levin B, Paik MC. Statistical Methods for Rates and Proportions: Wiley; 2013. 23. Rivals I, Personnaz L, Taing L, Potier M-C. Enrichment or depletion of a GO category within a class of
genes: which test? Bioinformatics. 2007;23(4):401-7. g
;
( )
24. Johnston SL, Pattemore PK, Sanderson G, Smith S, Lampe F, Josephs L, et al. Community study of role
f i l i f
ti
i
b ti
f
th
i 9 11
ld hild
BMJ 1995 310(6989) 1225 g
( )
24. Johnston SL, Pattemore PK, Sanderson G, Smith S, Lampe F, Josephs L, et al. Community study of role
of viral infections in exacerbations of asthma in 9-11 year old children. BMJ. 1995;310(6989):1225. y
(
)
25. Nicholson KG, Kent J, Ireland DC. Respiratory viruses and exacerbations of asthma in adults. Br Med
J. 1993;307(6910):982. 26. Su X, Ren Y, Li M, Zhao X, Kong L, Kang J. Prevalence of Comorbidities in Asthma and Nonasthma
Patients: A Meta-analysis. Medicine (Baltimore). 2016;95(22):e3459-e. 27. Comberiati P, Katial RK, Covar RA. Bronchoprovocation Testing in Asthma: An Update. Immunology
and Allergy Clinics of North America. 2018;38(4):545-71. 28. Bizzintino J, Lee WM, Laing IA, Vang F, Pappas T, Zhang G, et al. Association between huma
rhinovirus C and severity of acute asthma in children. Eur Respir J. 2011;37(5):1037-42. 29. Corne JM, Marshall C, Smith S, Schreiber J, Sanderson G, Holgate ST, et al. Frequency, severity, and
duration of rhinovirus infections in asthmatic and non-asthmatic individuals: a longitudinal cohort study. The
Lancet. 2002;359(9309):831-4. 30. Heymann PW, Carper HT, Murphy DD, Platts-Mills TAE, Patrie J, McLaughlin AP, et al. Viral
infections in relation to age, atopy, and season of admission among children hospitalized for wheezing. Journal
of Allergy and Clinical Immunology. 2004;114(2):239-47. gy
gy
;
( )
31. Johnston SL, Pattemore PK, Sanderson G, Smith S, Campbell MJ, Josephs LK, et al. The relationship
between upper respiratory infections and hospital admissions for asthma: a time-trend analysis. American
Journal of Respiratory and Critical Care Medicine. 1996;154(3):654-60. p
y
( )
32. Khetsuriani N, Lu X, Teague WG, Kazerouni N, Anderson LJ, Erdman DD. Novel human rhinoviruses
and exacerbation of asthma in children. Emerging infectious diseases. 2008;14(11):1793-6. 33. Miller EK, Edwards KM, Weinberg GA, Iwane MK, Griffin MR, Hall CB, et al. A novel group of
rhinoviruses is associated with asthma hospitalizations. References Fractal characterization of complexity in temporal
physiological signals. Physiol Meas. 2002;23(1):R1. 13. Amigó JM, Small M. Mathematical methods in medicine: neuroscience, cardiology and pathology. Philosophical transactions Series A, Mathematical, physical, and engineering sciences. 2017;375(2096):20170016. (
)
14. Glass L, Kaplan D. Time series analysis of complex dynamics in physiology and medicine. Medical
progress through technology. 1993;19:115-. 15. Glass L, Mackey MC. From clocks to chaos : the rhythms of life. Princeton, N.J.: Princeton University
Press; 1988. xvii, 248 p. p. . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was not
this version posted July 2, 2019. ;
https://doi.org/10.1101/684910
doi:
ioRxiv preprint . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was not
this version posted July 2, 2019. ;
https://doi.org/10.1101/684910
doi:
bioRxiv preprint 16. Goldberger AL, Amaral LAN, Glass L, Hausdorff JM, Ivanov PC, Mark RG, et al. PhysioBank,
PhysioToolkit, and PhysioNet : Components of a New Research Resource for Complex Physiologic Signals. Circulation. 2000;101(23):e215-e20. ;
(
)
17. Bélair J. Dynamical Disease: Mathematical Analysis of Human Illness: AIP Press; 1995. 18. Hall DM, Xu L, Drake VJ, Oberley LW, Oberley TD, Moseley PL, et al. Aging reduces adaptiv
capacity and stress protein expression in the liver after heat stress Journal of Applied Physiology 18. Hall DM, Xu L, Drake VJ, Oberley LW, Oberley TD, Moseley PL, et al. Aging reduces adaptive
capacity and stress protein expression in the liver after heat stress. Journal of Applied Physiology. 2000;89(2):749-59. 19. Orešic M, Vidal-Puig A. A systems biology approach to study metabolic syndrome. 2014. 20
Gan G Ma C Wu J Data Clustering: Theory Algorithms and Applications: Society for Ind 20. Gan G, Ma C, Wu J. Data Clustering: Theory, Algorithms, and Applications: Society for Industrial and
Applied Mathematics (SIAM, 3600 Market Street, Floor 6, Philadelphia, PA 19104); 2007. 20. Gan G, Ma C, Wu J. Data Clustering: Theory, Algorithms, and Applications: Society for Industrial and
Applied Mathematics (SIAM, 3600 Market Street, Floor 6, Philadelphia, PA 19104); 2007. Applied Mathematics (SIAM, 3600 Market Street, Floor 6, Philadelphia, PA 19104); 2007. 21. Agresti A. .
CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was not
this version posted July 2, 2019.
;
https://doi.org/10.1101/684910
doi:
bioRxiv preprint References It is made available
The copyright holder for this preprint (which was not
this version posted July 2, 2019. ;
https://doi.org/10.1101/684910
doi:
bioRxiv preprint 43. Rape M, Jentsch S. Taking a bite: proteasomal protein processing. Nature cell biology. 2002;4(5):E113-
6. 44. Zhu J, Message SD, Qiu Y, Mallia P, Kebadze T, Contoli M, et al. Airway inflammation and illness
severity in response to experimental rhinovirus infection in asthma. Chest. 2014;145(6):1219-29. y
p
p
( )
45. Miller MR, Hankinson J, Brusasco V, Burgos F, Casaburi R, Coates A, et al. Standardisation of
spirometry Eur Respir J 2005;26(2):319 38 Miller MR, Hankinson J, Brusasco V, Burgos F, Casaburi R, Coates A, et al. Standardisation of
ry. Eur Respir J. 2005;26(2):319-38. 45. Miller MR, Hankinson J, Brusasco V, Burgos F, Casaburi R, Coates A, et al. Standardisation of
spirometry. Eur Respir J. 2005;26(2):319-38. 46. Dweik RA, Boggs PB, Erzurum SC, Irvin CG, Leigh MW, Lundberg JO, et al. An official ATS clinical
practice guideline: interpretation of exhaled nitric oxide levels (FENO) for clinical applications. Am J Respir
Crit Care Med. 2011;184(5):602-15. 47. DeMore JP, Weisshaar EH, Vrtis RF, Swenson CA, Evans MD, Morin A, et al. Similar colds in subjects
with allergic asthma and nonatopic subjects after inoculation with rhinovirus-16. The Journal of allergy and
clinical immunology. 2009;124(2):245-52, 52 e1-3. 48. Gern JE, Calhoun W, Swenson C, Shen G, Busse WW. Rhinovirus infection preferentially increases
lower airway responsiveness in allergic subjects. Am J Respir Crit Care Med. 1997;155(6):1872-6. b
h
h d
ijk
l k
l
hi
i 49. Grunberg K, Sharon RF, Sont JK, In 't Veen JC, Van Schadewijk WA, De Klerk EP, et al. Rhinovirus-
induced airway inflammation in asthma: effect of treatment with inhaled corticosteroids before and during
experimental infection. Am J Respir Crit Care Med. 2001;164(10 Pt 1):1816-22. 50. Grunberg K, Timmers MC, de Klerk EP, Dick EC, Sterk PJ. Experimental rhinovirus 16 infection
causes variable airway obstruction in subjects with atopic asthma. Am J Respir Crit Care Med. 1999;160(4):1375-80. 51. Mallia P, Message SD, Kebadze T, Parker HL, Kon OM, Johnston SL. An experimental model of
rhinovirus induced chronic obstructive pulmonary disease exacerbations: a pilot study. Respir Res. 2006;7:116. 52. Traves SL, Proud D. Viral-associated exacerbations of asthma and COPD. Curr Opin Pharmacol. 2007;7(3):252-8. 53. Ravi A, Koster J, Dijkhuis A, Bal SM, Sabogal Piñeros YS, Bonta PI, et al. References Journal of Allergy and Clinical Immunology. 2009;123(1):98-104.e1. ;
( )
34. Miller EK, Poehling KA, Edwards KM, Zhu Y, Griffin MR, Hartert TV, et al. Rhinovirus-Associated
Hospitalizations in Young Children. The Journal of Infectious Diseases. 2007;195(6):773-81. 35. Murray CS, Poletti G, Kebadze T, Morris J, Woodcock A, Johnston SL, et al. Study of modifiable risk
factors for asthma exacerbations: virus infection and allergen exposure increase the risk of asthma hospital
admissions in children. Thorax. 2006;61(5):376. 36. Olenec JP, Kim WK, Lee W-M, Vang F, Pappas TE, Salazar LEP, et al. Weekly monitoring of children
with asthma for infections and illness during common cold seasons. The Journal of allergy and clinical
immunology. 2010;125(5):1001-6.e1. 37. Seemungal T, Harper-Owen R, Bhowmik A, Moric I, Sanderson G, Message S, et al. Respiratory
Viruses, Symptoms, and Inflammatory Markers in Acute Exacerbations and Stable Chronic Obstructive
Pulmonary Disease. American Journal of Respiratory and Critical Care Medicine. 2001;164(9):1618-23. 38. Smuts HE, Workman LJ, Zar HJ. Human rhinovirus infection in young African children with a
wheezing. BMC infectious diseases. 2011;11:65-. 39. Quint JK, Donaldson GC, Goldring JJ, Baghai-Ravary R, Hurst JR, Wedzicha JA. Serum IP-10 as a
biomarker of human rhinovirus infection at exacerbation of COPD. Chest. 2010;137(4):812-22. ( )
40. Wark PA, Bucchieri F, Johnston SL, Gibson PG, Hamilton L, Mimica J, et al. IFN-gamma-induced
protein 10 is a novel biomarker of rhinovirus-induced asthma exacerbations. The Journal of allergy and clinical
immunology. 2007;120(3):586-93. gy
( )
41. Bardin PG, Fraenkel DJ, Sanderson G, van Schalkwyk EM, Holgate ST, Johnston SL. Peak expiratory
flow changes during experimental rhinovirus infection. Eur Respir J. 2000;16(5):980-5. g
g
p
p
;
( )
42. de Kluijver J, Evertse CE, Sont JK, Schrumpf JA, van Zeijl-van der Ham CJ, Dick CR, et al. Are
rhinovirus-induced airway responses in asthma aggravated by chronic allergen exposure? Am J Respir Crit Care
Med. 2003;168(10):1174-80. . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was not
this version posted July 2, 2019. ;
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under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. ( )
Sokal RR, Rohlf FJ. The Comparison of Dendrograms by Objective Methods. Taxon. 1962;11(2):33-40. References ;
https://doi.org/10.1101/684910
doi:
bioRxiv preprint References Interferon-induced
epithelial response to rhinovirus-16 in asthma relates to inflammation and FEV1. Journal of Allergy and Clinical
Immunology. 2018. Levene H. Robust tests for equality of variances. Contributions to Probability and Statistics Essays in
r of Harold Hotelling. 1961:279-92. 55. Benjamini Y, Hochberg Y. Controlling the False Discovery Rate: A Practical and Powerful Approach to
Multiple Testing. Journal of the Royal Statistical Society Series B (Methodological). 1995;57(1):289-300. 56. Rubner Y, Tomasi C, Guibas LJ, editors. A metric for distributions with applications to image 55. Benjamini Y, Hochberg Y. Controlling the False Discovery Rate: A Practical and Powerful Approach to
Multiple Testing. Journal of the Royal Statistical Society Series B (Methodological). 1995;57(1):289-300. 55. Benjamini Y, Hochberg Y. Controlling the False Discovery Rate: A Practical and Powerful Approach to
Multiple Testing. Journal of the Royal Statistical Society Series B (Methodological). 1995;57(1):289-300. 56. Rubner Y, Tomasi C, Guibas LJ, editors. A metric for distributions with applications to image
databases. Computer Vision, 1998 Sixth International Conference on; 1998: IEEE. p
g
y
y
(
g
)
( )
56. Rubner Y, Tomasi C, Guibas LJ, editors. A metric for distributions with applications to image
databases. Computer Vision, 1998 Sixth International Conference on; 1998: IEEE. 57. Denlinger LC, Sorkness RL, Lee W-M, Evans MD, Wolff MJ, Mathur SK, et al. Lower Airway
Rhinovirus Burden and the Seasonal Risk of Asthma Exacerbation. American Journal of Respiratory and Critical
Care Medicine. 2011;184(9):1007-14. ;
( )
58. Sokal RR, Rohlf FJ. The Comparison of Dendrograms by Objective Methods. Taxon. 1962;11(2):33-40. . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was not
this version posted July 2, 2019. ;
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doi:
bioRxiv preprint . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
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this version posted July 2, 2019. Results Effectiveness of the viral inoculation: Each participant in the study was administered the same dose of the virus (100 TCD 50) through
the nose and every subject was tested for being positive for the virus after inoculation. False
positive results due to previous exposure to the virus was ruled out by strict inclusion criteria
of not having the titer of antibodies against RV16 > 1:8 in serum, measured at screening and
prior to inoculation. Positivity to viral inoculation was confirmed by either one of these three criteria 1) Positive test for antibodies against RV at the terminal visits of the participant
2) Positive RV PCR test from approximately 3rd day (second visit) post RV challenge The effect of the virus in every study participant is summarized below in Table 1 (Column 1
indicates the seroconversion of the antibodies against the virus at the end of the study, Column
2 reflects the response to the virus by the PCR product on the day 3 after challenge and finally
Column 3 shows the symptoms developed in the volunteers after the viral inoculation). Positive
response in either of the 3 categories was considered evidence for a successful viral challenge. Study
volunteers
Responders
by
seroconversion
Responders
by
RVPCR
Description
of
symptoms
01A
1
1
Running nose, blocked
nose and cough
02A
0
1
Sore throat, blocked
nose, full head,
coughing and sneezing. 04A
1
0
Slight symptoms of
cold, very mild
05A
0
1
No clear symptoms
06A
1
1
Symptoms of cold . CC-BY 4.0 International license
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bioRxiv preprint 07A
1
1
Minor sore throat,
dripping nose,
shortness of breath. Results 08A
1
0
Sore throat, Probably
very mild effect, no
other symptoms
09A
0
1
Running nose, head
ache, fever
10A
1
1
Very mild symptoms
11A
1
1
Very mild symptoms
12A
0
1
Cough, blocked nose
13A
0
1
No clear symptoms
01H
0
1
Sore throat
03H
0
1
Sore throat and
blocked nose
05H
1
1
Cough, blocked nose
and headache
06H
0
1
No clear symptoms
observed
07H
1
0
Sneezing, itchy eyes
and a little bit of a
cough
08H
1
1
Little bit sore throat
09H
1
1
Sore throat and little
cough
11H
0
0
Blocked nose and
sputum
12H
1
1
Running nose and
sneeze
13H
1
1
No clear symptoms
14H
0
1
Cough and blocked
nose
15H
1
0
No clear symptoms Table 1: Effectiveness of the viral challenge in asthmatic and healthy participants, respectively. A participant is
considered to have a successful viral inoculation if any one of the 3 tests is positive. 1 indicates positive response
and 0 indicates a failed response in the corresponding tests indicated in columns. Table 1: Effectiveness of the viral challenge in asthmatic and healthy participants, respectively. A participant is
considered to have a successful viral inoculation if any one of the 3 tests is positive. 1 indicates positive response
and 0 indicates a failed response in the corresponding tests indicated in columns. Percentage of eosinophils in nasal lavage fluid The corresponding dendrogram is depicted in Fig. 1 below. . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
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bioRxiv preprint Figure 1: Dendrogram obtained from clustering the participants’ tim
nasal lavage fluid using the EMD. Figure 1: Dendrogram obtained from clustering the participants’ time series of the percentage of eosinophils in
nasal lavage fluid using the EMD. Figure 1: Dendrogram obtained from clustering the participants’ time series of the percentage of eosinophils in
nasal lavage fluid using the EMD. . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
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bioRxiv preprint Figure 2: The boxplot to the left represents the distribution of cophenetic distances between time series
corresponding to infected healthy participants and their uninfected counterparts. Only time series belonging to
Cluster 2 in the clustering dendrogram obtained using the percentage of eosinophils in nasal lavage fluid (see Fig. 1 above) are contemplated here. The boxplot to the right represents the distribution of cophenetic distances between
time series corresponding to infected asthmatic participants and their uninfected counterparts. Only time series
belonging to Cluster 3 in the clustering dendrogram obtained using the percentage of eosinophils in nasal lavage
fluid (see Fig. 1 above) are contemplated here. The two distributions are statistically significantly different (p-
value= 8.96e-05, one-tailed Mann-Whitney-U-test, the cophenetic distances in Cluster 3 being, on average, higher
than the ones in Cluster 2. Percentage of eosinophils in nasal lavage fluid Only time series belonging to
Cluster 3 in the clustering dendrogram obtained using FeNo data (see Fig. 1 in the Main Manuscript) are
contemplated here. The two distributions are statistically significantly different (p-value=0.033, one-tailed Mann-
Whitney-U-test, the cophenetic distances in Cluster 3 being, on average, higher than the ones in Cluster 2. FeNO (Exhaled Nitric Oxide)
Figure 3: The boxplot to the left represents the distribution of cophenetic distances between time series
corresponding to infected healthy participants and their uninfected counterparts. Only time series belonging to
Cluster 2 in the clustering dendrogram obtained using FeNo data (see Fig. 1 in the Main Manuscript) are
contemplated here. The boxplot to the right represents the distribution of cophenetic distances between time series
corresponding to infected asthmatic participants and their uninfected counterparts. Only time series belonging to
Cluster 3 in the clustering dendrogram obtained using FeNo data (see Fig. 1 in the Main Manuscript) are
contemplated here. The two distributions are statistically significantly different (p-value=0.033, one-tailed Mann-
Whitney-U-test, the cophenetic distances in Cluster 3 being, on average, higher than the ones in Cluster 2. Figure 3: The boxplot to the left represents the distribution of cophenetic distances between time series
corresponding to infected healthy participants and their uninfected counterparts. Only time series belonging to
Cluster 2 in the clustering dendrogram obtained using FeNo data (see Fig. 1 in the Main Manuscript) are
contemplated here. The boxplot to the right represents the distribution of cophenetic distances between time series
corresponding to infected asthmatic participants and their uninfected counterparts. Only time series belonging to
Cluster 3 in the clustering dendrogram obtained using FeNo data (see Fig. 1 in the Main Manuscript) are
contemplated here. The two distributions are statistically significantly different (p-value=0.033, one-tailed Mann-
Whitney-U-test, the cophenetic distances in Cluster 3 being, on average, higher than the ones in Cluster 2. Percentage of eosinophils in nasal lavage fluid Figure 2: The boxplot to the left represents the distribution of cophenetic distances between time series
corresponding to infected healthy participants and their uninfected counterparts. Only time series belonging to
Cluster 2 in the clustering dendrogram obtained using the percentage of eosinophils in nasal lavage fluid (see Fig
1 above) are contemplated here. The boxplot to the right represents the distribution of cophenetic distances between
time series corresponding to infected asthmatic participants and their uninfected counterparts Only time series Figure 2: The boxplot to the left represents the distribution of cophenetic distances between time series
corresponding to infected healthy participants and their uninfected counterparts. Only time series belonging to
Cluster 2 in the clustering dendrogram obtained using the percentage of eosinophils in nasal lavage fluid (see Fig. 1 above) are contemplated here. The boxplot to the right represents the distribution of cophenetic distances between
time series corresponding to infected asthmatic participants and their uninfected counterparts. Only time series
belonging to Cluster 3 in the clustering dendrogram obtained using the percentage of eosinophils in nasal lavage
fluid (see Fig. 1 above) are contemplated here. The two distributions are statistically significantly different (p-
value= 8.96e-05, one-tailed Mann-Whitney-U-test, the cophenetic distances in Cluster 3 being, on average, higher
than the ones in Cluster 2. . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
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bioRxiv preprint FeNO (Exhaled Nitric Oxide) FeNO (Exhaled Nitric Oxide) FeNO (Exhaled Nitric Oxide)
Figure 3: The boxplot to the left represents the distribution of cophenetic distances between time series
corresponding to infected healthy participants and their uninfected counterparts. Only time series belonging to
Cluster 2 in the clustering dendrogram obtained using FeNo data (see Fig. 1 in the Main Manuscript) are
contemplated here. The boxplot to the right represents the distribution of cophenetic distances between time series
corresponding to infected asthmatic participants and their uninfected counterparts. Cell density in nasal lavage fluid: We found one cluster of size 25 enriched in pre-challenge time series (p= 0.041), and one cluster
of size 7 enriched in asthmatics (p=0.049). See Fig. 2 . CC-BY 4.0 International license
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bioRxiv preprint Figure 4: Dendrogram obtained from clustering the participants’ time serie
nasal lavage fluid using the EMD. Percentage of neutrophils in nasal lavage fluid Fi
4 D
d
bt i
d f
l
t i
th
ti i
t Figure 4: Dendrogram obtained from clustering the participants’ time series of cell density (millions per ml) in
nasal lavage fluid using the EMD. Percentage of neutrophils in nasal lavage fluid Figure 4: Dendrogram obtained from clustering the participants’ time series of cell density (millions per ml) in
nasal lavage fluid using the EMD. Percentage of neutrophils in nasal lavage fluid Figure 4: Dendrogram obtained from clustering the participants’ time series of cell density (millions per ml) in
nasal lavage fluid using the EMD. Figure 4: Dendrogram obtained from clustering the participants’ time series of cell density (millions per ml) in
nasal lavage fluid using the EMD. Percentage of neutrophils in nasal lavage fluid . CC-BY 4.0 International license
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bioRxiv preprint We found one cluster of size 10 enriched in time series from healthy participants (p= 0.036). Figure 5: Dendrogram obtained from clustering the participants’ time series of the percentage of neutrophils in
nasal lavage fluid using the EMD. Cell density in nasal lavage fluid: Moreover, no clusters were found showing a significant enrichment in or depletion of pre-
challenge or post-challenge time series. challenge or post-challenge time series. Figure 5: Dendrogram obtained from clustering the participants’ time series of the percentage of neutrophils in
nasal lavage fluid using the EMD. Figure 5: Dendrogram obtained from clustering the participants’ ti
nasal lavage fluid using the EMD. Figure 5: Dendrogram obtained from clustering the participants’ time series of the percentage of neutrophils in
nasal lavage fluid using the EMD. Figure 5: Dendrogram obtained from clustering the participants’ time series of the percentage of neutrophils in
nasal lavage fluid using the EMD. . CC-BY 4.0 International license
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bioRxiv preprint Lung function parameters Lung function parameters
Figure 6: Dendrogram obtained from clustering the participants’ time series of the normalized ratio FEV1/FVC
using the EMD. Figure 6: Dendrogram obtained from clustering the participants’ time series of the normalized ratio FEV1/FVC
using the EMD. . CC-BY 4.0 International license
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bioRxiv preprint e 7: Dendrogram obtained from clustering the participants’ time series Figure 7: Dendrogram obtained from clustering the participants’ time series of PEF (% predicted) using the EMD. . CC-BY 4.0 International license
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bioRxiv preprint Cell density in nasal lavage fluid: It is made available
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bioRxiv preprint re 8: Dendrogram obtained from clustering the participants’ time series of normalized FEV1 using the EMD. Figure 8: Dendrogram obtained from clustering the participants’ time series of normalized FEV1 using the EMD. Figure 8: Dendrogram obtained from clustering the participants’ time series of normalized FEV1 using the EMD. . CC-BY 4.0 International license
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bioRxiv preprint re 9: Dendrogram obtained from clustering the participants’ time series of ure 9: Dendrogram obtained from clustering the participants’ time series of normalized FVC using the EM Figure 9: Dendrogram obtained from clustering the participants’ time series of normalized FVC using the EMD. Figure 9: Dendrogram obtained from clustering the participants’ time series of normalized FVC using the EMD. . CC-BY 4.0 International license
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bioRxiv preprint . Sensitivity analysis of main clustering results: We investigated the sensitivity of the clustering of FeNO time series to changes in the data via
nonparametric bootstrapping. However, given that the post-viral challenge time series are very
short (eleven data points or fewer), resampling would be strongly affected by small sample size
effects [1]. Thus, we only applied bootstrapping to the pre-challenge time series. Moreover, we
resorted to soft bootstrapping [2] (see Methods below for more details) in order to increase the
likelihood that least frequent values in the time series would be chosen during the resampling
procedure. The results from 1000 soft bootstrapping iterations were as follows: In 100% of the resulting soft bootstrap dendrograms, Cluster 1 (“outliers” cluster) was found. Moreover, two additional clusters, one, Cluster 2’, significantly enriched in time series
stemming from heathy participants, and another, Cluster 3’, significantly enriched in time series
stemming from asthmatic participants were found in 100% of the resulting soft bootstrap
dendrograms. In other words, there was always a bootstrap counterpart to clusters 1,2, and 3 as
found in the dendrogram obtained using the original, unperturbed data (see Figure 1 in the Main
Manuscript). In 51.4% of the soft bootstrap dendrograms, Cluster 3’ (the cluster enriched in time series
stemming from asthmatic participants) contained a subcluster enriched in post-challenge time
series. Whereas, only in 7.9% of the soft bootstrap dendrograms, Cluster 2’ (the cluster
enriched in time series stemming from healthy participants) contained a subcluster enriched in
post-challenge time series. (cf. Table 3 in the Main Manuscript). Bootstrap distribution of mean cophenetic distances between the members of all pre- and post-
pairs contained in Cluster 2’: Min. 1st Qu. Median Mean 3rd Qu. Max. 6.358 11.017 11.954 13.527 14.947 26.654 Min. 1st Qu. Median Mean 3rd Qu. Max.
6.358 11.017 11.954 13.527 14.947 26.654 Min. 1st Qu. Median Mean 3rd Qu. Max. 6.358 11.017 11.954 13.527 14.947 26.654 Bootstrap distribution of mean cophenetic distances between the members of all pre- and post-
pairs contained in Cluster 3’: Min. 1st Qu. Median Mean 3rd Qu. Max. 7.036 21.581 33.691 29.321 35.610 49.080 In 80.6% of the soft bootstrap dendrograms, the mean cophenetic distances between the
members of all pre- and post-pairs contained in Cluster 2’ was smaller than the mean cophenetic
distances between the members of all pre- and post-pairs contained in Cluster 3’. . CC-BY 4.0 International license
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bioRxiv preprint Bootstrap distribution of the p-values resulting from the one-tailed Mann-Whitney-U-test
comparing the distribution of cophenetic distances between time series corresponding to
infected healthy participants and their uninfected counterparts in Cluster 2’, to the distribution
of cophenetic distances between time series corresponding to infected asthmatic participants
and their uninfected counterparts in Cluster 3’ (cf. Figure 3 above): Min. 1st Qu. Median Mean 3rd Qu. Max. 0.0005046 0.0163020 0.0397435 0.1563541 0.2208664 0.9537708 In 54.2% of the soft bootstrap dendrograms the resulting p-value was smaller or equal to 0.05. Bootstrap distribution of the percentage of neighboring pre- and post-pairs in Cluster 2’:
Min. 1st Qu. Median Mean 3rd Qu. Max. 0.00 14.29 19.90 19.13 23.53 42.86
Bootstrap distribution of the percentage of neighboring pre- and post-pairs in Cluster 3’:
Min. 1st Qu. Median Mean 3rd Qu. Max. 8.33 20.00 25.00 28.63 37.50 80.00 In 54.2% of the soft bootstrap dendrograms the resulting p-value was smaller or equal to 0.05. ootstrap distribution of the percentage of neighboring pre- and post-pairs in Cluster 2’: In 27.1% of the soft bootstrap dendrograms, the percentage of neighboring pre- and post-pairs
in Cluster 2’was bigger than the percentage of neighboring pre- and post-pairs in Cluster 3’ (cf. Table 2 in the Main Manuscript). Min. 1st Qu. Median Mean 3rd Qu. Max.
6.358 11.017 11.954 13.527 14.947 26.654 Bootstrap distribution of the empirical p-values resulting from the permutation test used for
establishing the statistical significance of the proportion of neighboring pre- and post-pairs
found in Cluster 2’: Min. 1st Qu. Median Mean 3rd Qu. Max. 0.00000 0.00596 0.03780 0.12118 0.15008 1.00000 Min. 1st Qu. Median Mean 3rd Qu. Max. 0.00000 0.00596 0.03780 0.12118 0.15008 1.00000 Min. 1st Qu. Median Mean 3rd Qu. Max. 0.00000 0.00596 0.03780 0.12118 0.15008 1.00000 In 52.7% of the soft bootstrap dendrograms, the resulting empirical p-value was smaller or
equal to 0.05. Bootstrap distribution of the empirical p-values resulting from the permutation test used for
establishing the statistical significance of the proportion of neighboring pre- and post-pairs
found in Cluster 3’: Min. 1st Qu. Median Mean 3rd Qu. Max. 0.000000 0.004287 0.014640 0.080993 0.080477 0.450480 0.000000 0.004287 0.014640 0.080993 0.080477 0.450480 . CC-BY 4.0 International license
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bioRxiv preprint In 57.5% of the soft bootstrap dendrograms, the resulting empirical p-value was smaller or
equal to 0.05. In 57.5% of the soft bootstrap dendrograms, the resulting empirical p-value was smaller or
equal to 0.05. Soft bootstrapping of the time series of the percentage of eosinophils in nasal lavage fluid
yielded comparable results (data not shown). Materials and Methods Materials and Methods 1. Participant cohort 24 study subjects were recruited among which 12 were healthy volunteers and the other 12
steroids naïve (or stopped using steroids 6 weeks prior to the study) mild to moderately
persistent asthmatics. The inclusion and exclusion criteria for participants to enter the study were as follows:
1.1 Inclusion criteria 1.1 Inclusion criteria Steroid-naïve, non-atopic participants who are currently not on any maintenance
(subjects using oral contraceptives can be accepted) Baseline FEV1 ≥ 80% of predicted • Negative acetyl-ß-methylcholine bromide (MeBr) challenge or PC20 ≥ 19.6 mg/ml • Negative skin prick test (SPT) to one or more of the 12 common aeroallergen extra • Negative history of pulmonary and any other relevant disease • Able to give written and dated informed consent prior to any study-specific procedure. 1.2 Exclusion criteria Potential subjects who meet any of the following criteria were excluded from participation in
the study: Potential subjects who meet any of the following criteria were excluded from participation in
the study: • Women who are pregnant, lactating or have a positive urine pregnancy test at baseline
visit • Participation in any clinical investigational drug treatment protocol within the preceding
5 half-lives (or 12 weeks, if the half-life is unknown) before the screening visit of this
study. • Concomitant disease or condition which could interfere with the conduct of the study,
or for which the treatment might interfere with the conduct of the study, or which would,
in the opinion of the investigator, pose an unacceptable risk to the patient Furthermore, the following additional exclusion criteria were used in phase 2 of the study: • RV16 titer > 1:8 in serum, measured at screening (visit 1) and also at the visit prior to
the challenge • History of clinically significant hypotensive episodes or symptoms of fainting,
dizziness, or light-headedness • History of an asthma exacerbation within the last 6 weeks prior to the study • Has had any acute illness, including a common cold, within 4 weeks prior to visit • Close contact with young children or with any immunosuppressed patients • Has donated blood or has had a blood loss of more than 450 mL within 60 days prior to
screening visit or plans to donate blood during the study • Positive for any virus in nasal lavage at visit immediately prior to rhinovirus challenge. 1.3 Sample size calculation 1.1 Inclusion criteria Asthma patients were selected using the following inclusion criteria: • Age 18-50 years • Age 18-50 years • History of episodic chest tightness and wheezing • Intermittent or mild to moderate persistent asthma according to the criteria by the Global
Initiative for Asthma (Global Initiative of Asthma. www.ginasthma.org) • Non-smoking or stopped smoking more than 12 months ago and 5 pack years or les • Non-smoking or stopped smoking more than 12 months ago and 5 pack years or less
• Clinically stable, no exacerbations within last six weeks prior to study • Steroid-naïve or those participants who are currently not on corticosteroids and have not
taken any corticosteroids by any dosing-routes within 6 weeks prior to the study or only
using on-demand reliever therapy • Baseline pre-bronchodilator FEV1 ≥ 70% of predicted [3] • Airway hyperresponsiveness, indicated by a positive acetyl-ß-methylcholine bromide
(MeBr)challenge with PC20£ 9.8 mg/ml [4] • Positive skin prick test (SPT) to one or more of the 12 common aeroallergen extracts,
defined as a wheal with an average diameter of ³ 3 mm • No other clinically significant abnormality on history and clinical examination • Able to give written and dated informed consent prior to any study-specific procedures
Healthy subjects were selected using the following inclusion criteria • Age 18-50 years . CC-BY 4.0 International license
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bioRxiv preprint • Non-smoking or stopped smoking more than 12 months ago and 5 pack years or less. 2.3 Baseline visit The medication history (also adverse events and concomitant medication) was carefully
recorded and thereby routine FeNO, and nasal lavage were measured. Pregnancy test was also
performed. 2.1 Screening Visit First, the subjects were asked to sign an informed consent form after which they were examined
for inclusion and exclusion criteria along with medical history and scoring of adverse events
and concomitant medication. Spirometry, Methacholine challenge and Skin Prick Test along
with physical examinations were performed meticulously. 1.3 Sample size calculation . CC-BY 4.0 International license
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bioRxiv preprint The study is explorative in nature and is based on some first estimates of fluctuating
inflammatory biomarkers. A definite sample size for all biomarkers measured was not possible
to be accurately determined. A sample size of 12/12, based on previous studies with lesser data points, was calculated to
identify biomarkers that have a strong, clinically relevant, sensitivity to the changes of disease
stability over time [5, 6]. The sample size will probably have missed effects on biomarkers
with weak effects, nevertheless, it has for all biomarkers screened provided evidence on long
time variability, correlation and cross-correlation between biomarker properties, and
susceptibility to strong viral external challenges. These three dimensions are a first estimate to
understand the degree of variability and complexity of any disease process. 2.2 Run-in Phase During this phase adverse events and medication were recorded along with physical
examination. Exhaled nitric oxide (FeNO), nasal lavage, measurements were performed to
acquaint the study subjects with the study procedures. Starting from the run-in, once-daily
home-monitoring by Asthma Control Questionnaires, symptom scores and twice daily
maneuvers by the pocket spirometer were executed. 2. Study Design The project represents a prospective observational, follow up study including patients with
asthma and healthy controls with an experimental rhinovirus intervention in between. 2.1 Screening Visit 2.5 End of study visit All measurements were repeated at the end of each phase. The schematic representation of the phases of the study is mentioned below in Fig. 10
Figure 10: Schematic representation of the study design. he schematic representation of the phases of the study is mentioned below in Fig. 10 The schematic representation of the phases of the study is mentioned below in Fig. 10 Figure 10: Schematic representation of the study design. 3.1.1 Asthma Control Questionnaire The Asthma Control Questionnaire (ACQ) was used in this study to assess the symptoms before
and after Rhinovirus challenge along with lung function coupled to a hand-held spirometer
device. Though widely used and well validated, it covers a 7-day time span and may not be
optimal for a challenge protocol wherein symptoms change daily. Hence ACQ was used on a
daily basis to record the daily changes in symptoms. Subjects complete the diary on rising in
the morning and retiring at bedtime. 2.4 Study period • Phase 1: The study participants were asked to visit the hospital clinic thrice weekly for the
aforementioned measurements on Monday, Wednesday and Fridays, during the first 60 days. • Phase 2: The participants were again followed up for the same measurements for the next 30
days with the same sampling frequency after being inoculated nasally with the common cold • Phase 1: The study participants were asked to visit the hospital clinic thrice weekly for the
aforementioned measurements on Monday, Wednesday and Fridays, during the first 60 days. y,
y
y ,
g
y
• Phase 2: The participants were again followed up for the same measurements for the next 30
days with the same sampling frequency after being inoculated nasally with the common cold . CC-BY 4.0 International license
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bioRxiv preprint virus Rhinovirus 16, thereby mimicking the trigger for loss of asthma control or a mild
exacerbation. virus Rhinovirus 16, thereby mimicking the trigger for loss of asthma control or a mild
exacerbation. All participants in the study were screened for the presence of respiratory viruses just before
the rhinovirus challenge, using Polymerase Chain Reaction confirmation on nasal lavage
samples. This was performed in order to rule out a concomitant infection of the respiratory
airways with viruses other than the one used in our study. 3.1.2 Skin prick test Allergy skin prick tests were performed based on the position paper by the European Academy
of Allergology and Clinical Immunology (EAACI) [7]. The test was done by placing a drop of . CC-BY 4.0 International license
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bioRxiv preprint each of the 12 solutions containing common aeroallergens on the skin, followed by needle
pricks by small SPT-lancets. Histamine was used as a positive control and saline solution as a
negative control. The test result was considered positive if the skin develops a red, itchy area,
called a ‘wheal’, with an average diameter of at least 3 mm, 15 minutes after the prick. The
outline of all wheals was marked with a water-soluble marker and transferred to a test result
from using adhesive tape, so as to facilitate measuring of the diameter of the wheal. All test
result forms were archived. If the patient has been using anti-allergic medications, they were
instructed to stop 3 days prior to the test in the clinic. 3.1.5 Methacholine challenge Methacholine is a cholinergic synthetic analogue of acetylcholine and acts directly on the
airway smooth muscle resulting in bronchoconstriction. Measurement of airway responsiveness
can be done using incremental inhaled doses of methacholine as bronchoprovocation test. This
challenge test was performed using MeBr (acetyl-β-methylcholine bromide) according to the
standardized tidal volume method that is operative in routine clinical diagnostics in the hospital
[4]. 3.1.4 Exhaled nitric oxide Measurements of fractionated exhaled nitric oxide (FENO) were performed (one measurement
per subject) using the NIOX MINO® (Aerocrine AB, Sweden). This analyser has an accuracy
of ± 5 ppb or max 10%. The precision is < 3 ppb of a measured value < 30 ppb and ± 10 % of
measured value > 30 ppb. Every individual subject was measured using a single measurement
as standardised in the hospital. Subjects were asked to perform a slow vital capacity manoeuvre with a standardised expiratory
flow of 50 ml/sec for as long as possible. Positive mouth pressure was applied to close the
velum and prevent contamination with NO from nose and paranasal sinuses. Expired gas was
sampled continuously at the mouth piece and mean FENO values at the end expiratory plateau
were calculated. PEF: Peak Expiratory Flow All the home recordings were monitored regularly (every 2 weeks) for uniformity and
consistency of data. Stringent quality control of home lung function measurements was done at
each visit. FEV1/FVC: Ratio of the amount of air exhaled in 1st second to the amount of air exhaled during
a maximal expiration. FEV1/FVC: Ratio of the amount of air exhaled in 1st second to the amount of air exhaled during
a maximal expiration. a maximal expiration. 3.1.3 Spirometry Spirometry was performed using a daily calibrated spirometer according to European
Respiratory Society (ERS) recommendations. Spirometry was done only once on the screening
visit at the clinic. There was no use of bronchodilators (pre/post measurements) in the study. The subjects were asked not to use reliever medications at least 6 hours before the test starts in
the clinic. An experienced lung function analyst from the AMC performed the lung function
tests throughout the study to reach optimal performance and to enhance reproducibility. Spirometry (FEV1) results were printed out and documented. Home monitoring of morning and
evening lung function were done by Peak Expiratory Flow Meter (PEF, Micro Diary,
CareFusion). Waking and bedtime FEV1 separately are of interest due to the exaggerated diurnal airflow
variation seen in asthmatics. Subjects were encouraged to measure FEV1 at consistent times
(upon waking between approximately 6am-9am and prior to bedtime between approximately
8pm-11pm, respectively – always prior to any bronchodilator therapy). Forced expiratory flow 25–75% (FEF 25–75) was measured. Patients were systematically
instructed to perform the home monitoring PEF measurements. The electronic PEF devices
were tested and compared to the lab spirometry measurements. Home monitoring: The morning and evening lung function measurements at home was done
by Peak Expiratory Flow Meter (PEF, Micro Diary, CareFusion). The following routine lung function indices were measured along with Asthma Control
Questionnaire FEV1: Forced Expiratory Volume in 1st second
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bioRxiv preprint 3.1.7 Blood venapunction: 15 ml of venous blood was collected at each scheduled visit (once weekly) to determine whether
circulating antibodies against RV16 are present, for hematology, and other immunological
biomarker assays. Collection of blood was done in standard EDTA and non-EDTA tubes for
specific purposes. 10 ml of it was immediately be followed by centrifugation to obtain plasma
[2000g for 10 minutes (min) at room temperature, RT] aiding in removal of RBCs and WBCs. The supernatants were aliquoted for biomarker hunt. 3.1.6.1 Cell and slide preparation: A cell suspension of 200,000 cells/ml were prepared in PBS. 2 sets of Cytospin apparatus
(Thermo Shandon Cytospin 4) were assembled and the cytospin filter was pre wet by spinning
at 550 rpm for 1 minute with 50 µl PBS. 100 µl of the cell suspension was added to each slide
and centrifuged at 450 rpm for 2 min. The quality of slides and the density of the cells were
evaluated using a phase contrast microscopy. If the cell density was too high or low, more slides
were prepared using an adjusted volume of the cell suspension. A target of 4 good slides with
no overlapping or clumping of cells was set to improve the overall quality and consistency. If the total cell count (for both slides together i.e. less than 7500 cells/slide) was less than or
equal to 15000 cells, then the sample was centrifuged and re-suspended in 100 µl and was
loaded onto a single block. The prepared slides were stained as soon as possible with Diff Quick 1 stain (Medion
Diagnostics), dried and mounted on Depex. 3.1.6.2 Nasal cell cytology: A differential cell count on a maximum of 400 inflammatory cells were performed. Numbers
of eosinophils, neutrophils, macrophages/monocytes, lymphocytes, columnar and squamous
epithelial cells were recorded. A report was prepared with the overall counts, the quality of
cytospin and additional aspects such as the presence of mucous, cell debris, inclusions,
eosinophil granules etc. 3.1.6 Nasal lavage Nasal lavage was collected from the study participants once weekly before rhinovirus challenge
and was up scaled to thrice weekly after the challenge at the clinic. . CC-BY 4.0 International license
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bioRxiv preprint Eight ml of saline solution was introduced into the nasal cavity by a catheter and maintained
for 10 min before recovery, followed by filtration and removal of mucous and cells by
centrifugation. Standardized washings collected from the nose was used for biomarker analyses. 3.1.6.1 Cell and slide preparation: 3.2 Rhinovirus challenge . CC-BY 4.0 International license
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bioRxiv preprint In this study we exposed the volunteers to a mild dose (100 TCID 50) of RV16 using a validated
approach which has been previously shown to be sufficient in inducing mild cold symptoms
and decrease in lung function. In this study we exposed the volunteers to a mild dose (100 TCID 50) of RV16 using a validated
approach which has been previously shown to be sufficient in inducing mild cold symptoms
and decrease in lung function. We used the GMP RV16 stock that has been tested previously by the medical ethical
commission at the hospital in AUMC, and also by the U-BIOPRED showing efficacy in terms
of cold symptoms and viral replication at 100 TCID50, which is part of the accompanying
IMPD. This GMP RV16 stock has been prepared from a seed virus in extensively characterized
human volunteers as described in METC 2010_310 and was expanded and aliquoted by Charles
River Laboratories (USA) under GMP conditions. This preparation was considered safe for in
vivo testing in human volunteers during a scientific advice meeting at BFarM (Bonn, Germany,
April 30, 2013). This testing was carried out as per the approved protocol from the Amsterdam
UMC medical ethical committee. 4. Statistical and computational Analysis All computations were done using R [8] version 3.5.2 together with the following R-packages:
car [9], reshape2 [10], openxlsx [11], lubridate [12], emdist [13], gplots [14], ape [15], ggdendro
[16], cluster [17], factoextra [18], philentropy [19], dendextend [20], and plyr [21]. Statistical
tests resulting in a p-value less or equal to 0.05 were regarded as significant. 4.1 Assessment of differences: Pre- vs. post-viral-challenge 4.1 Assessment of differences: Pre- vs. post-viral-challenge Consecutive measurements of a given biomarker prior to and after the viral challenge resulted
in pre- and post-challenge time series for each cohort participant. Except for the calculation of
transient changes, the time series were treated as empirical distributions, thus disregarding the
chronological order of the measurements. The empirical distribution of any biomarker before and after viral challenge was compared
using Kolmogorov Smirnov test. A participant is considered a responder with respect to a given
biomarker if the outcome of the Kolmogorov-Smirnov test results in a p-value <= 0.05. Differences in the variance between the pre- and post-challenge distributions were assessed
using Levene’s test. The empirical distributions of a given biomarker, prior to and after the viral challenge, were
compared to each other using the Earth Mover’s Distance (EMD), see Fig. 2 in the Main
Manuscript. The resulting pair-wise distances between distributions were then used for
hierarchical clustering of pre- and post-challenge distributions. Our clustering approach makes . CC-BY 4.0 International license
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bioRxiv preprint use of the entire collection (distribution) of values measured before and after the challenge,
respectively, and does not amalgamate the information into a single magnitude (e.g., the mean
value). This method unveils subtler differences and similarities between the participants’
measurements that are less likely to be captured by conventional methods based on averages. 4.2 Clustering approach In our approach, for each biomarker, the measurements collected before and after the viral
challenge were used to construct the individual empirical distribution of measurements for each
study participant, before and after the challenge, respectively. We performed agglomerative hierarchical clustering [22] of the aforementioned empirical
distributions of biomarkers. Within the hierarchical clustering algorithm, the distances between
the distributions were calculated using the Earth Mover’s Distance [23], and the agglomeration
procedure was done according to Ward’s minimum variance method [22]. Intuitively speaking,
the Earth Mover’s Distance contemplates the pair of distributions to be compared as piles of
sand and measures the effort that it would take to shovel one distribution into the shape and
position of the other (see below for a more detailed description of this method). 4. Statistical and computational Analysis Therefore, this part of our methodology is based on the distributional properties of each
participant’s measurements and neglects the time dimension. 4.3 Calculation of short-term/transient changes For each participant individually, throughout the entire period of observation, the relative
change of each biomarker taking place within time intervals of 10 days was calculated. This
was done throughout the entire period of observation considering all possible time intervals
consisting of 10 consecutive days, see Fig. 3 in the Main Manuscript. The rationale for this is
as follows. Any changes taking place within a period of 10 days that contained the day of the
viral challenge need to be interpreted and understood in the context of any changes taking place
within a period of about 10 days during the entire pre-challenge phase of the study. For each participant, the day of the challenge (i.e., the day of the inoculation) was labeled as
“day 0”. All days between recruitment and the day of the challenge were labeled with negative
integer numbers. All days between the day of the challenge and the participant’s final visit were
labeled with positive integer numbers. Given that measurements were not conducted every day
on a given participant (see study design above), for each biomarker considered in this study and . CC-BY 4.0 International license
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bioRxiv preprint for each participant separately, interpolation was used in order to have, for every biomarker one
value for every day. Except for the total cell count in nasal lavage fluid, and the percentages of
eosinophils and neutrophils in nasal lavage fluid, which were linearly interpolated, all
biomarker time series were interpolated using cubic splines with natural boundary conditions
(see, e.g., [24]). These interpolated values were only used for the assessment of the short-
term/transient response. for each participant separately, interpolation was used in order to have, for every biomarker one
value for every day. 4.3 Calculation of short-term/transient changes Except for the total cell count in nasal lavage fluid, and the percentages of
eosinophils and neutrophils in nasal lavage fluid, which were linearly interpolated, all
biomarker time series were interpolated using cubic splines with natural boundary conditions
(see, e.g., [24]). These interpolated values were only used for the assessment of the short-
term/transient response. The time interval of 10 days was decided based on prior knowledge where differences in signals
from biomarkers (lung function etc.) usually take not less than a week to subside As a sensitivity analysis, the same calculations were repeated using a 7-day time interval (data
not shown). The outcomes were very similar to the ones obtained using the 10-day interval. As a sensitivity analysis, the same calculations were repeated using a 7-day time interval (data
not shown). The outcomes were very similar to the ones obtained using the 10-day interval. Consequently, a 10-day time period was considered optimal to calculate the short term/transient
changes. Consequently, a 10-day time period was considered optimal to calculate the short term/transient
changes. 4.4 Characterization of the dendrogram clusters 4.4 Characterization of the dendrogram clusters 4.4 Characterization of the dendrogram clusters The clusters obtained using the clustering dendrogram were tested for enrichment in or
depletion of healthy or asthmatic participants, and/or for enrichment in or depletion of pre- or
post-challenge distributions. The relative location of leaves in the clustering dendrogram was quantitatively
evaluated using the cophenetic distance [25]. The cophenetic distance between two leaves of a
dendrogram is defined as the height of the dendrogram at which the two largest branches that
individually contain the two leaves merge into a single branch. For every cohort participant and any given biomarker there is a pre-challenge and a
post-challenge time series, which we call the participant’s pre- and post-pair. If the disruption
caused by the viral challenge is not strong enough, the pre- and post-challenge distributions of
a given participant will tend to cluster together. Therefore, a cluster in which pre- and post-
pairs are closely located in terms of the cophenetic distance within the dendrogram, represents
a subgroup of participants for which the viral challenge caused a relatively weaker disruption,
at least with respect to the biomarker under scrutiny. Two dendrogram leaves are called neighbors if their mutual cophenetic distance is equal
to the minimum of all cophenetic distances from one of the leaves to all the other leaves in the
dendrogram. If this condition is fulfilled for both leaves simultaneously, then the two leaves
form a two-element cluster in the dendrogram. If the condition is only fulfilled for one of the . CC-BY 4.0 International license
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bioRxiv preprint leaves, the two are still considered neighbors, even if this is not always visually obvious from
inspecting the dendrogram (see Fig. 11 below). leaves, the two are still considered neighbors, even if this is not always visually obvious from
inspecting the dendrogram (see Fig. 11 below). 4.4 Characterization of the dendrogram clusters Under the null-hypothesis that the branching in the dendrogram is the result of a purely random
process, the number of neighboring pre- and post-pairs to be expected just by chance within a
given cluster can be estimated by simply permuting the labels of the leaves in the dendrogram
and counting the number of neighboring pre- and post-pairs. This permutation test is used for
calculating the empirical p-values displayed in Tables 1 and 3 in the Main Manuscript. . CC-BY 4.0 International license
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bioRxiv preprint Figure 11: Panel A: Cluster dendrogram obtained via hierarchical clustering of the participants’ pre- an
challenge time series of FeNO. The distance between any two-time series was calculated using the
Rectangles mark the clusters identified. Panel B displays a more detailed view of the second cluster. Accor
the definition of neighboring leaves provided in the text above, the leaves P03H_Uninf and P03H_Inf are ne
in this dendrogram (highlighted in red). The reason for this is that the cophenetic distance from leaf P03H
to leaf P03H_Inf is equal to the minimum of all distances from leaf P03H_Uninf to all other leaves
dendrogram. This is not the case for the leaf P03H_Inf. However, the fact that this condition holds for at le
of the two leaves renders them neighboring. Leaves P08H_Inf and P08H_Uninf, and P11H_Inf and P11H_
are neighbors, respectively (the latter pair is not marked in color). P08A_Inf and P08A_Uninf are also nei
In this case the minimum condition is fulfilled by both leaves for of this leaf-pair This is why the two leav Figure 11: Panel A: Cluster dendrogram obtained via hierarchical clustering of the participants’ pre- and post-
challenge time series of FeNO. The distance between any two-time series was calculated using the EMD. Rectangles mark the clusters identified. Panel B displays a more detailed view of the second cluster. 4.4 Characterization of the dendrogram clusters According to
the definition of neighboring leaves provided in the text above, the leaves P03H_Uninf and P03H_Inf are neighbors
in this dendrogram (highlighted in red). The reason for this is that the cophenetic distance from leaf P03H_Uninf
to leaf P03H_Inf is equal to the minimum of all distances from leaf P03H_Uninf to all other leaves in the
dendrogram. This is not the case for the leaf P03H_Inf. However, the fact that this condition holds for at least one
of the two leaves renders them neighboring. Leaves P08H_Inf and P08H_Uninf, and P11H_Inf and P11H_Uninf,
are neighbors, respectively (the latter pair is not marked in color). P08A_Inf and P08A_Uninf are also neighbors;
In this case, the minimum condition is fulfilled by both leaves for of this leaf-pair. This is why the two leaves form
a two-element cluster in the dendrogram. Panel C displays a more detailed view of the third cluster. Analogous
information about neighboring leaves in Cluster 3 is highlighted in blue. However, as opposed to Cluster 2 . CC-BY 4.0 International license
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bioRxiv preprint (depicted in Panel B), the amount of neighboring leaves in Cluster 3 is not statistically significant (permutation
test, see Table 2 in the Main Manuscript). (depicted in Panel B), the amount of neighboring leaves in Cluster 3 is not statistically significant (permutation
test, see Table 2 in the Main Manuscript). 4.5 Soft Bootstrapping As elucidated in [2], when resampling with replacement from a given time series in order to
generate a bootstrap replicate of the same time series, the relative frequencies of the values in
the original time series were adjusted in the sense of soft bootstrapping as follows: 1) The relative frequencies of the values in the original time series were sorted in ascending
order: 𝑝" ≤𝑝$ ≤𝑝% ≤⋯≤𝑝', where m is the number of different values in the time
series. 2) The smallest (𝑝") and the strictly second-smallest (𝑝*) relative frequencies were
adjusted using a softness parameter (see [2]) 𝛿= 0.008 according to the following
formula: 𝑝0
1 = 𝑝0 + 𝛿 for 𝑖∈{1, … , 𝑞} 𝑝0
1 = 𝑝0 + 𝛿 for 𝑖∈{1, … , 𝑞} 3) The remaining relative frequencies were adjusted according to the following formula: 3) The remaining relative frequencies were adjusted according to the following formula: 3) The remaining relative frequencies were adjusted according to the following formula: 𝑝0
1 = 𝑝0 −
𝑞𝛿
𝑚−𝑞 for 𝑖> 𝑞 𝑝0
1 = 𝑝0 −
𝑞𝛿
𝑚−𝑞 for 𝑖> 𝑞 For time series with m<3 the corresponding relative frequencies were left unchanged during the
soft bootstrapping iterations. For time series with m<3 the corresponding relative frequencies were left unchanged during the
soft bootstrapping iterations. 4.6 The Earth Mover’s Distance The Earth Mover’s Distance [23] is a method for quantifying the dissimilarity between two
probability distributions. Intuitively speaking, the EMD contemplates the pair of distributions
to be compared as piles of sand and measures the minimal effort that it would take to shovel
one distribution into the shape and position of the other. In practice, two distributions will be given by two representative samples, which can be written
as lists of pairs {(v1 , w1),…, (vn , wn)} and {(c1 , f1),…, (cn , fm)}. Each pair (vi , wi) corresponds
to a value vi and its relative frequency wi in the sample. If we translate the above described
intuitive approach into numbers, the problem becomes a well-known transportation problem
[26]: Suppose that n suppliers are located at positions v1 ,…, vn, respectively, and each one has
a given amount of goods wi. Furthermore, they are required to supply m consumers, located at
positions c1 ,…, cm, respectively, whereas each one has a given specific demand fi. For each
supplier-consumer pair (vi , wi) and (cj , fj), the cost of transporting a single unit of goods is
determined by the distance d(vi , cj) between their locations. The transportation problem is then . CC-BY 4.0 International license
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bioRxiv preprint to find a least expensive pattern of flow of goods from suppliers to consumers that would satisfy
the consumers' demand. Once the optimal pattern of the goods’ flow has been found, the total
cost is the corresponding EMD. to find a least expensive pattern of flow of goods from suppliers to consumers that would satisfy
the consumers' demand. Once the optimal pattern of the goods’ flow has been found, the total
cost is the corresponding EMD. 4.6 The Earth Mover’s Distance Mathematically, this transportation problem can be formalized as a linear programming
problem, for which efficient solution algorithms were developed in the late 1940s (see, e.g. [27]). Discussion Utility of biomarker time series analysis Utility of biomarker time series analysis CC-BY 4.0 International license
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bioRxiv preprint References 1. Isaksson A, Wallman M, Göransson H, Gustafsson MG. Cross-validation and bootstrapping are
unreliable in small sample classification. Pattern Recognition Letters. 2008;29(14):1960-5. doi:
https://doi.org/10.1016/j.patrec.2008.06.018. p
g
j p
2. Mucha H-J, Bartel H-G, editors. Soft Bootstrapping in Cluster Analysis and Its Comparison with Other
Resampling Methods. Data Analysis, Machine Learning and Knowledge Discovery; 2014 2014//; Cham:
Springer International Publishing. 3. Quanjer PH, Tammeling GJ, Cotes JE, Pedersen OF, Peslin R, Yernault JC. Lung volumes and forced
ventilatory flows. Report Working Party Standardization of Lung Function Tests, European Community for Steel
and Coal. Official Statement of the European Respiratory Society. Eur Respir J Suppl. 1993;16:5-40. Epub
1993/03/01. PubMed PMID: 8499054. 4. Sterk PJ, Fabbri LM, Quanjer PH, Cockcroft DW, O'Byrne PM, Anderson SD, et al. Standardized
challenge testing with pharmacological, physical and sensitizing stimuli in adults. Eur Respir J. 1993;6 Suppl
16:53-83. Epub 1993/03/01. doi: 10.1183/09041950.053s1693. PubMed PMID: 24576917. p
5. Turner C, Spanel P, Smith D. A longitudinal study of ethanol and acetaldehyde in the exhaled breath of
healthy volunteers using selected-ion flow-tube mass spectrometry. Rapid Commun Mass Spectrom. 2006;20(1):61-8. Epub 2005/11/29. doi: 10.1002/rcm.2275. PubMed PMID: 16312013. ( )
p
6. Turner C, Spanel P, Smith D. A longitudinal study of breath isoprene in healthy volunteers using
selected ion flow tube mass spectrometry (SIFT-MS). Physiol Meas. 2006;27(1):13-22. Epub 2005/12/21. doi:
10.1088/0967-3334/27/1/002. PubMed PMID: 16365507. 7. Position paper: Allergen standardization and skin tests. The European Academy of Allergology and
Clinical Immunology. Allergy. 1993;48(14 Suppl):48-82. Epub 1993/01/01. PubMed PMID: 8342740. 8
R Core Team R: A Language and Environment for Statistical Computing Vienna Austria: R 7. Position paper: Allergen standardization and skin tests. The European Academy of Allergology and gy
gy
;
(
pp )
p
R Core Team. R: A Language and Environment for Statistical Computing Vienna, Austria: R Foundation for Statistical Computing; 2018. Available from: https://www.R-project.org. Fox J, Weisberg S. An {R} Companion to Applied Regression. Second ed: Thousand Oaks; 2011. ,
g
{ }
p
pp
g
;
10. Wickham H. Reshaping Data with the reshape Package. Journal of Statistical Software; Vol 1, Issue 12
(2007). 2007. (
)
11. Walker A. openxlsx: Read, Write and Edit XLSX Files. R package version 4.1.0. https://CRAN.R-
project.org/package=openxlsx. 2018. p
j
g p
g
p
12. Grolemund G, Wickham H. Dates and Times Made Easy with lubridate. Journal of Statistical Software;
Vol 1, Issue 3 (2011). 2011. 14. .
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bioRxiv preprint Utility of biomarker time series analysis Utility of biomarker time series analysis Do longitudinal measurements provide deeper insights into complex disease physiology as
compared to single measurements or average values? In order to answer this question, we tried
to reproduce the results obtained using each participant’s entire collections of pre- and post-
challenge biomarker measurements after collapsing them to the corresponding pre- and post-
challenge individual average. For example, in this cohort, FeNO time series have the ability to discriminate between healthy
and asthmatics, and, within the group of asthmatics, between infected and uninfected (see Fig. 1 in the Main Manuscript). In order to investigate whether the average value would have a
similar discriminative power, we calculated, for each participant, the average of their pre- and
of their post-challenge series and used the absolute value of the difference between averages as
a distance measure for clustering. The resulting dendrogram is depicted in Fig. 12 below. While
the discriminative power between healthy and asthmatics is still given, the ability to distinguish
between infected and uninfected within the group of asthmatics gets lost. . CC-BY 4.0 International license
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bioRxiv preprint Figure 12: Dendrogram obtained from clustering the participants’ pre- and post-challenge average value of Fe
using the EMD. Figure 12: Dendrogram obtained from clustering the participants’ pre- and p
using the EMD. Figure 12: Dendrogram obtained from clustering the participants’ pre- and post-challenge average value of FeNO
using the EMD. . References ;
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bioRxiv preprint Hillier FS, Lieberman GJ. Introduction to operations research. 9th ed. New York: McGraw-Hill Higher
n; 2010. xxiv, 1047 p. p. Normalized FEV1 Healthy group before viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. -2.7287 -1.5404 -0.9138 -1.0405 -0.7148 0.3588
Healthy group after viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. -3.0971 -1.9662 -1.3308 -1.3310 -0.8425 0.4305
Asthmatics group before viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. -2.4603 -2.1253 -1.0570 -1.3044 -0.8828 -0.1823
Asthmatics group after viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. -3.0350 -2.1484 -1.1480 -1.3369 -0.8418 0.5686 Healthy group before viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. -2.7287 -1.5404 -0.9138 -1.0405 -0.7148 0.3588 Asthmatics group after viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. -3.0350 -2.1484 -1.1480 -1.3369 -0.8418 0.5686 PEF (% predicted) PEF (% predicted)
Healthy group before viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. 63.31 84.63 91.52 92.16 100.97 119.01
Healthy group after viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. 63.42 78.42 91.92 90.44 102.39 118.84
Asthmatics group before viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. 57.62 82.45 88.64 86.77 94.14 105.09
Asthmatics group after viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. 47.67 78.49 88.44 85.01 95.21 109.98 References Warnes GR, Bolker B, Bonebakker L, Gentleman R, Liaw WHA, Lumley T, et al. gplots: Various R
programming tools for plotting data. 2014. 5. Paradis E, Claude J, Strimmer K. APE: analyses of phylogenetics and evolution in R language. ioinformatics. 2004;20:289-90. 15. Paradis E, Claude J, Strimmer K. APE: analyses of phylogenetics and evolution in R language. Bioinformatics. 2004;20:289-90. 16. ggdendro: Tools for extracting dendrogram and tree diagram plot data for use with ggplot. [Internet]. 16. ggdendro: Tools for extracting dendrogram and tree diagram plot data for use with ggplot. [Internet]. 2013. Available from: http://CRAN.R-project.org/package=ggdendro. 2013. Available from: http://CRAN.R-project.org/package=ggdendro. 7. Maechler M, Rousseeuw P, Struyf A, Hubert M, Hornik K. cluster: Cluster Analysis Basics and
xtensions. R package version 2.0.7-1. 2018. p
g
18. Kassambara A, Mundt F. factoextra: Extract and Visualize the Results of Multivariate Data Analyses. R
package version 1.0.5. https://CRAN.R-project.org/package=factoextra. 2017. 19. Drost H-G. philentropy: Similarity and Distance Quantification Between Probability Functions. R package version 0.2.0. https://CRAN.R-project.org/package=philentropy. 2018. package version 0.2.0. https://CRAN.R project.org/package philentropy. 2018. 20. Galili T. dendextend: an R package for visualizing, adjusting and comparing trees of hierarchic 20. Galili T. dendextend: an R package for visualizing, adjusting and comparing trees of hierarchical
clustering. Bioinformatics (Oxford, England). 2015;31(22):3718-20. Epub 2015/07/23. doi:
10.1093/bioinformatics/btv428. PubMed PMID: 26209431. 21. Wickham H. The Split-Apply-Combine Strategy for Data Analysis. Journal of Statistical Software; Vol
1, Issue 1 (2011). 2011. doi: 10.18637/jss.v040.i01. (
)
j
22. Gan G, Ma C, Wu J. Data Clustering: Theory, Algorithms, and Applications: Society for Industrial and
Applied Mathematics (SIAM, 3600 Market Street, Floor 6, Philadelphia, PA 19104); 2007. 23. Rubner Y, Tomasi C, Guibas LJ, editors. A metric for distributions with applications to image
databases. Computer Vision, 1998 Sixth International Conference on; 1998: IEEE. 24. Bartels RH, Beatty JC, Barsky BA. An introduction to splines for use in computer graphics and
geometric modeling: Morgan Kaufmann; 1995. 5. Sokal RR, Rohlf FJ. The Comparison of Dendrograms by Objective Methods. Taxon. 1962;11(2):3
0. doi: 10.2307/1217208. 40. doi: 10.2307/1217208. 26. Hitchcock FL. The Distribution of a Product from Several Sources to Numerous Localities. Journal of
Mathematics and Physics. 1941;20(1-4):224-30. doi: 10.1002/sapm1941201224. . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
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this version posted July 2, 2019. Normalized FVC Healthy group before viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. -2.19123 -1.87550 -1.66646 -1.34813 -0.83674 -0.02098
Healthy group after viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. -3.0841 -2.6730 -2.1174 -1.7966 -0.9592 0.2229
Asthmatics group before viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. -3.0455 -2.0029 -0.9102 -1.3416 -0.6539 -0.3614 . CC-BY 4.0 International license
under a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
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bioRxiv preprint Asthmatics group after viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. -3.726 -1.840 -1.319 -1.483 -0.668 0.194 Normalized FEV1/FVC Healthy group before viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. -1.4796 0.1851 0.6670 0.6940 1.3305 2.1131
Healthy group after viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. -0.6591 0.1088 1.3446 1.1083 1.9541 2.5255
Asthmatics group before viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. -1.2123 -0.5753 -0.3160 0.1677 1.2150 1.7450
Asthmatics group after viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. -1.5441 -0.4089 0.3237 0.4484 1.3195 2.5751 FeNO
Healthy group before viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. 9.435 12.779 13.872 14.608 15.594 22.227
Healthy group after viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. 7.545 10.807 16.143 15.841 19.350 25.909
Asthmatics group before viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. 16.57 35.67 46.21 61.97 55.68 181.30
Asthmatics group after viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. 18.36 29.80 42.59 61.42 58.73 181.18
Cell density in 106 per ml of nasal lavage fluid
Healthy group before viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. 0.1975 0.4308 0.5569 0.9327 1.2431 2.4600
Healthy group after viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. 0.3436 0.6409 1.4536 1.6078 2.3982 3.7255
Asthmatics group before viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. 0.0375 0.2490 0.8489 1.4183 1.8562 5.0444
Asthmatics group after viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. 0.2873 0.9698 1.8445 2.7841 4.0850 10.8618 FeNO Healthy group before viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. 9.435 12.779 13.872 14.608 15.594 22.227
Healthy group after viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. 7.545 10.807 16.143 15.841 19.350 25.909
Asthmatics group before viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. 16.57 35.67 46.21 61.97 55.68 181.30
Asthmatics group after viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. 18.36 29.80 42.59 61.42 58.73 181.18 Cell density in 106 per ml of nasal lavage fluid
Healthy group before viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. 0.1975 0.4308 0.5569 0.9327 1.2431 2.4600
Healthy group after viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. 0.3436 0.6409 1.4536 1.6078 2.3982 3.7255
Asthmatics group before viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. 0.0375 0.2490 0.8489 1.4183 1.8562 5.0444
Asthmatics group after viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. 0.2873 0.9698 1.8445 2.7841 4.0850 10.8618 Healthy group before viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. 0.1975 0.4308 0.5569 0.9327 1.2431 2.4600 . CC-BY 4.0 International license
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bioRxiv preprint Percentage of Eosinophils in nasal lavage fluid
Healthy group before viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. 0.00000 0.00000 0.04375 0.95683 0.36979 9.03750
Healthy group after viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. 0.0000 0.0000 0.0500 1.2977 0.2909 14.1273
Asthmatics group before viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. Percentage of Eosinophils in nasal lavage fluid
Healthy group before viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max.
0.00000 0.00000 0.04375 0.95683 0.36979 9.03750 Healthy group after viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. 0.0000 0.0000 0.0500 1.2977 0.2909 14.1273
Asthmatics group before viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. 0.4091 7.2031 14.3950 16.9439 28.9778 35.9111
Asthmatics group after viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. 0.000 7.218 10.227 13.186 13.935 59.736 FeNO 0.4091 7.2031 14.3950 16.9439 28.9778 35.9111
Asthmatics group after viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. 0.000 7.218 10.227 13.186 13.935 59.736
Percentage of Neutrophils in nasal lavage fluid
Healthy group before viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. 24.90 47.36 58.90 62.45 86.44 99.64
Healthy group after viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. 18.18 51.45 66.16 61.72 72.33 88.95
Asthmatics group before viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. 29.30 36.18 60.47 57.23 79.79 92.72
Asthmatics group after viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. 27.53 55.17 68.01 66.96 80.31 97.29 Percentage of Eosinophils in nasal lavage fluid
Healthy group before viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. 0.00000 0.00000 0.04375 0.95683 0.36979 9.03750 Percentage of Neutrophils in nasal lavage fluid Percentage of Neutrophils in nasal lavage fluid
Healthy group before viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. 24.90 47.36 58.90 62.45 86.44 99.64
Healthy group after viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. 18.18 51.45 66.16 61.72 72.33 88.95
Asthmatics group before viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. 29.30 36.18 60.47 57.23 79.79 92.72
Asthmatics group after viral challenge
Min. 1st Qu. Median Mean 3rd Qu. Max. 27.53 55.17 68.01 66.96 80.31 97.29 For each biomarker, plots of the time series of relative changes within 10 days can be found in
Supplementary Figures File 1. Plots of the time series of each biomarker can be found in Supplementary Figures File 2.
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Distinct assembly mechanisms underlie similar biogeographical patterns of rare and abundant bacteria in Tibetan Plateau grassland soils
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Distinct assembly mechanisms underlie similar
biogeographical patterns of rare and abundant
bacteria in Tibetan Plateau grassland soils biogeographical patterns of rare and abundant
bacteria in Tibetan Plateau grassland soils
Mukan Ji
Institute of Tibetan Plateau Research, Chinese Academy of Sciences
Weidong Kong
(
wdkong@itpcas.ac.cn
)
Institute of Tibetan Plateau Research Chinese Academy of Sciences
https://orcid.org/0000-0001-968
1484
James Stegen
Pacific Northwest National Laboratory
Linyan Yue
Institute of Tibetan Plateau Research Chinese Academy of Sciences
Fei Wang
Institute of Tibetan Plateau Research Chinese Academy of Sciences
Xiaobin Dong
Beijing Normal University
Don A Cowan
Department of Genetics, University of Pretoria
Belinda C Ferrari
UNSW Sydney Distinct assembly mechanisms underlie similar
biogeographical patterns of rare and abundant
bacteria in Tibetan Plateau grassland soils
Mukan Ji
Institute of Tibetan Plateau Research, Chinese Academy of Sciences
Weidong Kong
(
wdkong@itpcas.ac.cn
)
Institute of Tibetan Plateau Research Chinese Academy of Sciences
https://orcid.org/0000-0001-9682-
1484
James Stegen
Pacific Northwest National Laboratory
Linyan Yue
Institute of Tibetan Plateau Research Chinese Academy of Sciences
Fei Wang
Institute of Tibetan Plateau Research Chinese Academy of Sciences
Xiaobin Dong
Beijing Normal University
Don A Cowan
Department of Genetics, University of Pretoria
Belinda C Ferrari
UNSW Sydney
Research
Keywords: Soil bacteria, Rare biosphere, Community structure, Assembly processes
Posted Date: January 2nd, 2020
DOI: https://doi.org/10.21203/rs.2.19893/v1
License:
This work is licensed under a Creative Commons Attribution 4.0 International
License.
Read Full License Distinct assembly mechanisms underlie similar
biogeographical patterns of rare and abundant
bacteria in Tibetan Plateau grassland soils
Mukan Ji
Institute of Tibetan Plateau Research, Chinese Academy of Sciences
Weidong Kong
(
wdkong@itpcas.ac.cn
)
Institute of Tibetan Plateau Research Chinese Academy of Sciences
https://orcid.org/0000-0001-9682-
1484
James Stegen
Pacific Northwest National Laboratory
Linyan Yue
Institute of Tibetan Plateau Research Chinese Academy of Sciences
Fei Wang
Institute of Tibetan Plateau Research Chinese Academy of Sciences
Xiaobin Dong
Beijing Normal University
Don A Cowan
Department of Genetics, University of Pretoria
Belinda C Ferrari
UNSW Sydney
Research
Keywords: Soil bacteria, Rare biosphere, Community structure, Assembly processes
Posted Date: January 2nd, 2020
DOI: https://doi.org/10.21203/rs.2.19893/v1
License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Research Research
Keywords: Soil bacteria, Rare biosphere, Community structure, Assembly processes
Posted Date: January 2nd, 2020
DOI: https://doi.org/10.21203/rs.2.19893/v1
License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License
Version of Record: A version of this preprint was published at Environmental Microbiology on April 2nd, Version of Record: A version of this preprint was published at Environmental Microbiology on April 2nd,
2020. See the published version at https://doi.org/10.1111/1462-2920.14993. Page 1/23 Abstract Background: Bacteria in low abundance represent the majority of Earth’s biodiversity and perform vital
ecological functions, but little is known about their biogeography nor the ecological processes that drive
their community assembly in terrestrial ecosystems. Here, we investigated the community compositions
and phylogenies of rare (relative abundance < 0.1%) and abundant (> 1%) soil bacteria along a transect
containing three alpine grassland types (meadow, steppe, and desert) on the Tibetan Plateau. Results: Our results revealed similar biogeographical patterns of rare and abundant bacteria, with their
community compositions and phylogenies shifting gradually along the transect. The similar patterns,
however, were driven by contrasting community assembly processes, with rare subcommunity being more
heavily influenced by stochasticity (72%) than abundant (57%). The composition of abundant
subcommunity (80%) was better explained by local (including soil and vegetation factors), geospatial
and climatic factors than that of rare subcommunity (41%), while the phylogeny of the rare one (36%)
was better explained than that of the abundant one (29%). Variation partitioning analysis indicated that
pure local factors consistently explained a higher proportion of the community composition than
geospatial factors in both rare (12.3% and 8.7%, respectively) and abundant bacteria (18.3% and 14.1%,
respectively). In contrast, the phylogeny of rare subcommunity was explained by local and geospatial
factors equally (11.5% and 11.9%, respectively), while that of abundant subcommunity was more
explained by geospatial (22.1%) than local factors (11.3%). Furthermore, our results revealed a tighter
connection between the community phylogeny and composition in rare than in abundant bacteria. Conclusions: Our results revealed consistent biogeographical patterns of rare and abundant bacteria in
grassland soils, but their assembly processes were distinct. We further demonstrated that rare
subcommunity was less predictable than the abundant subcommunity by environmental and geospatial
factors. Rare and abundant bacteria responded differentially to factors, which was attributed to the
distinct life strategies. Our study provides novel insights into the assembly processes and biographical
patterns of rare and abundant bacteria in terrestrial ecosystems. Background The bacteria in low abundance represent the majority of Earth’s biodiversity [1], and these rare bacteria
contain an enormous pool of genetic novelty [2]. Accumulating evidence has confirmed the crucial
ecological functions of rare bacteria in terrestrial and aquatic ecosystems. For example, rare bacteria are
responsible for nitrogen and carbon assimilation in freshwater lakes [3–5], they enhance plant defence
against aphids [6], and provide insurance during ecological restoration [7–9]. Therefore, variation in these
low abundance, but functional important microorganisms could cause substantial consequences on an
ecosystem. Compared to rare bacteria, abundant bacteria present at much higher relative abundance globally, and
potentially exhibit greater tolerance to environmental stresses . Thus, rare bacteria have been identified to Page 2/23 Page 2/23 drive bacterial community structure changes both spatially and temporally [10–12]. Several studies have
examined the biogeography of rare and abundant bacteria in aquatic ecosystems, such as lake reservoirs
[13], subtropical bays [14], the Arctic ocean [15], and the Pacific ocean [16]. These studies have
consistently reported that the community compositions of rare and abundant bacteria were influenced by
geographical and environmental factors differentially, despite exhibiting similar biogeographical patterns
[13, 17]. Specifically, the community composition (typically measured using Bray-Curtis dissimilarity) of
rare bacteria was more strongly influenced by local environmental factors, while that of abundant
bacteria was predominately explained by geospatial factors [13, 14]. Compared to aquatic ecosystems,
the biogeographical patterns of rare and abundant bacteria in terrestrial ecosystems are less understood. Furthermore, as soils are non-fluidic and are much more heterogeneous compared to the aquatic
environment [18], the biogeographical pattern of rare bacteria in soil could be different from that of
abundant bacteria. Composition-based metrics have traditionally been used to examine microbial community structure
variations, but phylogeny-based community metrics, such as the β-mean nearest taxon distance (β-
MNTD) and UniFrac distance, are receiving increased attention [19, 20]. Compared to composition-based
community metrics, these phylogeny-based community metrics can be used to infer changes in microbial
community function [21–23]. This is based on the assumption that phylogenetically close-related
microorganisms have similar habitat associations [20]. Thus, using phylogeny-based community metrics,
together with the composition-based community metrics, could provide additional insights into the
ecogeographical patterns of rare and abundant subcommunities. Due to the strong environmental stress tolerance of abundant bacteria and the greater niche
differentiation in terrestrial ecosystems, we hypothesize that rare bacteria could exhibit distinct
biogeographical pattern compared to abundant bacteria in soils. Background Furthermore, the relative importance of
local environmental factors and geospatial factors on the biogeography of rare and abundant bacteria
could be different from that identified in aquatic ecosystems. To test these hypotheses, the bacterial
community structure was investigated using DNA-based amplicon sequencing targeting the 16S rRNA
gene, and both the community Bray-Curtis compositional dissimilarity and βMNTD phylogenetic distance
were calculated. These metrics were used to compare the community compositions and phylogenies of
rare and abundant bacteria along a 1200 km grassland transect on the Tibetan Plateau. Taxonomic compositions of rare and abundant bacteria A total of 7,890 OTUs were identified from the retained 806,358 high-quality sequences at 97% sequence
identity. Rare bacteria (relative abundance < 0.1%) comprised 87.7% of the total bacterial richness (6,923
OTUs), but their total relative abundance accounted for only 10.1% of the entire community. In contrast,
abundant bacteria (relative abundance > 1%) comprised only 1% of the total richness (114 OTUs), but
their relative abundance was 59.4%. Of the retained sequences, 91.3% were classified at the phylum level, Page 3/23 and 34, 13 and 34 bacterial phyla were identified from the rare, abundant and entire communities,
respectively (Fig. 1A). Across all samples, rare subcommunity was dominated by Proteobacteria (24.8%),
followed by unclassified bacteria (18.1%), Actinobacteria (15.4%), Bacteroidetes (14.9%), and
Planctomycetes (7.7%). In contrast, the abundant and entire communities were both dominated by
Actinobacteria (57.5% and 47.2%, respectively), and exhibited lower relative abundance of Proteobacteria
(12.2% and 18.2%, respectively) than the rare subcommunity. Community compositions of rare and abundant bacteria Both rare and abundant OTUs exhibited positive correlations between their relative abundance and the
number of samples they were identified in (Pearson r = 0.84 and 0.36, respectively, both P < 0.001,
Fig. 1B). The abundant OTUs were more widespread geographically than rare OTUs, with over 90% of the
abundant OTUs being detected in > 50% of samples and 54% of the abundant OTUs being detected from
> 90% of the samples. In contrast, the majority (97%) of rare OTUs were only detected in < 50% of samples
analysed. The community compositions of rare (Fig. 2A), abundant (Fig. 2B) and entire communities (Fig. 2C) were
significantly different among the three grassland types (PERMANOVA, all P < 0.001). The community
compositions of rare, abundant, and entire communities also exhibited gradual shifts along the grassland
transect (from meadow to steppe, and then to desert grassland). The measured local, climatic and
geospatial factors together explained 41%, 80%, and 60% of the community compositions for rare,
abundant, and entire communities, respectively (Figs. 3A, 3B and, 3C, Additional file 1: Table S1). VPA
analysis revealed that pure local factors consistently explained higher community composition than pure
geospatial factors for both rare and abundant subcommunities, while pure climatic factors explained the
lowest community composition (Figs. 3A, 3B and, 3C). Furthermore, soil pH, TOC, and NH4
+ explained a
higher proportion of the community variation in abundant over rare subcommunities (Additional file 1:
Table S1). Mantel tests revealed significant linear correlations of the community composition with local, climatic
and geospatial factors for the rare, abundant and entire communities (Figs. 3D, 3E, and 3F, Mantel tests revealed significant linear correlations of the community composition with local, climatic
and geospatial factors for the rare, abundant and entire communities (Figs. 3D, 3E, and 3F,
Supplementary Fig. 2, and Additional file 1: Table S2). However, partial Mantel tests indicated that the
correlations between climatic factors and the community compositional dissimilarity were non-
significant. Furthermore, local factors consistently exhibited stronger correlation strength (indicated by
Pearson r) than geospatial factors. Community phylogenies of rare and abundant bacteria The community phylogenies of rare (Fig. 4A), abundant (Fig. 4B) and entire bacterial communities
(Fig. 4C) differed significantly among the three grassland types (PERMANOVA, P < 0.001, = 0.003, and <
0.001, respectively). DistLM analysis revealed that the measured local, climatic and geospatial factors
together explained 36%, 29%, and 44% of the community phylogeny for rare, abundant and entire
communities, respectively (Figs. 5A, 5B, 5C, and Additional file 1: Table S3). VPA analysis indicated that Page 4/23 Page 4/23 the pure local and geospatial factors contributed equally to the community phylogeny of rare
subcommunity (Fig. 5A, 11.5% and 11.9%, respectively). In contrast, the geospatial factors explained a
larger proportion of community phylogeny (22.1%) than local factors (11.3%) for abundant
subcommunities (Fig. 5B). Consistent with that identified at the community compositional level, climatic
factors explained the lowest community phylogeny in rare, abundant, and entire communities (Figs. 5A,
5B, and 5C). Mantel tests revealed significant correlations of the community phylogeny with the measured local,
climatic and geospatial factors in rare subcommunity (all P < 0.001, Fig. 5D, Additional file 1: Table S4,
and Additional file 2: Fig. S3), but these correlations were not significant for abundant or entire
communities (Figs. 5E and 5F). Partial Mantel tests further revealed that the community phylogeny of
rare bacteria significantly correlated with the local and geospatial factors (P < 0.001 and 0.002,
respectively), but not with climatic factors (P = 0.133). Furthermore, a significant partial Mantel
correlation was detected between the geospatial factors and the community phylogeny of abundant
bacteria (P = 0.007). Correlation relationships between the community
composition and phylogeny Significant correlations between community composition and phylogeny were observed in rare, abundant,
and entire communities (Fig. 6). The correlations in rare and entire communities were much stronger than
that in abundant subcommunity (R2 = 0.65, 0.62 and 0.08, respectively, all P < 0.001). In each case, the
observed increase in the community composition was accompanied by a relatively weak change in the
community phylogeny until the community compositional dissimilarity further increased to
approximately 60%, 40%, and 40% for rare, abundant, and entire communities, respectively. After passing
these thresholds, exponential increases in the community phylogenetic distance were then occurred. This
observed delay in the community phylogenetic variation was smaller for rare subcommunity than that in
abundant subcommunity. approximately 60%, 40%, and 40% for rare, abundant, and entire communities, respectively. After passing
these thresholds, exponential increases in the community phylogenetic distance were then occurred. This
observed delay in the community phylogenetic variation was smaller for rare subcommunity than that in
abundant subcommunity. Community assembly processes of rare and abundant
bacteria The ecological processes shaping bacterial community assembly were explored using compositional
(modified stochasticity ratio, MST) and phylogenetic (β-Mean Nearest Taxon Index, βNTI) approaches. The MST of rare subcommunity was 72±18%, which was higher than that in abundant (57±20%) and
entire communities (66±22%; Additional file 2: Fig. S4). Furthermore, the within ecosystem community
variations were less influenced by stochasticity than those between ecosystems for rare and entire
communities. The βNTI of rare subcommunity varied from − 8.9 to 8.1, with 66%, 30%, and 4% of which being < -2,
between − 2 and 2, and > 2, respectively. In contrast, the βNTI of abundant and entire communities varied Page 5/23 Page 5/23 from − 8.8 to -4.5 and − 16.5 to 1.7, with 100% and 99% of βNTIs being < -2, respectively (Additional file 2:
Fig. S5). Significant positive linear correlations of βNTI values with local, climatic and geospatial factors
were detected in rare and entire community, whereas the βNTI values of the abundant subcommunity
only significantly correlated to local factors (Additional file 2: Fig. S6, Additional file 1: Table S5). Furthermore, partial Mantel tests indicated that the βNTI values of rare (P = 0.002), abundant (P < 0.001),
and entire communities significantly correlated only with local factors (P < 0.001), while the correlations
with climatic and geospatial factors were not significant. Rare and abundant bacteria exhibited similar biogeographical patterns, but were shaped by distinct
assembly processes The bacterial lineages that present at low abundance, yet potentially perform vital ecological
functions [9], could be more strongly influenced by stochasticity than abundant bacteria. Despite the
dominance of determinism in shaping the community composition of abundant bacteria, substantial
contributions from stochasticity were also observed (Additional file 2: Fig. S4), consistent with previous
studies [14, 33]. Despite the similar biogeographical patterns, the community compositions of rare and abundant
subcommunities were predominately influenced by stochasticity and determinism, respectively. It is
interesting that how two distinct assembly processes lead to consistent biogeographical patterns in rare
and abundant subcommunities. We propose that this may be caused by the co-variation of geospatial
and environmental heterogeneity. Our results showed that the geospatial and local environmental
variations were highly correlated (Additional file 2: Fig. S7). Thus the co-variation of stochastic
(geospatial) and deterministic (local environmental) processes could lead to the similar biogeographical
patterns of rare and abundant subcommunities observed. Rare and abundant bacteria exhibited similar biogeographical patterns, but were shaped by distinct
assembly processes Rare and abundant bacteria both exhibited similar community compositional shifts along the meadow,
steppe, and desert grassland transect (Figs. 2 and 4). The similar biogeographical patterns of rare and
abundant subcommunities have also been consistently reported from freshwater lakes [13, 24] and
marine environments [13, 14, 16, 24]. Thus, this consistency suggests that the similar biogeographical
patterns of rare and abundant bacteria are ubiquitous to both terrestrial and aquatic ecosystems. The stronger influence of stochasticity on rare subcommunity (Additional file 2: Fig. S4) was consistent
with its lower proportion of the community composition explained by environmental factors compared
with abundant subcommunity (Figs. 3A and 3B). The varied influence of stochasticity on rare and
abundant bacteria may be related to their different life strategies. The abundant subcommunity was
comprised of more copiotrophic than oligotrophic bacteria. For example, copiotrophic Actinobacteria
were presented at higher relative abundances in abundant than rare subcommunities, while oligotrophic
bacteria such as Acidobacteria and Planctomycetes were more abundant in rare subcommunity (Fig. 1A)
[25]. Copiotrophic bacteria (such as Actinobacteria) are more sensitive to carbon availability than
oligotrophic bacteria [26, 27], which is proposed to be due to a lack of carbon and energy regulatory
system in copiotrophs [28]. The higher sensitivity of abundant bacteria to soil nutrients was further
evidenced by the stronger contribution of soil nutrients (TOC and NH4
+) in explaining the community
composition in abundant than rare subcommunities (Additional file 1: Table S1). Rare subcommunity exhibited a stronger correlation between the community composition and geospatial
factors than abundant bacteria (Fig. 3D and 3E), indicating a stronger influence dispersal limitation [13]. Dispersal limitation is one of the most important stochastic factors influencing bacterial community
assembly, and is dependent on cell numbers [29]. Thus, the low relative abundance at less than < 0.1%
could be the major limiting factor for rare bacteria being dispersed across large geographic distance [9,
13, 14]. The stronger influence of dispersal limitation on rare over abundant bacteria at the compositional
level has also been reported in soil fungal community [30]. Page 6/23 Stochasticity has been reported to play less important role than determinism in shaping bacterial
community composition in grassland and polar desert soils [31, 32]. Our results further expand this
phenomenon, and show that the influence of stochasticity on community assembly could be lineage-
specific. Rare bacteria exhibited a greater community phylogenetic variation than abundant bacteria Phylogenetically close-related microorganisms have similar habitat associations, thus phylogeny-based
community metrics could infer potential community functional change [20]. The community phylogeny of
rare bacteria was better separated among the three grassland ecosystems than that of abundant bacteria
(Fig. 4A and 4B), potentially suggesting a stronger divergence of ecological functions [34]. Partial Mantel
results demonstrated that the community phylogenies of rare and abundant subcommunities both
exhibited significant correlations with geospatial factors (Figs. 5D and 5E, Additional file 1: Table S4). This contrasted to the lack of correlation between bacterial community phylogeny and geographical
distance reported in Wang et al. [35]. The difference could be attributed to the geographical scale, as the
sampling distance was less than 10 km in Wang et al. (2013), which could be too small for substantial
phylogenetic variation to be detected. The community phylogeny of abundant bacteria was more strongly influenced by dispersal limitation
than that of rare bacteria (Figs. 5A). This finding contradicts the observations at the community
compositional level (Figs. 3A and 3B), but is consistent with the previous phylogeny-based investigation
at the northwest Pacific Ocean [36]. This suggests the factors influence the community composition and
phylogeny could be different. The stronger influence of geospatial than environmental factors in shaping
the community phylogeny of abundant bacteria could be explained by their environmental adaptability. Abundant bacteria have been reported to exhibit strong tolerance to alkaline and saline conditions [37,
38]. This is also evidenced by the wide distribution of abundant bacteria across the transect in the
present study (Fig. 1B) and the dominance of homogenous selection in shaping their community
phylogeny (Additional file 2: Fig. S5). Thus, dispersal limitation could be the main factor constraining the Page 7/23 Page 7/23 closeness of community phylogeny in abundant subcommunity across the transect (Additional file 1:
Table S4). The equal importance of environmental and geospatial factors in shaping the community phylogeny of
rare subcommunity can be attributed to their life strategies [37]. Rare bacteria generally exhibit lower
environmental adaptability and smaller niche breadth [39] than abundant bacteria, which hinder their
colonization in new habitats after being dispersed. This resulted in their community phylogeny being
gradual shifted across environmental gradient (Fig. 4A) and the strong correlation between community
phylogeny and environmental heterogeneity (Additional file 2: Fig. S3), which was not detected in the
abundant subcommunity. Decoupled community compositional and phylogenetic variations in abundant bacteria A tighter connection (Fig. 6, larger R2 value) between the community composition and phylogeny was
observed in rare than abundant subcommunities. This is mainly due to the phylogeny of abundant
subcommunity being less sensitive to environmental changes (Figs. 4 and 5). The ability to maintain a
community’s phylogeny could reflect its ecological niche preservation capacity under changed
environmental conditions [20]. Thus, the decoupling between community composition and phylogeny
indicates that abundant bacteria could be better in preserving ecological niches than rare bacteria. This
could explain the easier loss of narrow niche functions (such as the degradation of toxic compounds,
which is typically performed by rare bacteria) than broad niche functions (such as the degradation of
organic compounds in general, which are performed by all bacteria) [8, 40]. Rare bacteria exhibited a greater community phylogenetic variation than abundant bacteria The critical contribution of environmental filtering on the community phylogeny
of rare bacteria is consistent with that observed in the marine environment [36]. However, unlike aquatic
environment, the non-fluidic soil [18] further constrain the chance of dispersal, thus geospatial factors
exhibited greater influence on the community phylogeny of rare subcommunity in soil than in marine
ecosystems [14, 36]. Conclusions Our study provides novel insights to explain the bacterial distribution patterns of rare and abundant
bacteria in alpine grassland soils. The similar biogeographical patterns of rare and abundant
subcommunities were consistent with that observed in aquatic ecosystems, highlighting their global
consistency. Our results also revealed that the similar biogeographical patterns of rare and abundant
subcommunities could be predominately influenced by stochastic and deterministic processes,
respectively. We propose that this could be due to the co-variation of stochastic (geospatial) and
deterministic (environmental) factors. This shall be further investigated to disentangle the contributions
of stochasticity and determinism in other ecosystems with inconsistent changing patterns of geospatia
and environmental factors. Materials And Methods Site description and soil sampling The study area is located on the Tibetan Plateau, between 31 and 33°N latitude and 79 and 93°E
longitude, with an average altitude above 4400 m a.s.l. Soil samples were collected along a 1200 km
transect (Additional file 2: Fig. S1) in July 2015. The 1200 km transect contained ecosystems classified
as alpine desert grassland (4 sites), alpine steppe (11 sites), and alpine meadow (4 sites) from west to
east, thereby forming a natural gradient with broad environmental conditions. At each sampling site, four
1 m × 1 m quadrats were randomly selected. In each quadrat, approximately 200 g of bulk soils (0–
10 cm) were collected after plant material being removed by sieving (2.0 mm), and were transported on
ice to laboratories at Beijing, then stored at -80 °C until processed. The plant Shannon diversity, plant
above-ground biomass, and plant below-ground biomass were calculated as described previously [41]. Soil physiochemical analysis Soil pH was measured in a 1:5 soil-to-water suspension using a pH meter (Sartorius, Goettingen,
Germany). Soil nitrate (NO3
−) and ammonium (NH4
+) were extracted with 2 M KCl (1:5) and their
concentrations were determined using a Smartchem200 Discrete Auto Analyzer (Alliance, Frépillon,
France). Total organic carbon (TOC) was measured in the solid-state using a TOC analyzer (Shimadzu,
Kyoto, Japan). The aridity index (potential evaporation/precipitation) of each site was obtained from the
Global Aridity Index dataset (www.cgiar-csi.org) [42]. The mean annual precipitation (MAP) and mean
annual temperature (MAT) were predicted using the meteorological data between 2003 and 2012 from 33
climatic stations (China meteorological Data Sharing Service System; http://cdc.cma.gov.cn/) using the
Kriging interpolation. DNA extraction, PCR and high-throughput 16S rRNA gene
sequencing DNA extraction, PCR and high-throughput 16S rRNA gene
sequencing Total DNA was extracted using the MO BIO Power Soil DNA extraction kit (Mo Bio Laboratories, Carlsbad,
CA, USA) according to manufacturer’s instructions. Universal primers 515F (5'- Total DNA was extracted using the MO BIO Power Soil DNA extraction kit (Mo Bio Laboratories, Carlsbad,
CA, USA) according to manufacturer’s instructions. Universal primers 515F (5'-
GTGCCAGCMGCCGCGGTAA-3') and 909r (5'-CCCCGYCAATTCMTTTRAGT-3') with 12 nt unique barcodes
were used to amplify the V4 and V5 hyper-variable regions of the 16S rRNA gene [43]. The PCR mixture
(25 ∝l) contained 1x PCR buffer, 1.5 mM of MgCl2, 0.4 ∝M each of deoxynucleoside triphosphate bases,
1.0 ∝M of each primer, 0.5 U of Ex Taq (TaKaRa, Dalian, China) and 20 ng of DNA template. The PCR
amplification program included an initial denaturation at 94 °C for 3 min, followed by 30 cycles of 94 °C
for 20 s, 56 °C for 30 s, and 72 °C for 45 s, and a final extension at 72 °C for 10 min. PCR products were
purified using a OMEGA Gel Extraction Kit (Omega Bio-Tek, Georgia, USA) following electrophoresis. PCR
products from different samples were pooled in equimolar amounts and then used for pair-end
sequencing (2 × 250 bp) on the Illumina MiSeq platform at the Chengdu Institute of Biology, Chinese
Academy of Sciences. Sequence data processing Page 9/23 Three samples were not sequenced successfully, generating zero reads. These samples were not re-
sequenced but removed from downstream analysis to avoid artificial community structure differences
caused by batch-to-batch sequencing variation. Raw sequence data of the remaining 73 samples were
processed using the MOTHUR pipeline (v. 1.34.3) [44]. Paired-end reads were merged and quality
screened by removing any reads with > 1 mismatch in the primer region, average quality < 30, ambiguous
bases > 0, and homopolymer length > 9 from downstream analysis. The remaining sequences were
aligned against the Silva reference alignment (release 128), which was trimmed to include only the same
region amplified, and those sequences that did not align were removed. Chimeric sequences were
identified using UCHIME [45] and removed. The remaining sequences were classified using the Bayesian
classifier against the Silva database (release 128), with a minimum confidence score of 80% [46]. All
archaeal and unknown sequences were removed using the remove.lineage command. DNA extraction, PCR and high-throughput 16S rRNA gene
sequencing The archaeal
sequences were removed due to a recent report showing that the currently used prokaryotic primers may
have poor coverage on the archaeal domain [47]. Finally, sequences were classified into operational
taxonomic units (OTUs) at 97% identity and singletons were removed. To correct for possible uneven
sequencing coverage between samples, samples were randomly sub-sampled without replacement to an
equal depth of 11,046, which was the smallest sample size in the dataset. This ended with a total of
806,358 high-quality bacterial sequences, with an average sequence length of 329 bp. Bacterial richness
was calculated using the summary.single command in Mothur [44]. Defining rare and abundant OTUs Various arbitrary thresholds have been used to define rare and abundant OTUs in literatures. For example,
abundant OTUs have been defined as OTUs with relative abundance > 1% or 0.1%, whereas rare OTUs
have been defined as OTUs with relative abundance < 0.1%, 0.001% or 0.006% [13, 24, 48–50]. Here, rare
OTUs were defined as the OTUs with relative abundance < 0.1% in all samples analysed, whereas
abundant OTUs were defined as the OTUs with a relative abundance > 1% in one or more samples. The
latter contained both OTUs that were abundant ubiquitously and occasionally across the transect (Shade
et al., 2014). Transformation and normalization of environmental and
geospatial factors Measured environmental factors were divided into locally measured (local) and climatic factors. The
local factors included soil pH, soil total organic carbon (TOC), ammonium (NH4
+) and nitrate (NO3
−)
concentrations, soil moisture, plant Shannon diversity, plant above-ground biomass, and plant below-
ground biomass. The climatic factors included mean annual temperature (MAT), mean annual
precipitation (MAP), and aridity index. The geographical distances between samples were calculated
from GPS coordinates. This geographical distance matrix was converted into a set of geospatial factors
using the principal coordinates of neighbour matrices analysis (PCNM) [14, 55]. The environmental factors (both local and climatic) and the derived geospatial factors were combined,
and then transformed to reduce data skewness. Specifically, TOC, NH4
+, plant above-ground biomass,
plant below-ground biomass, and aridity indices were log-transformed, whereas MAP, NO3
−, and soil
moisture were square-root transformed. The resulting environmental and geospatial factors were further
normalized (x-mean/standard derivation) to improve data normality for multivariate statistical analysis. Quantifying community structure variation and assembly
process The community structure variations of the rare, abundant and entire communities were calculated
separately. Bray-Curtis dissimilarity matrices were calculated using the square-root transformed
community abundance matrices, and were used to indicate community compositional dissimilarity
between samples. Weighted β-MNTD distance matrices were calculated to indicate community
phylogenetic distance between samples. The Bray-Curtis dissimilarity was calculated using Primer V6
[51], whereas the β-MNTD distance was calculated using the function ‘comdistnt’ in the package ‘Picant
in R (http//www.r-project.org), with the exclude.conspecific parameter being set to TRUE. Bray-Curtis dissimilarity-based modified stochasticity ratio (MST) index was calculated using the ‘NST’
package in R (http//www.r-project.org) to represent the contribution of stochasticity to community Page 10/23 Page 10/23 compositional assembly [52]. The MST index ranges from 0–100%, a 0% indicates zero contribution of
stochasticity, whereas 100% indicates the community assembly being completely stochasticity-driven. We also calculated the β-Nearest Taxon index (βNTI) to quantify the relative contributions of stochastic
and deterministic processes in shaping community phylogenetic assembly [20] using the script provided
in Swenson [53]. βNTI values < − 2 indicate the between-sample phylogenetic distance being significantly
lower than expected by chance (homogeneous selection); βNTI values > 2 indicate the phylogenetic
distance being significantly higher than expected (variable selection) [23, 54]. βNTI values between − 2
and 2 indicate that the observed phylogenetic distance does not significantly deviate from the null model
and the assembly process is not the result of deterministic selection, but due to stochastic processes. Transformation and normalization of environmental and
geospatial factors Consent for publication Not applicable. Authors' contributions WK conceived the study and developed the idea with MJ, LY, XD, and WK and LY sampled the field soils
and surveyed plants. MJ conducted the data statistical analysis. MJ and WK wrote the first draft of the
manuscript, JS, DC, and BF revised the manuscript substantially. All authors read and approved the final
manuscript. Conflict interests The authors declare no conflict of interest. Ethical approval This article does not contain any studies with human participants or animals performed by any of the
authors. Funding This project was financially supported by Chinese Academy of Sciences (XDA19070304, QYZDB-SSW-
DQC033 and XDA20050101), and National Natural Science Foundation of China (41771303). J.S. was
supported by the US Department of Energy (DOE), Office of Biological and Environmental Research, as
part of the Subsurface Biogeochemical Research Program’s Scientific Focus Area at the Pacific Northwest
National Laboratory (PNNL). PNNL is operated for DOE by Battelle Memorial Institute under contract DE-
AC06-76RLO 1830. Availability of data and material The raw sequencing reads generated in the present study supporting the conclusions of this article are
available in the National Center for Biotechnology Information (NCBI) Sequence Read Archive under
project ID PRJNA419993, available at https://www.ncbi.nlm.nih.gov/bioproject/PRJNA419993. Correlation and multivariate analyses Permutational analysis of variance (PERMANOVA) was used to test the significance of community
compositional and phylogenetic differences among the three grassland types [56]. Variation partitioning
analysis (VPA) based on distance-based linear model (DistLM) was used to partition the relative
contributions of local, climatic and geospatial factors, as well as the contribution of each individual
environmental factor (the pure effect) [56]. Then, distance-based redundancy analysis (dbRDA) ordination
plots were used to visualize the associations between bacterial communities and the measured
environmental/geospatial factors [56]. In addition to VPA, Mantel and partial Mantel tests were conducted
to explore the correlations between bacterial community composition dissimilarity (or phylogenetic
distance) and local /climatic/geospatial factors [57]. The PERMANOVA, DistLM and dbRDA ordination
analyses were performed using Primer V6 [51], whereas the Mantel and partial Mantel tests were
performed using the VEGAN package [55] in R (http//www.r-project.org). Page 11/23 Acknowledgments The authors greatly thank Dr. Xiagnzhen Li in the Chengdu Institute of Biology, Chinese Academy of
Sciences (CAS) for thoughtful discussions. We thank Dr. Jiangshan Lai in the Institute of Botany, CAS for
the statistical analysis assistance, and Dr. Yongqin Liu in the Institute of Tibetan Plateau Research, CAS
for the computational plantform in partial data processing. Correspondence to Weidong Kong. Correspondence to Weidong Kong. References 1. Naeem S, Li S. Biodiversity enhances ecosystem reliability. Nature. 1997;390:507-09. 1. Naeem S, Li S. Biodiversity enhances ecosystem reliability. Nature. 1997;390:507-09. 2. Vigneron A, Cruaud P, Alsop E, de Rezende JR, Head IM, Tsesmetzis N. Beyond the tip of the iceberg:
a new view of the diversity of sulfite- and sulfate-reducing microorganisms. ISME J. 2018;12:2096-
99. 3. Hua Z, Han Y, Chen L, Liu J, Hu M, Li S, et al. Ecological roles of dominant and rare prokaryotes in
acid mine drainage revealed by metagenomics and metatranscriptomics. ISME J. 2015;9:1280-94. 3. Hua Z, Han Y, Chen L, Liu J, Hu M, Li S, et al. Ecological roles of dominant and rare prokaryotes in
acid mine drainage revealed by metagenomics and metatranscriptomics. ISME J. 2015;9:1280-94. 4. Montoya JP, Holl CM, Zehr JP, Hansen A, Villareal TA, Capone DG. High rates of N2 fixation by
unicellular diazotrophs in the oligotrophic Pacific Ocean. Nature. 2004;430:1027-31. 5. Hausmann B, Knorr K-H, Schreck K, Tringe SG, del Rio TG, Loy A, et al. Consortia of low-abundance
bacteria drive sulfate reduction-dependent degradation of fermentation products in peat soil
microcosms. ISME J. 2016;10:2365-75. 6. Hol WHG, De Boer W, Termorshuizen AJ, Meyer KM, Schneider JHM, Van Dam NM, et al. Reduction of
rare soil microbes modifies plant–herbivore interactions. Ecol Lett. 2010;13:292-301. 7. Gibbons SM, Caporaso JG, Pirrung M, Field D, Knight R, Gilbert JA. Evidence for a persistent
microbial seed bank throughout the global ocean. Proc Natl Acad Sci U S A. 2013;110:4651-55. 8. Mouillot D, Bellwood DR, Baraloto C, Chave J, Galzin R, Harmelinvivien M, et al. Rare species support
vulnerable functions in high-diversity ecosystems. PLOS Biol. 2013;11:e1001569. 9. Jousset A, Bienhold C, Chatzinotas A, Gallien L, Gobet A, Kurm V, et al. Where less may be more: how
the rare biosphere pulls ecosystems strings. ISME J. 2017;11:853-62. 9. Jousset A, Bienhold C, Chatzinotas A, Gallien L, Gobet A, Kurm V, et al. Where less may be more: how
the rare biosphere pulls ecosystems strings. ISME J. 2017;11:853-62. 10. Alonso-Saez L, Diaz-Perez L, Moran XAG. The hidden seasonality of the rare biosphere in coastal
marine bacterioplankton. Appl Environ Microbiol. 2015;17:3766-80. 10. Alonso-Saez L, Diaz-Perez L, Moran XAG. The hidden seasonality of the rare biosphere in coastal
marine bacterioplankton. Appl Environ Microbiol. 2015;17:3766-80. 11. Gobet A, Boeer SI, Huse SM, van Beusekom JEE, Quince C, Sogin ML, et al. Corresponding authors Page 12/23 Page 12/23 Correspondence to Weidong Kong. References Zhou J, Deng Y, Zhang P, Xue K, Liang Y, Van Nostrand JD, et al. Stochasticity, succession, and
environmental perturbations in a fluidic ecosystem. Proc Natl Acad Sci U S A. 2014;111:E836-E45. 19. Lozupone C, Lladser ME, Knights D, Stombaugh J, Knight R. UniFrac: an effective distance metric for
microbial community comparison. Isme Journal. 2011;5:169-72. 19. Lozupone C, Lladser ME, Knights D, Stombaugh J, Knight R. UniFrac: an effective distance metric for
microbial community comparison. Isme Journal. 2011;5:169-72. 20. Stegen JC, Lin X, Konopka AE, Fredrickson JK. Stochastic and deterministic assembly processes in
subsurface microbial communities. ISME J. 2012;6:1653-64. 20. Stegen JC, Lin X, Konopka AE, Fredrickson JK. Stochastic and deterministic assembly processes in
subsurface microbial communities. ISME J. 2012;6:1653-64. 21. Stegen JC, Lin X, Fredrickson JK, Chen X, Kennedy DW, Murray CJ, et al. Quantifying community
assembly processes and identifying features that impose them. ISME J. 2013;7:2069-79. 22. Moroenyane I, Dong K, Singh D, Chimphango SBM, Adams JM. Deterministic processes dominate
nematode community structure in the Fynbos Mediterranean heathland of South Africa. Ecol Evol. 2016;30:685-701. 23. Dini-Andreote F, Stegen JC, van Elsas JD, Salles JF. Disentangling mechanisms that mediate the
balance between stochastic and deterministic processes in microbial succession. Proc Natl Acad Sci
U S A. 2015;112:E1326-E32. 24. Liao J, Cao X, Wang J, Zhao L, Sun J, Jiang D, et al. Similar community assembly mechanisms
underlie similar biogeography of rare and abundant bacteria in lakes on Yungui Plateau, China. Limnol Oceanogr. 2017;62:723-35. 25. Ho A, Di Lonardo DP, Bodelier PLE. Revisiting life strategy concepts in environmental microbial
ecology. FEMS Microbiol Ecol. 2017;93:fix006. 26. Leff JW, Jones SE, Prober SM, Barberán A, Borer ET, Firn JL, et al. Consistent responses of soil
microbial communities to elevated nutrient inputs in grasslands across the globe. Proc Natl Acad Sci
U S A 2015;112:10967-72. 27. Eilers KG, Lauber CL, Knight R, Fierer N. Shifts in bacterial community structure associated with
inputs of low molecular weight carbon compounds to soil. Soil Biol Biochem. 2010;42:896-903. 28 K
h AL Oli
t
h
i t
h
Bi
2001 23 657 61 27. Eilers KG, Lauber CL, Knight R, Fierer N. Shifts in bacterial community structure associated with
inputs of low molecular weight carbon compounds to soil. Soil Biol Biochem. 2010;42:896-903. 28. Koch AL. Oligotrophs versus copiotrophs. Bioessays. 2001;23:657-61. 29. Taylor TB, Buckling A. Competition and Dispersal in Pseudomonas aeruginosa. Am Nat. 2010;176:83-89. 29. References Diversity and dynamics of
rare and of resident bacterial populations in coastal sands. ISME J. 2012;6:542-53. 11. Gobet A, Boeer SI, Huse SM, van Beusekom JEE, Quince C, Sogin ML, et al. Diversity and dynamics of
rare and of resident bacterial populations in coastal sands. ISME J. 2012;6:542-53. 12. Hugoni M, Taib N, Debroas D, Domaizon I, Dufournel IJ, Bronner G, et al. Structure of the rare
archaeal biosphere and seasonal dynamics of active ecotypes in surface coastal waters. Proc Natl
Acad Sci U S A. 2013;110:6004-09. 12. Hugoni M, Taib N, Debroas D, Domaizon I, Dufournel IJ, Bronner G, et al. Structure of the rare
archaeal biosphere and seasonal dynamics of active ecotypes in surface coastal waters. Proc Natl
Acad Sci U S A. 2013;110:6004-09. 13. Liu L, Yang J, Yu Z, Wilkinson DM. The biogeography of abundant and rare bacterioplankton in the
lakes and reservoirs of China. ISME J. 2015;9:2068-77. 13. Liu L, Yang J, Yu Z, Wilkinson DM. The biogeography of abundant and rare bacterioplankton in the
lakes and reservoirs of China. ISME J. 2015;9:2068-77. 14. Mo Y, Zhang W, Yang J, Lin Y, Yu Z, Lin S. Biogeographic patterns of abundant and rare
bacterioplankton in three subtropical bays resulting from selective and neutral processes. ISME J. 2018;12:2198-210. 14. Mo Y, Zhang W, Yang J, Lin Y, Yu Z, Lin S. Biogeographic patterns of abundant and rare
bacterioplankton in three subtropical bays resulting from selective and neutral processes. ISME J. 2018;12:2198-210. 15. Galand PE, Casamayor EO, Kirchman DL, Lovejoy C. Ecology of the rare microbial biosphere of the
Arctic Ocean. Proc Natl Acad Sci USA. 2009;106:22427-32. 15. Galand PE, Casamayor EO, Kirchman DL, Lovejoy C. Ecology of the rare microbial biosphere of the
Arctic Ocean. Proc Natl Acad Sci USA. 2009;106:22427-32. Page 13/23 Page 13/23 16. Wu WX, Logares R, Huang BQ, Hsieh CH. Abundant and rare picoeukaryotic sub-communities present
contrasting patterns in the epipelagic waters of marginal seas in the northwestern Pacific Ocean. Environ Microbiol. 2017;19:287-300. 17. Galand PE, Casamayor EO, Kirchman DL, Lovejoy C. Ecology of the rare microbial biosphere of the
Arctic Ocean. Proc Natl Acad Sci U S A. 2009;106:22427-32. 18. Zhou J, Deng Y, Zhang P, Xue K, Liang Y, Van Nostrand JD, et al. Stochasticity, succession, and
environmental perturbations in a fluidic ecosystem. Proc Natl Acad Sci U S A. 2014;111:E836-E45. 18. References Taylor TB, Buckling A. Competition and Dispersal in Pseudomonas aeruginosa. Am Nat. 2010;176:83-89. 30. Oono R, Rasmussen A, Lefevre E. Distance decay relationships in foliar fungal endophytes are driven
by rare taxa. Environ Microbiol. 2017;19:2794-805. 30. Oono R, Rasmussen A, Lefevre E. Distance decay relationships in foliar fungal endophytes are driven
by rare taxa. Environ Microbiol. 2017;19:2794-805. 31. Cao P, Wang J, Hu H, Zheng Y, Ge Y, Shen J, et al. Environmental filtering process has more important
roles than dispersal limitation in shaping large-scale prokaryotic beta diversity patterns of grassland
soils. Microb Ecol. 2016;72:221-30. 31. Cao P, Wang J, Hu H, Zheng Y, Ge Y, Shen J, et al. Environmental filtering process has more important
roles than dispersal limitation in shaping large-scale prokaryotic beta diversity patterns of grassland
soils. Microb Ecol. 2016;72:221-30. Page 14/23 Page 14/23 32. Ferrari BC, Bissett A, Snape I, Van Dorst J, Palmer AS, Ji M, et al. Geological connectivity drives
microbial community structure and connectivity in polar, terrestrial ecosystems. Environ Microbiol. 2016;18:1834-49. 33. Evans S, Martiny JBH, Allison SD. Effects of dispersal and selection on stochastic assembly in
microbial communities.ISME J. 2016;11:176-85. 33. Evans S, Martiny JBH, Allison SD. Effects of dispersal and selection on stochastic assembly in
microbial communities.ISME J. 2016;11:176-85. 34. Cadotte MW. Phylogenetic diversity-ecosystem function relationships are insensitive to phylogenetic
edge lengths. Funct Ecol. 2015;29:718-23. 34. Cadotte MW. Phylogenetic diversity-ecosystem function relationships are insensitive to phylogenetic
edge lengths. Funct Ecol. 2015;29:718-23. 35. Wang J, Shen J, Wu Y, Tu C, Soininen J, Stegen JC, et al. Phylogenetic beta diversity in bacterial
assemblages across ecosystems: deterministic versus stochastic processes. ISME J. 2013;7:1310-
21. 35. Wang J, Shen J, Wu Y, Tu C, Soininen J, Stegen JC, et al. Phylogenetic beta diversity in bacterial
assemblages across ecosystems: deterministic versus stochastic processes. ISME J. 2013;7:1310-
21. 36. Wu W, Logares R, Huang B, Hsieh CH. Abundant and rare picoeukaryotic sub-communities present
contrasting patterns in the epipelagic waters of marginal seas in the northwestern Pacific Ocean. Environ Microbiol. 2017;19:287-300. 37. Bissett A, Richardson AE, Baker G, Wakelin S, Thrall PH. Life history determines biogeographical
patterns of soil bacterial communities over multiple spatial scales. Mol Ecol. 2010;19:4315-27. 38. Delgado-Baquerizo M, Oliverio AM, Brewer TE, Benavent-Gonzalez A, Eldridge DJ, Bardgett RD, et al. A
global atlas of the dominant bacteria found in soil. Science. 2018;359:320-25. 39. References Yan WK, Ma HM, Shi GT, Li YS, Sun B, Xiao X, et al. Independent shifts of abundant and rare bacterial
populations across East Antarctica glacial foreland. Front Microbiol. 2017;8:1534. 51. Clarke KR, Warwick RM. PRIMER v6: user manual/tutorial. 2 ed: PRIMER-E, Plymouth; 2006. 51. Clarke KR, Warwick RM. PRIMER v6: user manual/tutorial. 2 ed: PRIMER-E, Plymouth; 2006. 52. Ning DL, Deng Y, Tiedje JM, Zhou JZ. A general framework for quantitatively assessing ecological
stochasticity. Proc Natl Acad Sci U S A. 2019;116:16892-98. 52. Ning DL, Deng Y, Tiedje JM, Zhou JZ. A general framework for quantitatively assessing ecological
stochasticity. Proc Natl Acad Sci U S A. 2019;116:16892-98. 53. Swenson NG. Functional and phylogenetic ecology in R (Use R! series). Gentleman R, Hornik K,
Parmigiani G, editors. New York: Springer Science+Business Media; 2014. 144-45. 53. Swenson NG. Functional and phylogenetic ecology in R (Use R! series). Gentleman R, Hornik K,
Parmigiani G, editors. New York: Springer Science+Business Media; 2014. 144-45. 54. Stegen JC, Lin X, Fredrickson JK, Konopka AE. Estimating and mapping ecological processes
influencing microbial community assembly. Frontiers in Microbiology. 2015;6:370. 55. Dixon P. VEGAN, a package of R functions for community ecology. J Veg Sci. 2003;14:927-30. 56. Legendre P, Anderson MJ. Distance-based redundancy analysis: Testing multispecies responses in
multifactorial ecological experiments. Ecol Monogr. 1999;69:1-24. 57. Legendre P, Legendre L. Numerical ecology. 3rd ed. Amsterdam: Elsevier Science Ltd; 2012. 57. Legendre P, Legendre L. Numerical ecology. 3rd ed. Amsterdam: Elsevier Science Ltd; 2012. References Kurm V, van der Putten WH, de Boer W, Naus-Wiezer S, Hol WHG. Low abundant soil bacteria can be
metabolically versatile and fast growing. Ecology. 2017;98:555-64. 39. Kurm V, van der Putten WH, de Boer W, Naus-Wiezer S, Hol WHG. Low abundant soil bacteria can be
metabolically versatile and fast growing. Ecology. 2017;98:555-64. 40. Girvan MS, Campbell CD, Killham K, Prosser JI, Glover LA. Bacterial diversity promotes community
stability and functional resilience after perturbation. Environ Microbiol. 2005;7:301-13. 40. Girvan MS, Campbell CD, Killham K, Prosser JI, Glover LA. Bacterial diversity promotes community
stability and functional resilience after perturbation. Environ Microbiol. 2005;7:301-13. 41. Zhao K, Kong W, Wang F, Long X-E, Guo C, Yue L, et al. Desert and steppe soils exhibit lower
autotrophic microbial abundance but higher atmospheric CO2 fixation capacity than meadow soils. Soil Biol Biochem. 2018;127:230-38. 42. Zomer RJ, Trabucco A, Bossio DA, Verchot L. Climate change mitigation: a spatial analysis of global
land suitability for clean development mechanism afforestation and reforestation. Agric Ecosyst
Environ. 2008;126:67-80. 43. Tuan NN, Chang Y-C, Yu C-P, Huang S-L. Multiple approaches to characterize the microbial
community in a thermophilic anaerobic digester running on swine manure: A case study. Microbiol
Res. 2014;169:717-24. 44. Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, et al. Introducing mothur: open-
source, platform-Independent, community-supported software for describing and comparing
microbial communities. Appl Environ Microbiol. 2009;75:7537-41. 45. Edgar RC, Haas BJ, Clemente JC, Quince C, Knight R. UCHIME improves sensitivity and speed of
chimera detection. Bioinformatics. 2011;27:2194-200. 46. Wang Q, Garrity GM, Tiedje JM, Cole JR. Naïve Bayesian classifier for rapid assignment of rRNA
sequences into the new bacterial taxonomy. Appl Environ Microbiol. 2007;73:5261-67. 46. Wang Q, Garrity GM, Tiedje JM, Cole JR. Naïve Bayesian classifier for rapid assignment of rRNA
sequences into the new bacterial taxonomy. Appl Environ Microbiol. 2007;73:5261-67. Page 15/23 Page 15/23 47. Bahram M, Anslan S, Hildebrand F, Bork P, Tedersoo L. Newly designed 16S rRNA metabarcoding
primers amplify diverse and novel archaeal taxa from the environment. Environ Microbiol Rep. 2019;11:487-94. 48. Jiao S, Chen W, Wei G. Biogeography and ecological diversity patterns of rare and abundant bacteria
in oil-contaminated soils. Mol Ecol. 2017;26:5305-17. 49. Jiao S, Luo Y, Lu M, Xiao X, Lin Y, Chen W, et al. Distinct succession patterns of abundant and rare
bacteria in temporal microcosms with pollutants. Environ Pollut. 2017;225:497-505. 50. Figure 1 Taxonomic compositions and abundance-occupancy relationships. The taxonomic compositions of rare,
abundant and entire bacterial communities are shown in (A), and the relative abundance-occupancy
relationships of rare and abundant OTUs are shown in (B). Occupancy is the number of samples an OTU
being detected from. Taxonomic compositions and abundance-occupancy relationships. The taxonomic compositions of rare,
abundant and entire bacterial communities are shown in (A), and the relative abundance-occupancy
relationships of rare and abundant OTUs are shown in (B). Occupancy is the number of samples an OTU
being detected from. Page 17/23 Figures Page 16/23 Page 16/23 Figure 1 Figure 1
Taxonomic compositions and abundance-occupancy relationships. The taxonomic compositions of rare,
abundant and entire bacterial communities are shown in (A), and the relative abundance-occupancy
relationships of rare and abundant OTUs are shown in (B). Occupancy is the number of samples an OTU
being detected from. Figure 1 Page 17/23 Page 17/23 Figure 2
Community composition comparison for rare (A), abundant (B), and entire (C) bacterial communities. The
community compositional variation is quantified using Bray-Curtis dissimilarity. The proportion of
community variation explained by the measured local, climatic and geospatial factors are calculated
using DistLM analysis, and visualized on dbRDA ordination plots (only the top five environmental and
geospatial factors are displayed) TOC total organic carbon; PCNM1 5 are geographical distance derived Figure 2 Figure 2 Community composition comparison for rare (A), abundant (B), and entire (C) bacterial communities. The
community compositional variation is quantified using Bray-Curtis dissimilarity. The proportion of
community variation explained by the measured local, climatic and geospatial factors are calculated
using DistLM analysis, and visualized on dbRDA ordination plots (only the top five environmental and
geospatial factors are displayed). TOC, total organic carbon; PCNM1-5 are geographical distance derived
geospatial factors. Community composition comparison for rare (A), abundant (B), and entire (C) bacterial communities. The
community compositional variation is quantified using Bray-Curtis dissimilarity. The proportion of
community variation explained by the measured local, climatic and geospatial factors are calculated
using DistLM analysis, and visualized on dbRDA ordination plots (only the top five environmental and
geospatial factors are displayed). TOC, total organic carbon; PCNM1-5 are geographical distance derived
geospatial factors. Page 18/23 Figure 3
The variation of bacterial community composition explained by local, climatic and geospatial factors. Variation partitioning analysis for rare (A), abundant (B), and entire (C) bacterial communities. The
variation explained by pure local climatic and geospatial factors corresponds to the proportion of Figure 3 The variation of bacterial community composition explained by local, climatic and geospatial factors. Variation partitioning analysis for rare (A), abundant (B), and entire (C) bacterial communities. The
variation explained by pure local, climatic and geospatial factors corresponds to the proportion of
community composition explained without the effect of the others by the DistLM analysis. Mantel and
partial Mantel tests for the correlations between community composition and the measured local, Page 19/23 Page 19/23 climatic and geospatial factors using Pearson’s coefficients for rare (D), abundant (E), and entire (F)
communities. *: P < 0.01, **: P < 0.001, ns: not significant. Local factors include soil pH, soil total organic
carbon, NH4+ and NO3- concentrations, soil moisture, plant Shannon diversity, plant above-ground
biomass, and plant below-ground biomass; the climatic factors include mean annual temperature, mean
annual precipitation, and aridity index. – indicates negative or zero proportion of the community
composition explained. p
p
Figure 4 p
p Figure 4 Community phylogeny comparison for rare (A), abundant (B), and entire (C) bacterial communities. The
community phylogeny variation is quantified using the -mean nearest taxon distance (-MNTD). The
proportion of community phylogeny explained by the measured local, climatic and geospatial factors are
calculated using DistLM analysis, and visualized on dbRDA ordination plots (only the top five
environmental and geospatial factors are displayed). TOC, total organic carbon; PCNM1-5 are
geographical distance derived geospatial factors. Page 20/23 Figure 5
The variation of bacterial community phylogeny explained by local, climatic and geospatial factors. Variation partitioning analysis for rare (A), abundant (B), and entire (C) communities. The variation Page 21/23
Figure 5
The variation of bacterial community phylogeny explained by local, climatic and geospatial factors. Variation partitioning analysis for rare (A), abundant (B), and entire (C) communities. The variation
explained by pure local, climatic and geospatial factors corresponds to the proportion of community
phylogeny explained without the effect of the others by the DistLM analysis. Mantel and partial Mante
tests for the correlation between community phylogenetic distance and the measured local, climatic a Figure 5 The variation of bacterial community phylogeny explained by local, climatic and geospatial factors. Variation partitioning analysis for rare (A), abundant (B), and entire (C) communities. The variation
explained by pure local, climatic and geospatial factors corresponds to the proportion of community
phylogeny explained without the effect of the others by the DistLM analysis. Mantel and partial Mantel
tests for the correlation between community phylogenetic distance and the measured local, climatic and Page 21/23 geospatial factors using Pearson’s coefficients for rare (A), abundant (B), and entire (C) communities. *: P
< 0.01, **: P < 0.001, ns: not significant. Local factors included soil pH, soil total organic carbon, NH4+
and NO3- concentrations, soil moisture, plant Shannon diversity, plant above-ground biomass, and plant
below-ground biomass; the climatic factors included average annual temperature, annual precipitation,
and aridity index. – indicates negative or zero proportion of the community phylogeny explained. geospatial factors using Pearson’s coefficients for rare (A), abundant (B), and entire (C) communities. *: P
< 0.01, **: P < 0.001, ns: not significant. Local factors included soil pH, soil total organic carbon, NH4+
and NO3- concentrations, soil moisture, plant Shannon diversity, plant above-ground biomass, and plant
below-ground biomass; the climatic factors included average annual temperature, annual precipitation,
and aridity index. – indicates negative or zero proportion of the community phylogeny explained. Page 22/23
and NO3- concentrations, soil moisture, plant Shannon diversity
below-ground biomass; the climatic factors included average an
and aridity index. – indicates negative or zero proportion of the
Figure 6 Figure 6 Figure 6 Page 22/23 Correlations between the community composition and phylogeny for rare, abundant, and entire
communities. The compositional dissimilarity is measured using Bray-Curtis dissimilarity, whereas the
phylogenetic distance is measured using -mean nearest taxon distance. The correlations are fitted by
power law correlations. Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Additionalfile1.docx Additionalfile2.docx Page 23/23
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https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0080820&type=printable
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Complexity Reduction of Rate-Equations Models for Two-Choice Decision-Making
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PloS one
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cc-by
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Abstract We are concerned with the complexity reduction of a stochastic system of differential equations governing the dynamics of
a neuronal circuit describing a decision-making task. This reduction is based on the slow-fast behavior of the problem and
holds on the whole phase space and not only locally around the spontaneous state. Macroscopic quantities, such as
performance and reaction times, computed applying this reduction are in agreement with previous works in which the
complexity reduction is locally performed at the spontaneous point by means of a Taylor expansion. Citation: Carrillo JA, Cordier S, Deco G, Mancini S (2013) Complexity Reduction of Rate-Equations Models for Two-Choice Decision-Making. PLoS ONE 8(12):
e80820. doi:10.1371/journal.pone.0080820 Editor: Dante R. Chialvo, National Research & Technology Council, Argentina Editor: Dante R. Chialvo, National Research & Technology Council, Argentina Received July 17, 2013; Accepted October 15, 2013; Published December 5, 2013 Received July 17, 2013; Accepted October 15, 2013; Published December 5, 2013 Copyright: 2013 Carrillo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: J.A. Carrillo acknowledges support from the Royal Society by a Wolfson Research Merit Award and by the Engineering and Physical Sciences Research
Council grant with references EP/K008404/1. J.A. Carrillo was partially supported by the project MTM2011-27739-C04-02 DGI (Spain) and 2009-SGR-345 from
AGAUR-Generalitat de Catalunya. S. Cordier and S. Mancini acknowledge support by the ANR project MANDy, Mathematical Analysis of Neuronal Dynamics, ANR-
09-BLAN-0008-01. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: simona.mancini@univ-orleans.fr with a visual stimulus consisting of a field of randomly moving
dots. On any one trial, a fixed fraction of the dots, determined by
the experimentalist, moves coherently in one of two directions. The subject must discriminate in which direction the majority of
dots are moving. Electrophysiological recordings from awake
behaving monkeys performing this task have shown that trial-
averaged spiking activity of neurons in the lateral intraparietal
cortex (LIP) reflects the accumulation of information mentioned
above [4–6]. In fact, LIP cells receive excitatory drive from
direction-selective cells of extrastriate visual area MT. Introduction Goal-oriented
behavior
involves
the
constant
making
of
decisions between alternative choices. Hence, a thorough under-
standing
of
the
mechanisms
underlying
decision-making
is
fundamental in behavioral neurosciences. During the last years
tremendous
advances
in
neurophysiological
and
theoretical
neurosciences have started to reveal the neural mechanisms
underlying decision-making. Most of these studies are focused on
binary decision-making. In binary decision tasks, the subject is
asked to make a choice between two alternatives according to an
experimentally defined criterion based on sensory input informa-
tion. The difficulty of the making of the decision can be
manipulated by the level of uncertainty in the discrimination of
the sensory input information which can be influenced by
regulating the signal to noise ratio. For the same pattern of
stimulation the decision of the subject from trial-to-trial is
stochastic but such that its trial-to-trial average is determined by
the input. A computational model of the cortical circuit putatively
underlying LIP cell activity has been proposed and studied
numerically [7–9]. This model was able to explain qualitatively the
experimentally observed trial-averaged spiking activity. The model
consists of two populations of spiking neurons, within which
interactions are mediated by excitatory synapses and between
which interactions occur principally through an intermediary
population of inhibitory interneurons. Sensory input may bias the
competition in favor of one of the populations, potentially resulting
in a gradually developing decision in which neurons in the chosen
population exhibit increased activity while activity in the other
population is inhibited. When the activity of one of the two
populations exceeds a pre-defined threshold then a behavioral
response is generated. There are two possible mechanisms by
which the decision can be made which correspond to two different
dynamical regimes. In one mechanism, the appearance of the
stimulus destabilizes the so-called spontaneous state, in which all
neurons show low activity. In the other mechanism, even after In a wide range of such decision-making experiments, the
behavioral response, i.e. performance and reaction times, can be
properly described by a simple stochastic diffusion model [1]. In
the diffusion model evidences about one or the other choice are
accumulated continuously over time until a decision boundary is
reached. Hence, it is plausible to think that the underlying
neuronal system performs decision-making by accumulation of
evidences. Jose´ Antonio Carrillo1, Ste´phane Cordier2, Gustavo Deco3,4, Simona Mancini2* 1 Department of Mathematics, Imperial College London, London, United Kingdom, 2 Univ. Orle´ans, CNRS, MAPMO, UMR 7349 & FR 2964, Orle´ans, France, 3 ICREA
(Institucio´ Catalana de Recerca i Estudis Avanc¸ats), Barcelona, Spain, 4 Computational Neuroscience Unit, Universitat Pompeu Fabra, Barcelona, Spain Abstract The
difference in the firing rates of MT cells with opposing preferred
directions is a likely measure of the available visual evidence for
the direction of coherent motion of the moving dot stimulus,
further indicating that the LIP area is involved in this decision-
making task. December 2013 | Volume 8 | Issue 12 | e80820 Results In terms of wz the underlying dynamical system without noise
presents a subcritical pitchfork bifurcation whose bifurcation
diagram is shown in Figure 1. For values of the excitatory
coefficient around wz~1:4 the system pass from a single
asymptotically stable equilibria to a situation in which there are
three stable (continuous lines) and two unstable equilibria (hashed
lines). The second local bifurcation, where the central asymptotic
equilibria
disappears,
happens
at
around
wz~2:5695
for
Dl~10{3. y
y
g
y
g
One
dimensional Fokker-Planck effective
reductions
near
bifurcation points can be obtained applying the center manifold
reduction method as explained in [11]. In this method the
projection on the slow manifold is done asymptotically, i.e. order
by order in E, so that the resulting dynamics along the slow
manifold does not contain effects of different order. This method is
equivalent to the method used in [10], where the authors locally
approximate the evolution on the slow manifold at the bifurcation
point by Taylor expanding the nonlinearities with respect to the
slow variable based on the different time scales. In this way, they
obtain an effective potential of degree 4 for the one dimensional
Fokker-Planck dynamics on the slow manifold. We propose to
build on this strategy by not performing any Taylor expansion and
rather approximate the slow manifold in terms of the nonlinea-
rities defining the evolution using the slow-fast character of the
dynamical system, see [12]. Our method is similar to the one
presented in [11], but once the slow manifold is approximated we
then globally project the Fokker-Planck dynamics on it to obtain a
one dimensional Fokker-Planck reduction possibly valid beyond
the local character of the expansion in [10]. More importantly, we
recover an approximated full reduced potential and an explicit
formula for the stationary state distribution on the slow manifold. This effective reduced potential covers the other two stable
equilibrium points, and not only the central equilibrium point
changing its stability character as in [10]. For instance, in the case
of the subcritical Hopf bifurcation in Figure 1, we get a three well
approximated potential, while the approach in [10] leads to a
potential of degree 4 approximating an interval around the central
equilibrium point, see ([10], Figure 2). Results stimulus presentation, the network can still sustain low activity, but
random fluctuations eventually drive the collective activity of the
network to an activated state, in which a fraction of the neurons
fires at a high rate. Let us remark that the behavioral performance
of the model (i.e. the fraction of trials in which the ‘correct’
direction is chosen) and corresponding reaction time to make a
decision match well the behavioral data from both monkeys and
humans performing the moving-dot task [10]. Therefore, we
conjecture, that it may be possible to establish a direct link
between the neuronal and behavioral correlates of decision-
making. Here, we aim to establish this link between the underlying
physiology and the observed behavioral response in decision-
making tasks by performing a 1D reduction of the dynamics of the
neuronal circuit in such a way that we derive from the underlying
detailed neuronal dynamics a valid ‘‘nonlinear’’ diffusion model. We showcase our strategy in a particular example of a neuronal
population model with two pools with self-excitation and cross-
inhibition as in [13]. The firing rates n1 and n2 of the neuronal
networks are determined by the stochastic dynamical system: dn1~ {n1zQ(l1zwzn1{wIn2)
½
dtzbdW 1
t
dn2~ {n2zQ(l2{wIn1zwzn2)
½
dtzbdW 2
t
(
,
ð1Þ ð1Þ with n1,n2§0. Here the applied stimuli are, l1~33, l2~l1{Dl,
with the bias Dl[½0,10{3, the inhibitory connectivity coefficient is
wI~1:9, the standard deviation is b~3:10{3, and the excitatory
connectivity coefficient wz is the bifurcation parameter, its range
of values will be discussed later on. Moreover, the sigmoid
(response) function w(z) is given by: In order to perform the explicit link between a neuronal circuit
processing decision-making and a diffusion model able to describe
the behavioral level, we consider a reduced model of the
competitive cortical circuit solving the decision-making problem. In particular, we consider a mean-field reduction consisting of two
competing rate models. This system of Langevin equations can be
associated with a Fokker-Planck equation for the probability
distribution of the activities of the different neuronal populations. The nonlinear nature of the original equations, however, hinders
analytical progress in the Fokker-Planck framework. For this
reason, the main analysis of such noise driven probabilistic
decision-making systems remains based on numerical investiga-
tions, which are time consuming because of the need for
sufficiently many trials to generate statistically meaningful data. w(z)~
nc
1z exp ({bzza) , with nc~15, b~0:25, and a~11:1. Introduction Indeed, recent electrophysiological recordings from
awake behaving monkeys performing decision-making have shown
that trial-averaged spiking activity of some neurons shows a
gradual, nearly linear increase shortly after stimulus onset until just
before the response is made [2–6]. Let us consider for example the
so-called random-dot task. The random-dot task is a two-choice
visual decision-making task. In this task, the subject is confronted December 2013 | Volume 8 | Issue 12 | e80820 December 2013 | Volume 8 | Issue 12 | e80820 1 PLOS ONE | www.plosone.org Reduction of Rate-Equations for Decision-Making Results Our strategy is valid as long
as the approximated slow manifold lies fully in the relevant
biological range of positive rates, this is the reason we restrict the
range of the bifurcation parameter wz as explained later on. We next need to find an approximation of the slow manifold
joining the spontaneous S0 and the decision states S1 and S2, as
sketched in Figure 2. This curve is found by introducing the
linearization of the dynamical system around the spontaneous
equilibria S0 as a new set of variables defined by X~P{1(n{S0),
with X~(x,y)T, or equivalently n~S0zPX with n~(n1,n2)T. Here, P is the matrix diagonalizing the jacobian of the dynamical
system (1) at the spontaneous state S0, see (2) in the Materials and
Methods section for more details, or [14]. This change of variables, sketched in Figure 2, is a natural way
to introduce slow y and fast x variables in the system determined
by the eigenvalues of the linearization at the spontaneous state S0. By rewriting the dynamical system above (1) in these new variables
together with the new time variable t~Et, we have an evolution
governed by E dx
dt ~f (x,y)
dy
dt ~g(x,y)
8
>
>
<
>
>
:
, E dx
dt ~f (x,y)
dy
dt ~g(x,y)
8
>
>
<
>
>
:
, where E is a small parameter. Assuming that the scaling ratio is
zero, E~0, we find the implicit relation f (x,y)~0 that determines
the approximated slow manifold x(y). In Figure 3, we plot the
curves x(y) for different values of vz with Dl~10{3. We
remark that this restricts the validity of this particular example
since these curves exit the set of positive values for the rate
variables. This comes from the fact that there are some trajectories
of the dynamical system without noise leading to negative values
for the rate variables. In this example the approximated curves lie
on the positive quadrant for wz§1:9 approximately. dy~g x(y),y
ð
ÞdtzbydW 2
t , with by properly obtained in terms of b via the change of variables. The
effective
potential
is
determined
by
the
relation
LyG(y)~{g(x(y),y) . The 1D effective computed potential
G(y) for the biased case (Dl~10{3) with respect to the slow
variable y is plotted in Figure 4 for wz~2:0, . . . ,2:9 (left),
together with a zoom at the spontaneous state (right). We note that
increasing wz beyond the second bifurcation point, the sponta-
neous state pass from local minimum to local maximum. We
remind that the effective potential G(y) is given by formula (8)
based on the approximated slow manifolds computed in Figure 3. with by properly obtained in terms of b via the change of variables. The
effective
potential
is
determined
by
the
relation
LyG(y)~{g(x(y),y) . The 1D effective computed potential
G(y) for the biased case (Dl~10{3) with respect to the slow
variable y is plotted in Figure 4 for wz~2:0, . . . ,2:9 (left),
together with a zoom at the spontaneous state (right). We note that
increasing wz beyond the second bifurcation point, the sponta-
neous state pass from local minimum to local maximum. We
remind that the effective potential G(y) is given by formula (8)
based on the approximated slow manifolds computed in Figure 3. Concerning the comparison between the numerical solutions of
the 2D complete Fokker-Planck and those of the 1D reduction, we
refer the reader to [14]. We just note here that since the standard
deviation is rather small (b~3:10{3), the stable states tends to be
Dirac masses and the computational costs are unbearable to
compute 2D solutions approaching the steady state. Note also that
the 1D Fokker-Planck reduction can be solved numerically
applying
an
implicit
in
time
scheme,
drastically
reducing
computational time costs. The 1D effective computed potentials G(y) both for the
unbiased Dl~0 and the biased case Dl~10{4,10{3 with respect
to the slow variable y are plotted in Figure 5 for the three values of
wz
close
to
the
second
bifurcation
point
(wz~2:5685,
wz~2:5695, wz~2:5705). We note the asymmetry of the
effective potential G(y) when increasing the bias Dl and the
change in the nature of the spontaneous state when increasing the
excitatory coefficient wz. Results PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org December 2013 | Volume 8 | Issue 12 | e80820 December 2013 | Volume 8 | Issue 12 | e80820 2 Reduction of Rate-Equations for Decision-Making PLOS ONE | www.plosone.org
3
Decem PLOS ONE | www.plosone.org
3
December 2013 | Volume 8 | Issue 12 | e80820 December 2013 | Volume 8 | Issue 12 | e80820 December 2013 | Volume 8 | Issue 12 | e80820 PLOS ONE | www.plosone.org Reduction of Rate-Equations for Decision-Making Reduction of Rate-Equations for Decision-Making Figure 1. Bifurcation diagrams with respect to wz. A: the bias is Dl~0; B: the bias is Dl~10{4; C: the bias is Dl~10{3. The components of
the spontaneous state are traced by the black line. There are other two stable equilibria points which coordinates are represented by the blue and
light-blue lines. The red continuous and dashed lines correspond to the unstable equilibrium points. The small picture is a zoom on the bifurcation
point. d i 10 1371/j
l
0080820 001 doi:10.1371/journal.pone.0080820.g001 Figure 3. We note that yw0 is pointing toward S1 while yv0 is
pointing toward S2, see Figure 2. Therefore, the top left
(respectively low right) well whose minimum is attained at the
stable decision state S1 (respectively S2) in the 2D n-plane in
Figure 2 corresponds to the right (respectively left) well in Figure 5. Once we have found the slow manifold approximation x(y),
we can restrict the dynamics in (1) to a single effective Langevin
equation. This equation is determined by a potential G(y)
obtained from the evaluation of the dynamics over the slow
manifold approximation, leading to In Figure 6, we have computed the marginals along y of the 2D
numerical computations of the Fokker-Planck dynamics for
t~200 ms, compared with the solution of the 1D reduction
Fokker-Planck evolution equation (7) at t~200 ms. We observe
the errors committed by the 1D reductions and we also observe
that the slow-fast behavior is also present in the 1D reduced
Fokker-Planck equation. However, since the one dimensional
reduction is easily solved by numerical methods, then we can
achieve much larger computational times and compute explicitly
the approximated stationary state. dy~g x(y),y
ð
ÞdtzbydW 2
t , Bifurcation: Reaction Time and Performance Finally, once the 1D reduction is validated by the above
arguments, the biologically interesting quantities to compute are
the performance Pa and the reaction times RT. The first one This 1D effective potential can be considered as a good
approximation of the equilibrium state of the 2D Fokker-Planck
equation (associated to the system (1)) projected on the 1D-slow
manifold which is parameterized by the slow variable y, see Figure 3. Slow manifold. Variation of the approximated slow
manifold represented on the (n1,n2) plane for wz~2:0, . . . ,2:9 and
for the biased case Dl~10{3. For wzv1:9 the slow manifold crosses
the coordinate axes for small values of n1 and n2, becoming negative. The reduction theory is valid only for ranges of wzw1:9. doi:10.1371/journal.pone.0080820.g003 p
y
y,
Figure 2. Change of variables. Position of the new coordinate
system (x,y) with respect to the initial one (n1,n2). The origin is taken at
the central equilibrium point S0 which may stable or unstable
accordingly to the value of wz. S1 and S2 are two others equilibrium
points. The dashed line represents the slow manifold on which the
reduced dynamics takes place, and it contains all the equilibrium points
of the system. doi:10.1371/journal.pone.0080820.g002
Figure 3. Slow manifold. Variation of the approximated slow
manifold represented on the (n1,n2) plane for wz~2:0, . . . ,2:9 and
for the biased case Dl~10{3. For wzv1:9 the slow manifold crosses
the coordinate axes for small values of n1 and n2, becoming negative. The reduction theory is valid only for ranges of wzw1:9. doi:10.1371/journal.pone.0080820.g003
PLOS ONE | www.plosone.org
4
December 2013 | Volume 8 | Issue 12 | e80820 Figure 2. Change of variables. Position of the new coordinate
system (x,y) with respect to the initial one (n1,n2). The origin is taken at
the central equilibrium point S0 which may stable or unstable
accordingly to the value of wz. S1 and S2 are two others equilibrium
points. The dashed line represents the slow manifold on which the
reduced dynamics takes place, and it contains all the equilibrium points
of the system. doi:10.1371/journal.pone.0080820.g002 Figure 2. Change of variables. Position of the new coordinate
system (x,y) with respect to the initial one (n1,n2). The origin is taken at
the central equilibrium point S0 which may stable or unstable
accordingly to the value of wz. S1 and S2 are two others equilibrium
points. Bifurcation: Reaction Time and Performance The dashed line represents the slow manifold on which the
reduced dynamics takes place, and it contains all the equilibrium points
of the system. Figure 2. Change of variables. Position of the new coordinate
system (x,y) with respect to the initial one (n1,n2). The origin is taken at
the central equilibrium point S0 which may stable or unstable
accordingly to the value of wz. S1 and S2 are two others equilibrium
points. The dashed line represents the slow manifold on which the
reduced dynamics takes place, and it contains all the equilibrium points
of the system. Figure 3. Slow manifold. Variation of the approximated slow
manifold represented on the (n1,n2) plane for wz~2:0, . . . ,2:9 and
for the biased case Dl~10{3. For wzv1:9 the slow manifold crosses
the coordinate axes for small values of n1 and n2, becoming negative. The reduction theory is valid only for ranges of wzw1:9. doi:10.1371/journal.pone.0080820.g003 y
doi:10.1371/journal.pone.0080820.g002 December 2013 | Volume 8 | Issue 12 | e80820 PLOS ONE | www.plosone.org 4 Reduction of Rate-Equations for Decision-Making Figure 4. The 1D potential. A: Variation of the approximated 1D potential traced along the y variable, varying wz~2:0, . . . ,2:9 and for the biased
case Dl~10{3. B: A zoom around the spontaneous state. Increasing wz, the central equilibrium point passes from a minima to a maxima. doi:10.1371/journal.pone.0080820.g004
Reduction of Rate-Equations for Decision-Making Figure 4. The 1D potential. A: Variation of the approximated 1D potential traced along the y variable, varying wz~2:0, . . . ,2:9 and for the biased
case Dl~10{3. B: A zoom around the spontaneous state. Increasing wz, the central equilibrium point passes from a minima to a maxima. doi:10.1371/journal.pone.0080820.g004 corresponds to the ratio of good answers at equilibrium, and the
second one to the smallest time needed to give an answer, no
matter wether it is good or wrong. The computations of the
reaction time RT and performances Pa use the formulas in the
supplementary material in [10], which hold locally around
the spontaneous point and are based on the knowledge of the
effective potential G(y). potential gets tilted towards the preferred decision state, and thus
the performance of the solution gets higher. Accordingly, the
reaction time decreases as the bias increases. Discussion Here, we have reinforced the link between the underlying
physiology and the observed behavioral response in decision-
making tasks by developing a new strategy for the 1D reduction of
the dynamics of the neuronal circuit. In this way we have derived
from the underlying detailed neuronal dynamics a ‘‘nonlinear’’
diffusion model valid for a wide range of the bifurcation
parameter. We observe how our method recovers the reduced
one dimensional dynamics proposed by Roxin and Ledberg in
[10] close to the bifurcation point since we keep the same
qualitative behavior in terms of performance and reaction times. In Figure 7 we plot the performance Pa and reaction time RT
for b~3:10{3 as a functions of Dl and wz. They are computed
for the decision state with higher probability, the one for negative
values of the y-variable in Figure 5, and over an interval ½a{,az
where a{ and az correspond to the y-coordinates to the left and
right of the spontaneous state for which G(y) has a maximum. We
observe the same qualitative behavior as in the comparisons with
experimental data performed in [10]. As the bias increases, the December 2013 | Volume 8 | Issue 12 | e80820 PLOS ONE | www.plosone.org December 2013 | Volume 8 | Issue 12 | e80820 5 Reduction of Rate-Equations for Decision-Making Figure 5. Potential at the bifurcation parameter wz~2:5696. A: Dl~0, E: zoom for Dl~0 around the spontaneous state, B: Dl~10{4, F:
zoom for Dl~10{4 around the spontaneous state, C: Dl~10{3, G: zoom for Dl~10{3 around the spontaneous state. For large bias Dl the
potential becomes asymmetric, and for Dl~10{3 the spontaneous state changes its nature from a minima to a maxima. doi:10.1371/journal.pone.0080820.g005 Figure 5 Potential at the bifurcation parameter w ~2 5696 ~0, E: zoom for Dl~0 around the spontaneous state, B: Dl~10{4, F:
om for Dl~10{3 around the spontaneous state. For large bias Dl the
changes its nature from a minima to a maxima. ~0, E: zoom for Dl~0 around the spontaneous state, B: Dl~10{4, F:
m for Dl
10{3 around the spontaneous state For large bias Dl the Figure 5. Potential at the bifurcation parameter wz~2:5696. A: Dl~0, E: zoom for Dl~0 around the spontaneous state, B: Dl~10{4, F:
zoom for Dl~10{4 around the spontaneous state, C: Dl~10{3, G: zoom for Dl~10{3 around the spontaneous state. Discussion For large bias Dl the
potential becomes asymmetric, and for Dl~10{3 the spontaneous state changes its nature from a minima to a maxima. doi:10.1371/journal.pone.0080820.g005 We have also shown that our reconstructed effective potential
leads to a good approximation of the stationary state of the two
dimensional dynamics projected on the slow manifold. This
reduction allows for efficient computations of the dynamics as soon
as the approximated slow manifold is well-defined. {nzW(LzWn) is the drift. The linearized jacobian matrix at
any equilibrium point is given by: JF(z1,z2)~
{1zw11Q0(z1)
w12Q0(z1)
w21Q0(z2)
{1zw22Q0(z2)
,
ð2Þ ð2Þ E~{m2=m1 E~{m2=m1 where D~diag(m1,m2) is the associated diagonal matrix. We can
describe the coordinates n in the eigenvector basis and centered on
the saddle point S0 as follows: where D~diag(m1,m2) is the associated diagonal matrix. We can
describe the coordinates n in the eigenvector basis and centered on
the saddle point S0 as follows: In order to reduce the system we need to introduce new
variables based on the linearization of the problem. We will denote
by P the matrix containing the normalized eigenvectors of JF(S0)
and by P{1 its inverse matrix. Furthermore, using the Hartman-
Grobman theorem [15], we know that the solutions of the
dynamical system are topologically conjugate with its linearization
in the neighborhood of a hyperbolic fixed point. Let us write it as n~S0zPX, which gives the definition for the new variable X~(x,y)T, see
1 also Figure 2, X~P{1(n{S0) also Figure 2, X~P{1(n{S0) Figure 7. Performances and Reaction Times. A: the performances (Pa) computed for three values of the bifurcation parameter wz close to the
one of the spontaneous state and as a function of the bias Dl. When wz increases, the performance decreases, and it converges to 1 for large values
of the bias Dl. B: the reaction times (RT) computed for three values of the bifurcation parameter wz close to the one of the spontaneous state and
as a function of the bias Dl. When wz increases, the reaction time decreases. In both pictures the standard deviation is b~3:10{3. These behaviors
are in agreement with previous results and experimental data. doi:10.1371/journal.pone.0080820.g007 Figure 7. Performances and Reaction Times. A: the performances (Pa) computed for three values of
one of the spontaneous state and as a function of the bias Dl. When wz increases, the performance decre
of the bias Dl. B: the reaction times (RT) computed for three values of the bifurcation parameter wz clos
as a function of the bias Dl. When wz increases, the reaction time decreases. In both pictures the standa
are in agreement with previous results and experimental data. doi:10.1371/journal.pone.0080820.g007 Figure 7. Performances and Reaction Times. A: the performances (Pa) computed for three values of the bifurcation parameter wz close to the
one of the spontaneous state and as a function of the bias Dl. When wz increases, the performance decreases, and it converges to 1 for large values
of the bias Dl. Materials and Methods where we have denoted by zi the values zi : ~lizwi1n1zwi2n2. Since S0 is a hyperbolic fixed point (saddle point), the jacobian
JF(S0) has two real eigenvalues, m1 and m2 of opposite signs. Let us
denote by m1 the (large in magnitude) negative eigenvalue and by
m2 the (small) positive eigenvalue of JF(S0). The small parameter
E%1 responsible for the slow-fast behavior is determined by the
ratio In this section, we give more details of the 1D reduction of
system (1) presented above. Preliminary results of this strategy
were previously reported in [14] in the easier case of the
supercritical Hopf bifurcation. The slow-fast behavior of the
system (1) with no noise, i.e. b~0, can be characterized by the fact
that the jacobian of the linearized system at the unstable critical
point S0~(n
1,n
2)T has a large condition number. More precisely,
we write the deterministic part of the dynamical system (1) as
_n~F(n), where n~(n1,n2)T
is the rate vector and F(n)~ December 2013 | Volume 8 | Issue 12 | e80820 PLOS ONE | www.plosone.org 6 Reduction of Rate-Equations for Decision-Ma Reduction of Rate-Equations for Decision-Making Red PLOS ONE | www.plosone.org
7
December PLOS ONE | www.plosone.org
7
December 2013 | Volume 8 | Issue December 2013 | Volume 8 | Issue 12 | e80820 December 2013 | Volume 8 | Issue 12 | e80820 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org Reduction of Rate-Equations for Decision-Making Reduction of Rate-Equations for Decision-Making Figure 6. Distribution function comparison along y. The computed marginals of the 2D problem (blue line) at time t~200 ms and the
corresponding solution of the 1D reduced problem (black line) at the same time show a good agreement. In this test we have chosen the bifurcation
parameter wz~2:45, the standard deviation b~0:3 and the bias Dl takes the values 0 in A, 10{4 in B and 10{3 in C. doi:10.1371/journal.pone.0080820.g006 follows JF(S0)~PDP{1,
ð3Þ ð3Þ E~{m2=m1 E~{m2=m1 We can easily find it by defining the
associated potential G(y) being the antiderivative of the flux term
g x(y),y
ð
Þ. In other words, we can always define the potential
function: with
no-flux
boundary
conditions
on
y~+ym:
g x(y),y
ð
Þq{ 1
2 b2
yLyq~0 , see [16] and [14]. The 1D Fokker-
Planck dynamics (7) are given for large times by the stationary
solution given by the invariant measure for the stochastic
differential equation (6). We can easily find it by defining the
associated potential G(y) being the antiderivative of the flux term
g x(y),y
ð
Þ. In other words, we can always define the potential
function: Our claim is that the curve of equation f (x,y)~0 approximates
well when E%1 the slow manifold, that is the unstable manifold
that joins the spontaneous point S0 to the other equilibrium points. We refer the reader to the discussion in [12] on stochastic slow-fast
dynamics. Due to the non-linearity of the function f in (4), we
cannot expect an explicit formula for the solution of this equation. Nevertheless, since Lxf (0,0)=0, the resolution in the neighbor-
hood of the unstable equilibrium point (0,0) is insured by the
implicit function theorem. Hence we can define a curve: G(y)~{
ð y
0
g x(z),z
ð
Þdz
ð8Þ ð8Þ x~x(y),
ð5Þ ð5Þ Then, the stationary probability density of the 1D Fokker-
Planck dynamics (7) is given by Then, the stationary probability density of the 1D Fokker-
Planck dynamics (7) is given by such that f x(y),y
ð
Þ~0 in a neighborhood of (0,0). We note
also that, by construction the approximated slow manifold
S0zP x(y),y
ð
ÞT, implicitly defined by (5), intersects the exact
slow manifold at all equilibrium points, i.e. where both f and g
vanishes. Finally, we can conclude the slow-fast ansatz, replacing
the
complete
dynamics
by
the
reduced
dynamics
on the
approximated slow manifold, and obtain the reduced one
dimensional differential equation: qs(y)~ 1
Z exp {2G(y)=b2
y
, where Z is the normalization constant. As explained also in [17],
this stationary solutions are the asymptotic equilibrium states for
the solution of the Fokker-Planck equation. In other words, letting
time going to infinity, the solution q(t,y) to (7) must converge to
qs(y). We have shown in [17] that the decay to equilibrium for the
two dimensional problem was exponential. E~{m2=m1 B: the reaction times (RT) computed for three values of the bifurcation parameter wz close to the one of the spontaneous state and
as a function of the bias Dl. When wz increases, the reaction time decreases. In both pictures the standard deviation is b~3:10{3. These behaviors
are in agreement with previous results and experimental data. doi:10.1371/journal.pone.0080820.g007 December 2013 | Volume 8 | Issue 12 | e80820 PLOS ONE | www.plosone.org 8 Reduction of Rate-Equations for Decision-Making In these new coordinates x corresponds to the fast scale while y
is the slow varying variable. We can conclude that system (1) reads
in the X phase space as _X~H(X) where H(X)~(f (x,y),g(x,y))
is the two dimensional vector defined by H(X)~P{1F(S0zPX),
and thus, note that the noise term also changes since
ffiffiE
p dW 1
t ~dW 1
t and
Edt~dt. Therefore, we can deduce again that the reduced one
dimensional model must read: dy~g x(y),y
ð
Þdtzby dW 2
t ,
ð6Þ ð6Þ dx
dt ~f (x,y)~{x{(P{1S0)1z P{1W LzW(S0zPX)
ð
Þ
1
dy
dt ~g(x,y)~{y{(P{1S0)2z P{1W LzW(S0zPX)
ð
Þ
2
8
>
>
<
>
>
:
ð4Þ with by~b
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
a2
21za2
22
q
. Finally,
we
can
consider
the
Fokker-Planck
or
forward
Kolmogorov equation associated to the 1D stochastic differential
equation (6). This gives the reduced dynamics for the probability
density q(t,y) of finding neurons with rate determined by the rate
y[½{ym,zym
over
the
approximated
slow
manifold
S0zP x(y),y
ð
ÞT , for t§0. Actually, it must obey to the following
1D Fokker-Planck equation: Direct computations gives that JH(X)~P{1JF(S0zPX)P,
and using (3) and that X(S0)~0, we obtain that JH(0)~D. We
can choose a new time scale for the fast variable t~Et in such a
way that for t^1, then t^E{1. Then, the fast character of the
variable x is clarified and the system reads as LtqzLy g x(y),y
ð
Þq{ 1
2 b2
yLyq
~0,
ð7Þ ð7Þ E dx
dt ~f (x,y)
dy
dt ~g(x,y)
0
B
B
@ E dx
dt ~f (x,y)
dy
dt ~g(x,y)
0
B
B
@ with
no-flux
boundary
conditions
on
y~+ym:
g x(y),y
ð
Þq{ 1
2 b2
yLyq~0 , see [16] and [14]. The 1D Fokker-
Planck dynamics (7) are given for large times by the stationary
solution given by the invariant measure for the stochastic
differential equation (6). December 2013 | Volume 8 | Issue 12 | e80820 The authors thanks N. Berglund for many fruitful discussions. The authors thanks N. Berglund for many fruitful discussions. MY~
ð
V
Y(n)ps dn1dn2^
ð
Y S0zP x(y),y
ð
ÞT
qs(y)dy MY~
ð
V
Y(n)ps dn1dn2^
ð
Y S0zP x(y),y
ð
ÞT
qs(y)dy MY~
ð
V
Y(n)ps dn1dn2^
ð
Y S0zP x(y),y
ð
ÞT
qs(y)dy Reduction of Rate-Equations for Decision-Making Reduction of Rate-Equations for Decision-Making Fokker-Planck (9). More precisely, as E?0, p approaches a
concentrated density along the the curve n(y)~S0zP(x(y),y)T . Then, for any test function Y~Y((n1,n2)T) , the moment MY of
the stationary probability distribution function ps(n1,n2) as E?0
can be approximated by Fokker-Planck (9). More precisely, as E?0, p approaches a
concentrated density along the the curve n(y)~S0zP(x(y),y)T . Fokker-Planck (9). More precisely, as E?0, p approaches a
concentrated density along the the curve n(y)~S0zP(x(y),y)T . Then, for any test function Y~Y((n1,n2)T) , the moment MY of
the stationary probability distribution function ps(n1,n2) as E?0
can be approximated by Fokker-Planck (9). More precisely, as E?0, p approaches a
concentrated density along the the curve n(y)~S0zP(x(y),y)T . This formulae can be used to compute either classical moments
of ps or marginals. Then, for any test function Y~Y((n1,n2)T) , the moment MY of
the stationary probability distribution function ps(n1,n2) as E?0
can be approximated by References 1. Ratcliff R, Smith P (2004) A comparison of sequential sampling sampling models
for two-choice reaction time. Psychol Rev 111: 333–367. 11. Crawford J (1991) Introduction to bifurcation theory. Rev Mod Phys 63: 991–
1037. 1. Ratcliff R, Smith P (2004) A comparison of sequential sampling sampling models
for two-choice reaction time. Psychol Rev 111: 333–367. 1. Ratcliff R, Smith P (2004) A comparison of sequential sampling sampling models
for two-choice reaction time. Psychol Rev 111: 333–367. y
2. Romo R, Salinas E (2001) Touch and go: Decision-making mechanisms in
somatosensation. Annu Rev Neurosci 24: 107–137. 2. Romo R, Salinas E (2001) Touch and go: Decision-making mechanisms in
somatosensation. Annu Rev Neurosci 24: 107–137. 12. Berglund N, Gentz B (2005) Noise-induced phenomena in slow-fast dynamical
systems. a sample-paths approach. Springer, Probability and its Applications. 3. Romo R, Salinas E (2003) Flutter discrimination: neural codes, perception,
memory and decision-making. Nature Reviews Neuroscience 4: 203–218. 3. Romo R, Salinas E (2003) Flutter discrimination: neural codes, perception,
memory and decision-making. Nature Reviews Neuroscience 4: 203–218. y
p
p
pp
p
g
y
pp
13. Deco G, Martı´ D (2007) Deterministic analysis of stochastic bifurcations in
multi-stable neurody-namical systems. Biol Cybern 96: 487–496. multi-stable neurody-namical systems. Biol Cybern 96: 487–496. Shadlen M, Newsome W (2001) Neural basis of a perceptual decisi 4. Shadlen M, Newsome W (2001) Neural basis of a perceptual decision in the
parietal cortex (area lip) of the rhesus monkey. J Neurophysiol 86: 1916–1936. 4. Shadlen M, Newsome W (2001) Neural basis of a perceptual decision in the
parietal cortex (area lip) of the rhesus monkey. J Neurophysiol 86: 1916–1936. 14. Carrillo J, Cordier S, Mancini S (2013) One dimensional Fokker-Planck reduced
dynamics of decision making models in computational neuroscience. Comm
Math Sci 11: 523–540. 5. Roitman J, Shadlen M (2002) Response of neurons in the lateral intraparietal
area during a com-bined visual discrimination reaction time task. J Neurosci 22:
9475–9489. 15. Hartman P (1960) A lemma in the theory of structural stability of differential
equations. Proc Amer Math Soc 11: 610–620. 6. Huk A, Shadlen M (2005) Neural activity in macaque parietal cortex reects
temporal integration of visual motion signals during perceptual decision making. J Neurosci 25: 10420–10436. 6. Huk A, Shadlen M (2005) Neural activity in macaque parietal cortex reects
temporal integration of visual motion signals during perceptual decision making. J Neurosci 25: 10420–10436. 16. Author Contributions Wrote the paper: JAC SC GD SM. Wrote and ran the numerical code: SC
SM. Wrote the paper: JAC SC GD SM. Wrote and ran the numerical code: SC
SM. E~{m2=m1 This rate of conver-
gence is also true for the 1D reduction since the potential is a small
perturbation of convex potentials, see the well-know results in
[18,19]. We also recall that we are interested in the long time
behavior of the solutions and that the convergence in the slow
manifold given by the variable y is slow. Hence it is relevant to
have a direct computation of their asymptotic behavior without
need to solve the whole 1D or 2D Fokker-Planck equation. _y~g x(y),y
ð
Þ A similar treatment can be done in the presence of the noise
terms. By changing variables from n to X and since the new
variables x and y are linear combination of n1 and n2, then it is
standard to check We also remind that the solution of the system (1) is related to
the 2D Fokker-Planck or forward Kolmogorov equation: dx~f (x,y)dtzb
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
a2
11za2
12
q
dW 1
t
dy~g(x,y)dtzb
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
a2
21za2
22
q
dW 2
t
8
>
<
>
:
, with boundary conditions
F p{ b2
2 +p
! :n~0 on the bound-
2 ary of the domain ½0,nm2, see [16]. Let us remark that, once we
know the stationary state of the 1D reduced Fokker-Planck
equation (7), we can approximate the long-time dynamics of
all quantities of interest related to the system (1) and the 2D where (aij) are the elements of the matrix P{1 and W i
t are two
independent normalized white noise. Arguing as in the determin-
istic case, we can choose a fast time scale for the variable x and we PLOS ONE | www.plosone.org December 2013 | Volume 8 | Issue 12 | e80820 December 2013 | Volume 8 | Issue 12 | e80820 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 9 References Gardiner C (1985) Handbook of stochastic methods for physics, chemistry and
the natural sciences. Springer. 17. Carrillo J, Cordier S, Mancini S (2011) A decision-making Fokker-Planck model
in computational neuroscience. J Math Biol 63: 801–830. J
7. Wang X (2002) Probabilistic decision making by slow reverberation in cortical
circuits. Neuron 36: 955–968. 7. Wang X (2002) Probabilistic decision making by slow reverberation in cortical
circuits. Neuron 36: 955–968. 18. Holley R, Stroock D (1987) Logarithmic Sobolev inequalities and stochastic
Ising models. J Statist Phys 46: 1159–1194. 8. Wong K, Wang X (2006) A recurrent network mechanism of time integration in
perceptual decisions. J Neurosci 26: 1314–1328. 19. Arnold A, Markowich P, Toscani G, Unterreiter A (2001) On convex Sobolev
inequalities and the rate of convergence to equilibrium for Fokker-Planck type
equations. Comm Partial Differential Equations 26: 43–100. 9. Lo C, Wang X (2006) Cortico-basal ganglia circuit mechanism for a decision
threshold in reaction time tasks. Nat Neurosci 9: 956–963. 10. Roxin A, Ledberg A (2008) Neurobiological models of two-choice decision
making can be reduced to a one-dimensional nonlinear diffusion equation. PLoS
Computational Biology 4: 43–100. 10. Roxin A, Ledberg A (2008) Neurobiological models of two-choice decision
making can be reduced to a one-dimensional nonlinear diffusion equation. PLoS
Computational Biology 4: 43–100. December 2013 | Volume 8 | Issue 12 | e80820 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 10
|
https://openalex.org/W3164786371
|
https://journals.vgtu.lt/index.php/JEELM/article/download/14834/10542
|
English
| null |
GROUNDWATER QUALITY ASSESSMENT OF CHILANCHIL ABAY WATERSHED: THE CASE OF BAHIR-DAR CITY WASTE DISPOSAL SITE
|
Journal of environmental engineering and landscape management
| 2,021
|
cc-by
| 10,711
|
Journal of Environmental Engineering and Landscape Management
ISSN 1648–6897 / eISSN 1822-4199
2021 Volume 29 Issue 2: 123–134
https://doi.org/10.3846/jeelm.2021.14834 Journal of Environmental Engineering and Landscape Management
ISSN 1648–6897 / eISSN 1822-4199
2021 Volume 29 Issue 2: 123–134
https://doi.org/10.3846/jeelm.2021.14834 Special issue on Waste Valorisation for Sustainable Production, Process and Products Highlights Highlights
X The study assesses the spatial and temporal variations of groundwater quality. X Total coliform detected were above the permissible limit in all up and downstream
X Most of the parameters were registered to be significant temporally over spatially. X The result of the study classifies the status of the water shed as marginal. X The study classifies the sample points of ground water status by CCMEWQ. X The study assesses the spatial and temporal variations of groundwater quality. Total coliform detected were above the permissible limit in all up and downstream. Most of the parameters were registered to be significant temporally over spatially. X The result of the study classifies the status of the water shed as marginal. The study classifies the sample points of ground water status by CCMEWQ. Abstract. Improper dump sites have served many years as an ultimate disposal site for all types of waste; municipal solid
waste, industrial sewage and hazardous waste in developing countries such as Ethiopia. Physical, chemical and biological
processes interact simultaneously to bring about the overall decomposition of the wastes (chemically laden leachate). If
not, deal properly, such kind of dumping site can cause pollution to groundwater (because of Leachates) and surface wa-
ter (through contaminant transportation by flooding and groundwater movements). Therefore, this study investigates the
spatial and temporal variation of groundwater quality within the Chilanchil Abay watershed during dry and wet season
due to the waste disposal site of the Bahir Dar city. Water samples were collected from 6 sampling points of groundwater
from 30th March to 20th August 2019 by a monthly period. Over 10 water quality parameters such as pH, TDS, Electrical
conductivity, Turbidity, Temperature, DO, BOD, COD, TC, NO3- and PO43–, were analyzed. Moreover, the overall status of
the groundwater quality of the study area was evaluated by the Canadian Water Quality Index. Based on the result of this
model the status of the groundwater sample points throughout the study area were raged from 42 to 46.2 (marginal status). hir-Dar city Waste disposal site, CCMEWQI, Chillan Chile Abay watershed, groundwater, water pollution,
ation Keyword: Bahir-Dar city Waste disposal site, CCMEWQI, Chillan Chile Abay watershed, groundwater, wa
seasonal variation. *Corresponding author. E-mail: nigushabtu@gmail.com This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unre-
stricted use, distribution, and reproduction in any medium, provided the original author and source are credited. GROUNDWATER QUALITY ASSESSMENT OF CHILANCHIL ABAY
WATERSHED: THE CASE OF BAHIR-DAR CITY WASTE DISPOSAL SITE Dargie HAILE1, Nigus GABBIYE2* Dargie HAILE1, Nigus GA 1Department of Chemical Engineering, Institute of Technology Woldia University,
P.O. Box 400, Kombolcha, Ethiopia
2Department of Chemical Engineering, Faculty of Chemical and Food Engineering,
Bahir-Dar Institute of Technology Bahir-Dar University, P.O. Box 26, Bahir Dar, Ethiopia p
2Department of Chemical Engineering, Faculty of Chemical and Food Engineering,
Bahir-Dar Institute of Technology Bahir-Dar University, P.O. Box 26, Bahir Dar, Ethiopia Received 29 May 2020; accepted 14 April 2021 Received 29 May 2020; accepted 14 April 2021 Introduction water pollution, especially groundwater contamination,
and rapidly spread beyond human expectation because
of its flow (Afolayan et al., 2012). Wastes of different
types, mostly solid wastes are the major input of dump-
sites/landfills. With respect to the hydrological analysis of
groundwater, it flows from areas of higher topography to-
wards areas of lower topography, thereby bringing about
the examination of the degradable material which forms
leachate and contaminate the groundwater of the study
area. Unfortunately, groundwater is all too often consid-
ered out of sight but it requires great care. Groundwater is
not only abstracted for supply or river regulated purposes, Water is the most abundant resource on earth, but only
3% are accessible for human activities while the remaining
is present in the ocean as a salt water (Love & Luchsinger,
2014). It may be available in various forms and quantity,
but its use for various purposes is the subject of quality. Of all the environmental concerns that developing coun-
tries face, the lack of adequate and clean water remains the
most serious problem (Markandya, 2006). Once contami-
nated, groundwater may forever remain polluted without
remedy or treatment. Diseases may spring up through *Corresponding author. E-mail: nigushabtu@gmail.com This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativ
stricted use, distribution, and reproduction in any medium, provided the original author and source are credited. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unre-
stricted use, distribution, and reproduction in any medium, provided the original author and source are credited. D. Haile, N. Gabbiye. Groundwater quality assessment of Chilanchil abay watershed: the case of Bahir-dar city... 124 in that watershed, especially near this waste disposal site
to examine its effect on ground water quality. but it also naturally feeds surface -waters through springs
and passages into rivers and it is often important in sup-
porting wetlands and their ecosystems. Removal or diver-
sion of groundwater can affect total flow. A reduction in
either quality of quantity of the discharging groundwa-
ter can significantly influence surface water quality and
the attainment of water quality standards. Surface water
and groundwater are therefore intimately linked in the
water cycle, with many common issues. If groundwater
becomes polluted, it is difficult, if not impossible, to re-
habilitate. Introduction The slow rate of groundwater flow and low mi-
crobiological activity, limit any self-purification. Open and
improper dumping sites have been served many years as
the ultimate disposal site for all types of waste; municipal
solid waste, industrial sewage and hazardous waste in de-
veloping countries (Nathanson, 2015). Physical, chemical
and biological processes interact simultaneously to bring
about the overall decomposition of the wastes. One of the
by-products of this mechanism is chemically laden lea-
chate and it is a potentially hazardous waste generated
from waste disposal sites. If not deal with proper waste
management, such kind of dumping site can cause pol-
lution to groundwater (because of Leachates) and surface
water (through contaminant transportation by flooding,
run-off, wind and ground water movement from the open
dump sites). The Bahir Dar city open site is amongst the
improper open dump site and located in a place where
a number of people are living around. The communi-
ties who are living near the disposal site (in a position of
downstream and upstream) are using polluted ground and
surface water for their day-to-day activities. As a result of
this it becomes a great deal or threat to those communities
regarding the water quality aspect. Therefore, the focus of
this research was to assess and evaluate the water quality 1.2. Sample collection, preservation and laboratory
analyses The measure for scope F1 is calculated as follows: The measure for scope F1 is calculated as follows: 1
Number of failed vaiabbles
F
100. Total numbers of variables
=
×
(1) (1) Groundwater samples were collected from the selected
sample areas near the dumpsite called locally Zebir, to the
laboratory for the analysis. Those six sampling points were
chosen based on their accessibility, proximity to pollution
sources such as communal sites, cottage. Global Position-
ing System navigator (GPS etrex VISTA HCX) was used to
determine the actual positions of the sampling points and
referenced to ensure consistency in the sampling points
during subsequent sampling periods. The sampling points
were carefully selected to include the upstream, and the
downstream communities. Taking the sample was started
during the dry season from the month of March and con-
tinued with a wet season up in the month of August from
all the six sample points. Groundwater samples were col-
lected from the depth of 5–12 meter bore holes by using
an open water grab sampler 3L capacity prepared with a
simple pull - ring that allowed for sampling at various wa-
ter depths of a borehole for groundwater samples (Hamad,
2018). To assess the water quality, water samples were kept
in 1L polyethylene plastic bottles cleaned with metal-free
soap, rinsed with deionized water and finally soaked by
10% nitric acid for 24 hours, finally rinsed with ultra-pure
water. All water samples were stored in an icebox and de-
livered on the same day to the laboratory and kept at a
constant temperature of 4 °C (Clesceri et al., 1998). Wa-
ter quality parameters such as temperature, pH, electrical
conductivity, total dissolved solids, turbidity, salinity were
measured with handy YSI Pro 30 Multiparameter (model
HI 98130 HANNA). Other water quality parameters ana-
lyzed include, dissolved oxygen (DO) using HI 98193 dis-
solved oxygen meter BOD5 using BOD5 incubated in the
presence of NaOH, COD using COD analysis method in
the presence of standard potassium dichromate, sulfuric
acid reagent, ferroin indicator and standard ferrous am-
monium sulfate titrant and total coil form was done us-
ing a membrane filter method. Phosphates (PO42–) and
nitrate (NO3–) were determined using the method of
palintest Spectrophotometer (WAGTECH 8000). 1.1. Descriptions of the study area Eriamecharia waste disposal site is 5 km far away from
Bahir Dar city, center on the expressway to Addis Ababa –
Ethiopia and TIS Abay cascade road. According to the
Central Statistics Agency of Ethiopia (CSA 2007 G.C) 3053
females and 3348 males an aggregate of 6401 populaces is
found around the dumpsite. Its geological directions are:
scope 11.54, longitude 37.38 and height of 1803 meter at
and its rise above sea level is 1801 meter. The height and
width of this unpredictable shape removal site are 384 m
(by the side of the agrarian place that is known for Eri-
amecharia) and 174 m (by the roadside of sebatamit to-
wards Addis Ababa) separately. It has not been furnished
either with liners nor leachate assortment framework
since it started. Besides, no natural effect appraisal has
been done before the determination of this spot to be a
waste removal site. Trucks and separate vehicles from vari-
ous pieces of the city gather and carry the loss to this site
and dump in an anarchic manner. The waste is dumped
as such without isolation. Then again, the base measure of
strong waste which is created from the city and dumped in
the site is, private waste 12610 kg/day, business 4202 kg/
day, specialist co-op 988 kg/day, civil waste 1044 kg/day,
all out 22774 kg/day (source: strong waste portrayal and
evaluation of the Bahir Dar city report, 2007). At present
the base measure of waste dumped at the site is estimated
to be 31321 kg/day. The topographic map of the study
area along with the sampling site is presented in Figure 1. Figure 1. Topographic map of the study area Figure 1. Topographic map of the study area Journal of Environmental Engineering and Landscape Management, 2021, 29(2): 123–134
125 Journal of Environmental Engineering and Landscape Management, 2021, 29(2): 123–134
125 Journal of Environmental Engineering and Landscape Management, 2021, 29(2): 123–134 125 1.2. Sample collection, preservation and laboratory
analyses All the
methodologies for laboratory analysis were conducted ac-
cording to the Standard Methods for the Examination of
Water and Wastewater (Clesceri et al., 1998). The measure for frequency F2 is calculated as follows: 2
Number of failed tests
F
100. Total numbers of tests
=
×
(2) (2) The measure for amplitude, F3 is calculated as follows.h The excursion is the number of times by which an in-
dividual concentration is greater than (or less than, when
the objective is a minimum) the objective. When the test
value does not exceed the objective: Excursion =100–
–1. failed to values
objective
(3) (3) For cases in which the test value exceeds the objective. For cases in which the test value exceeds the objective. Excursion =
failed test value
100–
–1. objective
(4) (4) The collective amount by which individual tests are out
of compliance is calculated by summing the excursions of
individual tests from their objectives and dividing by the
total number of tests (both those meeting objectives and
those not meeting objectives). This variable, referred to as
the normalized sum of excursions (nse) is calculated as: 1
. i
Excursions
nse
number of tests
∞
=
=∑
(5) (5) F3 is then calculated by an asymptotic function that
scales the normalized sum of the excursions from objec-
tives (nse) to yield a range between 0 and 100. F3 =
3F
. 0.01
0.01
nse
nse
=
+
(6) (6) The water quality index (CCME WQI) is then calcu-
lated as: 2
2
2
1
1
3
CCMEWQI
100
. 1.732
F
F
F
+
+
=
−
(7) (7) The divisor 1.732 normalizes the resultant values to a
range between 0 and 100, where 0 represents the “worst”
water quality and 100 represents the “best” water quality. 2. Results and discussion The CCME WQI model consists of three measures of vari-
ance of selected water quality objectives (Scope; Frequen-
cy; Amplitude) (Khan et al., 2004). The “Scope (F1)” the
number of variables not meeting water quality objectives. The “Frequency (F2)” the number of times these objectives
are not met (“failed tests”). The “Amplitude (F3)” repre-
sents the amount by which failed tests do not meet their
objectives. These three factors combine to produce a unit-
less value between 0 and 100 that represents the overall
water quality. The formulation of the WQI as described in
the Canadian Water Quality Index 2001 technical Report
is as follows. 2.1. Spatial and temporal variation of groundwater
quality 2.1.1. Spatial and temporal variation in physical
groundwater quality parameters On The base of spatial vari-
ation in both dry and wet season the maximum values of
total dissolved solid were observed at SS5 and SS6 whereas
the minimum value for dry and wet season recorded at
SS2 (Figure 2, Table 1). This was due to the location of
those sample points (SS5 and SS6) were below the waste disposal site which is susceptible to more agricultural run-
off and discharged municipal waste flow freely from the
dump site while for SS2 above the dump site. h Temperature: The temperature of ground water sam-
ples analyzed have a mean temperature value of 26.54±
1.58 °C during the dry season with the highest tempera-
ture value of 28.73±1.36 °C at SS5.The mean temperature
during the wet season was 22.25±1.59 °C, with the highest
value of 23.5033±0.54 °Cat SS5 and SS6 (Table 1). From
Table 1, one can see that the mean temperature during the
dry season was higher than wet season due to the prevail-
ing atmospheric conditions. A higher number of sunshine
hours would naturally imply lower relative humidity, a
temperature increases of water bodies due to conduction
and convection processes by the earth crust. This shows
that the temperature of ground water is lower in the wet
season than in the dry season. WHO permissible limit
stipulates that water for drinking and domestic purposes
should have a temperature not exceeding 30–40 °C. There-
fore, temperature values recorded for ground water in the
study area were within the WHO permissible limit. In
most cases temperature changes affect pH, electrical con-
ductivity, sorption processes, complexation, speciation,
precipitation, redox reactions, flow rate, ion exchange ca-
pacity, solubility of gases and/or other compounds, just to
mention but a few. Also, increase in temperature decreas-
es the amount of dissolved oxygen, accelerates nitrification
and oxidation of ammonia to nitrates and leading to oxy-
gen deficient water environment. This increases toxicity of
pesticides and heavy metals in drinking water. Tempera-
ture increase in drinking water leads to a less palatable
water taste. Generally, the ANOVA result shows that all
the physical properties of ground water quality discussed
above were observed to be significantly (p < 0.05) affected
by the season. This means the difference regarding season
was real but the difference regarding the occurrence of
sample site was due to chance(not significant). Figure 2. 2.1.1. Spatial and temporal variation in physical
groundwater quality parameters The result of spatial and temporal variation of ground-
water physical parameters measured in all six sampling
points throughout the study time are presented in Table 1.h g
y
Total dissolved solid (TDS): The total dissolved solids
are an indication of the degree of dissolved substances D. Haile, N. Gabbiye. Groundwater quality assessment of Chilanchil abay watershed: the case of Bahir-dar city... 126 Table 1. Seasonal and temporal variation in physical quality parameters of groundwater
Sampling
sites (SS)
E.C
(µS/m)
TDS (mg/L)
Turbidity (NTU)
Temperature (°C)
Dry season
Wet season
Dry season
Wet season
Dry season
Wet season
Dry Season
Wet season
SS1
2037
±34.1
842.33
±32.5
1343.67
±32.72
585.83
±30.09
6.78
±0.705
24.71
±4.39
26.4
±0.36
21.87
±1.76
SS2
2183.33
±176.29
804.67
±16.80
1294.33
±6.35
560.5
±44.21
6.98
±0.60
27.12
±1.40
24.37
±3.88
21.63
±2.03
SS3
1939.33
±36.295
869.667
±71.93
1443.33
±36.94
717.67
±31.501
7.32
±0.43
18.94
±0.55
26.7
±0.56
20.97
±2.59
SS4
2211
±62.65
805.667
±56.37
1427.83
±26.71
617.33
±69.17
7.83
±0.38
22.12
±5.63
25.3
±2.022
23.23
±0.61
SS5
2271
±24.02
866.67
±37.541
1488
±12.12
843.67
±89.07
13.85
±3.35
32.33
±3.512
28.73
±1.36
22.3
±2.04
SS6
2220
±20.66
901.67
±114.18
1456
±38.31
755.67
±110.74
12.4
±4.82
31.09
±2.87
27.4
±1.42
23.5033
±0.54 Table 1. Seasonal and temporal variation in physical quality parameters of groundwater such as metal ions in the water sample. In this study, the
amount of dissolved solid in the ground water samples
were ranged from 1294.33±6.35 to 1488±12.12 mg/L
with a mean value of 1408.86±25.52 mg/L during
the dry season while in a wet season it ranges from
560.5±44.21 to 843.67±89.07 mg/L with a mean value of
680.11±62.46 mg/L (Figure 2, Table 1). The findings of the
study indicated that the measured mean values of total dis-
solved solid during the dry season (1408.86±25.52 mg/L)
and wet season (680.11±62.47 mg/L) were within the
WHO’s permissible limit (500–1500 mg/L). But on the
base of seasonal variation all values of total dissolved
solid during the dry season were higher than the values
of the wet season (Figure 2, Table 1). This could be due
to the higher temperatures observed during the dry sea-
son which facilitated dissolution, ion exchange capacity,
desorption and weathering processes. Also, during the
dry season borehole water evaporated and ionic con-
centrations increased. The significance of this temporal
variation was supported by the statistical result of one-way
ANOVA at 95% confidence. 2.1.1. Spatial and temporal variation in physical
groundwater quality parameters Seasonal variation in total dissolved solid of ground
water quality in the study area
–200
100
400
700
1000
1300
1600
SS1
SS2
SS3
SS4
SS5
SS6
Total dissolved solid(mg/L)
Sampling sites
Total dissolved solids of ground water
Dry season
Wet season
WHOstandard value
SS = Sampling site i
Electrical conductivity: Conductivity in groundwater
is affected by the geology of the area through which the Figure 2. Seasonal variation in total dissolved solid of ground
water quality in the study area Journal of Environmental Engineering and Landscape Management, 2021, 29(2): 123–134 127 Figure 3. Seasonal variation in Electrical conductivity of
groundwater quality in the study area
0
500
1000
1500
2000
2500
SS1
SS2
SS3
SS4
SS5
SS6
Electrical conductivity
( s/cm)
Sampling sites
Electrical conductivity of ground water
Dry season
Wet season
WHO standard value
SS = Sampling site water flows. In this study, the values of electrical conduc-
tivity at the six samples of groundwater during dry season
ranged from 1939.33±36.295 to 2271±24.02 µS/cm (Fig-
ure 3, Table 1) with a mean value of 2143.61±58.999 µS/
cm while in a wet season it varies from 804.67±16.80
to 901.667±114.18 µS/cm with a mean value of
848.44±54.88 µS/cm. The results indicate that the mean
values of electrical conductivity for the dry season were
higher than the electrical conductivity of the wet season
(Figure 3, Table 1). It appears that the values for the dry
season exceeds WHO’S standard limit of 1000 µS/cm for
drinkable water. This could be due to water evaporates
during the dry season and concentration of ions increases
hence electrical conductivity increases (the increase in
electrical conductivity was due to evaporation of water in
under groundwater channels which increased the concen-
trations of dissolved salts or conducting substances in the
borehole water systems). The findings of high-value E.C in
a dry season during the study period was similar with the
study of (Nsengimana et al., 2012) who have done the as-
sessment of heavy metals in the groundwater wells in the
vicinity of Nyanza municipal Solid waste in Kigali City-
Rwanda and found out the higher value of E.C during the
dry season. 2.1.1. Spatial and temporal variation in physical
groundwater quality parameters From the data of dry season, the maximum
values were obtained at SS5, SS6 andSS4 respectively with
a minimum value at SS3 while in a wet season maximum
values were recorded orderly at SS6, SS3 andSS5 with a
minimum value at SS2 (Figure 3, Table 1). These differenc-
es in all values of the dry and wet season were due to the
location of the sample points from the waste disposal site
and other sources of pollution. Generally, Electrical con-
ductivity values provide a useful indicator for spatial and/
or temporal changes in abstraction, salt water intrusion;
recharge mechanism, etc. leading to different groundwater
qualities in wet and dry seasons.hl Figure 3. Seasonal variation in Electrical conductivity of
groundwater quality in the study area other microscopic organisms. Mean turbidity values re-
corded gave an overview of the variations in groundwater
quality during wet and dry seasons. In the dry season,
the value of turbidity at all six samples of groundwater
were ranged between 6.78±0.705 to 13.85±3.35 NTU
with a mean value of 7.25±0.56 NTU while in the wet
season from18.94±0.55 to32.33±3.512 NTU with a mean
26.0517±3.056 NTU (Figure 4, Table 1). As one can see
in Figure 4, the mean values of turbidity recorded in the
wet season (26.0517±3.056 NTU) were higher than WHO
permissible limits of 5-25 NTU as well as from the mean
value of dry season (7.25±0.56 NTU). This may have been
due to the coagulation of dissolved substances in the bore-
holes, particles from weathering activities in the under-
ground waterways and surface runoff from agricultural
fields around the groundwater sources, dissolved clay and
mud materials into the groundwater through infiltration. This means during a rainfall, particles from the waste
disposal site and surrounding land are washed into the
water sample points and leaching into groundwater mak-
ing the water a muddy brown color, indicating water that
has higher turbidity values. Also, during the rainy season,
water velocities are faster and water volumes are higher,
which can more easily stir up and suspend material from
the stream bed, causing higher turbidities. The reason for
the dry season to be lower was because of the reduction in
runoff material, precipitates, suspended solids and colored
dissolved substances which contributed to the turbidity Turbidity: The measurement of turbidity reflects the
transparency in water. It is caused by the substances
present in water in suspension. 2.1.1. Spatial and temporal variation in physical
groundwater quality parameters This was due to the location of those
sample points (SS5 and SS6) were below the waste disposal
site which is susceptible for more agricultural runoff and
discharged municipal waste flow freely and a number of
people are using those sample points in common for their
domestic purpose, as a result, particles will not get enough
residence time for settling while SS1 and SS3 are located
above the dump site which is expected the effect of waste
disposal site is non-significant. Generally, the high tur-
bidities constitute a health risk for the children consuming
this water. High turbidity also indicates a higher amount
of total suspended solids which might include microor-
ganisms such as bacteria or parasites as well as an increase
in the concentration of minerals (Oram, 2015). Watershed
features, such as geology, chemical fertilizer and pesticide
runoff from agricultural activities, urban development
activities, topography, vegetation and precipitation events
can all greatly influence raw water turbidity in the study
area. 2.1.1. Spatial and temporal variation in physical
groundwater quality parameters In the base of
seasonal variation, the mean value of pH in groundwater
was lower in the wet season than in the dry season (Fig-
ure 5, Table 2) This may have been due to during wet sea-
son rainfall combines with carbon dioxide can influence
the water toward acidity, lower temperature hence lower
TDS values, adsorption processes and lower ion exchange
capacity taking place (Bogan et al., 2009). The findings
of lower value pH in a wet season during the study pe-
riod was similar with the work of (Idoko & Oklo, 2012)
who investigates seasonal variation in Physico-chemical
characteristics of rural groundwater of Benue state, Nige-
ria and found out lower value of pH during wet season. Therefore, carbon dioxide is the most common cause of
acidity in water. Photosynthesis, respiration and decom-
position all contribute to pH fluctuations due to their in-
fluences on CO2 levels. The pH values within the range of
5.5–7.5 were suitable for the normal range of irrigation
(Kay, 2001). The pH of the Groundwater falls above FAO
and WHO water quality guidelines for irrigation and use
as raw public water supply in dry season (Figure 5, Ta-
ble 2). According to (Kay, 2001) pH exceeds from 7.5 is
not advisable for irrigation purpose. Generally, the acidity
may have been due to high carbon dioxide concentrations
from eutrophication processes of organic matter, adsorp-
tion of metal anions and presence of some non-metallic
compounds such as fluorides in the groundwater sources. The low pH values which were common in the wet sea-
son are attributed to the anaerobic conditions that could
lead to the production of acidic substances such as organic
acids. Figure 4. Seasonal variation in turbidity of groundwater quality
in the study area of the sample points from the waste disposal site. This
higher value of turbidity in a wet season resembles the
work of (Makwe & Chup, 2013) who assesses the seasonal
variation in Physico-chemical properties of groundwater
around Karu abattoir. Figure 4 shows that, on the base of
spatial variation in both dry and wet season the maximum
values of turbidity were observed at SS5 and SS6 whereas
the minimum value at SS1 for the dry season and at SS3
for the wet season. 2.1.1. Spatial and temporal variation in physical
groundwater quality parameters In natural waters it is
caused by clay, silt, organic matter, phytoplankton and Table 2. Seasonal and temporal variation in chemical properties of ground water
Sampling
sites (SS)|
pH
DO (mg/L)
BOD (mg/L)
COD (mg/L)
Dry
season
Wet
season
Dry
season
Wet season
Dry
season
Wet season
Dry
season
Wet
season
SS1
6.67
±0.42
6.48
±0.67
2.16
±0.087
9.18
±0.17
2.23
±0.10
2.81
±0.37
61
±2.65
72.33
±5.51
SS2
8.67
±0.21
6.66
±0.61
2.51
±0.11
8.97
±0.09
2.80
±0.04
3.1
±0.12
61.67
±2.08
77.43
±4.18
SS3
8.96
±0.03
6.63
±0.77
2.34
±0.148
7.54
±1.14
2.79
±0.076
2.98
±0.15
60
± 2
77.3
±9.02
SS4
8.65
±0.92
6.59
±0.61
2.05
±0.09
7.83
±0.87
2.26
±0.06
2.72
±0.25
68.13
±1.70
73.27
±3.26
SS5
9.2
±0.16
7.59
±0.095
1.58
±0.12
2.7
±0.58
3.12
±0.07
3.91
±0.04
71.5
±1.10
80.63
±6.97
SS6
8.8
±0.27
7.32
±0.18
2.76
±0.08
7.92
±0.48
2.85
±0.01
3.02
±0.23
71.67
±1.53
83.1
±8.15 Table 2. Seasonal and temporal variation in chemical properties of ground water Table 2. Seasonal and temporal variation in chemical properties of ground water D. Haile, N. Gabbiye. Groundwater quality assessment of Chilanchil abay watershed: the case of Bahir-dar city... 128 Figure 4. Seasonal variation in turbidity of groundwater quality
in the study area
0
10
20
30
40
SS1
SS2
SS3
SS4
SS5
SS6
Turbidity (NTU)
Sample sites
Turbidity of ground water
Dry season
Wet season
WHOstandard value
SS = Sampling site points throughout the study time are presented in below
table.h Groundwater pH: The measurement of pH is one of
the most frequently used tests in water chemistry. The
pH results for the groundwater samples of the study areas
ranged from 6.67±0.42 to 9.2±0.16 with a mean value of
8.49±0.33 for the dry season and ranged from 6.48 ±0.67
to 7.59±0.095 (Figure 5) with a mean value of 6.88 ±0.49
for wet seasons. The analysis result of water samples shows
the pH of groundwater was above the WHO allowable
limit of 5.5–7.5 for drinking water for both wet and dry
season (Figure 5, Table 2). This could be due to run off
from the community (Humans contribute to elevated pH
primarily in the form of nutrient runoff most commonly
fertilizer). As from the Figure 5 below indicated atSS1
and SS5 registered the lowest and the highest pH values
throughout the period of study respectively. 2.1.2. Spatial and temporal variation in chemical
characteristics of groundwater qualityh Then at
the highest depth, air was not interacting more relatively
and the water that is near the sediment will be depleted
of oxygen. On the other hand, the maximum value of dis-
solved oxygen in dry season was measured at sampling
point 6 (Figure 6, Table 2) which is typically spring water. Although spring waters are considered as ground water,
they are simply absorbing oxygen from atmospheric air
as a result sampling point 6 has higher value as compared
to the other. Where as in the wet season the maximum
value occurs at sampling point1 (Figure 6, Table 2) due to
this sampling point is found far apart from the dump site
in the upstream direction (above the dump site) and the
minimum value at sampling point 5 similar to dry season. The oxidation of organic substances and reduced inorgan-
ic substances leads to lower oxygen content in groundwa-
ter. Generally, a high content of oxygen infiltration water
enriches the groundwater with oxygen. Because of most
groundwater recharge takes place in the wet season and
lower solubility of oxygen in warm water than in cold
water, higher contents of dissolved oxygen are measured
in the wet season. However, in wet season the measured
mean value of DO was above WHO water quality permis-
sible limit of 5 mg/L. On the account of this the statis-
tical analysis of variance (ANOVA) result indicates that
there was significant difference in mean DO concentration
between and within seasons during the study period (p
< 0.05). As dissolved oxygen levels in water drop below
5.0 mg/L, aquatic life is put under stress. The lower the
concentration, the greater the stress. Low dissolved oxygen
(DO) primarily results from excessive algae growth caused
by phosphate. As the algae die and decompose, the process
consumes dissolved oxygen. This can result in insufficient
amounts of dissolved oxygen available for aquatic life. Biological oxygen demand: From the study, the amount
f BOD f
ll
l
d
i
th d
d Figure 6. Seasonal variation in the dissolved oxygen of
groundwater quality in the study area into the aquifers because of the waste disposal site. 2.1.2. Spatial and temporal variation in chemical
characteristics of groundwater qualityh The result of spatial and temporal variation of ground-
water chemical parameters measured in all six sampling Figure 5. Seasonal variation in pH of groundwater quality in
the study area
0
2
4
6
8
10
SS1
SS2
SS3
SS4
SS5
SS6
pH
Sampling sites
pHof ground water
Dry season
Wet season
WHO standard value
SS = Sampling site Dissolved oxygen: The findings of the study show
that the values of dissolved oxygen were ranged between
2.05±0.09 to 2.76±0.08 mg/L during dry season (Figure 6,
Table 2) with the mean value of 2.24±0.102 mg/L where as
in the wet season from 2.7±0.58 to 9.18±0.17 mg/L with
the mean value of 7.36±0.56 mg/L (Figure 6, Table 2). The recorded results (Figure 6) indicate that the amount
of dissolved oxygen during wet season were higher than
the value of dry season. This might have been due to
temperature and rain fall. This was in line with the study
of (Makwe & Chup, 2013) who investigates the seasonal Figure 5. Seasonal variation in pH of groundwater quality in
the study area
Dry season
Wet season
WHO standard value Journal of Environmental Engineering and Landscape Management, 2021, 29(2): 123–134 129 Figure 6. Seasonal variation in the dissolved oxygen of
groundwater quality in the study area
0
1
2
3
4
5
6
7
8
9
10
SS1
SS2
SS3
SS4
SS5
SS6
Dissolved oxygen (mg/L)
Sampling sites
Dissolved oxygens of ground water
Dry season
Wet season
WHO standard value
SS = Sampling site Dissolved oxygens of ground water variations in physicochemical properties of groundwater
around Karuabattoir. As recorded above the amount of
temperature measured in a wet season was lower than dry
season and then it has a tendency to hold high dissolved
oxygen which leads dissolved oxygen to be higher in wet
season. Besides to those in wet season oxygen is mixed
in through rain, wave and wind as a result its value to be
recorded at the sample point was increase as compared to
dry season. Also, spatial variation among the six sample
points in both dry and wet season was observed. Figure 6,
and Table 2 shows that in dry season the minimum value
was measured at sampling point 5. This was due to sam-
pling point 5 was the highest in depth from all. 2.1.2. Spatial and temporal variation in chemical
characteristics of groundwater qualityh The
higher BOD observed in the rainy season implied that
high demand for oxygen was made to support the life
process (Clark, 1986) noted similar situations of high
BOD and conclude that it was due to higher organic waste
load experienced during the rainy season. (Odokuma &
Okpokwasili, 1993) also observed that BOD fluctuation
between season may be attributed to additional organic
matter introduced into groundwater as the result of run-
off and soil erosion caused by continuous rainfall in the
rain season. In the aspect of spatial variation, the maxi-
mum BOD value was recorded at sampling point 5 and
sampling point 6 respectively for both dry and wet season
(Figure 7, Table 2). This was due to the depth of boreholes
and the locations of sample points from the waste disposal
site (the pollutant source) since in the highest in-depth
and downstream from the pollutant source mostly charac-
terized by a deficiency of oxygen. Whereas the minimum
values were obtained at sampling point 1 and sampling
point 4 (Figure 7, Table 2) respectively for both dry and
wet season due to both are located above the dumping
site which is not rich with a significant amount of organic
waste that comes from the source (disposal site). Gener-
ally, the BOD values of groundwater samples throughout
the study period were below the EPA’S/WHO/ guideline
values of 5 mg/l (Figure 7, Table 2). This indicates that the
water was moderately good interims of BOD for different
household activities, because low BOD is an indicator of
good quality water, while a high BOD indicates polluted The oxidation of organic substances and reduced inorgan-
ic substances leads to lower oxygen content in groundwa-
ter. Generally, a high content of oxygen infiltration water
enriches the groundwater with oxygen. Because of most
groundwater recharge takes place in the wet season and
lower solubility of oxygen in warm water than in cold
water, higher contents of dissolved oxygen are measured
in the wet season. However, in wet season the measured
mean value of DO was above WHO water quality permis-
sible limit of 5 mg/L. On the account of this the statis-
tical analysis of variance (ANOVA) result indicates that
there was significant difference in mean DO concentration
between and within seasons during the study period (p
< 0.05). As dissolved oxygen levels in water drop below
5.0 mg/L, aquatic life is put under stress. 2.1.2. Spatial and temporal variation in chemical
characteristics of groundwater qualityh The lower the
concentration, the greater the stress. Low dissolved oxygen
(DO) primarily results from excessive algae growth caused
by phosphate. As the algae die and decompose, the process
consumes dissolved oxygen. This can result in insufficient
amounts of dissolved oxygen available for aquatic life. Figure 7. Seasonal variation in the BOD of groundwater quality
in the study area
0
1
2
3
4
5
6
SS1
SS2
SS3
SS4
SS5
SS6
BOD (mg/L)
Sampling sites
BOD of ground water
Dry season
Wet season
WHO standard value
SS = Sampling site BOD of ground water Biological oxygen demand: From the study, the amount
of BOD for all samples during the dry season were ranged
from 2.23±0.10 to 3.12±0.07 mg/L with a mean value of
2.68 ±0.07 mg/L while during the wet season the value
of BOD varies from 2.72±0.25 to 3.91±0.04 mg/L with a
mean value of 3.09±0.202 m/L (Figure 7, Table 2). The
findings of this study show that the mean value of the
wet season was higher than the mean value of the dry
season (Figure 7, Table 2). This might have been due to
percolation and infiltrations of biodegradable organic
matter and leaching of organic iron and or manganese Figure 7. Seasonal variation in the BOD of groundwater quality
in the study area D. Haile, N. Gabbiye. Groundwater quality assessment of Chilanchil abay watershed: the case of Bahir-dar city... 130 Figure 8. Seasonal variation in the COD of groundwater quality
in the study area
0
20
40
60
80
100
120
140
160
SS1
SS2
SS3
SS4
SS5
SS6
COD (mg/L)
Sampling sites
COD of ground water
Dry season
Wet season
WHO standard value
SS=Sampling site
SS = Sampling site water. From the ANOVA result, there was no possible sig-
nificant variation between the sampling points as well as
between the seasons that might lead the BOD beyond the
limit of WHO. In water with a BOD level of above 5 mg/L,
the water is considered somewhat polluted because there
is usually organic matter present and bacteria are decom-
posing this waste. The higher the BOD value, the greater
the amount of organic matter or food available for oxygen-
consuming bacteria. BOD values increase when nutrient
loads and accumulation of plant decaying matters in sam-
pling points increase. 2.1.2. Spatial and temporal variation in chemical
characteristics of groundwater qualityh When compared this finding with
other studies a higher range of COD values than those in
this study was obtained in the study that assessed the qual-
ity of boreholes located close to the dumpsite in Benin and
south-south Nigeria. The COD range in this study show
similar with COD range of 55–89 mg/L obtained in the
assessment of water quality of borehole around selected
landfills in Kanometroplis, north Nigeria, as well as some
selected boreholes in Umiahia in Abia state, southeast Ni-
geria, in that they are below the recommended value set
by WHO (Onwugara et al., 2013). 2.1.2. Spatial and temporal variation in chemical
characteristics of groundwater qualityh Chemical oxygen demand: COD is an indicator of or-
ganic pollution, which is caused by the inflow of domes-
tic waste, live stocks and industrial waste that contains an
elevated level of organic pollutants (Maitera et al., 2010). The investigations of the study show that chemical oxy-
gen demand in all the six groundwater samples ranged
from 60±2 to 71.67±1.53 mg/L during the dry season
with a mean value of 65.67±1.84 while in the wet season it
ranged from 72.233±5.51 to 83.1±8.15 mg/L with a mean
value of 77.34±6.18 mg/L (Figure 8, Table 2). From this
(Figure 8 and Table 2) one can see that the values of both
dry and wet season were below the EPA/WHO/ guideline
permissible limit. This could probably be because most
of the wastes that are being generated from the various
communities (available at the dumpsite) are biodegrad-
able or the observed pollutions of borehole water samples
studied may not be due to chemical oxidation of pollut-
ants present but due to aerobic degradations of organic
matter present by microorganism in the water samples. Even though all values of this study conform to the stand-
ard limit the mean value of COD during the wet season
was higher than the value of the dry season (Figure 8, Ta-
ble 2). This might have been due to the effect of household
chemical products such as detergents, soaps, shampoos,
preservatives, dyes, and cleaners available in the dumpsite
by the actions of rainfall. As a result of this, the prolong
accumulation of seeped organic leachates in the aquifer
can increase. The discoveries of the high value of COD
in a wet season during the examination time frame of
this research was comparable with the investigation of
(Gadhia et al., 2012) who have done the examinations of
seasonal variations in Physico-chemical characteristics
of Tapi estuary in Hazira industrial area and discovered Figure 8. Seasonal variation in the COD of groundwater quality
in the study area Figure 8. Seasonal variation in the COD of groundwater quality
in the study area higher estimation of COD during the wet season than the
dry season. In the base of spatial variation maximum and
minimum values were recorded at sampling point 6 and
sampling point 1 respectively for both dry and wet sea-
son (Figure 8, Table 2). 2.1.3. Spatial and temporal variation of nutrients and
micro biological parameters of groundwater quality The result of spatial and temporal variation of ground-
water nutrients and microbiological parameters measured
in all six sampling points throughout the study time are
presented in Table 3. Dynamics of nitrate in ground water: From the study,
the concentrations of nitrate during dry season varied
between 0.071±0.0011 to 0.08±0.0021 mg/L with a mean
value of 0.34±0.006 mg/L while in a wet season it ranged
from 0.51±0.02 to 0.94±0.056 mg/L with a mean value of Table 3. Spatial and temporal variation in measured values of nutrients and micro biological parameters
Sampling sites
(SS)
NO3– (mg/L)
PO43–(mg/L)
TC (CFU/100 ml)
Dry season
Wet season
Dry season
Wet season
Dry season
Wet season
SS1
0.071±0.0011
0.53±0.056
0.0058±0.003
0.0018±0.001
8.33±0.1.53
13±3
SS2
0.72±0.023
0.66±0.053
0.0088±0.0029
0.031±0.023
6.33±1.53
13.67±5.132
SS3
0.09±0.003
0.94±0.056
0.048±0.0023
0.0072±0.005
7.33±3.51
18.33±4.51
SS4
0.73±0.01
0.51±0.02
0.039±0.003
0.05±0.0075
5±1
16±1
SS5
0.073±0.0021
0.52±0.16
0.064±0.0042
0.264±0.0.34
11.67±2.52
23±6
SS6
0.08±0.0021
0.55±0.06
0.098±0.001
0.262±0.87
9±2
20±4.583 Journal of Environmental Engineering and Landscape Management, 2021, 29(2): 123–134 131 Figure 9. Seasonal variation in the nitrate of groundwater
quality in the study area
0
0.2
0.4
0.6
0.8
1
SS1
SS2
SS3
SS4
SS5
SS6
NO3
– (mg/L)
Sampling sits
Nitrates of ground water
Dry season
Wet season
SS=Sampling
site
SS = Sampling site
0
0.2
0.4
0.6
0.8
1
SS1
SS2
SS3
SS4
SS5
SS6
NO3
– (mg/L)
Sampling sits
Nitrates of ground water
Dry season
Wet season
SS=Sampling
site
SS = Sampling site 0.62±0.07 mg/L (Figure 9, Table 3). The result of Figure 9
shows that all values of the dry and wet season were ex-
tremely below the WHO standard value of 50 mg/L for
groundwater. This could probably be due to the absence
of DO significantly in the wastewater; a smaller amount of
nitrate is produced from ammonia as well as decomposi-
tion of food wastes and other sources of protein. Mostly
factors affecting the occurrence of nitrate in groundwater
boreholes are subsurface clay lenses and land use practice. The disclosures/findings/ of low values of nitrate (below
WHO standard) during the examination time frame was
similar with the work of (Akale et al., 2018) who have
assessed nitrate in wells and springs in the north-central
Ethiopian high lands and found value which is less than
WHO standard limit throughout the study period. 2.1.3. Spatial and temporal variation of nutrients and
micro biological parameters of groundwater quality Even
though all values were below the permissible limit the
mean concentrations of nitrate obtained during wet sea-
son were higher than the value of dry season (Figure 9,
Table 3). The disclosures of the high value of nitrate in
a wet season during the study period were similar to the
research of (Akale et al., 2017) who investigates ground-
water quality in an upland agricultural watershed in the
sub-humid Ethiopian highlands and found out the higher
value of nitrate during the wet season than the dry season. This might have been due to high nitrate concentration
in the wet season suggested that increased flush of ni-
trate causing components (mixed source such as decaying
plant or animal material, agricultural fertilizers, manure,
compost, human or animal waste, and domestic sewage)
derived from waste disposal site and the areas around dur-
ing storm event resulted in nitrate concentration or the
increased nitrate level was due to freshwater inflow and
terrestrial run-off during the wet season (Karuppasamy
& Perumal, 2000). Another possible way of nitrates en-
try is through oxidation of ammonia form of nitrogen
to nitrite formation (Rajasegar, 2003). Generally, during
rainy season groundwater is recharged through precipita-
tion via percolation leading to a general rise in the level
of the water. This makes them highly susceptible to pollu-
tion and runoff activities as elements in soils and rocks are
easily released into the water. The main source of nitrate
in these groundwater samples could be attributed use of
chemical fertilizers on farms, effluent discharges and run-
off from animal feedlots have been identified as one of the
main causes of nitrate in groundwater. Besides, improper
disposal of human and animal wastes on open land re-
sults in leaching of residual nitrate thereby causing high
nitrate concentration in groundwater in the wet season. In most case uses of chemical fertilizers; improper dis-
posal of human and animal wastes and effect of seasons Nitrates of ground water
Nitrates of ground water 0.62±0.07 mg/L (Figure 9, Table 3). The result of Figure 9
shows that all values of the dry and wet season were ex-
tremely below the WHO standard value of 50 mg/L for
groundwater. This could probably be due to the absence
of DO significantly in the wastewater; a smaller amount of
nitrate is produced from ammonia as well as decomposi-
tion of food wastes and other sources of protein. 2.1.3. Spatial and temporal variation of nutrients and
micro biological parameters of groundwater quality Mostly
factors affecting the occurrence of nitrate in groundwater
boreholes are subsurface clay lenses and land use practice. The disclosures/findings/ of low values of nitrate (below
WHO standard) during the examination time frame was
similar with the work of (Akale et al., 2018) who have
assessed nitrate in wells and springs in the north-central
Ethiopian high lands and found value which is less than
WHO standard limit throughout the study period. Even
though all values were below the permissible limit the
mean concentrations of nitrate obtained during wet sea-
son were higher than the value of dry season (Figure 9,
Table 3). The disclosures of the high value of nitrate in
a wet season during the study period were similar to the
research of (Akale et al., 2017) who investigates ground-
water quality in an upland agricultural watershed in the
sub-humid Ethiopian highlands and found out the higher
value of nitrate during the wet season than the dry season. This might have been due to high nitrate concentration
in the wet season suggested that increased flush of ni-
trate causing components (mixed source such as decaying
plant or animal material, agricultural fertilizers, manure,
compost, human or animal waste, and domestic sewage)
derived from waste disposal site and the areas around dur-
ing storm event resulted in nitrate concentration or the
increased nitrate level was due to freshwater inflow and
terrestrial run-off during the wet season (Karuppasamy
& Perumal, 2000). Another possible way of nitrates en-
try is through oxidation of ammonia form of nitrogen
to nitrite formation (Rajasegar, 2003). Generally, during
rainy season groundwater is recharged through precipita-
tion via percolation leading to a general rise in the level
of the water. This makes them highly susceptible to pollu-
tion and runoff activities as elements in soils and rocks are
easily released into the water. The main source of nitrate
in these groundwater samples could be attributed use of
chemical fertilizers on farms, effluent discharges and run-
off from animal feedlots have been identified as one of the
main causes of nitrate in groundwater. Besides, improper
disposal of human and animal wastes on open land re-
sults in leaching of residual nitrate thereby causing high
nitrate concentration in groundwater in the wet season. 2.1.3. Spatial and temporal variation of nutrients and
micro biological parameters of groundwater quality In most case uses of chemical fertilizers; improper dis-
posal of human and animal wastes and effect of seasons
are fundamental factors for sources of nitrogen-containing
compounds that are converted to nitrate in the soil. On
the other hand, the low mean value was recorded dur-
ing the dry season due to less freshwater inflow and high Figure 9. Seasonal variation in the nitrate of groundwater
quality in the study area maximum value occurs at sampling point 3 with a mini-
mum value at sampling point1 (Figure 9). The reason for
the values to be maximum might have been due to plant
decaying, manure, compost, and runoff since sampling
point 3 and sampling point 4 borehole samples are found
particularly at grazing land and gardens while for the min-
imum the reverse is true. From the ANOVA result, there
was no possible significant variation between the sampling
points as well as between the seasons that might lead the
concentration of nitrate above the limit of WHO.h Dynamics of phosphate in ground water: The concen-
tration of phosphate in the groundwater samples ranged
from 0.0088±0.0029 to 0.098±0.001 mg/L in the dry sea-
son and 0.002±0.001 to 0.264±0.0.34 mg/L in the rainy
season (Figure 10, Table 3). The findings of the study
show that higher levels of phosphate concentrations were
however registered during the wet seasons compared to
dry seasons in all the groundwater samples (Figure 10,
Table 3). This difference can be explained in terms of
increased moisture in the ground of waste disposal site
which contains human wastes, medicated shampoos,
food waste and cosmetics that can leach the phosphate
into groundwater. Generally, the value of phosphate dur-
ing the rainy season can increase due to the possible input
of phosphates from the external environment, improper
disposal of solid wastes and runoff due to excessive use of
chemical fertilizers into the water and natural decomposi-
tion of rocks and minerals that contain phosphates. Phos-
phates are usually highly adsorbed into the soil and can
be transported into the catchments and the recipient water Figure 10. Seasonal variation in the phosphate of groundwater
quality in the study area
0
0.05
0.1
0.15
0.2
0.25
0.3
SS1
SS2
SS3
SS4
SS5
SS6
PO4
3– (mg/L)
sampling sites
Concentrations of phosphates in ground water
Dry season
Wet season
WHOstandard value
SS = Sampling site Figure 10. 2.1.3. Spatial and temporal variation of nutrients and
micro biological parameters of groundwater quality This was eventu-
ally washed by rainwater as runoff towards groundwater
samples and then leaked into groundwater besides to mix
with the open boreholes and spring water via surface flow. The maximum values among the six groundwater samples
were found at sampling point 5 sampling point 6, sam-
pling point 1 and sampling point 3 with a minimum at
sampling point 4 during the dry season while in a wet
season the maximum values were obtained at sampling
point 5, sampling point 6, sampling point 3 and sampling
point 4 with a minimum at sampling point 1. The tem-
poral mean value of total coliform in the water at the six
sampling points of the study area during the dry and wet
season were shown in Figure 11. Total coliform: The results of the bacteriological analy-
sis of borehole water samples indicate that the amount
of total coliform during the dry season was ranged from
5±1 to 11.67±2.52 CFU/100 ml with a mean value of
7.9±2.01 CFU/100 ml whereas in a wet season the to-
tal coliform fluctuated from 13±3 to 23±6 CFU/100 ml
with a mean value of 17.3±4.04 CFU/100 ml (Figure 11,
Table 3). From the result, one can see that the bacterial
colony counts were all above the WHO guideline limit of
0 CFU/100 ml for drinking purposes. Therefore, ground-
water samples were contaminated with total coliform. This
must be due to wastes particularly human wastes which
are transported from Bahir Dar city and discharged di-
rectly to the open land (at the open waste disposal site). It can, therefore, be inferred that the boreholes were
mainly polluted by the drains emanating from the waste
site that empty their content directly into the open land. The measured mean value of total coliform during the wet
season was higher than that of the dry season. This could
be due to the discharged human wastes or fecal matters
are flushed/washed/ away by the actions of rainfall from
its source to the different water bodies. Then during its
flow, it joins surface waters and open boreholes besides
leaching into groundwater through percolation and in-
filtration. The transport of bare soil contaminated with
farces by the wind/rain/ into open bores as well as surface
runoff could also have accounted for the high bacterial
load during the wet season as compared to the dry season. 2.1.3. Spatial and temporal variation of nutrients and
micro biological parameters of groundwater quality Sample points that are nearest to the disposal site in the
downstream like sampling point 5 and sampling point 6
were more affected by the total coliform. Un expectedly
sample points which are located above the dumpsite were
polluted with total coliform. This might have been due to 2.1.3. Spatial and temporal variation of nutrients and
micro biological parameters of groundwater quality Seasonal variation in the phosphate of groundwater
quality in the study area Figure 10. Seasonal variation in the phosphate of groundwater
quality in the study area D. Haile, N. Gabbiye. Groundwater quality assessment of Chilanchil abay watershed: the case of Bahir-dar city... 132 bodies by the actions of rain and then accounted for the
high phosphate concentration in local water bodies. The
minimum value of phosphate was registered during the
dry season, this might have been due to the low solubility
of native phosphate minerals and the ability of soils to re-
tain phosphate. This is in line with the findings of (Gadhia
et al., 2012) who investigates seasonal variations in Phys-
ico-chemical characteristics of Tapi estuary in the Hazira
industrial area and found out a higher value of phosphate
during the wet season than dry. From the point of spatial
variation, the highest concentrations of phosphate were
recorded at sampling point 5 followed by sampling point
6 during the rainy season with a minimum value at sam-
pling point 1 while in the dry season the highest level was
at sampling point 6 with a minimum value at sampling
point 2 (Figure 10, Table 3). This was due to the loca-
tions of boreholes from the selected waste disposal site. From below obtained result (Figure 10) except sampling
point 5 and sampling point 6 the concentration of PO43–
in both dry and wet seasons were below WHO standards
limit (PO43– = 0.1 mg/L). On account of this, the statistical
analysis of variance (ANOVA) results indicates that there
was no significant difference in mean phosphate concen-
tration between and within sampling points as well as sea-
sons during the study period (p < 0.05). h Figure 11. Seasonal variation in total coliform of groundwater
quality in the study area
0
5
10
15
20
25
SS1
SS2
SS3
SS4
SS5
SS6
Total coliform (MPN/100ml)
Sampling sites
Total coliform of ground water
Dry season
Wet seasonSS=Sampling site
SS = Sampling site Figure 11. Seasonal variation in total coliform of groundwater
quality in the study area faeces and other wastes from anthropogenic sources (open
field defecation along the boreholes by humans and other
animals that graze along with the groundwater samples)
located in bushes close to boreholes. Reference Analysis in Chilanchil Abay watershed Table 4 above
and Figure 12 below shows the variation of WQI with
CCME standard level to evaluate the status of existing
groundwater quality in the study area. The calculated re-
sults obtained from all sampling points of groundwater
were showing four sample points as marginal and two
sample points as poor for 30th March 2019 to 20th August
2019. All the marginal status sample points are found in
the upstream of the waste disposal site. This indicates that
Water quality is frequently endangered or deteriorated;
conditions often deviate from natural or desirable levels. This could be resulted due to decaying of plants and ani-
mals, agricultural fertilizers and open defection activities. While the remaining two poor status sample points were
found in the downstream of the waste disposal site. This
indicates that the water quality of these sampling points
is always endangered or deteriorated; conditions usually
deviate from natural or desirable levels. Considering all
sample sites, SS5 and SS6 have shown the worst quality in
the context of CCMEWQI. The reasons may include the
migrations of leachate in downstream from the dumpsite
towards those two sample points. Afolayan, O., Ogundele, F., & Odewumi, S. (2012). Spatial vari-
ation in landfills leachate solution in urbanized area of Lagos
State, Nigeria. American International Journal of Contempo-
rary Research, 2(8), 178–184. http://www.aijcrnet.com/jour-
nals/Vol_2_No_8_August_2012/17.pdf Akale, A. T., Dagnew, D. C., Giri, S., Belete, M. A., Tilahun, S. A.,
Mekuria, W., & Steenhuis, T. S. (2017). Groundwater quality
in an upland agricultural watershed in the sub-humid Ethi-
opian highlands. Journal of Water Resource and Protection,
9(10), 1199. https://doi.org/10.4236/jwarp.2017.910078 Akale, A., Moges, M., Dagnew, D., Tilahun, S., & Steenhuis, T. (2018). Assessment of nitrate in wells and springs in the
North Central Ethiopian Highlands. Water, 10(4), 476. https://doi.org/10.3390/w10040476 Bogan, R. A., Ohde, S., Arakaki, T., Mori, I., & McLeod, C. W. (2009). Changes in rainwater pH associated with increasing
atmospheric carbon dioxide after the industrial revolution. Water, Air, and Soil Pollution, 196(1–4), 263–271. https://doi.org/10.1007/s11270-008-9774-0 https://doi.org/10.1007/s11270-008-9774-0
d Clark, R. B. (1986). Marine pollution (3rd ed.). Oxford Science
Press Publication, Calendon. B. Clesceri, L., Greenberg, A., & Eaton, A. (1998). Standard meth-
ods for the examination of water and wastewater (20th ed.). American Public Health Association, Washington, DC. http://srjcstaff.santarosa.edu/~oraola/Assets/APHA_SM_20.pdf http://srjcstaff.santarosa.edu/~oraola/Assets/APHA_SM_2 Gadhia, M., Surana, R., & Ansari, E. (2012). Seasonal variations
in physico-chemical characterstics of Tapi estuary in Hazira
industrial area. Reference Our Nature, 10(1), 249–257. https://doi.org/10.3126/on.v10i1.7811 Conflict of interest The Authors declare that there is no conflict of interest
regarding the publications of this paper. Acknowledgements The authors would like to thank Bahir Dar University, Ba-
hir Dar Institute of Technology for technical and financial
supports of this project. Figure 12. variations of water quality index in the study area at
different groundwater sample sites
CCMEWQI values 2.2. Assessing/Evaluating/ groundwater Quality of
Chilanchil Abay watershed through Water Quality
Index (CCMEWQI) In this study, the CCME Water Quality Index was applied
and tested for the Chilanchil Abay Watershed quality. The
index ranges from 0 to 100 and depending on the value;
the water quality is characterized as excellent, good, fair,
marginal and poor. The CCME WQI was calculated using
the method described by CCME 2001 guidelines (Eqs (1),
(2), (3), (4), (5), (6) and (7). The results obtained from the
application of CCME WQI concerning nutrients, heavy
metals, and Physico-chemical characteristics were pre-
sented in Table 4 and Figure 12. Table 4. variation of WQI of Chilanchil Abay watershed with
different sampling points
Sampling points
level
Status
SS1
46.2
Marginal
SS2
45.53
Marginal
SS3
45.52
Marginal
SS4
45.53
Marginal
SS5
42. Poor
SS6
42.2
Poor Table 4. variation of WQI of Chilanchil Abay watershed with
different sampling points Journal of Environmental Engineering and Landscape Management, 2021, 29(2): 123–134 133 Figure 12. variations of water quality index in the study area at
different groundwater sample sites
0
20
40
60
80
100
120
CCMEWQI values
CCMEWQI values of ground water
CCMEWQI values
SS=Sampling site
SS = Sampling site CCMEWQI values of ground water CCMEWQI values of ground water Conclusions The temporal and spatial variations of groundwater qual-
ity of Chilanchil Abay watershed was assessed following
WHO quality parameters standards and Canadian wa-
ter quality index. The analysis of physicochemical and
concentrations of nutrients recorded values, show that
BOD, COD, and NO3– were within the acceptable limit
for groundwater quality during the study period while the
remaining water quality characteristics fluctuated with the
seasons. The total coliform counts detected were above
the permissible limits for drinking water in all sample
points. According to the Canadian water quality index
(CCMEWQI), the water quality of Chilanchil Abay wa-
tershed was categorized under poor and marginal status. i Hamad, O. H. M. (2018). Occupational and environmental haz-
ards among workers in petroleum stations, Khartoum State,
Sudan (2013–2015). University of Gezira. http://repo uofg edu sd/handle/123456789/3483 http://repo.uofg.edu.sd/handle/123456789/3483 Idoko, M., & Oklo, A. (2012). Seasonal variation in physico-
chemical characteristics of rural groundwater of Benue State,
Nigeria. Journal of Asian Scientific Research, 2(10), 574–586. https://ideas.repec.org/a/asi/joasrj/2012p574-586.html Karuppasamy, P., & Perumal, P. (2000). Biodiversity of zooplank-
ton at Pichavaram mangroves, South India. Advances in the
Biosciences, 19(2), 23–32. https://www.sciencebase.gov/cata-
log/item/505772cee4b01ad7e027c1d2 Kay, M. (2001). Smallholder irrigation technology: Prospects for
sub-Saharan Africa. Food & Agriculture Organization of the
United Nations. http://www.fao.org/publications/card/en/c/
f391cd0a-8d84-5d6e-8d30-4de028cad7ae/ i From the findings of the study, it is assured that sample
points downstream the dumpsite were more affected than
the sample points of upstream. Khan, A. A., Paterson, R., & Khan, H. (2004). Modification and
application of the Canadian Council of Ministers of the Envi-
ronment Water Quality Index (CCME WQI) for the commu-
nication of drinking water quality data in Newfoundland and
Labrador. Water Quality Research Journal, 39(3), 285–293. https://doi.org/10.2166/wqrj.2004.039 Data suggested the importance of greater attention for
household contamination, environmental sanitation con-
trol and awareness about water contamination since the
open waste disposal site and practice of open defecation
have actually impacted the watershed of the study area. D. Haile, N. Gabbiye. Groundwater quality assessment of Chilanchil abay watershed: the case of Bahir-dar city... D. Haile, N. Gabbiye. Groundwater quality assessment of Chilanchil abay watershed: the case of Bahir-dar city... 134 Krishnamurthy, K., & Mani, P. (1989). Variation of phytoplank-
ton in a tropical estuary (Vellar estuary, Bay of Bengal, India). Internationale Revue der gesamten Hydrobiologie und Hydrog-
raphie, 74(1), 109–115. https://doi.org/10.1002/iroh.19890740109 Nsengimana, H., Bigirimana, B., Suwa, M., Mukubwa, A., De-
bruyn, W., & Kalisa, N. (2012). Conclusions Assessment of heavy metals
(Pb, Cu, Cr, Cd and Fe) in the groundwater wells in the vicin-
ity of Nyanza Municipal Solid waste in Kigali City-Rwanda. Rwanda Journal, 25(1), 3–21. https://doi.org/10.4314/rj.v25i1.1 Love, J., & Luchsinger, V. (2014). Sustainability and water re-
sources. Journal of Sustainability and Green Business, 2, 1–12. https://www.aabri.com/manuscripts/121354.pdf Odokuma, L. O., & Okpokwasili, G. C. (1993). Seasonal ecol-
ogy of hydrocarbon-utilizing microbes in the surface waters
of a river. Environmental Monitoring and Assessment, 27(3),
175–191. https://doi.org/10.1007/BF00548364 https://www.aabri.com/manuscripts/121354.pdf Maitera, O., Ogugbuaja, V., & Barminas, J. (2010). An assessment
of the organic pollution indicator levels of River Benue in
Adamawa State, Nigeria. Journal of Environmental Chemistry
and Ecotoxicology, 2(7), 110–116. https://academicjournals. org/article/article1379512159_Maitera%20et%20al.pdf Onwugara, N., Ajiwe, V., & Nnabuenyi, H. (2013). Physico-
chemical studies of water from selected boreholes in Umua-
hia north Local Government Area of in Abia State, Nigeria. International Journal of Pure & Applied Bioscience, 1(3), 34–
44. http://www.ijpab.com/form/2013%20Volume%201,%20
issue%203/IJPAB-2013-1-3-34-44.pdf Makwe, E., & Chup, C. (2013). Seasonal variation in physico-
chemical properties of groundwater around Karu abattoir. Ethiopian Journal of Environmental Studies and Management,
6(5), 489–497. https://doi.org/10.4314/ejesm.v6i5.6 Oram, B. (2015). Stream water quality – Importance of total sus-
pended solids/turbidity. https://www.water-research.net/index. php/stream-water-quality-importance-of-total-suspended-
solids-turbidity Markandya, A. (2006). Water quality issues in developing coun-
tries. In R. López & M. A. Toman (Eds.), Economic devel-
opment and environmental sustainability: New policy options
(pp. 307–344). https://doi.org/10.1093/0199298009.003.0011 Rajasegar, M. (2003). Physico-chemical characteristics of the
Vellar estuary in relation to shrimp farming. Journal of Envi-
ronmental Biology, 24(1), 95–101. https://pubmed.ncbi.nlm.nih.gov/12974418/ Nathanson, J. (2015). Solid-waste management. Encyclopaedia
Britannica.
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Delayed sleep phase cases and controls
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Journal of circadian rhythms
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Published: 29 April 2008 Published: 29 April 2008 Journal of Circadian Rhythms 2008, 6:6
doi:10.1186/1740-3391-6-6 This article is available from: http://www.jcircadianrhythms.com/content/6/1/6 This article is available from: http://www.jcircadianrhythm © 2008 Kripke et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. BioMed Central BioMed Central Abstract Background: Delayed sleep phase disorder (DSPD) is a condition in which patients have difficulty
falling asleep before the early morning hours and commonly have trouble awakening before late
morning or even early afternoon. Several studies have suggested that variations in habitual bedtime
are 40–50% heritable. Methods: We recruited a case series of 205 participants, along with 221 controls (DSPD-C) with
normal sleep, roughly matched for age, gender, and ancestry. A representative sample of San Diego
adults recruited some years before was already available to confirm the control group. Both DSPD
and DSPD-C provided blood or saliva samples for DNA and completed extensive questionnaires
about sleep habits, sleep history, family history, sleep quality, morningness-eveningness traits,
depression, mania, and seasonality of symptoms. The DSPD group wore wrist actigraphs for a
median of 13.2 days. The representative sample collected previously had undergone actigraphic
recordings, from which 48 hours of data were generally available. Results: The DSPD and DSPD-C samples showed almost no overlap on morningness-eveningness
scores. DSPD cases went to bed and arose about 3 hours later than the DSPD-C and the
representative sample. DSPD cases reported more difficulties with sleep, poorer sleep quality, and
more depression, but there was no significant difference in a history of mania. DSPD cases reported
more family history of late bedtimes, but female DSPD reported that their fathers' bedtimes were
later than the fathers of male DSPD. Conclusion: These results indicate a DSPD phenotype is familial and associated with unipolar
depression. Larks also tend to be "morning" types who are energetic
early in the morning, whereas owls are "evening" types
whose energy may increase late in the evening. These traits Larks also tend to be "morning" types who are energetic
early in the morning, whereas owls are "evening" types
whose energy may increase late in the evening. These traits Delayed sleep phase cases and controls Address: 1Department of Psychiatry, University of California, San Diego, La Jolla, California 92093-0667, USA, 2Scripps Clinic Sleep Center, 10666
North Torrey Pines Road, La Jolla, California 92037, USA, 3Department of Psychiatry 116A, VA San Diego Health System, 3350 La Jolla Village
Drive, San Diego, CA 92161, USA, 4The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA and
5Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, PO Box 210207, Tucson, Arizona, 85721-0207, USA Email: Daniel F Kripke* - Kripke.Daniel@ScrippsHealth.org; Katharine M Rex - KRex@popmail.ucsd.edu; Sonia Ancoli-
Israel - SAncoliIsrael@UCSD.edu; Caroline M Nievergelt - CNieverg@ucsd.edu; Walt Klimecki - Klimecki@pharmacy.Arizona.edu;
John R Kelsoe - JKelsoe@UCSD.edu * Corresponding author Received: 25 February 2008
Accepted: 29 April 2008 Received: 25 February 2008
Accepted: 29 April 2008 Journal of Circadian Rhythms Open Access Background g
Some people are characteristically "larks" who retire and
awaken early or "owls" who stay up late and awaken late. Page 1 of 14
(page number not for citation purposes) Page 1 of 14
(page number not for citation purposes) http://www.jcircadianrhythms.com/content/6/1/6 http://www.jcircadianrhythms.com/content/6/1/6 Journal of Circadian Rhythms 2008, 6:6 among reports [25-28]. Less replicated reports have noted
associations of DSPD, or eveningness, with other circa-
dian system polymorphisms [29-37]. Associations of
sleep timing with the T3111C polymorphism in the
CLOCK gene have been reported inconsistently, possibly
because all authors agree that these associations are weak,
and this polymorphism is in strong linkage disequilib-
rium with numerous other polymorphisms throughout
the CLOCK gene [29,38-41]. All reported associations of
polymorphisms with DSPD need further replication. Moreover, it appears plausible that genetic susceptibility
factors with important contributions to DSPD may not yet
have been discovered. have been described with morningness-eveningness scales
(MES) such as the Horne-Östberg Scale (HO) [1]. Though
mild variations in habitual bedtime are often easily
accommodated, extreme forms of morningness or
eveningness may produce social distress and disability. When distressing, the severe forms of morningness and
eveningness are termed Advanced Sleep Phase Disorder
(ASPD, the larks) and Delayed Sleep Phase Disorder
(DSPD, the owls) [2]. Patients with ASPD may retire at ~8
PM or earlier and likewise arise around ~4 AM or earlier. Such sleep times may resemble those of an agricultural
society without electric light. Early bedtimes often cause
only minimal social distress, so patients with ASPD rarely
complain to physicians. Patients with DSPD may be una-
ble to fall asleep early enough to rest adequately before it
is time for school or work, or they may be quite unable to
report to school or work on time. Consequently, DSPD
can be a disabling and socially isolating condition, unless
the patients are able to fit their habits into an accommo-
dating social milieu. A Norwegian study estimated the
prevalence of DSPD at 0.17% [3]. A Japanese study esti-
mated 0.13% [4]. The prevalence of definite DSPD among
adults ages 40–64 years was estimated in a representative
actigraphic survey of San Diego as less than 1%, though
either trouble falling asleep or trouble waking up was
reported by 24.6% and both symptoms by 3.1% [5]. Late
bedtime is also a common problem among adolescents. Background Adolescents with late bedtimes obtain less sleep and have
more trouble with school, more disciplinary problems,
and more depression [6,7], though it seems less than 1%
meet formal criteria for DSPD [8]. Bedtimes are also com-
monly late among college students: for example, average
bedtimes for samples at a Midwestern college ranged from
01:25 to 2:05 AM [9,10]. Some college populations in
Europe may retire even later, with 6.9% of men and 2.8%
of women reporting being extreme evening types [11]. It
seems that social, developmental and environmental fac-
tors modify the prevalence of DSPD. To further explore the genetic polymorphisms associated
with DSPD, we recruited self-selecting samples of DSPD
cases and a control group roughly matched for ancestral
origin, age, and gender. A representative sample of acti-
graphically-recorded San Diego adults was also available
from previous research [5,42,43]. In this report, we present a description of our DSPD sam-
ple and analyze how cases differed from the control sam-
ples. Identification of genetic differences between the case
and matched-control samples is currently under way, and
will be reported elsewhere. Delineation of a DSPD case sample encountered several
difficulties. The landmark descriptions of delayed sleep
phase disorder suggested very late bedtimes as diagnostic
criteria, e.g., after 0200. However, some patients with ear-
lier bedtimes were included in early case samples, because
they made similar complaints of inability to fall asleep at
the desired time and inability to arise at the desired wak-
ing time. It soon became evident that defining an explicit
bedtime criterion for DSPD would be almost impossible
because of age, social, and geographic variations in what
may be considered a "normal" and socially-desirable bed-
time [44]. Several studies have found that DSPD may be familial
[12]. Despite the social and developmental influences,
analyses have most commonly estimated the heritability
of bedtime at 40–50% [13-19], though in a Hutterite sam-
ple, the estimated heritability of bedtime was not signifi-
cant [20], and in the Framingham study it was only 22%
[21]. Evidently, genetic polymorphisms influence an
important component of a person's habitual bedtimes. ASPD is associated in some families with incompletely-
dominant single-nucleotide polymorphisms (SNPs)
within PER2 and CSNK1D, which are genes which com-
prise parts of the body's circadian clock [22-24]. Background Some
polymorphisms in the PER3 gene (also a circadian system
gene) have been described as associated with DSPD and
eveningness, though there have been inconsistencies Both the International Classification of Sleep Disorders
and DSM-IV rejected an explicit bedtime criterion for
DSPD, requiring instead a mismatch or misalignment
between the desired sleep and wake-up times and the
hours when a patient was able to sleep [2,45]. A misalign-
ment definition accommodates the diversity of patient
complaints without being confounded by a rigid bedtime
criterion, but this criterion has the scientific limitation
that it may be impractical to determine the extent of mis-
alignment or to define a patient's "desired and socially
acceptable time" for sleep [2]. By such contemporary def-
initions, the presence of delayed sleep rests entirely on the
clinician's interpretation of the patient's complaint. We
feared that mismatch criteria alone might not function
adequately for our scientific goals. Page 2 of 14
(page number not for citation purposes) http://www.jcircadianrhythms.com/content/6/1/6 http://www.jcircadianrhythms.com/content/6/1/6 http://www.jcircadianrhythms.com/content/6/1/6 Journal of Circadian Rhythms 2008, 6:6 As a compromise, we relied on the Horne-Östberg scale as
the primary method of identifying "evening types" com-
parable to DSPD. This had the advantage of an explicit
semi-numeric scale which incorporates subjective judg-
ments about when a patient feels energetic or active, but
the scale does not require distress or misalignment. The
Horne-Östberg criteria were originally calibrated with a
young-adult British sample, perhaps including many stu-
dents [1]. We noted that applying the original Horne-Öst-
berg criteria to mid-American adults, ASPD appeared
rampant but extreme eveningness was more rare [38],
which was inconsistent with clinical experience. Samples
from France, India and New Zealand have likewise called
for a modification of the original Horne-Östberg criteria
[44,46,47]. Eventually, with a goal of collecting a sample
primarily for genetic research which would emphasize the
heritable characteristics, we adopted a hybrid classifica-
tion approach relying on modified morningness-evening-
ness
scale
(MES)
criteria,
complaints
suggesting
misalignment of sleep propensity, and a past history sug-
gesting that the delayed pattern was constitutional rather
than acquired or environmental in origin or attributable
to comorbidities. social influences of youth culture or a college environ-
ment. Elderly participants were accepted if there was no
suggestion of dementia or other illness which might dis-
tort circadian rhythms, and if there was a life-long history
of delayed sleep. BALM The Basic Language Morningness scale or BALM [49] was
administered as a simplified-language revision of the 13-
item Composite Scale (CS) [50]. The CS was reported to
have superior psychometric properties to the HO scale as
well as greater brevity. PSQI The Pittsburgh Sleep Quality Index (PSQI) measures sub-
jective distress with sleep quality [51]. A total score above
5 suggests complaints of poor subjective sleep quality, but
does not necessarily correspond to curtailments or inter-
ruptions of sleep measured polysomnographically. DSPD sample recruitment Recruitment was targeted to the Southern California
region, particularly San Diego County, to enable the
investigators to make home visits when necessary. Recruit-
ment took place between June, 2004 and February, 2007. Recruitment of DSPD participants utilized contacts with
Southern California sleep physicians, word of mouth,
media contacts, newspaper advertising, late night radio
advertising, UCSD minority outreach programs, paid con-
text-based internet ads, and free internet advertising. A
web site was constructed to facilitate contacts, provide
potential volunteers with information, and allow poten-
tial volunteers to self-screen themselves versus the Horne-
Östberg Scale: those with scores ? 30 were encouraged to
volunteer for the full study. Health questionnaire
l h
l h A general health questionnaire (available from the
authors) was expanded from the version used in a previ-
ous representative survey [42] to included items about
sleep satisfaction, past-week bedtimes and waking times,
difficulties falling asleep too early or too late, lifetime
health including depression, mania, and sleep disorders,
medical treatments for sleep disorders or affective disor-
ders, and family history. This questionnaire included
items asking if participants went to bed earlier or later
than most people as a child and also earlier or later than
most people throughout adult life. Average adult bedtime
was also recorded. Medications a participant used were
recorded in broad categories. The investigators recognized that a sample of homogene-
ous ancestry would help to prevent false-positive genetic
findings due to sample stratification. On the other hand,
inclusion of participants of diverse ancestry may produce
more generalizable findings, may identify a larger number
of associated genetic polymorphisms, and may satisfy
institutional goals for fairness in medical research. Balanc-
ing these considerations, our target was to achieve a sam-
ple roughly representative of the San Diego County
ancestral origins. Background Potential participants were provided written and verbal
information about the study and signed written informed
consent under the supervision of the UCSD Human
Research Protection Program. Subjects referred or self-
referred as potential DSPD cases completed a series of
questionnaires, contributed a sample of blood or saliva
for DNA, and underwent a 2-week wrist activity and illu-
mination recording, using a wrist-activity recorder. Horne-Östberg morningness-eveningness questionnaire
An American idiom rephrasing was adopted for the
Horne-Östberg Scale [1,48]. This is a 19-item form yield-
ing scores from 16–30 (extreme eveningness) to above 70
(extreme morningness), as originally defined. DSPD-control recruitment While the DSPD case series was being assembled, the
investigators recruited DSPD-Controls (DSPD-C) who
claimed to have healthy and normal sleep. Control volun-
teers were recruited by word-of-mouth, outreach at health
fairs and community meetings, the internet, and by a cam-
pus poster seeking volunteers who "sleep like a baby."
Control recruitment was targeted, so far as possible, to
match the ancestry of the case series. Each DSPD-C received an explanation of the study and
signed written informed consent. DSPD-C completed all
the questionnaires described above and contributed a
sample of blood or saliva, but they were not asked to wear
the actigraphs or to provide sleep logs. Since control par-
ticipation usually required less than 2 hours, they were
reimbursed $25. The principal investigator (DFK) reviewed the record of
each participant volunteering as control. Based on their
questionnaires, control volunteers were retrospectively
rated as 1) certain DSPD, 2) possible DSPD, 3) neither, 4)
possible ASPD, or 5) certain ASPD. Only those in classes
3 or 4 were included in these DSPD-C analyses. Actigraph g
p
Each DSPD participant was provided an Actiwatch-L wrist
actigraph to wear for 2 weeks (Mini Mitter Respironics,
Bend, Oregon). This device recorded an integrated sum of
wrist accelerations every 1 minute (using an arbitrary
scale) and an average of the illumination of the wrist,
using a phototransducer comparable to a photometer. Participants also completed a sleep log every 24-hours,
estimating bed times, wake times, naps, and those times
when the Actiwatch was removed from the wrist. The par-
ticipant's activity patterns were then graphed, using the
manufacturer's software. In addition, each 2-week record-
ing was scored for sleep-wake minute by minute, using an
algorithm validated for a different actigraph [56], modi-
fied for the Actiwatch settings, and then corrected by hand
when the sleep log and behavioral cues, including the illu-
mination data, suggested that the computer scoring was
likely in error. This algorithm and hand scoring have not
been validated with the Actiwatch. A 24-hour cosine was
fit to each time series for sleep-wake, activity, and illumi-
nation, using a least-squares technique. The mesors (fitted
means) of the cosines were utilized as the best magnitude
estimates of daily sleep, activity, and illumination expo-
sures, since this procedure adjusted for off-wrist intervals,
incorporated data concerning wakes-after-sleep-onset
within the major sleep period, and reflected sleep epi-
sodes outside the major sleep period. The acrophases (fit-
ted peaks) of the cosines were the best estimates of
circadian timing. DSPD participants were reimbursed $100 for their time
and effort, which included completing the questionnaires,
providing a blood or saliva sample, wearing the Actiwatch
for 2 weeks, completing sleep logs, and associated travel. Clinical evaluation Once all data were assembled, the principal investigator
(DFK) reviewed the record of each participant who had
volunteered as a DSPD case and recorded the participant's
DSPD classification as 1) absolutely certain, 2) fairly cer-
tain, 3) questionable, 4) unlikely, or 5) very doubtful. The
main criterion for classification was the HO score, recog-
nizing that the criterion for definite evening type of ? 30
was too strict for the San Diego population. Confirmatory
classification criteria included the score on the BALM,
reported prior-week and adult-life bedtimes and awaken-
ing times, the actigraphic recordings, and whether the par- Mania history Participants completed the Mood Disorders Question-
naire, a lifetime screening scale for mania with very high
specificity [52]. Any occurrence of mania would indicate
the presence of bipolar disorder. Only participants 25 years of age or older were accepted
(with a few exceptions), to reduce the likelihood that par-
ticipants might acquire late hours entirely through the Page 3 of 14
(page number not for citation purposes) Page 3 of 14
(page number not for citation purposes) http://www.jcircadianrhythms.com/content/6/1/6 http://www.jcircadianrhythms.com/content/6/1/6 Journal of Circadian Rhythms 2008, 6:6 SPAQ The Seasonal Pattern Assessement Questionnaire (SPAQ)
includes a global seasonality scale, which reflects the sea-
sonality of mood and may suggest the presence of a sea-
sonal mood disorder e.g., winter depression, a condition
sometimes associated with a delayed sleep phase [55]. QIDS-SR ticipant reported going to sleep "somewhat later" or
"much later" than most people their age, both as a child
and as an adult. Whether the participant reported distress
about falling asleep, reported related social or vocational
problems, or had sought medical attention for a sleep
problem was also considered. The consistency of the data
supporting a classification of DSPD was evaluated,
together with the presence of depression, other mental ill-
nesses, or other sleep disorders which might confuse the
classification. However, if depression or other disorders
had their first incidence after the onset of delayed sleep
and did not appear to be causing the delay, these disorders
were not considered exclusionary. As many DSPD patients
cannot consistently report to work by 8–9 AM, evening or
night shift work was not considered exclusionary if the
history indicated that the delay in sleep occurred before
shift work was adopted, and the delay tended to persist
when the participant was off work. The Quick Inventory of Depression Symptomatology Self
Report (QIDS-SR) is a self-rating scale for current major
depression [53,54] with high correlation to the psychia-
trist-administered Hamilton Depression rating scale. Page 4 of 14
(page number not for citation purposes) Representative sample As part of an earlier research project, from 1990–1994,
our laboratory had recruited a representative population
sample of San Diego adults ages 40–64, using structured
random telephone dialing. A smaller representative sam-
ple of women ages 19–39 was recruited in parallel. More Page 4 of 14
(page number not for citation purposes) http://www.jcircadianrhythms.com/content/6/1/6 http://www.jcircadianrhythms.com/content/6/1/6 Journal of Circadian Rhythms 2008, 6:6 samples were 65%, 64%, and 60% female, respectively
(not significant). detail on the recruitment and characteristics of this sam-
ple has been reported in prior publications [5,42,43,57]. Representative sample participants had worn an actigraph
of different design for 3 nights and 2 days. Representative
participants had also completed a questionnaire which
included similarly-worded questions about bedtimes,
awakening times, and sleep disorders, but they did not
receive any MES scales. The values of this sample as an
additional control were its population representativeness
and the availability of actigraphic sleep measures, whereas
the disadvantages were numerous differences in the
recruitment procedures and data gathered. Recruitment of
the representative sample was completed over a decade
earlier in a narrower geographic targeting. No DNA had
been obtained from this earlier sample. Sample numbers and ages p
g
The available samples included 205 DSPD volunteers,
221 DSPD-C controls, and 318 participants in the repre-
sentative San Diego sample. The recruited DSPD volun-
teers included 10% who were retrospectively considered
unlikely or very doubtful to have true DSPD, as shown in
Table 1, and these 10% were excluded from certain analy-
ses. Of the DSPD-C, 202 were considered neither delayed
nor advanced. Ten DSPD-C controls that were judged pos-
sibly delayed were excluded from the analyses, but 9
judged possibly advanced were included. At least 3 of the
DSPD cases displayed free-running sleep-wake rhythms
(non-24-hour rhythms), and in several others, a masked
free-running component was suspected from inspection
of the 2-week actigraph. Because free-running subjects
were excluded from comparisons assuming synchronized
habits (e.g., bedtimes, acrophases), and because various
data (especially actigraphy) were missing in certain cases,
most comparisons involved somewhat fewer subjects
than the total groups. The mean age of the DSPD partici-
pants was 38.8 (SD 12.4, range 22–78) years, and the
mean age for DSPD-C was 40.8 years (SD 13.7, range 23–
82) years (not significantly different). The mean age for
the representative sample was 48.1 (SD 10.4, range 19–
64) years, which was significantly different from DSPD
and DSPD-C. The DSPD, DSPD-C, and representative Objectively, by actigraphic scoring, DSPD slept 417 ± SD
78 min. per 24 hr., while the representative sample slept
353 ± SD 71 min. per bedtime (P < 0.001, N = 194 and N
= 301, respectively). Actigraphic sleep times did not differ
by the certainty of the DSPD classification (classes 1 to 5). The DSPD data included daytime sleep and "naps", but
the representative sample had little sleep out of bed, << 15
min. in most cases, so this had not been scored. The mean
sleep acrophase (approximating the mid-sleep time) was
5:56 AM (SD 2.3 hours) for DSPD and 2:44 AM (SD 1.2
hours) for the representative sample (P < 0.001). Thus,
the DSPD group's sleep acrophase was more than 2 stand-
ard deviations later than the representative sample and
much more variable: Levene's Test rejected equality of var-
iances (P = 0.001.) As illustrated in Fig. Results
Sample num Results
Sample numbers and ages
The available samples included 205 DSPD
221 DSPD-C controls, and 318 participants
sentative San Diego sample. The recruited
teers included 10% who were retrospective
unlikely or very doubtful to have true DSPD
Table 1, and these 10% were excluded from
ses. Of the DSPD-C, 202 were considered ne
nor advanced. Ten DSPD-C controls that wer
sibly delayed were excluded from the an
judged possibly advanced were included. At
DSPD cases displayed free-running sleep-w
(non-24-hour rhythms), and in several othe
free-running component was suspected fro
of the 2-week actigraph. Because free-run
were excluded from comparisons assuming
habits (e.g., bedtimes, acrophases), and be
data (especially actigraphy) were missing in
most comparisons involved somewhat fe
than the total groups. The mean age of the D
pants was 38.8 (SD 12.4, range 22–78) y
mean age for DSPD-C was 40.8 years (SD 13
82) years (not significantly different). The
the representative sample was 48.1 (SD 10
64) years, which was significantly different
and DSPD-C. The DSPD, DSPD-C, and r
Table 1: Classification of DSPD cases
1 Absolutely Certain
2 Fairly Certain
3 Questionable
4 Unlikely
5 Very Doubtful
TOTAL Horne-Östberg scores As shown in Fig. 1, the HO scores of the DSPD volunteers
rated certain, fairly certain, or questionable DSPD largely
overlapped each other, but were very well separated from
those of the DSPD-C group. Those DSPD volunteers rated
unlikely or very doubtful to have delayed sleep phase dis-
order were intermediate, overlapping both the DSPD and
DSPD-C groups. The Spearman Rank Order Correlation of
reported age with the HO was r = 0.16 (P = 0.001), i.e.,
older participants reported slightly more morningness. Actigraphic results From DSPD participants, 193 actigraphic recordings were
available, ranging from roughly 5 to 14 days in duration. The median actigraphic recording was 13.2 days in dura-
tion, with the 10th percentile 10.4 days and 90th percentile
13.8 days. For the representative sample, there were 311
actigraphic data sets available. Although most of those
original representative recordings included 3 nights and 2
days of data, to avoid biasing circadian data by time of
day, 89% of these recordings were truncated to approxi-
mately 48 hours, but the rest were even shorter. Reported bedtimes and uptimes As shown in Fig. 3, 4, the bedtimes and uptimes reported
for the prior week by DSPD groups overlapped DSPD-C
somewhat more than did the HO scores or actigraphic
sleep acrophases. Nevertheless, as was the case for HO
scores and sleep acrophases, reported bedtimes and upti-
mes still differed very significantly between DSPD and
DSPD-C groups (p < 0.001), and the variances were signif-
icantly greater in the DSPD group (p < 0.001). Bedtime
and uptime distributions of the DSPD-C group and the
representative sample were almost superimposable. How-
ever, small percentages of the representative sample had
bedtimes earlier or later than the range for the DSPD-C,
reflecting the exclusion of suspected cases of ASPD or
DSPD and shiftworkers from among the DSPD-C group
but not from the representative sample. Among DSPD
and DSPD-C, the Spearman Rank Order Correlation of
reported bedtimes with age was not significant. Likewise, wrist activity acrophases were 16:55 (SD 2.1
hours) for DSPD and 13:47 (SD 1.4 hours) for the repre-
sentative sample (P < 0.001), Levene's test for equality of
variance P < 0.001. Similarly, the light acrophase was
15:07 (SD 1.9 hours) for DSPD and 12:59 (SD 1.5 hours)
for the representative sample (P < 0.001), Levene's test for
equality of variances not significant (NS). Note that
scored sleep and wrist activity displayed about the same
degree of delay among DSPD participants compared to
the representative sample, but their wrist light exposures
were not correspondingly as late. The mesor fitted to log10
[lux] for the DSPD participants was 0.93, whereas that for
the representative sample was 1.01 (P = 0.001, two-tailed
t test), but the representative sample had a greater range
(Levene's Test for Equality of Variances P = 0.001). This
difference corresponded to about 20% greater illumina-
tion for the representative sample, about 4% of the total
range of illumination exposures in the samples, the differ-
ence in samples representing an effect size of 0.31. Sample numbers and ages 2, subjects classi-
fied with DSPD with greater certainty had later sleep
acrophases, but even the groups rated 4 or 5 (probably or Page 5 of 14
(page number not for citation purposes)
Table 1: Classification of DSPD cases
Number
Percent
Cumulative Percent
1 Absolutely Certain
72
35.1
35.1
2 Fairly Certain
69
33.7
68.8
3 Questionable
44
21.5
90.2
4 Unlikely
16
7.8
98.0
5 Very Doubtful
4
2.0
100.0
TOTAL
205
100.0 Table 1: Classification of DSPD cases
Number
Percent
Cumulative Percent
1 Absolutely Certain
72
35.1
35.1
2 Fairly Certain
69
33.7
68.8
3 Questionable
44
21.5
90.2
4 Unlikely
16
7.8
98.0
5 Very Doubtful
4
2.0
100.0
TOTAL
205
100.0 Table 1: Classification of DSPD cases Journal of Circadian Rhythms 2008, 6:6 http://www.jcircadianrhythms.com/content/6/1/6 Distribution of morningness-eveningness scores
Figure 1
Distribution of morningness-eveningness scores. The percentiles of Horne-Östberg morningness-eveningness scores
are plotted for 5 groups: a) absolutely certain DSPD 1, b) fairly certain DSPD 2, b) questionable DSPD 3, d) unlikely or very
doubtful DSPD 4–5, and e) DSPD-C matched controls. Low HO scores indicate eveningness and high scores indicate greater
morningness. In this study, the best criterion separating DSPD and DSPD-C was a score of 41. MORNINGNESS-EVENINGNESS
0
10
20
30
40
50
60
70
80
90
100
10
20
30
40
50
60
70
80
evening type<-- HORNE - OSTBERG SCORE -->morning type
PERCENTILE
DSPD 1
DSPD 2
DSPD 3
DSPD-C
DSPD 4-5 MORNINGNESS-EVENINGNESS
0
10
20
30
40
50
60
70
80
90
100
10
20
30
40
50
60
70
80
evening type<
HORNE
OSTBERG SCORE
>morning type
PERCENTILE
DSPD 1
DSPD 2
DSPD 3
DSPD-C
DSPD 4-5 MORNINGNESS-EVENINGNESS Distribution of morningness eveningness scores
Figure 1
Distribution of morningness-eveningness scores. The percentiles of Horne-Östberg morningness-eveningness scores
are plotted for 5 groups: a) absolutely certain DSPD 1, b) fairly certain DSPD 2, b) questionable DSPD 3, d) unlikely or very
doubtful DSPD 4–5, and e) DSPD-C matched controls. Low HO scores indicate eveningness and high scores indicate greater
morningness. In this study, the best criterion separating DSPD and DSPD-C was a score of 41. definitely not DSPD) had substantially later sleep acro-
phases than most of the representative sample. Correlations of measures f
Among DSPD participants and the representative sample,
combined, the sleep acrophase was strongly correlated Page 6 of 14
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(page number not for citation purposes) Journal of Circadian Rhythms 2008, 6:6 http://www.jcircadianrhythms.com/content/6/1/6 Distribution of actigraphic sleep acrophases
Figure 2
Distribution of actigraphic sleep acrophases. The percentiles with an actigraphic sleep acrophase at or below each time
are plotted versus clock hours after midnight. The percentiles are plotted for 5 groups: a) absolutely certain DSPD 1, b) fairly
certain DSPD 2, c) questionable DSPD 3, d) unlikely or very doubtful DSPD 4–5, and e) a representative population sample of
San Diego adults. ACTIGRAPHIC SLEEP ACROPHASE
0
10
20
30
40
50
60
70
80
90
100
-2
0
2
4
6
8
10
12
14
HOURS AFTER MIDNIGHT
PERCENTILE
DSPD 1
DSPD 2
DSPD 3
DSPD 4-5
REPRESENTATIVE ACTIGRAPHIC SLEEP ACROPHASE
0
10
20
30
40
50
60
70
80
90
100
-2
0
2
4
6
8
10
12
14
HOURS AFTER MIDNIGHT
PERCENTILE
DSPD 1
DSPD 2
DSPD 3
DSPD 4-5
REPRESENTATIVE ACTIGRAPHIC SLEEP ACROPHASE Distributio
Figure 2 g
p
p
p
g
Distribution of actigraphic sleep acrophases. The percentiles with an actigraphic sleep acrophase at or below each time
are plotted versus clock hours after midnight. The percentiles are plotted for 5 groups: a) absolutely certain DSPD 1, b) fairly
certain DSPD 2, c) questionable DSPD 3, d) unlikely or very doubtful DSPD 4–5, and e) a representative population sample of
San Diego adults. tion of the HO and BALM scales was r = 0.96 (P < 0.001). Over this expanded range, the correlation of the HO scale
with reported bedtimes was r = -0.72 (P < 0.001), with
reported uptimes r = -0.66 (P < 0.001), and with usual
sleep time (since age 21) r = -0.74 (P < 0.001). The BALM
was slightly less strongly correlated with bedtimes, upti-
mes, and usual sleep times. with the wrist activity acrophase (r = 0.83, P < 0.001) and
somewhat less associated with the wrist illumination
acrophase (r = 0.44, P < 0.001). The objective sleep acro-
phases correlated r = 0.80 (P < 0.001) with reported bed-
times for the week before the questionnaire was
completed and r = 0.83 (P < 0.001) with reported upti-
mes. Discriminating DSPD from DSPD-C Only the DSPD group both had wrist activity recordings
and had completed the MES scales. It was surprising that
within the DSPD participant group (definite, probable, or
questionable), correlations of the Horne-Östberg scale
with the observed sleep acrophase and the reported bed-
times and uptimes were not significant, and the HO cor-
relation with the activity acrophase was quite weak (r = -
0.16, P < 0.05). The correlation of the HO scale with the
reported average time of going to sleep since age 21 was
somewhat greater (r = -0.31, P < 0.001). The correlation of
the HO and BALM MES scales within the restricted range
of the DSPD group was only r = 0.68 (P < 0.001). Contrasting the DSPD group (certain, probable, or ques-
tionable) with the DSPD-C, there were one DSPD and one
DSPD-C participant with scores of 41 on the HO MES. There was 1 DSPD-C out of N = 202 with score < 41, and
there were 2 DSPD (1–3) out of N = 183 with scores > 41. In the BALM, there were 5 DSPD with scores of 28 and 5
scoring > 28, whereas there were 5 DSPD-C with BALM of
28 and 5 < 28. Thus, the HO scale separated the groups
somewhat better than the BALM. With the HO scale alone, binary logistic regression cor-
rectly classified 180 of 183 DSPD cases (absolutely cer-
tain, fairly certain, or questionable) and 200 of 201
DSPD-C (certain). Adding the BALM as an independent However, expanding the range analyzed by including the
DSPD-C participants with the DSPD group, the correla- Page 7 of 14
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(page number not for citation purposes) Journal of Circadian Rhythms 2008, 6:6 http://www.jcircadianrhythms.com/content/6/1/6 Distribution of bedtimes
Figure 3
Distribution of bedtimes. The percentiles with a reported bedtime (week prior to completing the questionnaire) at or
below each time are plotted versus clock hours after midnight. The percentiles are plotted for 6 groups: a) absolutely certain
DSPD 1, b) fairly certain DSPD 2, c) questionable DSPD 3, d) unlikely or very doubtful DSPD 4–5, e) the DSPD-C, and f) a rep-
resentative sample of San Diego adults. Discriminating DSPD from DSPD-C BEDTIMES (PRIOR WEEK)
0
10
20
30
40
50
60
70
80
90
100
-6
-4
-2
0
2
4
6
8
10
12
HOURS AFTER MIDNIGHT
PERCENTILE
DSPD 1
DSPD 2
DSPD 3
DSPD-C
DSPD 4-5
REPRESENTATIVE BEDTIMES (PRIOR WEEK)
0
10
20
30
40
50
60
70
80
90
100
-6
-4
-2
0
2
4
6
8
10
12
HOURS AFTER MIDNIGHT
PERCENTILE
DSPD 1
DSPD 2
DSPD 3
DSPD-C
DSPD 4-5
REPRESENTATIVE BEDTIMES (PRIOR WEEK) BEDTIMES (PRIOR WEEK) g
Distribution of bedtimes. The percentiles with a reported bedtime (week prior to completing the questionnaire) at or
below each time are plotted versus clock hours after midnight. The percentiles are plotted for 6 groups: a) absolutely certain
DSPD 1, b) fairly certain DSPD 2, c) questionable DSPD 3, d) unlikely or very doubtful DSPD 4–5, e) the DSPD-C, and f) a rep-
resentative sample of San Diego adults. 7.2 (SD 1.5) hours and DSPD-C reported 7.4 (SD 1.1)
hours (P < 0.05). Likewise, DSPD reported 30 (SD 50)
min of being awake during the night, whereas DSPD-C
reported 18 (SD 38) min (P < 0.01), yet there was no sig-
nificant difference in reported trouble staying asleep. DSPD also reported snoring slightly more commonly and
awakening earlier than intended more commonly (P =
0.005). DSPD reported trouble waking up 5 nights per
week, whereas DSPD-C reported an average of 1 night a
week (P < 0.001). Reported bedtimes of DSPD for the
prior week averaged 2:00 AM whereas those of controls
averaged 22:45 (P < 0.001, see Fig. 3). Likewise, reported
uptimes averaged 10:00 for DSPD and 06:47 for controls
(P < 0.001, see Fig 4). Similarly, DSPD reported trouble
waking up in the morning 4.8 (SD 2.5) times per week,
while DSPD-C reported this problem only 1.0 (SD1.7)
times per week (P < 0.001). DSPD reported very slightly
more snoring (P < 0.005) and awakening gasping for
breath than controls (P = 0.02), but were very slightly less
likely to report being treated for sleep apnea (P = 0.01). There was no significant difference in reported leg twitch-
ing or kicking during sleep. In terms of reported sleep variable correctly classified one additional DSPD, but the
BALM by itself misclassified 14 participants. Reported
bedtime in the prior week by itself misclassified 36 partic-
ipants. Discriminating DSPD from DSPD-C The combination of HO, BALM, reported bedtime,
and age correctly classified all DSPD and DSPD-C. Sleep symptoms Based on sleep and health questionnaires, in one-way
contrasts, DSPD-C tended to report their sleep as "moder-
ately" or "very" satisfactory, whereas DSPD were more
likely to report sleep as "somewhat troubled" or "trou-
bled" (P < 0.001). DSPD-C were more likely to report get-
ting "just enough sleep" (P < 0.001). DSPD were more
likely to report that their sleep was "abnormal" (P <
0.001). DSPD were 10 times as likely to report "insomnia"
and 6 times more likely to have consulted a doctor for
help with a sleep problem (P < 0.0001). DSPD reported
trouble falling asleep 2.9 (SD 2.4) times per week,
whereas controls reported such trouble 0.7 times per week
(SD 1.3) (P < 0.001). DSPD-C reported a mean sleep
latency of 15 min (SD 12) and DSPD 37 min (SD 42) (P
< 0.001). DSPD reported a past-week total sleep time of Page 8 of 14
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(page number not for citation purposes) Journal of Circadian Rhythms 2008, 6:6 http://www.jcircadianrhythms.com/content/6/1/6 Distribution of wake-up times
Figure 4
Distribution of wake-up times. The percentiles with a reported wake-up times (week prior to completing the question-
naire) at or below each time are plotted versus clock hours after midnight. The percentiles are plotted for 6 groups: a) abso-
lutely certain DSPD 1, b) fairly certain DSPD 2, c) questionable DSPD 3, d) unlikely or very doubtful DSPD 4–5, e) the DSPD-
C, and f) a representative sample of San Diego adults. UPTIMES (PRIOR WEEK)
0
10
20
30
40
50
60
70
80
90
100
0
2
4
6
8
10
12
14
16
18
HOURS AFTER MIDNIGHT
PERCENTILE
DSPD 1
DSPD 2
DSPD 3
DSPD-C
DSPD 4-5
REPRESENTATIVE UPTIMES (PRIOR WEEK)
0
10
20
30
40
50
60
70
80
90
100
0
2
4
6
8
10
12
14
16
18
HOURS AFTER MIDNIGHT
PERCENTILE
DSPD 1
DSPD 2
DSPD 3
DSPD-C
DSPD 4-5
REPRESENTATIVE UPTIMES (PRIOR WEEK) UPTIMES (PRIOR WEEK) Distributio
Figure 4
i
i p
g
Distribution of wake-up times. The percentiles with a reported wake-up times (week prior to completing the question-
naire) at or below each time are plotted versus clock hours after midnight. Mood About 44% of DSPD but only 21% of DSPD-C reported
having seen a counsellor for treatment of emotional prob-
lems (P < 0.001), and likewise, 51% of DSPD vs. 23% of
controls reported that there had "been a time when you
were depressed or down most of the day nearly every day
for as long as two weeks," a common screening question
for major depression (P < 0.001.) The mean scores were
6.0 for DSPD and 3.4 for controls on the QIDS-SR depres-
sion scale (P < 0.001). Similarly, 4% of DSPD vs 2% of
controls reported having been hospitalized for depression
(NS). Also, 38% of DSPD vs. 26% of controls reported
"grandparents, parents, brothers, sisters, or children ever
had definite depression" (P < 0.02). Only about 1% of
each group reported having been treated for mania or
bipolar disorder (NS), and only about 10% of each group Sleep symptoms The percentiles are plotted for 6 groups: a) abso-
lutely certain DSPD 1, b) fairly certain DSPD 2, c) questionable DSPD 3, d) unlikely or very doubtful DSPD 4–5, e) the DSPD-
C, and f) a representative sample of San Diego adults. reported a family history of bipolar disorder (NS). Simi-
larly, the mean score on the Mood Disorder Question-
naire was 3.6 for DSPD and 3.0 for controls (NS). Using
this Mood Disorder Questionnaire, 9 DSPD and 6 con-
trols met screening criteria for a past history of mania
(NS). The SPAQ global seasonality score was not signifi-
cantly associated with DSPD (mean 5.47 SD 5.03 and
mean 4.70 SD 3.99 for DSPD and DSPD-C respectively, P
> 0.10). No controls but 8% of DSPD reported having
been treated for a circadian rhythm disorder or with light
treatment (P < 0.001). DSPD reported having taken hyp-
notics 2.7% of their lives, whereas controls reported only
0.2% on average (P < 0.001). Reporting no lifetime use of
hypnotics were 81% of DSPD and 96% of controls. Like-
wise, DSPD reported taking stimulants (amphetamines,
methylphenidate, or modafinil) 2% of their lives vs. 0.3%
for controls (P < 0.02). Similarly, DSPD reported taking
antidepressants 5.9% (SD 13.7%) of their lives vs. 1.5%
(SD 6.4%) for controls (P < 0.001). quality, DSPD had a mean PSQI of 7.3 (SD 3.8) indicating
poor sleep quality, whereas DSPD-C had a mean PSQI of
3.8 (SD 2.8) (P < 0.001, Mann-Whitney U test). Moreover,
of the DSPD-C, 88% had a PSQI lower than the median of
7 for DSPD and 17% of DSPD had a PSQI equal or lower
to the DSPD-C control median of 3. Eye color and ancestry Participants had self-described eye color, which might
influence the synchronizing efficiency of light exposures. Among the DSPD and DSPD-C combined, 29% were
blue-eyed, 9% grey or green eyed, 15% hazel eyed, and
47% brown-eyed. The DSPD and DSPD-C groups did not
differ significantly by eye color, either considering the dis-
tribution of 4 color categories, or considering them by
order of iris lightness. The groups were also generally sim-
ilar in aspects of reported ancestry. Developmental history In childhood, 50% of DSPD reported that they went to
sleep "much later" than other children their age and 32% Page 9 of 14
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(page number not for citation purposes) http://www.jcircadianrhythms.com/content/6/1/6 http://www.jcircadianrhythms.com/content/6/1/6 Journal of Circadian Rhythms 2008, 6:6 20.8% late, 12.3% very late, and 8.4% extremely late,
whereas DSPD-C reported 22.3%, 6.3%, and 1.1% respec-
tively (P = 0.005, Mann-Whitney U). Further, DSPD
reported mothers were 21.8% late, 13.3% very late, and
12.7% extremely late, whereas DSPD-C reported 23.8%,
4.9%, and 2.2% respectively (P < 0.001, Mann-Whitney
U). The lateness of fathers and mothers did not differ sig-
nificantly overall. However, DSPD female probands
reported that fathers were later than male probands
reported them (P = 0.02, Mann-Whitney U), but the
mothers were described with similar lateness by male and
female DSPD. DSPD reported that they had at least one
brother or sister who was very late (22.7%) or extremely
late (13.3%), whereas DSPD-C reported 14.7% and 7.9%
respectively (P = 0.005, Mann-Whitney U). At least one
child was reported very late or extremely late by 34.4% of
DSPD but only 20.3% of controls (P < 0.001, Mann-Whit-
ney U). reported "somewhat later," whereas the percentages for
controls were 3% and 19% (P < 0.001, Mann-Whitney U). Similarly, in adulthood, 72% of DSPD reported they went
to sleep "much later" than other people their age and 25%
"somewhat later," whereas the corresponding percentages
for controls were 4% and 20% (P < 0.001, Mann-Whitney
U). Height and BMI DSPD and DSPD-C did not differ significantly in height. For BMI, both gender (P = 0.025) and case/control (DSPD
vs DSPD-C, P = 0.002) were significant factors in
ANCOVA with age as a significant covariate (P = 0.001),
but there was no significant gender/diagnosis interaction. The BMI of DSPD averaged 33.0, and the BMI of DSPD-C
averaged 30.5. General health In the past month, antacids were used by 15% of DSPD
and only 5% of DSPD-C (Chi-Square = 11.31, P = 0.001). Antidepressants were used by 23.8% of DSPD and 10.4%
of DSPD-C (Chi-Square = 12.39, P < 0.001). Hypnotics
were used by 4.9% of DSPD and 0.5% of DSPD-C (Chi-
Square = 7.33, P = 0.007). Melatonin was used by 7% of
DSPD and 1% of DSPD-C (Chi-Square-9.45, P = 0.002). Over-the-counter hypnotics were used by 3.2% of DSPD
and 0.5% of DSPD-C (Chi-Square = 4.1, P < 0.05). Use of
anticonvulsants, antihypertensives, bronchodilators, cal-
cium channel blockers, cardiac medications, corticoster-
oids, cytotoxic drugs, diuretics, hormones (largely birth
control), insulin, laxatives, major tranquilizers, minor
tranquilizers, narcotics, and miscellaneous other medica-
tions did not differ significantly between DSPD and
DSPD-C. However, DSPD took an average of 0.84 of this
latter group of medications, whereas DSPD-C took an
average of 0.64 (t = 2.05, two-tailed, P < 0.05). There was almost no overlap of HO MES scores between
those classified as definite probable or possible DSPD
cases and those classified as DSPD-C. The two groups were
distinguished 98% by the HO criterion of 41. Likewise,
there was little overlap in BALM scores, but there was
somewhat more overlap in self-reported bedtimes and
uptimes. The superiority of the HO scale over bedtimes
for distinguished DSPD may be somewhat tautological,
resulting from the way in which recruitment and case-clas-
sification were achieved. Defining a DSPD case group pri-
marily by the HO scale, we could examine how bedtimes
and other characteristics compared in DSPD and DSPD-C. Discussion These data characterize a case series of DSPD volunteers
and compare them to normal controls (both DSPD-C and
a previously-collected representative sample). The DSPD-
C were well-matched by age, gender, and ancestry. The
sleep timing of DSPD-C was demonstrably normal in the
sense that their reported bedtimes and uptimes were
almost superimposable on the distributions of a repre-
sentative sample of San Diego adults. DSPD cases went to
bed and arose about 3 hours later than DSPD-C on aver-
age, but the DSPD group had a considerably wider distri-
bution of sleep times, reflecting a proportion who retired
and arose very late indeed. Subjectively reported bedtimes
and uptimes and MES scores for DSPD-C and activity
acrophases and the acrophases of sleep inferred from wrist
activity for the representative sample were reasonably
consistent in indicating the degree of delay experienced by
the DSPD cases. It must be recognized, however, that the
Horne-Östberg (HO) MES scores, bedtimes, and sleep
acrophases for cases and controls in San Diego might
occupy somewhat different numerical ranges from those
in other communities, where average bedtimes might be
later. Page 10 of 14
(page number not for citation purposes) Familiality It appears that
those suffering DSPD who must force their sleep schedule
into a biologically adverse time will habitually experience
lying in bed having trouble falling asleep. As a conse-
quence, they may develop fear of the bed and anxiety
about retiring. Even when able to retire at a biologically
appropriate time, this negative conditioning may lead to
psychophysiologic insomnia and sleep disturbance. Although these results supported many previous studies
which have observed that older adults tend to go to bed
and arise somewhat earlier than young adults, the effect
size was very small. It did not appear that adjustment for
age would be important in discriminating DSPD from
normal people. Page 11 of 14
(page number not for citation purposes)
Self reported comparison of bedtimes with other people
Figure 5
Self reported comparison of bedtimes with other
people. Self-reported bedtimes (in clock time after mid-
night) are reported for participants who stated that on aver-
age (after age 21) they went to sleep much earlier, somewhat
earlier, about the same time, somewhat later, or much later
that other people their age. Thick horizontal lines are medi-
ans. Red boxes show the 2nd and 3rd quartiles. Bars show the
upper and lower 5%. MUCH EARLIER
SOMEWHAT EARLIER
SAME
SOMEWHAT LATER
MUCH LATER
-4
-2
0
2
4
6
8
BEDTIME ( hours after midnight )
median, quartiles,
90% range MUCH EARLIER
SOMEWHAT EARLIER
SAME
SOMEWHAT LATER
MUCH LATER
-4
-2
0
2
4
6
8
BEDTIME ( hours after midnight )
median, quartiles,
90% range Over the broad range of DSPD cases and DSPD-C, the HO
and BALM scales were well correlated with bedtimes, upti-
mes, and sleep acrophases, but within the group of DSPD
cases, these correlations were poor. This suggests that
though the HO and BALM scales distinguished well
between cases and controls, MES scales may be an unsat-
isfactory indicator of the severity of DSPD within the
DSPD group. Perhaps bedtimes and especially uptimes
would be the best indicator of the extent to which DSPD
may be handicapping. Recently, there has been interest in MES scales which ask
participants to compare their morning-evening prefer-
ences to those of "most people" [59-61]. Our question
asking people to compare their bedtimes with most peo-
ple their age produced responses with considerable over-
lap in bedtimes (Fig. 5). Familiality When asked about biological relatives with a tendency to
go to bed late and get up late, DSPD reported that at least
one grandparent was characteristically late 16.5%, very
late 10.2%, or extremely late 3.9%, whereas DSPD-C
reported 7.5%, 5.5%, and 0.7% respectively (P = 0.001,
Mann-Whitney U). Similarly, DSPD reported fathers were Page 10 of 14
(page number not for citation purposes) Page 10 of 14
(page number not for citation purposes) http://www.jcircadianrhythms.com/content/6/1/6 http://www.jcircadianrhythms.com/content/6/1/6 Journal of Circadian Rhythms 2008, 6:6 That the HO scale distinguished somewhat better than the
BALM may reflect the investigators' classification meth-
ods, but since the HO was slightly better correlated with
bedtime, uptime, and actigraphic measures than the
BALM, it did appear that in these samples, the HO distin-
guished cases somewhat more effectively. Ultimately,
comparison with genetic findings may best reflect the rel-
ative validity of these two MES scales and determine
whether they serve classification of circadian disorders
better than indicators of bedtime or uptime. It is interest-
ing that in a case series based on clinical contacts, greater
overlap was observed for bedtimes and physiologic circa-
dian indicators between ASPD and normal groups and
between normals and DSPD [58]. Perhaps genetic find-
ings will also reflect whether MES scales or clinical mis-
alignment criteria better identify heritable circadian
abnormalities. In addition, genetic data may clarify
whether the 3% of the population reporting both trouble
falling asleep and trouble waking up suffer a milder
masked form of DSPD, with a misalignment of sleep time
and sleep propensity, even when their bedtimes may be
within the normal range [5]. normal bedtimes and in desired bedtimes. It remains to
be established whether misalignment between objective
sleep times and desired sleep times, perhaps indicative of
genetic polymorphisms, may best be recognized by refer-
ence to a person's individual desired sleep times, to the
perceived bedtimes of their peers, or to the bedtimes of
the overall surrounding community. There was considerable overlap between case and control
groups in measures of distress about sleep. Thus, 17% of
DSPD had a PSQI score as low as the control median,
whereas 12% of DSPD-C had a PSQI at least equal to the
DSPD median. Some DSPD cases were able to arrange
their sleep schedules well enough to avoid distressed
sleep, whereas others suffered persistent trouble falling
asleep and trouble arising in the morning. Conclusion Finally, these studies tend to confirm that a tendency to
being a nightowl is partly familial and presumably caused
in part by genetic susceptibilities. Family members of
DSPD cases were far more likely to be described as very
late in their habits than family members of DSPD-C, and
family members also had more depression. We do not
think the preponderance of women in our case series indi-
cates that DSPD is more common among women: indeed,
a variety of evidence suggests the contrary. Rather, we
would attribute the preponderance of women to their
greater willingness to participate in such the studies, since
a similar preponderance was found in the control groups. One finding of possible interest was that DSPD female
probands reported that fathers had later bedtimes and
uptimes than male probands reported. This might be
explained if DSPD is transmitted in part through an X
chromosome (and therefore, not from fathers to sons),
possibly as a recessive trait, but the evidence was not
robust enough to be persuasive. None of the initially-rec-
ognized circadian system genes are found on the X Chro-
mosome, but FMR1 is one X Chromosome gene which
might be a candidate for polymorphisms related to DSPD
[70]. DSPD seemed to experience less illumination per 24
hours than had the representative sample, but the effect
size was modest. Because the photometric measurement
devices used in the two studies were quite different in
design and performance, we do not know if this difference
was reliable. It would be logical that since DSPD arise late
and are up later at night than the normal population, they
would be out of bed less in bright daylight. Less daylight
exposure, in turn, could promote susceptibility to DSPD,
since daylight usually functions to advance circadian
rhythms, particularly soon after a person awakens. Increasing a patient's exposure to bright light soon after
awakening is an accepted treatment approach for DSPD
[62]. Depression comorbid with DSPD was noted in the pio-
neering description of DSPD [63] and has been described
by several observers [64-69]. Our data confirm that DSPD
cases had higher current depression ratings and a greater
life-time history of unipolar depression, depression treat-
ment, and anti-depressant use than DSPD-C. DSPD cases
also had a greater family history of depression, supporting
the possibility that DSPD and depression share genetic
susceptibility factors. Competing interests p
g
The authors declare that they have no competing interests. Familiality This would lead us to be sceptical
that subjective comparisons to other people would be an
optimal approach to discerning morningness-eveningness
traits. People who perceived their own bedtimes as earlier
or later than most people did not always retire corre-
spondingly early or late in reference to our representative
sample. Even within a single complex society, differences
in peer groups related to income, occupation, and social
background may produce marked differences in perceived Self repor
Figure 5 Self reported comparison of bedtimes with other people
Figure 5
Self reported comparison of bedtimes with other
people. Self-reported bedtimes (in clock time after mid-
night) are reported for participants who stated that on aver-
age (after age 21) they went to sleep much earlier, somewhat
earlier, about the same time, somewhat later, or much later
that other people their age. Thick horizontal lines are medi-
ans. Red boxes show the 2nd and 3rd quartiles. Bars show the
upper and lower 5%. Self reported comparison of bedtimes with other people
Figure 5
Self reported comparison of bedtimes with other
people. Self-reported bedtimes (in clock time after mid-
night) are reported for participants who stated that on aver-
age (after age 21) they went to sleep much earlier, somewhat
earlier, about the same time, somewhat later, or much later
that other people their age. Thick horizontal lines are medi-
ans. Red boxes show the 2nd and 3rd quartiles. Bars show the
upper and lower 5%. Page 11 of 14
(page number not for citation purposes) http://www.jcircadianrhythms.com/content/6/1/6 http://www.jcircadianrhythms.com/content/6/1/6 http://www.jcircadianrhythms.com/content/6/1/6 Journal of Circadian Rhythms 2008, 6:6 As a group, we found that the DSPD participants had very
irregular sleep patterns. They commonly had prolonged
midsleep awakenings. Many appeared to nap frequently
at all times of day. By comparing the actigraphic records
with the sleep logs, we gained the impression that some
DSPD participants were drowsing while watching televi-
sion late at night, a considerable time before they went to
bed and turned out the lights. Unfortunately, some acti-
graphic records were so interspersed with immobile inter-
vals that we regarded our minute-by-minute actigraphic
scoring of sleep as quite unreliable, though only in rare
cases did we lack confidence in the overall estimate of the
circadian pattern. Familiality However, because the representative sample were
older on average, because different actigraphs and scoring
algorithms were used in the two samples, and because of
numerous other details of the sleep measurements, we
would not consider this distinction in actigraphic sleep
times between DSPD cases and the representative sample
to be reliable. Note that DSPD-C subjectively reported
slightly more sleep than DSPD in the week prior to com-
pleting their questionnaires. – or perhaps because of the napping out of bed – it
appeared that DSPD cases got about an hour more acti-
graphic sleep each 24 hours than our representative sam-
ple. However, because the representative sample were
older on average, because different actigraphs and scoring
algorithms were used in the two samples, and because of
numerous other details of the sleep measurements, we
would not consider this distinction in actigraphic sleep
times between DSPD cases and the representative sample
to be reliable. Note that DSPD-C subjectively reported
slightly more sleep than DSPD in the week prior to com-
pleting their questionnaires. Conclusion Nevertheless, there may be behavio-
rally-mediated mechanisms for comorbidity between
DSPD and depression. For example, the lateness of DSPD
cases and their unusual hours may lead to social oppro-
brium and rejection, which might be depressing. Moreo-
ver, late awakenings and the rush to school or work might In future reports, we hope to extensively examine associa-
tions of DSPD cases with polymorphisms in several of the
circadian system genes. Familiality Despite the degree of sleep disturbance
– or perhaps because of the napping out of bed – it
appeared that DSPD cases got about an hour more acti-
graphic sleep each 24 hours than our representative sam-
ple. However, because the representative sample were
older on average, because different actigraphs and scoring
algorithms were used in the two samples, and because of
numerous other details of the sleep measurements, we
would not consider this distinction in actigraphic sleep
times between DSPD cases and the representative sample
to be reliable. Note that DSPD-C subjectively reported
slightly more sleep than DSPD in the week prior to com-
pleting their questionnaires. As a group, we found that the DSPD participants had very
irregular sleep patterns. They commonly had prolonged
midsleep awakenings. Many appeared to nap frequently
at all times of day. By comparing the actigraphic records
with the sleep logs, we gained the impression that some
DSPD participants were drowsing while watching televi-
sion late at night, a considerable time before they went to
bed and turned out the lights. Unfortunately, some acti-
graphic records were so interspersed with immobile inter-
vals that we regarded our minute-by-minute actigraphic
scoring of sleep as quite unreliable, though only in rare
cases did we lack confidence in the overall estimate of the
circadian pattern. Despite the degree of sleep disturbance lead to reduced morning daylight exposure, which may
predispose to depression. Conversely, the withdrawal and
fatigue associated with depression, by reducing outdoor
activities and daylight exposure, could lead to circadian
phase delay. Perhaps genetic studies will clarify these asso-
ciations. We attempted to carefully identify any associa-
tion of bipolar disorder (history of mania) with DSPD,
but we found no evidence of significant association with
bipolar disorder. Insofar as medications indicate health concerns, DSPD
took more medications for sleep and mood, more medi-
cations in general, and more antacids. Perhaps the circa-
dian misalignment produced by DSPD tends to cause
epigastric discomfort, as is often reported by shift workers,
which might lead to increased use of antacids and
increased snacking. On the other hand, DSPD may have
averaged relatively high BMI because of lack of exercise,
resulting from their schedules. – or perhaps because of the napping out of bed – it
appeared that DSPD cases got about an hour more acti-
graphic sleep each 24 hours than our representative sam-
ple. Authors' contributions DFK helped design the study, supervised the recruitment
and clinical classification of participants, and participated Page 12 of 14
(page number not for citation purposes) Page 12 of 14
(page number not for citation purposes) http://www.jcircadianrhythms.com/content/6/1/6 Journal of Circadian Rhythms 2008, 6:6 http://www.jcircadianrhythms.com/content/6/1/6 19. Heath AC, Kendler KS, Eaves LJ, Martin NG: Evidence for genetic
influences on sleep disturbance and sleep pattern in twins. Sleep 1990, 13:318-335. in data analyses and writing. KMR helped plan the study,
recruited most of the cases, and participated in data entry,
analyses, and writing. SAI helped design the study,
assisted in recruiting, and participated in manuscript
drafting. CMN helped design the study and participated in
manuscript preparation. WK helped design the study,
supervised DNA assays, and participated in manuscript
review. JRK helped design the study, supervised DNA
preparation, and participated in manuscript review. All
authors read and approved the final manuscript. p
20. Klei L, Reitz P, Miller M, Wood J, Maendel S, Gross D, Waldner T,
Eaton J, Monk TH, Nimgaonkar VL: Heritability of morningness-
eveningness and self-report sleep measures in a family-based
sample of 521 hutterites. Chronobiol Int 2005, 22:1041-1054. p
21. Gottlieb DJ, O'Connor GT, Wilk JB: Genome-wide association of
sleep and circadian phenotypes. BMC Med Genet 2007, 8 Suppl
1:S9. 22. Toh KL, Jones CR, He Y, Eide EJ, Hinz WA, Virshup DM, Ptacek LJ,
Fu YH: An hPer2 phosphorylation site mutation in familial
advanced sleep-phase syndrome. Science 2001, 291:1040-1043. p p
y
23. Xu Y, Padiath QS, Shapiro RE, Jones CR, Wu SC, Saigoh N, Saigoh K,
Ptacek LJ, Fu YH: Functional consequences of CKId mutation
causing familial advanced sleep phase syndrome. Nature 2005,
434:640-644. Acknowledgements This research was supported by the National Institutes of Health's NHLBI
HL071123 and by National Center on Minority Health and Health Dispar-
ities EXPORT grant P60MD00220, and by the Sam and Rose Stein Institute
for Research on Aging. Martin Schalling, M.D., Ph.D. contributed to the
design of this study. Mr. C. Im and Ms D. Vo recruited many of the cases
and control participants. 24. Xu Y, Toh KL, Jones CR, Shin JY, Fu YH, Ptacek LJ: Modeling of a
Human Circadian Mutation Yields Insights into Clock Regu-
lation by PER2. Cell 2007, 128:59-70. y
25. Ebisawa T, Uchiyama M, Kajimura N, Mishima K, Kamei Y, Katoh M,
Watanabe T, Sekimoto M, Shibui K, Kim K, Kudo Y, Ozeki Y, Sugishita
M, Toyoshima R, Inoue Y, Yamada N, Nagase T, Ozaki N, Ohara O,
Ishida N, Okawa M, Takahashi K, Yamauchi T: Association of struc-
tural polymorphisms in the human period3 gene with
delayed sleep phase syndrome. EMBO reports 2001, 2:342-346. References g
y
Mishima K, Nomura M, Ozaki N, Okawa M, Takahashi K, Yamauchi
T: Mutation screening of the human Clock gene in circadian
rhythm sleep disorders. Psychiatry Res 2002, 109:121-128. g
p
7. Chung KF, Cheung MM: Sleep-wake patterns and sleep distur-
bance among Hong Kong Chinese adolescents. Sleep 2008,
31:185-194. y
p
y
y
30. Hohjoh H, Takasu M, Shishikura K, Takahashi Y, Honda Y, Tokunaga
K: Significant association of the arylalkylamine N-acetyl-
transferase (AA-NAT) gene with delayed sleep phase syn-
drome. Neurogenetics 2003, 4:151-153. 8. Ohayon MM, Roberts RE, Zulley J, Smirne S, Priest RG: Prevalence
and patterns of problematic sleep among older adolescents. J Am Acad Child Adolesc Psychiatry 2000, 39:1549-1556. J
y
y
9. Pilcher JJ, Ott ES: The relationships between sleep and meas-
ures of health and well-being in college students: a repeated
measures approach. Behav Med 1998, 23:170-178. 31. Carpen JD, Archer SN, Skene DJ, Smits M, Schantz M: A single-
nucleotide polymorphism in the 5'-untranslated region of
the hPER2 gene is associated with diurnal preference. J Sleep
Res 2005, 14:293-297. pp
10. Pilcher JJ, Ginter DR, Sadowsky B: Sleep quality versus sleep
quantity: relationships between sleep and measures of
health, well-being and sleepiness in colleges students. J Psy-
chosom Res 1997, 42:583-596. 32. Carpen JD, Von SM, Smits M, Skene DJ, Archer SN: A silent poly-
morphism in the PER1 gene associates with extreme diurnal
preference in humans. J Hum Genet 2006. 11. Adan A, Natale V: Gender differences in morningness-evening-
ness preference. Chronobiol Intl 2002, 19:709-720. 33. Ebisawa T: Circadian rhythms in the CNS and peripheral clock
disorders: human sleep disorders and clock genes. J Pharmacol
Sci 2007, 103:150-154. p
12. Ancoli-Israel S, Schnierow B, Kelsoe J, Fink R: A pedigree of one
family with delayed sleep phase syndrome. Chronobiol Intl 2001,
18:831-840. 34. Hohjoh H, Takahashi Y, Hatta Y, Tanaka H, Akaza T, Tokunaga K,
Honda Y, Juji T: Possible association of human leucocyte anti-
gen DR1 with delayed sleep phase syndrome. Psychiat Clin Neu-
rosciences 1999, 53:527-529. 13. de Castro JM: The influence of heredity on self-reported sleep
patterns in free-living humans. Physiol Behav 2002, 76:479-486. 13. de Castro JM: The influence of heredity on self-reported sleep
patterns in free-living humans. Physiol Behav 2002, 76:479-486. 14. Vink JM, Groot AS, Kerkhof GA, Boomsma DI: Genetic analysis of
morningness and eveningness. Chronobiol Intl 2001, 18:809-822. 15. Page 13 of 14
(page number not for citation purposes) References 26. Archer S, Robilliard DL, Skene DJ, Smits M, Williams A, Arendt J, von
Schantz M: A length polymorphism in the circadian clock gene
per3 is linked to delayed sleep phase syndrome and extreme
diurnal preference. Sleep 2003, 26:413-415. 1. Horne JA, Östberg O: A self-assessment questionnaire to
determine morningness-eveningness in human circadian
rhythms. Int J Chronobiol 1976, 4:97-110. y
J
2. Winkelman J, Kotagal S, Olson E, Scammel T, Schenck C, Spielman A:
The International Classification of Sleep Disorders, Second Edition, Pocket
Version: Diagnostic and Coding Manual Westchester, IL, American
Academy of Sleep Disorders; 2006. 27. p
p
27. Pereira DS, Tufik S, Louzada FM, Benedito-Silva AA, Lopez AR, Lemos
NA, Korczak AL, Almeida VD, Pedrazzoli M: Association of the
length polymorphism in the human per3 gene with the
delayed sleep-phase syndrome: does latitude have an influ-
ence upon it? Sleep 2005, 28:29-32. y
p
3. Schrader H, Bovim G, Sand T: The prevalence of delayed and
advanced sleep phase syndrome. J Sleep Res 1993, 2:51-55. 28. Jones KH, Ellis J, Von SM, Skene DJ, Dijk DJ, Archer SN: Age-related
change in the association between a polymorphism in the
PER3 gene and preferred timing of sleep and waking activi-
ties. J Sleep Res 2007, 16:12-16. p p
y
J
p
,
4. Yazaki M, Shirakawa S, Okawa M, Takahashi K: Demography of
sleep disturbances associated with circadian rhythm disor-
ders in Japan. Psychiatry and Clinical Neurosciences 1999, 53:267-268. J p
y
y
5. Ando K, Kripke DF, Ancoli-Israel S: Delayed and advanced sleep
phase symptoms. Isr J Psychiatry Relat Sci 2002, 39:11-18. 6
f
C
S
i
f 29. 29. Iwase T, Kajimura N, Uchiyama M, Ebisawa T, Yoshimura K, Kamei Y,
Shibui K, Kim K, Kudo Y, Katoh M, Watanabe T, Nakajima T, Ozeki
Y, Sugishita M, Hori T, Ikeda M, Toyoshima R, Inoue Y, Yamada N,
Mishima K, Nomura M, Ozaki N, Okawa M, Takahashi K, Yamauchi
T: Mutation screening of the human Clock gene in circadian
rhythm sleep disorders. Psychiatry Res 2002, 109:121-128. 5. Ando K, Kripke DF, Ancoli Israel S: Delayed and advanced sleep
phase symptoms. Isr J Psychiatry Relat Sci 2002, 39:11-18. 6. Wolfson AR, Carskadon MA: Sleep schedules and daytime func-
tioning in adolescents. Child Development 1998, 69:875-887. p
y
p
J
y
y
6. Wolfson AR, Carskadon MA: Sleep schedules and daytime func-
tioning in adolescents. Child Development 1998, 69:875-887. References Page 13 of 14
(page number not for citation purposes) http://www.jcircadianrhythms.com/content/6/1/6 Journal of Circadian Rhythms 2008, 6:6 sense variation in human casein kinase I epsilon gene that
induces functional alteration and shows an inverse associa-
tion with circadian rhythm sleep disorders. Neuropharmacology
2004, 29:1901-1909. 56. Jean-Louis G, Kripke DF, Cole RJ, Assmus JD, Langer RD: Sleep
detection with an accelerometer actigraph: comparisons
with polysomnography. Physiol Behav 2001, 72:21-28. sense variation in human casein kinase I epsilon gene that
induces functional alteration and shows an inverse associa-
tion with circadian rhythm sleep disorders. Neuropharmacology
2004, 29:1901-1909. sense variation in human casein kinase I epsilon gene that
induces functional alteration and shows an inverse associa-
tion with circadian rhythm sleep disorders. Neuropharmacology
2004, 29:1901-1909. 57. Jean-Louis G, Kripke DF, Ancoli-Israel S, Klauber MR, Sepulveda RS,
Mowen MA, Assmus JD, Langer RD: Circadian sleep, illumina-
tion, and activity patterns in women: influences of aging and
time reference. Physiology & Behavior 2000, 68:347-352. 38. Katzenberg D, Young T, Finn L, Lin L, King DP, Takahashi JS, Mignot
E: A clock polymorphism associated with human diurnal
preference. Sleep 1998, 21:569-576. y
gy
58. Chang AM: Phenotypic Characterization and Genetic Analysis
of Human Circadian Rhythm Sleep Disorders. In Doctoral The-
sis Northewestern University; 2003:1-244. 39. Mishima K, Tozawa T, Satoh K, Saitoh H, Mishima Y: The 3111T/C
polymorphism of hClock is associated with evening prefer-
ence and delayed sleep timing in a Japanese population sam-
ple. American Journal of Medical Genetics Part B (Neuropsychiatric
Genetics) 2005, 133B:101-104. y
59. Di Milia L: A psychometric evaluation and validation of the
preferences scale. Chronobiol Int 2005, 22:679-693. p
60. Diaz-Morales JF, Sanchez-Lopez MP: Composite scales of morn-
ingness and preferences: preliminary validity data in Peru-
vian undergraduates. Ergonomics 2005, 48:354-363. 40. Robilliard DL, Archer SN, Arendt J, Lockley SW, Hack LM, English J,
Leger D, Smits MG, Williams A, Skene DJ, von Schantz M: The 3111
Clock gene polymorphism is not associated with sleep and
circadian rhythmicity in phenotypically characterized
human subjects. J Sleep Res 2002, 11:305-312. g
g
61. Smith CS, Folkard S, Schmieder RA, Parra LF, Spelten E, Almiral H,
Sen RN, Sahu S, Perez LM, Tisak J: Investigation of morning-
evening orientation in six countries using the preference
scale. Personality and Individual Differences 2002, 32:949-968. j
J
p
41. References Pedrazzoli M, Louzada FM, Pereira DS, edito-Silva AA, Lopez AR,
Martynhak BJ, Korczak AL, Koike B V, Barbosa AA, D'Almeida V,
Tufik S: Clock polymorphisms and circadian rhythms pheno-
types in a sample of the Brazilian population. Chronobiol Int
2007, 24:1-8. y
ff
62. Lack LC, Wright HR: Clinical management of delayed sleep
phase disorder. Behav Sleep Med 2007, 5:57-76. 63. Weitzman ED, Czeisler CA, Coleman RM, Spielman AJ, Zimmerman
JC, Dement WC: Delayed sleep phase syndrome. Arch Gen Psy-
chiatry 1981, 38:737-746. 42. Kripke DF, Ancoli-Israel S, Klauber MR, Wingard DL, Mason WJ, Mul-
laney DJ: Prevalence of sleep disordered breathing in ages 40-
64 years: A population-based survey. Sleep 1997, 20:65-76. y
64. Hirata FC, Lima MC, de B V, Nobrega PR, Wenceslau GP, de Bruin
PF: Depression in medical school: the influence of morning-
ness-eveningness. Chronobiol Int 2007, 24:939-946. y
p p
y
p
43. Jean-Louis G, Kripke DF, Ancoli-Israel S, Klauber MR, Sepulveda RS:
Sleep duration, illumination, and activity patterns in a popu-
lation sample: effects of gender and ethnicity. Biol Psychiatry
2000, 47:921-927. 65. Shirayama M, Shirayama Y, Iida H, Kato M, Kajimura N, Watanabe T,
Sekimoto M, Shirakawa S, Okawa M, Takahashi K: The psychologi-
cal aspects of patients with delayed sleep phase syndrome
(DSPS). Sleep Med 2003, 4:427-433. 44. Paine SJ, Gander PH, Travier N: The epidemiology of morning-
ness/eveningness: influence of age, gender, ethnicity, and
socioeconomic factors in adults (30-49 years). J Biol Rhythms
2006, 21:68-76. (
)
p
66. Shur-Fen Gau S, Soong WT, Merikangas KR: Correlates of sleep-
wake patterns among children and young adolescents in Tai-
wan. Sleep 2004, 27:512-519. 45. American Psychiatric Association: Diagnostic and Statistical Manual of
Mental Disorders: DSM-IV Fourth Edition, Text Revision edition. Washington, D.C., American Psychiatric Association; 2000. p
67. Taillard J, Philip P, Chastang JF, Diefenbach K, Bioulac B: Is self-
reported morbidity related to the circadian clock? J Biol
Rhythms 2001, 16:183-190. 46. Pati AK, Chandrawanshi A: Distribution of morningness-
eveningness and variation in behavior of a human population
of Indian sub-continent. Chronobiol Intl 2001, 18:1180-1181. y
68. Ong JC, Huang JS, Kuo TF, Manber R: Characteristics of insomni-
acs with self-reported morning and evening chronotypes. J
Clin Sleep Med 2007, 3:289-294. g J ,
g J ,
,
acs with self-reported morning and evening chronotypes. J
Clin Sleep Med 2007, 3:289-294. 47. References Taillard J, Philip P, Chastang JF, Bioulac B: Validation of Horne and
Ostberg morningness-eveningness questionnaire in a mid-
dle-aged population of French workers. J Biol Rhythms 2004,
19:76-86. p
69. Thorpy MJ, Korman E, Spielman AJ, Glovinsky PB: Delayed sleep
phase syndrome in adolescents. J Adolesc Health Care 1988,
9:22-27. 70. Zhang J, Fang Z, Kaasik K, Lee CC, Oostra BA, Nelson DL: FMR1
gene family regulates circadian clock in mammals. ASGH
2006. 48. Terman M, Rifkin JB, Jacobs J, White TM: Morningness-Evening-
ness Qeustionnaire (revised). New York, New York, New York
State Psychiatric Institute; 2001. y
49. Brown FM: Psychometric equivalence of an improved Basic
Language Morningness (BALM) Scale using industrial popu-
lation within comparisons. Ergonomics 1993, 36(1-3):191-197. p
g
(
)
50. Smith CS, Reilly C, Midkiff K: Evaluation of three circadian
rhythm questionnaires with suggestions for an improved
measure of morningness. J Appl Psychol 1989, 74:728-738. g
J
pp
y
51. Buysse DJ, Reynolds CF 3rd, Monk TH, Berman SR, Kupfer DJ: The
Pittsburgh Sleep Quality Index: a new instrument for psychi-
atric practice and research. Psychiatry Res 1989, 28(2):193-213. 52
H
hf ld RMA H l
C C l b
JR W
M R
d M D p
y
y
( )
52. Hirschfeld RMA, Holzer C, Calabrese JR, Weissman M, Reed M, Dav-
ies M, Frye MA, Keck P, McElroy S, Lewis L, Tierce J, Wagner KD,
Hazard E: Validity of the Mood Disorder Questionnaire: A
general population study. Am J Psychiatry 2003, 160:178-180. g
p p
J
y
y
53. Rush AJ, Trivedi MH, Ibrahim HM, Carmody TJ, Arnow B, Klein DN,
Markowitz JC, Ninan PT, Kornstein S, Manber R, Thase ME, Kocsis
JH, Keller MB: The 16-item Quick Inventory of Depressive
Symptomatology (QIDS), clinical rating (QIDS-C), and self-
report (QIDS-SR): a psychometric evaluation in patients
with chronic major depression. Biol Psychiatry 2003, 54:573-583. References Drennan MD, Selby J, Kripke DF, Kelsoe J, Gillin JC: Morningness/
eveningness is heritable
Soc Neurosci Abstr 1992 18:196 13. de Castro JM: The influence of heredity on self-reported sleep
patterns in free-living humans. Physiol Behav 2002, 76:479-486. 14. Vink JM, Groot AS, Kerkhof GA, Boomsma DI: Genetic analysis of
morningness and eveningness. Chronobiol Intl 2001, 18:809-822. patterns in free-living humans. Physiol Behav 2002, 76:479-486. 14. Vink JM, Groot AS, Kerkhof GA, Boomsma DI: Genetic analysis of
morningness and eveningness. Chronobiol Intl 2001, 18:809-822. 15. Drennan MD, Selby J, Kripke DF, Kelsoe J, Gillin JC: Morningness/
eveningness is heritable. Soc Neurosci Abstr 1992, 18:196. p
g
y
14. Vink JM, Groot AS, Kerkhof GA, Boomsma DI: Genetic analysis of
morningness and eveningness. Chronobiol Intl 2001, 18:809-822. 35. Johansson C, Willeit M, Smedh C, Ekholm J, Paunio T, Lichtermann D,
Praschak-Rieder N, Neumeister A, Nilsson LG, Kasper S, Peltonen L,
Adolfsson R, Schalling M, Partonen T: Circadian clock related pol-
ymorphisms in seasonal affective disorder and their rele-
vance to diurnal preference. Neuropsychopharmacol 2003,
28:734-739. g
g
15. Drennan MD, Selby J, Kripke DF, Kelsoe J, Gillin JC: Morningness/
eveningness is heritable. Soc Neurosci Abstr 1992, 18:196. 6
C
i
i g
16. Koskenvuo M, Hublin C, Partinen M, Heikkila K, Kaprio J: Heritabil-
ity of diurnal type: a nationwide study of 8753 adult twin
pairs. J Sleep Res 2007, 16:156-162. 36. Matsuo M, Shiino Y, Yamada N, Ozeki Y, Okawa M: A novel SNP in
hPer2 associates with diurnal preference in a healthy popu-
lation. Sleep and Biological Rhythms 2007, 5:141-145. p
J
p
17. Hur YM: Stability of genetic influence on morningness-
eveningness: a cross-sectional examination of South Korean
twins from preadolescence to young adulthood. J Sleep Res
2007, 16:17-23. p
g
y
37. Takano A, Uchiyama M, Kajimura N, Mishima K, Inoue Y, Kamel Y,
Kitajima T, Shibui K, Katoh M, Watanabe T, Hashimotodani Y, Naka-
jima T, Ozeki Y, Hori T, Yamada N, Toyoshima R, Ozaki N, Okawa
M, Nagai K, Yakahashi K, Isojima Y, Yamauchi T, Ebisawa T: A mis- 18. Hur YM, Bouchard TJ Jr., Lykken DT: Genetic and environmental
influence on morningness-eveningness. Personality and Individual
Differences 1998, 25:917-925. References Publish with BioMed Central and every
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scientist can read your work free of charge 54. Rush AJ, Bernstein IH, Trivedi MH, Carmody TJ, Wisniewski S, Mundt
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evaluation of the quick inventory of depressive symptoma-
tology and the hamilton rating scale for depression: a
sequenced treatment alternatives to relieve depression trial
report. Biol Psychiatry 2006, 59:493-501. p
y
y
55. Rosenthal NE, Kasper S, Schulz PM, Wehr TA: New concepts and
developments in seasonal affective disorder. In Seasonal Affec-
tive Disorder Edited by: Thompson C and Silverstone T. London, CNS
Publishers; 1989:97-132. p
y
y
55. Rosenthal NE, Kasper S, Schulz PM, Wehr TA: New concepts and
developments in seasonal affective disorder. In Seasonal Affec-
tive Disorder Edited by: Thompson C and Silverstone T. London, CNS
Publishers; 1989:97-132. Page 14 of 14
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https://openalex.org/W4230213853
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https://journals.vgtu.lt/index.php/JCEM/article/download/9078/7923
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English
| null |
DAMAGE-INDUCED STRESS-SOFTENING EFFECTS IN ELASTOMERIC AND BIOLOGICAL MEMBRANES
|
Journal of civil engineering and management
| 2,002
|
cc-by
| 4,455
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DAMAGE-INDUCED STRESS-SOFTENING EFFECTS IN
ELASTOMERIC AND BIOLOGICAL MEMBRANES Rasa Kazakevicifite-Makovska
Dept of Strength of Materials, Vilnius Gediminas Technical University,
Sauletekio a!. JJ, LT-2040 Vilnius, Lithuania. E-mail: rasa.kama@st. vtu.lt
Received 27 Dec 2001; accepted 10 Febr 2002 Abstract. The two-dimensional finite strain constitutive model for membranes is presented; it incorporates stress-soften-
ing behaviour typically observed in elastomeric and natural or biologically-derived soft membranes subjected to severe
deformations. It is assumed that the experimentally observed progressive degradation of a membrane stiffness under
monotonous and cycling loading can macroscopically be modelled by a scalar damage variable. The evolution of this
variable during the deformation process is specified by the kinetic law of damage growth, which together with the
constitutive equation for the surface stress tensor and the damage criteria completely determines the presented constitu-
tive model. It is shown that the general constitutive model can be specified for particular classes of problems under
certain additional assumptions. In particular, a remarkable simplification of the model is achieved assuming that the
state of strain at membrane points can be characterised by a single scalar variable, the so-called effective (equivalent)
strain. This assumption is combined with the hypothesis of maximum strain according to which the stress softening in
the membrane depends only on the maximum previous strain experienced during deformation history. Within these two
hypotheses the progressive degradation of membrane stiffness is completely described by a softening function which
determines the current value of damage variable in terms of maximum equivalent strain. Various specific forms of such
a softening function as well as different definitions of the effective strain are considered. Keywords: damage mechanics, elastomeric membranes, biological membranes, constitutive equations. 68 68 JOURNAL OF CIVIL ENGINEERING AND MANAGEMENT
http:/www.vtu.lt/englishledition
2002, Vol VIII, No 1, 68-72 1. Introduction reversible. However, the perfect elasticity is often a very
crude assumption. Membranes are widely used as structural elements
in many engineering fields. Fabric (pneumatic and ten-
sion) structures, automobile airbags, parachutes, meteo-
rological balloons are examples. Membranes have also
their natural appearance, eg soap films and bubbles, bio-
membranes and fluid films. One of the difficulties in mo-
delling membranes is to account for their highly non-
linear elastic and inelastic response to external loading. A typical inelastic effect observed in the inflation
and subsequent deflation of balloons is that of hysteresis
accompanied by stress-softening (Fig 1). These effects
are also observed in biological membranes. However, un- circumferential engineering strain E
Fig 1. Experimental study of latex balloon inflation/de-
flation [I] circumferential engineering strain E While not a problem of great technological impor-
tance, the inflation of a toy balloon provides a good il-
lustration of a highly non-linear behaviour of elastome-
ric membranes. When a balloon is inflated, its radius
increases continually until the internal pressure reaches
a maximum at a relatively small deformation. If the bal-
loon is further inflated, the pressure decreases, and later
rises again at very large strains. This behaviour of a bal-
loon results from combined effects of geometric and ma-
terial non-linearities and it is also observed in inflating
balloons of other shapes. Over years, the problem of in-
flation of membranes has been studied by many authors
under the assumption that the deformation is completely Fig 1. Experimental study of latex balloon inflation/de-
flation [I] e-Makovska I JOURNAL OF CIVIL ENGINEERING AND MANAGEMENT- 2002, Vol Vlll, No I, 68-72 R. KazakeviCiute-Makovska I JOURNAL OF CIVIL ENGINEERING AND MANAGEMENT- 2002, Vol Vlll, No I 69 like the polymeric membranes, they display highly ani-
sotropic behaviour. Also, stress-softening in such memb-
ranes is anisotropic; a load in one direction does not
produce the same degree of softening when applied in a
different direction (Fig 2). have no bending stiffness and hence they cannot support
transverse shear forces. Therefore, N(y) is the tangen-
tial surface tensor (the linear map of the tangent space
TYN into itself). Moreover, this tensor is symmetric by
virtue of the balance law of moments. Let m be the unit normal vector to N . The curva-
ture tensor of N is defined by K = - P grad 5m , where
P denotes the projection operator (see [9-12]). 1. Introduction (5) (5) The basic relations and equations summarized above are
formally similar to their counterparts in the three-dimen-
sional theory. However, it should be noted that all quan-
tities are defined here on curved surfaces and not in the
flat Euclidean space. This is seen from the derivation
leading to the equations (2) and (3). 1. Introduction The differ-
ential identity m • (div5 N) = N • K may next be used to
split the equilibrium equations (1) into the scalar equation Fig 2. Biaxial experimental tests on rat bladder wall [2] N•K+q=O,
q=m•q,
(2) (2) which represents the equilibrium condition of all forces
normal to N , and the vector equation in the tangent
space TYN, which represents the equilibrium condition of all forces
normal to N , and the vector equation in the tangent
space TYN, P(div5 N)+ Pq = 0,
(3) (3) which represent the equilibrium conditions of all forces
tangent to N at a given point. Relative to a fixed reference configuration M, the
deformation of the membrane is described by a smooth
mapping X: M ~ N with the tangential deformation
gradient F(x)= Grad5X(x) being the linear map of TxM
into TYN . This tensor is non-singular and hence it may
be written as the product of the surface stretch tensor
U(x) and the tangential rotation tensor R(x) [9-12]. Then the tangential Cauchy-Green deformation tensor
C(x) and the surface Green strain tensor E(x) are given
by Fig 2. Biaxial experimental tests on rat bladder wall [2] Elastomeric and biological membranes are often mo-
delled as elastic or viscoelastic ones ( eg [3-6]). Howe-
ver, standard elastic and viscoelastic formulations are not
able to predict the damage-induced stress-softening ef-
fects. In polymeric materials and membranes, this effect
is originated by internal damage in the form of progres-
sive de bonding of polymer chains. Physical mechanisms
underlying stress-softening in biological membranes is
more complex and actually poorly understood ( cf. [2, 7,
8]). Nevertheless, phenomenological modelling using two-
dimensional constitutive equations is possible and often
very successful. 2E=C-1,
(4) (4) where l(x) denotes the unit tensor on TxM. In the de-
scription of deformation relative to M , it is convenient
to introduce the surface stress tensor S(x) of the second
Piola-Kirchhoff type, which is defined by The aim of this work is to develop a constitutive
model which is capable to capture main effects of dama-
ge-induced stress-softening observed in polymeric and
biological membranes. This goal is achieved through the
use of certain concepts from the field of continuum da-
mage mechanics. As a final result, a simple description
of stress-softening effects is derived through successive
specification of the general constitutive equations. j == det F = .J det C . 4. Constitutive modelling stress-softening effect If the function ~ in the kinetic law of damage growth
(10) depends on the deformation tensor C only through
the effective strain ~=~(C), then this law takes a simple
form a = ~(~}(; . Moreover, this relation may be integra-
ted to give the damage variable in the explicit form a(t)= Z(~(t)), Z@t))= J;(~;~(~(s))d~(s). (11) In continuum damage mechanics, various so-called
equivalence hypotheses are postulated which considerably
simplify the constitutive modelling of damage phenome-
na. Adopting similar point of view for membranes, it
may be assumed that <1> = i(c,a,c),
S=S(C,a,c)
(6) (6) together with the following evolution equation for the
damage variable <i>(C,a)= (1-a)W(C),
(12) (12) (7) (7) where W(C) denotes a strain energy function of an un-
damaged membrane. Under this assumption, often refer-
red to as the strain equivalence hypothesis, the stress-
strain (8) relation takes the form Here i , S and 13 are given functions of their common
arguments. In general, these functions must satisfy the
principle of irreversibility (the second law of thermo-
dynamics). From this principle the following results can
be derived: (l) the elastic potential i
is independent of
C , Bt<i> = 0 , (2) the constitutive equation for the stress
is given by Here i , S and 13 are given functions of their common
arguments. In general, these functions must satisfy the
principle of irreversibility (the second law of thermo-
dynamics). From this principle the following results can
be derived: (l) the elastic potential i
is independent of
C , Bt<i> = 0 , (2) the constitutive equation for the stress
is given by S = (1-a)Oc W(C)
(13) (13) with a being given by (11 ). Within the assumptions le-
ading to ( 13) and ( 11 ), there remains only to specify the
damage softening function Z(~) and an appropriate da-
mage criteria. s =S(c,a,C)=28ci(c,a)+S*(c,a,c),
(8) (8) and (3) the following inequality and (3) the following inequality An idealized stress-softening membrane may be con-
sidered as an inelastic material having a selective me-
mory of only the maximum previous strain experienced
during its deformation. With the effective strain being
defined by ~(t) = ~( C(t)) , the maximum previous strain
is ~max (t) =max O!>s!>t ~(s) with t denoting current time. Moreover, the damage will grow only if ~>~max . 3. Constitutive modelling damaged membranes In the coordinate free description (absolute tensor
notation), the geometry, kinematics and dynamics of
membranes have been treated in detail in [9, 10]. Be-
low, only basic relations and equations are summarized
which are needed in the following considerations. In the theory of elastic membranes, the constitutive
equations relating to surface stresses and strains are typi-
cally formulated in terms of a strain energy function
which depends on the deformation tensor C alone. How-
ever, inelastic effects are path dependent and their mod-
elling should be based on the thermodynamical theory
of irreversible processes. In this paper thermal effects
are not considered but the restrictions implied by the
principle of irreversibility will be pointed out. In the deformed configuration N , assumed to be a
smooth surface in the space, the equilibrium equations
at each point y E N are div5 N +q = 0,
(1) (1) where N(y) denotes the surface stress tensor (Cauchy
type), q(y) is the surface force and div s stands for the
surface divergence operator [9,10]. Flexible membranes Stress-softening effects are generally attributed to
an internal damage of a material. On macroscopic scale,
this may be accounted for by a scalar damage variable keviCiiite-Makovska I JOURNAL OF CIVIL ENGINEERING AND MANAGEMENT·· 2002, Vol VIII, No 1, 68-72 R. KazakeviCiiite-Makovska I JOURNAL OF CIVIL ENGINEERING AND MANAGEMENT·· 2002, Vol VIII, No 1, 68-72 70 a(x,t) such that a= 0 in the undamaged (virgin) state
of the membrane. Here t is a time-like parameter. Ac-
cording to the general theory of irreversible processes,
the complete constitutive model of an inelastic membrane
requires that an elastic potential (the free energy func-
tion under isothermal conditions) <I> and the stress ten-
sor S are given by appropriate constitutive equations in
terms of C and a including possibly their surface and
time derivatives. For the purpose of this paper it will
suffice to consider the constitutive equations of the form 4. Constitutive modelling stress-softening effect Ac-
cordingly, the evolution of damage variable a is given
by a= Z(~) during loading from the undamaged state
and a= Z(~max) during reloading from the state of ma-
ximum effective strain. s*(c,a,t)•c-(oa<i>(c,a)}x 2: o
(9) (9) must be satisfied in every deformation process. This in-
equality simply states that the deformation of a mem-
brane is accompanied by the energy dissipation due to
viscous stresses s* (c, a, t) and the growth of damage
and thus it is an irreversible process. The constitutive equation (8) is general enough to
account for viscous effects. However, although the stress-
softening process depends on deformation history, strain
rate dependency often appears to be negligibly small [1,
7, 13]. Therefore, in the remaining part of this paper it
will be assumed that the viscous part s* of the stress
may be neglected. With the assumption that
s*(c,a,C)=o, the constitutive description of a dam-
aged membrane requires specification of two scalar func-
tions, namely of i(C,a) and 13(C,a,c). Moreover, the
assumption that viscous stresses can be neglected requires
that 13 is a homogenous function of degree one with
respect to C . This condition is satisfied if the evolution
law (7) is assumed in the special form 5. Isotropic membranes Further specifications of various damage models may
be obtained if the membrane exhibits certain symmetries
in its mechanical properties. The simplest case is that of
an isotropy. Elastomeric membranes have this property,
in which case the constitutive equation for the stress ten-
sor takes the form (see [10-12]) (14) S = S(C,a) = h0(i1 ,i2, a)l + h1 (i1 ,i2 ,a)C
(14) independently of various hypothesis considered in the pre-
vious chapter. Here h0 and h1 are given scalar func-
tions of the principal invariants of the deformation ten-
sor C . They are defined by a= 13(C,a,c)= ~(c.~)(;,
(10) (10) where
~(C,~) is a given constitutive function and
~=~(C) is the so-called effective or equivalent strain. y
i1 = trC,
i2 = detC. (15) (15) The stress-softening effects illustrated in Fig 1 and
Fig 2 is a property of a membrane material in which a
decrease in stress accompanies an increase in strain. This
is a rather special case of the general damage phenom-
ena for which simplifications of the general constitutive
equations are possible. The particular form (14) of the stress-strain relations will
be obtained in the next chapter, where a more general
situation is considered. Here it is worth to note that in
the case of isotropic membranes the stress tensor S and
the deformation tensor C have common principal direc-
tions which are determined by two mutually orthogonal The particular form (14) of the stress-strain relations will
be obtained in the next chapter, where a more general
situation is considered. Here it is worth to note that in
the case of isotropic membranes the stress tensor S and
the deformation tensor C have common principal direc-
tions which are determined by two mutually orthogonal R. KazakeviCiute-Makovska I JOURNAL OF CIVIL ENGINEERING AND MANAGEMENT- 2002, Vol VIII, No 1, 68-72 71 R. unit vectors u 1 and u 2 . Denoting by A1 and A2 the
associated principal stretches, the deformation tensor may
be written in the canonical form used together with the equilibrium equations (2) and (3)
in the analysis of stress-softening effects in membranes. However, due to path dependent nature of the damage
process, a numerical integration algorithm is required for
the evaluation of stresses and damage evolution throug-
hout the entire deformation history. The problem is sim-
plified considerably if the evolution of the damage va-
riable is given explicitly by the relation (11). i3 =A•C=a•Ca.
(19) (19) Denoting by <1>1, <1>2, <1> 3 the derivatives of <I> with res-
pect to the invariants (i1, i2 , i3 ), it is easy to shown (see
[12]) that the constitutive equation for the stress tensor
takes the form X
"'
E
c._
c._
1.2-r----;:::==========;-----------:l
experimental results
• Johnson and Beatty [19951
• Beatty [19871
a Har1·Smith [t 9661
' '
~ 0.8
' ' '
:::>
"' "'
~
c._
'0
(I)
-6 0.4
E
0
c:
analytical solutions
for different values
of material parameters
o+-~~~~~~==~~~~~_J
1.0
2.0
3.0
4.0
5.0
6.0
7.0
radial stretch A. Fig 3. Experimental results and analytical solutions for a
membrane modelled as Hart-Smith material X
"'
E
c._
c._
1.2-r----;:::==========;-----------:l
experimental results
• Johnson and Beatty [19951
• Beatty [19871
a Har1·Smith [t 9661
' '
~ 0.8
' ' '
:::>
"' "'
~
c._
'0
(I)
-6 0.4
E
0
c:
analytical solutions
for different values
of material parameters
o+-~~~~~~==~~~~~_J
1.0
2.0
3.0
4.0
5.0
6.0
7.0
radial stretch A. (20) (20) where h0 , h1 and h2 are functions of (i1, i2 , i3 ) and a
given by where h0 , h1 and h2 are functions of (i1, i2 , i3 ) and a
given by h0 = 2(<1>1 +i1<I> 2 ),
h1 = -2<1> 2 ,
h2 = 2<1> 3 • (21)
With <1> 3 = 0, the stress-strain relations (14) follow from
(20) and (21). In effect, this is a general procedure for
deriving the constitutive equations for other classes of
anisotropic membranes, each being completely determi-
ned by certain structure tensors. Such tensors has been
studied in [14] within the context of plane composites. (16) Moreover, in the deformed configuration of the memb-
rane the unit vectors along the principal directions are
determined by A1 v 1 = Fu1 and A2 v 2 = Fu2 . With the
use of these relations, the constitutive equation (14) may
be rewritten in terms of the Cauchy type stress tensor N = F 1f?.Hho +A~hl~l ®vl +A.Hho +A~h1~2 ®vj(l7)
Substituting (17) into (2) and (3), the equilibrium equa-
tions are obtained in the form particularly convenient in
the analysis of general constitutive equations. N = F 1f?.Hho +A~hl~l ®vl +A.Hho +A~h1~2 ®vj(l7)
Substituting (17) into (2) and (3), the equilibrium equa-
tions are obtained in the form particularly convenient in
the analysis of general constitutive equations. N = F 1f?.Hho +A~hl~l ®vl +A.Hho +A~h1~2 ®vj(l7) Substituting (17) into (2) and (3), the equilibrium equa-
tions are obtained in the form particularly convenient in
the analysis of general constitutive equations. One of the conceptual differences between problems
of the classical linear elasticity and those of the finite strain
deformation of membranes is that the specification of
the form of constitutive relations is generally part of pro-
blem in the theory of membranes whereas in the classi-
cal theory one needs only to specify the material cons-
tants occurring in the generalized Hooke's law. This is
not a trivial difficulty since the various natural and synt-
hetic membranes which can sustain large elastic and ine-
lastic deformations each behave somewhat differently. 6. Anisotropic membranes The isotropy cannot be assumed in modelling bio-
logical membranes. Without this assumption, the elastic
potential <1> must be considered as an arbitrary function
of the deformation tensor C and of the damage variable
a . Its general form is only restricted by the inequality
(9). However, there are experimental evidence that ma-
ny biological membranes may be modelled as orthotro-
pic ones (see [2, 7, 8]). This kind of an anisotropy may
be characterised at each point of the membrane by a unit
vector a along the preferred direction, usually called
the fibre direction. Moreover, in the case of the orthot-
ropic membranes a and - a are physically equivalent. Hence, not the vector a but the tensor A = a® a cha-
racterises this class of membranes. Moreover, it follows
from the theory of tensor function (see eg [14) that <I>
depends on C and A through their common isotropic
invariants, ie In the case of elastic membranes, the problem is re-
duced to the identification of material parameters for the
assumed form of a strain energy function W(C). This is
still a non-trivial task due to limited number of available
experimental results and difficulties in the interpretation
of the results obtained by different authors. This is illust-
rated in Fig 3. The presented analytical solutions were
obtained for the strain energy function W(C) derived from
the three-dimensional constitutive equation of Hart-Smith
[15] for rubber-like materials using the method [12]. In the analysis of damage phenomena in general and
of the stress-softening effects in particular, the problem
of identification of constitutive equations is even more
difficult. Even if the strain equivalence hypothesis (12)
is adopted and if a particular form of the strain energy
function W(C) is assumed, there still remains to specify
the effective strain ~ and the softening function Z(~). In general, a growth of damage in stress-softening memb-
ranes is only delimited by the inequality (9). Under the <I> = <l>(i1, i2 , i3, a),
( 18) ( 18) consisting of the principal invariants (15) of C and one
additional invariant defined by consisting of the principal invariants (15) of C and one
additional invariant defined by i3 =A•C=a•Ca. (19) 5. Isotropic membranes In the par-
ticular case of the inflation of spherical membranes, the
resulting equation follows from (17) and (2) while the
equilibrium equation (3) is trivially satisfied. The pro-
blem is thus reduced to a single algebraic for the radial
stretch. C=A~u 1 ®u 1 +A~u 2 ®u 2 . C=A~u 1 ®u 1 +A~u 2 ®u 2 . (16) (16) References assumption (12), this inequality takes the form
W(C)a ~ 0 and since W(C) ~ 0 by virtue of the basic
results in the non-linear elasticity, we must have a~ 0. In effect, the softening in the membrane response incre-
ases with increasing values of the effective strain ~ . Hen-
ce, Z(~) must be a monotonously increasing functions
of ~. A simple example of such a function is (cf. [13]) I. Johnson M. A., Beatty M. F. The Mullins effect in equi-
biaxial extension and its influence on the inflation of a
balloon. Int. J. Engng Sci., 33, 1995, p. 223-245. 2. Sacks M. S. Biaxial mechanical evaluation of planar bio-
logical materials. J. Elasticity, 61, 2000, p. 199-246. 3. Rand R. P. Mechanical properties of the red cell membra-
ne, II. Viscoelastic breakdown of the membrane. Biophy-
sical J., 4, 1964, p. 303-316. (22) Z(~) = 1- ~ exp( -11(~- ~0 )) ,
(22) 4. Hochmuth R. M., Hampel W. L. Surface elasticity and vis-
co-elasticity of red cell membranes. J. Rheology, 23, 1979,
p. 669-680. where ~ and 11 are parameters characterising growth of
damage and ~0 is an initial damage threshold. In general, Z(~) is given by (11) under the assump-
tion that the constitutive function ~ appearing in the ki-
netic law of damage growth (10) depends on C only
through the effective strain ~=~(C). It follows then that
a specific form of the softening function Z(~) is essen-
tially independent of possible material symmetries. In con-
trast, the effective strain is a function of the deformation
tensor C and thus its specific form should respect parti-
cular material symmetries. 5. Wineman A. On axisymmetric deformations of nonlinear
viscoelastic membranes. J. Non-Newtonian Fluid Mech.,
4, 1978, p. 249-260. 6. Feng W. W. Viscoelastic behavior of elastomeric membra-
nes. ASCE J. Appl. Mech., 59, 1992, p. S29-S34. 7. Fung Y. C. Biomechanics. Mechanical properties of living
tissues. New York: Springer-Verlag, 1981. 8. Sacks M. S. A structural constitutive model for chemical-
ly treated planar tissues under biaxial loading. Comput. Mech., 26, 2000, p. 243-249. In the case of isotropic membranes, the effective
strain may be defined as an isotropic function of C , ie
as a given function ~ = ~(i1 , i2 ) of the invariants (15). For orthotropic membranes, the corresponding general de-
finition would be ~=W1 ,i2 ,i2 ). References For illustration only, a
simple form of such a function may be assumed 9. Gurtin M. E., Murdoch A. I. A continuum theory of elas-
tic material surfaces. Arch. Rat. Mech. Anal., 57, 1975,
p. 291-323. I 0. Murdoch A. 1., Cohen H. Symmetry considerations for ma-
terial surfaces. Arch. Rat. Mech. Anal., 72, 1979, p. 61-98. ~ = Ji1
2 - 2i2 •
(23) (23) II. Kazakeviciiite-Makovska R. Structure of constitutive rela-
tions for isotropic elastic membranes with voids. Statyba,
Vol VII, No I. Vilnius: Technika, 2001, p. 23-28. With the use of the effective strain defined by (23), the
solutions for different values of the parameters appearing
in the softening function (22) are shown in Fig 4. It can
be seen from these simple calculations that various types
of stress-softening can be modelled by varying only the
parameters in the assumed damage softening function. Ho-
wever, a complete study of this problem is beyond the
aim of this paper. 12. Kazakeviciiite-Makovska R. Nonlinear response functions
for transversely isotropic elastic membranes. Statyba,
Vol VII, No 5. Vilnius: Technika, 2001, p. 345-351. 13. Beatty M. F., Krishnaswamy S. The Mullins effect in equi-
biaxial deformation. Z. angew. Math. Phys., 51, 2000,
p. 984-1016. 14. Zheng Q.-S., Betten J. The formulation of constitutive equ-
ations for fibre-reinforced composites in plae problems:
Part II. Arch. Appl. Mech., 65, 1995, p. 161-177. 7. Analysis of general models radial stretch A. The general constitutive model and its various spe-
cial versions derived in the previous chapters may be Fig 3. Experimental results and analytical solutions for a
membrane modelled as Hart-Smith material 72
R. KazakeviCiute-Makovska I JOURNAL OF CIVIL ENGINEERING AND MANAGEMENT- 2002, Vol Vll azakeviCiute-Makovska I JOURNAL OF CIVIL ENGINEERING AND MANAGEMENT- 2002, Vol Vlll, No 1, 68-72 72
R. KazakeviCiute-Makovska I JOURNAL OF CIVIL ENGINEERING AND MANAGEMENT- 2002, Vol Vlll, No 1, 68-72 72 1·2 .,---....,...e-xp_e.,.rim-e-nt=al=re-s""ul-ts====-;-------~
o Johnson and Beatty [1995]
X
"'
E
0. 0. ~ 0.8
:::J
"' "'
Q)
0. -o
Cl>
-a 0.4
E
0
c
! ..... -~ .............. .. . . • Beatty [1987]
a Hart-Smith [1966]
-.. ·7
--undamaged membrane
-------·damage membrane
•
,•
.~
•• ,:: .I
•
,.~ J
,~f
-__ ::::::::::::;~~::;:f;:::i>""
. . . . . . . . 2.0
3.0
4.0
5.0
6.0
7.0
radial s !retch A. Fig 4. Parametric study of softening effects 1·2 .,---....,...e-xp_e.,.rim-e-nt=al=re-s""ul-ts====-;-------~
o Johnson and Beatty [1995]
X
"'
E
0. 0. ~ 0.8
:::J
"' "'
Q)
0. -o
Cl>
-a 0.4
E
0
c
! ..... -~ .............. .. . . • Beatty [1987]
a Hart-Smith [1966]
-.. ·7
--undamaged membrane
-------·damage membrane
•
,•
.~
•• ,:: .I
•
,.~ J
,~f
-__ ::::::::::::;~~::;:f;:::i>""
. . . . . . . . 2.0
3.0
4.0
5.0
6.0
7.0
radial s !retch A. haviour, for polymeric and biological membranes most
of the available experimental data have been obtained in
biaxial experiments [1-4, 8, 15]. Although biaxial tests
alone are not sufficient to derive a three-dimensional
stress-strain relationships, for membranes they suffice to
yield the two-dimensional constitutive equations relating
to the surfaces stresses and surface strains. The general
results derived in this paper provide the conceptual fra-
mework within which specific forms of constitutive equ-
ations for membranes may be sought entirely indepen-
dently of any three-dimensional theory . Acknowledgement. This research was partially supported
by the Polish State Committee for Scientific Research under
grant KBN 7 T07 A 021 16. Fig 4. Parametric study of softening effects 8. Closing remarks 15. Hart-Smith L. J. Elasticity parameters for finite deforma-
tions of rubber-like materials. Z. angew. Math. Phys., 17,
1966, p. 608-626. While three-dimensional constitutive equations of
continuum mechanics may be specified for membranes
and thus they may be used to analyse their inelastic be-
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Reforestation challenges in Scandinavia
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Reforesta
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Anders Mattsson Dalarna University
Department of Energy, Forests and Built Environments Dalarna University
Department of Energy, Forests and Built Environments amn@du.se Scandinavia, Reforestation Challenges, Deforestation, Degradation, Erosion, Pine
Weevils, Lifting, Field Storage, Alternative Species, Climate Change Copyright: © 2016 Mattsson
Anders. This work is licensed under
a Creative Commons Attribution-
NonCommercial-ShareAlike 4.0
International License. Copyright: © 2016 Mattsson
Anders. This work is licensed under
a Creative Commons Attribution-
NonCommercial-ShareAlike 4.0
International License. This article is Chapter 4 of
establishing issue of Reforesta
Journal, edited in form of Thematic
Proceedings, by Vladan Ivetić and
Steven Grossnickle. Citation:
Mattsson A (2016) Restoration
challenges in Scandinavia.
Reforesta 1: 67-85.
DOI: http://dx.doi.org/10.21750/R
EFOR.1.05.5 Citation:
Mattsson A (2016) Restoration
challenges in Scandinavia. Reforesta 1: 67-85. DOI: http://dx.doi.org/10.21750/R
EFOR.1.05.5 In the keynote, major reforestation challenges in Scandinavia will be highlighted. The
following countries make up Scandinavia: Iceland, Norway, Sweden, Finland and
Denmark. For Iceland, with only a forest cover of 2%, a major reforestation challenge is
the deforestation and overgrazing in combination with land degradation and extensive
soil erosion. The challenges include the conflicts with livestock farmers. For centuries
the commons were used for sheep and horse grazing. However, more and more of
farmer grazing land have been fenced up, allowing the regeneration of birch and
plantations of other species to increase. With a forest cover of 37% and 69%
respectively, for decades a major reforestation challenge in Norway and Sweden has
been the risk of seedling damages from the pine weevil. Unprotected seedlings can have
a survival rate of less than 25% after being planted. Pine weevils feed on the bark of
planted young seedlings at regeneration sites. If the seedling is girdled, it will not
survive. In Sweden, and soon in Norway, pesticides have been forbidden. In the keynote,
new methods and technology will be presented based on non-chemical protection. In
Finland, with a forest cover of 75%, a major reforestation challenge is linked to the forest
structure. The structure of Finnish forestry includes many private forests in combination
with small regeneration sites. This implies a situation where logistics and methods for
lifting and field storage provide a major challenge in order to preserve seedling quality
until the planting date. Due to this situation, new logistic systems and technologies are
being developed in Finland, including new seedling cultivation programs (including
cultivation under Light Emitting Diodes (LEDs)) to match the access of fresh planting
stock to different planting dates. In Denmark, with a forest cover of 13%, a major
reforestation challenge is the possibility of future plantations based on a wide range of
relevant species. For this to become a realistic option, new methods and technology
have to be developed in reforestation activities that support this possibility. These
methods and technology should make it possible to not be limited to certain species
due to problems and restrictions during field establishment. This due to the prospect of
establishing stable, healthy, and productive stands of various forest species that can be
adapted to future climate change. Editor: Vladan Ivetić, Serbia
Received: 2016-02-07
Accepted: 2016-05-03
Published: 2016-05-25 Citation: Citation:
Mattsson A (2016) Restoration
challenges in Scandinavia. Reforesta 1: 67-85. DOI: http://dx.doi.org/10.21750/R
EFOR.1.05.5 REFORESTA (2016) 1:67-85 REFORESTA (2016) 1:67-85 Mattsson A Mattsson A Keywords Scandinavia, Reforestation Challenges, Deforestation, Degradation, Erosion, Pine
Weevils, Lifting, Field Storage, Alternative Species, Climate Change Reforesta Scientific Society Reforesta Scientific Society 67 REFORESTA (2016) 1:67-85 Mattsson A Contents
1
Definition of Scandinavia
68
2
Iceland
69
2.1
Forest facts
69
2.2
Reforestation challenges
69
3
Norway
71
3.1
Forest facts
71
3.2
Reforestation challenges
72
4
Sweden
73
4.1
Forest facts
73
4.2
Reforestation challenges
74
5
Finland
80
5.1
Forest facts
80
5.2
Reforestation challenges
81
6
Denmark
82
6.1
Forest facts
82
6.2
Reforestation challenges
83
7
References
84 Contents 5.1
Forest facts 6.1
Forest facts 6.2
Reforestation challenges 6.2
Reforestation challenges
7
References 7
References 2 Iceland 2.1 Forest facts Since the 1950s, afforestation by planting trees has been a priority in Iceland. Planting done by forestry societies increased greatly during the 1950s, reaching over 1.5
million seedlings per year in the early 1960s. The principal species planted, besides birch,
were exotic conifers like Norway spruce, Sitka spruce, Scots pine, Lodgepole pine and
Siberian larch. Figure 2. Wood from the thinning of a 50-year-old Sitka spruce forest planted in the early 1960s now destined for
chipping. Figure 2. Wood from the thinning of a 50-year-old Sitka spruce forest planted in the early 1960s now destined for
chipping. Planting declined in the middle of the 1960s and remained at 0.5-1 million
seedlings annual to the late 1980s. After that, afforestation through planting has
increased to 4 million seedlings annual throughout most of the 1990s. Today about 6
million seedlings are planted per year (Eysteinsson 2013). 1 Definition of Scandinavia In my keynote, major reforestation challenges in Scandinavia will be highlighted. In Scandinavia, the following countries are included: Iceland, Norway, Sweden, Finland
and Denmark (Fig. 1). Figure 1. Countries included in Scandinavia regarding this keynote. Figure 1. Countries included in Scandinavia regarding this keynote. Reforesta Scientific Society 68 REFORESTA (2016) 1:67-85 Mattsson A Reforesta Scientific Society Reforesta Scientific Society 3 Norway Almost 40% of the surface area in Norway is covered by forests (Fig. 5). The total
forested area amounts to 12 million hectares, including more than 7 million hectares of
productive forests. 15% of productive forests have been estimated as non-economic
operational areas due to difficult terrain (mostly steep) and long distance transport. This
means that economical forestry can only be operated in about 50% of the total forested
area. Regarding tree species in Norway, the most important are Norway spruce (47%)
and Scots pine (33%). Birch represents an important species (18%) (Nordic Forest
Owners´ Association www.nordicforestry.org). Figure 5. Typical Norwegian forest landscape in mountainous terrains. 2.2 Reforestation challenges Iceland once had a forest cover of up to 60%, mostly with birch forests whereas
today, forest coverage is around 2% (Fig. 3). Due to extensive logging, land degradation
and soil erosion, Iceland faces major reforestation challenge (Arnalds and Barkarson
2003; Eysteinsson 2013). Some of the most common species included in this challenge
are birch, Siberian larch, Sitka spruce and Lodgepole pine. The challenge also includes conflicts with livestock farmers (Arnalds and
Barkarson 2003; Halldorsson et al. 2008). For centuries commons were used for horse
and sheep grazing. However, more and more of the grazing lands have been fenced up
(Fig. 4) allowing the regeneration of birch and other species. Reforesta Scientific Society Reforesta Scientific Society 69 REFORESTA (2016) 1:67-85 Mattsson A Figure 3. Reforestation in Iceland, a real challenge. Figure 3 Reforestation in Iceland a real challenge Figure 3. Reforestation in Iceland, a real challenge. Figure 4. Grazing by sheep controlled by fencing. Figure 4. Grazing by sheep controlled by fencing. Regarding land degradation and eroded soils, forest regeneration often starts
by planting Siberian larch in order to establish a humus layer produced by the litter from
the needles falling of each year followed by, for example, the planting of Sitka spruce. Some forest nurseries have also been established in recent years adding to the picture
of a rising hope for increased future efforts regarding forest regeneration in Iceland. Reforesta Scientific Society 70 REFORESTA (2016) 1:67-85 Mattsson A 3 Norway
3.1 Forest facts
Almost 40% of the surface area in Norway is covered by forests (Fig. 5). The total
forested area amounts to 12 million hectares, including more than 7 million hectares of
productive forests. 15% of productive forests have been estimated as non-economic
operational areas due to difficult terrain (mostly steep) and long distance transport. This
means that economical forestry can only be operated in about 50% of the total forested
area. Regarding tree species in Norway, the most important are Norway spruce (47%)
and Scots pine (33%). Birch represents an important species (18%) (Nordic Forest
Owners´ Association www.nordicforestry.org). Figure 5. Typical Norwegian forest landscape in mountainous terrains. Standing volume of the forest is about 750 million m³, compared with 300
million m³ when the first national forest survey was carried out in 1919 The tremendous Figure 5. Typical Norwegian forest landscape in mountainous terrains. Standing volume of the forest is about 750 million m³, compared with 300
million m³ when the first national forest survey was carried out in 1919. The tremendous
increase is a result of a forest policy with the main objective of restoring forest
resources. The annual increment of Norwegian forests is approximately 25 million m³. The total annual harvest is less than 50% of this growth, about 10 million m³, which
means that the amount of trees in Norway forests increases significantly every year. In Norway, 84% of the forest is private property. The state and municipalities
own 12% and forest companies only own 4%. There are a total of 120,000 forest holdings
in Norway, with more than 2.5 hectares of forestland. The average size of a forest
holding is about 50 hectares. Small forest properties, steep terrain, varying terrain
conditions, and the varying production possibilities have created great variations within
the forest landscape. The forest along the coast is often very valuable, but is usually on a steep terrain. Therefore, logging operations are generally done using cable cranes (Samset 1985;
Torgersen and Lisland 2002) and the transportation of logs is done by helicopter (Fig. 6). Reforesta Scientific Society 71 REFORESTA (2016) 1:67-85 Mattsson A Figure 6. Logging by cable crane and transportation of logs from the site supported by helicopter. Figure 6. Logging by cable crane and transportation of logs from the site supported by helicopter Reforesta Scientific Society Reforesta Scientific Society 3.2 Reforestation challenges A major reforestation challenge in Norway has for decades been the risk of
seedling damages from pine weevils. Unprotected seedlings can have a survival rate of
less than 25% after being planted. The pine weevils feed on the bark of planted young
seedlings at regeneration sites. If the seedling is girdled, it will not survive (Hanssen 2010
and 2011). To reduce the damages from pine weevils, chemical protection has been used
in Norway for many years (Kohmann 1999). Figure 7. Planting operations with containerized seedlings and planting tubes. Bottom right: a pine weevil feeding on the
bark of a young seedling. Figure 7. Planting operations with containerized seedlings and planting tubes. Bottom right: a pine weevil feeding on the
bark of a young seedling. Reforesta Scientific Society 72 REFORESTA (2016) 1:67-85 Mattsson A In Sweden, and soon in Norway, major restrictions on the use of pesticides for
protection from pine weevil attacks have recently been introduced. Therefore, future
protection of seedlings against pine weevils has to be based on silvicultural or non-
chemical methods. Since seedling damage caused by pine weevils (Fig. 7) is one of the major
reforestation challenges in Sweden as well, impacts and possible methods to reduce
pine weevil damage will be discussed under the heading "Sweden". 4.1 Forest facts Sweden is the third largest country by area in Europe and has the biggest
afforested area, where productive forests cover about 27 million hectares. About 70%
of the country is covered in forests (Fig. 8). Swedish forests are primarily boreal. The
rotation period of the forest varies from 65-110 years, depending on the species (spruce
shorter than pine) and geographical location. Due to effective and far-sighted forest
management, the total standing volume in Sweden has increased by more than 60% in
the last hundred years. Now it amounts to 3,000 million m³ (Nordic Forest Owners´
Association www.nordicforestry.org). Norway spruce makes up 41% of the total forest mass, Scots pine - 40%, birch -
18%. The remaining 6% consists of other deciduous trees. Growth has been more rapid
in southern Sweden compared to the north, due to climatic and soil conditions. In recent
years, timber quantities have been between 85-90 million m³, whereas annual growth
amounts to about 120 million m³. In order to increase logging amounts, improved
forestry methods are required. Current studies indicate that wood production can be
increased by up to 20% by 2050 by means of improved forest management. Figure 8. Sweden has an extensive forest cover of mostly Norway spruce and Scots pine. Figure 8. Sweden has an extensive forest cover of mostly Norway spruce and Scots pine. Figure 8. Sweden has an extensive forest cover of mostly Norway spruce and Scots pi Reforesta Scientific Society 73 REFORESTA (2016) 1:67-85 Mattsson A In Sweden, private individuals own 50% of the forests. The average size of a
privately owned forest is about 50 hectares. Large forest companies own 25% and
another 25% is owned by the state and other public organizations. In total there are
355,000 forest owners in Sweden. The ownership conditions of a forest in Sweden vary
between regions. In the southern parts of the country, private persons mainly own
forests whereas in northern Sweden, companies own more significant amounts. In many parts of Sweden, especially in the south, we have forest stands with a
good volume growth. This implies intensive forest harvesting operations for delivery to
our pulp/saw mills (Fig. 9). In Sweden, about 80 million m³ is harvested each year and
used as pulp for paper production or as timber for wood constructions. Figure 9. 4.1 Forest facts Forest harvesting with a modern, single grip, harvester with a capacity of up to 50 m³ per machine hour. Figure 9. Forest harvesting with a modern, single grip, harvester with a capacity of up to 50 m³ per machine hour. Figure 9. Forest harvesting with a modern, single grip, harvester with a capacity of up to 50 Reforesta Scientific Society 4.2 Reforestation challenges To manage reforestation challenges after extensive harvesting operations,
Swedish forest nurseries produce about 400 million seedlings (Fig. 10) each year for
planting operations (Lindström and Mattsson 1994). The production is almost only
based on containerized seedlings, making us one of the largest producers in the world
of this plant type. Before discussing the mutual reforestation challenge in Norway and Sweden
regarding how to reduce seedling damage from pine weevil, the additional reforestation
challenge for Sweden in producing millions of seedlings each year for planting
operations will be discussed. Today seedlings are produced in large conventional plastic greenhouses limiting
the production time to the short vegetation period in Sweden. Also, environmental and
cost issues are involved in the discussion of how future production technology should
be outlined. As almost all greenhouses are heated by oil, the consumption for a greenhouse
is very high. This is based on the fact that production normally has to start in March for
requested volumes. At this time of the year, outside temperatures can be -20°C, Reforesta Scientific Society 74 REFORESTA (2016) 1:67-85 Mattsson A whereas the temperature inside the greenhouse has to be +20°C in order to reach high
seed germination. Figure 10. A conventional greenhouse at a Swedish forest nursery. Typical size: 20 meter wide and 100 meter long. whereas the temperature inside the greenhouse has to be +20°C in order to reach high
seed germination. whereas the temperature inside the greenhouse has to be +20°C in order to reach high
seed germination. tional greenhouse at a Swedish forest nursery. Typical size: 20 meter wide and 100 meter long. Figure 10. A conventional greenhouse at a Swedish forest nursery. Typical size: 20 meter wide a Therefore, Dalarna University in Sweden is involved in a EU-project (acronym
Zephyr) regarding new innovative technology for production of forest seedlings without
using conventional greenhouses (Hernandez Velasco and Mattsson 2014). This project
can really be seen as a major reforestation challenge. The technology (Fig. 11) is based on a short pre-cultivation period in a growth
chamber at high seedling density. For our major species, Scots pine and Norway spruce,
3,500 seedlings m-² followed by automatic transplanting to any optional container
system, and after transplanting final growth outdoors without using conventional
greenhouses. Reforesta Scientific Society 4.2 Reforestation challenges In addition the concept includes, among other things, LED lights providing
maintenance free, energy efficient, and low thermal emittance, as an alternative to
traditional light sources (Landis et al. 2013). The life span of a LED light is almost 10 times
longer compared to conventional lighting. The new technology also includes a
photovoltaic system and wireless sensors in order to control the climate and soil
conditions. With this system it would be possible for Sweden to produce forest seedlings
on a year-around basis without using conventional greenhouses. In co-operation between Dalarna University and one of the major forest
companies in Sweden, the technology with pre-cultivation followed by transplanting to
outdoor cultivation has been introduced commercially at one forest nursery. As can be
seen in Figure 12, the total annual production of 14 million seedlings can be pre-
cultivated on an area of about 100 m² without using conventional greenhouses. Reforesta Scientific Society Reforesta Scientific Society 75 REFORESTA (2016) 1:67-85 Mattsson A Figure 11. Outline of the Zephyr pre-cultivation incubator. Figure 12. Circulating multiple floor pre-cultivation chamber for large-scale production of Norway spruce and Scots pine
seedlings. Now back to the other reforestation challenge that we share with Norway -how
to reduce the effect of seedling damage caused by pine weevil (Eidmann and Lindelöw
1997). Pine weevils are attracted to fresh clear-cuts by the smell of monoterpenes and Figure 11. Outline of the Zephyr pre-cultivation incubator. Figure 11. Outline of the Zephyr pre-cultivation incubator. Figure 12. Circulating multiple floor pre-cultivation chamber for large-scale production of Norway spruce and Scots pine
seedlings. Figure 12. Circulating multiple floor pre-cultivation chamber for large-scale production of Norway spruce and Scots pine
seedlings. Figure 12. Circulating multiple floor pre-cultivation chamber for large-scale production of Norway spruce and Scots pine
seedlings. Now back to the other reforestation challenge that we share with Norway -how
to reduce the effect of seedling damage caused by pine weevil (Eidmann and Lindelöw
1997). Pine weevils are attracted to fresh clear-cuts by the smell of monoterpenes and
ethanol released upon cutting. After feeding on fresh slash, the weevils mate and the
female lays her eggs in the roots of the stumps. The new generation hatches the second
autumn, and start feeding on the fresh bark of the newly planted seedlings (Fig. 13). There is a potential risk of pine weevil attacks in many countries in Europe. The
magnitude of the problem is connected to the fact whether or not chemical treatment
is allowed in a specific country. Reforesta Scientific Society Reforesta Scientific Society 76 REFORESTA (2016) 1:67-85 Mattsson A Figure 13. Pine weevil life cycle. Figure 13. Pine weevil life cycle. Figure 14 show a risk ranking made in a unique European project (Långström
and Day 2004). As can be seen, there was no data available from most of the Balkan
countries at the time, so of course there could be a potential risk of attack in this part of
Europe as well. Figure 14. Risk ranking of pine weevil attacks in Europe. Figure 14. Risk ranking of pine weevil attacks in Europe. In Sweden, the pine weevil damage is estimated to a cost of about 30 million
USD per year. In recent years a lot of efforts and money have been allocated to the
problem and methods to reduce pine weevil damage without using chemicals have been
developed or are developing (Nilsson et al. 2010; Nordlander et al. 2011). These includes, as shown in Figure 15, both silvicultural methods (von Sydow
1997) and mechanical protection of single seedlings. These includes, as shown in Figure 15, both silvicultural methods (von Sydow
1997) and mechanical protection of single seedlings. Reforesta Scientific Society Reforesta Scientific Society 77 REFORESTA (2016) 1:67-85 Mattsson A Figure 15. Reforesta Scientific Society Silvicultural or mechanical (coating or tubes) methods for preventing damage from pine weevil attacks. Figure 15. Silvicultural or mechanical (coating or tubes) methods for preventing damage from pine weevil attacks. In the first category, scarification (Petersson and Örlander 2003) and delayed
planting have been used. Scarification has shown to be quite effective since pine weevils
do not find areas with pure mineral soil attractive, therefore, they don't invade seedlings
planted on that specific type of soil, compared to a seedling planted in an intact humus
layer that has more vegetation and humid conditions. Delayed planting can reduce the attacks since the amount of weevils will be
reduced each year due to lack of suitable feeding materials. The problem is the fast
introduction of weeds, making the establishment of young seedlings difficult due to
increasing competition for light, water and nutrients. The introduction of direct protection of each seedling by coating or mechanical
devices has shown to be a very promising method. These methods have now been
introduced in a large-scale in Swedish forest nurseries. Looking at how the proportion of chemical or mechanical protection of seedlings
(Fig. 16) used in forest regeneration in Forest Stewardship Council (FSC) holders in
Sweden (all major forest companies) has changed during the past four years, it is clear
that chemical treatments have decreased (Giurca and von Stedingk 2014). Still a large portion of all planted seedlings were left untreated during 2014. This
is due to the fact that in northern Sweden the pine weevil population is, due to climatic
conditions, rather small compared to southern Sweden that has much more favorable
conditions for an intensive forest yield (compared to the harsh conditions in northern
Sweden). Reforesta Scientific Society 78 REFORESTA (2016) 1:67-85 Mattsson A Figure 16. The proportion of all types of seedlings (untreated and mechanically/chemically treated) used in regeneration
by FSC holders during the past four years. Figure 16. The proportion of all types of seedlings (untreated and mechanically/chemically treated) used in regeneration
by FSC holders during the past four years. Data also shows that pesticide applications have been decreasing from the total
(chemical and mechanical treatment) proportion of treated seedlings (Fig. 17). According to predictions, all FSC holders are expected to completely cease pesticide
application by 2019 at the latest (Giurca and von Stedingk 2014). Figure 17. Proportions of chemical and mechanical treatment for all FSC holders. Dashed lines show predictions. Note that
untreated seedlings are not included in the calculation. Figure 17. Proportions of chemical and mechanical treatment for all FSC holders. Dashed lines show predictions. Note that
untreated seedlings are not included in the calculation. Figure 17. Proportions of chemical and mechanical treatment for all FSC holders. Dashed lines show predictions. Note that
untreated seedlings are not included in the calculation. Reforesta Scientific Society 79 REFORESTA (2016) 1:67-85 Mattsson A Reforesta Scientific Society 5 Finland Finland is Europe´s most heavily forested country with 75% of the land area (Fig. 18), representing 23 million hectares, under forest cover. There is an additional 3 million
hectares of sparsely wooded forest areas, treeless open mires, and rocky forestland. Taking all of this into consideration, you will see that forestry land accounts for 86% of
the land area. Nearly all of Finland belongs to the boreal coniferous forest zone, which
is characterized by a short growing season and limited number of species. Due to the
Gulf Stream, however, conditions in Finland are more favorable than in other places on
the same latitude. There are four coniferous species native to Finland and over twenty species of
deciduous trees. Just like in Norway and Sweden the most common species, which also
are economically most significant, are Scots pine, Norway spruce, and silver and downy
birch. Regarding these species forest cover in Finland includes 50% Scots pine and 30%
Norway spruce leaving 20% for the broadleaved species (mostly birch). In contrast to
Norway and Sweden, the amount of birch planting and birch stands are quite significant
in Finland. Beside forestry, Finland is also known as the land of the thousand lakes as
illustrated in Figure 18. Figure 18. Finland: a country of forests and lakes. Figure 18. Finland: a country of forests and lakes. The total volume of growing stock in Finnish forests amounts to about 2,200
million m³. Since the late 1960s, the volume and increment of the growing stock have
continuously risen and is now almost 50% higher than four decades ago. The annual
increment of Finnish forests is about 100 million m³ where the maximum annual harvest
is evaluated to be about 70 million m³ (Nordic Forest Owners´ Association
www.nordicforestry.org). Reforesta Scientific Society Reforesta Scientific Society Reforesta Scientific Society 80 REFORESTA (2016) 1:67-85 Mattsson A Private individuals own 60% of all forestland. The number of private forest
holdings of at least one hectare is about 440,000 and the number of individual private
forest owners is estimated to be around 920,000. This means that almost every fifth Finn
is a forest owner. The state and municipalities own 30%, while forest companies own
the remaining 10%. Finland has a long tradition in producing and planting birch seedlings. 5 Finland A forest
stand is often a mix of birch and spruce with birch as a nurse stand and Norway spruce
established as an understory (Valkonen and Valsta 2001). In Finland birch is known as
the mother of the spruce since the conditions, such as light and others, under a birch
stand are very good for spruce development (Fig. 19). Figure 19. A typical mixed birch and spruce forest stand in Finland. Figure 19. A typical mixed birch and spruce forest stand in Finland. Reforesta Scientific Society 5.2 Reforestation challenges A major reforestation challenge in Finland is linked to the forest structure. The
structure of Finnish forestry includes many private forest owners in combination with
small regeneration sites (Leppänen and Nuoro 2006) where one estate is in average 30
hectares. Private forestry plays a key role in Finland because 80% of Finnish wood used
by the forest industry comes from privately owned forests. In the southern part of
Finland with the most productive forest, private forest ownership is as high as 80%. This implies a situation where logistics and methods for lifting in combination
with field storage at the site provide a major reforestation challenge in order to preserve
seedling quality until the planting date (Lilja et al. 2010; Nilsson et al. 2010). Small
quantities of seedlings have to be delivered to each small regeneration site in a relatively
short planting season due to climate conditions. This implies problems to be able to take
care of the seedlings while maintaining their quality. Thus forest owners have to water
the plants at the field storage with portable water systems (Fig. 20). Reforesta Scientific Society 81 REFORESTA (2016) 1:67-85 Mattsson A Figure 20. Watering of seedlings at field storage by a regeneration site. Figure 20. Watering of seedlings at field storage by a regeneration site. Due to this situation, a major reforestation challenge for Finnish forestry is to
develop new logistic systems and technology, including new seedling cultivation
programs, to match the access of fresh planting stock to a lot of small regeneration sites
in combination with different planting dates. Reforesta Scientific Society 6.1 Forest facts Danish forests cover about 550,000 hectares which is about 13% of the total
land area (Fig. 21). Denmark is the northernmost of the central European temperate
forest zone, just south of the boreal coniferous forest zone. Therefore the climate
conditions allow more variations in forest species compared to other Scandinavian
countries. The most common tree species are Norway spruce (19%), beech (13%), Scots
pine (12%) and oak (9%). Species used as Christmas trees and greenery production,
mainly Nordmann and Noble firs, adds up to 6% (Nordic Forest Owners´ Association
www.nordicforestry.org). The total volume of the growing stock in Danish forests amounts to about 110
million m³. The growing stock and forest area have been constantly increasing from a
level of about 3% forest cover in the early 1800s. Today the annual increment is
calculated to be 5.5 million m³, whereas annual logging amounts to 2.2 million m³. In Denmark private persons own 65% of all forestland. Private foundations own
another 4%. The number of private forest holdings amounts to 25,000. The Danish state
owns 23% of the forestland. Mostly municipalities publicly own 8%. Reforesta Scientific Society 82 REFORESTA (2016) 1:67-85 Mattsson A Figure 21. The forest cover could be increased in Denmark due to favorable climatic conditions. Figure 21. The forest cover could be increased in Denmark due to favorable climatic conditions. Reforesta Scientific Society 7 References Arnalds O, Barkarson B H (2003) Soil erosion and land use policy in Iceland in relation to sheep grazing and
government subsidies. Environmental Science and Policy, volume 6, Issue 1: 105-113. Arnalds O, Barkarson B H (2003) Soil erosion and land use policy in Iceland in relation to sheep grazing and
government subsidies. Environmental Science and Policy, volume 6, Issue 1: 105-113. Bolte A, Ammer C, Löf M, Madsen P, Nabuurs G-J, Schall P, Spathelf P, Rock J (2009) Adaptive forest
management in central Europe: Climate change impacts, strategies and integrative concepts. Scandinavian Journal of Forest Research 24: 473-482. Bolte A, Ammer C, Löf M, Madsen P, Nabuurs G-J, Schall P, Spathelf P, Rock J (2009) Adaptive forest
management in central Europe: Climate change impacts, strategies and integrative concepts. Scandinavian Journal of Forest Research 24: 473-482. Eidmann H H, Lindelöw Å (1997) Estimates and measurements of pine weevil feeding on conifer seedlings:
their relationships and application. Canadian Journal of Forest Research 27: 1068-1073. Eysteinsson T (2013) Forestry in a treeless land. Iceland Forest Service, Egilsstadir Giurca A, von Stedingk H (2014) FSC report Pesticides policy in Sweden, 16 pp. http://se.fsc.org/download-
box.763.htm Halldorsson G, Oddsdottir E S, Sigurdsson B D (2008) AFFORNORD Effects of afforestation on ecosystems,
landscape and rural development. Tema Nord, The Nordic Council of Ministers, Copenhagen, pp
15-28. Hanssen K H (2010) Pine weevil damage in the west and north part of Norway. Report from Skog og
landskap: 13 pp (In Norwegian with English summary). Hanssen K H (2011) Pine weevil damage in the east and south part of Norway. Report from Skog og
landskap: 14 pp (In Norwegian with English summary). Hanssen K H (2011) Pine weevil damage in the east and south part of Norway. Report from Skog og
landskap: 14 pp (In Norwegian with English summary). Hernandez Velasco M, Mattssom A (2014) Silvicultural practices to facilitate forest restoration: a new
seedling cultivation technology for regeneration establishment. in: Parrotta J A, Moser C F,
Scherzer A J, Koerth N E, Lederle D R (eds). Sustaining Forests, Sustaining People: The role of Hernandez Velasco M, Mattssom A (2014) Silvicultural practices to facilitate forest restoration: a new
seedling cultivation technology for regeneration establishment. in: Parrotta J A, Moser C F, Hernandez Velasco M, Mattssom A (2014) Silvicultural practices to facilitate forest restoration: a new
seedling cultivation technology for regeneration establishment. 6.2 Reforestation challenges Although the conditions allow more variations (Fig. 22), spruce monocultures
have been established in many places mainly due to economic reasons. A major future
reforestation challenge for Denmark is the possibility to establish plantations based on
a wide range of relevant species (Jacobs et al. 2015). Figure 22. Climatic and soil conditions allow a wide range of species to be cultivated in Denmark. Figure 22. Climatic and soil conditions allow a wide range of species to be cultivated in Denmark 83 Reforesta Scientific Society REFORESTA (2016) 1:67-85 Mattsson A For this option, new methods and technology have to be developed in
reforestation activities that support this possibility. These methods and technology
should make it possible not to be limited to certain species due to problems and
restrictions during field establishment (Stanturf et al. 2014). In a forest with a wide range of species the risk of specific species being attacked
by, for example, a fungi disease will be limited. Also a wide range of species will in
general make the forest more stable, healthy and productive and in that way also more
adapted to future climate change (Bolte et al. 2009). With this I conclude my presentation of forestry in Scandinavia and the
reforestation challenges lying ahead. 7 References in: Parrotta J A, Moser C F,
Scherzer A J, Koerth N E, Lederle D R (eds). Sustaining Forests, Sustaining People: The role of
Research. XXIV World Congress, 5-11 October 2014, Salt Lake City, USA. The Commonwealth
Forestry Association, www.cfa-international.org 16 (5), pp 129. http://www.zephyr-project.eu/ Scherzer A J, Koerth N E, Lederle D R (eds). Sustaining Forests, Sustaining People: The role of
Research. XXIV World Congress, 5-11 October 2014, Salt Lake City, USA. The Commonwealth
Forestry Association, www.cfa-international.org 16 (5), pp 129. http://www.zephyr-project.eu/ Scherzer A J, Koerth N E, Lederle D R (eds). Sustaining Forests, Sustaining People: The role o Research. XXIV World Congress, 5-11 October 2014, Salt Lake City, USA. The Commonwealth Jacobs D F, Oliet J T, Aronson J, Bolte A, Bullock J M, Donoson P J, Landhausser S M, Madsen P, Peng S,
Rey-Benayas J M, Weber J C (2015) Restoring forest: What constitutes success in the twenty-first
century? New Forests 46: 601-614. Jacobs D F, Oliet J T, Aronson J, Bolte A, Bullock J M, Donoson P J, Landhausser S M, Madsen P, Peng S,
Rey-Benayas J M, Weber J C (2015) Restoring forest: What constitutes success in the twenty-first
century? New Forests 46: 601-614. Kohmann K (1999) Side-effects on formulations of permethrin and fenvalerate insecticides on frost
resistance and field performance of Picea abies seedlings. Scandinavian Journal of Forest
Research, Volume 14, Issue 4. Kohmann K (1999) Side-effects on formulations of permethrin and fenvalerate insecticides on frost
resistance and field performance of Picea abies seedlings. Scandinavian Journal of Forest
Research, Volume 14, Issue 4. Kohmann K (1999) Side-effects on formulations of permethrin and fenvalerate insecticides on frost
resistance and field performance of Picea abies seedlings. Scandinavian Journal of Forest
Research, Volume 14, Issue 4. Landis T D, Pinto J R, Dumroese R K (2013) Light-emitting diodes (LED): applications in forest and native
plant nurseries. Forest Nursery Notes 33: 5-13. Landis T D, Pinto J R, Dumroese R K (2013) Light-emitting diodes (LED): applications in forest and native
plant nurseries. Forest Nursery Notes 33: 5-13. Leppänen J, Nouro P (2006) Results and implications from a comparative study of Swedish and Finnish
forest sectors. Scandinavian Forest Economics 41: 147-159. Leppänen J, Nouro P (2006) Results and implications from a comparative study of Swedish and Finnish
forest sectors. Scandinavian Forest Economics 41: 147-159. 7 References Lilja A, Poteri M, Petäistö R-L, Rikala R, Kurkela T, Kasanen R (2010) Fungal diseases in forest nurseries in
Finland. Silva Fennica 44(3): 525-545. Lilja A, Poteri M, Petäistö R-L, Rikala R, Kurkela T, Kasanen R (2010) Fungal diseases in forest nurseries in
Finland. Silva Fennica 44(3): 525-545. Reforesta Scientific Society 84 REFORESTA (2016) 1:67-85 Mattsson A Lindström A, Mattsson A (1994) Cultivation of containerized seedlings in Sweden, ISHS Acta Horticulturae
361. Lindström A, Mattsson A (1994) Cultivation of containerized seedlings in Sweden, ISHS Acta Horticulturae
361. Långström B, Day K R (2004) Damage, control and management of weevil pests, especially Hylobius abietis. in: Lieutier F, Day K R, Battisti A, Grégoire J-C and Evans H F (eds.) Bark and wood boring insects
in living trees in Europe, a synthesis. Kluwer Academic Publishers, Dordrecht. pp 415-444. Långström B, Day K R (2004) Damage, control and management of weevil pests, especially Hylobius abietis. in: Lieutier F, Day K R, Battisti A, Grégoire J-C and Evans H F (eds.) Bark and wood boring insects
in living trees in Europe, a synthesis. Kluwer Academic Publishers, Dordrecht. pp 415-444. Nilsson U, Luoranen J, Kolström T, Örlander G, Puttonen P (2010) Reforestation with planting in northern
Europe. Scandinavian Journal of Forest research 25(4): 283-294 Nilsson U, Luoranen J, Kolström T, Örlander G, Puttonen P (2010) Reforestation with planting in northern
Europe. Scandinavian Journal of Forest research 25(4): 283-294 Nordic Forest Owners´ Association www.nordicforestry.org Nordic Forest Owners´ Association www.nordicforestry.org Norlander G, Hellqvist C, Johansson K, Nordenhem H (2011) Regeneration of European boreal forests:
Effectiveness of measures against seedling mortality caused by the pine weevil Hylobius abietis. Forest Ecology and Management 262: 2354-2363. Norlander G, Hellqvist C, Johansson K, Nordenhem H (2011) Regeneration of European boreal forests:
Effectiveness of measures against seedling mortality caused by the pine weevil Hylobius abietis. Forest Ecology and Management 262: 2354-2363. Petersson M, Örlander G (2003) Effectiveness of combinations of shelterwood, scarification and feeding
barriers to reduce pine weevil damage. Canadian Journal of Forest Research 33: 64-73 Petersson M, Örlander G (2003) Effectiveness of combinations of shelterwood, scarification and feeding
barriers to reduce pine weevil damage. Canadian Journal of Forest Research 33: 64-73 Samset I (1985) Winch and cable system. Forestry Sciences, Vol. 18, pp 540. Stanturf J A, Palik B A, Williams M A, Dumroese R K, Madsen P (2014) Forest restoration paradigms. Journal
of sustainable forestry 33: 161-194. Reforesta Scientific Society 7 References Stanturf J A, Palik B A, Williams M A, Dumroese R K, Madsen P (2014) Forest restoration paradigms. Journal
of sustainable forestry 33: 161-194. Torgersen H, Lisland T (2002) Excavator-based cable logging and processing system: A Norwegian case
study. Journal of forest engineering, Vol. 13 nr 1. Torgersen H, Lisland T (2002) Excavator-based cable logging and processing system: A Norwegian case
study. Journal of forest engineering, Vol. 13 nr 1. Valkonen S, Valsta L (2001) Productivity and economics of mixed two-storied spruce and birch stands in
southern Finland simulated with empirical models. Forest Ecology and Management, Volume
140, Issues 2-3: 133-149. Valkonen S, Valsta L (2001) Productivity and economics of mixed two-storied spruce and birch stands in
southern Finland simulated with empirical models. Forest Ecology and Management, Volume
140, Issues 2-3: 133-149. von Sydow F (1997) Abundance of pine weevils (Hylobius abietis) and damage to conifer seedlings in
relation to silvicultural practices. Scandinavian Journal of Forest Research 12: 157-167. von Sydow F (1997) Abundance of pine weevils (Hylobius abietis) and damage to conifer seedlings in
relation to silvicultural practices. Scandinavian Journal of Forest Research 12: 157-167. Reforesta Scientific Society Reforesta Scientific Society 85
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Caregiver Satisfaction with a Video Telehealth Home Safety Evaluation for Dementia
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International journal of telerehabilitation
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CAREGIVER SATISFACTION WITH A VIDEO
TELEHEALTH HOME SAFETY EVALUATION FOR 3DEPARTMENT OF PSYCHOLOGY, TUFTS UNIVERSITY, MEDFORD, MASSACHUSETTS, UNITED STATES
4AMERICAN OCCUPATIONAL THERAPY ASSOCIATION, BETHESDA, MARYLAND, UNITED STATES
5DEPARTMENT OF COMMUNITY HEALTH, TUFTS UNIVERSITY, MEDFORD, MASSACHUSETTS, UNITED STATES
6DEPARTMENT OF NEUROLOGY, HARVARD MEDICAL SCHOOL, BOSTON, MASSACHUSETTS, UNITED STATES International Journal of Telerehabilitation ABSTRACT Family caregivers are vital to telehealth-delivered dementia care. The objective of this mixed methods descriptive study
conducted in the VA Bedford Healthcare System was to examine caregiver satisfaction with a video telehealth dementia
home safety occupational therapy evaluation. Ten caregivers of Veterans with dementia participated. Ratings of caregiver
satisfaction, measured by nine Likert scale items including ability to see and hear, were examined in relation to person and
visit-related contextual factors extracted from research assistants’ field notes, to develop an in-depth understanding of
caregiver experience. Person factors included caregiver age and gender and Veteran cognitive status. Visit-related
contextual factors included occurrence of technical glitches. Caregiver visit satisfaction was overall positive, with exceptions
related to technological glitches and the presence of the person with dementia during the visit. Veteran cognitive status
appeared to influence caregiver satisfaction. Implications of the study are that proactively addressing technical glitches and
incorporating dementia stage-specific approaches may optimize caregivers’ telehealth experience. Keywords: Caregivers, Dementia, Safety, Telemedicine day functioning. The person domain embodies individuals’
roles, identities, and health status. The environment
includes physical structures and sociocultural factors, such
as the social network. Occupation refers to the tasks a
person wants and needs to do. Imbalance in any one
domain reduces occupational performance or day-to-day
functioning (Law et al., 1996). This model emphasizes the fit
between the individual and their preferred activities within
the home setting to optimize functional performance. Adjustments to any of the domains of PEO can influence
both safety and functional performance. Individuals with dementia experience progressive
cognitive decline affecting judgment, communication,
emotional, psychological, and motor functioning. These
changes lead to increased and evolving safety risks within
the home environment, necessitating home modifications. Home safety evaluations for dementia are performed in the
context of progressive cognitive deficits that affect individual
functioning. As such, they are ideally client-centered,
reflecting complex person-environment-occupation factors
affecting individuals with dementia and their caregivers
(Struckmeyer & Pickens, 2016). The Person-Environment-Occupation (PEO) model
highlights that everyday functioning is the result of optimal
interaction between the person, the environment, and the
occupation (target tasks). PEO describes these overlapping,
inextricably linked domains which are involved with day-to- For individuals with dementia, caregivers are central to
this PEO transaction. Caregivers increasingly speak for the
person with dementia, and are influential to their health and
well-being, particularly as cognitive status worsens. Similarly, the level of home safety risk due to dementia Vol. 12, No. International Journal of Telerehabilitation • telerehab.pitt.edu International Journal of Telerehabilitation • telerehab.pitt.edu PARTICIPANTS Video telehealth is care in which patient and provider
are in two locations synchronously connected via
videoconferencing. Video telehealth may increase access to
dementia care; however, in-home video telehealth for
dementia is undeveloped. Our scoping review of in-home
video telehealth identified primarily time-limited, protocolized
caregiver support programs (Gately et al., 2019). No study
included a home safety evaluation, which is a complex
intervention involving a room-by-room assessment. Though
video telehealth has been employed for home safety
(Renda, 2018; Sanford et al., 2009), no study has relied on
a caregiver of a person with dementia to operate technology
in the home. This study aims to address this gap by
examining caregiver satisfaction of a video telehealth
delivered home safety evaluation in relation to PEO factors. A convenience sample of self-identified family
caregivers of community-dwelling Veterans with dementia
with a scheduled visit at either the in-person or video
telehealth dementia management clinics at VA Bedford
Healthcare System participated. Caregivers could embody
one of a variety of roles (e.g., spouses, adult children,
friend) and were not required to live with the Veteran. Given
the cognitive and physical demands of the telehealth
evaluation (which involved navigating the home while
holding a portable computing device under clinician
instructions), caregivers, rather than Veterans with
dementia, were study participants. Caregivers needed to be
English-speaking, have basic computer skills (e.g., ability to
email), normal or corrected-to-normal vision and hearing,
and adequate mobility to navigate the home, which was
determined through self-report during the informed consent
process once the study procedures were described. There
were no other inclusion or exclusion criteria. To directly link the PEO model to caregivers’
satisfaction of a video telehealth home safety evaluation, we
draw from the work of Lee and Coughlin (2015) about
technology adoption by older adults. They identified
interrelated factors— Individual, Social, Technology, and
Delivery—found to influence older adults’ adoption of
technological solutions and strategies. Their Individual and
Social factors represent in our study, Person characteristics
of the caregiver and care recipient dyad that are relevant to
technology adoption. In our study, their Technology factor
represents the Environmental and Occupational
characteristics relevant to the video telehealth home safety
intervention, including use of technology. The Delivery factor
represents broader health care delivery, including clinician
and organizational factors that impinge upon or promote
successful adoption of technological innovations like video
telehealth. PARTICIPANTS For our study the PEO model guided our
selection and description of our study measures, and Lee
and Coughlin’s model guided suggested adaptations to
video telehealth home safety evaluations, based on our
findings (Lee & Coughlin, 2015). DESCRIPTION OF THE INTERVENTION Prior to the video telehealth home safety evaluation,
caregivers received a standard, in-person, non-technological
evaluation to expose caregivers to a typical home
evaluation. Trained graduate-level research assistants
(RAs) administered both types of evaluations under
supervision of the principal investigator (PI). In-person and
video evaluations followed similar procedures: a brief
interview to ascertain home safety concerns followed by
room-by-room assessment using a checklist ("Worksheet for
making the home safer for a person with memory loss,"
2019). The checklist included items related to whether
walkways and stairs were free of clutter and presence of
firearms, for example. For the video telehealth home safety
evaluations, caregivers navigated the home while holding a
laptop or tablet under verbal direction of the study staff (PI-
RARA) based at the hospital. During the evaluation,
caregivers went up and down stairs and outside the home,
as appropriate and feasible, under remote RA instruction. Study staff monitored participants for adverse events or
unforeseen hazards during procedures and were prepared ABSTRACT 2 Fall 2020
• (10.5195/ijt.2020.6337 35 International Journal of Telerehabilitation • telerehab.pitt.edu behaviors such as wandering, and the degree to which
caregivers are confident in promoting home safety, influence
the relative success of home safety strategies (Horvath et
al., 2005). All of these complex PEO factors are relevant to
delivery of dementia home safety evaluations. Though
occupational therapist-led home safety evaluations are the
gold standard (CDC, 2019; Maggi et al., 2018; Pighills et al.,
2016), in that occupational therapy (OT) practitioners are
trained to consider the complex interaction of PEO factors,
OT-led home safety evaluations are often not available (Lin
et al., 2015). Veterans with dementia. The primary aims of the initial study
were to examine feasibility of the telehealth encounter. Complete study details are reported elsewhere (Gately et
al., 2020). Here we examine caregiver satisfaction with the
video telehealth home safety evaluation, through in-depth
examination of caregiver satisfaction relative to person,
environment, and occupation (PEO) factors. The study was
approved by VA Bedford Healthcare System Institutional
Review Board. International Journal of Telerehabilitation METHODS This mixed methods descriptive analysis involves data
drawn from a previously published study of video telehealth
dementia home safety evaluations provided to caregivers of 6
International Journal of Telerehabilitation Vol. 12, No. 2 Fall 2020
• (10.5195/ijt.2020.6337) 36 nternational Journal of Telerehabilitation • telerehab.pitt.edu International Journal of Telerehabilitation • telerehab.pitt.edu to assist and/or notify emergency responders as needed. Video telehealth home evaluations were conducted
approximately four days after in-person home evaluations. determine the severity of an incident. Thus, potential scores
on the questionnaire range from 0 to undetermined with an
indeterminate maximum score. For this study, caregivers
were asked at baseline to provide the total number of
Veteran Risky Behaviors in the prior month. The Confidence in Caregiving Scale includes the 12-
item Home Safety sub-scale, created by researchers at VA
Bedford Healthcare System (Horvath et al., 2013). The
Home Safety sub-scale asks caregivers to rate on a scale of
0-100 their perceived confidence in preventing dementia
home safety behaviors such as wandering and eating non-
food items. FACTORS See Table 1 for a list of study variables by PEO factor. Caregiver age, gender, race, and relationship to the
Veteran, were gathered via standard demographic
questionnaire. Veteran factors, including gender, age, racial
and ethnic self-designations, and cognitive status, which
was represented by most recent Mini-Mental Status
Examination (MMSE) score, were gathered via chart review. MMSE scores to indicate dementia stage included 20 to 24
for mild dementia, 13 to 20 for moderate dementia, and less
than 12 indicating severe dementia (Folstein, 1975). Dementia-specific person factors included Veteran
dementia risky behaviors and caregiver confidence in
addressing home safety in dementia, which were gathered
at baseline using two standardized measures: Risky
Behaviors Questionnaire and Confidence in Caregiving
Scale. OUTCOME MEASURE
CAREGIVER SATISFACTION We employed an investigator-developed, nine-item visit
satisfaction questionnaire utilized by co-author, Moo, in her
study of in-home video telehealth for dementia (Moo et al.,
2014). Items gathered information about caregivers’ ability
to see, hear, communicate with, and understand the
provider; their comfort using technology and whether there
was enough technical assistance; whether the visit was
sufficiently private; whether the visit was an efficient use of
time; and their visit format preference. The questionnaire
employed a five-point Likert scale of agreement ranging
from Strongly Disagree (1) to Strongly Agree (5). For all
items, stronger agreement indicated a more positive
experience. Caregivers completed the questionnaire over
video telehealth or phone with the PI (without the RA
present) immediately following video telehealth home safety
evaluations. RAs also completed field notes immediately following
evaluations. Field notes included brief descriptions of who
was present during evaluations, any technological
difficulties, and other perceived challenges. Table 1
Summary of Variables by PEO Domain
Variable
Source
PEO
domain
Caregiver
demographics, e.g.,
age, gender
Demographic
questionnaire
P, E
Veteran demographics,
e.g., age, cognitive
status
Chart review
P
Veteran risky behaviors
Risky Behaviors
Questionnaire
P, E
Caregiver confidence in
home safety
Confidence in
Caregiving Scale
P, E
Visit details of the home
safety evaluation
Field notes
E
Caregiver satisfaction of
virtual home evaluation
Satisfaction
questionnaire
P, E, O
N t
Pl
M
h d f
h
h
i bl PERSON, ENVIRONMENT, OCCUPATION International Journal of Telerehabilitation DATA ANALYSIS The Risky Behaviors Questionnaire is a one-page, 22-
item checklist developed for use in the clinical trial of a
dementia Home Safety Toolkit (Horvath et al., 2013). This
questionnaire has demonstrated content validity and gathers
behaviors common in dementia, such as instances of
wandering and sleep disturbance. Designed to capture data
at baseline and biweekly for three months, the outcome is
the summed total of risky behaviors that occurred. Individual
behaviors are not weighted because it is difficult to Veteran and caregiver demographics, risky behaviors,
caregiver confidence, and caregiver visit satisfaction scores
were examined and summarized using descriptive statistics. Total scores or individual item scores were examined
participant-by-participant to explore systematic variation
among variables. Specifically, caregiver visit satisfaction
scores on each item were compared with caregiver and Vol. 12, No. 2 Fall 2020
• (10.5195/ijt.2020.6337 37 International Journal of Telerehabilitation • telerehab.pitt.edu Veteran demographic factors, risky behaviors, and caregiver confidence. Caregiver satisfaction scores were ordered from
lowest to highest by risky behavior scores and Veteran variables such as age and cognitive status. Any similarities,
differences, and patterns between satisfaction scores and person factors such as caregiver age and Veteran risky behaviors
were noted. RA field notes were analyzed using conventional content analysis (Hsieh & Shannon, 2005) whereby visit notes
were repeatedly read by the PI with the sole purpose of helping to explain the few instances of lower caregiver satisfaction. Note. Variables sorted by Veteran MMSE score. Identification numbers listed are from our manuscript related to technical
feasibility (Gately et al., 2020) . 1Total number of Veteran risky behaviors, e.g., instances of wandering, falls, etc., in the past
month. 2Average of 12-items on Home Safety Confidence in Caregiving sub-scale. Abbreviations: MMSE, Mini-Mental Status
Examination. PARTICIPANT CHARACTERISTICS Table 2 shows Veteran-caregiver characteristics ranked by Veteran’s MMSE score which ranged from 3 to 22 (out of 30). Six caregivers were spouses and four adult children. Caregiver age ranged from 54 to 71 (average 62.8 years old). Eight
caregivers were female and two were male. Most Veterans (90%) were male. All but two caregivers lived with the Veteran. Self-reported caregiving hours ranged from eight to 133 hours per week, with most caregivers (60%) providing over 100 hours
of caregiving per week, while caregiving duration ranged from 18 to 144 months. Veteran cognitive status varied, with most
Veterans (60%) in the mild-to-moderate stages of dementia, as indicated by MMSE score >12. Most caregivers (90%) reported
at least one Veteran risky behavior in the month prior to enrollment (range 0 to 81). Table 2 y person factor that appeared to relate to caregiver satisfaction was Veteran MMSE, in that caregivers of Veterans with severe dementia (MMSE <12) were more often
(Median across all items was 5 for three of four caregivers in this group, range 3-5) compared to caregivers of Veterans with mild-to-moderate dementia (Median across all
as 4 for five of six caregivers in this group, range 1-5). This pattern was observed even though Veterans with severe dementia had more risky behaviors than Veterans with
moderate dementia; Veterans with severe dementia (MMSE <12) had an average 38.3 risky behaviors (range 14-81) compared to those with mild-to-moderate dementia,
an average 15.2 risky behaviors (range 0-26) International Journal of Telerehabilitation • telerehab.pitt.edu Table 2 Table 2
Summary of Caregiver and Veteran Characteristics
ID
Veteran
MMSE
Veteran
Age
Veteran
1Risky
Behaviors
(Total)
Care-
giver
Age
Care-
giving
Hours/
Week
Care-
giving
Duration
(Months)
Caregiver
2Confidence
in
Home Safety
Caregiver
Gender
Caregiver
Role
2
3
69
38
67
133
120
98%
Female
Partner
7
9
70
14
63
120
18
80%
Female
Partner
5
9
67
81
62
114
50
93%
Female
Partner
3
11
90
20
57
120
96
86%
Male
Child
10
15
84
26
67
64
120
97%
Female
Partner
6
16
85
4
59
8
36
99%
Male
Child
4
17
83
16
54
48
96
56%
Female
Child
1
17
84
19
71
116
144
83%
Female
Partner
8
20
82
0
58
15
42
68%
Female
Child
9
22
74
26
70
133
24
91%
Female
Partner
Median 15.5
82.5
19.5
62.5
87.1
73
88.5
Note. Variables sorted by Veteran MMSE score. Identification numbers listed are from our manuscript related to technical
feasibility (Gately et al., 2020) . 1Total number of Veteran risky behaviors, e.g., instances of wandering, falls, etc., in the past
month. 2Average of 12-items on Home Safety Confidence in Caregiving sub-scale. Abbreviations: MMSE, Mini-Mental Status
Examination. Summary of Caregiver and Veteran Characteristics Note. Variables sorted by Veteran MMSE score. Identification numbers listed are from our manuscript related to technical
feasibility (Gately et al., 2020) . 1Total number of Veteran risky behaviors, e.g., instances of wandering, falls, etc., in the past
month. 2Average of 12-items on Home Safety Confidence in Caregiving sub-scale. Abbreviations: MMSE, Mini-Mental Status
Examination. 38
International Journal of Telerehabilitation
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• (10.5195/ijt.2020.6337) 38
International Journal of Telerehabilitation 38 International Journal of Telerehabilitation • telerehab.pitt.edu
GIVER SATISFACTION
le 3 shows caregiver satisfaction ratings. Most caregivers (80%) rated less than strong agreement (<5) on one or more visit satisfaction questions. However, low ratings
w, with all caregivers either strongly agreeing, agreeing, or being neutral about visit satisfaction for the following items: ease of communicating with the provider (Median = 4,
5); ability to understand provider (Median = 5, range 4-5); comfort with technology (Median = 5, range 4-5); and, enough technological assistance (Median = 4, range 3-5). CAREGIVER SATISFACTION Table 3 shows caregiver satisfaction ratings. Most caregivers (80%) rated less than strong agreement (<5) on one or more visit satisfaction questions. However, low ratings
were few, with all caregivers either strongly agreeing, agreeing, or being neutral about visit satisfaction for the following items: ease of communicating with the provider (Median = 4,
ange 3-5); ability to understand provider (Median = 5, range 4-5); comfort with technology (Median = 5, range 4-5); and, enough technological assistance (Median = 4, range 3-5). he only person factor that appeared to relate to caregiver satisfaction was Veteran MMSE, in that caregivers of Veterans with severe dementia (MMSE <12) were more often
atisfied (Median across all items was 5 for three of four caregivers in this group, range 3-5) compared to caregivers of Veterans with mild-to-moderate dementia (Median across all
ems was 4 for five of six caregivers in this group, range 1-5). This pattern was observed even though Veterans with severe dementia had more risky behaviors than Veterans with
mild-to-moderate dementia; Veterans with severe dementia (MMSE <12) had an average 38.3 risky behaviors (range 14-81) compared to those with mild-to-moderate dementia,
who had an average 15.2 risky behaviors (range 0-26) er Satisfaction with Video Telehealth Home Safety Evaluation
Veteran
MMSE
Able to
Hear
Able
to
See
Easy to
Communicate
Understood
Provider
Visit
Private
Enough
Comfort
Using
Tech
Enough
Technical
Assistance
Visit
Efficient
Use of Time
Prefer Video to
In-Person
Caregiver
Score
Across
Items
(Median)
3
3
3
3
4
4
4
4
5
3
4
9
5
5
4
5
5
5
4
5
3
5
9
5
5
5
5
5
5
5
5
3
5
11
5
5
5
5
5
—
—
5
3
5
15
5
2
5
5
5
5
5
5
3
5
16
4
4
5
5
4
5
3
5
3
4
17
5
4
4
5
1
5
4
4
1
4
17
4
4
4
4
4
4
4
4
2
4
20
2
4
3
4
5
4
3
4
3
4
22
4
4
4
4
4
4
5
2
2
4
n
4.5
4
4
5
4.5
5
4
5
3
4
Caregiver responses to visit satisfaction questionnaire. Responses were in five-point Likert scales of agreement ranging from Strongly Disagree (1) to Strongly Agree (5). ables sorted by Veteran MMSE. CAREGIVER SATISFACTION Identification numbers listed are from our manuscript relate to technical feasibility (Gately et al., 2020). Caregiver Satisfaction with Video Telehealth Home Safety Evaluation
ID
Veteran
MMSE
Able to
Hear
Able
to
See
Easy to
Communicate
Understood
Provider
Visit
Private
Enough
Comfort
Using
Tech
Enough
Technical
Assistance
Visit
Efficient
Use of Time
Prefer Video to
In-Person
Caregiver
Score
Across
Items
(Median)
2
3
3
3
3
4
4
4
4
5
3
4
7
9
5
5
4
5
5
5
4
5
3
5
5
9
5
5
5
5
5
5
5
5
3
5
3
11
5
5
5
5
5
—
—
5
3
5
10
15
5
2
5
5
5
5
5
5
3
5
6
16
4
4
5
5
4
5
3
5
3
4
4
17
5
4
4
5
1
5
4
4
1
4
1
17
4
4
4
4
4
4
4
4
2
4
8
20
2
4
3
4
5
4
3
4
3
4
9
22
4
4
4
4
4
4
5
2
2
4
Median
4.5
4
4
5
4.5
5
4
5
3
4
Note. Caregiver responses to visit satisfaction questionnaire. Responses were in five-point Likert scales of agreement ranging from Strongly Disagree (1) to Strongly Agree (5). All variables sorted by Veteran MMSE. Identification numbers listed are from our manuscript relate to technical feasibility (Gately et al., 2020). Caregiver Satisfaction with Video Telehealth Home Safety Evaluation Note. Caregiver responses to visit satisfaction questionnaire. Responses were in five-point Likert scales of agreement ranging from Strongly Disagree (1) to Strongly Agree (5). All variables sorted by Veteran MMSE. Identification numbers listed are from our manuscript relate to technical feasibility (Gately et al., 2020). International Journal of Telerehabilitation Vol. 12, No. 2 Fall 2020
• (10.5195/ijt.2020.6337 Vol. 12, No. 2 Fall 2020 39 International Journal of Telerehabilitation • telerehab.pitt.edu Three of the four instances of lower caregiver
satisfaction (disagree or strongly disagree) for caregivers 8,
9, and 10 appeared to relate to the environmental factor of
technological glitches. CAREGIVER SATISFACTION In the one report of difficulty hearing
the provider (caregiver 8), field notes indicated the visit
began with eight minutes of technical assistance for audio
difficulty, as “speakers on RA computer were not turned on.”
In the single report of difficulty seeing the provider (caregiver
10), field notes indicated the caregiver’s screen froze
towards the end of the visit and that “CG reported she did
not have view of RA for about 15 minutes.” The RA chose to
continue the visit, however, since she was still able to see
the caregiver and the home. Similarly, in the single report of
the visit not being an efficient use of time (caregiver 9), field
notes mentioned 70 minutes of technical assistance, which
included caregiver difficulty logging in. Field notes indicated
that the process of trouble-shooting “took a little over an
hour until RA was able to call CG successfully and connect,
but with no audio,” at which point the caregiver opted to use
a laptop for video and a phone for audio. Details of all
technological glitches encountered are reported elsewhere
(Gately et al., 2020). Returning to PEO, we found that the person factor of
cognitive status of the person with dementia appeared to
influence caregiver satisfaction ratings, as did the
environmental factors of the presence of the person with
dementia and technological glitches. Below are key findings
and strategies to optimize caregiver satisfaction, organized
by Lee and Coughlin’s (2015) technology adoption factors:
Individual and Social, Technology, and Delivery. Similar to
PEO, these factors structure potential ways to optimize
video telehealth. DISCUSSION To our knowledge, this is the first study to examine
satisfaction with video telehealth delivered dementia-
focused home safety evaluations, specifically employing a
caregiver of the person with dementia to operate
technology. Actively involving caregivers of persons with
dementia in video telehealth aligns with recent evidence
highlighting the importance of caregiver and family-centered
models in dementia technological approaches (Sriram et al.,
2019). Since most persons with dementia live in the
community, engaging family caregivers to assist with
telehealth may increase access to care when sending paid
care staff into the home is not feasible. It is also in
accordance with the need for dementia technologies that
reduce risk and prevent negative outcomes (Astell et al.,
2019). INDIVIDUAL AND SOCIAL Overall positive caregiver satisfaction across caregiver
age, role, gender, and caregiving duration, including
substantial caregiving hours, suggests that in-home video
telehealth may be appropriate for caregivers with a variety of
PEO profiles. This aligns with prior work indicating openness
of caregivers of persons with dementia to technological
strategies (Lindauer et al., 2017). The finding that greater
satisfaction occurred in caregivers of Veterans with more
severe dementia despite there being more Veteran risky
behaviors, may indicate caregiver resilience as dementia
progresses (Harris, 2008). It may also suggest that
caregivers are more willing to endure technological glitches
of a virtual home safety evaluation when there is greater
perceived home safety risk. Given indications that
implementing behavioral modifications in the early stages
may increase carry-over into later stages (Harris, 2002), we
recommend intervening early in dementia to minimize home
safety risk. For the remaining instance of strong disagreement, the
presence of the person with dementia during the home
safety evaluation appeared to influence caregiver
satisfaction. In the single report of the evaluation not being
private enough (caregiver 4), field notes revealed that the
Veteran was shadowing the caregiver (accompanying her to
each area of the home during the evaluation) and appeared
“annoyed and confused by the purpose of the visit.” Of note,
this evaluation occurred later in the day, when increased
confusion in people with dementia is common. This
caregiver also strongly disagreed with preferring the video
telehealth evaluation. High caregiver satisfaction in communication domains
underscores the importance of clear, effective verbal
communication, which is even more pronounced in a home
safety evaluation than in other less mobile video telehealth. We found the process of a video telehealth home safety
evaluation required constant cueing and directing (e.g., tilt
the camera, pan more slowly) to better see the home for
accurate assessment (Gately et al., 2020). This process
occurred while caregivers were physically walking around
the home and holding remote computing devices, a highly
complicated process. Balancing the need to maximize
visualization of the environment with not overly stressing
caregivers by bombarding them with directions may
increase caregiver satisfaction. REFERENCES This study had several limitations, including a small
convenience sample, participants’ racial and ethnic
homogeneity, and the fact that Veterans with dementia were
mostly male. Larger studies with persons with dementia and
caregivers from more diverse racial and ethnic backgrounds
will broaden our understanding of the relationship between
PEO factors and experience of telehealth. In terms of
methodology, we have limited qualitative data, and due to
sample size, cannot demonstrate statistical significance. The PI, who was present during the evaluation,
administered the caregiver satisfaction scale, introducing
possible response bias. We also did not ask Veterans for
their visit satisfaction, whereas persons with dementia
express interest in being included in technological studies
(Meiland et al., 2017). Astell, A. J., Bouranis, N., Hoey, J., Lindauer, A., Mihailidis, A.,
Nugent, C., Robillard, J. M. (2019) Technology and dementia:
The future is now. Dementia and Geriatric Cognitive Disorders,
47(3), 131-139. https://doi.org/10.1159/000497800 Centers for Disease Control (CDC). (2019). Stopping Elderly
Accidents, Death and Injury (STEADI) Algorithm. https://www.cdc.gov/steadi/pdf/STEADI-Algorithm-508.pdf Folstein, M. F., Folstein, S.E., McHugh, P.R. (1975). "Mini-mental
state." A practical method for grading the cognitive state of
patients for the clinician. Journal of Psychiatric Research,
12(3), 189-198. Gately, M. E., Trudeau, S. A., & Moo, L. R. (2019). In-home video
telehealth for dementia management: Implications for
rehabilitation. Current Geriatrics Reports, 8(3), 239-249. https://doi.org/10.1016/0022-3956(75)90026-6 Gately, M. E., Trudeau, S. A., & Moo, L. R. (2020). Feasibility of
telehealth-delivered home safety evaluations for caregivers of
clients with dementia. OTJR: Occupation, Participation and
Health., 40(1), 42-49. https://doi.org/10.1177/1539449219859935 CONCLUSION Recognition of person-environment-occupation factors
will ensure client-centered video telehealth that is well-
received by populations contending with even the most
complex chronic conditions. We found the detection of
patterns in home safety evaluation caregiver satisfaction
was expedited by employing the PEO framework. This
framework can be utilized in larger, more controlled studies
of video telehealth. International Journal of Telerehabilitation DELIVERY Our findings suggest several considerations for health
care systems planning to deliver in-home video telehealth
home safety evaluations for dementia. Timing the evaluation
to avoid behavioral disturbances such as commonly occur
later in the day may optimize the experience for both
caregiver and person with dementia. Also, for our study,
caregivers were consumed with operating the technology
and thus were unable to monitor or supervise the person
with dementia. This may result in safety concerns if the
person with dementia cannot be left unattended. This
highlights the potential need for contingency planning, (e.g.,
having another person present in the home). International Journal of Telerehabilitation • telerehab.pitt.edu •
Prepare before the visit by offering the caregiver a
technology trial and discuss possible contingencies
such as having another person present to occupy
the individual with dementia during the visit. impact of technological glitches. This makes it more
important to proactively address or anticipate technological
needs by providing training (Waller et al., 2017) and
conducting a test session ahead of time. Having technical
support available may optimize a caregiver’s experience. Since user-friendly, easy-to-use technology increases older
adults’ willingness to utilize technology (Kerssens et al.,
2015; Wang et al., 2019), allowing caregivers to use their
own technological devices may soften the learning curve. •
Effective communication is paramount, given the
nature of technology-mediated communication and
the dynamic nature of the intervention. •
Streamline the technological experience by training
caregivers ahead of time and ensuring technical
support is available. •
Consider visit timing and potential safety concerns
for the person with dementia when relying on
caregivers to operate technology. •
Consider visit timing and potential safety concerns
for the person with dementia when relying on
caregivers to operate technology. TECHNOLOGY The negative impact of technological glitches on
caregiver satisfaction aligns with research highlighting
problems with technology as a barrier to technology for older
adults (Vaportzis et al., 2017). Caring for someone with
dementia is demanding which may amplify the negative • Vol. 12, No. 2 Fall 2020
• (10.5195/ijt.2020.6337) International Journal of Telerehabilitation 40 International Journal of Telerehabilitation • telerehab.pitt.edu International Journal of Telerehabilitation • telerehab.pitt.edu Human Development, 67(1), 43-61. https://doi.org/
10.2190/AG.67.1.c Meiland, F., Innes, A., Mountain, G., Robinson, L., van der Roest,
H., Garcia-Casal, J. A., Gove, D., Thyrian, J. R., Evans, S.,
Droes, R. M., Kelly, F., Kurz, A., Casey, D., Szczesniak, D.,
Dening, T., Craven, M. P., Span, M., Felzmann, H., Tsolaki,
M., & Franco-Martin, M. (2017). Technologies to support
community-dwelling persons with dementia: A position paper
on issues regarding development, usability, effectiveness and
cost-effectiveness, deployment, and ethics. JMIR
Rehabilitation and Assistive Technologies, 4(1), e1. https://doi.org/10.2196/rehab.6376 Hercegovac, S., Kernot, J., & Stanley, M. (2020). How qualitative
case study methodology informs occupational therapy
practice: A scoping teview. OTJR: Occupation, Participation
and Health., 40(1), 6-16. https://doi.org/10.1177/1539449219850123 Horvath, K. J., Hurley, A. C., Duffy, M. E., Gauthier, M., Harvey, R. M., Trudeau, S. A., Cipolloni, P. B., & Smith, S. J. (2005). Caregiver competence to prevent home injury to the care
recipient with dementia. Rehabilitation Nursing, 30(5), 189-
196. https://doi.org/10.1002/j.2048-7940.2005.tb00109.x Moo, L. R., Jafri, Z., & Morin, P. J. (2014). Home-based video
telehealth for veterans with dementia. Federal Practitioner,
31(12), 36-38. https://www.ncbi.nlm.nih.gov/pubmed/29398887 Horvath, K. J., Trudeau, S. A., Rudolph, J. L., Trudeau, P. A., Duffy,
M. E., & Berlowitz, D. (2013). Clinical trial of a home safety
toolkit for Alzheimer's disease. International Journal of
Alzheimer’s Disease, 913606. https://doi.org/10.1155/2013/913606 Pighills, A., Ballinger, C., Pickering, R., & Chari, S. (2016). A critical
review of the effectiveness of environmental assessment and
modification in the prevention of falls amongst community
dwelling older people. British Journal of Occupational Therapy,
79(3), 133-143. https://doi.org/10.1177%2F0308022615600181 Hsieh, H. F., & Shannon, S. E. (2005). Three approaches to
qualitative content analysis. Qualitative Heath Research,
15(9), 1277-1288. https://doi.org/10.1177%2F1049732305276687 Renda M, L. J. (2018). Feasibility and effectiveness of telehealth
occupational therapy home modification interventions. International Journal of Telerehabilitation, 10(1), 3-14. https://doi.org/10.5195/ijt.2018.6244 Kerssens, C., Kumar, R., Adams, A. E., Knott, C. C., Matalenas, L.,
Sanford, J. A., & Rogers, W. A. (2015). Personalized
technology to support older adults with and without cognitive
impairment living at home. American Journal of Alzheimer's
Disease and Other Dementias, 30(1), 85-97. https://doi.org/10.1177/1533317514568338 Sanford, J. A., Hoenig, H., Griffiths, P. C., Butterfield, T.,
Richardson, P., & Hargraves, K. (2009). A comparison of
televideo and traditional in-home rehabilitation in mobility
impaired older adults. Physical & Occupational Therapy in
Geriatrics, 25(3), 1-18. https://doi.org/10.1080/J148v25n03_01 Law, M., Cooper, B., Strong, S., Stewart, D., Rigby, P., & Letts, L. (1996). The Person-Environment-Occupation Model: A
transactive approach to occupational performance. IMPLICATIONS FOR
OCCUPATIONAL THERAPY
PRACTICE Harris, P. B. (2002). The person with Alzheimer's disease :
pathways to understanding the experience. The Johns
Hopkins University Press. http://works.bepress.com/phyllis_harris/5/ Our study suggests the following implications for video
telehealth home safety evaluations for dementia: Harris, P. B. (2008). Another wrinkle in the debate about successful
aging: The undervalued concept of resilience and the lived
experience of dementia. International Journal of Aging and 41 Vol. 12, No. 2 Fall 2020
• (10.5195/ijt.2020.6337 International Journal of Telerehabilitation • telerehab.pitt.edu Canadian
Journal of Occupational Therapy, 63(1), 9-23. https://doi.org/10.1177/000841749606300103 Sriram, V., Jenkinson, C., & Peters, M. (2019). Informal carers'
experience of assistive technology use in dementia care at
home: A systematic review. BMC Geriatrics, 19(1), 160. https://doi.org/10.1186/s12877-019-1169-0 Stake, R. E. (1995). The art of case study research. SAGE
Publishers. Lee, C., & Coughlin, J. F. (2015). PERSPECTIVE: Older adults'
adoption of technology: An integrated approach to identifying
determinants and barriers. Journal of Product Innovation
Management, 32(5), 747-759. https://doi.org/10.1111/jpim.12176 Struckmeyer, L. R., & Pickens, N. D. (2016). Home modifications for
people with Alzheimer’s disease: A scoping review. American
Journal of Occupational Therapy, 70, 7001270020. https://doi.org/10.5014/ajot.2015.016089 Lin, V., Zhang, X., & Dixon, P. (2015). Occupational therapy
workforce in the United States: Forecasting nationwide
shortages. Physical Medicine and Rehabilitation 7(9), 946-954. https://doi.org/10.1016/j.pmrj.2015.02.012 Vaportzis, E., Clausen, M. G., & Gow, A. J. (2017). Older adults
perceptions of technology and barriers to interacting with tablet
computers: A focus group study. Frontiers in Psychology, 8,
1687. https://doi.org/10.3389/fpsyg.2017.01687 Lindauer, A., Seelye, A., Lyons, B., Dodge, H. H., Mattek, N.,
Mincks, K., Kaye, J., & Erten-Lyons, D. (2017). Dementia care
comes home: Patient and caregiver assessment via
telemedicine. Gerontologist, 57(5), e85-e93. https://doi.org/10.1093/geront/gnw206 Waller, A., Dilworth, S., Mansfield, E., & Sanson-Fisher, R. (2017). Computer and telephone delivered interventions to support
caregivers of people with dementia: A systematic review of
research output and quality. BMC Geriatrics, 17(1), 265. https://doi.org/10.1186/s12877-017-0654-6 Maggi, P., de Almeida Mello, J., Delye, S., Ces, S., Macq, J.,
Gosset, C., & Declercq, A. (2018). Fall determinants and home
modifications by occupational therapists to prevent falls. Canadian Journal of Occupational Therapy, 85(1), 79-87. https://doi/10.1177/0008417417714284. Wang, S., Bolling, K., Mao, W., Reichstadt, J., Jeste, D., Kim, H. C.,
& Nebeker, C. (2019). Technology to support aging in place:
Older adults' perspectives. Healthcare (Basel), 7(2). https://doi.org/10.3390/healthcare7020060 Worksheet for making the home safer for a person with memory
loss. (2019, March). A guide for families: Keeping the person
with memory loss safer at home. https://www.va.gov/Geriatrics/docs/HOME_SAFETY_BOOKLE
T_March_2019.pdf International Journal of Telerehabilitation 2
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https://link.springer.com/content/pdf/10.1007/s10340-022-01586-1.pdf
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English
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Spotted cucumber beetle (Diabrotica undecimpunctata howardi) is attracted to vittatalactone, the pheromone of striped cucumber beetle (Acalymma vittatum)
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Journal of pest science
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cc-by
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Journal of Pest Science (2023) 96:21–27
https://doi.org/10.1007/s10340-022-01586-1 Journal of Pest Science (2023) 96:21–27
https://doi.org/10.1007/s10340-022-01586-1 BRIEF REPORT Abstract Spotted cucumber beetle (Diabrotica undecimpunctata howardi; also known as southern corn rootworm) is a pest feeding
on many crops, including cucurbits, corn (maize), peanuts, beans, and other crops and wild plants in North America. On
cucurbit crops, where it co-occurs with striped cucumber beetle (Acalymma vittatum), both are key pests that vector Erwinia
tracheiphila, a destructive pathogen that causes bacterial wilt. Here we deployed synthetic vittatalactone, the male-produced
aggregation pheromone of A. vittatum, as lures in the field, to measure attraction of the target and non-target insects. Both
sexes of D. u. howardi were strongly attracted to the vittatalactone lures (5–7.5-fold compared to blank trap) throughout
the growing season in Maryland and Virginia, USA. Moreover, D. u. howardi was more attracted to live A. vittatum males
feeding on squash plants than to plants with females or without any beetles, confirming the genuine organismal cross-
attraction between these two key pests. The kairomonal attraction to vittatalactone suggests that it functions as a “keystone
semiochemical,” mediating the colonization of cucurbits by multiple herbivore species. Such cross attraction could allow
for use of vittatalactone in management of multiple pest species, with potential reduction of pesticide use in cucurbit crops. Keywords Kairomone · Behavioral control · Cucurbitaceae · Chrysomelidae · Diabroticina Key message • Use of vittatalactone as a kairomone in host-finding by
the spotted-cucumber beetle confirms the latter species’
evolutionary connection to cucurbit hosts i
evolutionary connection to cucurbit hosts • Vittatalactone, pheromone of striped cucumber beetle,
is highly attractive to both sexes of spotted cucumber
beetle, in eastern USA • Attractiveness of vittatalactone to both sexes of both spe-
cies of cucumber beetles offers the potential for advances
in sustainable pest management of both species Spotted cucumber beetle (Diabrotica undecimpunctata howardi)
is attracted to vittatalactone, the pheromone of striped cucumber
beetle (Acalymma vittatum) . Haber1 · Kayla Pasteur1,2 · Filadelfo Guzman1 · Sean M. Boyle1,3 · Thomas P. Kuhar3 ·
d C. Weber1 Received: 27 September 2022 / Revised: 28 November 2022 / Accepted: 8 December 2022 / Published online: 3 January 2023
This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2022 Introduction Communicated by Cesar Rodriguez-Saona. * Ariela I. Haber
ariela.haber@usda.gov
* Donald C. Weber
don.weber@usda.gov
1
Invasive Insect Biocontrol and Behavior Laboratory, USDA
Agricultural Research Service, Beltsville, MD 20705, USA
2
Present Address: Comparative Pathobiology Dept., College
of Veterinary Medicine, Purdue University, West Lafayette,
IN 47907, USA
3
Department of Entomology, Virginia Tech, Blacksburg,
VA 24061, USA Communicated by Cesar Rodriguez-Saona. Communicated by Cesar Rodriguez-Saona. * Ariela I. Haber
ariela.haber@usda.gov
* Donald C. Weber
don.weber@usda.gov
1
Invasive Insect Biocontrol and Behavior Laboratory, USDA
Agricultural Research Service, Beltsville, MD 20705, USA
2
Present Address: Comparative Pathobiology Dept., College
of Veterinary Medicine, Purdue University, West Lafayette,
IN 47907, USA
3
Department of Entomology, Virginia Tech, Blacksburg,
VA 24061, USA Field experiments testing attraction to synthetic
vittatalactone Synthesis of the vittatalactone mixture containing eight
diastereomers with 2R, 3R configuration was performed
by method of Chauhan and Paraselli (2017) by contract of
USDA ARS with Chemveda Life Sciences, San Diego, CA. Gray rubber septa (1-F SS 1888 GRY, West Pharmaceutical
Services, Lititz, PA) were washed with hexane in a Sox-
hlet apparatus, and dried in an exhaust hood overnight. The
washed septa were then placed in a one-neck Norton flask,
and a hexane solution with 1 mg of vittatalactone mixture
and 0.6 ml hexane per septum added. The flask was rotated
in a rotary evaporator (without vacuum) until the liquid was
completely soaked into septa. Septa were then dried in an
exhaust hood for 1 h before deployment in the field. This lure
was shown to be highly attractive to A. vittatum in earlier
field trials (Weber 2018). Acalymma vittatum has a male-produced aggregation
pheromone, vittatalactone (Smyth and Hoffmann 2003;
Morris et al. 2005), that also attracts the cucurbit special-
ist squash bugs Anasa tristis and A. armigera (Brzozowski
et al. 2022; Weber et al. 2022). In the course of determining
the attractiveness of vittatalactone in various habitats and
seasons to A. vittatum and to nontarget insects, we noted
the attraction of D. u. howardi to vittatalactone synthetic
lures. In this report, we document this strong cross-species
kairomonal attraction both to synthetic lures and to live A. vittatum males on host cucurbit plants. i
Traps in all experiments were double-sided clear sticky
traps (STKY Dual Panel Adhesive Traps, Trécé Inc., Adair,
OK, USA), 15 × 30 cm, attached vertically with a large metal
binder clip to wooden stakes (2 × 2 cm) at ~ 45 cm height
(middle of trap above ground). Lures were affixed to the
“lure” treatment traps with a small metal binder clip, hung
from the large binder clip. Traps with and without lures
were paired for each assay, with traps collected and rerand-
omized, and lures changed each week. Traps were deployed
5 May–1 December 2020 on BARC South Farm, Beltsville,
Maryland (39.0175° N, 76.9455° W), and 19 May–1 Octo-
ber on Virginia Tech Kentland Farm, Whitethorn, Virginia
(37.2006° N, 80.5657° W). Communicated by Cesar Rodriguez-Saona. Spotted cucumber beetle (Diabrotica undecimpunctata
howardi; also known as southern corn rootworm) is a poly-
phagous pest feeding on cucurbits, corn (maize), peanuts,
beans, and many other horticultural and ornamental crops
in North America. Although adults are generalist feeders,
they prefer certain hosts, especially cucurbits (Cranshaw and
Shetlar 2018). This preference may arise from the fact that
D. u. howardi sequesters cucurbitacins, bitter defensive com-
pounds present in cucurbits that elicit a compulsive feed-
ing response in D. u. howardi. In addition to cucurbitacins
being used by D. u. howardi as a defense against predators * Ariela I. Haber
ariela.haber@usda.gov * Donald C. Weber
don.weber@usda.gov 1
Invasive Insect Biocontrol and Behavior Laboratory, USDA
Agricultural Research Service, Beltsville, MD 20705, USA 2
Present Address: Comparative Pathobiology Dept., College
of Veterinary Medicine, Purdue University, West Lafayette,
IN 47907, USA 3
Department of Entomology, Virginia Tech, Blacksburg,
VA 24061, USA (0121 (0123
1 3 Journal of Pest Science (2023) 96:21–27 22 collecting) at the borders of the previous season’s (2021)
squash fields, and from a newly planted squash field at the
USDA Beltsville Agricultural Research Center (BARC)
North Farm (Beltsville, Maryland, USA; 39.0328° W,
76.9316° W). Following field collection, we identified the
sex of each beetle and kept male and female beetles in a
greenhouse under a natural photoperiod (25–30 °C) in sepa-
rate cages provisioned with potted squash plants. We col-
lected beetles no more than two days prior to the start of an
experiment. (Metcalf and Lampman 1989), male D. u. howardi provide
them as a component of nuptial gifts to females (Tallamy
et al. 2000), helping to protect their eggs which are laid
underground. Diabrotica undecimpunctata howardi over-
winters as adults and is highly mobile, colonizing northern
latitudes over the course of the growing season. Its wide-
spread abundance, mobility and polyphagy pose major
challenges to pest management (Hoffmann and Zitter 1994;
Diver and Hinman 2008). A better understanding of D. u. howardi host location behavior is therefore needed to effec-
tively manage this pest. Volatile cues have shown promise for managing a number
of agricultural pests (e.g., Bruce et al. 2005; Weber et al. 2014; Morrison et al. 2016), and evidence suggests that vola-
tile cues could be used to also manipulate D. u. howardi
behavior for pest management. Diabrotica undecimpunc-
tata howardi males are attracted to a female-produced sex
pheromone, R-10-methyl-2-tridecanone (Guss et al. Communicated by Cesar Rodriguez-Saona. 1983),
and there is evidence that females, at least at close range,
base mating preferences on male-specific compounds (Brodt
et al. 2006). Both sexes are also attracted to cucurbit floral
compounds including 1,2,4-trimethoxybenzene and trans-
cinnamaldeyde (Jackson et al. 2005). They share this attrac-
tion with striped cucumber beetle (Acalymma vittatum), a
cucurbit specialist and key pest with which D. u. howardi
often co-occurs. Field experiments testing attraction to synthetic
vittatalactone At each site, four randomized
complete blocks, consisting of one trap baited with 1 mg
mixed vittatalactone, and one unbaited trap, were set up
adjacent to former and current year’s cucurbit crops, at a
separation of 10 m within blocks (pairs) and ≥ 10 m between
blocks. Fields were not treated with any insecticide or fun-
gicide applications. In the Maryland experiment, we identi-
fied captured D. u. howardi to sex and recorded the number
of males and females captured each week. In the Virginia
experiment, we recorded the total number of D. u. howardi
captured each week. 1 3 Plants and insects For all experiments, beetle collecting, and beetle feeding, we
used potted summer squash (Cucurbita pepo) cv. ‘Yellow
Crookneck’ (Johnny’s Selected Seeds, Fairfield, ME). We
sowed seeds directly into 2.8 L plastic pots (3 seeds per pot)
with peat/bark based growing medium (Pro-mix BK55, Pre-
mier Tech Horticulture, Rivière-du-Loup, Québec, Canada). Plants were kept in a greenhouse (25–30 °C, 16 L: 8 D) prior
to experimental set-up. We used A. vittatum collected in June 2022 from pot-
ted squash plants that we placed (exclusively for beetle 1 3 1 3 23 Journal of Pest Science (2023) 96:21–27 Field experiments testing attraction to volatiles
from live Acalymma vittatum howardi, A. vittatum, A. tristis, and A. armigera captured on
each sticky trap. howardi, A. vittatum, A. tristis, and A. armigera captured on
each sticky trap. We conducted two experiments in 2022 to test attraction
of D. u. howardi to olfactory cues produced by live striped
cucumber beetles. All treatments included potted squash
plants bagged with paint strainers (600 μm polyester mesh,
with c. 18 L maximum volume, Trimaco, Inc., Morrisville,
NC, USA) to prevent beetles from leaving plants and so
that insect and plant-produced cues were exclusively olfac-
tory. Each of these bait plants was placed inside of a cylin-
drical wire cage staked to the ground with two flanking
wire frames. To catch attracted beetles, we attached two
clear sticky traps horizontally using metal binder clips,
one to each of the frames (Figure S1).i Attraction to synthetic vittatalactone Diabrotica undecimpunctata howardi activity exhibits a
nonuniform pattern throughout the season; therefore, we
evaluated attraction to the vittatalactone lure versus the
unbaited lure based on the season-long sum of D. u. how-
ardi captured. We first used log-linear models in the Catmod
procedure in SAS 9.4 (SAS Institute 2016) with sex, lure
treatment, and block as variables for the Maryland study
and lure treatment and block as variables for the Virginia
study. All two- and three-way interactions with block were
nonsignificant. Therefore, we proceeded with binomial
exact tests to determine the statistical significance and
magnitude (with 95% confidence interval) of the treatment
effect (Lowry 2022; StatPages 2009). In addition, we used
Fisher 2 × 2 Exact tests (Lowry 2022) to test for interaction
of treatment*sex for Maryland captures, and for interaction
of treatment*location for total Virginia versus Maryland
captures. The first experiment took place from 10 to 21 June and
included four treatments: (1) four males on plant, (2) four
females on plant, (3) plant without beetles, and (4) blank
pot with growing medium. Because our early-season bee-
tle collection from the field was male-biased, this experi-
ment was set up as a completely randomized design with
seven replicates of the male treatment, five replicates
of the female treatment, and four replicates each of the
uninfested plant and blank pot treatments. These plants
were randomized along the borders of two fields on BARC
North Farm (10 plants per field). One field (39.0333° N,
76.9344° W) had been planted in the 2021 season with
yellow crookneck squash, and the other (39.0284° N,
76.9333° W) with zucchini (C. pepo). Potted plants were
spaced 10 m apart within each field. All potted plants were
late in their vegetative stage upon experimental set-up,
and most had begun flowering at the conclusion of the
experiment. To evaluate attractiveness of each treatment,
we counted the number of D. u. howardi, as well as A. vit-
tatum, the squash bug Anasa tristis, and the horned squash
bug A. armigera on each sticky trap. Attraction to volatiles from live Acalymma vittatum To determine whether the number of captured D. u. howardi
and A. vittatum differed by bait plant treatment, we con-
ducted separate generalized linear mixed models for each
species using SAS Proc Glimmix with treatment as the fixed
effect (four and five treatments total for the completely ran-
domized and blocked experiments, respectively). For the
completely randomized experiment, field was included as a
random effect, and for the blocked experiment, spatiotempo-
ral block was a random effect. Because we had count data,
both models assumed a negative binomial distribution and
used a log link function. When models indicated a significant
overall treatment effect, we followed up with Tukey–Kramer
tests at a Bonferroni-adjusted alpha level to determine which
pairwise treatments were significantly different at an experi-
mentwise α = 0.05. For the low numbers of Anasa spp. also
captured, we employed the Kolmogorov–Smirnov one-sam-
ple test (Lowry 2022) to test total captures. The second experiment took place from 28 June to 15
July along the border of the previous season’s zucchini field
(the latter field above). Because we speculated that floral
volatiles might play a role in D. u. howardi attraction, this
experiment included five treatments: (1) four males on veg-
etative plant, (2) four females on vegetative plant, (3) veg-
etative plant without beetles, (4) flowering plant without
beetles, (5) blank pot with growing medium. The vegetative
plants were in the cotyledon stage at the start of the experi-
ment, and had true leaves but had not yet started producing
flower buds at the experiment’s completion. Each flowering
plant had 1–3 flowers at a time throughout the experiment. This experiment was set up as eight spatiotemporal blocks:
four spatial blocks from 28 June-6 July, and another four
spatial blocks from 6 to 15 July. For the second temporal
block, we rerandomized plants in each spatial block and
replaced the sticky traps. We counted the number of D. u. Attraction to synthetic vittatalactone In Maryland, we captured a total of 450 male and 338 female
D. u. howardi across all traps. The sex*lure interaction was 1 3 3 Journal of Pest Science (2023) 96:21–27 24 unbaited traps (Fig. 1). In Virginia, we captured a total of
120 D. u. howardi (both sexes combined) across all traps. There were over 7.5 times as many D. u. howardi on vit-
tatalactone-baited traps as there were on unbaited traps
(P < 0.0001; Fig. 1). The site*lure interaction was nonsig-
nificant (Fisher Exact test P = 0.34), indicating that D. u. howardi response to the lure did not differ between Mary-
land and Virginia locations. nonsignificant (Fisher Exact test P = 0.77), indicating that
attraction to the vittatalactone versus unbaited lure was inde-
pendent of sex. There were over 5 times as many of both
male D. u. howardi (P < 0.0001) and female D. u. howardi
(P < 0.0001) at traps baited with vittatalactone relative to Fig. 1 Captures of Diabrotica undecimpunctata howardi on clear
sticky traps baited with 1 mg lure of mixed vittatalactone, versus
captures on unbaited clear sticky traps, during entire growing season
2020, in four randomized complete blocks at both Beltsville, Mary-
land, and Whitethorn, Virginia, USA. Error bars and ratio confidence
intervals are 95% exact binomial CIs; comparisons between sexes
(Maryland) and between sites are both non-significant by Fisher 2 × 2
Exact tests Fig. 2 Least squares mean
captures of Diabrotica undec-
impunctata howardi at caged
plants with either male or
female Acalymma vittatum, or
no beetles, or a blank pot. Error
bars show 95% confidence lim-
its; different letters above bars
indicate pairwise significant
differences based on Bonfer-
roni-corrected Tukey–Kramer
tests, P < 0.05. a: 10–21 June
2020, in completely randomized
design. b: 28 June through 15
July 2020, in four randomized
complete blocks Discussion Our results demonstrate that the eastern subspecies of spot-
ted cucumber beetle (D. u. howardi) is attracted to vittata-
lactone, the male-produced aggregation pheromone of the
striped cucumber beetle (A. vittatum). At all experimental
sites, traps baited with vittatalactone caught more D. u. how-
ardi than unbaited traps. In addition, squash plants with live
A. vittatum males attracted more D. u. howardi than plants
with A. vittatum females or with no beetles, confirming that
D. u. howardi is in fact attracted to the male-produced phero-
mone and not any different isomer present in the synthetic
lures. Furthermore, the stronger attraction of A. vittatum to
squash plants with A. vittatum males relative to other bait
plant treatments is consistent with previous work (Smyth
and Hoffmann 2003) and confirms that the male A. vittatum
on our bait plants produced a detectable pheromone sig-
nal. Although we captured only a few Anasa tristis and A. armigera individuals, captures were significantly higher on
plants with male A. vittatum, providing further support to
prior work demonstrating cross attraction of both species to
vittatalactone (Brzozowski et al. 2022; Weber et al. 2022). Here, we show vittatalactone attracts yet another cucurbit
pest, indicating the potential for use of vittatalactone as a
sustainable management strategy to manipulate the behavior
of multiple pests simultaneously. g g
p
p
To our knowledge, this is the first study to document
attraction of a generalist herbivore (D. u. howardi) to the
pheromone of a co-occurring specialist (A. vittatum). These
results likely reflect the selective advantage to D. u. howardi
of feeding on cucurbitacins. Our results suggest that vittata-
lactone has an inordinate influence on the pest complex of
cucurbits in North America, constituting a “keystone semio-
chemical” with effects on at least four important herbivores. This kairomonal attraction, and the availability of synthetic
vittatalactone, present a new opportunity for efficient man-
agement of multiple pests that minimizes both insecticide
use and nontarget effects. We suggest that vittatalactone be
further evaluated for potential attractiveness to other cucur-
bit pests, as well as for deployment in commercial cucurbit
fields as part of sustainable pest management strategy. The attraction of D. u. howardi to vittatalactone seems to
reflect the continued special role of plant hosts in the Cucur-
bitaceae in the life history of this very generalist feeder. Attraction to volatiles from live Acalymma vittatum The generalized linear model for the completely randomized
experiment indicated a significant treatment effect on D. u. howardi attraction (F3,15 = 6.06, P = 0.007). The number of
D. u. howardi captured on the male treatment was signifi-
cantly greater than the number captured at either the female
or blank treatment, with over six times as many captured in
the male treatment relative to the female treatment and over
seven times as many captured in the male treatment relative
to the blank treatment (Fig. 2a). Over four times as many D. u. howardi were captured in the male treatment relative to
the plant treatment, although this pairwise difference was
not statistically significant (Fig. 2a). i
Diabrotica undecimpunctata howardi attraction in the
blocked experiment also differed by treatment (F4,28 = 8.66,
P < 0.0001). The number of D. u. howardi attracted to the
male treatment was over three times greater than the number
attracted to the female treatment, over four times greater
than the number attracted to the flowering plant treatment,
and over twice as many as the number attracted to the veg-
etative plant and blank treatments each (Fig. 2b). Acalymma
vittatum attraction exhibited a strong treatment effect in Fig. 1 Captures of Diabrotica undecimpunctata howardi on clear
sticky traps baited with 1 mg lure of mixed vittatalactone, versus
captures on unbaited clear sticky traps, during entire growing season
2020, in four randomized complete blocks at both Beltsville, Mary-
land, and Whitethorn, Virginia, USA. Error bars and ratio confidence
intervals are 95% exact binomial CIs; comparisons between sexes
(Maryland) and between sites are both non-significant by Fisher 2 × 2
Exact tests 1 3
Fig. 2 Least squares mean
captures of Diabrotica undec-
impunctata howardi at caged
plants with either male or
female Acalymma vittatum, or
no beetles, or a blank pot. Error
bars show 95% confidence lim-
its; different letters above bars
indicate pairwise significant
differences based on Bonfer-
roni-corrected Tukey–Kramer
tests, P < 0.05. a: 10–21 June
2020, in completely randomized
design. b: 28 June through 15
July 2020, in four randomized
complete blocks 1 3 Journal of Pest Science (2023) 96:21–27 25 both experiments (completely randomized: F3,15 = 13.48,
P = 0.0002; blocked: F4,28 = 16.09, P < 0.0001), with attrac-
tion to the male treatment significantly greater than attrac-
tion to all other treatments (Figure S2). Discussion Genetic analyses estimate that the genera Acalymma and
Diabrotica diverged 45 (± 8, 95%CI) million years ago, and
that the polyphagous fucata group of Diabrotica, of which
D. u. howardi is a member, diverged from the oligophagous
virgifera group 17 (± 4) Mya (Eben and Espinosa de los
Monteros 2013). Although a D. u. howardi male-produced
pheromone has not been identified, the continued affinity
of D. u. howardi and other polyphagous Diabrotica spe-
cies for cucurbitacin consumption and sequestration (Eben
2022) parallels with our findings of kairomonal attraction
to the pheromone of the cucurbit specialist Acalymma. The
eavesdropping kairomonal cross-attraction provides, through Attraction to volatiles from live Acalymma vittatum In the completely
randomized experiment, we also captured only seven total A. tristis, and zero A. armigera. In the blocked experiment we
captured ten A. tristis and seven A. armigera. In each case, in
which Anasa were captured, numbers for the male treatment
were significantly higher than all other treatments (P < 0.01,
Kolmogorov–Smirnov one-sample test); see Figure S3. the related specialist beetles, a mechanism “tethering” the
polyphagous beetles to their ancestral plant hosts. Such
eavesdropping is likely favored by natural selection given
the value of cucurbitacins as defensive compounds against
fungi, nematodes, and various other natural enemies to all
life stages (e.g., Ferguson and Metcalf 1985; Tallamy et al. 1998; Eben 2022). The very strong evolutionarily-conserved bond between
the generalist Diabrotica and their ancestral cucurbit hosts
offers an opportunity for behavioral pest management using
a combined bait of the volatile vittatalactone as a distance
attractant and cucurbitacins as arrestants and feeding stimu-
lants. Baits containing cucurbitacins and small amounts of
pesticides have been shown to be effective at controlling D. virgifera (Metcalf et al. 1987; Lance and Sutter 1990; Chan-
dler 2003), but distance attractants such as vittatalactone
have never been included in these baits. We note that we
have not yet determined the vittatalactone loading neces-
sary to ensure that baits are more attractive than A. vittatum
aggregations in the field, and this is worthy of future study. In addition, diversity and host affiliations among the many
Diabrotica species are still not well-defined (Derunkov et al. 2015; Eben 2022), and it remains to be determined whether
there are other species with attraction to vittatalactone or
other semiochemicals of Acalymma species. However, the
discovery that two species of Anasa (squash bugs; Hemip-
tera: Coreidae) are also attracted to vittatalactone (Brzozo-
wski et al. 2022; Weber et al. 2022), shows that cross-attrac-
tion to vittatalactone is not limited to the Chrysomelidae or
even Coleoptera. This merits further study of vittatalactone’s
attractiveness among generalist and specialist cucurbit pests.i Declarations Conflict of interest The authors declare they have no competing in-
terests. Hoffmann MP, Zitter TA (1994). Cucumber beetles, corn rootworms,
and bacterial wilt in cucurbits. Vegetable MD Online, Cornell
Cooperative Extension. http://vegetablemdonline.ppath.cornell.
edu/factsheets/Cucurbit_Beetles.htm Ethical approval This article does not contain any studies with verte-
brates performed by the authors. SAS Institute (2016) SAS® 9.4. SAS Institute Inc, Cary, NC. Jackson DM, Sorensen KA, Sorenson CE, Story RN (2005) Monitoring
cucumber beetles in sweetpotato and cucurbits with kairomone-
baited traps. J Econ Entomol 98:159–170. https://doi.org/10.1093/
jee/98.1.159 Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article's Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article's Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Lance DR, Sutter GR (1990) Field-cage and laboratory evaluations of
semiochemical-based baits for managing western corn rootworm
(Coleoptera: Chrysomelidae). J Econ Entomol 83:1085–1090. https://academic.oup.com/jee/article-pdf/83/3/1085/2581214/
jee83-1085.pdf Lowry R (2022) VassarStats: website for statistical computation. http://
vassarstats.net Metcalf RL, Ferguson JE, Lampman R, Andersen JF (1987) Dry
cucurbitacin-containing baits for controlling Diabroticite bee-
tles (Coleoptera: Chrysomelidae). J Econ Entomol 80:870–875. https://academic.oup.com/jee/article-pdf/80/4/870/19233206/
jee80-0870.pdf Author contributions DCW conceived the study and designed synthetic vittatalac-
tone experiments, FG made vittatalactone lures, TPK and
SMB conducted experiments in Virginia, KP conducted syn-
thetic vittatalactone experiments in Maryland, AIH designed 1 3 Journal of Pest Science (2023) 96:21–27 26 Cranshaw W, Shetlar D (2018) Garden insects of North America: the
ultimate guide to backyard bugs. Second ed. Princeton University
Press. and conducted live A. vittatum experiments, AIH and DCW
analyzed data and wrote the first draft of the manuscript, and
all other authors read and approved the manuscript. Derunkov A, Prado LR, Tishechkin AK, Konstantinov AS (2015)
New species of Diabrotica Chevrolat Coleoptera: Chrysomelidae
Galerucinae and a key to Diabrotica and related genera results of
a synopsis of North and Central American Diabrotica species. J
Insect Biodivers 3(2):1–55. https://doi.org/10.12976/jib/2015.3.2 Supplementary Information The online version contains supplemen-
tary material available at https://doi.org/10.1007/s10340-022-01586-1. y
p
p
ect Biodivers 3(2):1–55. https://doi.org/10.12976/jib/2015. Acknowledgements We thank Ashot Khrimian (Research Chemist,
USDA ARS IIBBL) for overseeing the vittatalactone synthesis. We
are grateful to Alex Bier, Megan Herlihy, and Daniel Wilczek for col-
lection of a portion of the Maryland field traps, and to George Meyers
and the BARC-West Farm Crew for planting and cultivation of the
experimental fields. We also thank Brian Currin and Lucas Raymond
for collection and deployment of a portion of traps at the Virginia site. Diver S, Hinman T (2008) Cucumber beetles: organic and biorational
integrated pest management. National center for appropriate tech-
nology. www.attra.ncat.org Eben A (2022) Ecology and evolutionary history of Diabrotica bee-
tles—overview and update. Insects 13:156. https://doi.org/10.
3390/insects13020156 Eben A, de Los E, Monteros A (2013) Tempo and mode of evolution-
ary radiation in Diabroticina beetles (genera Acalymma, Cero-
toma, and Diabrotica). ZooKeys 332:207–321. https://doi.org/10.
3897/zookeys.332.5220 Funding This work was funded in part by Southern SARE research
grant # LS20-337. Funding This work was funded in part by Southern SARE research
grant # LS20-337. Ferguson JE, Metcalf RL (1985) Cucurbitacins: Plant derived defense
compounds for Diabroticites (Coleoptera: Chrysomelidae). J
Chem Ecol 11:311–318. https://doi.org/10.1007/BF01411417i Data availability The data supporting the results of this study will be
made available in the Dryad repository. Guss PL, Tumlinson JH, Sonnet PE, McLaughlin JR (1983) Identifica-
tion of a female-produced sex pheromone from the southern corn
rootworm, Diabrotica undecimpunctata howardi Barber. J Chem
Ecol 9:1363–1375. https://doi.org/10.1007/BF00994805f References Metcalf RL, Lampman RL (1989) The chemical ecology of Diabrotic-
ites and Cucurbitaceae. Experientia 45:240–247. https://doi.org/
10.1007/BF01951810f Brodt JF, Tallamy DW, Ali J (2006) Female choice by scent recognition
in the spotted cucumber beetle. Ethology 112:300–306. https://
doi.org/10.1111/j.1439-0310.2006.01158.x Morris BD, Smyth RR, Foster SP, Hoffmann MP, Roelofs WL, Franke
S, Francke W (2005) Vittatalactone, a beta-lactone from the
striped cucumber beetle, Acalymma vittatum. J Nat Prod 68:26–
30. https://doi.org/10.1021/np049751v Bruce TJ, Birkett MA, Blande J, Hooper AM, Martin JL, Khambay
B, Prosser I, Smart LE, Wadhams LJ (2005) Response of eco-
nomically important aphids to components of Hemizygia petiolata
essential oil. Pest Manag Sci 61:1115–1121. https://doi.org/10.
1002/ps.1102 Morrison WR, Lee DH, Short BD, Khrimian A, Leskey TC (2016)
Establishing the behavioral basis for an attract-and-kill strategy
to manage the invasive Halyomorpha halys in apple orchards. J
Pest Sci 89:81–96. https://doi.org/10.1007/s10340-015-0679-6 Brzozowski LJ, Weber DC, Wallingford AK, Mazourek M, Agrawal
AA (2022) Trade-offs and synergies in management of two co-
occurring specialist squash pests. J Pest Sci 95:327–338. https://
doi.org/10.1007/s10340-021-01379-y Smyth RR, Hoffmann MP (2003) A male-produced aggregation
pheromone facilitating Acalymma vittatum [F.] (Coleoptera:
Chrysomelidae) early-season host plant colonization. J Insect Beh
16:347–359. https://doi.org/10.1023/A:1024824025210i Chandler LD (2003) Corn rootworm areawide management program:
United States department of agriculture-agricultural research ser-
vice. Pest Manag Sci 59:605–608. https://doi.org/10.1002/ps.683 StatPages (2009) Exact binomial and poisson confidence intervals. https://statpages.info/confint.html Chauhan KR, Paraselli BR (2017) Eight diastereomers of vittatalactone
and methods of making, and methods of attracting Acalymma vit-
tatum. 2017 Aug 1. U.S. patent 9,718,797. i
Tallamy DW, Whittington DP, Defurio F, Fontaine DA, Gorski PM,
Gothro TW (1998) Sequestered cucurbitacins and pathogenicity
of Metarhizium anisopliae (Monilialeas Moniliaceae) on spotted 1 3 Journal of Pest Science (2023) 96:21–27 27 cucumber beetle eggs and larvae (Coleoptera: Chrysomelidae). Environ Entomol 27:366–372. https://doi.org/10.1093/ee/27.2.366 cucumber beetle eggs and larvae (Coleoptera: Chrysomelidae). Environ Entomol 27:366–372. https://doi.org/10.1093/ee/27.2.366 p
g
Tallamy DW, Gorski PM, Burzon JK (2000) Fate of male-derived
cucurbitacins in spotted cucumber beetle females. J Chem Ecol
26:413–427. https://doi.org/10.1023/A:1005461522609 p
g
Weber DC (2018) Field attraction of striped cucumber beetles to a
synthetic vittatalactone mixture. J Econ Entomol 111:2988–2991. https://doi.org/10.1093/jee/toy283 Publisher's Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations. p
g
j
y
Weber DC, Cabrera Walsh G, DiMeglio AS, Athanas MM, Leskey TC,
Khrimian A (2014) Attractiveness of harlequin bug, Murgantia
histrionica, aggregation pheromone: field response to isomers,
ratios, and dose. Weber DC, Haber AI, Pasteur K, Boyle SM, Kuhar TP, Cornelius M
(2022) Both the squash bug Anasa tristis and horned squash bug
Anasa armigera are attracted to vittatalactone, the aggregation
pheromone of striped cucumber beetle. Environ Entomol. https://
doi.org/10.1093/ee/nvac079 Publisher's Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations. References J Chem Ecol 40:1251–1259. https://doi.org/10.
1007/s10886-014-0519-9 1 3 1 3 3
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Mimicking the Neurotrophic Factor Profile of Embryonic Spinal Cord Controls the Differentiation Potential of Spinal Progenitors into Neuronal Cells
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Abstract Recent studies have indicated that the choice of lineage of neural progenitor cells is determined, at least in part, by
environmental factors, such as neurotrophic factors. Despite extensive studies using exogenous neurotrophic factors, the
effect of endogenous neurotrophic factors on the differentiation of progenitor cells remains obscure. Here we show that
embryonic spinal cord derived-progenitor cells express both ciliary neurotrophic factor (CNTF) and brain-derived
neurotrophic factor (BDNF) mRNA before differentiation. BDNF gene expression significantly decreases with their
differentiation into the specific lineage, whereas CNTF gene expression significantly increases. The temporal pattern of
neurotrophic factor gene expression in progenitor cells is similar to that of the spinal cord during postnatal development. Approximately 50% of spinal progenitor cells differentiated into astrocytes. To determine the effect of endogenous CNTF on
their differentiation, we neutralized endogenous CNTF by administration of its polyclonal antibody. Neutralization of
endogenous CNTF inhibited the differentiation of progenitor cells into astrocytes, but did not affect the numbers of neurons
or oligodendrocytes. Furthermore, to mimic the profile of neurotrophic factors in the spinal cord during embryonic
development, we applied BDNF or neurotrophin (NT)-3 exogenously in combination with the anti-CNTF antibody. The
exogenous application of BDNF or NT-3 promoted the differentiation of these cells into neurons or oligodendrocytes,
respectively. These findings suggest that endogenous CNTF and exogenous BDNF and NT-3 play roles in the differentiation of
embryonic spinal cord derived progenitor cells into astrocytes, neurons and oligodendrocytes, respectively. Citation: Nakamura M, Tsuji O, Bregman BS, Toyama Y, Okano H (2011) Mimicking the Neurotrophic Factor Profile of Embryonic Spinal Cord Controls the
Differentiation Potential of Spinal Progenitors into Neuronal Cells. PLoS ONE 6(6): e20717. doi:10.1371/journal.pone.0020717 Editor: Cesario V Borlongan, University of South Florida, United States of America Received March 9, 2011; Accepted May 8, 2011; Published June 17, 2011 Copyright: 2011 Nakamura et al. This is an open-access article distributed under the terms of the Creative Commons Attribut
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Funding: This work was supported by grants from Grants-in-Aid for Scientific Research from JSPS and the Ministry of Education, Culture, Sports, Science and
Technology of Japan (MEXT), the project for realization of regenerative medicine and support for the core institutes for iPS cell research from MEXT; Research
Fellowships for Young Scientists from the Japan Society for the Promotion of Science; Keio Gijuku Academic Development Funds; and by a Grant-in-aid for the
Global COE program from MEXT to Keio University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: masa@sc.itc.keio.ac.jp Mimicking the Neurotrophic Factor Profile of Embryonic
Spinal Cord Controls the Differentiation Potential of
Spinal Progenitors into Neuronal Cells Masaya Nakamura1*, Osahiko Tsuji1, Barbara S. Bregman2, Yoshiaki Toyama1, Hideyuki Okano3
1 Department of Orthopaedic Surgery, Keio University, Tokyo, Japan, 2 Department of Neuroscience, Georgetown University Medical Center, Washington, D. C., United
States of America, 3 Department of Physiology, Keio University, Tokyo, Japan Citation: Nakamura M, Tsuji O, Bregman BS, Toyama Y, Okano H (2011) Mimicking the Neurotrophic Factor Profile of Embryonic Spinal Cord Controls the
Differentiation Potential of Spinal Progenitors into Neuronal Cells. PLoS ONE 6(6): e20717. doi:10.1371/journal.pone.0020717 June 2011 | Volume 6 | Issue 6 | e20717 Editor: Cesario V Borlongan, University of South Florida, United States of America Neutralization of endogenous CNTF inhibited the
astrocytic differentiation of spinal progenitor cells To examine the role of endogenous CNTF in differentiation of
spinal progenitor cells, we studied the effect of neutralization of
endogenous CNTF on the fate of differentiating spinal progenitor
cells. Different
concentrations
(0–500 mg/ml)
of
anti-CNTF
antibody were applied to the differentiation medium at the time
of plating. Neutralization of endogenous CNTF by polyclonal
antibody (100 mg/ml and more) significantly decreased the
number of astrocytes to approximately 20% of the total number
of cells compared to 52% in control conditions (100 mg/ml normal
rat IgG) (Fig. 4C, D). In contrast, neutralization of endogenous
CNTF did not change the number of neurons or oligodendrocytes
(Fig. 4A, B, E, F). There are two possible explanations for these
findings. One is that neutralization of endogenous CNTF had an
effect
on the survival or proliferation of progenitor
cells. Alternatively, neutralization of CNTF inhibited the differentiation
of progenitor cells into astrocytes. Neurotrophic factor gene profile of spinal cord during
normal development The development and maturation of progenitor cells depend
not only on their genetic programming, but also on sequences and
contributions of various environmental signals such as neuro-
trophic factors that are appropriate to their developmental stages. How
neurotrophic
factors
precisely
control
cell
fate
and
orchestrate the generation of neurons, astrocytes and oligoden-
drocytes in vivo is not clearly understood. To determine how
neurotrophic factor gene expression is regulated in the spinal cord
during normal development, we examined the neurotrophic factor
gene expression profiles of developing spinal cord between E14
and P17 by RNase protection assay. A high level of NT-3 gene expression, a moderate level of
CNTF and a low level of BDNF were observed in E14 spinal cord,
but the bands for NGF, GDNF or NT-4 were undetectable
(Fig. 1A). These neurotrophic factors showed distinct temporal
patterns of their gene expression during embryonic and postnatal
development. The high level of NT-3 gene expression at E14
decreased sharply by birth and subsequently there was almost no
expression of NT-3 at P17 (Fig. 1D). BDNF gene expression,
which was low at E14, increased between E19 and P10 (Fig. 1C). Following the increase of BDNF gene expression, NGF and
GDNF gene expression increased (Fig. 1B, F). The temporal
pattern of NGF and GDNF gene expression was similar to that of
BDNF, but the magnitude of increase of NGF and GDNF gene
expression was less than that of BDNF. Levels of NGF, BDNF and
GDNF gene expression decreased simultaneously between P10
and P17. Consistent with this decrease of BDNF, GDNF and NGF
gene expression, CNTF gene expression increased significantly at
P17 (Fig. 1G). Neurotrophic factor gene profiles of spinal progenitor
cells during differentiation Progenitor cells were isolated from E14 rat spinal cord and
mechanically dissociated in culture medium. These cells expanded
to form ‘‘neurospheres’’ in the presence of EGF/FGF2 and over
95% of these cells were nestin-immunoreactive (Fig. 2A, B). BrdU-
labeling of these clusters confirmed that about 90% of the cells
proliferated as determined by nuclear BrdU corporation (Fig. 2C). At 7 div, these neurospheres were centrifuged and dissociated
mechanically. Single cells were subsequently re-seeded into EGF/
FGF2-containing medium. This procedure resulted in a second
generation of neurospheres. After 2–4 passages, these cells were
seeded into the PEI-coated flasks or coverslips with differentiation
medium (EGF/FGF2-free and 1%FBS) following dissociation. At
1 div over 90% of these cells were still nestin immunoreactive
(Fig. 2D, E) and there were also trkB and/or CNTFRa
immunoreactive (Fig 2F, G). In the differentiation medium, these
cells slowed or stopped dividing. At 7 div about 30% of these cells Spinal Progenitors and Endogenous Neurotrophin Spinal Progenitors and Endogenous Neurotrophin In the present study, we used ribonuclease (RNase) protection
assay to detect the expression of multiple neurotrophic factors
genes in both developing spinal cord and embryonic spinal cord
derived-progenitor cells. We used triple epitope immunocyto-
chemistry to determine the phenotypic fate of progenitor cells in
vitro under different treatments, including neutralization of
endogenous CNTF and/or administration of exogenous BDNF
or NT-3. The data indicate that endogenous CNTF and exogenous
BDNF and NT-3 play crucial roles in the differentiation of E14
spinal cord derived- progenitor cells into astrocytes, neurons and
oligodendrocytes, respectively. were labeled with BrdU during differentiation (Fig. 2H), and about
80% of these cells differentiated into neurons, astrocytes or
oligodendrocytes (Fig. 2I, J, K ). Quantitative analysis revealed that
majority of differentiated cells was astrocytes (52%), while fewer
cells differentiated into oligodendrocytes (23%) and neurons (4%). This suggests that conditioning of the medium by the differenti-
ating progenitor cells, especially endogenous neurotrophic factors
may enhance glial cell differentiation. y
g
To determine how neurotrophic factor gene expression is
regulated during the differentiation of E14 spinal cord derived
progenitor cells, we examined the neurotrophic factor gene
expression profiles of these cells by multi-probe RNase protection
assay. BDNF and CNTF gene expression were observed in spinal
progenitor cells before differentiation (0 div) (Fig. 3). During their
differentiation BDNF gene expression declined and then became
undetectable at 7 div. In contrast, CNTF gene expression
increased significantly at 3 div and by 7 div reached the levels 3-
fold greater than that of before differentiation (0 div). The
expression
of
NGF,
NT-3,
NT-4
or
GDNF
mRNA
was
undetectable at any time point examined. Thus, E14 spinal cord
derived progenitor cells showed a different pattern of neurotrophic
factor gene expression during differentiation compared to the
normal embryonic developing spinal cord (Fig. 1), including up-
regulation of CNTF gene expression, down-regulation of BDNF
gene expression and a lack of NT-3 gene expression. Interestingly,
this pattern of neurotrophic factor gene expression is similar to
that observed during postnatal development, and correlated
temporally to active gliogenesis. Furthermore, spinal progenitor
cells expressed CNTFR-immunoreactivity during differentiation. Taken together, these findings have led us to propose that CNTF
acts as an autocrine and/or paracrine signal involved in the
astrocytic differentiation from spinal progenitor cells. Introduction induces neuronal differentiation from embryonic striatum, subep-
endymal
zone
and
hippocampus
derived
progenitor
cells
[4,9,17,18]. Neurotrophin-3 (NT-3) induces neuronal differentia-
tion from embryonic cortex and hippocampus derived progenitor
cells [18,19]. Despite these extensive studies using the exogenous
application of neurotrophic factors, little is known about the effect
of endogenous
neurotrophic factors on the
differentiation
of
progenitor
cells. We
suggest
that
differentiation
of
neural
progenitor cells is regulated not only by exogenous level of growth
factors but rather, the endogenous expression of growth factors also
play crucial roles in the differentiation of neural progenitor cells. In this study, we sought to determine the extent to which
differentiation of spinal progenitor cells is altered by manipulation
of endogenous as well as exogenous neurotrophic factors in vitro. We
focused on the spinal progenitor cells because spinal progenitor
cells may be distinct from cortical progenitor cells [20]. A better
understanding of the control mechanism of spinal progenitor cell’s
fate should enhance efforts to develop effective transplantation
strategies aimed at restoring functional connectivity in the injured
spinal cord. Neural stem/progenitor cells are an ideal source of tissue for
neural transplantation, since these cells can be expanded in vitro,
maintained in an undifferentiated state and retain the capacity to
differentiate into neurons, astrocytes and oligodendrocytes [1,2,3]. Previous in vitro studies, however, showed that when neural
progenitor cells were permitted to differentiate, they gave rise to
mainly glial cells and only smaller fraction developed into neurons
[4,5,6,7,8]. Although the culture systems used by various authors
have differed with regard to the species, anatomic location, and
developmental age of the harvested tissue, there is growing
evidence to suggest that the choice of lineage is determined, at
least in part, by environmental factors, among which neurotrophic
factors play a potential modulating role in differentiation of
progenitor cells [2,9,10]. For example, the exogenous addition of
ciliary neurotrophic factor (CNTF) and subsequent gp130-signal
activation promotes the differentiation of astrocytes from embry-
onic hippocampus, cortex and spinal cord derived progenitor cells
[11,12,13,14,15,16]. Brain-derived neurotrophic factor (BDNF) PLoS ONE | www.plosone.org PLoS ONE | www.plosone.org PL June 2011 | Volume 6 | Issue 6 | e20717 June 2011 | Volume 6 | Issue 6 | e20717 1 PLoS ONE | www.plosone.org Neutralization of endogenous CNTF did not affect the
survival or proliferation of spinal progenitor cells doi:10 1371/journal pone 0020717 g001 expression profile during their differentiation compared to that of
the normal spinal cord during embryonic development: 1) there is
a down-regulation of endogenous BDNF gene expression and
2) there is a lack of endogenous NT-3 gene expression in the spinal
progenitor cells. In order to mimic the neurotrophic factor profile
of spinal cord during embryonic development, we applied
exogenous BDNF or NT-3 to culture cells in combination with
anti-CNTF polyclonal antibody. numbers between control and neutralization groups at 7 div
(Fig. 5A). Moreover, there were no significant differences in the
percentages of BrdU-positive cells between control and neutral-
ization groups at 7 div (Fig. 5B). In contrast, the percentage of
nestin-positive cells of the neutralization group was significantly
higher than that of control group at 7 div (p,0.05) (Fig. 5C). These
findings suggest that neutralization of endogenous CNTF did not
influence the survival or proliferation of the progenitor cells, but
rather inhibited the astrocytic differentiation of these cells,
resulting in the increase of the number of nestin-positive
progenitor cells. The administration of exogenous BDNF alone increased the
number of neurons, but did not change the number of either
astrocytes or oligodendrocytes (Fig. 6A, D, G). The administration
of exogenous NT-3 alone increased the number of oligodendro-
cytes, but did not change the number of either neurons or
astrocytes (Fig. 6A, D, G). However, the administration of
exogenous BDNF or NT-3 did not have any apparent effect on
the survival or proliferation of spinal progenitor cells (data not
shown). In combination with anti-CNTF antibody, the adminis-
tration of exogenous BDNF not only decreased the number of
astrocytes (Fig. 6D, E) but also increased the number of neurons
(Fig. 6A, B). There was, however, no significant change in the
number of oligodendrocytes (Fig. 6G, H). In combination with
anti-CNTF antibody, the administration of exogenous NT-3 not Neutralization of endogenous CNTF did not affect the
survival or proliferation of spinal progenitor cells To determine the effect of neutralization of endogenous CNTF
on survival and proliferation of spinal progenitor cells, we
examined the number of total cells and the percentage of nestin-
positive or BrdU-positive cell number to the total cell number. Since the application of 100 mg/ml anti-CNTF antibody signifi-
cantly decreased the number of astrocytes as mentioned above, the
subsequent experiments were performed for neutralization of
endogenous CNTF using administration of 100 mg/ml anti-CNTF
antibody. There was no significant difference in the total cell PLoS ONE | www.plosone.org June 2011 | Volume 6 | Issue 6 | e20717 2 Spinal Progenitors and Endogenous Neurotrophin Figure 1. Neurotrophic factor gene profile of spinal cord during development. Spinal cord tissues were harvested from embryos or
neonates at each developmental age. Total RNA (10 mg) was loaded at each lane for RNase protection assay (A). There were three different temporal
patterns of neurotrophic factor gene expression. NT-3 gene expression peaked at E14 and then declined sharply (D). BDNF gene expression (C),
followed by NGF (B) and GDNF (F), increased between E19 and P10. Initial moderate expression of CNTF increased significantly at P10 and P17 (G). Each value represented mean 6 SEM of % GAPDH obtained from four independent experiments. GAPDH was used as an internal control to normalize
for loading differences. * Significant difference compared to the other developmental ages except (*), # Significant difference compared to the other
developmental ages including (*) (p,0.05, Tukey post hoc test). doi:10.1371/journal.pone.0020717.g001 Figure 1. Neurotrophic factor gene profile of spinal cord during development. Spinal cord tissues were harvested from embryos or
neonates at each developmental age. Total RNA (10 mg) was loaded at each lane for RNase protection assay (A). There were three different temporal
patterns of neurotrophic factor gene expression. NT-3 gene expression peaked at E14 and then declined sharply (D). BDNF gene expression (C),
followed by NGF (B) and GDNF (F), increased between E19 and P10. Initial moderate expression of CNTF increased significantly at P10 and P17 (G). Each value represented mean 6 SEM of % GAPDH obtained from four independent experiments. GAPDH was used as an internal control to normalize
for loading differences. * Significant difference compared to the other developmental ages except (*), # Significant difference compared to the other
developmental ages including (*) (p,0.05, Tukey post hoc test). PLoS ONE | www.plosone.org Neutralization of endogenous CNTF and administration
of exogenous BDNF/NT-3 promote the differentiation
into neurons/oligodendrocytes Neutralization of endogenous CNTF inhibited the differentia-
tion of progenitor cells into astrocytes, but did not affect the
differentiation into neurons or oligodendrocytes, resulting in the
increase of the undifferentiated progenitor cells. These progenitor
cells
may
require
additional
environmental
cues
for
their
differentiation into neurons or oligodendrocytes. Spinal progenitor
cells show two major differences in their neurotrophic factor gene PLoS ONE | www.plosone.org June 2011 | Volume 6 | Issue 6 | e20717 June 2011 | Volume 6 | Issue 6 | e20717 3 Spinal Progenitors and Endogenous Neurotrophin Figure 2. Characterization of E14 spinal cord-derived progenitor cells. Progenitor cells obtained from E14 fetal spinal cord formed sphere in
the growth medium containing EGF/FGF2 (A: phase contrast image) and they were nestin (B: identical field to A) and BrdU immunoreactive (C). After
dissociation, these cells were seeded on PEI-coated coverslips in no EGF/FGF2 and 1%FBS containing culture medium (D: phase contrast image). At
1div, over 90% of the dissociated progenitor cells were still nestin immunoreactive (E: identical field to D) and there were also trkB (F) and/or CNTFRa
immunoreactive (G). These cells slowed or stopped dividing and during differentiation about 30% of the cells were labeled with BrdU at 7 div (H). At 7
div, these cells were triple-stained with anti-TuJ1 for neuron (I), GFAP for astrocyte (J) and O4 for oligodendrocyte (K). Approximately 50% of these
cells differentiated into astrocytes. doi:10.1371/journal.pone.0020717.g002 Figure 2. Characterization of E14 spinal cord-derived progenitor cells. Progenitor cells obtained from E14 fetal spinal cord formed sphere in
the growth medium containing EGF/FGF2 (A: phase contrast image) and they were nestin (B: identical field to A) and BrdU immunoreactive (C). After
dissociation, these cells were seeded on PEI-coated coverslips in no EGF/FGF2 and 1%FBS containing culture medium (D: phase contrast image). At
1div, over 90% of the dissociated progenitor cells were still nestin immunoreactive (E: identical field to D) and there were also trkB (F) and/or CNTFRa
immunoreactive (G). These cells slowed or stopped dividing and during differentiation about 30% of the cells were labeled with BrdU at 7 div (H). At 7
div, these cells were triple-stained with anti-TuJ1 for neuron (I), GFAP for astrocyte (J) and O4 for oligodendrocyte (K). Approximately 50% of these
cells differentiated into astrocytes. doi:10.1371/journal.pone.0020717.g002 only decreased the number of astrocytes (Fig. 6D, F) but also
increased the number of oligodendrocytes (Fig. 6G, I). Discussion Multipotent and self-renewing progenitor cells that generate
neurons and macroglia are found in most areas of the developing
nervous system [1]. Using a neurosphere culture system that allows
us to follow the enriched expansion of spinal neural progenitor
cells [5,21], we examined their fate when exposed to conditions
that mimic the environment of late embryonic spinal cord in terms
of neurotrophic factor profile: neutralization of endogenous CNTF
and administration of exogenous BDNF or NT-3. Our results
indicate that endogenous CNTF plays a role in differentiation of
spinal progenitor cells into astrocytes. Furthermore, the additional
administration of exogenous BDNF or NT-3 promotes their
differentiation into neurons or oligodendrocytes, respectively. This pattern of neurotrophic factor gene expression of spinal
progenitor cells in vitro is similar to that of the spinal cord during
postnatal development correlated with active gliogenesis, but
different
from
that
of
the
spinal
cord
during
embryonic
development correlated with neurogenesis. In fact, we demonstrated the increase of NGF, BDNF and
GDNF gene expression within the developmental spinal cord,
which corresponds temporally with the period of differentiation
and maturation of neurons [23–24], and also the simultaneous
down-regulation of NGF, BDNF and GDNF gene expression and
up-regulation of CNTF gene expression, which are coincident
with postnatal gliogenesis [24,25]. Neutralization of endogenous CNTF and administration
of exogenous BDNF/NT-3 promote the differentiation
into neurons/oligodendrocytes This
treatment did not affect the number of neurons (Fig. 6A, C). There
was a significant increase in the number of oligodendrocytes in the
cultures treated by NT-3 plus anti-CNTF antibody compared to
NT-3 alone (Fig. 6G). endogenous neurotrophic factors may play a role in regulating the
differentiation of spinal progenitor cells as well [9]. To the best of
our knowledge, this is the first demonstration that spinal
progenitor cells express a distinct profile of endogenous neurotrophic
factor gene expression during their differentiation. We demon-
strated that before differentiation, spinal progenitor cells expressed
prominent CNTF and BDNF mRNAs in vitro. During their
differentiation, endogenous BDNF gene expression significantly
decreased, whereas CNTF gene expression significantly increased. Mimicking of the neurotrophic factor profile of spinal
cord during embryonic development inhibits the
differentiation of spinal progenitor cells into astrocytes,
and promotes the differentiation into neurons and
oligodendrocytes Mimicking of the neurotrophic factor profile of spinal
cord during embryonic development inhibits the
differentiation of spinal progenitor cells into astrocytes,
and promotes the differentiation into neurons and
oligodendrocytes Under baseline culture conditions approximately 50% of E14
spinal cord-derived progenitor cells differentiated into astrocytes. This differentiation of spinal progenitor cells towards an astrocytic
phenotype may reflect autocrine and/or paracrine interactions
between these cells. Such an autocrine mechanism of neurotrophic
factor (NT-3 and BDNF) appears to regulate neurogenesis in
progenitor cells isolated from the E14 mouse cortex [19,22]. Thus, Spinal progenitor cells were prepared from E14 embryos, a time
when active neurogenesis occurs [23,24]. Neurotrophic factor
gene expression of spinal progenitor cells in vitro differed from that
of the embryonic spinal cord during normal development in vivo. The spinal progenitor cells had higher levels of CNTF, and lower PLoS ONE | www.plosone.org June 2011 | Volume 6 | Issue 6 | e20717 4 Spinal Progenitors and Endogenous Neurotrophin Figure 3. Neurotrophic factor gene profile of E14 spinal cord derived-progenitor cells during differentiation in vitro. After 2–4
passages, cells were seeded on the PEI-coated culture flasks at a density of 26105 cells/ml in the differentiation medium (no EGF/FGF2, 1% FBS). Culture cells were harvested at 1, 3 and 7 days in vitro (div). As a control cells were harvested immediately after dissociation (0 div). Total RNA (5 mg)
was loaded at each lane for RNase protection assay. Before differentiation (0 div), there were two prominent gene expression of BDNF and CNTF. While BDNF gene expression decreased significantly at 3 and 7 div, CNTF gene expression increased significantly at 3 and 7 div. Each value
represented mean 6 SEM of % GAPDH obtained from three independent experiments. GAPDH was used as an internal control to normalize for
loading differences. * Significant difference compared to 0 div (p,0.05, Tukey post hoc test). doi:10.1371/journal.pone.0020717.g003 Figure 3. Neurotrophic factor gene profile of E14 spinal cord derived-progenitor cells during differentiation in vitro. After 2–4
passages, cells were seeded on the PEI-coated culture flasks at a density of 26105 cells/ml in the differentiation medium (no EGF/FGF2, 1% FBS). Culture cells were harvested at 1, 3 and 7 days in vitro (div). As a control cells were harvested immediately after dissociation (0 div). Total RNA (5 mg)
was loaded at each lane for RNase protection assay. Mimicking of the neurotrophic factor profile of spinal
cord during embryonic development inhibits the
differentiation of spinal progenitor cells into astrocytes,
and promotes the differentiation into neurons and
oligodendrocytes Before differentiation (0 div), there were two prominent gene expression of BDNF and CNTF. While BDNF gene expression decreased significantly at 3 and 7 div, CNTF gene expression increased significantly at 3 and 7 div. Each value
represented mean 6 SEM of % GAPDH obtained from three independent experiments. GAPDH was used as an internal control to normalize for
loading differences. * Significant difference compared to 0 div (p,0.05, Tukey post hoc test). doi:10.1371/journal.pone.0020717.g003 in vivo, it is difficult to determine the function of endogenous
CNTF by these knockout mice studies. Our in vitro data indicate
that endogenous CNTF plays a role in inducing the differentiation of
spinal progenitor cells into astrocytes, but does not effect their
survival or proliferation. A possible explanation of this discrepancy
between previous knockout mice studies and our in vitro study is
that the redundancy of neurotrophic factors in vivo was not
apparent in our cultures because these progenitor cells became
more homogenous, at least morphologically, after 2-4 passages. levels of BDNF gene expression and failed to express NT-3. We
suggest that these differences in the balance of neurotrophic factors
environment may promote the differentiation of spinal progenitor
cells in vitro into astrocytes. We hypothesize that by altering the
neurotrophic factor profile that neural progenitor cells are exposed
to in vitro to mimic that of the embryonic spinal cord in situ would
inhibit astrocytic differentiation and promote neural differentiation
of the spinal progenitor cells. First, we showed that neutralization of endogenous CNTF
inhibited
the differentiation of spinal progenitor
cells into
astrocytes, resulting in the increase of the number of undifferen-
tiated progenitor cells that were nestin-positive. Neutralization of
endogenous CNTF did not affect the survival or proliferation of
spinal
progenitor
cells. Consistently,
previous
studies
have
demonstrated that administration of exogenous CNTF stimulates
the two signal-transducing subunits of the CNTFRa-leukemia
inhibitory factor receptor b (LIFRb) and gp130 and induces the
differentiation of progenitor cells into astrocytes [11,15], and that
there was a significant reduction in the numbers of neurons and
astrocytes within the central nervous system of CNTFRa [26],
LIFRb [27] or gp130 [14] knockout mice. In contrast to mice
lacking
CNTFRa,
LIFRb
or
gp130,
mice
lacking
CNTF
developed in a normal manner and displayed only mild motor
neuron problems later in adulthood without any major neurolog-
ical abnormalities [28]. Mimicking of the neurotrophic factor profile of spinal
cord during embryonic development inhibits the
differentiation of spinal progenitor cells into astrocytes,
and promotes the differentiation into neurons and
oligodendrocytes Because there is a redundancy of CNTF
related neurotrophic factors such as LIF, IL-6 and Oncostatin M Although CNTF was previously shown to promote survival of
several kinds of neurons such as motor neurons [29,30],
hippocampal neurons [31] and sensory neurons [32] and also
survival and maturation of oligodendrocytes [33,34], neutraliza-
tion of endogenous CNTF did not affect the number of neurons or
oligodendrocytes in our cultures. Nakashima et al. demonstrated
that in gp130 knockout mice there was a significant neuronal loss
at E18.5, but not at E14.5[14]. Their findings suggest that
signaling through gp130 is essential for survival of subgroups of
differentiated neurons and that this effect depends on the
developmental stage of neurons. In our short-term culture system,
at least, neutralization of endogenous CNTF did not have
apparent effects either on the differentiation of spinal progenitor
cells into neurons or oligodendrocytes or on their survival. Further
studies using long-term culture system will be needed to determine
whether there are any effects of neutralization of endogenous
CNTF on their long-term survival and maturation. PLoS ONE | www.plosone.or June 2011 | Volume 6 | Issue 6 | e20717 June 2011 | Volume 6 | Issue 6 | e20717 5 PLoS ONE | www.plosone.org Spinal Progenitors and Endogenous Neurotrophin Figure 4. Neutralization of endogenous CNTF inhibited the differentiation of spinal progenitor cells into astrocytes. After 2–4
passages, cells were seeded on PEI-coated coverslips at a density of 26105 cells/ml in the differentiation medium with different concentrations of
anti-CNTF antibody (0–500 mg/ml). At 7div the culture cells were triple-stained with TuJ1 (A), GFAP (C) and O4 (E). Neutralization of endogenous CNTF
by polyclonal antibody (100 mg/ml and more) significantly decreased the number of astrocytes (D), but did not affect the numbers of neurons (B) or
oligodendrocytes (F). * Significant difference compared to the control (p,0.05, Tukey post hoc test). doi:10.1371/journal.pone.0020717.g004 Figure 4. Neutralization of endogenous CNTF inhibited the differentiation of spinal progenitor cells into astrocytes. After 2–4
passages, cells were seeded on PEI-coated coverslips at a density of 26105 cells/ml in the differentiation medium with different concentrations of
anti-CNTF antibody (0–500 mg/ml). At 7div the culture cells were triple-stained with TuJ1 (A), GFAP (C) and O4 (E). Neutralization of endogenous CNTF
by polyclonal antibody (100 mg/ml and more) significantly decreased the number of astrocytes (D), but did not affect the numbers of neurons (B) or
oligodendrocytes (F). Mimicking of the neurotrophic factor profile of spinal
cord during embryonic development inhibits the
differentiation of spinal progenitor cells into astrocytes,
and promotes the differentiation into neurons and
oligodendrocytes * Significant difference compared to the control (p,0.05, Tukey post hoc test). doi:10.1371/journal.pone.0020717.g004 Interestingly, while neutralization of endogenous CNTF inhibited
the differentiation of spinal progenitor cells into astrocytes, this led
to an increase in the number of undifferentiated progenitor cells,
rather than differentiation into the other cellular phenotypes. This
suggests that these cells need additional environmental cues to
promote the differentiation of spinal progenitor cells into neurons
or oligodendrocytes. Second, we showed that administration of
exogenous BDNF or NT-3 combined with neutralization of
endogenous CNTF not only decreased the number of astrocytes,
but also increased the number of neurons or oligodendrocytes,
respectively. Previously BDNF and NT-3 were shown to promote
neuronal differentiation of progenitor cells isolated from E14
striata [4], E14 cortex [19] and E16 hippocampus [18], and
CNTF were shown to promote the astrocytic differentiation from
NSCs
in
collaboration
with
Notch
signals
[35]. We
also
demonstrated that treatment of BDNF alone promoted the
neuronal differentiation of spinal progenitor cells, but did not
affect the glial differentiation. Treatments of BDNF plus anti-
CNTF antibody promoted neuronal differentiation and also
inhibited the astrocytic differentiation. There was no significant
difference in the number of neurons between the cultures treated
by BDNF plus anti-CNTF antibody and BDNF alone. Treatment of NT-3 alone promoted the differentiation of spinal progenitor
cells into oligodendrocytes, but not into neurons. Treatment of
NT-3 plus anti-CNTF antibody promoted the differentiation into
oligodendrocytes and inhibited the astrocytic differentiation. Furthermore, there was a significant difference in the number of
oligodendrocytes between the cultures treated by NT-3 plus anti-
CNTF antibody and NT-3 alone. Rao and colleagues demon-
strated that there are heterogeneous populations of E13.5 spinal
cord derived progenitor cells including multipotent, neuronal- and
glial- restricted progenitor cells [16,36]. Jean and colleagues
reported that in the chemical demyelination model of rat CNS,
treatment of NT-3 increased mature oligodendrocyte population
and influenced directly on the oligodendrocyte lineage cells to
enhance remyelination [37]. A possible explanation of our findings
is that neutralization of endogenous CNTF inhibits the differen-
tiation of multipotent and/or glial- restricted progenitor cells into
astrocytes, and that they differentiate into oligodendrocytes in the
presence of NT-3 and into neurons in the presence of BDNF. June 2011 | Volume 6 | Issue 6 | e20717 Developmental study Thoracic spinal cord tissues were harvested from Sprague-
Dawley rats (Zivic Laboratories, Zelienople, PA) at Embryonic
Days 14 (E14) and E19 and Postnatal days 3 (P3), P7 and P17. Following perfusion with ice-cold saline, tissue was frozen on dry-
ice quickly and stored at 280 uC until ribonuclease protection
assay. Spinal Progenitor Cell Culture and Passage p
g
g
Progenitor cells were cultured using the procedures of Weiss
et al (1996b). Thoracic spinal cord tissues were removed from E14
embryos and mechanically dissociated with a fire-polished pipette
in culture medium composed of a 1:1 mixture of DMEM and F-12
nutrient plus 50 U/ml penicillin and 50 mg/ml streptomycin (Life
Technologies, Rockville, MD). After dissociation, cells were seeded
in culture flasks (Nunclon) at a concentration of 26105 cells/ml
with growth medium. The growth medium contained DMEM and
F-12 nutrient (1:1), 50 U/ml penicillin, 50 mg/ml streptomycin,
0.6% glucose, 2 mM glutamine, 3 mM sodium bicarbonate,
5 mM HEPES buffer, 25 mg/ml insulin, 100 mg/ml transferrin
(Life Technologies), 20 nM progesterone, 60 mM putrescine,
30 nM selenium chloride (Sigma, St Louis, MO), and 20 ng/ml
EGF and FGF2 (Pepro Tech, Rocky Hill, NJ). After 7 days in vitro
(div), cells formed well-developed floating clusters (spheres). To
passage spheres, we pelleted 7 div spheres after centrifugation at
4006g for 5 min, resuspended them in fresh medium, and
mechanically dissociated them with a fire-polished Pasteur pipette. Single cells were seeded into growth medium in culture flasks at a
concentration of 26105 cells/ml. This procedure resulted in a
second generation of spheres. Spinal Progenitors and Endogenous Neurotrophin Spinal Progenitors and Endogenous Neurotrophin Spinal Progenitors and Endogenous Neurotrophin [41]. These endogenous stem/ progenitor cells may contribute the
glial scar formation after spinal cord injury. CNTF gene
expression increased significantly, but BDNF and NT-3 gene
expression does not increase after spinal cord injury in the adult
[42,43]. This notion is strongly supported by the previous study
[44] showing that transplantation of fibroblasts producing BDNF
or NT-3 into contused spinal cord promoted the differentiation of
endogenous
progenitor
cells
into
oligodendrocytes. It
was
noteworthy that the blocking of CNTF at the beginning of SCI
provides a more favorable environment for the differentiation of
transplanted NSC and the regeneration of host axons, thereby
promoting the functional recovery after SCI [45]. This strategy
could be applied to cell transplantation of ES- and iPS-cell derived
NSC for SCI. Figure 5. Effect of neutralization of endogenous CNTF on the
survival and proliferation of spinal progenitor cells. Compari-
sons of total cell number (A) and the percentage of BrdU (B) and nestin
(C) positive cells to total cell number between control and neutraliza-
tion groups (anti-CNTF antibody, 100 mg/ml). Neutralization of endog-
enous CNTF did not affect the survival or proliferation of spinal
progenitor cells, but increased the number of undifferentiated
progenitor cells expressing nestin (C). * Significant difference between
two groups (p,0.05, Mann-Whitney U-test). doi:10.1371/journal.pone.0020717.g005 Mimicking of the neurotrophic factor profile of spinal
cord during embryonic development inhibits the
differentiation of spinal progenitor cells into astrocytes,
and promotes the differentiation into neurons and
oligodendrocytes Previous studies demonstrated that there were also endogenous
neural stem/progenitor cells in adult spinal cord [21,38,39], but
after injury these endogenous stem/progenitor cell proliferated
[40], migrated to the lesion site and differentiated into astrocytes PLoS ONE | www.plosone.org June 2011 | Volume 6 | Issue 6 | e20717 6 Treatments of Cultures After
2–4
passages,
neurospheres
were
centrifuged
and
dissociated in EGF/FGF2-free medium. Cells were plated on
the
polyethyleneimine
(PEI)-coated
flasks
at
a
density
of
26105 cells/ml in EGF/FGF2-free and 1% fetal bovine serum
(FBS) culture medium (differentiation medium). The culture flasks
were coated by 0.1% PEI (sodium tetraborate decahyrate 0.15 M,
pH 8.5) overnight at room temperature. At 1, 3 and 7 div, these
cells were harvested for RNase protection assays of neurotrophic
factor gene expression profiles. As a control, cells were also
harvested before differentiation following dissociation (0 div). After
2–4
passages,
neurospheres
were
centrifuged
and
dissociated in EGF/FGF2-free medium. Cells were plated on
the
polyethyleneimine
(PEI)-coated
flasks
at
a
density
of
26105 cells/ml in EGF/FGF2-free and 1% fetal bovine serum
(FBS) culture medium (differentiation medium). The culture flasks
were coated by 0.1% PEI (sodium tetraborate decahyrate 0.15 M,
pH 8.5) overnight at room temperature. At 1, 3 and 7 div, these
cells were harvested for RNase protection assays of neurotrophic
factor gene expression profiles. As a control, cells were also
harvested before differentiation following dissociation (0 div). For immunocytochemistry, after 2–4 passages neurospheres
were centrifuged and dissociated in EGF/FGF2-free medium. Cells were plated onto PEI-coated 12 mm coverslips (Fisher
Scientific, Pittsburgh, PA) in 24-well plates at a density of
26105 cells/ml with 1 ml differentiation medium. The role of Figure 5. Effect of neutralization of endogenous CNTF on the
survival and proliferation of spinal progenitor cells. Compari-
sons of total cell number (A) and the percentage of BrdU (B) and nestin
(C) positive cells to total cell number between control and neutraliza-
tion groups (anti-CNTF antibody, 100 mg/ml). Neutralization of endog-
enous CNTF did not affect the survival or proliferation of spinal
progenitor cells, but increased the number of undifferentiated
progenitor cells expressing nestin (C). * Significant difference between
two groups (p,0.05, Mann-Whitney U-test). doi:10.1371/journal.pone.0020717.g005 For immunocytochemistry, after 2–4 passages neurospheres
were centrifuged and dissociated in EGF/FGF2-free medium. Cells were plated onto PEI-coated 12 mm coverslips (Fisher
Scientific, Pittsburgh, PA) in 24-well plates at a density of
26105 cells/ml with 1 ml differentiation medium. The role of June 2011 | Volume 6 | Issue 6 | e20717 June 2011 | Volume 6 | Issue 6 | e20717 PLoS ONE | www.plosone.org 7 Spinal Progenitors and Endogenous Neurotrophin Figure 6. Effects of additional application of exogenous neurotrophic factors on the differentiation of spinal progenitor cells. Treatments of Cultures Treatments of cultures were as follows in each bar graph: (1) control (normal rat IgG 100 mg/ml); (2) BDNF (20 ng/ml); (3) NT-3 (20 ng/ml); (4) anti-
CNTF (100 mg/ml); (5) anti-CNTF +BDNF; (6) anti-CNTF + NT-3. At 7 div after each treatment, cells were triple-stained with TuJ1 for neuron, GFAP for
astrocyte and O4 for oligodendrocyte. Combined treatments of anti-CNTF plus exogenous BDNF (B, E, H) or NT-3 (C, F, I) not only inhibited the
astrocytic differentiation (lanes 5 and 6 in D, E, F), but also promoted the differentiation of spinal progenitor cells into neurons (lane 5 in A, B) or
oligodendrocytes (lane 6 in G, I), respectively. The application of exogenous BDNF alone (lane 2 in A) or NT-3 alone (lane 3 in G) also increased the
numbers of neuron or oligodendrocytes, respectively. However, the differentiation of progenitor cells into astrocytes was not inhibited by these
treatments (lanes 2 and 3 in D). Compared to the cultures treated by NT-3 alone (lane 3 in G), there was a significant increase in the number of
oligodendrocytes in the cultures treated by anti-CNTF plus NT-3 (lane 6 in G). * Significant difference compared to the control (lane 1 in A, D, G)
(p,0.05, Tukey post hoc test). # Significant difference compared to NT-3 alone (lane 3 in G) (p,0.05, Tukey post hoc test). doi:10.1371/journal.pone.0020717.g006 Figure 6. Effects of additional application of exogenous neurotrophic factors on the differentiation of spinal progenitor cells. Treatments of cultures were as follows in each bar graph: (1) control (normal rat IgG 100 mg/ml); (2) BDNF (20 ng/ml); (3) NT-3 (20 ng/ml); (4) anti-
CNTF (100 mg/ml); (5) anti-CNTF +BDNF; (6) anti-CNTF + NT-3. At 7 div after each treatment, cells were triple-stained with TuJ1 for neuron, GFAP for
astrocyte and O4 for oligodendrocyte. Combined treatments of anti-CNTF plus exogenous BDNF (B, E, H) or NT-3 (C, F, I) not only inhibited the
astrocytic differentiation (lanes 5 and 6 in D, E, F), but also promoted the differentiation of spinal progenitor cells into neurons (lane 5 in A, B) or
oligodendrocytes (lane 6 in G, I), respectively. The application of exogenous BDNF alone (lane 2 in A) or NT-3 alone (lane 3 in G) also increased the
numbers of neuron or oligodendrocytes, respectively. However, the differentiation of progenitor cells into astrocytes was not inhibited by these
treatments (lanes 2 and 3 in D). Treatments of Cultures Compared to the cultures treated by NT-3 alone (lane 3 in G), there was a significant increase in the number of
oligodendrocytes in the cultures treated by anti-CNTF plus NT-3 (lane 6 in G). * Significant difference compared to the control (lane 1 in A, D, G)
(p,0.05, Tukey post hoc test). # Significant difference compared to NT-3 alone (lane 3 in G) (p,0.05, Tukey post hoc test). doi:10.1371/journal.pone.0020717.g006 gen, San Diego, CA). Briefly, a rat neurotrophin template set
(45028P, PharMingen) was labeled with (a-32P) uridine triphos-
phate. 4.66105 cpm of labeled probe and 5 mg total RNA were
used for hybridization. After RNase treatments, the protected
probes were resolved on a 5% urea-polyacrylamide gel. Autora-
diographs were produced by exposing the labeled gels to Biomax
film (Kodak, Rochester, NY) with intensifying screens at 280uC. The gels were exposed to a phosphor screen and analyzed using a
phosphoimaging system for quantification (Molecular Dynamics,
Sunnyvale, CA). GAPDH was used as an internal control to
normalize for loading differences. CNTF in differentiation of spinal progenitor cells was investigated
by neutralizing the endogenous CNTF with polyclonal antibody
against CNTF IgG. Rat anti-CNTF antibody (Sigma) was applied
to the differentiation medium at various concentrations (0–
500 mg/ml) at the time of plating. In the control group, we added
normal rat IgG (100 mg/ml, Sigma) to the differentiation medium. For analysis of the potential effects of exogenous BDNF or NT-3
on the differentiation of spinal progenitor cells, BDNF or NT-3
(20 ng/ml each, gift from Regeneron Pharmaceuticals, Inc.) was
added to the medium at the time of plating and added every 2–3
days. At
7
div,
these
cultured
cells
were
processed
for
immunocytochemistry. CNTF in differentiation of spinal progenitor cells was investigated
by neutralizing the endogenous CNTF with polyclonal antibody
against CNTF IgG. Rat anti-CNTF antibody (Sigma) was applied
to the differentiation medium at various concentrations (0–
500 mg/ml) at the time of plating. In the control group, we added
normal rat IgG (100 mg/ml, Sigma) to the differentiation medium. For analysis of the potential effects of exogenous BDNF or NT-3
on the differentiation of spinal progenitor cells, BDNF or NT-3
(20 ng/ml each, gift from Regeneron Pharmaceuticals, Inc.) was
added to the medium at the time of plating and added every 2–3
days. At
7
div,
these
cultured
cells
were
processed
for
immunocytochemistry. PLoS ONE | www.plosone.org References 1. Gage F, Ray J, Fisher L (1995) Isolation, characterization, and use of stem cells
from the CNS. Annu Rev Neurosci 18: 159–192. 1. Gage F, Ray J, Fisher L (1995) Isolation, characterization, and use of stem cells
from the CNS. Annu Rev Neurosci 18: 159–192. 15. Rajan P, McKay R (1998) Multiple routes to astrocytic differentiation in the
CNS. J Neurosci 18: 3620–3629. 2. McKay R (1997) Stem cells in the central nervous system. Science 276: p66–71. 2. McKay R (1997) Stem cells in the central nervous system. Science 276: p66–71 16. Rao M, Noble M, Mayer-Proschel M (1998) A tripotential glial precursor cell is
present in the developing spinal cord. Proc Natl Acad Sci U S A 95: 3996–4001. 3. Weiss S, Reynolds B, Vescovi A, Morshead C, Craig C, et al. (1996) Is there a
neural stem cell in the mammalian forebrain? [see comments]. Trends Neurosci
19: 387–393. 17. Kirschenbaum B, Goldman S (1995) Brain-derived neurotrophic factor
promotes the survival of neurons arising from the adult rat forebrain
subependymal zone. Proc Natl Acad Sci U S A 92: 210–214. 4. Ahmed S, Reynolds B, Weiss S (1995) BDNF enhances the differentiation but
not the survival of CNS stem cell-derived neuronal precursors. J Neurosci 15:
5765–5778. p
y
18. Vicario-Abejon C, Johe K, Hazel T, Collazo D, McKay R (1995) Functions of
basic fibroblast growth factor and neurotrophins in the differentiation of
hippocampal neurons [see comments]. Neuron 15: 105–114. 5. Reynolds B, Weiss S (1992) Generation of neurons and astrocytes from isolated
cells of the adult mammalian central nervous system [see comments]. Science
255: 1707–1710. pp
p
[
]
19. Ghosh A, Greenberg M (1995) Distinct roles for bFGF and NT-3 in the
regulation of cortical neurogenesis [see comments]. Neuron 15: 89–103. 6. Rosser A, Tyers P, ter BM, Dunnett S, Svendsen C (1997) Co-expression of
MAP-2 and GFAP in cells developing from rat EGF responsive precursor cells. Brain Res Dev Brain Res 98: 291–295. 20. Kalyani A, Hobson K, Rao M (1997) Neuroepithelial stem cells from the
embryonic spinal cord: isolation, characterization, and clonal analysis. Dev Biol
186: 202–223. 7. Svendsen C, Fawcett J, Bentlage C, Dunnett S (1995) Increased survival of rat
EGF-generated CNS precursor cells using B27 supplemented medium. Exp
Brain Res 102: 407–414. 21. Weiss S, Dunne C, Hewson J, Wohl C, Wheatley M, et al. References (1996) Multipotent
CNS stem cells are present in the adult mammalian spinal cord and ventricular
neuroaxis. J Neurosci 16: 7599–7609. 8. Von VJ, Yeon D, Oh T, Markelonis G (1994) Differentiation and maturation of
astrocytes derived from neuroepithelial progenitor cells in culture. Exp Neurol
128: 34–40. 22. Barnabe-Heider F, Miller FD (2003) Endogenously produced neurotrophins
regulate survival and differentiation of cortical progenitors via distinct signaling
pathways. J Neurosci 23: 5149–5160. 9. Islam O, Loo TX, Heese K (2009) Brain-derived neurotrophic factor (BDNF)
has proliferative effects on neural stem cells through the truncated TRK-B
receptor, MAP kinase, AKT, and STAT-3 signaling pathways. Curr Neurovasc
Res 6: 42–53. 23. Nornes H, Das G (1974) Temporal pattern of neurogenesis in spinal cord of rat. I. An autoradiographic study--time and sites of origin and migration and settling
patterns of neuroblasts. Brain Res 73: 121–138. p
24. Frederiksen K, McKay R (1988) Proliferation and differentiation of rat
neuroepithelial precursor cells in vivo. J Neurosci 8: 1144–1151. 10. Lillien L (1998) Neural progenitors and stem cells: mechanisms of progenitor
heterogeneity. Curr Opin Neurobiol 8: 37–44. 25. Ling E (1976) Study in the changes of the proportions and numbers of the
various glial cell types in the spinal cord of neonatal and young adult rats. Acta
Anat 96: 188–195. 11. Bonni A, Sun Y, Nadal-Vicens M, Bhatt A, Frank D, et al. (1997) Regulation of
gliogenesis in the central nervous system by the JAK-STAT signaling pathway. Science 278: 477–483. 26. De CT, Vejsada R, Poueymirou W, Acheson A, Suri C, et al. (1995) Mice
lacking the CNTF receptor, unlike mice lacking CNTF, exhibit profound motor
neuron deficits at birth. Cell 83: 313–322. 12. Johe KK, Hazel TG, Muller T, Dugich-Djordjevic MM, McKay RD (1996)
Single factors direct the differentiation of stem cells from the fetal and adult
central nervous system. Genes Dev 10: 3129–3140. 27. Ware C, Horowitz M, Renshaw B, Hunt J, Liggitt D, et al. (1995) Targeted
disruption of the low-affinity leukemia inhibitory factor receptor gene causes
placental, skeletal, neural and metabolic defects and results in perinatal death. Development 121: 1283–1299. 13. Lachyankar MB, Condon PJ, Quesenberry PJ, Litofsky NS, Recht LD, et al. (1997) Embryonic precursor cells that express Trk receptors: induction of
different cell fates by NGF, BDNF, NT-3, and CNTF. Exp Neurol 144:
350–360. 28. Masu Y, Wolf E, Holtmann B, Sendtner M, Brem G, et al. Statistics Each value of RNase protection assays represented mean 6
SEM of percentage of each neurotrophic factor mRNA expression
to GAPDH obtained from three independent experiments. Statistical comparisons were made using one-way ANOVA with
a Tukey post hoc test on each neurotrophic factor among different
time points. Each value of quantitative analysis of the immuno-
cytochemistry represented mean 6 SEM of percentage of
immunopositive cell number to total cell number obtained from
three independent experiments. Statistical comparisons were made
using one-way ANOVA with a Tukey post hoc test or Mann-
Whitney U-test among different treatment groups. Statistical
significance was set at p,0.05. Immunocytochemistry Immunocytochemical procedures were performed on spinal
progenitor cells at 7 div using a slight modification of the
procedures of Weiss et al (1996b). In brief, these cells were fixed with 4% paraformaldehyde for
20 min and washed three times successively with phosphate
buffered saline (PBS, pH 7.4). For triple labeling of TuJ1, GFAP
and O4, cells were incubated with the primary antibodies for TuJ1
and GFAP diluted in PBS containing 5% normal goat serum and
0.01% Triton-X100 at the same time at 4uC overnight. After
washing, cells were incubated with the secondary antibodies of
TRITC-conjugated anti-mouse IgG and Cy5-conjugated anti-
rabbit IgG at 37uC for 30 min. After washing, cells were incubated
with the primary antibody for O4 diluted in PBS containing 5%
normal goat serum and 0.01% Triton-X100 at 37uC for 2 hr. After washing, cells were incubated with the secondary antibody of
FITC-conjugated anti-mouse IgM at 37uC for 30 min. After
washing, the cells were mounted with Fluorsave (Calbiochem, La
Jolla, CA). The numbers of immuno-reactive cells and total cells
per each field were counted over 206 fields (four coverslips, six
random fields per each coverslip) after capturing each image by a
confocal laser-scanning microscope (Fluoview, Olympus, Melville,
NY). Each immunoreactive cell was counted only when the
nucleus was detectable on the 206 field investigated. Spinal Progenitors and Endogenous Neurotrophin Spinal Progenitors and Endogenous Neurotrophin CA). Appropriate secondary antibodies were purchased from
Jackson ImmunoResearch (West Grove, PA). cells was performed as described previously by Ahmed et al. (1995). BrdU (1 mM; Sigma) was added to each of the wells
containing progenitor cells (26105 cells/ml) in 1 ml differentiation
medium with or without anti-CNTF antibody (100 mg/ml) at the
time of plating for 7 div. The presence of BrdU did not modify
total cell number. Cells were stained with ZYMED BrdU staining
kit (Zymed, South San Francisco, CA) and counter stained with
hematoxylin according to the supplier’s instructions. The percent-
ages of BrdU positive cells to total cells of the examined field (four
coverslips, six random fields per each coverslip) were calculated. Proliferation Assay To determine the effect of neutralization of endogenous CNTF
on the proliferation of progenitor cells, BrdU labeling of dividing Author Contributions Conducted the cell cultures and most of the in vitro experiments: MN. Performed immunocytochemical analysis: OT. Provided instruction and
supervised all the experiments: BSB YT. Supervised the whole project:
HO. Wrote the manuscript HO MN. RNase protection assay Primary antibodies for indirect immunocytochemistry included
a mouse monoclonal antibody to TuJ1 (BAbCO, West Grove,
PA), rabbit antiserum to glial fibrillary acidic protein (GFAP,
Dako, Carpinteria, CA), mouse-monoclonal antibody to O4
(Boeringer
Mannheim,
Indianapolis,
IL),
mouse
anti-nestin
monoclonal antibody (Chemicon, Temecula, CA), antibodies for
trkB and CNTF receptor a (CNTFRa, Santa Cruz, Santa Cruz, Total RNA from each sample was extracted using TRIzol
Reagent (Life Technologies) according to the supplier’s instruc-
tions. RNase protection assays for relative quantification of
neurotrophic factor mRNAs [nerve growth factor (NGF), BDNF,
NT-3, NT-4, glial-derived neurotrophic factor (GDNF), CNTF]
were performed according to the supplier’s instructions (PharMin- PLoS ONE | www.plosone.org June 2011 | Volume 6 | Issue 6 | e20717 8 Spinal Progenitors and Endogenous Neurotrophin comparison with other neurotrophic factors and cytokines. J Neurosci 10:
3507–3515. comparison with other neurotrophic factors and cytokines. J Neurosci 10:
3507–3515. 38. Horner P, Power A, Kempermann G, Kuhn H, Palmer T, et al. (2000)
Proliferation and differentiation of progenitor cells throughout the intact adult
rat spinal cord. J Neurosci 20: 2218–2228. 30. Martinou J, Martinou I, Kato A (1992) Cholinergic differentiation factor (CDF/
LIF) promotes survival of isolated rat embryonic motoneurons in vitro. Neuron
8: 737–744. p
J
39. Johansson C, Momma S, Clarke D, Risling M, Lendahl U, et al. (1999)
Identification of a neural stem cell in the adult mammalian central nervous
system. Cell 96: 25–34. 31. Ip N, Li Y, van dSI, Panayotatos N, Alderson R, et al. (1991) Ciliary
neurotrophic factor enhances neuronal survival in embryonic rat hippocampal
cultures. J Neurosci 11: 3124–3134. 40. Namiki J, Tator C (1999) Cell proliferation and nestin expression in the
ependyma of the adult rat spinal cord after injury. J Neuropathol Exp Neurol 58:
489–498. 32. Thaler C, Suhr L, Ip N, Katz D (1994) Leukemia inhibitory factor and
neurotrophins support overlapping populations of rat nodose sensory neurons in
culture. Dev Biol 161: 338–344. 41. Frisen J, Johansson C, Torok C, Risling M, Lendahl U (1995) Rapid,
widespread, and longlasting induction of nestin contributes to the generation
of glial scar tissue after CNS injury. J Cell Biol 131: 453–464. 33. Louis J, Magal E, Takayama S, Varon S (1993) CNTF protection of
oligodendrocytes against natural and tumor necrosis factor-induced death. Science 259: 689–692. 42. Nakamura M, Bregman BS (2001) Differences in neurotrophic factor gene
expression profiles between neonate and adult rat spinal cord after injury. Exp
Neurol 169: 407–415. 34. Mayer M, Bhakoo K, Noble M (1994) Ciliary neurotrophic factor and leukemia
inhibitory factor promote the generation, maturation and survival of
oligodendrocytes in vitro. Development 120: 143–153. 43. Oyesiku N, Wilcox J, Wigston D (1997) Changes in expression of ciliary
neurotrophic factor (CNTF) and CNTF-receptor alpha after spinal cord injury. J Neurobiol 32: 251–261. 35. Nagao M, Sugimori M, Nakafuku M (2007) Cross talk between notch and
growth factor/cytokine signaling pathways in neural stem cells. Mol Cell Biol 27:
3982–3994. J
44. McTigue D, Horner P, Stokes B, Gage F (1998) Neurotrophin-3 and brain-
derived neurotrophic factor induce oligodendrocyte proliferation and myelina-
tion of regenerating axons in the contused adult rat spinal cord. J Neurosci 18:
5354–5365. 36. References (1993) Disruption of
the CNTF gene results in motor neuron degeneration. Nature 365: 27–32. 14. Nakashima K, Wiese S, Yanagisawa M, Arakawa H, Kimura N, et al. (1999)
Developmental requirement of gp130 signaling in neuronal survival and
astrocyte differentiation. J Neurosci 19: 5429–5434. 29. Arakawa Y, Sendtner M, Thoenen H (1990) Survival effect of ciliary
neurotrophic factor (CNTF) on chick embryonic motoneurons in culture: PLoS ONE | www.plosone.org June 2011 | Volume 6 | Issue 6 | e20717 June 2011 | Volume 6 | Issue 6 | e20717 9 Spinal Progenitors and Endogenous Neurotrophin Mayer-Proschel M, Kalyani A, Mujtaba T, Rao M (1997) Isolation of lineage-
restricted neuronal precursors from multipotent neuroepithelial stem cells. Neuron 19: 773–785. 45. Ishii K, Nakamura M, Dai H, Finn TP, Okano H, et al. (2006) Neutralization of
ciliary neurotrophic factor reduces astrocyte production from transplanted
neural stem cells and promotes regeneration of corticospinal tract fibers in spinal
cord injury. J Neurosci Res 84: 1669–1681. 37. Jean I, Lavialle C, Barthelaix-Pouplard A, Fressinaud C (2003) Neurotrophin-3
specifically increases mature oligodendrocyte population and enhances remye-
lination after chemical demyelination of adult rat CNS. Brain Res 972: 110–118. PLoS ONE | www.plosone.org June 2011 | Volume 6 | Issue 6 | e20717 10
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Qeios, CC-BY 4.0 · Review, May 5, 2023 Qeios ID: 49NZOE · https://doi.org/10.32388/49NZOE Review of: "[Empirical Paper] Product Development of Digital
Platform for Mental Health During Pregnancy Period: ‘Get
Well Mom’" Irma Nurbaeti1 Irma Nurbaeti1 1 Universitas Islam Jakarta Potential competing interests: No potential competing interests to declare. This paper is similar with previous mobile applications articles. Please revise with add the novelty of this paper, methods
you used to develop, results and discussion. However, Less references underpin to support this paper. Please add
current previous articles. Qeios ID: 49NZOE · https://doi.org/10.32388/49NZOE 1/1
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MAKTABGACHA TA'LIM TASHKILOTLARIDA TASHKIL ETILADIGAN TA'LIM - TARBIYA JARAYONLARI
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PEDAGOGS international research journal PEDAGOGS international research journal Mamayoqubova Shahlo Obloqulovna
Samarqand davlat chet tillar instituti. Xorijiy til va adabiyoti:
ingliz tili ll fakulteti o’qituvchisi
Cho'tboyeva Muslima
Samarqand davlat chet tillar instituti. Xorijiy til va adabiyoti:
ingliz tili ll fakulteti ikkinchi bosqich talabasi Mamayoqubova Shahlo Obloqulovna
Samarqand davlat chet tillar instituti. Xorijiy til va adabiyoti:
ingliz tili ll fakulteti o’qituvchisi Mamayoqubova Shahlo Obloqulovna
Samarqand davlat chet tillar instituti. Xorijiy til va adabiyoti:
ingliz tili ll fakulteti o’qituvchisi
Cho'tboyeva Muslima
Samarqand davlat chet tillar instituti. Xorijiy til va adabiyoti:
ingliz tili ll fakulteti ikkinchi bosqich talabasi Cho tboyeva Muslima
Samarqand davlat chet tillar instituti. Xorijiy til va adabiyoti:
ingliz tili ll fakulteti ikkinchi bosqich talabasi Samarqand davlat chet tillar instituti. Xorijiy til va adabiyoti:
ingliz tili ll fakulteti ikkinchi bosqich talabasi Maktabgacha ta‘lim sohasi uzluksiz ta‘lim tizimining birlamchi bo’g’ini
hisoblanib, u har tomonlama sog’lomva barkamol bola shaxsini tarbiyalash va
maktabga tayyorlashda muhim ahamiyat kasb etadi. Mustaqillik yillarida
respublikamizda ta‘lim-tarbiya tizimi va barkamol avlodni tarbiyalash davlat
siyosatining asosiy ustuvor yo’nalishlari darajasiga ko’tarildi. Har qanday
davlatning rivojlanishi, buguni kechasidan ham istiqbolli bo’lishi, avvalo, uning
kelajak avlodiga bog’liq. Bu har bir davrda o’z isbotini topib kelayotgan asos. Aynan
bizning mamlakatimizda ham bu yo’lda olib borilayotgan siyosat amalda o’zini
to’laqonli isbotlamoqda. Yosh avlodni jismonan sog’lom, ma‘nan yetuk qilib tarbiyalash davlat siyosati
darajasidagi vazifa bo’lib, bu ishlar yildan-yilga takomillashib bormoqda. Bu borada
Prezidentimizning ― 2017 – 2021 yillarda maktabgacha ta‘lim tizimini yanada
takomillashtirish chora-tadbirlari to’g’risida‖gi qarori ta‘lim-tarbiya maskanlari
zimmasiga yanada mas‘uliyatli vazifalarni yukladi. Qarorda maktabgacha ta‘lim
tizimini yanada takomillashtirish, moddiy-texnika bazasini mustahkamlash,
maktabgacha ta‘lim tashkilotlari tarmog’ini kengaytirish, malakali pedagog-kadrlar
bilan ta‘minlash, bolalarni maktab ta‘limiga tayyorlash darajasini tubdan yaxshilash,
ta‘lim-tarbiya jarayoniga zamonaviy ta‘lim dasturlari va texnologiyalarini tatbiq
etish, bolalarni har tomonlama intellektual, axloqiy, estetik va jismoniy rivojlantirish www.pedagoglar.uz Volume-4, Issue-1, February–2022 213 PEDAGOGS international research journal uchun shart-sharoitlar yaratish ko’zda tutilgan. Shu jihatdan maktabgacha ta‘lim
tashkilotlari (MTT)da tashkil etiladigan mashg’ulotlarda tarbiyalanuvchilarning
faolligini ta‘minlash, ularni mustaqil va erkin fikrlashga o’rgatish hamda muloqotga
kirishishlari uchun zaruriy shart-sharoitlarni yaratib berishning ahamiyati ta‘lim-
tarbiya jarayonlarining samaradorligini ta‘minlashda o’z ifodasini topadi. MTT tarbiyalanuvchilarida mustaqil va erkin fikrlash ko’nikmalari va boy
ma‘naviy dunyoqarashning shakllanishi nafaqat maxsus tashkil etiladigan
pedagogik jarayonlarda, balki ular yashayotgan va ta‘lim olayotgan muhit, ta‘lim
tashkiloti jamoasi a‘zolarining o’zaro munosabatlari va shaxslararo munosabatlar
jarayonlarida vujudga kelayotgan ta‘limiy muhitda ham amalga oshadi. Shuningdek,
MTT rahbar-xodimlari va tarbiyachilarining shaxsiy sifatlari, xattiharakati,
dunyoqarashi, tajribasi, bilim, ko’nikma va malakalari tarbiyalanuvchilar shaxsining
rivojlanishiga o’ziga xos ta‘sir ko’rsatadi. MTT tarbiyalanuvchilari shaxsining
rivojlanishida ularning mustaqil faoliyati, fikrlashi, o’rganishi va ma‘lumotlar olishi
hamda xulosalari asosida o’zini o’zi tarbiyalashi muhim ahamiyatga ega. www.pedagoglar.uz Mamayoqubova Shahlo Obloqulovna
Samarqand davlat chet tillar instituti. Xorijiy til va adabiyoti:
ingliz tili ll fakulteti o’qituvchisi
Cho'tboyeva Muslima
Samarqand davlat chet tillar instituti. Xorijiy til va adabiyoti:
ingliz tili ll fakulteti ikkinchi bosqich talabasi Demak,
MTTda tashkil etiladigan turli shakllardagi mashg’ulotlar va boshqa pedagogik
jarayonlar samaradorligini ta‘minlashda tarbiyalanuvchilar shaxsini rivojlantirishga
asos sifatida mustaqil ta‘lim, o’zini o’zi tarbiya, mustaqil ma‘lumot, rivojlanish va
tarbiyaviy munosabatlar jarayonlarini yaxlit pedagogik tizim komponentlari sifatida
inobatga olish zarur. MTTda tashkil etiladigan pedagogik jarayon komponenti sifatida o’zaro ta‘sir
jarayonining ilmiy va amaliy ahamiyati inson shaxsining rivojlanishida ham o’z
ifodasini topadi. Bunday o’zaro ta‘sir jarayonida erishish mumkin bo’lgan quyidagi natijalar
tarbiyalanuvchilar shaxsining rivojlanishida o’ziga xos ahamiyatga ega: – tarbiyalanuvchilar faolligi va tashabbuskorligining rivojlanishi; – tarbiyalanuvchilar shaxsida
yoshga bog’liq bo’lmagan psixologik
xususiyatlarning o’zgarishi; – tarbiyalanuvchilar shaxsida
yoshga bog’liq bo’lmagan psixologik
xususiyatlarning o’zgarishi; Volume-4, Issue-1, February–2022 214 PEDAGOGS international research journal PEDAGOGS international research journal PEDAGOGS international research journal PEDAGOGS international research journal PEDAGOGS international research journal –
tarbiyalanuvchilarning
ma‘naviy-madaniy
dunyoqarashi
va
tushunchalarining rivojlanishi; va – tarbiyalanuvchilarda o’zini o’zi anglash va o’zini o’zi baholash kabi
ko’nikmalarning shakllanishi va rivojlanishi; – tarbiyalanuvchilarda o’zini o’zi anglash va o’zini o’zi baholash kabi
ko’nikmalarning shakllanishi va rivojlanishi; – MTT rahbar-xodimlari va tarbiyachilarning kasbiy kompetentlilik
darajasining ortishi; – MTT rahbar-xodimlari va tarbiyachilarning kasbiy kompetentlilik
darajasining ortishi; – rahbar va pedagog xodimlarning hamkorlikdagi faoliyati vujudga kelishi; – MTTda tarbiyachi va tarbiyalanuvchilar o’rtasida axborotlar almashinuvi
samaradorligining ortishi; – yosh pedagog va tarbiyachilarning kasbiy faoliyatga hamda guruhlarga yangi
kelgan tarbiyalanuvchilarning ijtimoiy-ta‘limiy muhitga psixologik moslashuvining
tezlashuvi. Pedagogik qonuniyatlarga ko’ra, pedagogik tizim o’zaro aloqador va uzviy
bog’liq bo’lgan hamda o’zaro ta‘sir ko’rsatuvchi xususiyatlarga ega komponentlar
majmuyidan iborat bo’ladi. MTTda tashkil etiladigan turli shakllardagi
mashg’ulotlar,
to’garak
mashg’ulotlari,
sayr,
ekskursiya,
davra
suhbati,
uchrashuvlar, turli yo’nalishlarda tashkil etiladigan musobaqa va tanlovlar, san‘at
festivallari va boshqa turli yo’nalishlardagi tadbirlarni, tarbiyalanuvchi va
tarbiyachilarning o’zlari tomonidan amalga oshiriladigan mustaqil ta‘lim, o’zini o’zi
tarbiyalash, mustaqil ma‘lumot, mustaqil ishlarni, shuningdek, shaxslararo
munosabatlar jarayonida bevosita vujudga keluvchi tarbiyaviy munosabatlar
jarayonlarining maqsadi, yo’nalishi, mazmun-mohiyati va ahamiyatiga e‘tibor
qaratadigan bo’lsak, ularning o’zaro aloqadorligi va bog’liqligini, ya‘ni ularda
tizimga xos xususiyatlar mavjudligini ko’rishimiz mumkin. Chunki mazkur
pedagogik jarayonlar deb atalayotgan jarayonlarning ixtiyoriy birining o’zgarishi
ikkinchisi, uchinchisi va boshqalarining o’zgarishiga o’z ta‘sirini ko’rsatadi. Bizning fikrmizcha, MTTda ta‘lim-tarbiya bir necha kichik tizimlar doirasida, ya‘ni
o’quv mashg’ulotlarida, o’yin mashg’ulotlarida, sayr va ekskursiya jarayonlarida
hamda tarbiyaviy ahamiyatga ega bo’lgan tadbirlarda amalga oshirilishi Volume-4, Issue-1, February–2022 www.pedagoglar.uz 215 PEDAGOGS international research journal mumkinligini inobatga olgan holda, mazkur jarayonlarni hamda o’zaro bog’liq
bo’lgan tashkil etuvchi qismlarning ichki tabiatiga xos bo’lgan jarayonlarni o’z
ichiga olgan va o’zaro ta‘sir jarayoni mavjud bo’lgan shaxslararo munosabatlar
jarayonlari majmuyini ham pedagogik tizim sifatida keltirish mumkin. Pedagogik
tizim MTTning maqsadi, vazifasi, ta‘lim tashkiloti tomonidan pedagogik
maqsadlarni amalga oshirish yo’nalishlarida tanlangan model, strategiyalari orqali
tavsiflanishi ham mumkin. Ya‘ni pedagogik tizim nafaqat MTTda tashkil etiladigan
ta‘lim-tarbiya jarayonlari, balki pedagogik jarayonlar subyektlari faoliyatini
muvofiqlashtirish, tarbiyalanuvchilar, tarbiyachilar va rahbar xodimlarning
funksional vazifalarini amalga oshirish va boshqarishda tashqi muhit bilan o’zaro
ta‘sir ko’rsatuvchi, tarbiyaviy ahamiyat kasb etuvchi va alohida xususiyatlarga ega
bo’lgan o’zaro bog’liq qismlarning yaxlit holda vujudga kelishini ham qamrab oladi. Pedagogik tizimning tashqi muhit va boshqa tizimlar bilan o’zaro ta‘sir etish
xususiyatlariga ega ekanligini inobatga olib, MTTda tashkil etiladigan turli
yo’nalishlardagi tadbirlar, mustaqil ta‘lim, o’zini o’zi tarbiyalash, mustaqil
ma‘lumot, tarbiyaviy munosabatlar jarayonlari pedagogik tizimning asosiy
komponentlarini tashkil etadi deb aytishimiz mumkin. Foydalanilgan adabiyotlar: 1. Mirziyoyev Sh.― Maktabgacha ta‘lim tizimi boshqaruvini tubdan
takomillashtirish chora-tadbirlari to’g’risidagi PF-5198-sonli farmon. 2017-yil 30-
sentabr. 2. Turg’unov S.T., Maksudova L.A. Pedagogik jarayonlarni tashkil etish va
boshqarish. – T.: Fan, 2009. 3. Shoymardonov T.T. Pedagog kadrlar malakasini oshirishi va kasbiy faoliyati
monitoringini tashkil etishning elektron tizimi. // Pedagogika fanlari bo’yicha
falsafa doktorligi dissertatsiyasi. – T.: TDPU, 2017. www.pedagoglar.uz Volume-4, Issue-1, February–2022 216
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High Performance Textiles
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MDPI eBooks
| 2,022
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cc-by
| 93,740
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Edited by
Performance
es
k M. Abou Elmaaty and Maria Rosaria Plutino
nted Edition of the Special Issue Published in Polymers
mers Edited by
nce
aria Plutino
d in Polymers Contents About the Editors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Tarek AbouElmaaty, Shereen A. Abdeldayem, Shaimaa M. Ramadan, Khaled Sayed-Ahmed
and Maria Rosaria Plutino
Coloration and Multi-Functionalization of Polypropylene Fabrics with Selenium Nanoparticles
Reprinted from: Polymers 2021, 13, 2483, doi:10.3390/polym13152483 . . . . . . . . . . . . . . . . 1
Li-Hua Lyu, Wen-Di Liu and Bao-Zhong Sun
Electromagnetic Wave-Absorbing and Bending Properties of Three-Dimensional Honeycomb
Woven Composites
Reprinted from: Polymers 2021, 13, 1485, doi:10.3390/polym13091485 . . . . . . . . . . . . . . . . 15
Mary Morris, Xiaofei Philip Ye and Christopher J. Doona
Dyeing Para-Aramid Textiles Pretreated with Soybean Oil and Nonthermal Plasma Using
Cationic Dye
Reprinted from: Polymers 2021, 13, 1492, doi:10.3390/polym13091492 . . . . . . . . . . . . . . . . 31
Kabir Hossain, Thennarasan Sabapathy, Muzammil Jusoh, Mahmoud A. Abdelghany,
Ping Jack Soh, Mohamed Nasrun Osman, Mohd Najib Mohd Yasin, Hasliza A. Rahim and
Samir Salem Al-Bawri
A Negative Index Nonagonal CSRR Metamaterial-Based Compact Flexible Planar Monopole
Antenna for Ultrawideband Applications Using Viscose-Wool Felt
Reprinted from: Polymers 2021, 13, 2819, doi:10.3390/polym13162819 . . . . . . . . . . . . . . . . 49
Tarek Abou Elmaaty, Hanan G. Elsisi, Ghada M. Elsayad, Hagar H. Elhadad,
Khaled Sayed-Ahmed and Maria Rosaria Plutino
Fabrication of New Multifunctional Cotton/Lycra Composites Protective Textiles through
Deposition of Nano Silica Coating
Reprinted from: Polymers 2021, 13, 2888, doi:10.3390/polym13172888 . . . . . . . . . . . . . . . . 67
Sarianna Palola, Farzin Javanshour, Shadi Kolahgar Azari, Vasileios Koutsos and Essi Sarlin
One Surface Treatment, Multiple Possibilities: Broadening the Use-Potential of Para-Aramid
Fibers with Mechanical Adhesion
Reprinted from: Polymers 2021, 13, 3114, doi:10.3390/polym13183114 . . . . . . . . . . . . . . . . 79
Ana Mar´ıa Rodes-Carbonell, Josue´ Ferri, Eduardo Garcia-Breijo, Ignacio Montava
and Eva Bou-Belda
Influence of Structure and Composition of Woven Fabrics on the Conductivity of Flexography
Printed Electronics
Reprinted from: Polymers 2021, 13, 3165, doi:10.3390/ polym13183165 . . . . . . . . High Performance Textiles Editors
Tarek M. Abou Elmaaty
Maria Rosaria Plutino MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin Editors
Tarek M. Abou Elmaaty
Galala University
Egypt Editors
Tarek M. Abou Elmaaty
Galala University
Egypt Maria Rosaria Plutino
Istituto per lo Studio dei Materiali Nanostrutturati
Consiglio Nazionale delle Ricerche (ISMN-CNR)
Italy Editorial Office
MDPI
St. Alban-Anlage 66
4052 Basel, Switzerland This is a reprint of articles from the Special Issue published online in the open access journal
Polymers (ISSN 2073-4360) (available at: https://www.mdpi.com/journal/polymers/special issues/
High Perform Text). For citation purposes, cite each article independently as indicated on the article page online and as
indicated below: LastName, A.A.; LastName, B.B.; LastName, C.C. Article Title. Journal Name Year, Volume Number,
Page Range. Page Range. ISBN 978-3-0365-3851-8 (Hbk)
ISBN 978-3-0365-3852-5 (PDF) ISBN 978-3-0365-3851-8 (Hbk)
ISBN 978-3-0365-3852-5 (PDF) Cover image courtesy of Maria Rosaria Plutino © 2022 by the authors. Articles in this book are Open Access and distributed under the Creative
Commons Attribution (CC BY) license, which allows users to download, copy and build upon
published articles, as long as the author and publisher are properly credited, which ensures maximum
dissemination and a wider impact of our publications. The book as a whole is distributed by MDPI under the terms and conditions of the Creative Commons
license CC BY-NC-ND. About the Editors Tarek M. Abou Elmaatyz Tarek M. Abou Elmaaty is Galala University’s first Vice President for academic affairs. He
co-supervised in establishing Galala University within his role as a member of the higher committee
of establishing national universities, MOHE. He’s also a Professor of Textile Dyeing, Damietta
University. Professor Tarek Abou El-Maaty earned a Bachelor’s degree in Science as well as a Masters
and a Doctorate in Organic Chemistry. He started his academic career as a lecturer in Mansoura
University in 1998 before being appointed Dean of the Faculty of Applied Arts, Damietta University. He served as a member of the scientific committee for promotion of professors of Applied Art in
the Supreme Council of Universities before becoming the Vice President of Damietta University for
academic affairs. Prof. Abou El-Maaty’s research projects revolve around three themes; heterocyclic
chemistry, the development of new dyes, and the modification of fabrics and colorant to produce
multifunctional textiles. During the past 15 years, he worked on the synthesis of new functional
dyes as well as the modification of fabrics to produce multifunctional textiles to be introduced to the
market. He succeeded in creating several new dyes and modified fabrics which have antibacterial
activity and multifunctional properties. And currently, his group is specialized in dyeing & finishing
of textiles under supercritical carbon dioxide medium and this is the only group in the Middle East
working with this technology. Also, they established the international joint China-Egypt supercritical
dyeing in 2018. Prof. Tarek Abou El-Maaty received a number of awards including the best researcher
Award, Publication Award and recently he won the gold medal (textile field) in Cairo Innovates for
two consecutive cycles in 2018 & 2019. He has three patents in the field of textile chemical technology
registered at ASRT. He also has over 60 publications and 5 books. Contents . . . . . . . . 91
Hany El-Hamshary, Mehrez E. El-Naggar, Tawfik A. Khattab and Ayman El-Faham
Preparation of Multifunctional Plasma Cured Cellulose Fibers Coated with Photo-Induced
Nanocomposite toward Self-Cleaning and Antibacterial Textiles
Reprinted from: Polymers 2021, 13, 3664, doi:10.3390/polym13213664 . . . . . . . . . . . . . . . . 109
Tarek Abou Elmaaty, Sally Raouf, Khaled Sayed-Ahmed and Maria Rosaria Plutino
Multifunctional Dyeing of Wool Fabrics Using Selenium Nanoparticles
Reprinted from: Polymers 2022, 14, 191, doi:10.3390/polym14010191 . . . . . . . . . . . . . . . . . 121 Deposition of Nano Silica Coating v Caleb Metzcar, Xiaofei Philip Ye, Toni Wang and Christopher J. Doona
Soybean Oil-Based Biopolymers Induced by Nonthermal Plasma to Enhance the Dyeing of
Para-Aramids with a Cationic Dye
Reprinted from: Polymers 2022, 14, 628, doi:10.3390/polym14030628 . . . . . . . . . . . . . . . . . 137
Tarek Abou Elmaaty, Khaled Sayed-Ahmed, Radwan Mohamed Ali, Kholoud El-Khodary
and Shereen A. Abdeldayem
Simultaneous Sonochemical Coloration and Antibacterial Functionalization of Leather with
Selenium Nanoparticles (SeNPs)
Reprinted from: Polymers 2022, 14, 74, doi:10.3390/polym14010074
. . . . . . . . . . . . . . . . . 157 vi vi Maria Rosaria Plutino Recently, Dr. Plutino has set-up green and eco-friendly synthesis
protocols starting from natural substances or waste, which lead to the obtainment of functional
materials that can also be recycled and re-used. The research activity of Dr. Plutino is documented
by more than 50 papers published in journals with international circulation and two patents in the
evaluation phase. She is also a guest-editor and reviewer of different international scientific journals. Part of the results obtained are the outcome of numerous national and international multidisciplinary
scientific collaborations, and are included in various research financed projects (“Blue Growth” and
“Cultural Heritage” theme lines in the PO FESR 2014–2020 and PON 2017–2020, where she is the
scientific CNR coordinator); they have also been presented at national and international congresses
in the form of oral communications and posters. industrial and urban pollution. Her main research interest is the rational design, development,
and structural study of organometallic complexes of transition metals, supramolecular systems,
and nanostructured functional hybrid materials, for applications in the field of optoelectronics,
sensors, catalysis, environmental remediation, and biomedicine. She has acquired specific skills in
the use of different techniques, such as UV-Vis and Fluorescence Spectrophotometry, NMR (homo-
and heteronuclear, mono- and two-dimensional) and FT-IR spectroscopy, Mechanical Calculations
and organometallic syntheses in anaerobic and high empty. In particular, the research activity of
Dr. Plutino is focused on the development of new innovative and advanced, multi-component
and multifunctional, nano-hybrid systems (nanohybrids and nanocomposites), obtained thanks to
the use of sol-gel and polymerization techniques carried out in the presence of organic/inorganic
hybrid silanes or polymeric precursors and organic and inorganic functional nanofillers, which show
implemented physical and surface chemical and physical properties, and which can present potential
applications in different sectors such as building, naval, textile, environmental, cultural heritage,
biomedical, sensoristic, catalytic. Recently, Dr. Plutino has set-up green and eco-friendly synthesis
protocols starting from natural substances or waste, which lead to the obtainment of functional
materials that can also be recycled and re-used. The research activity of Dr. Plutino is documented
by more than 50 papers published in journals with international circulation and two patents in the
evaluation phase. She is also a guest-editor and reviewer of different international scientific journals. Maria Rosaria Plutino Maria Rosaria Plutino gas received her Ph.D. in Chemical Sciences at the Univ. of Messina (IT) in
1997. In March 1996 she won a 24-month post-doctoral grant (Human Capital and Mobility), funded
by the European Community at the Dep. Oorganisk1 of the Lund Chemical Center (Sweden). In
1999 she had a temporary research contract at the Institute of Chemistry and Technology of Natural
Products (ICTPN-CNR), Messina. Since 2001 she has a permanent researcher position at the Institute
for the Study of Nanostructured Materials of the CNR (ISMN) in Palermo (c/o Dip ChiBioFarAm,
Univ. of Messina). Dr. Plutino is also co-founder, President, and Scientific Manager of the recently
constituted ATHENA Green Solutions S.r.l., an Innovative Start-Up, and Joint and not attended
Spin-off by the National Research Council (CNR) and by the Univ. of Messina. The basis of the
Innovative Start-up is the Arginare entrepreneurial/patent idea, which has participated in various
local, regional, and national business competitions, achieving excellent placements and receiving
awards and prizes. The company operates in the field of Environmental Technologies, offering in
addition to the materials/prototypes of ArgiNaRe, other products, methodologies, consultancy and
services in multidisciplinary sectors and the perspective of innovation, sustainability, and recycling. In particular, the mission and objectives of the company ATHENA Green Solutions are to research
and production systems and/or prototypes for the resolution of problems deriving from high
environmental impact activities with particular reference to seagoing vessels and marine/coastal, vii industrial and urban pollution. Her main research interest is the rational design, development,
and structural study of organometallic complexes of transition metals, supramolecular systems,
and nanostructured functional hybrid materials, for applications in the field of optoelectronics,
sensors, catalysis, environmental remediation, and biomedicine. She has acquired specific skills in
the use of different techniques, such as UV-Vis and Fluorescence Spectrophotometry, NMR (homo-
and heteronuclear, mono- and two-dimensional) and FT-IR spectroscopy, Mechanical Calculations
and organometallic syntheses in anaerobic and high empty. In particular, the research activity of
Dr. Plutino is focused on the development of new innovative and advanced, multi-component
and multifunctional, nano-hybrid systems (nanohybrids and nanocomposites), obtained thanks to
the use of sol-gel and polymerization techniques carried out in the presence of organic/inorganic
hybrid silanes or polymeric precursors and organic and inorganic functional nanofillers, which show
implemented physical and surface chemical and physical properties, and which can present potential
applications in different sectors such as building, naval, textile, environmental, cultural heritage,
biomedical, sensoristic, catalytic. Maria Rosaria Plutino Part of the results obtained are the outcome of numerous national and international multidisciplinary
scientific collaborations, and are included in various research financed projects (“Blue Growth” and
“Cultural Heritage” theme lines in the PO FESR 2014–2020 and PON 2017–2020, where she is the
scientific CNR coordinator); they have also been presented at national and international congresses
in the form of oral communications and posters. viii polymers Article https://doi.org/10.3390/
polym13152483 Keywords: selenium nanoparticles; polypropylene; coloration; antibacterial; conductivity; UV protection Article AbouElmaaty 1,*, Shereen A. Abdeldayem 2, Shaimaa M. Ramadan 2, Khaled Sayed-Ahmed 3
Maria Rosaria Plutino 4 Tarek AbouElmaaty 1,*, Shereen A. Abdeldayem 2, Shaimaa M. Ramadan 2, Khaled Sayed-Ahmed 3
and Maria Rosaria Plutino 4 1
Department of Material Art, Galala University, Galala 43713, Egypt 2
Department of Textile Printing, Dyeing and Finishing, Faculty of Applied Arts, Damietta University, 2
Department of Textile Printing, Dyeing and Finishing, Faculty of Applied Arts, Damietta University,
Damietta 34512 Egypt; shereen abdeldayem 82@gmail com (S A A ); designer shemo@yahoo com (S M R ) p
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Damietta 34512, Egypt; shereen.abdeldayem.82@gmail.com (S.A.A.); designer_shemo@yahoo.com (S.M.R.) gyp
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Department of Agricultural Chemistry, Faculty of Agriculture, Damietta University, Damietta 34512, Egypt
dr_khaled@yahoo.com 3
Department of Agricultural Chemistry, Faculty of Agriculture, Damietta University, Damietta 34512, Egypt
dr_khaled@yahoo.com 4
Stituto per lo Studio dei Materiali Nanostrutturati, Consiglio Nazionale delle Ricerche, Vill. S. Agata
98166 Messina, Italy; plutino@pa.ismn.cnr.it 4
Stituto per lo Studio dei Materiali Nanostrutturati, Consiglio Nazionale delle Ricerche, Vill. S. Agata,
98166 Messina, Italy; plutino@pa.ismn.cnr.it *
Correspondence: tasaid@gu.edu.eg Abstract: In this study, we developed a new approach for depositing selenium nanoparticles (SeNPs)
into polypropylene (PP) fabrics via a one-step process under hydrothermal conditions by using an
IR-dyeing machine to incorporate several functionalities, mainly coloration, antibacterial activity
and ultraviolet (UV) protection. The formation, size distribution, and dispersion of the SeNPs
were determined using X-ray diffraction (XRD), ultraviolet-visible (UV/Vis), transmission electron
microscopy (TEM) and the color strength, fastness, antibacterial properties, and UV protection of the
treated fabrics were also explored. The UV-Vis spectra and TEM analysis confirmed the synthesis of
spherical well-dispersed SeNPs and the XRD analysis showed the successful deposition of SeNPs
into PP fabrics. The obtained results demonstrate that the SeNPs-PP fabrics is accompanied by a
noticeable enhancement in measurements of color strength, fastness, and UV-protection factor (UPF),
as well as excellent antibacterial activity. Viability studies showed that SeNPs-PP fabrics are non-toxic
against wi-38cell line. In addition, the treated SeNPs-PP fabrics showed an increase in conductivity. The obtained multifunctional fabrics are promising for many industrial applications such as the new
generation of curtains, medical fabrics, and even automotive interior parts. Citation: AbouElmaaty, T.;
Abdeldayem, S.A.; Ramadan, S.M.;
Sayed-Ahmed, K.; Plutino, M.R. Coloration and
Multi-Functionalization of
Polypropylene Fabrics with Selenium
Nanoparticles. Polymers 2021, 13,
2483. Citation: AbouElmaaty, T.;
Abdeldayem, S.A.; Ramadan, S.M.;
Sayed-Ahmed, K.; Plutino, M.R.
Coloration and
Multi-Functionalization of
Polypropylene Fabrics with Selenium
Nanoparticles. Polymers 2021, 13,
2483. https://doi.org/10.3390/
polym13152483 1. Introduction Numerous washable or disposable healthcare and hygiene textile products are used in
hospitals either for the protection of staff and patients (drapes, beddings, masks, uniforms,
wound dressings, bandaging materials, etc.) [1]. Due to the large surface areas, textiles
have superior abilities to retain warmth, moisture, and nutrients from spillages and ex-
udates, making them ideal substrates for microorganisms to grow on [2]. Some studies
have suggested that healthcare textiles can act as reservoirs and vehicles for the spread of
microorganisms in hospitals [3]. With the development of nanotechnology, some inorganic
nanoparticles (NPs), such as silver, copper, and zinc, have been identified as promising can-
didates in combating pathogenic microorganisms [4–9]. Nanotechnology-driven therapies
with metal and metal oxide nanoparticles (NPs) are emerging as a promising alternative
to antibiotics. The high reactivity of these NPs, due to large surface-to-volume ratio, re-
sults in intrinsic targeted antimicrobial efficiency even when they are applied in small
amounts [10]. Proven activity of metal and metal oxide NPs against wide range of micro-
organisms including bacteria, fungi, viruses, and other eukaryotic microorganisms [11] was
inspiring for researchers to immobilize them onto textiles [12–15]. Selenium nanoparticles
(SeNPs) has become the new research target because they are found to possess excellent Publisher’s Note: MDPI stays neutral
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iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article
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conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). 1 https://www.mdpi.com/journal/polymers Polymers 2021, 13, 2483. https://doi.org/10.3390/polym13152483 Polymers 2021, 13, 2483 bioavailability, low toxicity, and contribute to a wide spectrum of health promotion, as
well as disease prevention and treatment activities [16]. Biswas et al. [17] prepared silver
or selenium nanoparticles on polymeric scaffolds and compared the cytotoxicity of the
scaffolds towards mouse fibroblasts using an indirect contact method; the results indicated
that the Ag-loaded scaffolds showed high cytotoxicity, while the Se-loaded scaffolds were
not toxic to the cells. The low cytotoxicity of SeNPs indicates the great potential for use in
biomedical applications [3]. The research on SeNPs as antimicrobial agents is still limited. Several studies have pointed out the ability of SeNPs to exhibit anticancer [18], antioxi-
dant [19], antibacterial and anti-biofilm [20] properties. 2.1. Fabric and Chemicals Polypropylene fabric (100%) was supplied by Shikisen-sha Company (Osaka, Japan)
with crystallinity (50.6%), melting enthalpy (105.8 J/g) [32], density (0.91 g/c.c), moisture
regain (0%) and tenacity (3.5–8.0 g/den). Sodium hydrogen selenite, ascorbic acid and
polyvinylpyrrolidone (PVP) were purchased from LobaChemie, India. Other chemicals were
commercial grade. 2.2. Green Synthesis of Selenium Nanoparticles (SeNPs) 2.2. Green Synthesis of Selenium Nanoparticles (SeNPs) SeNPs were synthesized via a redox reaction based on the method reported by AbouEl-
maatyet al. with an improved modification [31]. PVP (6g) was dissolved in 100 mL of
sodium hydrogen selenite solution at a concentration of 100 mmol/L. Then, ascorbic acid
was added to the mixture at the same concentration and volume ratio of 1:1 under magnetic
stirring. The solution changed from colorless to orange to dark orange, indicating the for-
mation of SeNPs [33]. Moreover, the prepared SeNPs colloidal solution at the concentration
of 50 mmol/L was used in this treatment process. 1. Introduction So far, remarkable antimicrobial
activity of these nanoparticles has been evidenced against pathogenic bacteria, fungi, and
yeasts [21–23], which inspired researchers to immobilize them onto textiles. y
p
Polypropylene (PP) fabric has excellent physical and mechanical properties [24]. It is
a hydrophobic fabric, and several surface modification techniques are adopted to improve
wetting, adhesion to polymer surfaces by introduction of a variety of polar groups [25–27]. PP advantages include a great supply, good process, low energy demand, low cost and
high chemical stability [28]. There is a great demand for antibacterial PP fabric to be used
in different medical applications [29,30]. To our knowledge, there are no reports dealing
with coloring PP by using SeNPs without any additives and studying the antibacterial and
UV-protection properties, cytotoxicity, and electrical conductivity of the resulted SeNPs-PP
fabrics. In this study, SeNPs are prepared in one step process under a simple redox system
based on the method mentioned by Abou Elmaaty et al. [31] followed by application of the
SeNPs to PP fabric in one-step process under hydrothermal conditions. The characteris-
tics of SeNPs in solution phase were studied by UV-visible spectrophotometer (UV/Vis),
transmission electron microscopy (TEM) and X-ray diffraction (XRD). Moreover, scanning
electron microscopy (SEM), colour characteristics and antibacterial and UV-protection prop-
erties, cytotoxicity and electrical conductivity of the SeNPs-PP fabrics were also evaluated. In summary, we developed a simple, green, and feasible route to produce green coloration
of PP-based fabric for multifunctional applications. The low cytotoxicity of SeNPs and
antibacterial properties indicates their great potential for use in biomedical applications. Raman Spectroscopy Analysis Raman Spectroscopy Analysis The types of bonds present in the blank PP and SeNPs-PP fabrics were determined
using confocal Raman microscope (Jasco NRS-4500, Tokyo, Japan) which covered the range
from 200 to 4000 cm−1. Raman data acquisition and processing were performed using
Jasco spectroscopy suite software. 2.4. Characterization 2.4.1. Characterization of Selenium Nanoparticles (SeNPs) Transmission Electron Microscopy (TEM) Analysis The size and morphology of SeNPs were characterized using JEM-2100 Transmission
Electron Microscope with an acceleration voltage of 200 kV. A drop of colloidal solution
containing SeNPs was dripped onto a carbon coated copper grid and dried at room
temperature for TEM analysis. X-ray Diffraction (XRD) Analysis X-ray diffraction (XRD) analysis was conducted for synthesized SeNPs and SeNPs-PP
fabrics using an X-ray diffractometer system (Bruker D8 ADVANCE, Karlsruhe, Germany). While SeNPs solution was dried at 130 ◦C until completely dryness before the XRD analysis. UV/Vis Spectroscopy Analysis UV/Vis Spectroscopy Analysis SeNPs were further characterized via ultraviolet-visible spectrophotometer (Alpha-
1860, Indianapolis, IN, USA), and their formation was confirmed by the maximum absorp-
tion peak which attributed to their surface plasmon resonance. 2.4.2. Characterization of Poly Propylene (PP) Fabrics Scanning Electron Microscopy (SEM) Analysis The surface morphology of blank PP and SeNPs-PP fabric was characterized via
scanning electron microscope (JEOL JSM-6510LB with field emission gun, Tokyo, Japan). The deposition of SeNPs into PP fabrics was confirmed using surface energy dispersive
x-ray (EDX) analysis unit (EDAX AMETEK analyzer) attached to SEM device. 2.3. Treatment Method The PP fabrics were treated using an infrared dyeing machine. The machine includes
12 beakers fixed in a rotating carrying wheel. Heating was obtained by IR, cooling by air,
and automation by the microprocessor programmer DC4 F/R. The highest temperature
used in this device was120 ◦C, the highest rate of heating was 2 ◦C per minute, and the
highest rate of cooling was 6 ◦C per minute. First, a solution of SeNPs (50 mmol/L)
was prepared. The PP fabrics were then immersed in the solution with liquor ratio (LR) 2 2 Polymers 2021, 13, 2483 of 1:50. Next, the device was set at the following treatment temperatures periods. The
treatment was performed at different temperatures (70 ◦C, 100 ◦C and 120 ◦C) and different
periods (1, 2 and 3 h). The treated PP fabrics were rinsed with distilled water and allowed
to dry at room temperature after the treatment. The obtained fabrics were coded with
SeNPs-PP fabrics. 2.4. Characterization Colorimetric Study The colorimetric parameters such as lightness (L*), redness-greenness (a*), yellowness-
blueness (b*) and the color uptake which is expressed as the color strength (K/S) of
the obtained SeNPs-PP fabrics were determined using a spectrophotometer (CM3600A;
Konica Minolta, Tokyo, Japan). K/S values were evaluated at the wavelength of maximum
absorption (λmax) of the color’s reflectance curve at390 nm. Exhaustion of SeNPs into PP Fabric 3.1. Characterization of SeNPs 3.1.1. Transmission Electron Microscopy (TEM) Analysis 3.1.1. Transmission Electron Microscopy (TEM) Analysis TEM micrographs confirmed the formation of spherical Se-NPs in the range of 31–
79 nm. Furthermore, the synthesized SeNPs were well-dispersed with no aggregation and
deformation as shown in Figure 1. g
Histogram bins are10-nm wide and centered at 35, 45, 55, 65 and 75 nm. All SeNPs
with diameters from 40 nm to 50 nm were considered to have a size of 45 nm. Furthermore, TEM was used to examine the adsorption of SeNPs as shown in Figure 1. The TEM images illustrated that SeNPs were sufficiently monodispersed and adsorbed on
the SeNPs-PP fabric surface. Cytotoxicity Test of SeNPs-PP Fabric Cytotoxicity Test of SeNPs-PP Fabric The cytotoxicity of SeNPs-PP fabric was tested against wi-38 cell line. This type of cells
is diploid human cell line, including fibroblasts from lung tissue of a 3-month-gestation
female fetus. The SeNPs-PP fabric treated at the optimum conditions was sterilized, cut,
and plated on the bottom surface of a six-well tissue culture plate. The plate was inoculated
with 1 × 105 cells/mL (100 μL/well) and incubated at 37 ◦C for 24 h. Additionally, the
growth medium was decanted, and the cell monolayer was washed twice with washing
media. Cells were checked for any physical signs of toxicity. Moreover, the tissue was
picked up and 20 μL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye
(MTT) prepared in phosphate buffer saline (BIO BASIC CANADA INC, Markham, Ontario,
Canada) was added to each well at a concentration of 5mg/mL and shaken for 5 min. The wells were incubated at 37 ◦C and 5% CO2 for 1–5 h. After dumping the media, the
formazan as MTT metabolic product in the dry plate was resuspended in 200 μL dimethyl
sulfoxide and shaken for 5 min. Then, the optical density was read at 560 nm, while the
background was measured at 620 nm and subtracted. Electrical Conductivity Measurement The electrical conductivity of both the PP and SeNPs-PP fabrics were measured using
LRC-bridge (Hioki model 3531zHi Tester, Nagano, Japan). Antibacterial Activity The antibacterial activity of the SeNPs-PP fabric was evaluated using AATCC (147-
2004) test [37]. Antibacterial tests were carried out against G+ve bacteria (Staphylococcus
aureus and Bacillus cereus) as well as G-ve bacteria (Escherichia coli and Pseudomonas aerugi-
nosa) and the growth inhibition zone (mm) was determined. UV-Protection Properties UV-Protection Properties The UV protection factor (UPF) and UV-blocking activities of the SeNPs-PP fab-
ric were determined using Standards Australia and Standards New Zealand (AS/NZS)
4399:1996 tests and UV protection properties were expressed as good, very good, or excel-
lent for UPF values of 15–24, 25–39, and >40, respectively. Electrical Conductivity Measurement 3. Results and Discussion 3.1. Characterization of SeNPs 3.1. Characterization of SeNPs Exhaustion of SeNPs into PP Fabric The treatment colloidal solution was sampled before and after treatment to measure
the exhaustion of SeNPs. The absorbance of the SeNPs colloidal solution was measured
using an UV/Vis spectrophotometer (Alpha-1860, Indianapolis, IN, USA). Physical Properties of SeNPs-PP Fabric The fastness of the SeNPs-PP fabrics determines the fixation of SeNPs into the fabric. They were determined using AATCC (61-1972), (8-1972), and (16A-1972) [34] tests for
washing, rubbing and lightfastness, respectively. The tensile strength tests of the PP and
SeNPs-PP fabrics were performed using a universal testing machine (Tinius Olsen EN ISO 3 Polymers 2021, 13, 2483 13934-1;1999-model H25KT) [35]. Additionally, the durability to washing was evaluated
according to AATCC61(2A)-1996 test [36] after five washing cycles. 13934-1;1999-model H25KT) [35]. Additionally, the durability to washing was evaluated
according to AATCC61(2A)-1996 test [36] after five washing cycles. Cytotoxicity Test of SeNPs-PP Fabric 3.1.2. X-ray Diffraction (XRD) Analysis XRD confirmed the formation of SeNPs and their deposition into the treated PP fabric
based on the crystallinity of SeNPs. As illustrated in Figure 2, SeNPs in a colloidal solution
or into the PP fabric surface were highly crystalline. Additionally, the diffraction peaks at
24.28◦, 29.24◦, 43.64◦and 64.28◦were corresponding to 100, 101, 102 and 210 crystal planes,
respectively, based on the JCPDS 86-2246 international database [38]. Moreover, the peaks 4 Polymers 2021, 13, 2483 at 13,66, 16.56, 18.18, and 25.38 can be observed for PP fabric, corresponding to the planes
of (110), (040), (130), and (060), respectively [39]. at 13,66, 16.56, 18.18, and 25.38 can be observed for PP fabric, corresponding to the planes
of (110), (040), (130), and (060), respectively [39]. at 13,66, 16.56, 18.18, and 25.38 can be observed for PP fabric, corresponding to the planes
of (110), (040), (130), and (060), respectively [39]. Figure 1. (a) TEM image (b) size distributions histogram of prepared SeNPs, (c,d) SeNPs-PP fab-
ric surface. Figure 1. (a) TEM image (b) size distributions histogram of prepared SeNPs, (c,d) SeNPs-PP fab-
ric surface. Figure 2. XRD patterns of the prepared SeNPs and SeNPs-PP fabric. Figure 2. XRD patterns of the prepared SeNPs and SeNPs-PP fabric. 3.1.3. UV/Vis Spectroscopy Analysis The formation of SeNPs was confirmed from the UV/Vis spectra based on their SPRs. The solution changed from colorless to orange to dark orange, indicating the complete
reduction of sodium hydrogen selenite to SeNPs [40]. As shown in Figure 3, the SeNPs colloidal solution showed an absorption peak at
263 nm, confirming the formation of the spherical SeNPs [41]. 5 Polymers 2021, 13, 2483 Figure 3. UV/Vis spectra of SeNPs at different concentrations. Figure 3. UV/Vis spectra of SeNPs at different concentrations. Figure 3. UV/Vis spectra of SeNPs at different concentrations. 3.2. Characterization of Poly Propylene (PP) Fabrics 3.2. Characterization of Poly Propylene (PP) Fabrics f
y
py
3.2.1. Scanning Electron Microscopy (SEM) Analysis The SEM micrographs of the PP fabric revealed that the surface was clear with clean
scales and typical fibrous structure as displayed in Figure 4. On the other hand, the SEM
micrographs of the SeNPs-PP fabric show a coated layer of SeNPs into the PP fabric. Additionally, SeNPs were well distributed into the fabric. The SEM micrographs of the PP fabric revealed that the surface was clear with clean
scales and typical fibrous structure as displayed in Figure 4. On the other hand, the SEM
micrographs of the SeNPs-PP fabric show a coated layer of SeNPs into the PP fabric. Additionally, SeNPs were well distributed into the fabric. Figure 4. SEM micrographs of (a) the PP fabric, (b,c) SeNPs-PP fabric, (d) EDX analysis of the SeNPs-PP fabric. The chemical elements found on the surface of the treated PP fabric were analy
using EDX. The peaks around 1 and 11 Kev are attributed to SeNPs. The carbon and oxy
peaks were belonged to the native PP fabric. However, other elements were monitore
low concentration, such as Si, Ca, and Fe. Those traces of elements can be attribute Figure 4. SEM micrographs of (a) the PP fabric, (b,c) SeNPs-PP fabric, (d) EDX analysis of the SeNPs-PP fab The chemical elements found on the surface of the treated PP fabric were analyzed
using EDX. The peaks around 1 and 11 Kev are attributed to SeNPs. The carbon and oxygen
peaks were belonged to the native PP fabric. However, other elements were monitored at
low concentration, such as Si, Ca, and Fe. Those traces of elements can be attributed to The chemical elements found on the surface of the treated PP fabric were analyzed
using EDX. 3.1.3. UV/Vis Spectroscopy Analysis The peaks around 1 and 11 Kev are attributed to SeNPs. The carbon and oxygen
peaks were belonged to the native PP fabric. However, other elements were monitored at
low concentration, such as Si, Ca, and Fe. Those traces of elements can be attributed to Polymers 2021, 13, 2483 using IR-dyeing technique [42]. In addition, the Sulphur element was monitored at low
concentration because EDX is an elemental detection technique with a certain small error. Both SEM micrographs and EDX analysis confirmed the deposition of SeNPs into the PP
fabric surface as displayed in Figure 4. 3.2.2. Raman Spectroscopy Raman analysis revealed the chemical bonds inside the PP fabric and between the
SeNPs and PP surface. This analysis is important to compare the chemical structure of
Se-NPs-PP fabric and PP fabric as shown in Figure 5. The peaks at 2960 as well as 2888 cm−1
were corresponding to the asymmetric stretch of methyl group [43]. While the peak at
984 cm−1 was associated with asymmetrical stretching of C-C bond [44]. Furthermore,
Raman analysis revealed the presence of SeNPs into the SeNPs-PP fabric. The treated
fabric showed an obvious peak at 236 cm−1, corresponding to the symmetric stretching of
SeNPs [45]. On the other hand, no peak was observed in this region in the case of PP fabric
without SeNPs. Figure 5. Raman spectrums of (a) PP fabric and (b) SeNPs-PP fabric. Figure 5. Raman spectrums of (a) PP fabric and (b) SeNPs-PP fabric. 3.2.3. Colorimetric Study The color parameters of the SeNPs-PP fabric were analyzed using a Konica Minolta
spectrophotometer (CM-3600A). Figure 6 and Table 1 show the L*a*b* values of the fabric,
where (L*) values represent color lightness, (a*) is the red/green coordinate, and (b*) is
the yellow/blue coordinate [46]. These values indicated that SeNPs-PP fabric is darker
according to the color lightness values L*, less red and less yellow according to a*, b*
values, respectively. Effect of Treatment Time on Color Strength (K/S) The relationship between the K/S value of the SeNPs-PP fabric and treatment time (1,
2, and 3 h) is shown in Figure 7. Notably, the K/S value of PP fabric treated with SeNPs
(concentration of 50 mmole/L, L.R 1:50 and a temperature of 100 ◦C) increased with an
increase in the treatment time. The increase in K/S value reflected the positive effect that
increasing the treatment time had on the uniformity of PP adsorption of the SeNPs, and on
the uniformity of the penetration and diffusion of the SeNPs into the fabric; these effects in
turn contributed to an increase in the SeNPs uptake by the fabric [47,48]; which is indicated
by the highest K/S value observed at treatment time of 3 h. 7 Polymers 2021, 13, 2483 Figure 6. Lab colour space. Table 1. Optical measurements. Type
Sample
Colour Parameters
L*
a*
b*
C*
h
K/S
SeNPs-PP
fabric
47.39
23.45
17.09
29.02
36.08
4.34
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ϰ
ϱ
ϲ
Ϭ
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Figure 7. Effect of treatment time on color strength (K/S). Effect of Treatment Temperature on Color Strength (K/S)
Fi
8 h
th
l ti
hi
b t
t
t
t t
t
d th
l
t k Figure 6. Lab colour space. Table 1. Optical measurements. Type
Sample
Colour Parameters
L*
a*
b*
C*
h
K/S
SeNPs-PP
fabric
47.39
23.45
17.09
29.02
36.08
4.34 Figure 6. Lab colour space. Table 1. Optical measurements. Figure 6. Lab colour space. Table 1. Optical measurements. Ϭ
ϭ
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Figure 7. Effect of treatment time on color strength (K/S). Effect of Treatment Temperature on Color Strength (K/S) Figure 7. Effect of treatment time on color strength (K/S). Effect of Treatment Temperature on Color Strength (K/S) Effect of Treatment Temperature on Color Strength (K/S) Figure 8 shows the relationship between treatment temperature and the color uptake
(K/S) of the SeNPs-PP fabric with a (concentration of 50 mmol/L, L.R 1:50 and a treatment
time of 3h). K/S increased linearly with an increase in temperature from 70◦C to 120◦C and
increased considerably at low temperatures until the color approached to an equilibrium
point above 100◦C. The molecular structure opens, which facilitates the uptake of NPs as
the temperature increases. Hence, a high K/S value is obtained. Effect of Treatment Time on Color Strength (K/S) This can be attributed to
an increase in temperature, which improves the macromolecular chains of PP. Moreover, Figure 8 shows the relationship between treatment temperature and the color uptake
(K/S) of the SeNPs-PP fabric with a (concentration of 50 mmol/L, L.R 1:50 and a treatment
time of 3h). K/S increased linearly with an increase in temperature from 70◦C to 120◦C and
increased considerably at low temperatures until the color approached to an equilibrium
point above 100◦C. The molecular structure opens, which facilitates the uptake of NPs as
the temperature increases. Hence, a high K/S value is obtained. This can be attributed to
an increase in temperature, which improves the macromolecular chains of PP. Moreover, 8 Polymers 2021, 13, 2483 large pores and/or channels suitable for NPs penetration and diffusion are formed. Hence,
the optimum temperature was set at 100 ◦C [49–52]. large pores and/or channels suitable for NPs penetration and diffusion are formed. Hence,
the optimum temperature was set at 100 ◦C [49–52]. Ϭ
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ϴϬ
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Figure 8. Effect of treatment temperature on color strength (K/S). Figure 8. Effect of treatment temperature on color strength (K/S). 3.2.4. Exhaustion of SeNPs into PP Fabric 3.2.4. Exhaustion of SeNPs into PP Fabric The treatment solution was sampled before and after treatment to measure the SeNPs
exhaustion. Moreover, the absorbance of SeNPs solution was measured by using UV/VIS
spectrophotometer-model: Alpha-1860. Figure 9 showed the absorbance of SeNPs con-
centration before and after exhaustion by PP fabric in the wavelength ranges from 200 to
700 nm. The absorption spectrum of SeNPs-PP fabric before exhaustion shows a sharp
absorption band at 263 nm, indicating the presence of SeNPs. After exhaustion, remarkable
decrease in the absorbance of the treatment solution can be attributed to the low ratio of
SeNPs as it was absorbed by PP fabric.
Figure 9. Absorbance of SeNPs before and after treatment. Figure 9. Absorbance of SeNPs before and after treatment. 3.2.5. Physical Properties of SeNPs-PP Fabric The fastness of the SeNPs-PP fabric treated under optimum conditions was evaluated
mainly in washing, rubbing, and lightfastness. In addition, the tensile strength (elongation
and maximum force) was also evaluated. 3.2.6. Cytotoxicity of the SeNPs-PP Fabric 3.2.6. Cytotoxicity of the SeNPs-PP Fabric The cytotoxicity of the SeNPs-PP fabric was evaluated against healthy human cells
(wi-38) by the MTT assay. The viability of cells and the cytotoxicity of SeNPs-PP fabric
were evaluated against wi-38 cell lines. The viability of cells of SeNPs-PP fabric was 99.46%
of that of negative control. Whereas over 70% is the mean relative cell viability [54] Effect of Treatment Time on Color Strength (K/S) According to the results listed in Table 2, it
can be concluded that there is a decrease in elongation and a little increase in maximum
force of SeNPs-PP fabric without causing a significant damage to the structure of the yarn
indicating no significant change between PP and SeNPs-PP fabrics. The washing and
rubbing fastness were excellent, even after five washing cycles. Additionally, the light
fastness of SeNPs-PP fabric was also found to be in the field of good to very good, this 9 Polymers 2021, 13, 2483 indicates that the fixation of SeNPs onto PP fabric may be attributed to the generation of
metal chelates [53]. indicates that the fixation of SeNPs onto PP fabric may be attributed to the generation of
metal chelates [53]. Table 2. Properties of the PP and SeNPs-PP fabrics under optimum conditions. Table 2. Properties of the PP and SeNPs-PP fabrics under optimum conditions. Sample
Wash Fastness
Rubbing Fastness
Light
Fastness
Tensile Strength
St. Alt. Dry
Wet
Force, N
Elongation, %
PP fabric
-
-
-
-
-
1090
30.80
SeNPs-PP fabric
5
5
5
4–5
5
1102
27.36
SeNPs-PP fabric after
5 washing cycles
(durability test)
5
5
5
4–5
5
-
- 3.2.8. UV-Protection Properties of the SeNPs-PP Fabric Table 4 lists the results of the UV light protection characterization. The SeNPs-PP
fabric effectively blocked UV radiation; based on the AATCC test criteria: if UPF of any
fabric is more than 40as the fabric is a UV-defensive material [55]. 3.2.9. Electrical Conductivity Measurement The treatment of PP fabrics with SeNPs led to a slight increase in the electrical conduc-
tivity of PP fabrics. Samples treated with the optimum concentration of SeNPs showed
EC value of 5.84 × 10−11Ω−1 Cm−1 compared to untreated fabric which had EC value of
1.06 × 10−11 Ω−1 cm−1. 4. Conclusions In this paper, we propose a novel approach for coloring and incorporating new
functionalities to PP fabrics via one-step process using SeNPs. PP fabrics were colored
from light to dark orange depending on the treatment time and concentration of sodium
hydrogen selenite. The obtained results show that the deposition of SeNPs into PP fabric
is accompanied by a considerable improvement in UV-protection. The obtained colored
fabrics effectively blocked UV radiations, providing excellent UV-protection. Additionally,
the treated fabrics exhibited outstanding washing, rubbing and light fastness. Moreover,
the colored PP fabric showed excellent antibacterial activity against Staphylococcus aureus,
Bacillus cereus, and Escherichia coli and very good antibacterial activity against Pseudomonas
aeruginosa compared with the standard drugs such as tetracycline and ciprofloxacin. The
tensile strength of the colored fabrics increased slightly accompanied by a slight decrease
in elongation. This novel, and economical approach can be employed in the industry
for coloration and multifunctionalization of PP fabrics instead of traditional dyeing and
finishing processes. Author Contributions: Conceptualization, T.A. and S.A.A.; methodology, S.A.A.; software, S.A.A. validation, S.M.R., K.S.-A. and T.A.; formal analysis, K.S.-A.; investigation, S.M.R.; resources, S.M.R.;
data curation, S.A.A.; writing—original draft preparation, S.A.A.; writing—review and editing, T.A. and M.R.P.; visualization, M.R.P.; supervision, T.A.; project administration, T.A. All authors have
read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the
corresponding author. Conflicts of Interest: The authors declare no conflict of interest. 3.2.7. Antibacterial Activity of the SeNPs-PP Fabric 3.2.7. Antibacterial Activity of the SeNPs-PP Fabric Table 3 lists the antibacterial activity of the SeNPs-PP fabric using four bacterial strains,
(Staphylococcus aureus and Bacillus cereus) as Gram-positive bacteria and (Escherichia coli
and Pseudomonas aeruginosa) as Gram-negative bacteria. Tetracycline and ciprofloxacin
were used as standard drugs. The results reveal that the SeNPs-PP fabric exhibits excellent
antibacterial activity against Escherichia coli, Bacillus cereus, as well as Staphylococcus aureus
and very good antibacterial activity against Pseudomonas aeruginosa, which is indicated by
a clear zone diameter of bacterial colonies. The obtained results indicate the presence of
broad-spectrum antibacterial activity. The mechanism of action of SeNPs on bacteria is
still unclear. In this study, we suggest the following phenomena: (a) the release of ions
along with physical interaction with the bacterial cell wall peptidoglycan layer damages
the double-stranded structure of DNA. (b) the formation of reactive oxygen species and
inhibition of DNA replication. (c)nanoparticles can be in better contact with bacterial or
fungal cells than colloidal form [53]. Table 3. Clear zone (mm) of the PP and SeNPs-PP fabrics. Table 3. Clear zone (mm) of the PP and SeNPs-PP fabrics. Table 3. Clear zone (mm) of the PP and SeNPs-PP fabrics. Substrate
Antibacterial Activity
Diameter of Clear Zone (mm)
Staphylococcus
aureus
(G+)
Bacillus cereus
(G+)
Escherichia coli
(G-)
Pseudomonas
aeruginosa
(G-)
Ciprofloxacin
24
15
23
17
Tetracycline
21
14
20
15
Blank
_
_
_
_
SeNPs-PP fabric
20.9
22.7
23.2
11.3 3.2.8. UV-Protection Properties of the SeNPs-PP Fabric 3.2.8. UV-Protection Properties of the SeNPs-PP Fabric Table 4 lists the results of the UV light protection characterization. The SeNPs-PP
fabric effectively blocked UV radiation; based on the AATCC test criteria: if UPF of any
fabric is more than 40as the fabric is a UV-defensive material [55]. Table 4 lists the results of the UV light protection characterization. The SeNPs-PP
fabric effectively blocked UV radiation; based on the AATCC test criteria: if UPF of any
fabric is more than 40as the fabric is a UV-defensive material [55]. 10 Polymers 2021, 13, 2483 Table 4. UPF of the PP and SeNPs-PP fabrics. Sample
UVA
315–400 nm
UVB
290–315 nm
UPF Value
Blank PP fabric
35.89
29.49
3.20
SeNPs-PP fabric
0.11
0.11
920.19
3 2 9 El
t i
l C
d
ti it
M
t Table 4. UPF of the PP and SeNPs-PP fabrics. Table 4. UPF of the PP and SeNPs-PP fabrics. 3.
Wang, Q.; Barnes, L.-M.; Maslakov, K.I.; Howell, C.A.; Illsley, M.J.; Dyer, P.; Savina, I.N. In situ synthesis of silver or selenium
nanoparticles on cationized cellulose fabrics for antimicrobial application. Mater. Sci. Eng. C 2021, 121, 111859. [CrossRef]
4.
Beyth, N.; Houri-Haddad, Y.; Domb, A.; Khan, W.; Hazan, R. Alternative Antimicrobial Approach: Nano-Antimicrobial Materials.
Evid Based Complement Altern Med 2015 2015 246012 [CrossRef] [PubMed] 1.
Markovi´c, D.; Tseng, H.-H.; Nunney, T.; Radoiˇci´c, M.; Ilic-Tomic, T.; Radeti´c, M. Novel antimicrobial nanocomposite based
on polypropylene non-woven fabric, biopolymer alginate and copper oxides nanoparticles. Appl. Surf. Sci. 2020, 527, 146829.
[CrossRef] 2.
Gupta, D.; Khare, S.K.; Laha, A. Antimicrobial properties of natural dyes against Gram-negative bacteria. Color. Technol. 2004,
120, 167–171. [CrossRef] p
[
] [
]
5.
Abou Elmaaty, T.; Abdeldayem, S.; Elshafai, N. Simultaneous Thermochromic Pigment Printing and Se-NP Multifunctional
Finishing of Cotton Fabrics for Smart Childrenswear. Cloth. Text. Res. J. 2020, 38, 182–195. [CrossRef] References A review on the application of inorganic nano-structured materials in the modification of textiles:
Focus on anti-microbial properties. Colloids Surf. B 2010, 79, 5–18. [CrossRef] .; Montazer, M. A review on the application of inorganic nano-structured materials in the modification of t
ti-microbial properties. Colloids Surf. B 2010, 79, 5–18. [CrossRef] 12. Dastjerdi, R.; Montazer, M. A review on the application of inorganic nano-structured materials in the
Focus on anti-microbial properties. Colloids Surf. B 2010, 79, 5–18. [CrossRef] Montazer, M. A review on the application of inorganic Dastjerdi, R.; Montazer, M. A review on the application of inorganic nano-struct
Focus on anti-microbial properties. Colloids Surf. B 2010, 79, 5–18. [CrossRef] 13. Simonˇciˇc, B.; Klemenˇciˇc, D. Preparation and performance of silver as an antimicrobial agent for textiles: A review. Text. Res. J. 2015, 86, 210–223. [CrossRef] 14. Radeti´c, M. Functionalization of textile materials with silver nanoparticles. J. Mater. Sci. 2013, 48, 95–107. [CrossRef]
15
R d ti´ M M
k
i´ D N
fii hi
f
ll l
t
til
t
i l
ith
d
id
ti l
C ll l
2019 Functionalization of textile materials with silver nanoparticles. J. Mater. Sci. 2013, 48, 95–107. [CrossRef] 14. Radeti´c, M. Functionalization of textile materials with silver nanoparticles. J. Mater. Sci. 2013, 48, 95–10 15. Radeti´c, M.; Markovi´c, D. Nano-finishing of cellulose textile materials with copper and copper oxide nan
26, 8971–8991. [CrossRef] 16. Yip, J.; Liu, L.; Wong, K.-H.; Leung, P.H.M.; Yuen, C.-W.M.; Cheung, M.-C. Investigation of antifungal and antibacterial effects of
fabric padded with highly stable selenium nanoparticles. J. Appl. Polym. Sci. 2014, 131. [CrossRef] 17. Biswas, D.P.; O’Brien-Simpson, N.M.; Reynolds, E.C.; O’Connor, A.J.; Tran, P.A. Comparative study of novel in situ decorated
porous chitosan-selenium scaffolds and porous chitosan-silver scaffolds towards antimicrobial wound dressing application. J. Colloid Interface Sci. 2018, 515, 78–91. [CrossRef] [PubMed] f
18. Yu, B.; Zhang, Y.; Zheng, W.; Fan, C.; Chen, T. Positive Surface Charge Enhances Selective Cellular Uptake and Anticancer Efficacy
of Selenium Nanoparticles. Inorg. Chem. 2012, 51, 8956–8963. [CrossRef] 19. Forootanfar, H.; Adeli-Sardou, M.; Nikkhoo, M.; Mehrabani, M.; Amir-Heidari, B.; Shahverdi, A.R.; Shakibaie, M. Antioxidant
and cytotoxic effect of biologically synthesized selenium nanoparticles in comparison to selenium dioxide. J. Trace Elem. Med. Biol. 2014, 28, 75–79. [CrossRef] 20. Shakibaie, M.; Forootanfar, H.; Golkari, Y.; Mohammadi-Khorsand, T.; Shakibaie, M.R. References Anti-biofilm activity of biogenic selenium
nanoparticles and selenium dioxide against clinical isolates of Staphylococcus aureus, Pseudomonas aeruginosa, and Proteus mirabilis. J. Trace Elem. Med. Biol. 2015, 29, 235–241. [CrossRef] [PubMed] an, H.; Al-Harbi, N.; Karuppiah, P.; Rajaram, S.K. Microbial synthesis of selinium nanocomposite using Sacc 21. Hariharan, H.; Al-Harbi, N.; Karuppiah, P.; Rajaram, S.K. Microbial synthesis of selinium nanocompo
cerevisiae and its antimicrobial activity against pathogens causing nosocomial infection. Chalcog. Lett. 2 Hariharan, H.; Al-Harbi, N.; Karuppiah, P.; Rajaram, S.K. Microbial synthesis of selinium nanocomposite usi
erevisiae and its antimicrobial activity against pathogens causing nosocomial infection Chalcog Lett 2012 9 5 pp
j
y
p
g
e and its antimicrobial activity against pathogens causing nosocomial infection. Chalcog. Lett. 2012, 9, 509–515 22. Shakibaie, M.; Salari Mohazab, N.; Ayatollahi Mousavi, S.A. Antifungal Activity of Selenium Nanoparticles Synthesized by
B
ill
i
M h 1
i
t A
ill
f
i
t
d C
did
lbi
J
di h
J Mi
bi l 2015 8
26381 [C
R f] 22. Shakibaie, M.; Salari Mohazab, N.; Ayatollahi Mousavi, S.A. Antifungal Activity of Selenium Nanoparticles Synthesized by
Bacillus species Msh-1 against Aspergillus fumigatus and Candida albicans. Jundishapur J. Microbiol. 2015, 8, e26381. [CrossRef] 23. Beheshti, N.; Soflaei, S.; Shakibaie, M.; Yazdi, M.H.; Ghaffarifar, F.; Dalimi, A.; Shahverdi, A.R. Efficacy of biogenic selenium
nanoparticles against Leishmania major: In vitro and in vivo studies. J. Trace Elem. Med. Biol. 2013, 27, 203–207. [CrossRef]
[PubMed] [
]
24. Gawish, S.M.; Mosleh, S.E.-S.; Ramadan, A. Review Improvement of Polypropylene Properties by Irradiation/Grafting and Other
Modifications. Egypt. J. Chem. 2019, 62, 29–48. [CrossRef] 25. Gawish, S.; Ramadan, A.; Mosleh, S. Improvement of Polypropylene (PP) Dyeing by Modification Methods. Egypt. J. Chem. 2018,
62, 49–62. [CrossRef] 26. Tao, G.; Gong, A.; Lu, J.; Sue, H.-J.; Bergbreiter, D.E. Surface Functionalized Polypropylene: Synthesis, Characterization, and
Adhesion Properties. Macromolecules 2001, 34, 7672–7679. [CrossRef] 27. Goddard, J.M.; Hotchkiss, J.H. Polymer surface modification for the attachment of bioactive compounds. Prog. Polym. Sci. 2007,
32, 698–725. [CrossRef] D.; Tarafdar, A.; Panda, A.B.; Pramanik, P. Surface modification of low-density polyethylene films by a no 28. Bandopadhay, D.; Tarafdar, A.; Panda, A.B.; Pramanik, P. Surface modification of low-dens
solution base chemical process. J. Appl. Polym. Sci. 2004, 92, 3046–3051. [CrossRef] dopadhay, D.; Tarafdar, A.; Panda, A.B.; Pramanik, P. Surface modification of low d
ion base chemical process. J. Appl. Polym. Sci. 2004, 92, 3046–3051. [CrossRef] 29. 34.
American Association of Textile Chemists and Colorists. AATTCC 16A-1972: Colorfastness to Light: Carbon-arc Lamp; American
Association of Textile Chemists and Colorists: Research Triangle Park, NC, USA, 1972. References 1. Markovi´c, D.; Tseng, H.-H.; Nunney, T.; Radoiˇci´c, M.; Ilic-Tomic, T.; Radeti´c, M. Novel antimicrobial nanocomposite based
on polypropylene non-woven fabric, biopolymer alginate and copper oxides nanoparticles. Appl. Surf. Sci. 2020, 527, 146829. [CrossRef] 1. Markovi´c, D.; Tseng, H.-H.; Nunney, T.; Radoiˇci´c, M.; Ilic-Tomic, T.; Radeti´c, M. Novel antimicrobial nanocomposite based
on polypropylene non-woven fabric, biopolymer alginate and copper oxides nanoparticles. Appl. Surf. Sci. 2020, 527, 146829. [CrossRef] [
]
2. Gupta, D.; Khare, S.K.; Laha, A. Antimicrobial properties of natural dyes against Gram-negative bacteria. Color. Technol. 2004,
120, 167–171. [CrossRef] 3. Wang, Q.; Barnes, L.-M.; Maslakov, K.I.; Howell, C.A.; Illsley, M.J.; Dyer, P.; Savina, I.N. In situ synthesis of silver or selenium
nanoparticles on cationized cellulose fabrics for antimicrobial application. Mater. Sci. Eng. C 2021, 121, 111859. [CrossRef] 11 Polymers 2021, 13, 2483 6. Abou Elmaaty, T.; Mostafa, S.; Nasr Eldin, S.; Elgamal, G.M. One Step Thermochromic Pigment Printing a
Functional Finishing of Cotton and Cotton/PET Fabrics. Fibers Polym. 2018, 19, 2317–2323. [CrossRef] 7. Abou Elmaaty, T.; Abdelaziz, E.; Nasser, D.; Abdelfattah, K.; Elkadi, S.; El-Nagar, K.I. Microwave and nanotechnology advanced
solutions to improve ecofriendly cotton’s coloration and performance properties. Egypt. J. Chem. 2018, 61, 493–502. [CrossRef] p
y
p
p
p
gyp
J
,
,
[
]
8. Abou Elmaaty, T.; Elnnagar, K.; Raouf, S.; Abdelfattah, K.; El-Kadi, S.; Abdelaziz, E. One-step green approach for functional
printing and finishing of textiles using silver and gold NPs RSC Adv 2018 8 25546–25557 [CrossRef] 8. Abou Elmaaty, T.; Elnnagar, K.; Raouf, S.; Abdelfattah, K.; El-Kadi, S.; Abdelaziz, E. One-step green approach for functional y
g
p g
pp
d finishing of textiles using silver and gold NPs. RSC Adv. 2018, 8, 25546–25557. [CrossRef] y
g
p g
printing and finishing of textiles using silver and gold NPs. RSC Adv. 2018, 8, 25546–25557. [CrossRef] g
g
g
g
9. Abou Elmaaty, T.; Mandour, B. ZnO and TiO2 Nanoparticles as Textile Protecting Agents against UV Radiation: A Review. Asian
J. Chem. Sci. 2018, 4, 1–14. [CrossRef] 10. Gold, K.; Slay, B.; Knackstedt, M.; Gaharwar, A.K. Antimicrobial Activity of Metal and Metal-Oxide Based Nanoparticles. Adv. Ther. 2018, 1, 1700033. [CrossRef] 11. Roy, A.; Bulut, O.; Some, S.; Mandal, A.K.; Yilmaz, M.D. Green synthesis of silver nanoparticles
organizations targeting antimicrobial activity. RSC Adv. 2019, 9, 2673–2702. [CrossRef] organizations targeting antimicrobial activity. RSC Adv. 2019, 9, 2673 2702. [CrossRef]
12. Dastjerdi, R.; Montazer, M. References Perkas, N.; Shuster, M.; Amirian, G.; Koltypin, Y.; Gedanken, A. Sonochemical immobilization of silver nanoparticles on porous
polypropylene. J. Polym. Sci. Part A Polym. Chem. 2008, 46, 1719–1729. [CrossRef] p
yp
py
J
y
y
[
]
30. Gawish, S.M.; Mosleh, S. Antimicrobial Polypropylene Loaded by Silver Nano Particles. Fibers Polym. 2020, 21, 19–23. [CrossRef]
31. Elmaaty, T.A.; Raouf, S.; Sayed-Ahmed, K. Novel One Step Printing and Functional Finishing of Wool Fabric Using Selenium
Nanoparticles Fibers Polym 2020 21 1983–1991 [CrossRef] p
yp
py
J
y
y
30. Gawish, S.M.; Mosleh, S. Antimicrobial Polypropylene Loaded by Silver Nano Particles. Fibers Polym. 2020, 21, 19–23. [CrossRef] p
yp
py
J
y
y
30. Gawish, S.M.; Mosleh, S. Antimicrobial Polypropylene Loaded by Silver Nano Particle p
yp
py
y
y
30. Gawish, S.M.; Mosleh, S. Antimicrobial Polypropylene Loaded by Silver Nano Particles. Fibers Polym. 2020, 21, 19–23. [CrossRef]
31
El
t
T A R
f S S
d Ah
d K N
l O
St
P i ti
d F
ti
l Fi i hi
f W
l F b i U i
S l
i yp
py
y
y
31. Elmaaty, T.A.; Raouf, S.; Sayed-Ahmed, K. Novel One Step Printing and Functional Finishing of Woo
Nanoparticles. Fibers Polym. 2020, 21, 1983–1991. [CrossRef] 32. Brandrup, J.; Immergut, E.H.; Grulke, E.A.; Abe, A.; Bloch, D.R. Polymer Handbook; Wiley: New York, NY, USA, 1999; p. 89. 33. Malhotra, S.; Jha, N.; Desai, K. A superficial synthesis of selenium nanospheres using wet chemical approach. Int. J. Nanotechnol. Appl. 2013, 3, 7–14. 34. American Association of Textile Chemists and Colorists. AATTCC 16A-1972: Colorfastness to Light: Carbon-arc Lamp; American
Association of Textile Chemists and Colorists: Research Triangle Park, NC, USA, 1972. 12 Polymers 2021, 13, 2483 35. International Organization for Standardization. ISO 13934-1:1999: Tensile Properties of Fabrics; ISO: Vern g
p
f
36. American Association of Textile Chemists and Colorists. AATTCC 61(2A)-1996: Colorfastness to Laundering; American Association
of Textile Chemists and Colorists: Research Triangle Park, NC, USA, 1996. 36. American Association of Textile Chemists and Colorists. AATTCC 61(2A)-1996
of Textile Chemists and Colorists: Research Triangle Park, NC, USA, 1996. 37. American Association of Textile Chemists and Colorists. AATTCC 147-2004: Antimicrobial Activity Assessment of Textile Materials;
American Association of Textile Chemists and Colorists: Research Triangle Park, NC, USA, 2004. 38. Vieira, A.; Stein, E.; Andreguetti, D.; Cebrián-Torrejón, G.; Domènech, A.; Colepicolo, P.; Da Costa Ferreira, A.M. References “Sweet
Chemistry”: A Green Way for Obtaining Selenium Nanoparticles Active against Cancer Cells. J. Braz. Chem. Soc. 2017, 28,
2021–2027. [CrossRef] 39. Wang, S.; Ajji, A.; Guo, S.; Xiong, C. Preparation of Microporous Polypropylene/Titanium Dioxide Composite Membranes with
Enhanced Electrolyte Uptake Capability via Melt Extruding and Stretching. Polymers 2017, 9, 110. [CrossRef] 40. Alagesan, V.; Venugopal, S. Green Synthesis of Selenium Nanoparticle Using Leaves Extract of Wi
Biological Applications and Photocatalytic Activities. BioNanoScience 2019, 9, 105–116. [CrossRef] Alagesan, V.; Venugopal, S. Green Synthesis of Selenium Nanoparticle Using Leaves Extract of Withan 40. Alagesan, V.; Venugopal, S. Green Synthesis of Selenium Nanoparticle Using Leaves Extract of
Biological Applications and Photocatalytic Activities. BioNanoScience 2019, 9, 105–116. [CrossRef] Biological Applications and Photocatalytic Activities. BioNanoScience 2019, 9, 105–116. [CrossRef] 41. Satgurunathan, T.; Bhavan, P.; Komathi, S. Green synthesis of selenium nanoparticles from sodium selenite using garlic extract
and its enrichment on Artemia nauplii to feed the fresh water prawn Macrobrachium rosenbergii post-larvae. Res. J. Chem. Environ. 2017, 21, 1–12. 42. El-Kheir, A.; El-Ghany, N.; Fahmy, M.; Aboras, S.; El-Gabry, L. Functional Finishing of Polyester
Nano-Particles. Egypt. J. Chem. 2019, 63. [CrossRef] Nano-Particles. Egypt. J. Chem. 2019, 63. [CrossRef]
43. Arruebarrena de Báez, M.; Hendra, P.J.; Judkins, M. The Raman spectra of oriented isotactic polypropylene. Spectrochim. Acta Part
A Mol Spectrosc 1995 51 2117 [CrossRef] ez, M.; Hendra, P.J.; Judkins, M. The Raman spectra of oriented isotactic polypropylene. Spectrochim. Acta Part
95, 51, 2117. [CrossRef] 43. Arruebarrena de Báez, M.; Hendra, P.J.; Judkins, M. The Raman spectra of oriented isotactic polypropyle
A Mol. Spectrosc. 1995, 51, 2117. [CrossRef] 44. Martin, J.; Ponçot, M.; Hiver, J.M.; Bourson, P.; Dahoun, A. Real-time Raman spectroscopy measurements to study the uniaxial
tension of isotactic polypropylene: A global overview of microstructural deformation mechanisms. J. Raman Spectrosc. 2013, 44,
776–784. [CrossRef] A.; Montazer, M.; Mahmoudi Rad, M. Low toxic antibacterial application with hydrophobic properties
gh facile and clean fabrication of nano copper with fatty acid. Mater. Sci. Eng. C 2019, 97, 177–187. [CrossRef] 45. Bashiri Rezaie, A.; Montazer, M.; Mahmoudi Rad, M. Low toxic antibacterial application with hyd
polyester through facile and clean fabrication of nano copper with fatty acid. Mater. Sci. Eng. C 2019, 9 46. Khalifa, M.E.; Abdel-Latif, E.; Gobouri, A.A. Disperse Dyes Based on 5-Arylazo-thiazol-2-ylcarbamoyl-thiophenes: Synthesis,
Antimicrobial Activity and Their Application on Polyester. J. Heterocycl. Chem. 2015, 52, 674–680. [CrossRef] 47. Long, J.-J.; Ma, Y.-Q.; Zhao, J.-P. 36.
American Association of Textile Chemists and Colorists. AATTCC 61(2A)-1996: Colorfastness to Laundering; American Association
of Textile Chemists and Colorists: Research Triangle Park, NC, USA, 1996. Article
Electromagnetic Wave-Absorbing and Bending Properties of
Three-Dimensional Honeycomb Woven Composites Li-Hua Lyu 1,2,*, Wen-Di Liu 2 and Bao-Zhong Sun 1 Li-Hua Lyu 1,2,*, Wen-Di Liu 2 and Bao-Zhong Sun 1 1
Key Laboratory of High Performance Fibers & Products, Ministry of Education, Donghua University,
Shanghai 201620, China; sunbz@dhu.edu.cn 1
Key Laboratory of High Performance Fibers & Products, Ministry of Education, Donghua University
Shanghai 201620, China; sunbz@dhu.edu.cn 2
School of Textile and Material Engineering, Dalian Polytechnic University, Dalian 116034, China;
18108210003147@xy.dlpu.edu.cn
*
Correspondence: lvlh@dlpu.edu.cn 2
School of Textile and Material Engineering, Dalian Polytechnic University, Dalian 116034, China;
18108210003147@xy.dlpu.edu.cn
*
Correspondence: lvlh@dlpu.edu.cn *
Correspondence: lvlh@dlpu.edu.cn Abstract: To avoid the delamination of the traditional three-dimensional (3-D) honeycomb electro-
magnetic (EM) absorbing composites and improving the defects of low mechanical properties, the
3-D honeycomb woven fabrics were woven on the ordinary loom by practical design. The fabrication
of 3-D honeycomb woven EM absorbing composites was based on carbon black/carbonyl iron
powder/basalt fiber/carbon fiber/epoxy resin (CB/CIP/BF/CF/EP) by the vacuum-assisted resin
transfer molding (VARTM) process. A CB/CIP composite absorbent study showed that CB/CIP com-
posite absorbent belongs to a magnetic loss type absorbent. Adding CB/CIP significantly improved
the absorption performance of composite, increased the absorption peak and the effective absorp-
tion bandwidth (EAB), but the bending performance decreased. The normalization analysis results
showed that when the thickness was 15 mm, the mechanical properties and EM wave-absorbing
properties of the 3-D honeycomb woven composite were the best matches. The morphological
characteristics and displacement load curves of the composite after fracture were analyzed. The
bending failure modes were brittle fracture of the fiber bundle, matrix cracking, and typical shear
failure. Despite the above failure mechanism, the 3-D honeycomb woven EM absorbing composites
still has good integrity without delamination. Wave-Absorbing and Bending
Properties of Three-Dimensional
Honeycomb Woven Composites. Polymers 2021, 13, 1485. https://
doi.org/10.3390/polym13091485 Keywords: 3-D honeycomb woven fabric; CB/CIP; mechanical property; EM wave-absorbing property Academic Editor: Tarek M. Abou Elmaaty Academic Editor: Tarek M. Abou Elmaaty References Investigations on the level dyeing of fabrics in supercritical carbon dioxide. J. Supercrit. Fluids
2011, 57, 80–86. [CrossRef] 48. Hou, A.; Dai, J. Kinetics of dyeing of polyester with CI Disperse Blue 79 in supercritical carbon dioxide. Coloration Technol. 2005,
121, 18–20. [CrossRef] 49. Al-Etaibi, A.M.; Alnassar, H.S.; El-Apasery, M.A. Dyeing of polyester with disperse dyes: Part 2. Synthesis and dyeing
characteristics of some azo disperse dyes for polyester fabrics. Molecules 2016, 21, 855. [CrossRef] 50. Farizadeh, K.; Yazdanshenas, M.; Montazer, M.; Malek, R.; Rashidi, A. Kinetic studies of adsorption o
various models. Text. Res. J. 2010, 80, 847–855. [CrossRef] 51. Kim, S.; Wairkar, Y.P.; Daniels, R.W.; DiAntonio, A. The novel endosomal membrane protein Ema interacts with the class C
Vps–HOPS complex to promote endosomal maturation. J. Cell Biol. 2010, 188, 717–734. [CrossRef] p
p
p
52. Miah, L.; Ferdous, N.; Azad, D. Textiles Material Dyeing with Supercritical Carbon Dioxide (CO2) without using Water. Chem. Mater. Res. 2013, 3, 38–40. 53. Pandit, P.; Teli, M.D.; Therani Nadathur, G.; Maiti, S.; Singha, K.; Maity, S. Green synthesis of nanoparticle and its application on
cotton fabric using Sterculia foetida fruit shell extract. J. Text. Eng. Fash. Technol. 2020, 6. [CrossRef] 54. Kangwansupamonkon, W.; Lauruengtana, V.; Surassmo, S.; Ruktanonchai, U. Antibacterial effect of apatite-coated titanium
dioxide for textiles applications. Nanomed. Nanotechnol. Biol. Med. 2009, 5, 240–249. [CrossRef] pp
55. Mousa, M.A.; Khairy, M. Synthesis of nano-zinc oxide with different morphologies and its application on fabrics for UV protection
and microbe-resistant defense clothing. Text. Res. J. 2020, 90, 2492–2503. [CrossRef] 13 13 Citation: Lyu, L.-H.; Liu, W.-D.; Sun,
B.-Z. Electromagnetic
Wave-Absorbing and Bending
Properties of Three-Dimensional
Honeycomb Woven Composites.
Polymers 2021, 13, 1485. https://
doi.org/10.3390/polym13091485
Academic Editor: Tarek M.
Abou Elmaaty
Received: 5 April 2021
Accepted: 30 April 2021
Published: 5 May 2021 polymers polymers 1. Introduction Therefore, the traditional honeycomb
structure EM absorbing composites have difficulty achieving load-bearing and a broadband
EM energy absorbing capability. gy
g
p
y
However, three-dimensional (3-D) honeycomb woven composites are composites
which are made of 3-D honeycomb woven fabric as reinforcement and resin or other
polymers as the matrix. Three-dimensional honeycomb woven fabric is a kind of 3-D
structure textile material. In the fabric structure of the textile material, the concept of
vertical yarn was introduced along the thickness direction of the material, and the vertical
yarn was used to connect the warp yarn and the weft yarn. There were fiber bundles
and reinforcement structures through the thickness direction to enhance the integrity of
the textile materials. Therefore, 3-D honeycomb woven composites have good integrity
and structural stability and can solve the interlayer problems of traditional honeycomb
structure EM absorbing composites [11]. Three-dimensional honeycomb woven fabrics
have been applied in a number of practical applications, for example fibrous porous
media [12]. Bayraktar et al. [13] prepared a 3-D honeycomb woven composite and carried
out low-speed impact tests on fabric samples using a drop-weight impact tester. The
experimental results showed that the energy absorbed by the honeycomb structure was
more significant than that absorbed by the plate sample. The honeycomb woven fabric
showed good mechanical properties. Lv et al. [14] prepared 3-D honeycomb structure
composites using glass fiber filament yarn/BF filament yarn on the ordinary loom and
tested their bending properties, providing a reference structural optimization design and
performance analysis of 3-D honeycomb woven composites. Zahid et al. [15] wove three
kinds of 3-D honeycomb woven fabrics with different honeycomb sizes, and the breaking
strength and elongation at break of the 3-D honeycomb woven fabrics were measured. The
effect of honeycomb size on the mechanical properties should be considered in the design
and weaving of 3-D honeycomb woven fabrics. g
y
Current studies have shown that 3-D honeycomb woven composites have good anti-
delamination abilities. However, there was no research on the EM absorbing property of
3-D honeycomb woven composites. Therefore, the low-cost design and preparation of
3-D honeycomb woven EM absorbing composites with the integration of load-bearing
and absorbing structures and functions can realize the collaborative design of mechanical
properties and EM absorbing properties the composite. 1. Introduction The problem of electromagnetic (EM) interference and radiation has stimulated the
research interest in EM absorbing composites with thin thickness, low density, broadband
and strong absorption [1]. The research of EM absorbing composites has gradually turned
to structural EM absorbing composites, which have the functions of load-bearing and a
broadband EM energy absorbing capability. At present, the research of structural absorb-
ing composites, such as foam EM absorbing composites and honeycomb EM absorbing
composites, is being studied to overcome the disadvantages of traditional EM absorbing
composites, such as high density, weak absorption and narrow bandwidth [2]. Compared
with other structures, the honeycomb structure can not only be used as a part of the main
load-bearing structure, but also as a carrier of EM absorbing composites and absorbing
medium [3,4]. Li et al. [5] prepared carbon-coated honeycomb absorbing composites and
analyzed in detail the effects of honeycomb aperture, honeycomb height, coating thickness,
dielectric constant, and dielectric loss factor on EM absorption. Wang et al. [6] integrated
shorted carbon fibers into honeycomb frames as a dual-functional material for radar absorp-
tion and structural reinforcement, which showed excellent EM absorption at frequencies
ranging from 2–18 GHz. Sun et al. [7] used a honeycomb structure of aramid paper im-
pregnated with EM absorbent as the core layer and quartz fiberboard as the skin to form
a composite sandwich structure with EM absorption ability bonding. The experimental Received: 5 April 2021
Accepted: 30 April 2021
Published: 5 May 2021 Publisher’s Note: MDPI stays neutral
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conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). 15 https://www.mdpi.com/journal/polymers Polymers 2021, 13, 1485. https://doi.org/10.3390/polym13091485 Polymers 2021, 13, 1485 results showed that the minimum reflection loss (RLmin) was −29.5 dB, and the EAB was
13.1 GHz. At present, honeycomb structure EM absorbing composites are mostly prepared by the
lamination method, which has some disadvantages which were under high temperature
and high humidity environments or alternating external forces, the honeycomb structure
EM absorbing composites are easy to break [8–10]. 1. Introduction p
p
g p
p
p
Therefore, in this study, three different thicknesses (7.5 mm, 15 mm, 22.5 mm) of
3-D honeycomb fabrics with ordinary looms were woven through practical design. The
CB/CIP/EP as the matrix and the 3-D honeycomb woven EM absorbing composites were
prepared by the VARTM process. Then, the coaxial method was used to study the EM
properties of CB/CIP. The 3-D honeycomb woven EM absorbing composite used the United
States Naval Research Laboratory (NRL) arch method to test the RL on 2–18 GHz. Next,
the bending properties were tested by a microcomputer-controlled electronic universal
testing machine. Finally, it discussed particles and absorbent material thickness absorbing
the composites’ performance and the influence of mechanical properties, and experimental
results obtained from the failure modes. An effective method to coordinate the design of
absorbing and mechanical properties was proposed. 2. Materials and Methods 2. Materials and Methods 2.1. Materials and Equipment 2.1. Materials and Equipment EP JC-02A and the solidification reagent JC-02B (Changshu Jiafa Chemical Co., Ltd.,
Changshu City, China). 800 tex CF filament SYT49 (Zhongfu Shenying Carbon Fiber Co.,
Ltd., Lianyungang City, China). 800 tex BF filament (Zhejiang Shijin Basalt Fiber Co., Ltd., 16 Polymers 2021, 13, 1485 Hangzhou City, China). Ordinary loom SGA 598 (Jiangyin Tong Yuan spinning machine
Co., Ltd., Wuxi City, China). The universal testing machine TH-8102S (Suzhou Tuobo
Machinery Equipment Co., Ltd., Suzhou City, China). 2.2. Preform Design and Weaving The process involved
placing the one-layer of 3-D honeycomb woven fabric (200 mm × 200 mm × 7.5 mm),
the two-layer of 3-D honeycomb woven fabric (200 mm × 200 mm × 15 mm), and the
three-layer of 3-D honeycomb woven fabric (200 mm × 200 mm × 22.5 mm) into their
molds, closing the molds, and checking for leaks. Then, the resin solution was injected into
the mold. The injection process was continued until a sufficient resin volume was seen in
the resin trap to indicate that the mold had been completely filled with resin. The mold was
isolated from the resin pot and the resin trap and then put into an air-circulating oven. The
manufacturer-recommended cure cycle was employed: the first step of the cure cycle was
2 h at 90 ◦C, with the second step being 1 h at 130 ◦C, and the final step being 4 h at 150 ◦C,
as shown in Figure 3b. The 3-D honeycomb woven EM absorbing composite without
adding CB/CIP, with a thickness of 7.5 mm, was recorded as S0. The 3-D honeycomb
woven EM absorbing composite with added CB/CIP, with a thickness of 7.5 mm, was
recorded as S1. The 3-D honeycomb woven EM absorbing composite with added CB/CIP,
with a thickness of 15 mm, was recorded as S2. The 3-D honeycomb woven EM absorbing
composite with added CB/CIP, with a thickness of 22.5 mm, was recorded as S3. Table 1. Weaving parameters of 3-D honeycomb woven fabrics. Thickness (mm)
Linear Density/Tex
Layer Number of Yarns
Weaving Density (Yarn/10 cm)
Warp/Weft Yarns
Warp Density
Weft Density
7.5
800
1
180
95
15
2
300
22.5
3
420 Table 1. Weaving parameters of 3-D honeycomb woven fabrics. 2.3. Fabrication of the 3-D Honeycomb Woven EM Absorbing Composites The ratio of the EP, CIP, solidification reagent, and CB was 4:4:3.2:0.03. VARTM
manufactured the 3-D honeycomb woven EM absorbing composites. Figure 3a showed the
schematic diagram of the VARTM process. The CB, CIP, and EP were mixed in a beaker
and mechanically stirred until the CB/CIP could be uniformly dispersed in the EP without
significant settling. After vacuum defoaming for 1 h in a vacuum drying oven at 60 ◦C,
the CB/CIP/EP mixed solution without bubbles was obtained. 2.2. Preform Design and Weaving After vacuum defoaming for 1 h in a vacuum drying oven at 60 ◦C,
the CB/CIP/EP mixed solution without bubbles was obtained. The process involved
placing the one-layer of 3-D honeycomb woven fabric (200 mm × 200 mm × 7.5 mm),
the two-layer of 3-D honeycomb woven fabric (200 mm × 200 mm × 15 mm), and the
three-layer of 3-D honeycomb woven fabric (200 mm × 200 mm × 22.5 mm) into their
molds, closing the molds, and checking for leaks. Then, the resin solution was injected into
the mold. The injection process was continued until a sufficient resin volume was seen in
the resin trap to indicate that the mold had been completely filled with resin. The mold was
isolated from the resin pot and the resin trap and then put into an air-circulating oven. The
manufacturer-recommended cure cycle was employed: the first step of the cure cycle was
2 h at 90 ◦C, with the second step being 1 h at 130 ◦C, and the final step being 4 h at 150 ◦C,
as shown in Figure 3b. The 3-D honeycomb woven EM absorbing composite without
adding CB/CIP, with a thickness of 7.5 mm, was recorded as S0. The 3-D honeycomb
woven EM absorbing composite with added CB/CIP, with a thickness of 7.5 mm, was
recorded as S1. The 3-D honeycomb woven EM absorbing composite with added CB/CIP,
with a thickness of 15 mm, was recorded as S2. The 3-D honeycomb woven EM absorbing
composite with added CB/CIP, with a thickness of 22.5 mm, was recorded as S3. Table 1. Weaving parameters of 3-D honeycomb woven fabrics. Thickness (mm)
Linear Density/Tex
Layer Number of Yarns
Weaving Density (Yarn/10 cm)
Warp/Weft Yarns
Warp Density
Weft Density
7.5
800
1
180
95
15
2
300
22.5
3
420
2.3. Fabrication of the 3-D Honeycomb Woven EM Absorbing Composites
The ratio of the EP, CIP, solidification reagent, and CB was 4:4:3.2:0.03. VARTM
manufactured the 3-D honeycomb woven EM absorbing composites. Figure 3a showed the
schematic diagram of the VARTM process. The CB, CIP, and EP were mixed in a beaker
and mechanically stirred until the CB/CIP could be uniformly dispersed in the EP without
significant settling. After vacuum defoaming for 1 h in a vacuum drying oven at 60 ◦C,
the CB/CIP/EP mixed solution without bubbles was obtained. 2.2. Preform Design and Weaving The warp structural drawings of 3-D honeycomb woven fabrics with three different
layers are shown in Figure 1. As shown in Figure 1, the bottom warp and weft yarns were
made entirely of 800 tex CF filament, and the rest of the warp and weft yarns were wholly
made of 800 tex BF filament. (a)
(b)
(c)
Figure 1. (a) Warp structural drawings of 7.5 mm 3D honeycomb woven fabric; (b) warp structural drawings of 15 mm 3D
honeycomb woven fabric; (c) warp structural drawings of 22.5 mm 3D honeycomb woven fabric. (b) (c) (a) (a) (c) (b) Figure 1. (a) Warp structural drawings of 7.5 mm 3D honeycomb woven fabric; (b) warp structural drawings of 15 mm 3D
honeycomb woven fabric; (c) warp structural drawings of 22.5 mm 3D honeycomb woven fabric. Figure 1. (a) Warp structural drawings of 7.5 mm 3D honeycomb woven fabric; (b) warp structural drawings of 15 mm 3D
honeycomb woven fabric; (c) warp structural drawings of 22.5 mm 3D honeycomb woven fabric. The chain drafts of 3-D honeycomb woven fabrics with three different thicknesses
were drawn up according to the warp structural drawings, and they are shown in Figure 2. The weaving parameters of 3-D honeycomb woven fabrics are shown in Table 1. (a)
(b)
(c)
Figure 2. (a) Chain draft of 7.5 mm 3-D honeycomb woven fabric; (b) chain draft of 15 mm 3-D
honeycomb woven fabric; (c) chain draft of 22.5 mm 3-D honeycomb woven fabric. (a)
(b)
(c) Figure 2. (a) Chain draft of 7.5 mm 3-D honeycomb woven fabric; (b) chain draft of 15 mm 3-D
honeycomb woven fabric; (c) chain draft of 22.5 mm 3-D honeycomb woven fabric. 17 Polymers 2021, 13, 1485 Table 1. Weaving parameters of 3-D honeycomb woven fabrics. Thickness (mm)
Linear Density/Tex
Layer Number of Yarns
Weaving Density (Yarn/10 cm)
Warp/Weft Yarns
Warp Density
Weft Density
7.5
800
1
180
95
15
2
300
22.5
3
420
2.3. Fabrication of the 3-D Honeycomb Woven EM Absorbing Composites
The ratio of the EP, CIP, solidification reagent, and CB was 4:4:3.2:0.03. VARTM
manufactured the 3-D honeycomb woven EM absorbing composites. Figure 3a showed the
schematic diagram of the VARTM process. The CB, CIP, and EP were mixed in a beaker
and mechanically stirred until the CB/CIP could be uniformly dispersed in the EP without
significant settling. 2.2. Preform Design and Weaving The process involved
placing the one-layer of 3-D honeycomb woven fabric (200 mm × 200 mm × 7.5 mm),
the two-layer of 3-D honeycomb woven fabric (200 mm × 200 mm × 15 mm), and the
three-layer of 3-D honeycomb woven fabric (200 mm × 200 mm × 22.5 mm) into their
molds, closing the molds, and checking for leaks. Then, the resin solution was injected into
the mold. The injection process was continued until a sufficient resin volume was seen in
the resin trap to indicate that the mold had been completely filled with resin. The mold was
isolated from the resin pot and the resin trap and then put into an air-circulating oven. The
manufacturer-recommended cure cycle was employed: the first step of the cure cycle was
2 h at 90 ◦C, with the second step being 1 h at 130 ◦C, and the final step being 4 h at 150 ◦C,
as shown in Figure 3b. The 3-D honeycomb woven EM absorbing composite without
adding CB/CIP, with a thickness of 7.5 mm, was recorded as S0. The 3-D honeycomb
woven EM absorbing composite with added CB/CIP, with a thickness of 7.5 mm, was
recorded as S1. The 3-D honeycomb woven EM absorbing composite with added CB/CIP,
with a thickness of 15 mm, was recorded as S2. The 3-D honeycomb woven EM absorbing
composite with added CB/CIP, with a thickness of 22.5 mm, was recorded as S3. (a)
Figure 3. Cont. Figure 3. Cont. 18 Polymers 2021, 13, 1485 (b)
Figure 3. (a) Schematic diagram of VARTM process; (b) high-temperature curing process diagram of
the 3-D honeycomb woven EM absorbing composite. (b) Figure 3. (a) Schematic diagram of VARTM process; (b) high-temperature curing process diagram of
the 3-D honeycomb woven EM absorbing composite. 2.4. Characterization Methods The EM parameters of the CB/CIP were tested by the coaxial method. The specimens
were prepared by homogeneously mixing the CB/CIP with the paraffin in a mass ratio of
0.03:4:4; the mixture was pressed into a toroidal shape with an inner diameter of 3.04 mm
and an outer diameter of 7 mm. The schematic of the experimental setup is shown in
Figure 4a. g
The morphology of the absorbent was observed with the scanning electron micro-
scope JSM-7800F (JEOL Ltd., Guangzhou City, China) in the high-vacuum. The applied
accelerating voltage was 15 kV and the working distance was 10 mm. The United States NRL arch measurement system was used to evaluate the broadband
EM characteristics of the 3-D honeycomb woven EM absorbing composites with different
layers with a reference standard of GJB2038-2011; the schematic of the experimental setup
is shown in Figure 4b. Three-point bending with a universal testing machine followed GB/T 9341-2008. The
loading rates were 10 mm/min for the bending test. The schematic of the experimental
setup is shown in Figure 4c. 19 Polymers 2021, 13, 1485 (a)
(b)
(c)
Figure 4. (a) Schematic diagram of the dielectric property test equipment; (b) schematic diagram of
the EM reflection loss test equipment; (c) schematic diagram of the three-point bending test. 3. Results and Discussion (c) Figure 4. (a) Schematic diagram of the dielectric property test equipment; (b) schematic diagram of
the EM reflection loss test equipment; (c) schematic diagram of the three-point bending test. 3 R
lt
d Di
i 3.1. EM Properties The micro-structure of CIP and CB is shown in Figure 5a,b, respectively. As shown
in Figure 5a, the microscopic morphology of CIP was spherical particles with a sphere
structure, with an average particle size of about 3.5 μm. Such a spherical structure was
also conducive to its uniform distribution in the matrix [16]. As shown in Figure 5b, the
microscopic morphology of CB particles was approximately spherical, with a sphere size
of about 20–30 nm. Under the electron microscope, the CB particles were agglomerated,
caused by the high surface energy of nanoparticles. The agglomerated form made it easy
for CB particles to form the conductive path in the matrix, which significantly improved
CB’s conductivity. y
The EM parameters of CB/CIP tested are shown in Figure 6a. From Figure 6a, it can
be seen that the real part of the complex dielectric constant of CB/CIP fluctuates very little
in the range of 2–18 GHz test frequency, generally distributed in the range of 3.5–3.75,
and the imaginary part of the same dielectric constant ranges from 0 to 0.25, showing a
weak dielectric loss performance. The real part μ′ and imaginary part μ” of the complex
permeability decreased gradually with the increase in frequency and were distributed in
the range of 1–1.5 and 0.25–0.5, respectively. Figure 6b shows the curve of the loss tangent
of CB/CIP. It can be seen that the dielectric loss tangent of CB/CIP tends to decrease in the
measured range and eventually fluctuates around 0. These data indicated that CB/CIP has
a weak dielectric loss performance. The magnetic loss tangent of CB/CIP decreased at first
and then increased, which indicated that CB/CIP has good magnetic loss performance and
is an excellent magnetic loss absorbent. 20 Polymers 2021, 13, 1485 (a)
(b)
Figure 5. (a) Scanning electron microscope image of CIP; (b) scanning electron m
image of CB. (a) (a) (a) (a) (b) Figure 5. (a) Scanning electron microscope image of CIP; (b) scanning electron microscope
image of CB. 21 Polymers 2021, 13, 1485 (a)
(b)
Figure 6. (a) EM parameters of CB/CIP particles; (b) loss tangent of CB/CIP particles. A
h
i
Fi
7 th S b
d
f
t th
l
t
ti (EM)
f (a) (b) (b) Figure 6. (a) EM parameters of CB/CIP particles; (b) loss tangent of CB/CIP particles. 3.1. EM Properties As shown in Figure 7, the S-band refers to the electromagnetic (EM) wave frequency
range between 2 GHz and 4 GHz. The C-band refers to the EM wave segment with
a frequency between 4 GHz and 8 GHz. The X-band refers to the EM wave segment
with a frequency between 8 GHz and 12 GHz. The Ku-band refers to the EM wave
segment with frequencies between 12 GHz and 18 GHz. The S0 reached the RLmin of
−11.4 dB at a Ku-band (15.66 GHz). The S1 reached the RLmin of −15.06 dB at Ku-band
(15.48 GHz). Therefore, we found that CB/CIP can improve the absorbing loss capacity of
the whole composite. 22 Polymers 2021, 13, 1485 Figure 7. EM wave absorption performance of S0 and S1. Figure 7. EM wave absorption performance of S0 and S1. The BF/EP (basalt fiber and epoxy resin) in the 3-D honeycomb woven EM absorbing
composite were wave-permeable materials, which have no absorption ability to EM waves. The continuous CF layer mainly reflected the EM wave, and the EM wave that was not
lost and was reflected twice after reaching the CF layer to improve the loss efficiency. CB/CIP, as an absorber of EM waves, was distributed in the EP matrix and mainly played
the role of magnetic loss. CB/CIP can promote the formation of the absorbing network
structure in the matrix, which improved the impedance matching and attenuation loss of
the 3-D honeycomb woven EM absorbing composite, and the absorbing loss ability of the
whole composite was enhanced. Compared to the two curves, we also found that with
the addition of CB/CIP, the EM absorption intensity of the 3-D woven honeycomb EM
absorbing composites increases, and the peak value of RLmin moved to the low-frequency
direction. The RL curve showed the phenomenon that the absorption peak matches
the frequency to move to the low frequency, which can be shown through the quarter
wavelength theoretical explanation [17]: fm =
nc
4d√εrμr
(n = 1, 3, 5 . . .)
(1) (1) fm: the absorption peaks match frequencies (GHz); c: the speed of light (m/s); d: the thickness of the 3-D woven honeycomb EM absorbing composite (mm);
εr: the relative dielectric constant; d: the thickness of the 3-D woven honeycomb EM absorbing composite (mm);
th
l ti
di l
t i
t
t d: the thickness of the 3-D wov μr: the magnetic permeability. 3.1. EM Properties (a) RL curves of S1, S2, S3; (b) 3-D RL of S1, S2, S3; (c) two-dimensional plane RL of S1, S2, S3; (d) E 3.1. EM Properties It can be concluded that the matching frequency of the 3-D woven honeycomb EM
absorbing composite was related to its thickness and electromagnetic parameters. Accord-
ing to the effective medium theory [18], it can be seen that the equivalent electromagnetic
parameters of the mixture of absorbent and resin were related to the content of absorbent. When the thickness of the 3-D woven honeycomb EM absorbing composite was constant,
the increase in CB/CIP content in Equation (1) led to the increase in equivalent EM param-
eters of absorbent honeycomb, and the corresponding matching frequency of absorption
peak decreased; in the final step, the absorption peak moved to low frequency. p
p
p
p
q
y
The RL curve of the 3-D honeycomb woven EM absorbing composites is shown in
Figure 8. The RLmin of S1 was -15.06 dB (Figure 8a,b), and the EBA of the Ku–band was 23 Polymers 2021, 13, 1485 3.66 GHz (Figure 8a,c,d), and the corresponding absorption band accounts for 61%. For S2,
RLmin was -23.5 dB (Figure 8a,b). Although the EBA of the C–band (Figure 8a,c,d) was only
0.5 GHz and the corresponding absorption band occupies 12.5%, the EBA of the X-band
was 2.52 GHz, and the corresponding absorption band occupies 63.8%. S3 obtained an
excellent absorption intensity of -25.7 dB. In particular, the EAB of C–band (4–8 GHz) and
X-band (8–12 GHz) low-frequency bands were 3.26 GHz and 0.19 GHz, respectively, and
the corresponding absorption band occupancy rates were 81.5% and 19%, respectively,
indicating that the C-band has excellent EM absorption performance. By comparing the
three curves in Figure 8a, we also found that the EM absorption intensity of the 3-D woven
honeycomb composites increases, and RL curves’ peak value moves to the low-frequency
direction with the increase in thickness. The quarter wavelength theory can explain it. According to Equation (1), the thickness was inversely proportional to the absorbing
frequency. That is to say, the thicker the thickness, the lower the absorbing frequency. (a)
(b)
(c)
(d)
Figure 8. (a) RL curves of S1, S2, S3; (b) 3-D RL of S1, S2, S3; (c) two-dimensional plane RL of S1, S2, S3; (d) EAB of S1, S2, S3. (b) (a) (b) (a) (c) (d) (d) (c) curves of S1, S2, S3; (b) 3-D RL of S1, S2, S3; (c) two-dimensional plane RL of S1, S2, S3; (d) EAB of S1, S2, S3. Figure 8. 3.2. Bending Properties The three-point bending load–displacement curves of the 3-D honeycomb woven
EM absorbing composites are shown in Figure 9. The maximum bending load of S0 was
1002.03 N. The maximum bending load of S1 was 846.38 N. The maximum bending load
of S2 was 2271.07 N. The maximum bending load of S3 was 2843.03 N. The reason for the
maximum bending load decrease in S1 was that when the composites was subjected to
bending load, CB/CIP agglomerated in the resin matrix, and a large number of microcracks 24 Polymers 2021, 13, 1485 gathered around the agglomerated particles, which could easily lead to macroscopic
cracking caused by stress concentration. The maximum bending load increased with the
thickness due to the more fiber bearing force. Therefore, the maximum bending load
increased. Figure 9 shows that the curve was divided into three stages. In the initial
loading stage, the material as a whole bears the bending load and performs well within
the initial displacement, and the bending load–displacement curve also increases linearly. Therefore, the composite material has good linear elastic properties. With the increase in
displacement, the composites bore more extrusion pressure. The shape variables were
different between the fiber and the resin, the upper fiber bundle was subjected to the
extrusion, while the lower fiber bundle was subjected to stretching so that the fiber in the
composite material was pulled out and then destroyed the interface between the fiber and
matrix. In this way, the modulus and flexural stiffness of the composite decreased, and the
matrix cracked. Finally, the bending load–displacement curve showed a peak load and
then began to decline. The load–displacement curve was convex on the whole. (a)
(b)
Figure 9. (a) Load–displacement curves of S0 and S1; (b) load–displacement curves of S1, S2 and S3. (b) (a) (b) (a) Figure 9. (a) Load–displacement curves of S0 and S1; (b) load–displacement curves of S1, S2 and S3. The bending strength can be calculated according to the width, thickness, span, and
displacement of the sample, and its equation is as follows: σ = 3PL
2bd2
(2) σ = 3PL
2bd2 (2) σ: the bending strength (MPa); σ: the bending strength (MPa); P: the load (N); L: the span (mm); b: the width (mm); d: the thickness (mm), When the thickness was 7.5 mm, without CB/CIP, the bending strength was 106.88 MPa. 3.2. Bending Properties When the thickness was 7.5 mm, with CB/CIP, the bending strength was 90.28 MPa. When the thickness was 15 mm, the bending strength was 121 MPa. When the thickness
was 22.5 mm, the bending strength was 101.09 MPa. However, by comparing the load–
displacement curve and bending strength of S0 and S1, it was found that the maximum
load and bending strength of S1 were reduced. This may be due to the mutual attraction of
the CB/CIP surface, resulting in the particle agglomeration phenomenon, resulting in the
spatial barrier effect, and CB/CIP will also lead to the decrease in the crosslinking density
of EP, resulting in the increase in defects, and the continuity of the EP matrix will be broken. As a result, the strength of the interface between the EP and the fibers weakens, eventually
leading to direct cracking and failure of the composites at the CB/CIP aggregation sites. By
comparing the bending strength of S1, S2, and S3, it was found that the bending strength 25 Polymers 2021, 13, 1485 of 3-D woven honeycomb EM absorbing composites increased first and then decreased
with the increase in layers. This was mainly because, in the bending test, the upper BF was
subjected to the action of compression force, the middle layer was subjected to the action
of shear force, the lower CF was subjected to the action of tensile force. When the number
of BF layers was two, the volume content of BF did not change much because the number
of BF layers did not increase much. The bending strength of S2 was eventually increased. Subsequently, the number of layers of BF continued to increase, and the thickness of the 3-D
woven honeycomb EM absorbing composites also increased. According to Equation (2), the
bending strength was inversely proportional to the square of the thickness and positively
proportional to the first power of the span. In contrast, the effect of thickness on bending
strength was more obvious. Thus, S3 showed a decrease in bending strength. Additionally,
with the increase in the number of BF layers, the volume content of BF gradually increased. Therefore, the effect of the strength of BF was also more obvious. As the strength of BF
was lower than that of CF, the bending strength of S3 decreased. The results of the 3-D
honeycomb woven EM absorbing composites with different thicknesses were similar. 3.2. Bending Properties Thus,
only the 3-D honeycomb woven EM absorbing composite with S2 was used as an example. Figure 10a–d shows the overall view, partial side view, partial bottom view and partial
top view of S2, respectively. It can be seen from Figure 10 that the bending failure modes
had a brittle fracture of the fiber bundle, cracking of the matrix, and a typical shear failure. However, the 3-D honeycomb woven EM absorbing composite had good integrity, and
there was no delamination. (a)
(b)
(c)
(d)
Figure 10. (a) Overall view of S2; (b) partial side view of S2; (c) partial bottom view of S2; (d) partial top view of S2. (a) (a) (b) (d) ( )
(c) (b) (d) (c) Figure 10. (a) Overall view of S2; (b) partial side view of S2; (c) partial bottom view of S2; (d) partial top vi iew of S2; (b) partial side view of S2; (c) partial bottom view of S2; (d) partial top view of S2. 3.3. Mechanical Properties and EM Properties Match It was necessary to evaluate the comprehensive properties of the composite materials,
which met the requirements of the mechanical properties and met the requirements of the
absorbing properties. Table 2 shows the mechanical properties and absorbing properties of
the 3-D honeycomb woven EM absorbing composites. Table 2. The mechanical properties and absorbing properties of the 3-D honeycomb woven EM
absorbing composites. Table 2. The mechanical properties and absorbing properties of the 3-D honeycomb woven EM
absorbing composites. Specimen
Mechanical Property
EM Properties
Bending Strength (MPa)
RLmin (dB)
EAB (GHz)
S0
106.88
−11.4
0.3
S1
90.28
−15.06
3.66
S2
121
−23.5
3.02
S3
101.09
−25.7
3.26 To more intuitively represent the variation trend and amplitude of different thick-
nesses, mechanical properties, and EM absorbing properties and quickly match the optimal
thickness, the concept of the normalized factor β was introduced [19]. Pure BF/CF/EP
composite absorbing material without adding CB/CIP particles was regarded as the me- 26 Polymers 2021, 13, 1485 chanical performance standard, and the minimum requirement of RL value −10 dB was
regarded as the absorbing performance standard. The normalization factors of both were
shown in Equations (3) and (4). The data in Table 3 were obtained after the normalization
of the data in Table 2. chanical performance standard, and the minimum requirement of RL value −10 dB was
regarded as the absorbing performance standard. The normalization factors of both were
shown in Equations (3) and (4). The data in Table 3 were obtained after the normalization
of the data in Table 2. β1 =
σf −σ0
σ0
( f = 1, 2, 3)
(3) β1 =
σf −σ0
σ0
( f = 1, 2, 3)
(3)
β2 =
R f −(−10)
−10
( f = 1, 2, 3)
(4) (3) β2 =
R f −(−10)
−10
( f = 1, 2, 3)
(4) (4) β1: Normalization factor of mechanical properties; β1: Normalization factor of mechanical properties; β1: Normalization factor of mechanical properties;
σf : Mechanical properties of S1, S2 and S3;
σ0: Mechanical properties of S0;
β2: Normalization factor of EM absorbing properties;
Rf: EM absorbing properties of S1, S2 and S3. σ0: Mechanical properties of S0;
β2: Normalization factor of EM absorbing properties;
Rf: EM absorbing properties of S1, S2 and S3. β2: Normalization factor of EM absorbing properties; Table 3. The mechanical properties and absorbing properties of normalized the 3-D honeycomb
woven EM absorbing composites. 3.3. Mechanical Properties and EM Properties Match Specimen
Mechanical Properties
EM Properties
Bending Strength β
RLmin β
EAB (GHz)
S0
0
14%
0.3
S1
−15.53%
50.6%
3.66
S2
13.21%
135%
3.02
S3
−5.79%
157%
3.26 It can be seen from Table 3 that the bending strength of S1 and S3 decreased, so S2
conforms to the principle of matching the absorbing property and mechanical property,
and this specimen met the requirements of both absorbing property and bearing capacity. 4. Conclusions To meet the requirements of modern absorbing composites, which are the integration
of load-bearing and EM wave absorption, and improve the low mechanical properties
and overall performance of traditional honeycomb absorbing composites, the 3-D woven
honeycomb EM absorbing composite was designed. To determine the effects of CB/CIP
and thickness on the EM and mechanical properties of the 3-D honeycomb EM absorb-
ing composites, the 3-D honeycomb EM absorbing composite without adding CB/CIP
was designed. Three kinds of 3-D honeycomb EM absorbing composites with different
thicknesses were prepared. In the aspect of EM absorbing properties, the CB/CIP can promote the formation of
the absorbing network structure in the matrix, which improved the impedance match-
ing and attenuation loss of the 3-D honeycomb woven EM absorbing composite, so the
absorbing loss ability of the whole composite was enhanced. When the thickness of the
3-D honeycomb EM absorbing composites was 15 mm and 22.5 mm, respectively, the
composites’ absorptivity in the X-band and C-band was 63.8% and 81.5%, respectively. This
was because with the increase in thickness, the fiber layer number was increased, and the
hole was increased, the electromagnetic wave occurred in the material reflection, and the
number of refractions was increased. Thereby, there was an increase in the electromagnetic
waves absorbed. Besides, with CB/CIP and the increase in thickness, the RL peak of 3-D
woven honeycomb EM absorbing composites moved to low frequency. This fit the quarter
wavelength theory. In terms of mechanical properties, the EP matrix’s continuity was broken easily due
to the large amount of agglomeration of CB/CIP, and the strength between the resin and
dimensional interface was weakened, thus reducing the mechanical properties of the 3-D
honeycomb EM absorbing composites. Additionally, the results showed that the bending
load of the 3-D honeycomb EM absorbing composites increased with the increase in the 27 Polymers 2021, 13, 1485 thickness of the composites, and the bending strength increased first and then decreased
with the increase in the thickness of the composites. The 3-D honeycomb EM absorbing
composites’ failure modes were mainly fiber bundle brittle fracture, matrix cracking, and
typical shear failure. During the experiment, the 3-D honeycomb EM absorbing composite
had no delamination and splitting phenomenon, and the overall performance was good. After normalizing the mechanical and absorbing properties of the 3-D honeycomb EM
absorbing composites, the optimal load/absorbing ratio was S2. References 1. Luo, H.; Chen, F.; Wang, X.; Dai, W.Y.; Xiong, Y.; Yang, J.J.; Gong, R. A novel two-layer honeycomb sandwich structure absorber
with high-performance microwave absorption. Compos. Part A 2019, 119, 1–9. [CrossRef] 2. Liu, W.D.; Lyu, L.H. Research Progress of Three-dimensional Woven Wave-absorbing Composite Materials. Cotton Text. Technol. 2020, 48, 81–84. , L.; Sun, H.M.; Gu, Z.Z. Design and development of irregular hexagonal honeycomb absorbing materials. Comp
2, 67–71. 3. Guo, X.S.; Chen, L.; Sun, H.M.; Gu, Z.Z. Design and development of irregular hexagonal honeycomb abs
Sci. Eng. 2018, 12, 67–71. 4. Kim, P.C.; Lee, D.G. Composite sandwich constructions for absorbing the electromagnetic waves. Compos. Struct. 2009, 87,
161–167. [CrossRef] 5. Li, W.C.; Xu, L.Y.; Zhang, X.; Gong, Y.; Ying, Y.; Yu, J.; Zheng, J.; Qiao, L.; Che, S. Investigating the effect of honeycomb structure
composite on microwave absorption properties. Compos. Commun. 2020, 19, 182–188. [CrossRef] H.; Xiu, X.; Wang, Y.; Xue, Q.; Ju, W.B.; Che, W.Q.; Liao, S.; Jiang, H.; Tang, M.; Long, J.; et al. Paper-based comp 6. Wang, H.; Xiu, X.; Wang, Y.; Xue, Q.; Ju, W.B.; Che, W.Q.; Liao, S.; Jiang, H.; Tang, M.; Long, J.; et al. Paper-based composites as a
dual-functional material for ultralight broadband radar absorbing honeycomb. Compos. Part B 2020, 202, 1–9. [CrossRef] 6. Wang, H.; Xiu, X.; Wang, Y.; Xue, Q.; Ju, W.B.; Che, W.Q.; Liao, S.; Jiang, H.; Tang, M.; Long, J.; et al. Paper based composites as a
dual-functional material for ultralight broadband radar absorbing honeycomb. Compos. Part B 2020, 202, 1–9. [CrossRef] nctional material for ultralight broadband radar absorbing honeycomb. Compos. Part B 2020, 202, 1–9. [CrossR
W
L M W
T H
J Sh
X W Ch
W S
l
i
i
i
f h d
i
f
d
bl
l 7. Sun, P.C.; Wang, L.M.; Wang, T.; Huang, J.; Sha, X.W.; Chen, W. Structural optimization of the design of a double-layer absorbing
honeycomb composite. J. Beijing Univ. Chem. Technol. 2019, 46, 58–64. 7. Sun, P.C.; Wang, L.M.; Wang, T.; Huang, J.; Sha, X.W.; Chen, W. Structur
honeycomb composite. J. Beijing Univ. Chem. Technol. 2019, 46, 58–64. V.G.K.; Vuppalapati, R.R.; Chandrashekhara, K. Manufacturing and mechanical performance evaluation of res
mb composites. J. Reinf. Plast. Compos. 2012, 31, 415–423. [CrossRef] 8. Menta, V.G.K.; Vuppalapati, R.R.; Chandrashekhara, K. Manufacturing and mechanical perform
honeycomb composites. J. Reinf. Plast. Compos. 2012, 31, 415–423. [CrossRef] 9. 4. Conclusions The thickness was 15 mm,
the maximum bending load was 2271.07 N, the bending strength was 121 MPa, the RLmin
was −23.5 dB, and the EAB was 3.02 GHz; the composites satisfied with both load-bearing
and EM wave absorption capacity. p
p
y
This work provides a simple way to improve the mechanical properties and EM
absorption capacity of structural EM absorbing composites with the 3-D woven honeycomb
structure. However, we only investigated the EM absorbing property of the composites
in regard to adding absorbent and different thicknesses, and we need to design further a
3-D woven honeycomb composite with different aperture sizes to achieve EM absorbing so
that 3-D woven honeycomb composite can be used in weapon systems, such as fighters,
warships, and missiles. Author Contributions: L.-H.L. and W.-D.L. designed the experiments. W.-D.L. wrote the manuscript
and performed the experiments and analyzed the data. B.-Z.S. contributed to discussion. All authors
have read and agreed to the published version of the manuscript. Funding: This research was funded by the Key Laboratory of High Performance Fibers and Products,
Ministry of Education, Donghua University, Shanghai, China and the National Science Foundation of
Liaoning Province (2019-MS-017). Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the
corresponding author. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. 14.
Lv, L.H.; Huang, Y.L.; Cui, J.R. Bending Properties of Three-dimensional Honeycomb Sandwich Structure Composites: Experiment
and Finite Element Method Simulation. Text. Res. J. 2018, 88, 2024–2031. [CrossRef] 15.
Zahid, B.; Jamshaid, H.; Rajput, A.W. Effect of Cell Size on Tensile Strength and Elongation Properties of Honeycomb Weave. Ind.
Text. 2019, 70, 133–138. [CrossRef] 13.
Bayraktar, G.B.; Kianoosh, A.; Bilen, D. Fabrication of Woven Honeycomb Structures for Advanced Composites. Text. Leather Res.
2018, 1, 114–119. [CrossRef] 16.
Liu, L.; Duan, Y.; Ma, L. Microwave absorption properties of a wave-absorbing coating employing carbonyl-iron powder and
carbon black. Appl. Surf. Sci. 2010, 257, 842–846. [CrossRef] pp
f
17.
Dai, X.H. Research on Electromagnetic Wave Absorption Performance of Honeycomb Structure Composites. Master’s Thesis,
Dalian University of Technology, Dalian, China, June 2019. y
19.
Zhang, X.F. Study on Preparation and Properties of Structural Wave Absorbing Composite Materials via VARI Process. Master’s
Thesis, Nanjing University of Aeronautics and Astronautics, Nanjing, China, March 2019. Article
Dyeing Para-Aramid Textiles Pretreated with Soybean Oil and
Nonthermal Plasma Using Cationic Dye Mary Morris 1, Xiaofei Philip Ye 1,* and Christopher J. Doona 2,3 Mary Morris 1, Xiaofei Philip Ye 1,* and Christopher J. Doona 2,3 1
Department of Biosystems Engineering and Soil Science, The University of Tennessee, Knoxville, TN 37996, USA;
maramorr@vols.utk.edu
2
U.S. Army Combat Capabilities Development Command—Soldier Center, Natick, MA 01760, USA; 1
Department of Biosystems Engineering and Soil Science, The University of Tennessee, Knoxville, TN 37996, USA;
maramorr@vols.utk.edu 2
U.S. Army Combat Capabilities Development Command—Soldier Center, Natick, MA 01760, US
doonac@mit.edu 3
Research Affiliate, Massachusetts Institute of Technology—Institute for Soldier Nanotechnologie
77 Massachusetts Ave., NE47-4F, Cambridge, MA 02139, USA 3
Research Affiliate, Massachusetts Institute of Technology—Institute for Soldier Nanotechnologies,
77 Massachusetts Ave., NE47-4F, Cambridge, MA 02139, USA
*
Correspondence: xye2@utk.edu; Tel.: +1-(865)-974-7129 *
Correspondence: xye2@utk.edu; Tel.: +1-(865)-974-7129 Abstract: The increasing use of functional aramids in a wide array of applications and the inert nature
of aramids against conventional dye and print methods requires developing new dyeing methods. This study aims to use environmentally friendly method with a cationic dye as an alternative for
dyeing para-aramid fabrics. Experiments used a multi-factorial design with functions of pretreatment,
dye solvent (water and/or glycerol) and auxiliary chemical additives (swelling agent and surfactant)
and a sequential experimentation methodology. The most effective dyeing procedures involved the
following steps: (i) pretreatments of the fabrics with soybean oil and nonthermal plasma (NTP),
(ii) using water at T = 100 ◦C as the dye solvent, and (iii) omitting other chemical additives. With a
commercial cationic dye, these conditions achieved a color strength in K/S value of 2.28, compared to
~1 for untreated samples. FTIR analysis revealed that a functional network formed on the fibers and
yarns of the fabrics by chemical reactions of excited plasma species with double bonds in the soybean
oil molecules was responsible for significantly improving the color strength. These results extend
the potential uses of a renewable material (soybean oil) and an environmentally friendly technology
(NTP) to improve the dyeing of para-aramid textiles and reduce the use of harsh dye chemicals. Keywords: soybean oil; glycerol; nonthermal plasma; para-aramid textiles; cationic dye Academic Editors: Tarek M. Abou Elmaaty and Maria
Rosaria Plutino Citation: Morris, M.; Ye, X.P.; Doona,
C.J. Dyeing Para-Aramid Textiles
Pretreated with Soybean Oil and
Nonthermal Plasma Using Cationic
Dye. Polymers 2021, 13, 1492. https://
doi.org/10.3390/polym13091492 References Ji, G.F.; Ouyang, Z.Y.; Li, G.Q. Debonding and impact tolerant sandwich panel with hybrid foam core. Compos. Struct. 2013, 103,
143–150. [CrossRef] 10. Xiong, J.; Zhang, M.; Stocchi, A. Mechanical behaviors of carbon fiber composite sandwich columns with three dimensional
honeycomb cores under in-plane compression. Compos. Part B 2014, 60, 350–358. [CrossRef] 11. Wei, F.; Li, D.D.; Li, J.L.; Li, J.Z.; Yuan, L.J.; Xue, L.L.; Sun, R.-J.; Meng, J.-G. Electromagnetic properties of three-dimensional
woven carbon fiber fabric/epoxy composite. Text. Res. J. 2018, 88, 2353–2361. p
y
p
12. Xiao, B.Q.; Huang, Q.W.; Chen, H.X.; Chen, X.B.; Long, G.B. A fractal model for capillary flow through a single tortuous capillary
with roughened surfaces in fibrous porous media. Fractals 2021, 29, 2150017. [CrossRef] 28 Polymers 2021, 13, 1485 J
,
,
[
]
15. Zahid, B.; Jamshaid, H.; Rajput, A.W. Effect of Cell Size on Tensile Strength and Elongation Properties of Honeycomb Weave. Ind. Text. 2019, 70, 133–138. [CrossRef] 16. Liu, L.; Duan, Y.; Ma, L. Microwave absorption properties of a wave-absorbing coating employing carbonyl-iron powder and
carbon black. Appl. Surf. Sci. 2010, 257, 842–846. [CrossRef] pp
f
17. Dai, X.H. Research on Electromagnetic Wave Absorption Performance of Honeycomb Structure Composites. Master’s Thesis,
Dalian University of Technology, Dalian, China, June 2019. 17. Dai, X.H. Research on Electromagnetic Wave Absorption P
Dalian University of Technology, Dalian, China, June 2019. y
gy
18. Matttsine, S.M.; Hock, K.M.; Liu, L. Shift of resonance frequency of long conducting fibers embed
Phys. 2003, 94, 1146–1154. [CrossRef] 18. Matttsine, S.M.; Hock, K.M.; Liu, L. Sh
Phys. 2003, 94, 1146–1154. [CrossRef] y
,
,
[
]
19. Zhang, X.F. Study on Preparation and Properties of Structural Wave Absorbing Composite Materials via VARI Process. Master’s
Thesis, Nanjing University of Aeronautics and Astronautics, Nanjing, China, March 2019. 29 polymers polymers polymers https://www.mdpi.com/journal/polymers 1. Introduction Received: 18 April 2021
Accepted: 3 May 2021
Published: 6 May 2021 Aramid (portmanteau combining aromatic and polyamide) fibers are high-performance
fibers with properties of a high tensile strength, high melting point, chemical resistance to a
range of organic solvents and exceptional flame resistance [1]. Consequently, aramid fibers
have found wide applications in various technical areas, including textiles for apparel
such as body armor (bullet-proof vests and helmets), puncture–resistant correctional wear,
fire-protective clothing and sportswear and other applications such as brake pads, gaskets,
hot-air filters, industrial belts and ropes, reinforced composites, tire cords and the strength
member in fiber optics. Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. There are two main types of aramids: meta-aramids and para-aramids, and these
two groups have different properties due to differences in their molecular structures. At
the fundamental level, para-aramid fibers consist of poly(p-phenylene terephthalamide)
molecules estimated to be 230 nm long, with stiff para-linked aromatic rings and densely
arranged hydrogen bond donors and acceptors throughout their backbones. This inherent
molecular rigidity, combined with strong intermolecular hydrogen bonding interactions,
enables the molecules to achieve excellent alignment with their neighbors, resulting in
a highly anisotropic unit cell consisting of covalent bonds, hydrogen bonds and van der
Waals interactions along each fundamental axis, forming a highly crystalline structure [1]. The building-block molecules of meta-aramid are poly(m-phenylene isophthalamide) that Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article
distributed
under
the
terms
and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). 31 https://www.mdpi.com/journal/polymers Polymers 2021, 13, 1492. https://doi.org/10.3390/polym13091492 Polymers 2021, 13, 1492 bind via meta-linked aromatic rings to result in a semi-crystalline fiber with the molecular
chain oriented along the fiber axis. bind via meta-linked aromatic rings to result in a semi-crystalline fiber with the molecular
chain oriented along the fiber axis. With the increasing array of applications using aramids, there is also the need to find
alternative methods for dyeing aramid textiles. It has been a significant challenge to dye
or print aramid fabrics to a high color strength using conventional dyeing and printing
methods, especially for continuous filament para-aramids due to their highly crystalline
structure and chemical inertness. 1. Introduction Currently, the color of woven aramid materials uses
primarily solution dyeing methods, in which the coloring of the yarns in the woven or
knit fabric is determined by adding colorant to the polymer dope at the time the aramid
filament is produced, thereby limiting the color options for the fabrics and their use in
potential new applications. Various surface modification methods have been attempted to
improve the dyeing of aramids, including chemical treatments with strong acids [2,3] and
auxiliary additives [4], physical approaches using UV/O3 irradiation [5] or nonthermal
plasma [6], and chemical grafting using poly(acrylic acid) [7] or a diblock copolymer
derived from methacryloyloxy-ethyl-trimethylammonium chloride [8], which claimed
improved dyeability with different types of dyes. Most of these methods focused on
the surface modification of meta-aramids, and few reported successes in dyeing para-
aramids [9–11]. In terms of the dye characteristics suited for dyeing aramids, disperse dyes were
developed to dye synthetic fibers and worked well on nylon [12], which were aliphatic
or semi-aromatic polyamides, but limited success had been reported in dyeing aramids
without harsh chemical treatments [4]. In solution dyeing, the pigments are added to the
sulfuric acid solution just before spinning to achieve coloration of the resultant para-aramid
fibers [13]. Further, disperse dyes are so-called because they tend to have low solubility
in water and require special dispersing agents to effectuate coloration. Another type of
dye, the cationic dyes, are exclusively used in solution dyeing, with the help of special
dyeing auxiliaries and/or solvents in the case of meta-aramid fibers. In contrast, cationic
dyes can dissociate into positively charged chromophore ions in aqueous solution and
interact with the negative groups on the fiber molecules to form salts, which can become
firmly attached to the fibers for dyeing. Using conventional dyeing methods to investigate
dyeing characteristics of meta-aramid fibers with some commercial dyes, Kim and Choi [14]
reported that the cationic dyes showed comparatively higher exhaustion yield comparing
to those of disperse dyes and acid dyes, and under acidic conditions in the range of pH 3
to 5, the stability of cationic dyes could be enhanced, leading to higher adsorption. We hypothesize that surface modification of para-aramids via nonthermal plasma
(NTP) treatment, particularly an oxygen-containing plasma method, would introduce
anionic dyeing sites on the surface to facilitate dyeing. 1. Introduction Therefore, the objective of this
study is to dye para-aramid textiles with a cationic dye using renewable materials and
an environmentally friendly technology (NTP). Additionally, since the trend in the textile
dyeing industry is to reduce or avoid the use and disposal of environmentally-unfriendly
chemical additives [15], glycerol is used in this study as an alternative solvent to water
or ethanol due to glycerol’s non-toxicity, low volatility and high boiling point (290 ◦C),
all of which make glycerol environmentally safe and suitable for use in the textile dyeing
industry [16,17]. Further, glycerol is also used in this study, because there is a current
worldwide effort to valorize the excessive amount of crude glycerol that has been generated
as a result of massive biodiesel production [18–21]. Textile dyeing processes commonly add surfactants to the dyeing media, to ensure
the uniform dispersion of dye in the media that promotes penetration of the dye into the
fiber matrix [22]. Swelling agents or dyeing accelerators swell the fibers to facilitate the
penetration of dye into the fibers. Swelling agents are particularly useful for increasing
the dye substantivity in some instances with highly crystalline synthetic and blended
fibers [23,24]. In this study, the nonionic surfactant and emulsifier Polysorbate 80 (TWEEN
80) and the swelling agent benzyl alcohol, both of which have been reported to have
benefits in dyeing aramids [25,26], were tested as additives to improve dyeing. 32 Polymers 2021, 13, 1492 2.1. Materials The textiles tested in these experiments consisted of continuous filament 300 Denier
para-aramid yarns in a tightly woven plain weave construction, as provided by the U.S. Army Combat Capabilities Development Command—Soldier Center (Natick, MA, USA). The cationic dye Basic Blue 11 (Victoria Blue R, CAS Number 2185-86-6) was purchased
from Sigma-Aldrich (St. Louis, MO, USA); glycerol (99.5% purity) and acetic acid (glacial)
were purchased from Fisher Scientific (Waltham, MA, USA); Polysorbate 80 (TWEEN
80) and benzyl alcohol were purchased from Chem Center @ Amazon.com; and food
grade refined soybean oil (100 g contains approximately 16 g of saturated fat, 23 g of
monounsaturated fat, and 58 g of polyunsaturated fat) and laundry detergent (ECOS® Plus
with Stain-Fighting Enzymes) were purchased from a local supermarket. All reagents and
materials were used as-is without further purification. 2.2. Experimental Methods The sequential experimentation methodology used in this study (Experiments A–F)
was directed toward the goal of finding the combination of experimental factors that
achieve the highest color strength of para-aramid fabrics dyed with a cationic dye. The
experimental design used sequential factorial designs to quickly identify critical factors
and conditions that improved dyeing. The factorial functions consisted of: (i) pretreatment
with acetic acid, soybean oil and/or NTP treatment, (ii) dye solvent using water versus
glycerol and (iii) the addition of the auxiliary additives of swelling agent and surfactant. The experimental design and subsequent analysis were carried out using specially designed
software Design-Expert 6.0 (Stat-Ease, Inc., Minneapolis, MN, USA). 2.5. Color Strength Analysis Prior to determining the color strength, the fabric samples were washed with laundry
detergent using a homemade tumbler, in accordance with the ISO standard 105-C10:2006
protocol [28], then rinsed and dried as described in Section 2.4 above. Estimates of the color strength for each of the dyed fabric samples were determined
at the wavelength of maximum absorbance for Victoria Blue R dye (λmax = 615 nm) by
measuring percent spectral reflectance (%R) in the visible range with a SPECTRO 1 spec-
trophotometer (Variable Inc., Chattanooga, TN, USA). Results were expressed as the
samples’ absorption (K) and scattering (S) characteristics as its K/S value, which varies ap-
proximately linearly with colorant concentration, according to the Kubelka-Monk equation
(Equation (1)) [29]. K/S = (1 −0.01R)2
2(0.01R)
(1) (1) 2.3. Pretreatment (Acetic Acid, Soybean Oil, NTP) Para-aramid textile fabric samples (~645 mm2 or ~1 in2) were pretreated by simply
immersing the fabric samples into a Petri dish containing either 20 wt.% aqueous acetic acid
solution or soybean oil, according to an experimental design. The samples were removed
after 15 h, transferred onto a paper towel and hand-pressed with a roller to force out any
excess liquid. q
For fabric samples subjected to NTP exposure, the NTP treatment was carried out
using an in-house made Surface Dielectric Barrier Discharge (SDBD) system. The SDBD
method was chosen because it generates a higher density of micro-discharges that are
limited to the sample surface compared to the Volume Dielectric Barrier Discharge (VDBD)
method. The SDBD therefore avoids the formation of pinholes in the para-aramid fabric
samples, which is commonly associated with VDBD and caused by hot electron bombard-
ment of the para-aramid fabrics. Teflon-coated aramids have been made wettable after a
30 s exposure to this SDBD. p
The SDBD system has been described previously for dyeing with a disperse dye [27]
and is summarized here. The SDBD apparatus uses ambient air as the feed gas and
comprises two electrodes that are separated by an alumina dielectric plate (dimensions =
108 × 95 × 1 mm3 thick) and powered by a high voltage power source with a sinusoidal
high voltage of 9.2 kV that was tuned to a 23.2 kHz resonance frequency (see Figure 1). Embedded at the top of the alumina plate was an induction electrode made of rectangular
copper tape, and at the bottom of the alumina plate was a discharge electrode (17 tungsten
strips interconnected). Fabric samples were placed on the rotating stage and the SDBD
plate was lowered to a height 1 mm above the sample. When the power was switched
on, the plasma emissions, which included reactive oxygen and nitrogen species (RONS)
and other high-energy radicals, interacted with the fabric samples (typically for 1–2.5 min). Then, the power was turned off and the sample was removed for dyeing. 33 Polymers 2021, 13, 1492 Figure 1. (A) Diagram of the NTP system and (B) photo showing a sample under glowing plasma discharge. Figure 1. (A) Diagram of the NTP system and (B) photo showing a sample under glowing plasma discharg 2.4. Dyeing Experimental Procedures The cationic dye Victoria Blue R is soluble in water and in glycerol, so distilled water,
glycerol and aqueous glycerol solutions were tested and compared as the dye solvent. The
aqueous glycerol solutions are expressed as their volumetric percent of glycerol. The dye
solvents were moderately heated (T ≈50 ◦C) with vigorous stirring using a magnetic stirrer,
and appropriate amounts of Victoria Blue R dye, acetic acid, surfactant and swelling agent
were added to the respective solvents in accordance with the design of the experiment. The
dye bath liquid was dispensed into separate vials that were simultaneously heated and
stirred on a Reacti-Therm system (ThermoFisher Scientific, Waltham, MA, USA). Fabric
samples were loaded into the vials for dyeing. The dye bath and fabric samples took
about 30 min to reach a designated temperature, held at the temperature for 1 h, then
cooled for 20 min with the Reacti-Therm system shut off. After the dyeing, samples were
rinsed in flowing warm tap water for 2 min, rinsed in cold water for 2 min, then dried in
a programmable convective oven at T = 150 ◦C for 2 min (after starting at T = 30 ◦C and
increasing the temperature at a rate of 30 ◦C/min) to fix the dye. Table 1. Experiment A: experimental design and results @. Table 1. Experiment A: experimental design and results @. p
p
g
Sample
Dye
Solvent
Pretreatment NTP Time (s)
Swelling Agent
K/S
K/S
Std *
A1
Water
None
60
None
1.26
0.009
A2
Water
None
0
None
1.20
0.054
A3
Water
Acetic acid
60
None
1.22
0.011
A4
Water
Acetic acid
0
None
1.12
0.018
A5
Water
None
60
Benzyl–OH
1.44
0.003
A6
Water
None
0
Benzyl–OH
1.56
0.026
A7
Water
Acetic acid
60
Benzyl–OH
1.74
0.021
A8
Water
Acetic acid
0
Benzyl–OH
1.59
0.016
A9
Glycerol
None
60
None
0.42
0.005
A10
Glycerol
None
0
None
0.39
0.009
A11
Glycerol
Acetic acid
60
None
0.45
0.004
A12
Glycerol
Acetic acid
0
None
0.40
0.005
A13
Glycerol
None
60
Benzyl–OH
0.41
0.002
A14
Glycerol
None
0
Benzyl–OH
0.42
0.003
A15
Glycerol
Acetic acid
60
Benzyl–OH
0.48
0.019
A16
Glycerol
Acetic acid
0
Benzyl–OH
0.47
0.004
@ Experiment conducted at soaking time in 20 wt.% acetic acid solution = 15 h, dye concentration = 0.1 wt.%,
Benzyl alcohol concentration = 2 wt.%, T = 140 ◦C, dyeing time = 1 h. * Values represent mean and standard error
of 3 replicates. @ Experiment conducted at soaking time in 20 wt.% acetic acid solution = 15 h, dye concentration = 0.1 wt.%,
Benzyl alcohol concentration = 2 wt.%, T = 140 ◦C, dyeing time = 1 h. * Values represent mean and standard error
of 3 replicates. Overall, the highest K/S value achieved in Experiment A is only 1.74 (sample A7)
through the combination of water as solvent, pretreatments with acetic acid and NTP (60 s)
and the addition of swelling agent benzyl alcohol. The Analysis of Variance (ANOVA) fac-
torial model terms were selected based on the half-normal probability plot, which indicated
that all the factors and some of their interactions were significant (t-tests for coefficients,
p < 0.02). Since all of the factors are involved in interactions, statistical interpretations of
only the significant interaction terms are shown in Figure 2. Evidently in Figure 2, the dye solvent had the most significant influence on the color
strength (K/S value), with water performing significantly better than glycerol. Adding
benzyl alcohol to the dye bath increased the color strength, and the effect of benzyl alcohol
was slightly enhanced by the acetic acid pretreatment with water as the dye solvent. 2.6. FTIR Analysis In order to monitor some of the chemical changes occurring during the pretreatment
and dyeing processes, Attenuated Total Reflection—Fourier Transform Infrared (ATR-FTIR)
spectroscopy was used to analyze the fabric samples at different stages of the pretreatment
and dyeing processes with the Victoria Blue R dye. In summary, fabric samples were placed
on a potassium bromide (KBr) plate, pressed under the germanium crystal of ATR (UMA
400, Varian Inc., Palo Alto, CA, USA) and scanned in the mid-IR region (500–4000 cm−1
with a 4 cm−1 resolution) with an FTIR spectrometer (Excalibur 3100, Varian Inc., Palo Alto, 34 Polymers 2021, 13, 1492 CA, USA) equipped with an Attenuated Total Reflection (ATR) accessory. The ATR-FTIR
spectra were displayed in absorbance units with each spectrum representing an average
of 128 scans and taking into account the background spectrum acquired using a blank
KBr plate. 3.1. Experiments A–F: Analysis 3.1. Experiments A–F: Analysis Experiment A was a 4-factor, 2-level, full factorial experiment designed to probe
the effects of dye solvent (water or glycerol), pretreatment with acetic acid and/or NTP
treatment for 60 sec and the addition of the swelling agent benzyl alcohol. The dyeing
results are also shown in Table 1. Table 1. Experiment A: experimental design and results @. The
effects of pretreatment with acetic acid and NTP were not obvious, slightly improving the
color strength with their synergy. A significant loss of water was observed in the dye bath during Experiment A due to
the evaporation of water at the dyeing temperature T = 140 ◦C. The use of glycerol as the
dye solvent effectuated dyeing at T = 140 ◦C without the concomitant evaporative loss of
solvent from the dye bath but reached significantly lower values of color strengths. The
cationic dye might not be well-dissociated in glycerol as the solvent, and the high viscosity
of pure glycerol compared to water and the occurrence of hydrogen bonding between the 35 Polymers 2021, 13, 1492 dye and glycerol might hinder the mobility of the cationic dye and retard its diffusion onto
the para-aramids. dye and glycerol might hinder the mobility of the cationic dye and retard its diffusion onto
the para-aramids. Figure 2. Statistical inference of Experiment A: significant factorial interactions. Figure 2. Statistical inference of Experiment A: significant factorial interactions. To overcome the drawbacks of using glycerol as the dye solvent, full factorial Exper-
iment B was designed using 50% or 80% aqueous glycerol solutions as the dye solvent. Additional factors were investigated and included a pretreatment of soaking the para-
aramid fabrics in soybean oil and/or applying further treatment with NTP and adding
benzyl alcohol as the swelling agent. Table 2 lists the detailed experimental design and
resultant K/S values. Even though using 50% or 80% aqueous glycerol solutions as the dye
solvent suppressed the loss of the dye bath solution, the highest K/S value achieved in this
experiment was only 0.94 (sample B11) with 50% aqueous glycerol solution as the solvent
and soybean oil and NTP (60 s) as the pretreatment. Table 2. Experiment B: experimental design and results @. Table 2. Experiment B: experimental design and results @. Table 2. Experiment B: experimental design and results @. @ Experiment carried out with soaking in soybean oil time = 15 h, dye concentration = 0.1 wt.%, Benz
concentration = 2 wt.%, T = 140 ◦C and dyeing time = 1 h. * Mean and standard error of 3 replicates. Table 1. Experiment A: experimental design and results @. Sample
Dye Solvent
Pretreatment
Swelling
Agent
NTP
Time (s)
K/S
K/S
Std *
B1
80% Glycerol
Soy oil
Benzyl–OH
60
0.70
0.015
B2
80% Glycerol
Soy oil
Benzyl–OH
0
0.57
0.012
B3
80% Glycerol
Soy oil
None
60
0.79
0.014
B4
80% Glycerol
Soy oil
None
0
0.56
0.004
B5
80% Glycerol
None
Benzyl–OH
60
0.55
0.003
B6
80% Glycerol
None
Benzyl–OH
0
0.53
0.004
B7
80% Glycerol
None
None
60
0.46
0.017
B8
80% Glycerol
None
None
0
0.44
0.013
B9
50% Glycerol
Soy oil
Benzyl–OH
60
0.84
0.005
B10
50% Glycerol
Soy oil
Benzyl–OH
0
0.67
0.009
B11
50% Glycerol
Soy oil
None
60
0.94
0.001
B12
50% Glycerol
Soy oil
None
0
0.92
0.005
B13
50% Glycerol
None
Benzyl–OH
60
0.81
0.014
B14
50% Glycerol
None
Benzyl–OH
0
0.70
0.010
B15
50% Glycerol
None
None
60
0.72
0.006
B16
50% Glycerol
None
None
0
0.82
0.013
@ Experiment carried out with soaking in soybean oil time = 15 h, dye concentration = 0.1 wt.%, Benzyl alcohol
concentration = 2 wt.%, T = 140 ◦C and dyeing time = 1 h. * Mean and standard error of 3 replicates. 36 Polymers 2021, 13, 1492 The analysis of variance (ANOVA) of this factorial design indicates that the main effect
of all the factors and some of their interactions are significant (p < 0.005). The half-normal
probability plot was used to select the ANOVA model terms, and only the significant
interaction terms of the model are presented in Figure 3. Figure 3. Statistical inference of Experiment B: significant factorial interactions. Figure 3. Statistical inference of Experiment B: significant factorial interactions. The 50% aqueous glycerol solution performed better than the 80% aqueous glycerol
solution as the dye solvent (Figure 3). Benzyl alcohol appeared to be incompatible with
aqueous glycerol or soybean oil, which might hinder its diffusion and swelling functions
and consequently limited the benefits of soybean oil on dyeing. Soaking the para-aramids
in soybean oil slightly improved the dyeing color strength, while soaking the para-aramids
in soybean oil with subsequent NTP treatment for 60 s increased the color strength signifi-
cantly. Without a pretreatment of soybean oil pretreatment, NTP had negligible effect on
the dyeing strength. Table 1. Experiment A: experimental design and results @. y
g
g
The full factorial design for Experiment C used a lower dyeing temperature (T = 90 ◦C),
compared 50% aqueous glycerol solution with water as the dye solvent and used soybean
oil pretreatments and NTP treatment times of 90 s. The experimental design and dyeing
results are compiled in Table 3. The highest K/S value achieved in this experiment is
1.72 (sample C11), which is higher than the highest value obtained in Experiment B (0.94). Experiment B was carried out at a higher temperature (T = 140 ◦C vs. T = 90 ◦C for
Experiment C), used soybean oil with a shorter NTP treatment time (60 s vs. 90 s for
Experiment C) and used 50% or 80% aqueous glycerol solutions as the dye solvent (vs. water or 50% aqueous glycerol in Experiment C). The half-normal probability plot was used to select the ANOVA model terms and re-
sulted in a significant model (F-test, p < 0.0001). All of the terms included in the model were
significant (t-tests, p < 0.007), except for the main effect of swelling agent (t-test, p = 0.59). Since all of the factors were involved in interactions, only the significant interaction terms
are interpreted in Figure 4. 37 Polymers 2021, 13, 1492 Table 3. Experiment C: experimental design and the results @. Table 3. Experiment C: experimental design and the results @. Table 1. Experiment A: experimental design and results @. Sample
Dye Solvent
Pretreatment
Swelling
Agent
NTP
Time (s)
K/S
K/S
Std *
C1
50% Glycerol
Soy oil
Benzyl–OH
90
0.91
0.021
C2
50% Glycerol
Soy oil
Benzyl–OH
0
0.60
0.002
C3
50% Glycerol
Soy oil
None
90
1.06
0.045
C4
50% Glycerol
Soy oil
None
0
0.57
0.006
C5
50% Glycerol
None
Benzyl–OH
90
0.96
0.009
C6
50% Glycerol
None
Benzyl–OH
0
0.74
0.015
C7
50% Glycerol
None
None
90
1.04
0.027
C8
50% Glycerol
None
None
0
0.66
0.013
C9
Water
Soy oil
Benzyl–OH
90
1.66
0.262
C10
Water
Soy oil
Benzyl–OH
0
1.15
0.008
C11
Water
Soy oil
None
90
1.72
0.072
C12
Water
Soy oil
None
0
0.86
0.008
C13
Water
None
Benzyl–OH
90
1.68
0.024
C14
Water
None
Benzyl–OH
0
1.31
0.104
C15
Water
None
None
90
1.09
0.034
C16
Water
None
None
0
0.92
0.012
@ Experiment was carried out with soaking in soybean oil for time = 15 h, dye concentration = 0.1 wt.%, Benzyl
alcohol concentration = 2 wt.%, T = 90 ◦C and dyeing time = 1 h. * Mean and standard error of 3 replicates. Figure 4. Statistical inference of Experiment C: significant factorial interactions. 38 Figure 4. Statistical inference of Experiment C: significant factorial interactions. 38 Polymers 2021, 13, 1492 According to the half-normal probability plot, the solvent had the most influence on
color strength among all of the factors, with water as the solvent performing significantly
better than the 50% aqueous glycerol solution. Soybean oil pretreatment followed by NTP
significantly improved the color strength, although NTP treatment without the soybean
oil pretreatment also improved the color strength, but to a lesser degree. Benzyl alcohol
performed better in water than in 50% aqueous glycerol solution as the solvent, another
indication that the benzyl alcohol was not compatible with glycerol in Experiment B. Further, the effect of benzyl alcohol was minimal on the samples pretreated with soybean oil,
but benzyl alcohol significantly improved dyeing without the soybean oil pretreatment, also
indicating that benzyl alcohol was not compatible with soybean oil. Without a subsequent
NTP treatment, soybean oil soaking resulted in slightly lower color strength, which seems
contradictory to the results in Experiment B. However, Experiment C was carried out at
a much lower temperature (T = 90 ◦C vs. T = 140 ◦C in experiment B with water or 50%
aqueous glycerol solution (vs. Table 1. Experiment A: experimental design and results @. 50% or 80% aqueous glycerol in Experiment B in the dye
bath, and these factors may have limited the beneficial effects of the soybean oil in achieving
dyeing without an NTP treatment. The longer NTP treatment time in Experiment C (90 s
vs. 60 s in Experiment B also significantly improved dyeing even without the soaking in
soybean oil pretreatment. y
p
From the results of the previous Experiments A–C, it is clear that the use of glycerol in
the dye solvent does not help with dyeing, and there is a potential for improving dyeing
using a pretreatment consisting of soaking in soybean oil followed by NTP. Accordingly,
Experiment D used a lower dyeing temperature (T = 90 ◦C), water as the dye solvent, a
wider range of NTP treatment times (t = 30–150 s), eliminated the swelling agent benzyl
alcohol as a factor and added surfactant as a new factor using TWEEN 80, because of
its reported benefit in dyeing aramids [25,26]. Results of the 3-factor 2-level full factorial
design are presented in Table 4. Table 4. Experiment D: experimental design and the results @. p
p
g
Sample
Surfactant
Pretreatment
NTP Time (s)
K/S
K/S Std *
D1
None
None
30
0.81
0.006
D2
None
Soy oil
30
1.01
0.008
D3
None
None
150
0.97
0.008
D4
None
Soy oil
150
1.20
0.017
D5
TWEEN
None
30
0.61
0.010
D6
TWEEN
Soy oil
30
0.60
0.002
D7
TWEEN
None
150
0.79
0.003
D8
TWEEN
Soy oil
150
0.84
0.005
@ Experiment carried out with soaking in soybean oil for time = 15 h, dye solvent = water, dye concentration = 0.1
wt.%, T = 90 ◦C and dyeing time = 1 h. * Mean and standard error of 3 replicates. ANOVA for the factorial design indicated a significant model (F-test, p < 0.0001). All
three of the factors and their two-way interactions were significant (t-test, p < 0.0002). Since all of the factors are involved in interactions, only the interaction plots are shown
in Figure 5. g
According to the half-normal probability plot, the surfactant TWEEN 80 had the
highest impact on the dyeing color strength, followed by NTP treatment time, then soybean
oil pretreatment in descending order. The use of TWEEN 80 resulted in weaker color
strength (Figure 5). Table 1. Experiment A: experimental design and results @. Experiment F: experimental design and the results @. @ Experiment carried out with soaking in soybean oil for time = 15 h, dye solvent = water, dye concentration =
0.1 wt.%, dyeing time = 1 h, Additive concentrations = 1 wt.% Acetic acid and/or 2 wt.% Benzyl alcohol and
the NTP treatment time = 120 s prior to or post soaking in soybean oil for 15 h. * Mean and standard error of
3 replicates. # The change in K/S value was calculated by subtracting the K/S after the 1st detergent wash from
the K/S value measured after the 2nd detergent wash. The half-normal probability plot indicated that impact of the factors is in the order
of Dye temperature > NTP > Acetic acid. Even though the overall ANOVA model was
significant (p < 0.0001), the coefficient for benzyl alcohol was not significant (t-test, p = 0.63). The only significant interaction term is Temperature × NTP (t-test, p = 0.0003), while
the Temperature × Benzyl–OH (Benzyl alcohol) interaction was marginally significant
(t-test, p = 0.085). Figure 7 plots out the main effects of all the factors along with the
two interactions. Acetic acid was the only factor not involved in any significant interactions (Figure 7A). The addition of 1 wt.% acetic acid in the dye bath slightly decreased the dyeing color
strength. All of the other three factors were involved in the two interactions, so their effects
are discussed in the context of their interactions (Figure 7B). The dyeing temperature and NTP after soaking in soybean oil significantly impacted
the dyeing results, whereas the use of benzyl alcohol did not significantly influence the dye-
ing color strength. In Experiment F, the largest K/S value achieved was K/S = 2.28 (sample
F15) in dyeing conditions with T = 100 ◦C in a water-based dye bath, with a pretreatment
of soaking in soybean oil followed by a 120 s NTP treatment and no other additives of
benzyl alcohol or acetic acid in the dye bath. It is noteworthy that T = 100 ◦C is the highest
temperature that can be used for the water-based dye bath at atmospheric pressure without
significant loss of water due to evaporation. In general, dyeing at T = 100 ◦C was better
than dyeing at T = 70 ◦C. Table 1. Experiment A: experimental design and results @. The surfactant, although helping to form a dye dispersion, might also
hinder the diffusion of the dye onto the para-aramid fiber fabrics, even in the presence of
soybean oil. The soybean oil pretreatment improved dyeing, which was further improved
by longer subsequent NTP treatment times. Evidently, the longer NTP treatment time
of 150 s, comparing to the treatment time of 30 s, resulted in higher color strength in the
presence or the absence of TWEEN 80 surfactant. 39 Polymers 2021, 13, 1492 Figure 5. Statistical inference of Experiment D: significant factorial interactions. Figure 5. Statistical inference of Experiment D: significant factorial interactions. It appeared that the NTP treatment time is crucial to the dyed color strength, especially
for the soybean oil pretreated samples. Therefore, Experiment E was designed to optimize
the NTP treatment time. In Experiment E, samples were soaked in soybean oil for 15 h,
water was used as the dye solvent with a 0.1 wt.% dye concentration and with no other
additives and the dyeing temperature was T = 90 ◦C for 1 h. y
g
p
Results of the one-factor factorial design with NTP treatment time varied from 0–150 s
are presented in Figure 6. The effects of NTP treatment at these experimental conditions
show (Figure 6) that an NTP treatment time of 120 s provided the optimal result in dyeing
the soybean oil pretreated samples in terms of color strength as represented by K/S values. NTP treatment times shorter than 120 s achieved dyeing, but to a lesser extent and NTP
treatment times longer than 120 s decreased the K/S value, probably due to the degradation
of the soybean oil network by the increased exposure to the NTP treatment. Figure 6. Effect of NTP time on K/S value of soybean oil-soaked para-aramids. Figure 6. Effect of NTP time on K/S value of soybean oil-soaked para-aramids. Table 1. Experiment A: experimental design and results @. Based on the results of Experiments A–E, Experiment F was designed to optimize
dyeing performance by developing a suitable recipe that included auxiliary additives Based on the results of Experiments A–E, Experiment F was designed to optimize
dyeing performance by developing a suitable recipe that included auxiliary additives 40 Polymers 2021, 13, 1492 (swelling agent and acetic acid) in the dye bath and a suitable dyeing process that in terms
of the dyeing temperature (T = 70 ◦C or 100 ◦C) and the effects of a 120 s NTP treatment
prior to versus post soaking in soybean oil. The addition of acetic acid into the dye bath
brought down the pH to about 3, which should make the conditions favorable for cationic
dyes [14]. Results of the 4-factor full factorial design are shown in Table 5. Table 5. Experiment F: experimental design and the results @. Sample
Temperature Acetic Acid
Swelling
Agent
NTP
K/S
K/S
Std *
ΔK/S #
F1
70 ◦C
Yes
Benzyl–OH
Post
1.08
0.016
−0.08
F2
70 ◦C
Yes
Benzyl–OH
Prior
0.91
0.022
−0.13
F3
70 ◦C
Yes
None
Post
0.99
0.005
−0.17
F4
70 ◦C
Yes
None
Prior
0.91
0.045
−0.27
F5
70 ◦C
No
Benzyl–OH
Post
1.18
0.008
−0.15
F6
70 ◦C
No
Benzyl–OH
Prior
1.12
0.025
−0.27
F7
70 ◦C
No
None
Post
0.96
0.023
−0.06
F8
70 ◦C
No
None
Prior
0.93
0.007
−0.31
F9
100 ◦C
Yes
Benzyl–OH
Post
1.55
0.012
−0.04
F10
100 ◦C
Yes
Benzyl–OH
Prior
1.15
0.016
−0.20
F11
100 ◦C
Yes
None
Post
1.30
0.007
−0.10
F12
100 ◦C
Yes
None
Prior
1.06
0.020
−0.17
F13
100 ◦C
No
Benzyl–OH
Post
1.45
0.010
−0.12
F14
100 ◦C
No
Benzyl–OH
Prior
1.21
0.068
−0.16
F15
100 ◦C
No
None
Post
2.28
0.064
−0.09
F16
100 ◦C
No
None
Prior
1.01
0.013
−0.17
@ Experiment carried out with soaking in soybean oil for time = 15 h, dye solvent = water, dye concentration =
0.1 wt.%, dyeing time = 1 h, Additive concentrations = 1 wt.% Acetic acid and/or 2 wt.% Benzyl alcohol and
the NTP treatment time = 120 s prior to or post soaking in soybean oil for 15 h. * Mean and standard error of
3 replicates. # The change in K/S value was calculated by subtracting the K/S after the 1st detergent wash from
the K/S value measured after the 2nd detergent wash. Table 5. Table 1. Experiment A: experimental design and results @. While NTP treatment (120 s) prior to soaking in soybean oil
did not show a significant effect on dyeing, NTP treatment after soaking in soybean oil
significantly improved the color strength, especially when the dyeing was carried out in
the higher temperature regime of T = 100 ◦C. This clearly points out that the improved
dyeing effect should be attributed to the chemical reactions between soybean oil and NTP. 41 Polymers 2021, 13, 1492 Figure 7. Analysis of Experiment F. (A) Main effects of all of the factors and (B) the significant interactions. Figure 7. Analysis of Experiment F. (A) Main effects of all of the factors and (B) the significant interactions. All samples were laundered with a detergent wash for a second time according to the
same protocol described in Section 2.5, to determine colorfastness in terms of the change in
K/S values (denoted as ΔK/S in Table 5). A negative sign of the ΔK/S value indicates a
decrease in K/S value after the second wash with detergent. Generally, higher temperature
and NTP treatment after soaking in soybean oil resulted in better dyeing strength and more
durable dyeing (colorfastness) after laundering. For the purposes of providing a visual demonstration comparing the dyeing strengths
of different treatments, select samples were imaged using a flatbed scanner (Figure 8). Compared with the previous investigation of dyeing para-aramids with this method and
using a disperse dye [27], it is demonstrated that the pretreatment of soybean oil followed
by NTP treatment method is compatible with dyeing with both disperse dye and cationic
dye. Further, this method enables dyeing to a significantly high color strength, although
the disperse dye requires glycerol as a dispersant, but water as the solvent is better suited
for the use of cationic dye. Taken together, these results demonstrate the potential for this
method to replace hazardous chemicals currently used in dyeing practices with renewable
materials and environmentally friendly (“green”) technologies, to improve the dyeing of
para-aramid textiles. 3.2. FTIR Analysis NTP treatment of the para-aramid sample (line
III) did not induce discernible changes in the observed FTIR peaks. A similar result was
observed in Kašparová et al. [36], which also reported some changes in meta-aramids
after NTP treatment and may reflect the different internal molecular structures between
meta-aramids and para-aramids that make para-aramids more difficult to dye even with
an NTP treatment. The sample in line IV for the para-aramid fabric soaked in soybean oil shows peaks
characteristic of soybean oil and of the para-aramids. Subsequent NTP treatment induces
some important changes in the observed spectrum (line V). First, the unsaturated fatty
acids peak at 3009 cm−1 decreases, but the other major characteristic peaks of soybean
oil were still visible. This observation suggests that the double bonds in the unsaturated
fatty acids were rapidly consumed by the NTP treatment, presumably by reaction with the
plasma’s reactive oxygen and nitrogen species (RONS). Second, there were changes in the
1380–1250 cm−1 region, including the formation of a small peak appearing at 1275 cm−1,
which is assigned to the C–C stretching vibration of the C–C(=O)–C groups in aliphatic
ketone molecule [33]. These results suggest that the plasma’s RONS attack the C=C
double bonds, concomitant to the decrease in the peak at 3009 cm−1 (=C–H stretching
of aliphatic alkenes). However, the ketone molecule is unstable. A likely explanation is
that high-energy reactive oxygen species in the NTP (generated with ambient air as the
feedgas) attack the electron rich double bonds in the unsaturated fatty acids and form
epoxides, although the characteristic oxirane absorption peak typically observed at ~822
cm−1 would be obscured by the strong para-aramid absorption peak at 820 cm−1. The
–C=O triglycerides carbonyl stretching and C–O stretching in the esters at 1746 cm−1 and
1160 cm−1, respectively, remain strong in line V, indicating that the NTP did not break the
ester bonds in soybean oil. y
Strong acids such as phosphoric acid and sulfuric acid have been used to pretreat
para-aramids for the improvement of dyeing or interfacial bonding; a new absorption peak
at ~3440 cm−1 (which is ascribed to the hydroxyl group O–H) or the shift and broadening
of the 3313 cm−1 (–N–H stretching) band was reported, along with some decrease in
mechanical strength. This distinct change was explained as a consequence of the increased
number of –OH groups in the modified para-aramids due to hydrolysis [2,3]. 3.2. FTIR Analysis As shown in Figure 9, the following characteristic peaks of soybean oil (line I) are
well represented: 3009 cm−1 (=C–H stretching of aliphatic alkenes) in unsaturated fatty
acids, 2922 cm−1 (–CH2 asymmetric stretching), 2852 cm−1 (–CH3 symmetric stretching),
1746 cm−1 (–C=O triglycerides carbonyl stretching), 1462 cm−1 (–CH2 antisymmetric
deformation) and 1160 cm−1 (C–O stretching in the esters) [30–33]. 42 Polymers 2021, 13, 1492 Figure 8. Scanned images comparing dyed samples with the undyed fabric (labels correspond to the
sample numbers in Tables 1–5; F15 (W2) denotes sample F15 after second detergent wash). Figure 8. Scanned images comparing dyed samples with the undyed fabric (labels correspond to the
sample numbers in Tables 1–5; F15 (W2) denotes sample F15 after second detergent wash). Figure 9. FTIR spectra of the para-aramid fabrics, materials and after certain treatments as follows: (I) Soybean oil; (II) Para-
aramid fabric (untreated); (III) Para-aramid fabric treated with NTP; (IV) Para-aramid fabric after soaking in soybean oil;
(V) Para-aramid fabric after soaking in soybean oil followed by a subsequent NTP treatment; (VI) Para-aramid fabric dyed
with NTP as the only pretreatment; (VII) Para-aramid fabric dyed after soaking in soybean oil (without an NTP treatment);
(VIII) Para-aramid fabric dyed after soaking in soybean oil and an NTP treatment; and (IX) Victoria Blue R cationic dye. Figure 9. FTIR spectra of the para-aramid fabrics, materials and after certain treatments as follows: (I) Soybean oil; (II) Para-
aramid fabric (untreated); (III) Para-aramid fabric treated with NTP; (IV) Para-aramid fabric after soaking in soybean oil;
(V) Para-aramid fabric after soaking in soybean oil followed by a subsequent NTP treatment; (VI) Para-aramid fabric dyed
with NTP as the only pretreatment; (VII) Para-aramid fabric dyed after soaking in soybean oil (without an NTP treatment);
(VIII) Para-aramid fabric dyed after soaking in soybean oil and an NTP treatment; and (IX) Victoria Blue R cationic dye. The spectrum of untreated para-aramids (line II) has main peaks at 3313 cm−1 (–N–H
stretching), 1638 cm−1 (amide I C=O stretching) and 1538 cm−1 (–N–H deformation) [34]. The absorption bands at 1513 cm−1 (amide II), at 1017 cm−1 and at 820 cm−1 deriving from
the C–H bonds on the para-aromatic rings and the absorption band at 1305 cm−1 deriving 43 Polymers 2021, 13, 1492 from the C–N bond stretch of amide III of the para-aramid fiber fabrics can be considered
as internal standards for para-aramid [35]. 3.2. FTIR Analysis However,
this effect is not observed for the para-aramid sample pretreated with acetic acid in this
study, and the acetic acid pretreatment did not significantly improve the color strength
of dyeing. y
g
FTIR spectra for dyed para-aramid fabric samples (Figure 9) are shown in line VI
(with only NTP pretreatment), line VII (with soybean oil soaking but no NTP) and line
VIII (with soybean oil soaking and NTP). The three spectra all have a new peak emerging
at 786 cm−1, which is typically the region of C–H bending for 1,3-disubstituted or 1,2,3-
trisubstituted aromatic rings, possibly indicating a new bond forming on the para-aromatic
rings. Otherwise, the spectrum of the sample dyed only with NTP pretreatment (line VI)
is not much different from that of undyed para-aramids (line II or III). The para-aramid
fabric sample in line VII (pretreated with soybean oil soaking and without subsequent NTP
treatment, showed peaks characteristic of soybean oil that were significantly diminished. NTP treatment after oil soaking induced further decreases in the signal intensity of soybean
oil (line VIII). In addition to reactions with NTP, there are two possible explanations for
the diminished oil signal: (1) unfixed oil could be washed off after dyeing and subsequent
washing with detergent; and (2) the triacylglycerol could be hydrolyzed at the dyeing
conditions resulting in the destruction of ester bonds, as indicated by the diminished peaks
at 1746 cm−1 and 1160 cm−1. The FTIR spectrum of the Victoria Blue R is also shown in Figure 9 (line IX). The
para-aramid fabric samples showed a low uptake of dye, and most of the dye peaks were
obscured by the characteristic peaks of the para-aramids and the oil. Accordingly, no 44 Polymers 2021, 13, 1492 significant dye peaks were detected in the dyed samples, except for a small shoulder at
1355 cm−1 in the spectra of the dyed samples (lines VII and VIII). p
y
p
It is likely that the soybean oil well diffused throughout the tightly woven fabric
and adsorbed onto the surfaces of yarns and fibers, and the NTP treatment induced the
formation of a polymerized network in situ, enabling dyeing to a higher color strength. The unsaturated fatty acids in soybean oil play important roles in the dyeing process. 3.2. FTIR Analysis Cross-linking/polymerization by the action of reactive plasma species are possible, be-
cause hydrogenation, nitration and epoxidation reactions have been observed along with
polymerization under atmospheric NTP [37]. In a study on the tribological properties of
air plasma polymerized soybean oil, Zhao et al. [33] concluded that the free radicals in the
long-chain oil molecules, which were formed by the opening of the double bonds under the
plasma conditions, could capture the reactive O and N species to produce and incorporate
carbonyl, organic amine and nitrogen heterocyclic groups into the polymerized oil network. Moreover, depending on the duration of the NTP treatment in the dyeing procedure, the
soybean oil might undergo processes such as complex oxidations, decomposition or frag-
mentation, to form reactive intermediate sites (e.g., ethers, furans, peroxides, carboxylic
acids or 1,2,4-trioxolane [38,39]) on the para-aramid fiber fabrics that serve as catalysts or
ligands to bind dye molecules. Informed Consent Statement: Not applicable. 4. Conclusions Using a cationic dye and an environmentally friendly alternative dyeing method, we
found that the best formulation for dyeing para-aramid textiles involved (i) pre-treating
the para-aramid fiber fabrics with soybean oil followed by a brief NTP treatment, and
(ii) using water at T = 100 ◦C as the dye solvent and omitting auxiliary chemical additives. Dyeing temperature and NTP treatment time were the most important factors, with the
dyeing temperature of T = 100 ◦C resulting in higher color strength (larger K/S values),
and the optimized NTP treatment time of 120 s for soybean oil pretreated para-aramids. These conditions achieved a K/S value up to 2.28, which is significantly larger than the
K/S ~1 for untreated samples. For comparison, NTP alone or the use of the auxiliary
additives of acetic acid and benzyl alcohol only slightly improved dyeing with the cationic
dye. As a dye solvent, glycerol was inferior to water for dyeing with the cationic dye,
and the addition of the surfactant TWEEN 80 in the dye bath negatively impacted the
dyeing performance. In the present study, FTIR analysis revealed that NTP induced chemical reactions
in soybean oil on the surface of para-aramid fabrics were responsible for significantly
improving the color strength. These chemical reactions likely involved high-energy, short-
lived reactive plasma species that first attacked the double bonds in the unsaturated
fatty acids. While the results of this study provide ample evidence that the pretreatment
of soaking in soybean oil and ensuing NTP treatment improved dyeing color strength,
determining the chemical pathways that led to better dyeing warrant further research, to
control these processes with other dyestuffs. Future research along this line of sustainable
dyeing method will aim at deriving more functionalities of soybean oil on the surface of
para-aramids to further improve the dyeing strength and colorfastness. Author Contributions: Conceptualization, C.J.D. and X.P.Y.; methodology, X.P.Y. and C.J.D.; valida-
tion, X.P.Y. and M.M.; formal analysis, C.J.D. and X.P.Y.; investigation, M.M. and X.P.Y.; resources,
C.J.D. and X.P.Y.; data curation, M.M.; writing—original draft preparation, X.P.Y.; writing—review
and editing, X.P.Y. and C.J.D.; visualization, M.M. and X.P.Y.; supervision, X.P.Y.; project administra-
tion, C.J.D. and X.P.Y.; funding acquisition, C.J.D. and X.P.Y. All authors have read and agreed to the
published version of the manuscript. Funding: This work was partially supported by the Department of Agriculture HATCH project No. TEN00521. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. References 1. Roenbeck, M.R.; Sandoz-Rosado, E.J.; Cline, J.; Wu, V.; Moy, P.; Afshari, M.; Reichert, D.; Lustig, S.R.; Strawhecker, K.E. Probing
the internal structures of Kevlar® fibers and their impacts on mechanical performance. Polymer 2017, 128, 200–210. [CrossRef] p
p
y
2. Zhao, J. Effect of surface treatment on the structure and properties of para-aramid fibers by phosphoric acid. Fibers Polym. 2013,
14, 59–64. [CrossRef] L.J. Sulfuric Acid Treatment of Aramid Fiber for Improving the Cationic Dyeing Performance. Adv. Mater. R
247. [CrossRef] 3. Xia, D.; Wang, L.J. Sulfuric Acid Treatment of Aramid Fiber for Improving the Cationic Dyeing Pe
2012, 627, 243–247. [CrossRef] 4. Vassiliadis, A.A.; Roulia, M.; Boussias, C.M. Disperse Dyeing Systems for p-aramid Fibers. In Proceedings of the 37th International
Symposium on Novelties in Textiles, Ljubljana, Slovenia, 15–17 June 2006. 4. Vassiliadis, A.A.; Roulia, M.; Boussias, C.M. Disperse Dyeing Systems for p-aramid Fibers. In Proceedings
S
i
N
l i
i
T
il
Lj blj
Sl
i
15 17 J
2006 iliadis, A.A.; Roulia, M.; Boussias, C.M. Disperse Dyeing Systems for p-aramid Fibers. In Proceedings of the 37th y
p
j
j
5. Dong, Y.; Jang, J. The enhanced cationic dyeability of ultraviolet/ozone-treated meta-aramid fabrics. Color. Technol. 2011, 127,
173–178. [CrossRef] review of low-temperature plasma treatment of textile materials. J. Mater. Sci. 2015, 50, 5913–5943. [CrossRef 6. Jelil, R.A. A review of low-temperature plasma treatment of textile materials. J. Mater. Sci. 2015, 50, 591 7. Vu, N.; Michielsen, S. Near room temperature dyeing of m-aramid fabrics. J. Appl. Polym. Sci. 2019, 136, 48190. [CrossRef] 8. Han, S.Y.; Jaung, J.Y. Acid dyeing properties of meta-aramid fiber pretreated with PEO45-MeDMA derived from [2-
(methacryloyloxy)ethyl] trimethylammonium chloride. Fibers Polym. 2009, 10, 461–465. [CrossRef] 9. Xi, M.; Li, Y.-L.; Shang, S.-Y.; Li, D.-H.; Yin, Y.-X.; Dai, X.-Y. Surface modification of aramid fiber by air
pressure with continuous on-line processing. Surf. Coat. Technol. 2008, 202, 6029–6033. [CrossRef] L.; Shang, S. Y.; Li, D. H.; Yin, Y. X.; Dai, X. Y. Surface modification of aramid fiber by air DBD plasma
th continuous on-line processing. Surf. Coat. Technol. 2008, 202, 6029–6033. [CrossRef] p
p
g
f
10. Sun, Y.; Liang, Q.; Chi, H.; Zhang, Y.; Shi, Y.; Fang, D.; Li, F. The application of gas plasma technologies in surface modification of
aramid fiber. Fibers Polym. 2014, 15, 1–7. [CrossRef] y
11. Su, M.; Gu, A.; Liang, G.; Yuan, L. References The effect of oxygen-plasma treatment on Kevlar fibers and the properties of Kevlar
fibers/bismaleimide composites. Appl. Surf. Sci. 2011, 257, 3158–3167. [CrossRef] p
pp
f
12. Abou Elmaaty, T.; Eltaweel, F.; Elsisi, H. Water-free Dyeing of Polyester and Nylon 6 Fabrics with Novel 2-Oxoacetohydrazonoyl
Cyanide Derivatives under a Supercritical Carbon Dioxide Medium. Fibers Polym. 2018, 19, 887–893. [CrossRef] aty, T.; Eltaweel, F.; Elsisi, H. Water-free Dyeing of Polyester and Nylon 6 Fabrics with Novel 2-Oxoacetohydra Abou Elmaaty, T.; Eltaweel, F.; Elsisi, H. Water-free Dyeing of Polyester and Nylon 6 Fabrics with Novel 2-O
C
id D
i
i
d
S
i i
l C
b
Di
id M di
Fib
P l
2018 19 887 893 [C y,
;
,
;
,
y
g
y
y
Cyanide Derivatives under a Supercritical Carbon Dioxide Medium. Fibers Polym. 2018, 19, 887–893. [C yanide Derivatives under a Supercritical Carbon Dioxide Medium. Fibers Polym. 2018, 19, 887–893. [CrossRe 13. Kaul, B.L. Mass pigmentation and solution dyeing of synthetic fibres. In Synthetic Fibre Dyeing; Hawkyard, C., Ed.; Society of
Dyers and Colourists: Bradford, West Yorkshire, UK, 2004; p. 230. p
oi, J.-H. Dyeing Properties and Color. Fastness of 100% meta-Aramid Fiber. Fibers Polym. 2011, 12, 484–4 14. Kim, E.-M.; Choi, J.-H. Dyeing Properties and Color. Fastness of 100% meta-Aramid Fiber. Fibers P
[CrossRef] [CrossRef]
15. Ferrero, F.; Periolatto, M.; Rovero, G.; Giansetti, M. Alcohol-assisted dyeing processes: A chemical substitution study. J. Clean. Prod 2011 19 1377 1384 [C
R f] 15. Ferrero, F.; Periolatto, M.; Rovero, G.; Giansetti, M. Alcohol-assisted dyeing processes: A chemical substitution study. J. Clean. Prod. 2011, 19, 1377–1384. [CrossRef] 16. Ferrero, F.; Periolatto, M. Glycerol in comparison with ethanol in alcohol-assisted dyeing. J. Clean. Prod. 2012, 33, 127–131. [CrossRef] ]
Pawar, S.S.; Maiti, S.; Biranje, S.; Kulkarni, K.; Adivarekar, R.V. A novel green approach for dyeing polyeste
d
t
ti
l
t
d
i
di
H li
2019 5
01606 [C
R f] [P bM d] iti, S.; Biranje, S.; Kulkarni, K.; Adivarekar, R.V. A novel green approach for dyeing polyester using glycer 17. Pawar, S.S.; Maiti, S.; Biranje, S.; Kulkarni, K.; Adivarekar, R.V. A novel green approach for dyeing polyester using glycerine
based eutectic solvent as a dyeing medium. Heliyon 2019, 5, e01606. [CrossRef] [PubMed] 17. Pawar, S.S.; Maiti, S.; Biranje, S.; Kulkarni, K.; Adivarekar, R.V. A novel green approach for dyein
based eutectic solvent as a dyeing medium. 4. Conclusions Informed Consent Statement: Not applicable. 45 Polymers 2021, 13, 1492 Data Availability Statement: The data presented in this study are available on request from the
corresponding author. Data Availability Statement: The data presented in this study are available on request from the
corresponding author. Acknowledgments: We thank the support by the U.S. Department of Agriculture HATCH project
No. TEN00521. Conflicts of Interest: The authors declare no conflict of interest. References Heliyon 2019, 5, e01606. [CrossRef] [PubMed] j
g
pp
y
utectic solvent as a dyeing medium. Heliyon 2019, 5, e01606. [CrossRef] [PubMed] 18. Cheng, L.; Liu, L.; Ye, X.P. Acrolein Production from Crude Glycerol in Sub- and Super-Critical Water. J. Am. Oil Chem. Soc. 2013,
90, 601–610. [CrossRef] 19. Liu, L.; Ye, X.P. Simultaneous production of lactic acid and propylene glycol from glycerol using solid catalysts without external
hydrogen. Fuel Process. Technol. 2015, 137, 55–65. [CrossRef] 20. Liu, L.; Ye, X.P.; Bozell, J.J. A Comparative Review of Petroleum-Based and Bio-Based Acrolein Productio
1162–1180. [CrossRef] ell, J.J. A Comparative Review of Petroleum-Based and Bio-Based Acrolein Production. ChemSusChem 2012, 5,
ef] 21. Zou, B.; Ren, S.; Ye, X.P. Glycerol Dehydration to Acrolein Catalyzed by ZSM-5 Zeolite in Supercritical Carbon Dioxide Medium. ChemSusChem 2016, 9, 3268–3271. [CrossRef] 22. Baliarsingh, S.; Jena, J.; Das, T.; Das, N.B. Role of cationic and anionic surfactants in textile dyeing with natural dyes extracted
from waste plant materials and their potential antimicrobial properties. Ind. Crops Prod. 2013, 50, 618–624. [CrossRef] 23. Gur–Arieh, Z.; Ingamells, W. The Role of Benzyl Alcohol in the Solvent-assisted Dyeing of Acrylic Fibres. J. Soc. Dye. Colour. 2008,
90, 8–11. [CrossRef] 24. Shao, D.; Xu, C.; Wang, H.; Du, J. Enhancing the Dyeability of Polyimide Fibers with the Assistance of Swelling Agents. Materials
2019, 12, 347. [CrossRef] [PubMed] sbach, V. The Carrier Effect in the m-Aramid Fiber/Cationic Dye/Benzyl Alcohol System. Text. Res. J. 1999, 69,
] 25. Nechwatal, A.; Rossbach, V. The Carrier Effect in the m-Aramid Fiber/Cationic Dye/Benzyl Alcohol Sys
635–641. [CrossRef] 26. Zheng, H.-D.; Zhang, J.; Yan, J.; Zheng, L.-J. Investigations on the effect of carriers on meta-aramid fabric dyeing properties in
supercritical carbon dioxide. RSC Adv. 2017, 7, 3470–3479. [CrossRef] 46 Polymers 2021, 13, 1492 27. Morris, M.; Ye, X.P.; Doona, C.J. Soybean Oil and Nonthermal Plasma Pretreatment to Dye Para-Aram
Disperse Dye Using a Glycerol-Based Dye Bath. J. Am. Oil Chem. Soc. 2021, 98, 463–473. [CrossRef] y
g
y
y
d, ISO 105-C10:2006. Colour Fastness to Washing with Soap or Soap and Soda. In Textiles—Tests for Colour F 28. Standard, ISO 105-C10:2006. Colour Fastness to Washing with Soap or Soap and Soda. In Textiles—Tests for Colour Fastness. In
I
l i
l S
d
di
i
O
i
i
I
l i
l S
d
di
i
O
i
i
G
S
i
l
d 2006 28. Standard, ISO 105-C10:2006. Article
A Negative Index Nonagonal CSRR Metamaterial-Based
Compact Flexible Planar Monopole Antenna for Ultrawideband
Applications Using Viscose-Wool Felt Kabir Hossain 1,2, Thennarasan Sabapathy 1,2,*, Muzammil Jusoh 1,2, Mahmoud A. Abdelghany 3,4,*,
Ping Jack Soh 1,5 , Mohamed Nasrun Osman 1,2, Mohd Najib Mohd Yasin 1,2, Hasliza A. Rahim 1,2
and Samir Salem Al-Bawri 6 1
Advanced Communication Engineering (ACE), Centre of Excellence, Universiti Malaysia Perlis (UniMAP),
Jalan Tiga, Pengkalan Jaya Business Centre, Kangar 01000, Malaysia; hossain.kabir42@gmail.com (K.H.);
muzammil@unimap.edu.my (M.J.); pingjack.soh@oulu.fi(P.J.S.); nasrun@unimap.edu.my (M.N.O.); 1
Advanced Communication Engineering (ACE), Centre of Excellence, Universiti Malaysia Perlis (UniMAP),
Jalan Tiga, Pengkalan Jaya Business Centre, Kangar 01000, Malaysia; hossain.kabir42@gmail.com (K.H.);
muzammil@unimap.edu.my (M.J.); pingjack.soh@oulu.fi(P.J.S.); nasrun@unimap.edu.my (M.N.O.); najibyasin@unimap.edu.my (M.N.M.Y.); haslizarahim@unimap.edu.my (H.A.R.) 2
Faculty of Electronic Engineering Technology, Kampus Alam UniMAP Pauh Putra Faculty of Electronic Engineering Technology, Kampus Alam y
g
g
gy,
p
Universiti Malaysia Perlis (UniMAP), Arau 02600, Malaysia 3
Electrical Engineering Department, College of Engineering, Prince Sattam Bin Abdulaziz University,
Wadi Addwasir 11991, Saudi Arabia 4
Department of Electrical Engineering, Faculty of Engineering, Minia University, Minia 61519, Egypt
5
Centre for Wireless Communications (CWC), University of Oulu, P.O. Box 4500, 90014 Oulu, Finland
6
Space Science Centre, Climate Change Institute, Universiti Kebangsaan Malaysia, Bangi 43600, Malaysia;
s.albawri@gmail.com Citation: Hossain, K.; Sabapathy, T.;
Jusoh, M.; Abdelghany, M.A.; Soh,
P.J.; Osman, M.N.; Yasin, M.N.M.;
Rahim, H.A.; Al-Bawri, S.S. A
Negative Index Nonagonal CSRR
Metamaterial-Based Compact
Flexible Planar Monopole Antenna
for Ultrawideband Applications
Using Viscose-Wool Felt. Polymers
2021, 13, 2819. https://doi.org/
10.3390/polym13162819
Academic Editors: Tarek M. Abou Elmaaty and Maria
Rosaria Plutino
Received: 11 August 2021
Accepted: 20 August 2021
Published: 22 August 2021 Citation: Hossain, K.; Sabapathy, T.;
Jusoh, M.; Abdelghany, M.A.; Soh,
P.J.; Osman, M.N.; Yasin, M.N.M.;
Rahim, H.A.; Al-Bawri, S.S. A
Negative Index Nonagonal CSRR
Metamaterial-Based Compact
Flexible Planar Monopole Antenna
for Ultrawideband Applications
Using Viscose-Wool Felt. Polymers
2021, 13, 2819. https://doi.org/
10.3390/polym13162819 *
Correspondence: thennarasan@unimap.edu.my (T.S.); abdelghany@mu.edu.eg (M.A.A.) Abstract: In this paper, a compact textile ultrawideband (UWB) planar monopole antenna loaded
with a metamaterial unit cell array (MTMUCA) structure with epsilon-negative (ENG) and near-zero
refractive index (NZRI) properties is proposed. The proposed MTMUCA was constructed based
on a combination of a rectangular- and a nonagonal-shaped unit cell. The size of the antenna was
0.825 λ0 × 0.75 λ0 × 0.075 λ0, whereas each MTMUCA was sized at 0.312 λ0 × 0.312 λ0, with respect
to a free space wavelength of 7.5 GHz. The antenna was fabricated using viscose-wool felt due to its
strong metal–polymer adhesion. Article
A Negative Index Nonagonal CSRR Metamaterial-Based
Compact Flexible Planar Monopole Antenna for Ultrawideband
Applications Using Viscose-Wool Felt A naturally available polymer, wool, and a human-made polymer,
viscose, that was derived from regenerated cellulose fiber were used in the manufacturing of the
adopted viscose-wool felt. The MTMUCA exhibits the characteristics of ENG, with a bandwidth
(BW) of 11.68 GHz and an NZRI BW of 8.5 GHz. The MTMUCA was incorporated on the planar
monopole to behave as a shunt LC resonator, and its working principles were described using an
equivalent circuit. The results indicate a 10 dB impedance fractional bandwidth of 142% (from 2.55 to
15 GHz) in simulations, and 138.84% (from 2.63 to 14.57 GHz) in measurements obtained by the
textile UWB antenna. A peak realized gain of 4.84 dBi and 4.4 dBi was achieved in simulations and
measurements, respectively. A satisfactory agreement between simulations and experiments was
achieved, indicating the potential of the proposed negative index metamaterial-based antenna for
microwave applications. Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. Keywords: metamaterials; high-performance textiles; wearable antenna; textile antennas; polymer References Colour Fastness to Washing with Soap or Soap and Soda. In Textiles—Tests for Colour Fastness. In
Internaltional Standardisation Organisation; Internaltional Standardisation Organisation: Geneva, Switzerland, 2006. g
g
29. Etters, J.N.; Hurwitz, M.D. Opaque reflectance of translucent fabric. Text. Chem. Color. 1986, 18, 19–26. 29. Etters, J.N.; Hurwitz, M.D. Opaque reflectance of translucent fabric. Text. Chem. Color. 1986, 18, 19–26. 30. Lumakso, F.; Rohman, A.M.H.; Riyanto, S.; Yusof, F. Detection and quantification of soybean and corn oils as adulterants in
avocado oil using fourier transform mid infrared (FT-MIR) spectroscopy aided with multivariate calibration. J. Teknol. 2015, 77,
251–255. [CrossRef] 31. Man, Y.; Rohman, A. Analysis of Canola Oil in Virgin Coconut Oil Using FTIR Spectroscopy and Chemometrics. J. Food Pharm. Sci. 2013, 1, 5–9. 32. Sienkiewicz, A.M.; Czub, P. The unique activity of catalyst in the epoxidation of soybean oil and followin
product with bisphenol A. Ind. Crops Prod. 2016, 83, 755–773. [CrossRef] p
p
p
33. Zhao, X.; Yang, J.; Tao, D.; Xu, X. Synthesis and Tribological Properties of Air Plasma Polymerized Soybean Oil with N-Containing
Structures. J. Am. Oil Chem. Soc. 2014, 91, 827–837. [CrossRef] 34. Haijuan, K.; Hui, S.; Jin, C.; Haiquan, D.; Xiaoma, D.; Mengmeng, Q.; Muhuo, Y.; Youfeng, Z. Improvement of adhesion of
kevlar fabrics to epoxy by surface modification with acetic anhydride in supercritical carbon dioxide. Polym. Compos. 2019, 40,
E920–E927. [CrossRef] 35. Mukherjee, M.; Kumar, S.; Bose, S.; Das, C.; Kharitonov, A. Study on the Mechanical, Rheological, and Morphological Properties
of Short Kevlar™Fiber/s-PS Composites. Polym. Plast. Technol. Eng. 2008, 47, 623–629. [CrossRef] 36. Kašparová, M.; Šašková, J.; Gregr, J.; Wiener, J. Using of DSCBD plasma for treatment of Kevlar and Nomex fibers. Chem. Listy
2008, 102, 1515–1518. 37. Ximena, V.Y. Characterization and Analysis of High Voltage Atmospheric Cold Plasma Treatment of Soilbean Oil. Ph.D. Thesis,
Purdue University, West Lafayette, IN, USA, 2020. 38. Van Durme, J.; Nikiforov, A.; Vandamme, J.; Leys, C.; De Winne, A. Accelerated lipid oxidation using non-thermal plasma
technology: Evaluation of volatile compounds. Food Res. Int. 2014, 62, 868–876. [CrossRef] 39. Zou, X.; Xu, M.; Pan, S.; Gan, L.; Zhang, S.; Chen, H.; Liu, D.; Lu, X.; Ostrikov, K.K. Plasma Activated Oil: Fast Production,
Reactivity, Stability, and Wound Healing Application. ACS Biomater. Sci. Eng. 2019, 5, 1611–1622. [CrossRef] [PubMed] 47 47 Citation: Hossain, K.; Sabapathy, T.;
Jusoh, M.; Abdelghany, M.A.; Soh,
P.J.; Osman, M.N.; Yasin, M.N.M.;
Rahim, H.A.; Al-Bawri, S.S. A
Negative Index Nonagonal CSRR
Metamaterial-Based Compact
Flexible Planar Monopole Antenna
for Ultrawideband Applications
Using Viscose-Wool Felt. Polymers
2021, 13, 2819. https://doi.org/
10.3390/polym13162819 polymers polymers polymers 1. Introduction Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article
distributed
under
the
terms
and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). Flexible substrates including organic substances, such as polymers, paper, plastics,
textiles, and fabrics, have become increasingly important to enable increased flexibility in
wearable sensors/antennas [1]. Flexible antennas consist of a dielectric material (which
works as the substrate) and a conductive material (which can be used as a radiating element
and/or ground plane). Pure metals, metals mixed with fabrics, and conductive inks [2]
are examples of materials that can be used as conductive materials. Meanwhile, polymers 49 49 https://www.mdpi.com/journal/polymers Polymers 2021, 13, 2819. https://doi.org/10.3390/polym13162819 Polymers 2021, 13, 2819 such as foam, paper textile fabrics, plastics, and soft printed circuit boards (PCBs) are all
common dielectric polymer materials. Other than dielectric polymers, extensive research
has been conducted on conductive polymers. They are explored for various applications
such as solar energy harvesting [3], tissue engineering [4], supercapacitor design [5–7],
gas sensors [8], and immunosensors [9]. In particular, flexible conductive polymers were
also proposed by [10,11]. However, in antenna designs, more concerns are directed to
the dielectric of the antenna since the antenna performance is mainly determined by the
electrical characteristic and the mechanical flexibility of the dielectric substrate. Recent
developments in manufacturing techniques for flexible polymer antennas are appealing
due to the low permittivity with low losses [12]. In flexible antenna designs, dielectric
polymer materials which are commonly used as substrates are classified either as natural
polymers (e.g., rubber, silk, wool) or synthetic/human-made polymers (e.g., polystyrene,
polyvinylchloride, nylon) [1]. Textile structural composites (which are considered as
natural materials) are versatile in terms of their exceptional physical and mechanical
properties which can be adopted in particular engineering applications to meet the desired
requirements [13]. Ultrawideband (UWB) technology has triggered enormous research attention in wire-
less communications, especially after the allocation of the unlicensed frequency band
(3.1 to 10.6 GHz) by the Federal Communications Commission (FCC) in 2002 [14]. A UWB
antenna has the capability of providing high-speed data transmission with low-power
spectral densities compared to conventional wireless communication systems within short
distances. The application of UWB has been expanded into the wireless body area network
(WBAN) domain based on the IEEE 802.15.6 WBAN standard [15,16]. 1. Introduction However, the near-zero refractive index (NZRI) property can enhance 50 Polymers 2021, 13, 2819 the gain, as reported in [31]. Several metamaterial structures have been proposed in terms
of complementary split-ring resonators (CSRR) [26,32], split-ring resonators (SRRs) [33],
planar patterns, and capacitance-loaded strips (CLSs) [29]. Some other MTM structures
such as electromagnetic bandgaps (EBGs) and artificial magnetic conductors (AMCs) were
discussed in [27]. Such metamaterial-based UWB antennas have been reported in the
literature with proven antenna performance enhancements [27,28]. In [34], MTMs were
loaded into UWB wearable antennas for non-invasive skin cancer detection. Likewise,
in [35], the proposed MTM UWB antenna was used for breast cancer detection. Despite
the different designs, antennas for wearable applications should be compact, low cost,
lightweight, and able to be integrated into circuits with ease [28]. When constructing
metamaterials for metamaterial-enhanced devices, it is crucial to take into consideration
the fabrication difficulty. Therefore, when developing textile-based metamaterials, extra
care should be taken in each design phase [21,36]. This paper proposes a compact textile antenna incorporated with an MTM unit cell
array (MTMUCA) structure, with an in-depth analysis. A polymer-based viscose-wool felt
was adopted as the dielectric material of the antenna. The felt is a composite material that is
developed from a naturally available polymer, wool, and a human-made polymer, viscose,
that is derived from regenerated cellulose fiber. An equivalent circuit model was developed
to present the working principles of the overall structure. Its structure was simulated and
validated experimentally from 1 to 15 GHz. First, the transmission–reflection (RTR) method
was used to extract the effective parameters of the MTMs in this work. The simulation
results indicate that the MTM unit cell (MTMUC) and the MTMUCA are almost identical
in performance, with an ENG BW of at least 11.53 GHz and an NZRI BW of 8.5 GHz. To the
best of the authors’ knowledge, the design of such textile-based ENG/ NZRI incorporated
with a flexible MTM array antenna is yet to be reported in the literature. A comparison of
the proposed antenna with similar designs in the literature is presented in Table 1. Table 1. Comparison of the proposed design with relevant previous work in the literature. 1. Introduction Recent techno-
logical developments have resulted in compact and smart biomedical sensors/antennas
for implementation on the human body. These antennas and sensors are most ideal for
implementation in WBAN-type networks, as they are useful in sectors such as wearable
computing, health monitoring, rescue systems, and patient tracking [17,18]. These appli-
cations require wireless devices to be placed close to the human body, which demands
antennas and sensors to be developed using flexible materials. To prolong their usage near
or on the body and, at the same time, ensure the safety and comfort of the user, they are
best to be integrated onto clothing. Recently, conductive textiles have been introduced
commercially, spurring the design of antennas for WBAN using textiles [16]. y
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Sensors and smart devices have been the subject of extensive research over the past
decade, with the goal of making them more easily integrated onto the human body [19]. Fabrics have been used as a natural and comfortable substrate for wearable electronic
devices. Fabrics can now contain electrical functionality due to miniaturization of electronic
components and innovative technologies [20]. There has been a lot of recent research on
cloth fabrics, including sewn textiles, embroidered textiles, nonwoven textiles, knitted
fabrics, woven fabrics, printed fabrics, braiding, laminated fabrics, spinning, and chemically
treated fabrics [21]. Developing modern textile-based sensors has become a substantial
undertaking in recent years, with numerous studies focusing on applications such as
athletic training [22], emergency rescue and law enforcement [23], fitness monitoring [24],
and other fields. Metamaterials (MTMs) are artificial composite structures with exotic electromagnetic
properties which can be used for potential groundbreaking applications (e.g., in antenna
design, subwavelength imaging) [25]. Consequently, MTMs are suitable to be applied
to improve WBAN antennas in terms of gain, radiation patterns, bandwidth (BW), and
size compactness [26–28]. The characteristics of MTMs can be single negative (SNG) or
double negative (DNG) based on the dielectric permittivity (ε) or magnetic permeability
(μ). For SNG MTMs, either ε or μ can be negative, and for DNG MTMs, both ε and μ are
negative. For SNG MTMs, if ε is negative, they are called epsilon-negative (ENG) MTMs,
and if μ is negative, they are called mu-negative (MNG) MTMs [17,29]. Furthermore, the
refractive index of a material depends on the ε and μ, which defines the extent of reflection
and refraction [30]. 1. Introduction Reference
Size (mm3)
λ0 = 40 mm
Operating
Frequency Range
(GHz)
Metamaterial
Struc-
ture/Technique
Fractional
Bandwidth
(FBW) (%)
Antenna Peak
Gain (dBi)
Remarks
[37]
52.5 × 52.5 × 20
(1.313 λ0 × 1.313 λ0 × 0.5 λ0)
2.5–13.8
BW = 11.3
Flexible AMC
metamaterial
138.65
9
Huge gap between the antenna
and separate MTM layer made
the overall antenna size bigger,
complicated, and
practically unusable. [38]
43 × 40 × 2
(1.075 λ0 × 1 λ0 × 0.05 λ0)
1.8–10
BW=8.2
Semicircular ring
resonator in the
patch
139
5.09
Comparatively low BW and
large antenna size. [39]
20 × 12 × 0.8
(0.5 λ0 × 0.3 λ0 × 0.02 λ0)
2.4–10
BW = 7.6
Metamaterial
122.58
3.456
Antenna is compact in terms of
size, but the BW and peak gain
are not superior. [17]
105 × 91 × 7.9
(2.625 λ0 × 2.275 λ0 × 0.1975 λ0)
3.5–12.40
BW=8.9
Metasurface
114
9.1
Antenna size is large when
including MTM. Increased
design complexity with the use
of multiple layered substrates. [40]
50 × 43 × 14.95
(1.25 λ0 × 1.075 λ0 × 0.374 λ0)
2.3–16
BW = 13.7
MNG
metamaterial
149.73
8
The antenna has separate MNG
MTM layer. Multilayer antenna
design and huge gap between
the antenna and MTM layer
made the design complicated. [34]
48 × 36 × 6
(1.2 λ0 × 0.9 λ0 × 0.15 λ0)
8.2–13
BW=4.8
AMC
metamaterial
45
7.04
Low BW and does not cover
required FCC BW. Large
antenna size incorporated on a
separate AMC layer. Proposed
work
33 × 30 × 3
(0.825 λ0 × 0.75 λ0 × 0.075 λ0)
2.55–15
BW= 12.45
SNG/NZRI
metamaterial
142
4.84
Compact size and wide
operational bandwidth
and FBW. Table 1. Comparison of the proposed design with relevant previous work in the literature. 2. Flexible Polymer-Based Textile Antenna Design with Metamaterial 2. Flexible Polymer-Based Textile Antenna Design with Metamaterial In this work, the proposed MTM antenna was simulated and fabricated on textile
materials. Shieldit SuperTM with a thickness of 0.17 mm and a conductivity value of
1.18 × 105 S/m was used as the ground plane and the radiator. Meanwhile, a 3 mm
viscose-wool felt substrate with a dielectric constant of 1.44 and loss tangent of 0.044 was 51 Polymers 2021, 13, 2819 employed. The choice of the viscose-wool felt was mainly due its strong Shieldit SuperTM–
polymer adhesion. 1. Introduction This type of flexible polymer is also easily available on the market; thus,
no special treatment was required in developing the material in the lab, as required by
other polymers such as polydimethylsiloxane (PDMS) [2]. The felt contained 30% viscose
and 70% wool that formed a good composition of fibers with a density of 0.25 gm/CC. This
property can help the Shieldit SuperTM easily iron out and attach to the polymer felt [36,41]. Computer Simulation Technology’s (CST) Microwave Studio Suite (MWS) was used to
model and simulate the MTM and MTM-integrated antenna over the frequencies of interest
from 1 to 15 GHz. The analyses of these structures are reported in the following subsections. 2.1. Metamaterial Design The proposed MTMUCA was designed based on a CSRR structure and is illustrated in
Figure 1a. Its overall size was 12.5 × 12.5 × 3 mm3, and other dimensions are summarized
in Table 2. A square loop and a nonagonal-shaped structure were combined to form the
MTMUC structure. To characterize the metamaterial unit cell, the MTMUC structure was
placed between two waveguide ports on the positive and negative z-axis and was excited
with a transverse electromagnetic (TEM) wave, as depicted in Figure 2a. It was bounded
by a perfect electric conductor (PEC) boundary at the ±x-axis and a perfect magnetic
conductor (PMC) boundary at the ±y-axis. A frequency solver with a tetrahedral mesh
scheme was utilized in simulations over the frequency of interest. In this metamaterial
simulation, the unit cell was without a conductive layer at the bottom [42]. Figure 1. Schematic diagram of the proposed antenna and the MTMUCA structure (flexible polymer
in gray and flexible conductive element in blue): (a) front view; (b) rear view. Figure 1. Schematic diagram of the proposed antenna and the MTMUCA structure (flexible polymer
in gray and flexible conductive element in blue): (a) front view; (b) rear view. 52 52 Polymers 2021, 13, 2819 Table 2. Parameter dimensions of the proposed antenna. Para. Value (mm)
Para. Value (mm)
Para. Value (mm)
Ls
33.00
c
3.68
x
6.50
Ws
30.00
d
2.60
y
5.50
Wp
17.20
e
4.20
r
1.75
Lp
13.40
f
1.81
g2
3.00
Wg
13.70
g
1.56
g3
2.60
Lg
10.00
g1
0.5
Wf
2.80
Figure 2. (a) 3D view of the MTMUCA simulation setup. (b) Topology of the MTMUC structure
and its equivalent circuit model, where C = capacitance of MTMUCA, L/2 = each inductance (blue
indicates the metallized areas). Table 2. Parameter dimensions of the proposed antenna. Figure 2. (a) 3D view of the MTMUCA simulation setup. (b) Topology of the MTMUC structure
and its equivalent circuit model, where C = capacitance of MTMUCA, L/2 = each inductance (blue
indicates the metallized areas). The MTMUC structure and its equivalent circuit model are depicted in Figure 2b,
where ohmic losses are unaccounted for [43]. The MTM modeled on a transverse plane acts
as an LC resonator, which can be excited by the orthogonal electric field. Conversely, this
structure behaves similarly to an electric dipole when excited by an axial electric field. 2.1. Metamaterial Design The
primary resonance can also be excited by the external magnetic field along the y-axis, as
the CSRR can also exhibit a magnetic behavior [43,44]. The SNG properties can be tailored
by appropriately modeling the CSRR gaps into the design. The surface current distribution
of the proposed MTM was extracted for further study based on the setup exhibited in
Figure 2a. g
The simulated S-parameter of the MTMs is shown in Figure 3. In prior analyses, the
material effective parameters (e.g., permittivity, refractive index) and MTM’s stopband
behavior were investigated. The transmission coefficient (S21) of the MTMUC structure
ranged from 1 to 4.65 GHz, and from 7.53 to 11.71 GHz, and the reflection coefficient
(S11) ranged from 6.34 to 6.68 GHz, and from 13.83 to 14.44 GHz. On the other hand, the
MTMUCA showed an operational S21 from 1 to 4.45 GHz, and from 7.84 to 11.18 GHz,
whereas the S11 was from 6.34 to 7.01 GHz, and from 13.65 to 14.26 GHz. Hence, the
stopband behaviors clearly satisfied the S21 ≤−10 dB requirement. 53 Polymers 2021, 13, 2819 Figure 3. S-parameter of the MTMUC and MTMUCA structures. f h
d Figure 3. S-parameter of the MTMUC and MTMUCA structures. The RTR [28,45] method was employed to extract the effective parameters from the
normal incident scattering parameters using (1) to (7), starting with calculating S11 and S21
from Equations (1) and (2) as follows: S11 = ( R01(1 −ei2nk0d)
1 −R012ei2nk0d )
(1)
S21 = ((1 −R012)eink0d
1 −R012ei2nk0d )
(2) S11 = ( R01(1 −ei2nk0d)
1 −R012ei2nk0d )
(1) (1) S21 = ((1 −R012)eink0d
1 −R012ei2nk0d )
(2) (2) where η is the refractive index, the wave vector in free space is denoted as k0, the pro-
totype/slab thickness is denoted as d, and R01 = z−1
z+1. Solving (1) and (2) results in (3)
as follows:
where η is the refractive index, the wave vector in free space is denoted as k0, the pro-
totype/slab thickness is denoted as d, and R01 = z−1
z+1. Solving (1) and (2) results in (3)
as follows: z = ±
(1 + S11)2 −S212
(1 −S112) −S212
(3)
eink0d = X ± i
1 −X2
(4) (3) (4) where X =
1
2S21(1−S112+S212). As the material is deemed to be a passive medium, the
impedance imaginary part should be greater than or equal to zero. 2.1. Metamaterial Design Additionally, the real
part of the refractive index (η) should be greater than or equal to zero. The η value of the
material can be obtained from (5): n =
1
k0d[{imaginary(ln eink0d) + 2mπ} −i{real(ln eink0d)}]
(5) (5) where m is an integer value or the branch index of the real part of η in other studies [46]. It
can be noted that in this extraction method, m = 0 was considered [36]. The values of ε and
μ can be determined from the following expression [46,47]: where m is an integer value or the branch index of the real part of η in other studies [46]. It
can be noted that in this extraction method, m = 0 was considered [36]. The values of ε and
μ can be determined from the following expression [46,47]: ε = n
z
(6)
μ = nz
(7) (6) (7) μ = nz 54 54 Polymers 2021, 13, 2819 Figure 4 illustrates the real part of the permittivity and refractive index. The MTMUC
shows an ENG characteristic (εr < 0) from 1 to 6.45 GHz, from 7.48 to 13.52 GHz, and from
14.96 to 15 GHz, whereas its NZRI characteristic (η < 0) is featured from 1 to 6.16 GHz, and
from 9.35 to 13.44 GHz. On the other hand, the MTMUCA displays an ENG characteristic
from 1 to 6.75 GHz, from 7.70 to 13.36 GHz, and from 14.73 to 15 GHz, whereas the
NZRI characteristic is featured from 1 to 5.74 GHz, 6.21 to 6.37 GHz, 9.48 to 12.08 GHz,
and 12.49 to 13.19 GHz. Based on the S-parameters and the MTM characteristics, the
proposed MTMUC and MTMUCA can be used for stopband applications and microwave
applications [28], e.g., C-band and Ku-band. Figure 4. Permittivity and refractive index results of the MTMUC and MTMUCA structures. Figure 4. Permittivity and refractive index results of the MTMUC and MTMUCA structures. 2.2. Metamaterial Working Principle To gain a further understanding of the MTM structure, a parametric study to analyze
the effects of the nonagonal-shaped inner split conductor was performed, and its results
are presented in Figure 5. In the absence of the inner conductor, a relatively narrower NZRI
region can be observed for both the MTMUC and MTMUCA structures. Besides that, the
structures’ inner nonagonal-shaped split ring was further evaluated when rotated at 0◦, 90◦,
180◦, and 270◦angles. 2.1. Metamaterial Design The results are summarized in Table 3. When positioned at 0◦and
270◦rotation angles, the structure shows identical results for both MTMUC and MTMUCA. g
To provide further insight into the working principles and properties of the proposed
metamaterial, the simulated current distribution was analyzed and discussed. Figure 6
visualizes the surface current distribution at 3 GHz, 6.5 GHz, 10 GHz, and 12 GHz for the
different types of MTM structures. The surface current density and direction are indicated
by the colors and arrows, respectively. The concentration of the surface current at 3 GHz
is almost indistinguishable, as shown in Figure 6a–e for MTMUC and Figure 6f–j for
MTMUCA. On the contrary, when the nonagonal-shaped inner split conductor is absent in
Figure 6a,f, a lower surface current density at 6.5 GHz, 10 GHz, and 12 GHz is observed
compared to the structures with an inner conductor. Stronger surface currents are also
observed at the edges of the rectangles and the nonagonal-shaped slot. For the MTMUC
structures (in Figure 6a–e), the concentration of the surface current in Figure 6c shows
the strongest surface current distribution. Likewise, among the MTMUCA structures (in
Figure 6f–j), Figure 6h indicates the strongest surface current distribution. It is observed
that in both cases, the position of the inner nonagonal slot shown in Figure 6c,h facilitated
the achievement of stronger surface currents compared to the rest of the structures. 55 Polymers 2021, 13, 2819 Figure 5. Analysis of relative permittivity and refractive index of (a) MTMUC and (b) MTMUCA. Figure 5. Analysis of relative permittivity and refractive index of (a) MTMUC and (b) MTMUCA. Figure 5. Analysis of relative permittivity and refractive index of (a) MTMUC and (b) MTMUCA. Comparison of the structures with different rotation angles indicated that the 90◦ro-
tated structure showed the strongest surface current distribution for both MTMUC and
MTMUCA. This structure was chosen over the 0◦or 270◦rotated structure despite the
latter being slightly better in terms of ε and NZRI BW. Therefore, the chosen structure was
implemented in the UWB antenna to be studied further in the following section. Comparison of the structures with different rotation angles indicated that the 90◦ro-
tated structure showed the strongest surface current distribution for both MTMUC and
MTMUCA. This structure was chosen over the 0◦or 270◦rotated structure despite the
latter being slightly better in terms of ε and NZRI BW. 2.1. Metamaterial Design Therefore, the chosen structure was
implemented in the UWB antenna to be studied further in the following section. Comparison of the structures with different rotation angles indicated that the 90◦ro-
tated structure showed the strongest surface current distribution for both MTMUC and
MTMUCA. This structure was chosen over the 0◦or 270◦rotated structure despite the
latter being slightly better in terms of ε and NZRI BW. Therefore, the chosen structure was
implemented in the UWB antenna to be studied further in the following section. 56 Polymers 2021, 13, 2819 Table 3. Negative permittivity and negative refractive index results of different types of MTM structures. MTM Structure
Negative Permittivity
Band (GHz)
Negative Refractive
Index Band (GHz)
Array Structure
Inner
Nonagonal-Shaped
Split Ring Rotation
Angle (Degrees)
1 × 1 (MTMUC)
–
1–6.58
8.2–15
1–6.33
12.17–15
0
1–6.50
7.78–15
1–6.21
10.39–13.33
13.78–15
90
1–6.45
7.48–13.52
14.96–15
1–6.16
9.35–13.44
180
1–6.57
8.02–15
1–6.31
10.79–13.26
14.80–15.00
270
1–6.50
7.78–15
1–6.21
10.39–13.33
13.78–15
2 × 2 (MTMUCA)
–
1–6.88
8.57–15
1–5.79
6.28–6.56
11.79–13.19
13.74–15
0
1–6.80
8.11–15
1–5.76
6.23–6.43
10.39–12.5
13.39–15
90
1–6.75
7.70–13.36
14.73–15
1–5.74
6.21–6.37
9.48–12.08
12.49–13.19
180
1–6.88
8.33–15
1–5.81
5.56–6.29
10.8–13.28
14.83–15
270
1–6.8
8.11–15
1–5.76
6.23–6.43
10.39–12.5
13.39–15 Table 3. Negative permittivity and negative refractive index results of different types of MTM structures. MTM Structure
Negative Permittivity
Band (GHz)
Negative Refractive
Index Band (GHz)
Array Structure
Inner
Nonagonal-Shaped
Split Ring Rotation
Angle (Degrees)
1 × 1 (MTMUC)
–
1–6.58
8.2–15
1–6.33
12.17–15
0
1–6.50
7.78–15
1–6.21
10.39–13.33
13.78–15
90
1–6.45
7.48–13.52
14.96–15
1–6.16
9.35–13.44
180
1–6.57
8.02–15
1–6.31
10.79–13.26
14.80–15.00
270
1–6.50
7.78–15
1–6.21
10.39–13.33
13.78–15
2 × 2 (MTMUCA)
–
1–6.88
8.57–15
1–5.79
6.28–6.56
11.79–13.19
13.74–15
0
1–6.80
8.11–15
1–5.76
6.23–6.43
10.39–12.5
13.39–15
90
1–6.75
7.70–13.36
14.73–15
1–5.74
6.21–6.37
9.48–12.08
12.49–13.19
180
1–6.88
8.33–15
1–5.81
5.56–6.29
10.8–13.28
14.83–15
270
1–6.8
8.11–15
1–5.76
6.23–6.43
10.39–12.5
13.39–15 Table 3. Negative permittivity and negative refractive index results of different types of MTM structures. 2 × 2 (MTMUCA)
–
1–6.88
8.57–15
1–5.79
6.28–6.56
11.79–13.19
13.74–15
0
1–6.80
8.11–15
1–5.76
6.23–6.43
10.39–12.5
13.39–15
90
1–6.75
7.70–13.36
14.73–15
1–5.74
6.21–6.37
9.48–12.08
12.49–13.19
180
1–6.88
8.33–15
1–5.81
5.56–6.29
10.8–13.28
14.83–15
270
1–6.8
8.11–15
1–5.76
6.23–6.43
10.39–12.5
13.39–15 57 57 Polymers 2021, 13, 2819 Figure 6. Surface current distribution for different MTM structures. 2.1. Metamaterial Design MTMUC (1×1 array) (a) without the nonagonal-
shaped inner split ring, and with (b) 0◦, (c) 90◦, (d) 180◦, and (e) 270◦rotation angles of the nonagonal-shaped inner split
ring. MTMUCA (2 × 2 array) (f) without the nonagonal-shaped inner split ring, and with (g) 0◦, (h) 90◦, (i) 180◦, and
(j) 270◦rotation angles of the nonagonal-shaped inner split ring. Figure 6. Surface current distribution for different MTM structures. MTMUC (1×1 array) (a) without the nonagonal-
shaped inner split ring, and with (b) 0◦, (c) 90◦, (d) 180◦, and (e) 270◦rotation angles of the nonagonal-shaped inner split
ring. MTMUCA (2 × 2 array) (f) without the nonagonal-shaped inner split ring, and with (g) 0◦, (h) 90◦, (i) 180◦, and
(j) 270◦rotation angles of the nonagonal-shaped inner split ring. 2.3. Antenna Design Geometry and Configurations As it has previously been mentioned, the structure of the proposed antenna inte-
grated with the MTMUCA is depicted in Figure 1. The planar monopole antenna was
designed with a combination of rectangular and half elliptical-shaped patches, whereas
two MTMUCAs were located 0.4 mm from both sides of the planar feedline. A partial
ground plane was implemented on the reverse side of this feedline, and a 50 Ω SMA
connector was connected at the end of the feedline. The overall dimension of the antenna
was 33 × 30 × 3 mm3 (0.825λ0 × 0.75λ0 × 0.075λ0, where λ0 is the free space wavelength at
7.5 GHz, with the MTMUCA sized at 12.5 × 12.5 mm2 (0.312λ0 × 0.312λ0)). All dimensions
are summarized in Table 2. Figure 7 depicts the surface current distribution at 3 GHz, 6.5 GHz, 10 GHz, and
12 GHz with and without the MTMUCA structure integrated into the antenna. It is evident
that the MTMUCA improved the current intensity. The circuit model of the structure
was modeled based on [44,48–50], where the conventional planar monopole antenna
model is illustrated in Figure 8a. The planar monopole antenna’s input impedance can
be represented as an RLC circuit resonator near its resonance frequency, whereas the
microstrip feedline can be expressed as a series inductor [44,48,50], resulting in the overall
circuit model shown in Figure 8b. The CSRR-type MTMUC was modeled as a shunt RLC 58 Polymers 2021, 13, 2819 resonator tank (Rm, Lm, and Cm) [44], which was designed to work at the frequencies
of interest. The resistor Rm indicates the dielectric and conductor losses, whereas the
capacitance and inductance of the MTMCU are denoted as Lm and Cm, respectively. The
short distance between the metallic ground plane and the MTMUCA was modeled as
a capacitance, expressed as CMG [51], whereas CMP represents the capacitive coupling
between the MTMUCA and microstrip feedline and/or patch resonator. Figure 7. Surface current distribution. (a) Without the integration of the MTMUCA structure. (b) With the integration of the
MTMUCA structure. Figure 7. Surface current distribution. (a) Without the integration of the MTMUCA structure. (b) With the integration of the
MTMUCA structure. Figure 7. Surface current distribution. (a) Without the integration of the MTMUCA structure. (b) With the integration of the
MTMUCA structure. Figure 8. Equivalent circuit model. (a) Conventional planar monopole antenna with microstrip
feedline antenna [48–50]. 2.3. Antenna Design Geometry and Configurations (b) Proposed antenna loaded with the MTMUCA demonstrated in Figure 1. Figure 8. Equivalent circuit model. (a) Conventional planar monopole antenna with microstrip
feedline antenna [48–50]. (b) Proposed antenna loaded with the MTMUCA demonstrated in Figure 1. 59 Polymers 2021, 13, 2819 The microstrip feed line behaves as an inductance, denoted as LIN. The CSRR acts as
an electric dipole, and it mainly propagates along the xy plane within the substrate and
radiates in the vicinity of the antenna. To be more specific, the metamaterial’s EM energy could be coupled to the planar
monopole antenna through CMP, whereas the radiation of the antenna was modeled using
the radiation resistance Rp [44]. The same applies to the coupling between the feedline and To be more specific, the metamaterial’s EM energy could be coupled to the planar
monopole antenna through CMP, whereas the radiation of the antenna was modeled using
the radiation resistance Rp [44]. The same applies to the coupling between the feedline and
the MTMUCA. The coupling effect can be seen in Figure 7b, where a significantly increased
current concentration is observed. Therefore, the implementation of the MTMUCA enables
the operation of the antenna at the frequencies of interest and will be demonstrated in the
next section. p
the MTMUCA. The coupling effect can be seen in Figure 7b, where a significantly increased
current concentration is observed. Therefore, the implementation of the MTMUCA enables
the operation of the antenna at the frequencies of interest and will be demonstrated in the
next section. 3. Results and Discussion Figure 9 depicts the evolution process of the conventional antenna design in this study. In the first stage, as shown in Figure 9a, a full grounded patch with a combination of
rectangular and half elliptical-shaped patches produces relatively narrow frequency bands,
as shown in Figure 9b. In order to achieve a wider impedance bandwidth, the ground
plane of the antenna was modified, as illustrated in the second stage of Figure 9b. From
Figure 9b, it can be seen that the second stage has a wider impedance bandwidth compared
to the first stage. Furthermore, in the third stage, a 0.4 mm gap was chosen to avoid a
short circuit between the 50 Ω connector and the feedline of the planar monopole antenna. The truncation at the ground plane improved the impedance bandwidth slightly, where
S11 resulted in being lower than −10 dB, showing over 2.33–2.6 GHz, and 8.52–12.3 GHz,
which means FBW = 47% at the frequencies of interest. It can be noticed that the attained
impedance bandwidth does not cover the complete UWB band allocated by the FCC. However, integration of the proposed MTMUCA with the conventional antenna can
improve the overall performance. The related evidence can be found in the following
results and discussion. Figure 9. Evolution process of the conventional antenna in this study. (a) Evolution steps; (b) S11 results. Figure 9. Evolution process of the conventional antenna in this study. (a) Evolution steps; (b) S11 results. The fabricated prototype was used to validate this work, which is shown in Figure 10. The dielectric polymer material (viscose-wool felt) and conductive material (Shieldit
SuperTM) of the prototype were dimensioned by a laser cutting machine as a part of
the fabrication process. A great deal of study has already been carried out on a variety
of materials that have characteristics that render them appropriate for use as a substrate
for conductive materials for antennas, conductive threads [52], conductive polymers [53],
and conductive textiles [54]. However, in this study, viscose-wool felt was adopted since
it provides easier fabrication with sufficient flexibility and enables strong adhesion with
the conductive textile Shieldit SuperTM. S11 measurement was performed using an Agi-
lent E5071C Network Analyser (Agilent Technologies, Bayan Lepas, Penang, Malaysia) 60 Polymers 2021, 13, 2819 to ensure the simulated findings are accurate. The comparison of the proposed antenna
between the simulated and measured S11 illustrated in Figure 11 indicates a good agree-
ment. 3. Results and Discussion The simulation indicates a 10 dB impedance bandwidth from 2.55 to 15 GHz, which
corresponds to an FBW of 142%. The measurement results indicate that this range is from
2.63 to 14.57 GHz, with an FBW of 138.84%. On the other hand, the antenna without the
MTMUCA in Figure 9b (third iteration) shows a simulated FBW of 47%. Although the
conventional antenna (considering the third iteration in Figure 9) does not work within
the FCC region, by utilizing the unique characteristics of the metamaterial, the antenna
performance could be enhanced. By utilizing the MTM on the conventional antenna, the
antenna element’s radiation efficiency, gain, and overall performance can be improved. Figure 10. Prototype of the designed antenna: (a) front view, and (b) rear view. Figure 10. Prototype of the designed antenna: (a) front view, and (b) rear view. Figure 10. Prototype of the designed antenna: (a) front view, and (b) rear view. Figure 11. Reflection coefficients of the proposed antenna. To rigorously analyze the performance of the proposed MTM-driven antenna proto-
type, body evaluation was further carried out with the help of a male volunteer (with a
height of 1.72 m and weight of 84 kg), as shown in Figure 12. The prototype antenna was
placed on two different places on the body (chest and arm). The related S11 results are
presented in Figure 11, which indicate an excellent performance of the proposed antenna
for on-body application. It can be noted that a 6 dB reflection coefficient is frequently used
in the manufacturer’s specification, as reported in [55,56]; hence, the obtained performance
is sufficient for practical application. Figure 11. Reflection coefficients of the proposed antenna. To rigorously analyze the performance of the proposed MTM-driven antenna proto-
type, body evaluation was further carried out with the help of a male volunteer (with a
height of 1.72 m and weight of 84 kg), as shown in Figure 12. The prototype antenna was
placed on two different places on the body (chest and arm). The related S11 results are
presented in Figure 11, which indicate an excellent performance of the proposed antenna
for on-body application. It can be noted that a 6 dB reflection coefficient is frequently used
in the manufacturer’s specification, as reported in [55,56]; hence, the obtained performance
is sufficient for practical application. 3. Results and Discussion To rigorously analyze the performance of the proposed MTM-driven antenna proto-
type, body evaluation was further carried out with the help of a male volunteer (with a
height of 1.72 m and weight of 84 kg), as shown in Figure 12. The prototype antenna was
placed on two different places on the body (chest and arm). The related S11 results are
presented in Figure 11, which indicate an excellent performance of the proposed antenna
for on-body application. It can be noted that a 6 dB reflection coefficient is frequently used
in the manufacturer’s specification, as reported in [55,56]; hence, the obtained performance
is sufficient for practical application. 61 Polymers 2021, 13, 2819 Figure 12. On-body measurement setup. Figure 13 shows the antenna gain and total efficiency over the frequency. In simula-
tions, an average realized gain of 3.3 dBi was achieved with the MTMUCA, whereas it was
2.7 dBi without the MTMUCA. A maximum gain of 4.83 dBi and 3.64 dBi could be obtained
with the proposed antenna with and without the MTMUCA, respectively. On the other
hand, the average measured gain of the proposed antenna is 3.04 dBi, and the maximum
peak gain is 4.4 dBi. The maximum total efficiency achieved is 87% and 79.5% for the
antenna with and without MTMUCA implementation, respectively, in simulations. The
attainable average total efficiency of the proposed antenna is approximately 73%, whereas
the antenna without the MTM indicated levels of around 69%. The measurements show
a maximum total efficiency of 80%, and the average efficiency is 68.2%, for the proposed
integrated antenna. Figure 12. On-body measurement setup. Figure 12. On-body measurement setup. Figure 13 shows the antenna gain and total efficiency over the frequency. In simula-
tions, an average realized gain of 3.3 dBi was achieved with the MTMUCA, whereas it was
2.7 dBi without the MTMUCA. A maximum gain of 4.83 dBi and 3.64 dBi could be obtained
with the proposed antenna with and without the MTMUCA, respectively. On the other
hand, the average measured gain of the proposed antenna is 3.04 dBi, and the maximum
peak gain is 4.4 dBi. The maximum total efficiency achieved is 87% and 79.5% for the
antenna with and without MTMUCA implementation, respectively, in simulations. The
attainable average total efficiency of the proposed antenna is approximately 73%, whereas
the antenna without the MTM indicated levels of around 69%. 3. Results and Discussion The measurements show
a maximum total efficiency of 80%, and the average efficiency is 68.2%, for the proposed
integrated antenna. Figure 13. The proposed antenna’s realized gain and radiation efficiency. Figure 13. The proposed antenna’s realized gain and radiation efficiency. Figure 14 illustrates the radiation characteristics of the proposed antenna. The sim-
ulated and measured radiation patterns of the E-plane (yz-plane) and H-plane (xz-plane)
were performed at four different frequencies, i.e., 3 GHz, 6.5 GHz, 10 GHz, and 12 GHz,
indicating very good agreement. An omnidirectional radiation pattern can be observed
at 3 GHz. Meanwhile, at 6.5 GHz, 10 GHz, and 12 GHz, an omnidirectional pattern Figure 14 illustrates the radiation characteristics of the proposed antenna. The sim-
ulated and measured radiation patterns of the E-plane (yz-plane) and H-plane (xz-plane)
were performed at four different frequencies, i.e., 3 GHz, 6.5 GHz, 10 GHz, and 12 GHz,
indicating very good agreement. An omnidirectional radiation pattern can be observed
at 3 GHz. Meanwhile, at 6.5 GHz, 10 GHz, and 12 GHz, an omnidirectional pattern 62 Polymers 2021, 13, 2819 can be seen in the H-plane, whereas the E-plane shows a bidirectional radiation pattern. Slight discrepancies between the simulations and measurements can be observed due to
fabrication inaccuracies. Figure 14. Radiation patterns of the proposed antenna at: (a) 3 GHz, (b) 6.5 G
(d) 12 GHz. Figure 14. Radiation patterns of the proposed antenna at: (a) 3 GHz, (b) 6.5 GHz, (c) 10 GHz, and
(d) 12 GHz. Figure 14. Radiation patterns of the proposed antenna at: (a) 3 GHz, (b) 6.5 GHz, (c) 10 GHz, and
(d) 12 GHz. 63 Polymers 2021, 13, 2819 4. Conclusions A compact textile UWB planar monopole antenna integrated with an MTMUCA
featuring ENG and NZRI properties was proposed and studied. The MTM-integrated
antenna prototype was fabricated using flexible polymer viscose-wool felt as the substrate
(since it provides easier fabrication with sufficient flexibility) and Shieldit SuperTM as the
conductive element. The proposed MTMUC structure consists of a unique combination
of square- and nonagonal-shaped CSRR-type MTMs. The proposed MTMUCA and MT-
MUC exhibited SNG properties in different frequency bands. The MTMUCA structure
featured an 11.68 GHz ENG bandwidth and an 8.5 GHz NZRI bandwidth. This work also
presented an equivalent circuit design to illustrate the principles of the overall structure. The implementation of the MTMUCA into the antenna design was proven experimentally
to improve the antenna gain, efficiency, and bandwidth. Measurements showed an FBW of
up to 138.84%, from 2.63 to 14.57 GHz, with a peak gain and total efficiency of 4.4 dBi and
80%, respectively. The proposed design can potentially be applied for wearable applica-
tions, where future research could be carried out to investigate the feasibility of on-body
application, specifically for breast cancer detection. Author Contributions: Conceptualization, K.H., M.J. and T.S.; design methodology, K.H., M.N.O. and P.J.S.; software, K.H. and H.A.R.; validation, K.H. and T.S.; formal analysis, K.H. and M.N.M.Y.;
investigation, K.H.; design optimization, S.S.A.-B.; metamaterial characterization, K.H. and M.N.O.;
numerical calculation and data interpretation, M.A.A. and H.A.R.; resources, M.N.O.; writing—
original draft preparation, K.H., T.S. and M.J.; writing—review and editing, T.S., P.J.S. and S.S.A.-B.;
visualization, K.H. and M.N.M.Y.; supervision, T.S. and M.J.; project administration, M.N.M.Y.; fund-
ing acquisition, M.A.A. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the Deanship of Scientific Research at Prince Sattam bin
Abdulaziz University, Saudi Arabia. nstitutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: This study did not report any data. Data Availability Statement: This study did not report any data. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. f
y
g
p
10.
Jiang, X.; Xu, C.; Gao, T.; Bando, Y.; Golberg, D.; Dai, P.; Hu, M.; Ma, R.; Hu, Z.; Wang, X.-B. Flexi
composite materials based on strutted graphene foam. Compos. Commun. 2021, 25, 100757. [CrossRef] ypy
y
9.
Zia, T.U.H.; Ali Shah, A.U.H. Understanding the adsorption of 1 NLB antibody on polyaniline nanotubes as a function of zeta
potential and surface charge density for detection of hepatitis C core antigen: A label-free impedimetric immunosensor. Colloids
Surfaces A Physicochem. Eng. Asp. 2021, 626, 127076. [CrossRef] 8.
Bibi, S.; Ullah, H.; Ahmad, S.M.; Ali Shah, A.-H.; Bilal, S.; Tahir, A.A.; Ayub, K. Molecular and Electronic
Polypyrrole Gas Sensors. J. Phys. Chem. C 2015, 119, 15994–16003. [CrossRef] References 1. Ali Khan, M.U.; Raad, R.; Tubbal, F.; Theoharis, P.I.; Liu, S.; Foroughi, J. Bending Analysis of Polymer-Based Flexible Antennas for
Wearable, General IoT Applications: A Review. Polymers 2021, 13, 357. [CrossRef] pp
y
2. Kirtania, S.G.; Elger, A.W.; Hasan, M.R.; Wisniewska, A.; Sekhar, K.; Karacolak, T.; Sekhar, P.K. Flexible Antennas: A Review. Micromachines 2020, 11, 847. [CrossRef] [PubMed] .A.; Krewer, U.; Shah, A.U.H.A.; Bilal, S. Efficient photocatalysis through conductive polymer coated FTO 3. Farooq, S.; Tahir, A.A.; Krewer, U.; Shah, A.U.H.A.; Bilal, S. Efficient photocatalysis through conduct 3. Farooq, S.; Tahir, A.A.; Krewer, U.; Shah, A.U.H.A.; Bilal, S. Efficient photocatalysis through conductive polymer coated FTO
counter electrode in platinum free dye sensitized solar cells. Electrochim. Acta 2019, 320, 134544. [CrossRef] 3. Farooq, S.; Tahir, A.A.; Krewer, U.; Shah, A.U.H.A.; Bilal, S. Efficient photocatalysis through conductive polymer coated FTO
counter electrode in platinum free dye sensitized solar cells. Electrochim. Acta 2019, 320, 134544. [CrossRef] q
p
y
g
counter electrode in platinum free dye sensitized solar cells. Electrochim. Acta 2019, 320, 134544. [Cross n platinum free dye sensitized solar cells. Electrochim. Acta 2019, 320, 134544. [CrossRef] p
y
4. Rahman, S.U.; Bilal, S.; ul Haq Ali Shah, A. Synthesis and Characterization of Polyaniline-Chito 4. Rahman, S.U.; Bilal, S.; ul Haq Ali Shah, A. Synthesis and Characterization of Polyaniline-Chitosan Patches with Enhanced
Stability in Physiological Conditions. Polymers 2020, 12, 2870. [CrossRef] ,
;
,
;
q
,
y
y
Stability in Physiological Conditions. Polymers 2020, 12, 2870. [CrossRef] in Physiological Conditions. Polymers 2020, 12, 2870. [C Stability in Physiological Conditions. Polymers 2020, 12 y
y
g
5. Rahman, S.U.; Röse, P.; Surati, M.; Shah, A.U.H.A.; Krewer, U.; Bilal, S. 3D Polyaniline Nanofibers Anch 5. Rahman, S.U.; Röse, P.; Surati, M.; Shah, A.U.H.A.; Krewer, U.; Bilal, S. 3D Polyaniline Nanofibers Anchored on Carbon Paper for
High-Performance and Light-Weight Supercapacitors. Polymers 2020, 12, 2705. [CrossRef] [PubMed]
6. ur Rahman, S.; Röse, P.; ul Haq Ali Shah, A.; Krewer, U.; Bilal, S. An Amazingly Simple, Fast and Green Synthesis Route to
Polyaniline Nanofibers for Efficient Energy Storage. Polymers 2020, 12, 2212. [CrossRef] High-Performance and Light-Weight Supercapacitors. Polymers 2020, 12, 2705. [CrossRef] [PubMed] g
g
g
p
p
y
[
] [
]
6. ur Rahman, S.; Röse, P.; ul Haq Ali Shah, A.; Krewer, U.; Bilal, S. An Amazingly Simple, Fast and Green Synthesis Route to
Polyaniline Nanofibers for Efficient Energy Storage. f
y
g
p
10.
Jiang, X.; Xu, C.; Gao, T.; Bando, Y.; Golberg, D.; Dai, P.; Hu, M.; Ma, R.; Hu, Z.; Wang, X.-B. Flexible conductive polymer
composite materials based on strutted graphene foam. Compos. Commun. 2021, 25, 100757. [CrossRef] References Polymers 2020, 12, 2212. [CrossRef] 6. ur Rahman, S.; Röse, P.; ul Haq Ali Shah, A.; Krewer, U.; Bilal, S. An Amazingly Simple, Fast and G q
g y
p
Polyaniline Nanofibers for Efficient Energy Storage. Polymers 2020, 12, 2212. [CrossRef] 7. Ur Rahman, S.; Röse, P.; ul Haq Ali Shah, A.; Krewer, U.; Bilal, S.; Farooq, S. Exploring the Functional Properties of Sodium
Phytate Doped Polyaniline Nanofibers Modified FTO Electrodes for High-Performance Binder Free Symmetric Supercapacitors. Polymers 2021, 13, 2329. [CrossRef] Ahmad, S.M.; Ali Shah, A.-H.; Bilal, S.; Tahir, A.A.; Ayub, K. Molecular and Electronic Structure Elucidation of
nsors. J. Phys. Chem. C 2015, 119, 15994–16003. [CrossRef] 9. Zia, T.U.H.; Ali Shah, A.U.H. Understanding the adsorption of 1 NLB antibody on polyaniline nanotubes as a function of zeta
potential and surface charge density for detection of hepatitis C core antigen: A label-free impedimetric immunosensor. Colloids
Surfaces A Physicochem. Eng. Asp. 2021, 626, 127076. [CrossRef] f
y
g
p
10. Jiang, X.; Xu, C.; Gao, T.; Bando, Y.; Golberg, D.; Dai, P.; Hu, M.; Ma, R.; Hu, Z.; Wang, X.-B. Flexible conductive polymer
composite materials based on strutted graphene foam. Compos. Commun. 2021, 25, 100757. [CrossRef] 64 Polymers 2021, 13, 2819 12. Mohamadzade, B.; Hashmi, R.M.; Simorangkir, R.B.V.B.; Gharaei, R.; Ur Rehman, S.; Abbasi, Q.H. Recent Advances in Fabrication
Methods for Flexible Antennas in Wearable Devices: State of the Art. Sensors 2019, 19, 2312. [CrossRef] [
]
13. Jamshaid, H.; Mishra, R.; Zeeshan, M.; Zahid, B.; Basra, S.A.; Tichy, M.; Muller, M. Mechanical Performance of Knitted Hollow
Composites from Recycled Cotton and Glass Fibers for Packaging Applications. Polymers 2021, 13, 2381. [CrossRef] [PubMed] 13. Jamshaid, H.; Mishra, R.; Zeeshan, M.; Zahid, B.; Basra, S.A.; Tichy, M.; Muller, M. Mechanical Performance of Knitted Hollow
Composites from Recycled Cotton and Glass Fibers for Packaging Applications Polymers 2021 13 2381 [CrossRef] [PubMed] 13. Jamshaid, H.; Mishra, R.; Zeeshan, M.; Zahid, B.; Basra, S.A.; Tichy, M.; Muller, M. Mechanical Performance of Knitted Hollow J
y
Composites from Recycled Cotton and Glass Fibers for Packaging Applications. Polymers 2021, 13, 238 14. Commission, F.C. FCC Report and Order for Part 15 Acceptance of Ultra Wideband (UWB) Systems from 3.1–10.6 GHz; FCC: Washington,
DC, USA, 2002; pp. 1–10. 15. Samal, P.B.; Soh, P.J.; Zakaria, Z. Compact Microstrip-Based Textile Antenna for 802.15.6 WBAN-UWB with Full Ground Plane. Int. J. Antennas Propag. 2019, 2019, 1–12. [CrossRef] p g
16. References Gain Enhancement of Wideband Metamaterial-Loaded
T h l
C
l d A
Sh
d D
IEEE T
A
P
65 2090 2095 [C
R f] 26. Ahdi Rezaeieh, S.; Antoniades, M.A.; Abbosh, A.M. Gain Enhancement of Wideband Metamaterial-
Tightly Coupled Arc-Shaped Directors. IEEE Trans. Antennas Propag. 2017, 65, 2090–2095. [CrossRe Tightly Coupled Arc-Shaped Directors. IEEE Trans. Antennas Propag. 2017, 65, 2090–2095. [CrossRef] Coupled Arc-Shaped Directors. IEEE Trans. Antennas Propag. 2017, 65, 2090–2095. [CrossRef] g
y
p
p
p g
27. Islam, M.M.T.; Islam, M.M.T.; Samsuzzaman, M.; Faruque, M.R.I. Compact metamaterial antenna for UWB applications. Electron. Lett. 2015, 51, 1222–1224. [CrossRef] ,
,
[
]
28. Al-Bawri, S.S.; Hwang Goh, H.; Islam, M.S.; Wong, H.Y.; Jamlos, M.F.; Narbudowiczm, A.; Jusoh, M.; Sabapathy, T.; Khan, R.;
I l
M T C
t Ult
Wid b
d M
l A t
L
d d
ith M t
t
i l S
2020 20 796 [C
R f] [P bM d] 28. Al-Bawri, S.S.; Hwang Goh, H.; Islam, M.S.; Wong, H.Y.; Jamlos, M.F.; Narbudowiczm, A.; Jusoh, M.; Sabapathy, T.; Khan, R.;
Islam, M.T. Compact Ultra-Wideband Monopole Antenna Loaded with Metamaterial. Sensors 2020, 20, 796. [CrossRef] [PubMed] 29. Khandelwal, M.K.; Arora, A.; Kumar, S.; Kim, K.W.; Choi, H.C. Dual band double negative (DNG) metamaterial with small
frequency ratio. J. Electromagn. Waves Appl. 2018, 32, 2167–2181. [CrossRef] 29. Khandelwal, M.K.; Arora, A.; Kumar, S.; Kim, K.W.; Choi, H.C. Dual band dou
frequency ratio. J. Electromagn. Waves Appl. 2018, 32, 2167–2181. [CrossRef] 30. Singh, H.; Sohi, B.S.; Gupta, A. A compact CRLH metamaterial with wide band negative index characteristics. Bull. Mater. Sci. 2019, 42, 1–11. [CrossRef] ruque, M.R.; Islam, M.T. Design and absorption analysis of a new multiband split-S-shaped metamaterial. Sci. 2017, 24, 139–148. [CrossRef] 31. Islam, S.S.; Iqbal Faruque, M.R.; Islam, M.T. Design and absorption analysis of a new multiband split-S-s
Eng. Compos. Mater. 2017, 24, 139–148. [CrossRef] g
p
32. Hossain, K.; Sabapathy, T.; Jusoh, M.; Soh, P.J.; Osman, M.N.; Al-Bawri, S.S. A Compact Wideband CSRR Near Zero Refractive
Index and Epsilon Negative Metamaterial for Wearable Microwave Applications. J. Phys. Conf. Ser. 2021, 1962, 012019. [CrossRef]
33
Alemaryeen A ; Noghanian S Crumpling effects and specific absorption rates of flexible AMC integrated antennas IET Microw 32. Hossain, K.; Sabapathy, T.; Jusoh, M.; Soh, P.J.; Osman, M.N.; Al-Bawri, S.S. A Compact Wideband CSRR Near Zero Refractive
Index and Epsilon Negative Metamaterial for Wearable Microwave Applications. J. References Samal, P.B.; Soh, P.J.; Xu, H.; Vandenbosch, G.A.E. Microstrip-based all-textile unidirectional UWB antenna with full ground
plane. In Proceedings of the 8th European Conference on Antennas and Propagation, EuCAP 2014, Hague, The Netherlands,
6–11 April 2014; pp. 1408–1412. [CrossRef] p
pp
[
]
17. Yalduz, H.; Koç, B.; Kuzu, L.; Turkmen, M. An ultra-wide band low-SAR flexible metasurface-enabled antenna for WBAN
applications. Appl. Phys. A Mater. Sci. Process. 2019, 125, 1–11. [CrossRef] pp
pp
y
18. Gao, G.P.; Hu, B.; Wang, S.F.; Yang, C. Wearable Circular Ring Slot Antenna with EBG Structure for Wire
IEEE Antennas Wirel. Propag. Lett. 2018, 17, 434–437. [CrossRef] e
as
e
opag
e
0 8,
, 3
3
[C oss e ]
19. Tian, X.; Lee, P.M.; Tan, Y.J.; Wu, T.L.Y.; Yao, H.; Zhang, M.; Li, Z.; Ng, K.A.; Tee, B.C.K.; Ho, J.S. Wireless body sensor networks
based on metamaterial textiles. Nat. Electron. 2019, 2, 243–251. [CrossRef] 19. Tian, X.; Lee, P.M.; Tan, Y.J.; Wu, T.L.Y.; Yao, H.; Zhang, M.; Li, Z.; Ng, K.A.; Tee, B.C.K.; Ho, J.S. Wirele
based on metamaterial textiles. Nat. Electron. 2019, 2, 243–251. [CrossRef] 20. Moradi, B.; Fernández-García, R.; Gil, I. E-Textile Embroidered Metamaterial Transmission Line for Signal Propagation Control. Materials 2018, 11, 955. [CrossRef] 21. Zhang, K.; Soh, P.J.; Yan, S. Meta-Wearable Antennas—A Review of Metamaterial Based Antennas in Wireless Body Area
Networks. Materials 2020, 14, 149. [CrossRef] 22. Wu, J.F.; Qiu, C.; Wang, Y.; Zhao, R.; Cai, Z.P.; Zhao, X.G.; He, S.S.; Wang, F.; Wang, Q.; Li, J.Q. Human limb motion detection with
novel flexible capacitive angle sensor based on conductive textile. Electronics 2018, 7, 192. [CrossRef] 23. Tartare, G.; Zeng, X.; Koehl, L. Development of a wearable system for monitoring the firefighter’s physiological state. In
Proceedings of the 2018 IEEE Industrial Cyber-Physical Systems ICPS 2018, St. Petersburg, Russia, 15–18 May 2018; pp. 561–566. [CrossRef] 24. Saenz-Cogollo, J.F.; Pau, M.; Fraboni, B.; Bonfiglio, A. Pressure mapping mat for tele-home care applications. Sensors 2016, 16, 365. [CrossRef] 25. Gric, T.; Hess, O. Metamaterial Cloaking. In Phenomena of Optical Metamaterials; Gric, T., Hess, O., Eds.; Elsevier: Amsterdam, The
Netherlands, 2019; pp. 175–186. ISBN 9780128138960. S.; Antoniades, M.A.; Abbosh, A.M. Gain Enhancement of Wideband Metamaterial-Loaded Loop Antenna w
d Arc-Shaped Directors. IEEE Trans. Antennas Propag. 2017, 65, 2090–2095. [CrossRef] Ahdi Rezaeieh, S.; Antoniades, M.A.; Abbosh, A.M. 37.
Wang, F.; Arslan, T. A wearable ultra-wideband monopole antenna with flexible artificial magnetic conductor. In Proceedings of
the 2016 Loughborough Antennas & Propagation Conference LAPC 2016, Loughborough, UK, 14–15 November 2016; pp. 3–7.
[CrossRef] References Sabapathy, T.; Soh, P.J.; Jusoh, M. A Three-Year Improvement Assessment of Project-Based Learn
Propagation Course [Education Corner]. IEEE Antennas Propag. Mag. 2020, 62, 76–84. [CrossRef] p g
p g
g
42. Hossain, T.M.; Jamlos, M.A.F.; Jamlos, M.A.F.; Soh, P.J.; Islam, M.I.; Khan, R. Modified H-shaped DNG metamaterial for multiband
microwave application. Appl. Phys. A Mater. Sci. Process. 2018, 124, 183. [CrossRef] 42. Hossain, T.M.; Jamlos, M.A.F.; Jamlos, M.A.F.; Soh, P.J.; Islam, M.I.; Khan, R. Modified H
microwave application. Appl. Phys. A Mater. Sci. Process. 2018, 124, 183. [CrossRef] 43. Baena, J.D.; Bonache, J.; Martín, F.; Sillero, R.M.; Falcone, F.; Lopetegi, T.; Laso, M.A.G.; García-García, J.; Gil, I.; Portillo, M.F.; et al. Equivalent-circuit models for split-ring resonators and complementary split-ring resonators coupled to planar transmission lines. IEEE Trans. Microw. Theory Tech. 2005, 53, 1451–1460. [CrossRef] y
44. Dong, Y.; Toyao, H.; Itoh, T. Design and characterization of miniaturized patch antennas loaded with complementary split-ring
resonators. IEEE Trans. Antennas Propag. 2012, 60, 772–785. [CrossRef] p g
[
]
45. Chen, X.; Grzegorczyk, T.M.; Wu, B.I.; Pacheco, J.; Kong, J.A. Robust method to retrieve the constitutive effective parameters of
metamaterials. Phys. Rev. E Stat. Physics Plasmas Fluids Relat. Interdiscip. Top. 2004, 70, 7. [CrossRef] 46. Islam, S.S.; Faruque, M.R.I.; Islam, M.T. A new direct retrieval method of refractive index for the metamaterial. Curr. Sci. 2015,
109, 337–342. 47. Hossain, K.; Sabapathy, T.; Jusoh, M.; Soh, P.J.; Fazilah, A.F.M.; Halim, A.A.A.; Raghava, N.S.; Podilchak, S.K.; Schreurs, D.;
Abbasi, Q.H. ENG and NZRI Characteristics of Decagonal-Shaped Metamaterial for Wearable Applications. In Proceedings of the
2020 International Conference on UK-China Emerging Technologies (UCET), Glasgow, UK, 20–21 August 2020; pp. 1–4. nal Conference on UK-China Emerging Technologies (UCET), Glasgow, UK, 20–21 August 2020; pp. 1–4. g
g
g
g
g
pp
48. Moradikordalivand, A.; Rahman, T.A.; Ebrahimi, S.; Hakimi, S. An equivalent circuit model for broadband modified rectangular
microstrip-fed monopole antenna. Wirel. Pers. Commun. 2014, 77, 1363–1375. [CrossRef] 49. Wang, Y.; Li, J.Z.; Ran, L.X. An equivalent circuit modeling method for ultra-wideband antennas. Prog. Electromagn. Res. 2008, 82,
433–445. [CrossRef] 50. Balanis, C.A. Antenna Theory: Analysis and Design; John Wiley & Sons, 2016; ISBN 1118642066. 51. Navarro-Cía, M.; Beruete, M.; Agrafiotis, S.; Falcone, F.; Sorolla, M.; Maier, S.A. Broadband spoof plasmons and subwavelength
electromagnetic energy confinement on ultrathin metafilms. Opt. Express 2009, 17, 18184. [CrossRef] [PubMed] 51. References Phys. Conf. Ser. 2021, 1962, 012019. [CrossRef]
33. Alemaryeen, A.; Noghanian, S. Crumpling effects and specific absorption rates of flexible AMC integrated antennas. IET Microw. Antennas Propag. 2018, 12, 627–635. [CrossRef] p
g
pp
J
y
f
[
]
33. Alemaryeen, A.; Noghanian, S. Crumpling effects and specific absorption rates of flexible AMC integrated antennas. IET Microw. Antennas Propag. 2018, 12, 627–635. [CrossRef] 34. Mersani, A.; Osman, L.; Ribero, J.M. Flexible UWB AMC antenna for early stage skin cancer identification. Prog. Electromagn. Res. M 2019, 80, 71–81. [CrossRef] 35. Islam, M.M.; Islam, M.T.; Faruque, M.R.I.; Samsuzzaman, M.; Misran, N.; Arshad, H. Microwave imaging sensor using compact
metamaterial UWB antenna with a high correlation factor. Materials 2015, 8, 4631–4651. [CrossRef] Sabapathy, T.; Jusoh, M.; Soh, P.J.; Jamaluddin, M.H.; Al-Bawri, S.S.; Osman, M.N.; Ahmad, R.B.; Rahim, H.A. 36. Hossain, K.; Sabapathy, T.; Jusoh, M.; Soh, P.J.; Jamaluddin, M.H.; Al-Bawri, S.S.; Osman, M.N.; Ahmad, R.B.; Rahim, H.A.; Mohd
Yasin, M.N.; et al. Electrically Tunable Left-Handed Textile Metamaterial for Microwave Applications. Materials 2021, 14, 1274. [CrossRef] p
y
Yasin, M.N.; et al. Electrically Tunable Left-Handed Textile Metamaterial for Microwave Applications. Materials 2021, 14, 1274. [CrossRef] 37. Wang, F.; Arslan, T. A wearable ultra-wideband monopole antenna with flexible artificial magnetic conductor. In Proceedings of
the 2016 Loughborough Antennas & Propagation Conference LAPC 2016, Loughborough, UK, 14–15 November 2016; pp. 3–7. [CrossRef] 37. Wang, F.; Arslan, T. A wearable ultra-wideband monopole antenna with flexible artificial magnetic conductor. In Proceedings of
the 2016 Loughborough Antennas & Propagation Conference LAPC 2016, Loughborough, UK, 14–15 November 2016; pp. 3–7. [CrossRef] 65 Polymers 2021, 13, 2819 38. Gogikar, S.; Chilukuri, S. A Compact Wearable Textile Antenna with Dual Band-Notched Characteristics for UWB Applications. In Proceedings of the 2019 9th IEEE-APS Topical Conference on Antennas and Propagation in Wireless Communications APWC
2019, Granada, Spain, 9–13 September 2019; 94, pp. 426–430. [CrossRef] p
p
pp
39. Elwi, T.A. Novel UWB printed metamaterial microstrip antenna based organic substrates for RF-energy harvesting applications. AEU Int. J. Electron. Commun. 2019, 101, 44–53. [CrossRef] 40. Negi, D.; Khanna, R.; Kaur, J. Design and performance analysis of a conformal CPW fed wideband
metamaterial for wearable applications. Int. J. Microw. Wirel. Technol. 2019, 11, 806–820. [CrossRef] pp
41. Sabapathy, T.; Soh, P.J.; Jusoh, M. A Three-Year Improvement Assessment of Project-Based Learning for an Antennas and
Propagation Course [Education Corner]. IEEE Antennas Propag. Mag. 2020, 62, 76–84. [CrossRef] 41. Tarek Abou Elmaaty 1,*, Hanan G. Elsisi 2, Ghada M. Elsayad 3, Hagar H. Elhadad 3, Khaled Sayed-Ahmed 4
and Maria Rosaria Plutino 5 Tarek Abou Elmaaty 1,*, Hanan G. Elsisi 2, Ghada M. Elsayad 3, Hagar H. Elhadad 3, Khaled Sayed-Ahmed 4
and Maria Rosaria Plutino 5 1
Department of Material Art, Galala University, Galala 43713, Egypt 2
Department of Textile Printing, Dyeing & Finishing, Faculty of Applied Art, Damietta University,
Damietta 34512, Egypt; hanan.gamal58@yahoo.com 2
Department of Textile Printing, Dyeing & Finishing, Faculty of Applied Art, Damietta University,
Damietta 34512, Egypt; hanan.gamal58@yahoo.com p
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Damietta 34512, Egypt; hanan.gamal58@yahoo.com gyp
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3
Department of Spinning, Weaving and Knitting, Faculty of Applied Art, Damietta University,
Damietta 34512, Egypt; drghada3rm@yahoo.com (G.M.E.); hagarelhadad9@gmail.com (H.H.E.) gyp
g
y
g
g
4
Department of Agricultural Chemistry, Faculty of Agriculture, Damietta University, Damietta 34512, Egypt
dr_khaled@yahoo.com y
5
Stituto per lo Studio dei Materiali Nanostrutturati, Consiglio Nazionale delle Ricerche, Vill. S. Agata,
98166 Messina Italy; plutino@pa ismn cnr it 5
Stituto per lo Studio dei Materiali Nanostrutturati, Consiglio Nazionale delle Ricerche, Vill. S. Agata,
98166 Messina, Italy; plutino@pa.ismn.cnr.it *
Correspondence: tasaid@gu.edu.eg Abstract: This study aims to develop multifunctional pile cotton fabrics by implementing different
compositions of lycra yarns with different densities of the cotton fabric under study. Highly dispersed
silica nanoparticles (SiO2 NPs) with small sizes—in the range of 10–40 nm—were successfully pre-
pared and were analyzed using scanning electron microscopy (SEM). The particle size distribution of
nano silica was determined via dynamic laser scattering (DLS) and measurements of its zeta potential. Cotton/lycra fabrics were treated using prepared SiO2 NPs in presence of ethylenediaminetetraacetic
acid (EDTA) as a crosslinking agent. Energy dispersive X-ray (EDX) analysis and scanning electron
microscopy (SEM) were used to characterize the nano-treated fabrics and assure homogeneous dis-
persion of SiO2 NPs on the cotton/lycra composites. Additionally, the nanoparticles were screened
for their in vitro antibacterial activity against human pathogens such as Gram-positive Staphylococcus
aureus and Bacillus cereus and Gram-negative Escherichia coli and Pseudomonas aeruginosa strains. The
functional properties of the new composite pile cotton fabrics include excellent antibacterial, highly
self-cleaning, and excellent UV protection factor (UPF) properties. Citation: Abou Elmaaty, T.; Elsisi,
H.G.; Elsayad, G.M.; Elhadad, H.H.;
Sayed-Ahmed, K.; Plutino, M.R. Fabrication of New Multifunctional
Cotton/Lycra Composites Protective
Textiles through Deposition of Nano
Silica Coating. Polymers 2021, 13, 2888. https://doi.org/10.3390/
polym13172888 Keywords: cotton/lycra composites; silica nanoparticles; antibacterial activity; UV protection;
self-cleaning Academic Editor: Giulio Malucelli Academic Editor: Giulio Malucelli Citation: Abou Elmaaty, T.; Elsisi,
H.G.; Elsayad, G.M.; Elhadad, H.H.;
Sayed-Ahmed, K.; Plutino, M.R.
Fabrication of New Multifunctional
Cotton/Lycra Composites Protective
Textiles through Deposition of Nano
Silica Coating. Polymers 2021, 13, 2888.
https://doi.org/10.3390/
polym13172888 References Navarro Cía, M.; Beruete, M.; Agrafiotis, S.; Falcone, F.; Sorolla, M.; Maier, S.A. Broadband spoof plasmons and subwavelength
electromagnetic energy confinement on ultrathin metafilms. Opt. Express 2009, 17, 18184. [CrossRef] [PubMed] 52. Huang, X.; Leng, T.; Zhang, X.; Chen, J.C.; Chang, K.H.; Geim, A.K.; Novoselov, K.S.; Hu, Z. Binder-free highly conductiv
graphene laminate for low cost printed radio frequency applications. Appl. Phys. Lett. 2015, 106, 203105. [CrossRef] g
g
g
J
g
g
y
graphene laminate for low cost printed radio frequency applications. Appl. Phys. Lett. 2015, 106, 203105. [CrossRef] 53. Sayem, A.S.M.; Simorangkir, R.B.V.B.; Esselle, K.P.; Hashmi, R.M. Development of Robust Transparent Conformal Antennas
Based on Conductive Mesh-Polymer Composite for Unobtrusive Wearable Applications. IEEE Trans. Antennas Propag. 2019, 67,
7216–7224. [CrossRef] 54. Corchia, L.; Monti, G.; Tarricone, L. Durability of Wearable Antennas Based on Nonwoven Conductive Fabrics: Experimental
Study on Resistance to Washing and Ironing. Int. J. Antennas Propag. 2018, 2018, 1–8. [CrossRef] y
g
g
J
p g
55. Guo, Z.; Tian, H.; Wang, X.; Luo, Q.; Ji, Y. Bandwidth enhancement of monopole uwb antenna with
IEEE Antennas Wirel. Propag. Lett. 2013, 12, 1550–1553. [CrossRef] y
g
g
p g
55. Guo, Z.; Tian, H.; Wang, X.; Luo, Q.; Ji, Y. Bandwidth enhancement o
IEEE Antennas Wirel. Propag. Lett. 2013, 12, 1550–1553. [CrossRef] 56. Bialkowski, M.E.; Razali, A.R.; Boldaji, A. Design of an ultrawideband monopole antenna for portable radio transceiver. IEEE
Antennas Wirel. Propag. Lett. 2010, 9, 554–557. [CrossRef] 66 66 polymers polymers polymers Article
Fabrication of New Multifunctional Cotton/Lycra Composites
Protective Textiles through Deposition of Nano Silica Coating maaty 1,*, Hanan G. Elsisi 2, Ghada M. Elsayad 3, Hagar H. Elhadad 3, Khaled Sayed-Ahmed 4
aria Plutino 5 1. Introduction Textiles are fundamental to a country’s development and industrialization. As the
demand for modern functional textiles grows, new materials and technology are being
used. New multifunctional protective and smart textiles have been developed in response
to growing technical breakthroughs, new standards, and a customer demand for textiles
that are not only attractive but also practical. As a result, high-tech materials and well-
considered fabric constructions will enhance wearer comfort while also providing unique
features [1]. Textile products made from natural fibers such as cotton are good carriers of
a variety of bacteria, which can cause health issues to the wearer [2]. Some of the most
serious problems are “skin diseases,” which can be developed by wearing contaminated
clothes over a short time with the cross-transmission of bacteria found in air and on ground
surfaces. Additionally, presence of these bacteria on textiles can result in undesirable
damage to the fabric, such as fading, staining, a reduction in mechanical characteristics,
and the material’s deterioration [3]. Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article
distributed
under
the
terms
and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). Cotton is a widely used raw material in the manufacture of many fabrics, especially
pile fabrics with many functional properties, such as water absorbency, humidity, durability 67 https://www.mdpi.com/journal/polymers Polymers 2021, 13, 2888. https://doi.org/10.3390/polym13172888 Polymers 2021, 13, 2888 when wet, and acceptable properties of skin friction, as well as the ability to withstand
stress caused by washing and regular use [4]. Pile fabrics have a brush-like surface, which
is created by tufts of warp or weft cut threads. A series of threads that protrude at right
angles from a foundation or ground structure and form a pile or loop on the surface create
the brush-like surface. It differs from other fabrics in regard to the surface texture (loops
or cut ends) due to the extra warp and weft threads that appear at a certain height on
the surface of the fabric according to the purpose of use, and the pile of these textiles
comprises three threads [5]. 1. Introduction Pile fabrics are distinguished from other textiles by their ability
to illustrate and confirm the functional trends of the fabric’s third dimension, represented
by the thickness and height of the pile [6]. Obtaining the pile fabrics constitutes the greatest
and most common part of the production of warp pile fabrics. These fabrics consist of
two systems of warp threads for weaving warp pile fabrics (pile and ground warp), and
one system of weft threads. There are methods of producing warp pile fabrics, including
wire pile structures, that are used in upholstery, apparel wear, medical fabrics, ihram outfit
clothes, curtains, etc. However, the use of pure pile cotton fabrics does not achieve the required purpose of
comfort and resistance to bacteria, which affects the performance of fabrics, attracts dust,
and causes skin problems. Ahmed et al., 2020 [7] endeavored to solve the problems encoun-
tered by cotton fabrics using a facile fabrication of multifunctional cotton–modal–recycled
aramid blended with protective textiles through the deposition of a three-dimensional
tetrakis(hydroxymethyl)phosphonium chloride (THPC)-urea polymer coating. The results
exhibited high antibacterial properties and superior water repellency. g
y
On the other hand, synthetic fibers that can be used in the manufacture of pile fabrics
such as lycra (Spandex) are seldom used alone. It is stretchy since it is a combination of
nylon, cotton, or other fabrics [8]. Further, the use of lycra in woven fabrics provides a
superior fit on the body, acting as a second skin, with good form retention and no distortion
over the garment’s lifetime. Badr 2017 [2] investigated the influence of fabric structure
(Rib 4:1 Plain)/lycra combination on the antibacterial and mechanical properties. The
findings revealed that the material type and fabric structure had an impact on the survival
of Escherichia coli and staphylococcus aureus in socks. Lycra’s further characteristics include
less moisture absorption and resistance to both sunlight and industrial chemicals. The most
common option among the textile is a combination of lycra yarn with different densities
to impart a considerable level of stretch and recovery and to improve comfort due to its
capacity to stretch, beyond that which can be achieved by cotton alone [9]. The elastomeric
properties of a lycra and cotton blend are crucial in determining the elastic product’s
end-use [10,11]. Hence, we have focused on improving the functional performance of
cotton fabrics by incorporating lycra yarns with different densities. 1. Introduction Performance apparel is exposed to a wide range of external conditions, including sun-
light. Different factors promote the growth of melanoma and non-melanoma skin cancers,
with UV radiation exposure being one of the most important. As a result, researchers have
focused their efforts on altering fabric qualities as a layer to protect the skin from damaging
radiation. Modifying the surface of fabrics to protect against UV radiation is crucial [12,13]. The relevance of self-cleaning arises when the fabric is continually exposed to dust and
there is not enough time to wash them. One of the advantages of self-clean finishing is the
elimination of traditional laundry procedures [14]. Nanotechnology has been successfully applied to various commercial products. It has
also gained attention in the textile industry. Currently, it has been used in the processing
and finishing of textiles to impart functional benefits [15]. Polymeric nanostructures, metal
oxides, carbon nanotubes, clay nanoparticles (NPs), carbon black, graphite nanofibers, and
other nanomaterials provide unprecedented textile performance, such as being hydrophilic
and hydrophobic, antistatic, wrinkle-resistant, antimicrobial, antiodor, self-cleaning, and
antiUV [16–21]. Inorganic NPs, especially TiO2, ZnO, SiO2, Cu2O, CuO, Al2O3, and
reduced graphene oxide NPs, are more commonly used than organic NPs due to their
thermal and chemical durability at high temperatures, permanent stability under UV rays, 68 Polymers 2021, 13, 2888 and non-toxicity [22,23]. Nano silica has been proven to be a promising material due to its
low density and good mechanical stability. Silica (SiO2NPs) penetrate easily into a cotton
fiber’s interior and attach to the fiber structure tightly. As a result, the hydroxyl group of
cellulose and SiOH in SiO2NPs form a covalent bond [24–26]. This has recently been a
significant research topic in both the scientific and industrial sectors. The addition of SiO2
NPs to materials’ surfaces increases their mechanical properties and durability, as well as
influences their function, activity, and stability [27–29]. This study aims to examine the influence of both different fabric structure and imple-
mentation lycra yarns on the functional properties of the fabrics, as well as the treatment
of the new fabric structure with highly dispersed SiO2 NPs. During these processes,
ethylenediaminetetraacetic acid (EDTA) was used as a crosslinking agent. It has numerous
advantages: it is low cost, locally available, non-toxic, and provides the binding between
SiO2 NPs and cotton/lycra composites [23,30]. Table 2. Specifications of the machine used for preparing samples. Table 2. Specifications of the machine used for preparing samples. Type of Loom
Picanol Nova 600
Manufacturing Country
Italy
Date of Manufacturing
1996
Reed count
12 dent/cm
Denting
2 Ends/Dent
Weft Insertion Device
Rapiers 1. Introduction The SiO2 treated cotton/lycra composites
were also characterized, and their UV protective properties, antibacterial activities, and
self-cleaning qualities were measured. 2. Materials and Methods
2.1. Fabric The cotton fabric (100%), cotton/lycra (90.8/9.2%), and cotton/lycra (95.5/5.5%)
employed have specifications given in Table 1. The weave structure used to prepare the
samples was plain weave 1/2. The fabrics were prepared at (Cotton MISR Inc; Almahalla
Al-Kubra, Egypt) using an electronic jacquard loom with the specification shown in Table 2. The samples were cleaned for 1 h at a boil with 2 g/L soap and 2 g/L sodium carbonate
(Loba Chemie Pvt. Ltd., Boisar, India). The samples were washed in distilled water and
air-dried. Table 1. Specification of the prepared samples. Table 1. Specification of the prepared samples. Fabric Specification
Material
Density/cm
Count (Ne)
Application Method
Warp specification
For ground warp: cotton is used for
nine samples
For Pile warp: cotton is used for
nine samples
24 ends/cm (12 ground
ends+12 pile ends)
24/2 Ne for both
ground and pile
warp ends
Wire pile
Weft Specification
Cotton (100%): for samples (A, B, and C)
Cotton/Lycra (90.8/9.2%): for samples (D,
E, and F)
Cotton/Lycra (95.5/5.5%): for samples (G,
H, and I)
(15, 19, 22) picks/cm
16/1 Ne
Table 2. Specifications of the machine used for preparing samples. Type of Loom
Picanol Nova 600
Manufacturing Country
Italy
Date of Manufacturing
1996
Reed count
12 dent/cm
Denting
2 Ends/Dent
Weft Insertion Device
Rapiers Table 1. Specification of the prepared samples. Fabric Specification
Material
Density/cm
Count (Ne)
Application Method
Warp specification
For ground warp: cotton is used for
nine samples
For Pile warp: cotton is used for
nine samples
24 ends/cm (12 ground
ends+12 pile ends)
24/2 Ne for both
ground and pile
warp ends
Wire pile
Weft Specification
Cotton (100%): for samples (A, B, and C)
Cotton/Lycra (90.8/9.2%): for samples (D,
E, and F)
Cotton/Lycra (95.5/5.5%): for samples (G,
H, and I)
(15, 19, 22) picks/cm
16/1 Ne 2.2. Chemicals Sodium metasilicate (Na2SiO3.9H2O, 95%, Merk KGaA, Darmstadt, Germany), cetyl
trimethyl ammonium bromide (CTAB) (C19H42BrN, 98%, Loba Chemie Pvt. Ltd., Boisar,
India), (HCl) (37%, Merk KGaA, Darmstadt, Germany), ethylene diamine tetra acetic acid
(EDTA, Merk KGaA, Darmstadt, Germany), dihydrate, (C10H14N2Na2O8.2H2O, 99.5%, J.T. Baker, Phillipsburg, NJ, USA), silver nitrate (AgNO3, Merk KGaA, Darmstadt, Germany),
and sodium chloride (NaCl, Merk KGaA, Darmstadt, Germany) were used in pure forms. 69 Polymers 2021, 13, 2888 2.3. Preparation of Silica Nanoparticles (SiO2 NPs) 2.3. Preparation of Silica Nanoparticles (SiO2 NPs) SiO2 NPs were prepared by adding 6 g sodium metasilicate to (394 mL) distilled water
in a flask. The temperature was adjusted to 55 ◦C under constant stirring at a 300 rpm speed. CTAB (4 g) was added with stirring until complete dissolution for 15 min. The diluted
HCl (200 mL) was added dropwise to the solution in two steps. In the first step, HCl was
added dropwise to the mixture until the pH reached 9–9.5. With no further addition of HCl,
the solution was agitated for another 10 min. More HCl was added until the pH reached
3–3.5 in the second step. Then, 10 mL of 10% NaCl aqueous solution was added to the
reaction mixture at the acquired pH and agitated for an additional 20 min. The precipitated
wet-gel silica was aged at 50 ◦C for 24 h, and the produced wet-gel silica was centrifuged
and rinsed with distilled water until a negative response for chloride ions was observed,
as measured with a 0.1 M AgNO3 solution. The gel was dried at 40 ◦C via a microwave
heating process. The total drying time was 70 min for each sample, applied seven times
in an intermittent interval of 10 min. The dried gel was calcined at 650 ◦C for 3 h in a
muffle furnace to obtain the white powder of nano SiO2 [31,32]. The obtained micrographs
showed that the SiO2 NPs size was in the range of 10–40 nm, whereas their shape was
spherical (Figure 1). Moreover, most SiO2 NPs had a uniform size, and no aggregation was
observed in scanning electron microscopy (SEM) analysis (JEOL JSM-6510LB with field
emission gun, Tokyo, Japan). On the other hand, IR spectra were obtained for KBr pellets
on a JASCO 410 spectrometer (Jasco, Tokyo, Japan) with only selected absorptions recorded
in the range of 2000–200 cm−1. Figure 2 shows that the peaks at 1020–1110 and 807 cm−1
are attributed to Si-O-Si a symmetric and symmetric stretching style, respectively [33]. Figure 1. SEM micrographs of the amorphous SiO2 NPs at different bar scales (a) 1 μm, and (b) 0.5 μm. Figure 1. SEM micrographs of the amorphous SiO2 NPs at different bar scales (a) 1 μm, and (b) 0.5 μm. Figure 2. FTIR spectra of the amorphous silica prepared with CTAB. 2.4. Treatment of Fabric with SiO2 NPs 2.4. Treatment of Fabric with SiO2 NPs The pad-dry-cure procedure was used to incorporate SiO2 NPs into the cotton/lycra
composites. EDTA (1 g, as a crosslinking agent) was added to 19 mL distilled water to
prepare a nano silica solution. Afterwards, the mixture was agitated with a magnetic stirrer. Drops of silica amounting to 0.2 g were added to the liquid and stirred constantly until
complete dissolution. The solution was sonicated for 30 min at room temperature. The
fabric was soaked in the solution for 60 min followed by drying in an oven at 100 ◦C until
completely dried, then cured at 120 ◦C for 60 min. 2.5. Characterization and Functional Properties of Fabrics Treated with Metal NPs 2.3. Preparation of Silica Nanoparticles (SiO2 NPs) 70 Polymers 2021, 13, 2888 Dynamic Light Scattering (DLS) and Zeta Potential Analysis Dynamic Light Scattering (DLS) and Zeta Potential Analysis Zeta potential and dynamic light scattering (DLS) were measured using a Malvern
Zetasize Nano-zs90. As shown in Figure 3, DLS analysis confirmed the preparation of SiO2
particles in nano scale at approximately 90 nm. The obtained size from the DLS analysis
is more than that obtained from SEM micrographs, which is caused by the formation of
hydrogen bonds between SiO2 NPs because of water addition [34]. Furthermore, the zeta
potential value of the colloidal SiO2 NPs solution was −19.4 mV with a single peak. Figure 3. Dynamic light scattering (DLS) (a) and zeta potential (b) of the obtained SiO2 NPs. Figure 3. Dynamic light scattering (DLS) (a) and zeta potential (b) of the obtained SiO2 NPs. 2.5.4. Self-Cleaning The blank and SiO2NP/cotton/lycra composites were tested for self-cleaning by dye-
ing in a methylene blue dye solution (0.2 g/L) for 5 min and then drying it off. A one-half
portion of each stain on the fabric was exposed to sunlight for 24 h to determine their color
strength (K/S), whereas the other half portion was covered with black paper to prevent
irradiation with sunlight [17]. The K/S value was evaluated using a Spectrophotometer
CM-3600A. According to the following equation, decreasing K/S values refer to the level
of dye degradation, which is stated as the self-cleaning capacity (SCC): (1) SCC = [(K/S) b −(K/S) a]/(K/S) b × 100
(1) SCC = [(K/S) b −(K/S) a]/(K/S) b × 100 where (K/S) a: color strength after exposing to daylight, and (K/S) b: color strength before. 2.5.1. Scanning Electron Microscopy (SEM) Analysis 2.5.1. Scanning Electron Microscopy (SEM) Analysis The surface morphologies of blank cotton/lycra composites and SiO2 NP/cotton/lycra
composite fabrics were characterized via scanning electron microscope (JEOL JSM-6510LB
with field emission gun, Tokyo, Japan). The deposition of SiO2 NPs into cotton/lycra composites was confirmed using a
surface energy dispersive X-ray (EDX) analysis unit (EDAX AMETEK analyzer, Mahwah,
NJ, USA) attached to an SEM device. On the other hand, the elemental mapping analysis
also reflects the presence of SiO2 NPs on the surface of the treated cotton/lycra composites. 71 Polymers 2021, 13, 2888 2.5.3. UV Protection Properties The blank and SiO2NP/cotton/lycra composites were examined for UV protection
functionality, expressed as UV protection factor (UPF), according to the Australian/New
Zealand standard (AS/NZS 4399: 1996), and rated as follows: excellent protection (UPF > 40),
very good protection (UPF: 25–39), and good protection (UPF: 15–24) [18,19]. 2.5.2. Antibacterial Activity The antibacterial activity of the blank and SiO2NP/cotton/lycra composite fabrics
against Gram-positive bacteria Staphylococcus aureus and Bacillus cereus and Gram-negative
bacteria Escherichia coli and Pseudomonas aeruginosa was assessed qualitatively using the
AATCC Test Method (147-1988) expressed as the zone of growth (mm) [18,35]. 3. Results and Discussion A new method for developing high performance functional pile cotton fabric was
designed. First, the functional performance of pile cotton fabrics was improved by in-
corporating a different percentage of lycra yarns with different densities according to the
following parameters: •
Weft material: Cotton/lycra of six samples: three samples (90.8% cotton and 9.2%
lycra), three samples (95.5% cotton and 5.5% lycra), and three cotton samples for the
standard comparative. p
•
Pile density: 15, 19, 22 picks/cm. •
Pile density: 15, 19, 22 picks/cm. Second, SiO2 NPs were successfully synthesized and then applied on samples under
study in a finishing bath containing an aqueous nano-SiO2 dispersion and EDTA. Statistical Analysis All the data of antibacterial activity and self-cleaning were analyzed using descriptive
statistics based on mean and standard deviation. 3.1. Characterization of Metal NPs Fabrics 3.1.1. SEM and EDX Analysis 3.1.1. SEM and EDX Analysis The SEM images of both untreated cotton/lycra (95.5/5.5%) fabric of 15 pick/cm,
cotton/lycra (90.8/9.2%) fabric of 19 pick/cm, and cotton (100%) fabric of 22 pick/cm
and cotton/lycra (95.5/5.5%) fabric of 15 pick/cm, cotton/lycra (90.8/9.2%) fabric of
19 pick/cm, and cotton (100%) fabric of 22 pick/cm treated with SiO2 NPs are shown
in Figure 4A–F, respectively. The results indicated that SiO2 NPs are well-distributed
on the surface of the fabric, and the presence of SiO2 NPs on the fabric surfaces was
confirmed from EDX data. The elemental EDX data collected for the fabrics under study
were presented in Figure 5. The peaks were allocated at 1.9 keV with 1.69% weight and
0.85% atomic absorption of the analyzed spot in the cotton (100%) fabric of 22 pick/cm,
and at 1.9 keV with 1.08% weight and 0.54% atomic absorption of the analyzed spot in
the cotton/lycra (95.5/5.5%) fabric of 15 pick/cm, as well as at 1.9 keV with 1.61% weight
and 0.81% atomic absorption of the analyzed spot in the cotton/lycra (90.8/9.2%) fabric of 72 Polymers 2021, 13, 2888 19 pick/cm fiber surface—characteristic of Si. The presence of these peaks confirmed that
the SiO2 NPs were entirely composed of SiO2. The C and O signals originated from the
cellulose polymer. Furthermore, the appearance of SiO2 NPs on treated fabrics were also
investigated by elemental mapping analysis, as illustrated in Figure 5E–G. It is apparent
from the mapping images that silicon was distributed well on the surface confirming the
uniform coating on cotton/lycra composites using SiO2 NPs as a sufficient layer. Figure 4. SEM images of (A–C) untreated samples. SEM images of cotton/lycra (95.5/5.5%) fabric of 15 pick/cm (D),
cotton/lycra (90.8/9.2%) fabric of 19 pick/cm (E), and cotton (100%) fabric of 22 pick/cm (F) treated with SiO2 NPs. Figure 4. SEM images of (A–C) untreated samples. SEM images of cotton/lycra (95.5/5.5%) fabric of 15 pick/cm (D),
cotton/lycra (90.8/9.2%) fabric of 19 pick/cm (E), and cotton (100%) fabric of 22 pick/cm (F) treated with SiO2 NPs. 3.1.2. Antibacterial Activity of Blank and SiO2-NP/Cotton/Lycra Composite Fabrics The antibacterial properties of treated and untreated samples are summarized in
Table 3. Ciprofloxacin is used as a standard to correlate the lead samples from the series
(4A–I). It is an antibiotic, causing the production of oxidative radicals and bacterial cell
death [36]. The results indicated that the untreated samples were not affected, and there
were no inhibition areas. 3.1.1. SEM and EDX Analysis On the other hand, the SiO2-NP/cotton/lycra composite fabrics
exhibited excellent antibacterial activities against S. aureus, Bacillus cereus, E. coli, and
P. aeruginosa compared with ciprofloxacin. This may be attributed to the fact that the mode
of action of nanoparticles (NPs) is a direct interaction with the bacterial cell wall without
the need to penetrate the cell. Most antibiotic resistance mechanisms can enhance the
immunity of bacteria causing a lesser response to the antibacterial agent [37]. Samples E
and G showed excellent activity against both Gram-positive and Gram-negative bacteria
among all treated samples as compared to untreated samples. However, Sample (B) showed
good activity against the four model bacteria. Samples (D) and (I) revealed good activity
against both Gram-positive and Gram-negative bacteria in comparison to sample (A),
which exhibited the lowest activity against Gram-negative (P. aeruginosa). Samples (F) and
(H) showed good activity against the four model bacteria. However, sample (C) exhibited
the lowest activity Gram-positive against (Bacillus cereus). In general, Samples (E) and (G)
exhibited significantly high antibacterial activity compared to the all other samples in the
series (4A–I) and with the standard. Additionally, the density of the pile 19 pick/cm is 73 Polymers 2021, 13, 2888 better than the other densities, as shown in Figure 6. This relies on the fact that there is
more trapped air inside this structure (19 pick/cm) than in other densities. This trapped
air helps in extending the zone of inhibition around the sample to 19 pick/cm, rendering
it more resistant against bacterial attack. SiO2 NPs’ extraordinary antibacterial activity is
attributed to their large surface area, which allows for better interaction with microbes [29]. Figure 5. EDX spectrum of (A) SEM image (B) cotton/lycra (95.5/5.5%) fabric of 15 pick/cm, (C) cotton/lycra (90.8/9.2%)
fabric of 19 pick/cm, and (D) cotton (100%) fabric of 22 pick/cm treated with SiO2 NPs, and EDX mapping of cotton/lycra
(90.8/9.2%) fabric of 19 pick/cm corresponding to (E) carbon, (F) oxygen, (G) silicon, and (H) overlap at 20 μm bar scale. Figure 5. EDX spectrum of (A) SEM image (B) cotton/lycra (95.5/5.5%) fabric of 15 pick/cm, (C) cotton/lycra (90.8/9.2%)
fabric of 19 pick/cm, and (D) cotton (100%) fabric of 22 pick/cm treated with SiO2 NPs, and EDX mapping of cotton/lycra
(90.8/9.2%) fabric of 19 pick/cm corresponding to (E) carbon, (F) oxygen, (G) silicon, and (H) overlap at 20 μm bar scale. Table 3. Antibacterial Activity of the Blank and SiO2NP/Cotton/Lycra Composites. 3.1.1. SEM and EDX Analysis Cotton (100%) 15 pick/cm (A), cotton
(100%) 19 pick/cm (B), Cotton (100%) 22 pick/cm (C), cotton/lycra (90.8/9.2%) 15 pick/cm (D), cotton/lycra (90.8/9.2%)
19 pick/cm (E), cotton/lycra (90.8/9.2%) 22 pick/cm (F), cotton/lycra (94.5/5.5%) 19 pick/cm (G), cotton/lycra (94.5/5.5%)
22 pick/cm (H), cotton/lycra (94.5/5.5%) 15 pick/cm (I). Table 3. Antibacterial Activity of the Blank and SiO2NP/Cotton/Lycra Composites. Cotton (100%) 15 pick/cm (A), cotton
(100%) 19 pick/cm (B), Cotton (100%) 22 pick/cm (C), cotton/lycra (90.8/9.2%) 15 pick/cm (D), cotton/lycra (90.8/9.2%)
19 pick/cm (E), cotton/lycra (90.8/9.2%) 22 pick/cm (F), cotton/lycra (94.5/5.5%) 19 pick/cm (G), cotton/lycra (94.5/5.5%)
22 pick/cm (H), cotton/lycra (94.5/5.5%) 15 pick/cm (I). Type of NPs
Sample No. Zone of Inhibition in mm
Gram Positive Bacteria
Gram Negative Bacteria
Mean
SD
S. aureus
Bacillus cereus
E. coli
P. aeruginosa
Untreated
1
0
0
0
0
0
0
Treated using SiO2 NPs
A
22
13
21
5
15.25
7.93
B
28
12
24
20
21
6.83
C
28
13
27
28
24
7.35
D
25
21
21
15
20.5
4.12
E
30
30
30
26
29
2.00
F
29
26
30
29
28.5
1.73
G
29
27
31
31
29.5
1.91
H
28
25
27
29
27.25
1.71
I
19
17
23
22
20.25
2.75
Control Ciprofloxacin
as antibiotic
J
21
22
26.7
23.3
23.25
2.49 74 74 Polymers 2021, 13, 2888 Figure 6. Photograph of Antibacterial Activity of the Blank and SiO2 NP/Cotton/Lycra Composites Figure 6. Photograph of Antibacterial Activity of the Blank and SiO2 NP/Cotton/Lycra Composites. y
p
Table 4 showed the UPF values of the blank and SiO2-NP/cotton/lycra composite
fabrics. The results illustrated that both the treated and untreated samples have higher
values of UPF compared with 100% cotton fabrics. The cover factor was expected to have a
favorable impact on UV protection, since traditional cotton fabrics have a lower cover factor
than sun protective woven or knitted materials. A main determining component of cover
factor is the fabric construction parameter (ends/inch and picks/inch or courses/inch and
wales/inch). Because threads are frequently interlaced, woven fabrics offer a larger cover
factor than knitted fabrics, as shown in the cotton/lycra) composites under study. The
pores between the yarns are small, allowing more radiation to penetrate. The threads in
woven cotton/lycra composites are entirely opaque to UV radiation and the pores between
the yarns are quite small. 3.1.1. SEM and EDX Analysis UV transmission is proportional to the porosity of the ideal
fabric since light can only pass via the pores. The distances between pile fabrics, material
thickness, and weight have an impact on the degree of protection. On the other hand, the
higher weight and cover factor rate provide 95% of the best protection. The calculation of
total UV percent transmittance for a fabric specimen is the ratio of the amount of radiation
transmitted to the amount of radiation directed perpendicular to the fabric specimen surface. The cotton/lycra composites generally have no transmission when measuring. The term
penetration or erythema weighted transmittance (EWT) is the inverse value of UPF Equation
(2) and is frequently used to assign the degree of UVR protection of fabrics. EWT has a range of
values between 0 and 1 (or 0 and 100%). The greater the sun protection provided by the fabric,
the smaller the percentage of EWT. The results of EWT showed excellent protection (0.0002). Table 4 showed the UPF values of the blank and SiO2-NP/cotton/lycra composite
fabrics. The results illustrated that both the treated and untreated samples have higher
values of UPF compared with 100% cotton fabrics. The cover factor was expected to have a
favorable impact on UV protection, since traditional cotton fabrics have a lower cover factor
than sun protective woven or knitted materials. A main determining component of cover
factor is the fabric construction parameter (ends/inch and picks/inch or courses/inch and
wales/inch). Because threads are frequently interlaced, woven fabrics offer a larger cover
factor than knitted fabrics, as shown in the cotton/lycra) composites under study. The
pores between the yarns are small, allowing more radiation to penetrate. The threads in
woven cotton/lycra composites are entirely opaque to UV radiation and the pores between
the yarns are quite small. UV transmission is proportional to the porosity of the ideal
fabric since light can only pass via the pores. The distances between pile fabrics, material
thickness, and weight have an impact on the degree of protection. On the other hand, the
higher weight and cover factor rate provide 95% of the best protection. The calculation of
total UV percent transmittance for a fabric specimen is the ratio of the amount of radiation
transmitted to the amount of radiation directed perpendicular to the fabric specimen surface. The cotton/lycra composites generally have no transmission when measuring. 3.1.1. SEM and EDX Analysis The term
penetration or erythema weighted transmittance (EWT) is the inverse value of UPF Equation
(2) and is frequently used to assign the degree of UVR protection of fabrics. EWT has a range of
values between 0 and 1 (or 0 and 100%). The greater the sun protection provided by the fabric,
the smaller the percentage of EWT. The results of EWT showed excellent protection (0.0002). 75 Polymers 2021, 13, 2888 (2) Table 4. UPF Values of the Blank and SiO2 NP-Cotton/Lycra Composites. Table 4. UPF Values of the Blank and SiO2 NP-Cotton/Lycra Composites. Table 4. UPF Values of the Blank and SiO2 NP-Cotton/Lycra Composites. Sample
UPF Values
100% Cotton
30
Untreated Cotton/Lycra (94.5/5.5%) 19 pick/cm
464.9
Treated Cotton/Lycra (94.5/5.5%) 19 pick/cm
2690.4
Untreated Cotton/Lycra (90.8/9.2%) 19 pick/cm
666.7
Treated Cotton/Lycra (90.8/9.2%) 19 pick/cm
4676.8
Note. UPF = UV protection factor. Note. UPF = UV protection factor. The UPF values of SiO2-NP-cotton/lycra composite fabrics exhibited excellent UV
protection compared to those of blank fabrics, according to the Australian/New Zealand
standard. Therefore, these results confirmed the UV refection ability of SiO2 NPs, which
can effectively decrease aging and reduce human skin damage caused by harmful UV
radiation. However, the UPF value of the cotton/lycra (90.8/9.2%) 19 pick/cm is higher
than that of the cotton/lycra (94.5/5.5%) 19 pick/cm sample. This may be the result of the
finding that, the greater the percentage of lycra in the fabric, the greater the fabric cover
factor and, consequently, the greater the UPF values. 3.1.4. Self-Cleaning of Blank and SiO2-NP–Cotton/Lycra Composite Fabrics Table 5 showed that the K/S values of SiO2NP/cotton/lycra composite fabrics exposed
to sunlight decreased compared to blank fabrics. Compared to the untreated samples, the
results of both SiO2-NP/cotton/lycra (94.5/5.5%) fabric of 19 pick/cm and cotton/lycra
(90.8/9.2%) fabric of 19 pick/cm samples demonstrated greater self-cleaning activity,
attributable to the fact that transition metal oxides and their composites, such as SiO2
NPs, have strong photocatalytic activity for the photodegradation of organic pollutants, as
illustrated in Figure 7. Metal oxide-based nanomaterials with well-controlled structural,
crystalline, and surface characteristics behave as semiconductors with broadband gaps and
have desirable attributes, including non-toxicity and stability. Figure 7. Self-cleaning of the blank and SiO2NP/cotton/lycra composite fabrics before and after
light exposure. Figure 7. Self-cleaning of the blank and SiO2NP/cotton/lycra composite fabrics before and after
light exposure. 76 Polymers 2021, 13, 2888 Table 5. Self-cleaning of the blank and SiO2-NP/cotton/lycra composite fabrics. Sample
K/S Value Unexposed
SD
K/S Value Exposed
SD
SCC
Blank
25.8
0.32
11.9
0.25
53.8
Cotton/Lycra (94.5/5.5%) 19 pick/cm
20.7
0.31
0.63
0.02
96.9
Blank
25.3
0.32
16.7
0.33
33.9
Cotton/Lycra (90.8/9.2%) 19 pick/cm
21.1
0.23
0.73
0.03
96.5
Note. K/S = (Color strength). 4 Conclusions Table 5. Self-cleaning of the blank and SiO2-NP/cotton/lycra composite fabrics. 4. Conclusions This research demonstrated the effect of both adding lycra yarns with different densi-
ties and implementation of SiO2 NPs on the functional properties of plain pile cotton fabrics
to produce composites. The SiO2 NP coated cotton/lycra composites showed significant
self-cleaning activity with 24 h of exposure. The self-cleaning results are identical in both
the cotton/lycra (94.5/5.5%) and cotton/lycra (90.8/9.2%) 19 pick/cm samples (96.9 and
96.5 values), respectively. Besides the intrinsic antibacterial properties, the inclusion of
SiO2 NPs will simultaneously lead to the effective antibacterial activity of the fabricated
surface. The pile density of 19 pick/cm provides higher bactericidal activity (30–31 mm)
compared to other densities. The composite positively influenced the UV protection char-
acteristics compared to blank samples. However, cotton/lycra (90.8/9.2%) 19 pick/cm
sample showed higher UPF (4676.8) than did cotton/lycra (94.5/5.5%) 19 pick/cm sample
(2690.4). In summary, the cotton/lycra (90.5/9.2%) 19 pick/cm sample showed the best
results in terms of antibacterial activity, UV protection, and self-cleaning. Author Contributions: Conceptualization, T.A.E. and G.M.E.; methodology, H.G.E.; software, G.M.E. validation, H.G.E., H.H.E. and T.A.E.; formal analysis, K.S.-A.; investigation, H.G.E.; resources,
H.G.E.; data curation, H.G.E.; writing—original draft preparation, H.G.E.; writing—review and
editing, T.A.E. and M.R.P.; visualization, M.R.P.; supervision, T.A.E.; project administration, T.A.E. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Funding: This research received no external funding. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the
corresponding author. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. 9.
Kundu, S.; Chowdhary, U. Effect of fiber content on comfort properties of cotton/spandex, rayon/spandex, and polyester/spandex
single jersey knitted fabrics. Int. J. Polym. Text. Eng. 2018, 5, 33–39. [CrossRef] References [CrossRef] 10. Prakash, C. Established the Effect of Loop Length on Dimensional Stability of Single Jersey Knitted
ton/Lycra Core Spun Yarn. Indian J. Sci. Technol. 2010, 3, 287–289. [CrossRef] 11. Abd-Elfattah, M.A.; Abou-Taleb, E.M.; Emara, A.I. Improving the performance of terry fabrics to be used as pilgrimage Clothes. Int. Des. J. 2020, 10, 397–413. [CrossRef] J
12. Slater, K. Protection of or by textiles from environmental damage, in Environmental Impact of Textiles-Production, Processes and
Protection, United Kingdom. Text. Inst. 2003, 163–166. [CrossRef] 13. Beenu, S.; Manisha, G.; Rani, A. Development of UV Protective Clothing for College Going Girls. Int. J. Curr. Microbiol. Appl. Sci. 2019, 8, 1614–1621. [CrossRef] 14. Rildaa, Y.; Syukria, F.; Alifa, A.; Aziza, H.; Chandrenb, S.; Nurc, H. Self-cleaning TiO2-SiO2 clusters on cotton textiles prepared by
dip-spin coating process. J. Teknol. Sci. Eng. 2016, 78, 113–120. [CrossRef] p p
g p
g
15. Sobha, K.; Surendranath, K.; Meena, V.; Jwala, T.K.; Swetha, N.; Latha, K.S.M. Emerging trends in nanob
Mol. Biol. Rev. 2010, 5, 1–12. [CrossRef] g p
g
ndranath, K.; Meena, V.; Jwala, T.K.; Swetha, N.; Latha, K.S.M. Emerging trends in nanobiotechnology. Biotech
010, 5, 1–12. [CrossRef] 16. Abou Elmaaty, T.; Abdeldayem, S.; Ramadan, S.; Sayed-Ahmed, K.; Plutino, M. Coloration and Multi-Functionalization of
Polypropylene Fabrics with Selenium Nanoparticles. Polymers 2021, 13, 2483. [CrossRef] yp
py
p
y
17. Ibrahim, N.; Eid, B.; Abd El-Aziz, E.; Abou Elmaaty, T.; Ramadan, S. Loading of chitosan Nano metal oxide hybrids onto
cotton/polyester fabrics to impart permanent and effective multifunctions. Int. J. Biol. Macromol. 2017, 105, 769–776. [CrossRef] p
y
p
p
18. Abou Elmaaty, T.; El-Nagare, K.; Raouf, S.; Abdelfattah, K.; El-Kadi, S.; Abdelazi, E. One-step green approach for functional
printing and finishing of textiles using silver and gold NPs. RSC Adv. 2018, 8, 25546–25557. [CrossRef] u Elmaaty, T.; El-Nagare, K.; Raouf, S.; Abdelfattah, K.; El-Kadi, S.; Abdelazi, E. One-step green approach
ing and finishing of textiles using silver and gold NPs. RSC Adv. 2018, 8, 25546–25557. [CrossRef] 19. Cuk, N.; Sala, M.; Gorjanc, M. Development of antibacterial and UV protective cotton fabrics using plant food waste and alien
invasive plant extracts as reducing agents for the in-situ synthesis of silver nanoparticles. Cellulose 2021, 28, 3215–3233. [CrossRef]
20. Attia, G.H.; Alyami, H.S.; Orabi, M.A.A.; Gaara, A.H.; El Raey, A.M. Antimicrobial activity of silver and zinc nanoparticles 19. Cuk, N.; Sala, M.; Gorjanc, M. References Development of antibacterial and UV protective cotton fabrics using plant food waste and alien
invasive plant extracts as reducing agents for the in-situ synthesis of silver nanoparticles. Cellulose 2021, 28, 3215–3233. [CrossRef] 20. Attia, G.H.; Alyami, H.S.; Orabi, M.A.A.; Gaara, A.H.; El Raey, A.M. Antimicrobial activity of silver and zinc nanoparticles
mediated by eggplant green calyx. Int. J. Pharmacol. 2020, 16, 236–243. [CrossRef] 21. Filipic, J.; Glazar, D.; Jerebic, S.; Kenda, D.; Modic, A.; Roskar, B.; Vrhovski, I.; Stular, D.; Golja, B.; Smolej, S.; et al. Tailoring of
antibacterial and UV protective cotton fabric by an in-situ synthesis of silver particles in the presence of a sol–gel matrix and
sumac leaf extract. Tekstilec 2020, 63, 4–13. [CrossRef] 22. Ahmad, S.; Fatma, A.; Manal, E.; Ghada, A.M. Applications of Nanotechnology and Advancements in Smart Wearable Textiles:
An Overview. Egypt. J. Chem. 2020, 63, 2177–2184. [CrossRef] 23. Zhang, Y.Y.; Xu, Q.B.; Fu, F.Y.; Liu, X.D. Durable antimicrobial cotton textiles modified with inorganic
2016, 23, 2791–2808. [CrossRef] B.; Fu, F.Y.; Liu, X.D. Durable antimicrobial cotton textiles modified with inorganic nanoparticles. Cellulose
. [CrossRef] 24. Das, D.; Yang, Y.; Obrien, J.; Breznan, D.; Nimesh, S.; Bernatchez, S.; Hill, M.; Sayari, A.; Vincent, R.; Kumarathasan, P. Synthesis
and physicochemical characterization of mesoporous SiO2 nanoparticles. J. Nanomater. 2014, 2014, 62. [CrossRef] 25. Aya, R.; Abdel Rahim, A.; Amr, H.; El-Amir, M. Improving Performance and Functional Properties of Different Cotton Fabrics by
Silicon Dioxide Nanoparticles. J. Eng. Sci. 2019, 4, 1–17. [CrossRef] 26. Bae, G.Y.; Jeong, Y.G.; Min, B.G. Superhydrophobic PET fabrics achieved by silica nanoparticles and water-repellent agent. Fibers
Polym. 2010, 11, 976–981. [CrossRef] 27. Chakraborty, J.N.; Yashdeep, S. Comparative Performance of Synthesised Silica Nanoparticles for Enhanced Hydrophilic
Properties on Cotton. Tekstilec 2019, 62, 278–287. [CrossRef] 28. Smiechowicz, E.; Niekraszewicz, B.; Strzelinska, M.; Zielecka, M. Antibacterial fibers containing nano
silver nanoparticles. AUTEX Res. J. 2020, 20, 441–448. [CrossRef] p
29. Riaz, S.; Ashraf, M.; Hussain, T.; Hussain, M.T. Modification of silica nanoparticles to develop highly durable superhydrophobic
and antibacterial cotton fabrics. Cellulose 2019, 26, 5159–5175. [CrossRef] [
]
30. Abbas, R.; Khereby, M.A.; Sadik, W.A.; El Demerdash, A.M. Fabrication of durable and cost effective superhydrophobic cotton
textiles via simple one step process. Cellulose 2015, 22, 887–896. [CrossRef] 31. Maurice, A.R.; Faouzi, H. Synthesis and characterization of amorphous silica nanoparitcles from aqueous silicates using cationic
surfactants. J. Met. Mater. Miner. 2014, 24, 37–42. [CrossRef] 32. References 1. Toprak, T.; Anis, P. Textile industry’s environmental effects and approaching cleaner production and sustainability, an overview. J. Text. Eng. Fash. Technol. 2017, 2, 429–442. [CrossRef] J. Text. Eng. Fash. Technol. 2017, 2, 429–442. [CrossRef g
2. Badr, A. Anti-microbial and durability characteristics of socks made of cotton and regenerated cellulosic. Alex. Eng. J. 2018, 57,
3367–3373. [CrossRef] brahim, H.M.; Zaghloul, S.; Hashem, M.; El-Shafei, A. A green approach to improve the antibacterial prope
d f b i
i
M
i
l if
t
t
f il
ti l
C ll l
2021 28 549 564 [C 3. Ibrahim, H.M.; Zaghloul, S.; Hashem, M.; El-Shafei, A. A green approach to improve the antibacterial properties of cellulose
based fabrics using Moringa oleifera extract presence of silver nanoparticles. Cellulose 2021, 28, 549–564. [CrossRef] ased fabrics using Moringa oleifera extract presence of silver nanoparticles. Cellulose 2021, 28, 549–564. [Cros 4. Mesfin, S.; Sampath, V.R. Investigation on Mechanical and Comfort Properties of Denim Woven Fabrics Made from Different
Spandex Percentage for Sports Wear. Curr. Trends Fash. Technol. Text. Eng. 2021, 7, 49–61. [CrossRef] n, S.; Sampath, V.R. Investigation on Mechanical and 4. Mesfin, S.; Sampath, V.R. Investigation on Mechanical and Comfort Properties of Denim Woven Fabrics Made from Different
Spandex Percentage for Sports Wear. Curr. Trends Fash. Technol. Text. Eng. 2021, 7, 49–61. [CrossRef] p
g
p
Spandex Percentage for Sports Wear. Curr. Trends Fash. Technol. Text. Eng. 2021, 7, 49–61. [CrossRef] Alndijany, N. Evaluation of the comfort properties of pile fabrics. Int. Des. J. 2020, 10, 521–530. [CrossRef] 6. Tomar, R. Handbook of Worsted Wool and Blended Suiting Process; Woodhead Publishing India Pvt. Ltd.: New Delhi, India, 2010. [CrossRef] 7. Ahmed, M.; Morshed, M.; Farjana, S.; Ana, S. Fabrication of new multifunctional cotton–modal–recycled aramid blended
protective textiles through deposition of a 3D-polymer coating: High fire retardant, water repellent and antibacterial properties. New J. Chem. 2020, 44, 12122–12133. [CrossRef] 8. Singha, K. Analysis of Spandex/Cotton Elastomeric Properties Spinning and Applications. Int. J. Compos. Mater. 2012, 2, 11–16. [CrossRef] 9. Kundu, S.; Chowdhary, U. Effect of fiber content on comfort properties of cotton/spandex, rayon/spandex, and polyester/spandex
single jersey knitted fabrics. Int. J. Polym. Text. Eng. 2018, 5, 33–39. [CrossRef] 77 Polymers 2021, 13, 2888 10. Prakash, C. Established the Effect of Loop Length on Dimensional Stability of Single Jersey Knitted Fabric Made from Cot-
ton/Lycra Core Spun Yarn. Indian J. Sci. Technol. 2010, 3, 287–289. Article
One Surface Treatment, Multiple Possibilities: Broadening the
Use-Potential of Para-Aramid Fibers with Mechanical Adhesion Sarianna Palola 1,*, Farzin Javanshour 1, Shadi Kolahgar Azari 2, Vasileios Koutsos 2 and Essi Sarlin 1 a 1,*, Farzin Javanshour 1, Shadi Kolahgar Azari 2, Vasileios Koutsos 2 and Essi Sarlin 1 1
Materials Science and Environmental Engineering Unit, Faculty of Engineering and Natural Sciences,
Tampere University, FI-33014 Tampere, Finland; farzin.javanshour@tuni.fi(F.J.); essi.sarlin@tuni.fi(E.S.)
2
School of Engineering, Institute for Materials and Processes, The University of Edinburgh, The King’s
Buildings, Robert Stevenson Road, Edinburgh EH9 3FB, UK; s.kolahgarazari@ed.ac.uk (S.K.A.);
vasileios.koutsos@ed.ac.uk (V.K.) 1
Materials Science and Environmental Engineering Unit, Faculty of Engineering and Natural Sciences,
Tampere University, FI-33014 Tampere, Finland; farzin.javanshour@tuni.fi(F.J.); essi.sarlin@tuni.fi(E.S.)
2
School of Engineering, Institute for Materials and Processes, The University of Edinburgh, The King’s
ld
b
S
d
d
b
h
k l h
d
k (S
A ) 1
Materials Science and Environmental Engineering Unit, Faculty of Engineering and Natural Sciences,
Tampere University, FI-33014 Tampere, Finland; farzin.javanshour@tuni.fi(F.J.); essi.sarlin@tuni.fi(E.S.)
2
School of Engineering, Institute for Materials and Processes, The University of Edinburgh, The King’s
Buildings, Robert Stevenson Road, Edinburgh EH9 3FB, UK; s.kolahgarazari@ed.ac.uk (S.K.A.);
vasileios.koutsos@ed.ac.uk (V.K.) Tampere University, FI-33014 Tampere, Finland; farzin.javanshour@tuni.fi(F.J.); essi.sarlin@tuni.fi(E.S.)
2
School of Engineering, Institute for Materials and Processes, The University of Edinburgh, The King’s
Buildings, Robert Stevenson Road, Edinburgh EH9 3FB, UK; s.kolahgarazari@ed.ac.uk (S.K.A.);
vasileios.koutsos@ed.ac.uk (V.K.) *
Correspondence: sarianna.palola@tuni.fi Abstract: Aramid fibers are high-strength and high-modulus technical fibers used in protective
clothing, such as bulletproof vests and helmets, as well as in industrial applications, such as tires and
brake pads. However, their full potential is not currently utilized due to adhesion problems to matrix
materials. In this paper, we study how the introduction of mechanical adhesion between aramid fibers
and matrix material the affects adhesion properties of the fiber in both thermoplastic and thermoset
matrix. A microwave-induced surface modification method is used to create nanostructures to the
fiber surface and a high throughput microbond method is used to determine changes in interfacial
shear strength with an epoxy (EP) and a polypropylene (PP) matrix. Additionally, Fourier transform
infrared spectroscopy, atomic force microscopy, and scanning electron microscopy were used to
evaluate the surface morphology of the fibers and differences in failure mechanism at the fiber-matrix
interface. We were able to increase interfacial shear strength (IFSS) by 82 and 358%, in EP and PP
matrix, respectively, due to increased surface roughness and mechanical adhesion. Article
One Surface Treatment, Multiple Possibilities: Broadening the
Use-Potential of Para-Aramid Fibers with Mechanical Adhesion Also, aging
studies were conducted to confirm that no changes in the adhesion properties would occur over time. Citation: Palola, S.; Javanshour, F.;
Kolahgar Azari, S.; Koutsos, V.; Sarlin,
E. One Surface Treatment, Multiple
Possibilities: Broadening the
Use-Potential of Para-Aramid Fibers
with Mechanical Adhesion. Polymers
2021, 13, 3114. https://doi.org/
10.3390/polym13183114 Citation: Palola, S.; Javanshour, F.;
Kolahgar Azari, S.; Koutsos, V.; Sarlin,
E. One Surface Treatment, Multiple
Possibilities: Broadening the
Use-Potential of Para-Aramid Fibers
with Mechanical Adhesion. Polymers
2021, 13, 3114. https://doi.org/
10.3390/polym13183114
Academic Editors: Tarek M. Abou Elmaaty and Maria
Rosaria Plutino
Received: 23 August 2021
Accepted: 12 September 2021
Published: 15 September 2021 Keywords: aramid fibers; surface modification; adhesion; interphase; interfacial shear strength Article Article References Abou Elmaaty, T.; Elsisi, H.; Negm, E.; Ayad, S.; Sofan, M. Novel Nano Silica Assisted Synthesis of Azo pyrazole for the Sustainable
Dyeing and Antimicrobial Finishing of Cotton Fabrics in Supercritical Carbon Dioxide. J. Supercrit. Fluid 2021, in press. 34. Ruiz-Cañas, M.C.; Corredor, L.M.; Quintero, H.I.; Manrique, E.; Bohórquez, A.R.R. Morphological and Structural Properties
of Amino-Functionalized Fumed Nanosilica and Its Comparison with Nanoparticles Obtained by Modified Stöber Method. Molecules 2020, 25, 2868. [CrossRef] 35. Abou Elmaaty, T.; Kasem, A.; Mona Elsalamony, M.; Hanan, E. A Green Approach for One Step Dyein
Fabric with Natural Pigment Extracted from Streptomyces Thinghirensis. Egypt. J. Chem. 2020, 63, 1999 aty, T.; Kasem, A.; Mona Elsalamony, M.; Hanan, E. A Green Approach for One Step Dyeing and Finishing o
Natural Pigment Extracted from Streptomyces Thinghirensis. Egypt. J. Chem. 2020, 63, 1999–2008. [CrossRef] Fabric with Natural Pigment Extracted from Streptomyces Thinghirensis. Egypt. J. Chem. 2020, 63, 1999–2008. [CrossRef]
36. Masadeh, M.M.; Alzoubi, K.H.; Khabour, O.F.; Al-Azzam, S.I. Ciprofloxacin-induced antibacterial activity is attenuated by
phosphodiesterase inhibitors. Curr. Ther. Res. 2014, 77, 14–17. [CrossRef] 36. Masadeh, M.M.; Alzoubi, K.H.; Khabour, O.F.; Al-Azzam, S.I. Ciprofloxacin-induced antibacterial activity is attenuated by
phosphodiesterase inhibitors. Curr. Ther. Res. 2014, 77, 14–17. [CrossRef] 37. Wang, L.; Chen Hu, C.; Shao, L. The antimicrobial activity of nanoparticles: Present situation and prospects for the future. Int. J. Nanomed. 2017, 12, 1227–1249. [CrossRef] [PubMed] 78 https://www.mdpi.com/journal/polymers Citation: Palola, S.; Javanshour, F.;
Kolahgar Azari, S.; Koutsos, V.; Sarlin,
E. One Surface Treatment, Multiple
Possibilities: Broadening the
Use-Potential of Para-Aramid Fibers
with Mechanical Adhesion. Polymers
2021, 13, 3114. https://doi.org/
10.3390/polym13183114 polymers polymers polymers 1. Introduction Para-aramid, poly (p-phenylene terephthalamide), fibers are highly crystalline syn-
thetic fibers with high tensile strength, excellent chemical and abrasion resistance and high
melting point. They even outrank carbon fiber in impact and wear resistance while also
having higher a strength-to-weight ratio [1–3]. Aramid is used in fibrous form as well as in
woven textile and pulp form, as reinforcement in demanding composite applications rang-
ing from protective clothing (helmets, bulletproof vests, and fire protection) to automotive
and industrial applications (gaskets, brake pads, tires, conveyor belts, and hoses). Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. However, the full use-potential of aramid fiber is hindered due to adhesion issues to
matrix materials. To achieve the high strength-to-weight ratio, outstanding mechanical
performance and durability characteristic of advanced composite materials [1–3], strong
adhesion between the reinforcing fibers and the matrix material is critical. The adhesion
issues with aramid fibers arise from the surface structure of the fiber, which is very smooth
and chemically inert, lacking in reactive side groups [4,5]. To overcome this phenomenon,
surface treatments are used, which traditionally promote either physical or chemical
adhesion with the matrix. For example, a plasma treatment increases the surface energy
of the fiber by increasing hydrogen bonds at the fiber surface, thus enabling a physical
bond to be formed between the fiber and matrix [6–8]. On the other hand, with a chemical
surface treatment, reactive side groups are grafted to the fiber surface, which can react
with the matrix material and create a strong covalent bond between the fiber and the
matrix [9,10]. However, these methods are often suitable for only one type of matrix Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article
distributed
under
the
terms
and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). 79 https://www.mdpi.com/journal/polymers Polymers 2021, 13, 3114. https://doi.org/10.3390/polym13183114 Polymers 2021, 13, 3114 material [11–13], may lose their effectiveness rapidly during storage [7] and may drastically
reduce the mechanical properties of the fibers [14–16]. Thus, new approaches are needed,
and research is increasingly directed towards utilizing mechanical adhesion between fibers
and matrix [17]. Typically, mechanical adhesion is considered a lesser form of adhesion in composites,
but it has some major advantages, such as independence of chemical compatibility. 1. Introduction With
mechanical adhesion or interlocking as the prominent adhesion mechanism, a wider range
of material combinations could be used in composite applications, including thermoplas-
tics. Thermoplastic materials are a desirable group of matrix materials for composite
applications due to their lower toxicity and easier recyclability when compared to ther-
mosetting materials. However, they are a challenging material group in terms of adhesion. Another benefit of mechanical adhesion at the fiber-matrix interface is that composite
production becomes more economical as the same surface treatment can be used with
multiple matrix types. p
yp
Mechanical adhesion or interlocking can be formed between the fiber and matrix,
for example, by adding nanowires [18–20], nanoparticles [21–23], nanotubes [13,24–26],
or nanofibers [27–29] to the fiber surface. These structures simultaneously increase the
surface area and the surface roughness of the fiber. For example, by increasing mechanical
adhesion together with chemical interactions, Nasser et al. [30] have been able to increase
short beam strength of laser-induced graphene-coated aramid fabric by 70% in epoxy
matrix. Lv et al. [13] have achieved similar results with in-situ polymer grafting and
carbon nanotubes on aramid in the epoxy matrix, but they concluded the increase in
interfacial shear strength (IFSS) to be due to increased polarity rather than topography. However, by purely increasing mechanical adhesion with adsorbed aramid nanofibers,
Nasser et al. [27] have been able to increase short beam strength by 26% and IFSS by 70%
in epoxy, which shows what the imminent potential mechanical adhesion has in terms of
composite applications. However, to fully benefit from mechanical adhesion, the attached medium (i.e.,
nanofibers or particles) needs also to be strongly adhered to the fiber surface, as Gonzalez-
Chi et al. [24] and Ehlert et al. [20] have demonstrated. Also, the unique skin-core structure
of the highly crystalline para-aramid fiber may lower the overall adhesion properties of
the fiber even if strong interphase is formed between the fiber and matrix [31]. As force
is applied to the interphase, the top layer of the fiber may fibrillate and be sheared off
completely. By applying a “new skin” layer of graphene to the fiber, Cheng et al. [32]
have been able to reconfigure the phenomenon and change the failure mechanism from
fibrillation of the fiber “skin” to clean fracture at the interface, while increasing the IFSS by
75% in epoxy. 1. Introduction In this paper, we study the effect of mechanical adhesion as the main adhesion mecha-
nism at the fiber-matrix interface. This is done by adding nanoscale deposits onto aramid
fiber surface that increase surface area and topography and thus, enable mechanical adhe-
sion at the fiber-matrix interphase. The concept of nanoscale deposit addition to increase
adhesion in macroscale has been proven effective in our previous study [33]. However, the
question remained whether the increased adhesion was purely due to mechanical adhesion
or a combined effect (i.e., secondary entanglement) and would the result really be effective
with other matrix materials as well. In this paper, we aim to address these questions and
show that the effect is universal and does work with multiple matrix material types, and
that the adhesion increase is purely due to increased mechanical adhesion. Also, we show
that the effect is similar across different length scales ranging from micro to macroscale. Both thermoplastic and thermoset matrices were used to evaluate reliably the behavior of
the nanodeposit decorated fiber surface in different matrix types, which have significantly
different chemical and physical properties. Micromechanical testing is applied so that the
failure mode and mechanism of the interphase can be monitored more closely and the
effect of secondary artefacts, which may be present in macroscopic bulk material testing,
such as fiber entanglement, can be eliminated from the results. For this, a high throughput 80 Polymers 2021, 13, 3114 microbond test system [34] was used to measure the IFSS of these nano-deposit decorated
fibers. This test method was chosen over the more traditional fiber fragmentation test be-
cause fiber fragmentation test is unsuitable for aramid fibers due to their high-strain tensile
failure mode [31]. Also, the microbond test method can be applied more easily to both
thermoplastic and thermoset matrix materials. In order to focus on the effect of mechanical
adhesion, polypropylene (PP) was chosen as the thermoplastic matrix material. PP has
very limited hydrogen bonding interactions with the fiber surface, thus making it ideal
for this type of investigation. Epoxy (EP) was used as the thermoset matrix because of its
availability and wide use in polymer composites across the field. Fourier transform infrared
spectroscopy (FTIR), scanning electron microscopy (SEM), and atomic force microscopy
(AFM) were used to characterize the nanostructures, study the fiber-matrix interphase
and identify the failure mechanism. 1. Introduction Further, it was also investigated how well the widely
debated microbond methodology represents macroscale properties of the composite by
comparison to the previous results [33]. Also, the influence of aging during storage is
studied, and what effect it has on the effectiveness of the surface treatment. 2.1. Aramid Surface Modification Para-aramid fibers, Twaron 2201 (Teijin, Amsterdam, The Netherlands; properties
according to the supplier: tensile strength 2.1 N/tex, linear density 1610 dtex, ~0.15 w-%
sizing), were used as the fiber material. Prior to the surface treatment, the fibers were
washed with mild detergent and rinsed with ethanol to remove the water-soluble, EO
and PO alcohol component containing, surface sizing, and finally dried thoroughly. These
fibers are denoted with suffix W, as ‘washed’. Microwave-induced surface treatment was
applied to a section of the washed fibers. This was done by first carburizing the fibers with
Agar Turbo carbon evaporator B7230 (Agar Scientific, Stansted, UK) and then placing them
into a glass container together with reactive chemicals (1:1 graphite and ferrocene). The
container is then sealed and placed into a microwave oven, as described in the previous
study [33]. Irradiation time of 14 s is used as it has been [33] determined to yield the
best distribution and coverage of the fiber surface with the nanodeposits. These fibers are
denoted with suffix MW. To investigate the storage properties of the MW—fibers, some of
the fibers were kept in air at room temperature, protected from light, for 48 months prior
to testing. Sample nomenclature is presented in Table 1. Table 1. Sample nomenclature used in the study. Sample Name
Matrix Material
Washing
Microwave Treatment
EP_W
epoxy
YES
NO
EP_MW
epoxy
YES
YES
PP_W
polypropylene
YES
NO
PP_MW
polypropylene
YES
YES
2.2. Interfacial Shear Strength Table 1. Sample nomenclature used in the study. 2.3. Microscopy Field emission gun SEM Zeiss ULTRAplus (Zeiss, Oberkochen, Germany) was used
for detailed imaging of the fiber surface and of the failed interface after IFSS testing. To
minimize charging and to improve image quality, the samples were attached to aluminum
sample holders with carbon glue and coated with carbon and tiny amount of gold. Surface topography of the fibers was studied with an AFM MultiMode Nanoscope
IIIa (Bruker, Santa Barbara, CA, USA). The measurements were done with tapping mode
to gain information about possible phase shifts together with high spatial resolution while
limiting the effect of artifacts and sample damage. Antimony (n) doped silicon tips of
0.01–0.025 Ohm-cm (model: NTESPA, Bruker, Santa Barbara, CA, USA) were used, which
had a reflective aluminum coating on the backside. The imaging was done in air at room
temperature. For the imaging process, samples were attached to a magnetic disc with
double-sided adhesive. Data was analyzed with Gwyddion software [36]. 2.2. Interfacial Shear Strength 2.2. Interfacial Shear Strength 2.2. Interfacial Shear Strength IFSS was measured with the microbond test method [35], in which single fiber mi-
crocomposite samples are prepared and tested. The samples are prepared by depositing
droplets of the matrix material onto single fiber filaments and allowed to cure or cool down
depending on the material type used. The droplets are then individually loaded with
microvise blades until the droplet is detached from the fiber. To calculate the IFSS, the load
required to detach the droplet (Fmax) is then compared with the area of the fiber surface
embedded by the droplet (Aemb), as described by Equation (1). Fmax
Aemb
= IFSS
(1) (1) 81 81 Polymers 2021, 13, 3114 For the IFSS testing in thermoset matrix, a low-viscosity epoxy (EP) resin system
Araldite LY 5052/Aradur 5052 (Huntsman, Ratingen, Germany) was used, with a mixing
ratio of 100/38, respectively. The resin was cured for 24 h at room temperature, followed
by post-curing at 60 ◦C for 12 h. For the IFSS testing in thermoplastic matrix, a high melt
flow heterophasic copolymer, polypropylene (PP) BJ380MO (Borealis AG, Vienna, Austria),
was used. Epoxy and PP droplets were dispensed onto the fibers with FIBROdrop (Fibrobotics
Oy, Tampere, Finland) setup. A computer-controlled aluminum heating element was used
to achieve optimum melt flow during PP droplet deposition. To prevent oxidation and
thermal degradation of the PP melt during the droplet deposition, the FIBROdrop device
was placed into an air-tight cabinet filled with nitrogen (N2) gas. Also, a new batch of
polymer melt was prepared for each fiber. High-throughput FIBRObond (Fibrobotics
Oy, Tampere, Finland) [34] device was used for microbond measurements with a 1 N
S-beam load cell and stainless steel sample holder. Testing was done in air at room
temperature. Five fibers per sample type with 20–40 droplets per fiber were tested, resulting
in approximately 100–200 data points for each sample type. 2.4. FTIR Spectroscopy The aramid fiber surface was analyzed with FTIR spectroscope Spectrum One (Perkin-
Elmer, Buckinghamshire, UK). The Universal Attenuated Total Reflectance (ATR) sam-
pling accessory of FTIR had a Diamond/ZeSe crystal with a 1.66 μm depth of penetra-
tion. Transmittance spectra were recorded within the 4000 to 600 cm−1 range and a
0.5 cm−1 resolution. 3. Results and Discussion 83 83 Polymers 2021, 13, 3114 The increased Z-contrast also implies that the nanostructures are mostly carbon-based
compounds with traces of iron from ferrocene used in the microwave treatment. As a
heavier element, iron would show up lighter in the EsB image, as seen in Figure 2. The
iron molecule in ferrocene acts as a nucleation site for the carbon atoms as it is heated up
during the treatment process [38] and thus can accumulate into the nanostructures. As
seen from the figures, the irregular shape and varying size of the nanostructures increases
the surface area of the fiber efficiently. This increases frictional forces at the fiber-matrix
interface as well as adhesion through mechanical interlocking. The FTIR spectrum (Figure 3) of the washed aramid fibers reveals characteristic peaks for
para-aramid at 3312 cm−1 (–NH, hydrogen bond association states), 1637 cm−1 (C=O stretching
vibration band of amide), 1537 cm−1 (N–H curved vibration), and 1305 cm−1 (N–H bending
vibration) [39–41]. Compared to the FTIR spectrum of W-fibers, the hydrogen band peak
of MW-fibers has broadened and moved to a lower wavenumber of 3305 cm−1 indicat-
ing increased hydrogen bonding at the surface and weakened hydrogen bonding in the
polymer chains of the aramid fiber skin layer [40]. This means that the intense heat during
the surface treatment causes some damage to the fiber surface but not to a degree that
would affect the tensile properties of the fibers, as shown previously [33], or be visible in
SEM. Also, a new peak is present at 2870 cm−1, indicating CH2/CH groups at the fiber
surface [40]. The same peak is also present in ferrocene and graphite [42,43]. This confirms
that the nanostructures are decomposition products of ferrocene and graphite, formed
during the microwave irradiation treatment. Figure 3. FTIR transmittance spectrum of washed (W) and microwave surface-treated (MW)
aramid fibers. Figure 3. FTIR transmittance spectrum of washed (W) and microwave surface-treated (MW)
aramid fibers. IFSS was calculated with linear regression using the slope of load versus embedded
area (Aemb) for each tested fiber separately. The IFSS for each sample type was then taken
by calculating the average of the IFSS values of the separate fibers of that sample type. 3. Results and Discussion The fiber surface after the microwave irradiation treatment revealed an abundance of
nanostructures covering the surface. As seen in Figure 1, the nanostructures are of irregular
shape and that the topography of the fibers has changed due to the surface treatment
significantly, but no visible voids are generated on the fiber surface. This is in line with
our previous findings stating that the treatment has no negative effect on the mechanical
properties of the fibers [33] and highlights the repeatability of the surface treatment method. The AFM studies supported the SEM findings depicting clearly defined protrusions
on the fiber surface. The phase contrast image highlights the structural and chemical
difference between the bulk fiber and the nanostructures. As the color gradient in AFM
phase contrast image is a combination of topographical details as well as changes in
mechanical and adhesive properties, a contrast in color is created when the chemical and
physical properties change in the imaged area. As the nanostructures appear brighter
than the fiber surface, it can be deduced that they are not the same material as the fiber
surface. Additionally, when using an Energy selective Backscattered (EsB) detector with
SEM, the nanostructures also appear lighter than the bulk fiber itself, as seen in Figure 2. 82 Polymers 2021, 13, 3114 The EsB detector reduces edge contrast in the image and thus, the apparent color difference
between the bulk fiber and the nanostructures is due to increased Z-contrast [37] between
the two. This, together with the AFM findings, means that the nanostructures are of
different material and added to the surface during the microwave surface treatment rather
than coming from the bulk fiber itself due to wrinkling or surface degradation. igure 1. SEM images and corresponding AFM images of the aramid fibers before and after the microwave
urface treatment. Figure 1. SEM images and corresponding AFM images of the aramid fibers before and after the microwave
surface treatment. Figure 1. SEM images and corresponding AFM images of the aramid fibers before and after the microwave
surface treatment M images and corresponding AFM images of the aramid fibers before and after the microwave Figure 2. SEM EsB image of the microwave treated fiber surface highlighting the Z-contrast between
the fiber surface and nanostructures. Figure 2. SEM EsB image of the microwave treated fiber surface highlighting the Z-contrast between
the fiber surface and nanostructures. 3. Results and Discussion From
the data in Figure 4, it can be seen that the load required to debond a droplet is higher with
samples that are covered with nanostructures than with those that are not, even though the
effective embedded area is similar. This implies that protrusions as small as nanoscale, can
significantly alter the properties of the fiber-matrix interface in a way that can be detected
with a microscale method. This same trend can be seen with both EP and PP matrix. As
the behavior is similar in both thermoset and thermoplastic matrix, it emphasizes the
importance of mechanical adhesion as a major adhesion mechanism that is independent of
chemical compatibility. By increasing mechanical adhesion with the nanostructures, the
maximum load increased by 56 and 395% in MW_EP and MW_PP, respectively. Although,
the scattering of data appears to increase due to the surface treatment in MW_PP as
compared to W_PP, this is not the case. Relative standard deviation (RSD) in both data 84 Polymers 2021, 13, 3114 sets is similar (~14%), which means that the data is highly comparable. Also, the R2 value
for all measured samples ranged between 0.82–0.98, meaning high compatibility with the
linear fit and thus, highly reliable measurement results. Figure 4. Load vs. embedded area for all measured droplets for (a) W_EP, (b) MW_EP, (c) W_PP, and
(d) MW_PP. Figure 4. Load vs. embedded area for all measured droplets for (a) W_EP, (b) MW_EP, (c) W_PP, and
(d) MW_PP. The average IFSS results are presented in Figure 5 for W_EP, MW_EP, W_PP, and
MW_PP together with macroscopic fiber bundle pull-out test results for the same surface
treatment in rubber [33]. It is evident that the IFSS increases alongside with the increased
surface topography of the fibers. Moreover, the increase in IFSS follows a similar trend
with the bundle pull-out test in rubber. The results show that the IFSS increases in a similar
fashion with thermoplastic, thermoset, and elastomeric matrices, even though the potential
for chemical interaction of these matrix types is very different. For example, with EP, an
increase in interfacial adhesion can be achieved through covalent bonding with the fiber
surface during curing or by creating higher frictional force with the cured and cross-linked
resin [13]. Mercaptan compounds, Lewis acid, and alkali products can be used to achieve
such covalent bonds with EP. 3. Results and Discussion However, as none of them are grafted to the fiber surface
in this case, what remains, is the increase in friction. This is also the case with PP. The
chemical composition of PP provides only limited hydrogen interaction, which could affect
favorably to interfacial adhesion with aramid. The main attribute towards the adhesion is
mostly compressive forces due to favorable transcrystallization [31,39] occurring during
the cooling process of the polymer melt. This was noted by Wang et al. [44]. They showed
that small grooves and protrusions will increase thermal stress due to increased stress
concentration during the PP crystallization when the melt is cooling. This localized stress
concentration will further on enhance the nucleation ability of PP and, thus, promote
transcrystallization leading to enhanced interfacial adhesion. The nanostructures created 85 Polymers 2021, 13, 3114 to the aramid fiber surface in this study, will act as such protrusions as described by Wang
et al., and thus, lead to increased mechanical adhesion between the fiber surface and PP. In both EP and PP matrix, the nanostructures also increase stress transferability, which in
turn, increases the IFSS in a similar fashion in both matrix types. These findings indicate
that the primary adhesion mechanism between the fiber and polymer in this case, is indeed
mechanical adhesion. Figure 5. The IFSS results and SEM images of the corresponding fracture surfaces for (a) W_EP and MW_EP, where the arrow
indicates fibrillation of the fiber surface, and (b) W_PP and MW_PP. The fiber bundle pull-out force and corresponding
optical images are presented in (c) for W_rubber and MW_rubber [33]. Figure 5. The IFSS results and SEM images of the corresponding fracture surfaces for (a) W_EP and MW_EP, where the arrow
indicates fibrillation of the fiber surface, and (b) W_PP and MW_PP. The fiber bundle pull-out force and corresponding
optical images are presented in (c) for W_rubber and MW_rubber [33]. The measured IFSS value for W_PP is 5.7 MPa, which is similar to other studies [24]
done with microdroplet test and aramid/PP combination. This shows that the high-
throughput microbond method is highly suitable for IFSS evaluation also with thermo-
plastic matrix. Overall, the IFSS increased from 29.8 to 54.2 MPa (82% increase) and from
5.7 to 25.9 MPa (358% increase), in EP and PP matrix, respectively, due to the surface
treatment. 3. Results and Discussion This is very significant as it shows that the surface treatment is suitable for both
thermoplastic and thermoset materials and that it has a similar effect in them both. Also, 86 Polymers 2021, 13, 3114 it is worthwhile noting that with this straightforward and fast surface treatment process,
the IFSS of aramid/EP combination could be brought to the same level as with other more
complicated methods reported only recently [32]. SEM images of the failed fiber-matrix interphase and visual observation during mi-
crobond testing supported the IFSS results. The failure mechanism during testing changed
from pure shear at the interphase to a combination of peeling and shear as the surface
topography was introduced (see videos in Supplementary Materials: Supporting Informa-
tion files S1 and S2). With no surface treatment, the fiber surface after debonding appears
smooth and unscathed, with only a minor amount of matrix residue remaining, as seen in
Figure 5. This indicates that the fiber-matrix interphase has failed as the matrix droplet
is sheared off. Also, the detachment site of the droplet shows a clean break with a small
gap between the fiber surface and matrix, indicating a weak interphase. With the surface-
treated fibers, the detachment site of the droplet shows no gap and is more uneven, as
seen in Figure 6a,b, meaning that a stronger fiber-matrix interphase has been created. The
debonded surface is rougher even with some fibrillation of the fiber skin structure, which
indicates that the failure has shifted from purely occurring at the fiber-matrix interphase to
a combination of fiber surface fibrillation and peeling together with interfacial shearing. The change in the appearance of the debonded surface is very clear with the harder EP
matrix, as seen in Figures 5a and 6a, where red arrows point to sections of fibrillated fiber
surface. Whereas with the softer PP, the matrix is rather fibrillating itself and clinging to
the nanostructures than cleaving bits off from the fiber surface. The PP strands clinging
to the fiber can be seen clearly underneath the fiber in Figure 6b. This indicates that the
fiber/matrix adhesion is higher than the cohesive strength of the matrix. It also means, that
the nanostructures are strongly attached to the fiber surface, as delamination occurs jointly
from the skin-core interphase and skin-matrix interphase. As a result, it can be said that
the main adhesion mechanism contributing towards the increased interfacial adhesion is
mechanical adhesion. 3. Results and Discussion Figure 6. SEM images of the facture surfaces of (a) MW_EP, where red arrows indicate the fibrillation and peeling of the
fiber surface, and (b) MW_PP after IFSS testing. Figure 6. SEM images of the facture surfaces of (a) MW_EP, where red arrows indicate the fibrillation and peeling of the
fiber surface, and (b) MW_PP after IFSS testing. 87 87 Polymers 2021, 13, 3114 Additionally, the IFSS results follow very closely the same trend observed with the
macroscopic fiber bundle pull-out test in rubber [33]. By increasing the amount of nanode-
posits on the fiber surface, the adhesion and the strength of the interphase can be increased
in both micro and macroscale. This is in line with findings of previous studies, such as
the ones made by Beter et al. [45]. It also suggests that the high-throughput microbond
method produces reliable data, which can indeed be used to evaluate adhesion properties
in macroscopic composite structures. This type of composite research and development
process can be made more economical and efficient. Storage properties, and more precisely, aging, of the nanostructure covered fiber
surface was also investigated. Some of the surface-treated fibers were taken aside and kept
for 48 months at room temperature and protected from light. The IFSS of these fibers was
measured with the microbond procedure in EP, and visual changes in the fiber surface
were studied with SEM. The results revealed only minimal decrease in IFSS (~2%), which
is well within the deviation range, compared to newly surface-treated fibers. Also, no
change in the appearance of the fiber surface was observed. Thus, no significant decrease
in the interfacial properties of the fibers has occurred, and the surface treatment can be
considered durable enough to withstand storage over long periods of time. 4. Conclusions This work explored the effect of nanostructures on the interfacial adhesion of aramid
fiber in both a thermoplastic and a thermoset matrix and related the results also to an
elastomeric matrix from a previous study. Our findings demonstrated that a significant
increase in IFSS can be achieved in both thermoplastic (+358%) and thermoset (+82%)
matrix, while maintaining mechanical and storage properties of the fibers. The increase
in IFSS was noted to be due to enhanced mechanical adhesion between the fiber surface
and matrix material caused by the addition of nanostructures to the fiber surface. The
positive effect of the nanostructures on interfacial strength was observed both in micro and
macroscale tests. The failure mechanism of the fiber-matrix interphase changes from clean
shear to combined shear and peeling, as the level of mechanical adhesion increases, proving
that the nanostructures are strongly attached to the fiber surface. These results highlight
the significance of mechanical adhesion as the main adhesion mechanism and expand the
use-potential of aramid fibers to multiple matrix material types and applications with just
one fiber surface treatment. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10
.3390/polym13183114/s1, Supporting Information file S1: A video of debonding of PP droplet on
W_PP. Supporting Information file S2: A video of debonding of PP droplet on MW_PP. Author Contributions: Conceptualization, S.P., V.K. and E.S.; Methodology, S.P.; Formal analysis,
S.P.; Investigation, S.P., F.J., S.K.A. and E.S.; Data curation, S.P.; Writing—original draft preparation,
S.P. and F.J.; Writing—review and editing, S.P., F.J., V.K. and E.S.; Supervision, V.K. and E.S.; Project
administration, E.S.; Funding acquisition, S.P. All authors have read and agreed to the published
version of the manuscript. Funding: This research was funded by FINNISH CULTURAL FOUNDATION, grant number
00170813, TUTKIJAT MAAILMALLE and OTTO MALM FOUNDATION. References 1. Karger-Kocsis, J.; Mahmood, H.; Pegoretti, A. Recent advances in fiber/matrix interphase engineering for polymer composites. Prog. Mater. Sci. 2015, 73, 1–43. [CrossRef] 2. Jesson, D.A.; Watts, J.F. The Interface and Interphase in Polymer Matrix Composites: Effect on Mechanica
for Identification. Polym. Rev. 2012, 52, 321–354. [CrossRef] J.F. The Interface and Interphase in Polymer Matrix Composites: Effect on Mechanical Properties and Methods
Polym. Rev. 2012, 52, 321–354. [CrossRef] 3. Tam, T.; Bhatnagar, A. High-performance ballistic fibers and tapes. In Lightweight Ballistic Composites, 2nd ed.; Bhatnagar, A., Ed.;
Woodhead Publishing: Duxford, UK, 2016; pp. 1–39. g
pp
4. Chang, K.K.; Fibers, A. ASM Handbooks; Miracle, D.B., Donaldson, S.L., Eds.; Composites, ASM International: Materials Park, OH,
USA, 2001; Volume 21, pp. 41–45. pp
5. Qi, G.; Zhang, B.; Du, S.; Yu, Y. Estimation of aramid fiber/epoxy interfacial properties by fiber bundle tests and multiscale
modeling considering the fiber skin/core structure. Compos. Struct. 2017, 167, 1–10. [CrossRef] 5. Qi, G.; Zhang, B.; Du, S.; Yu, Y. Estimation of aramid fiber/epoxy interfacial properties by fiber bun
modeling considering the fiber skin/core structure Compos Struct 2017 167 1 10 [CrossRef] 5. Qi, G.; Zhang, B.; Du, S.; Yu, Y. Estimation of aramid fiber/epoxy interfacial properties by fiber
modeling considering the fiber skin/core structure. Compos. Struct. 2017, 167, 1–10. [CrossRef] modeling considering the fiber skin/core structure. Compos. Struct. 2017, 167, 1–10. [CrossRef] 6. Luo, S.; Ooij, W.J.V. Surface modification of textile fibers for improvement of adhesion to polymeric matrices: A review. J. Adhes. Sci. Technol. 2002, 16, 1715–1735. [CrossRef] 7. Sun, J.; Yao, L.; Sun, S.; Qiu, Y. ESR study of atmospheric pressure plasma jet irradiated aramid fibers. 205, 5312–5317. [CrossRef] , S.; Qiu, Y. ESR study of atmospheric pressure plasma jet irradiated aramid fibers. Surf. Coat. Technol. 2011,
ossRef] 8. Wang, J.; Chen, P.; Xiong, X.; Jia, C.; Yu, Q.; Ma, K. Interface characteristic of aramid fiber reinforced poly(phthalazinone ether
sulfone ketone) composite. Surf. Interface Anal. 2017, 49, 788–793. [CrossRef] )
p
f
f
9. Tie-Min, L.; Yuan-Suo, Z.; Jie, H. Surface modification of Aramid fibers with new chemical method for improving interfacial
bonding strength with epoxy resin. J. Appl. Polym. Sci. 2010, 118, 2541–2552. 10. Gao, B.; Zhang, R.; Gao, F.; He, M.; Wang, C.; Liu, L.; Zhao, L.; Cui, H. Interfacial Microstructure and Enhanced Mechanical
Properties of Carbon Fiber Composites Caused by Growing Generation 1–4 Dendritic Poly(amidoamine) on a Fiber Surface. Funding: This research was funded by FINNISH CULTURAL FOUNDATION, grant number
00170813, TUTKIJAT MAAILMALLE and OTTO MALM FOUNDATION. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the
corresponding author. Acknowledgments: Authors would like to thank Teijin Aramid Bv (Amsterdam, The Netherlands)
for kindly providing the aramid fibers. This work made use of Tampere Microscopy Center facilities
at Tampere University (33014 Tampere, Finland). 88 Polymers 2021, 13, 3114 Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results. p
p
g
25.
Hazarika, A.; Deka, B.K.; Kim, D.; Park, Y.; Park, H.W. Microwave-induced hierarchical iron-carbon nanotubes nanostructures
anchored on polypyrrole/graphene oxide-grafted woven Kevlar® fiber. Compos. Sci. Technol. 2016, 129, 137–145. [CrossRef] References Langmuir 2016, 32, 8339–8349. [CrossRef] [PubMed] g
11. Sa, R.; Yan, Y.; Wei, Z.; Zhang, L.; Wang, W.; Tian, M. Surface Modification of Aramid Fibers by Bio-Inspired Poly(dopamine) and
Epoxy Functionalized Silane Grafting. ACS Appl. Mater. Interfaces 2014, 6, 21730–21738. [CrossRef] [PubMed] g, C.; Wang, Y.; Li, P.; Yu, Y.; Jia, X.; Liu, H.; Yang, X.; Xue, Z.; Ryu, S. Interface correlation and toughness match 12. Li, G.; Zhang, C.; Wang, Y.; Li, P.; Yu, Y.; Jia, X.; Liu, H.; Yang, X.; Xue, Z.; Ryu, S. Interface correlation and toughness matching of
phosphoric acid functionalized Kevlar fiber and epoxy matrix for filament winding composites. Compos. Sci. Technol. 2008, 68,
3208–3214. [CrossRef] 13. Lv, J.; Cheng, Z.; Wu, H.; He, T.; Qin, J.; Liu, X. In-situ polymerization and covalent modification on aramid fiber surface via
direct fluorination for interfacial enhancement. Compos. Part B Eng. 2020, 182, 107608. [CrossRef] 14. Yue, C.Y.; Padmanabhan, K. Interfacial studies on surface modified Kevlar fibre/epoxy matrix composites. Compos. Part B Eng. 1999, 30, 205–217. [CrossRef] 15. Zhang, S.; Li, M.; Cheng, K.; Lu, S. A facile method to prepare PEG coatings on the fiber surface by the reconstruction of hydrogen
bonds for enhancing the interfacial strength of fibers and resins. Colloids Surf. A Physicochem. Eng. Asp. 2020, 589, 124426. [CrossRef] 16. Loureiro, L.; Carvalho, V.H.; Bettini, S.H.P. Reuse of p-aramid from industrial waste as reinforcement fiber in polyamide 6.6. Polym. Test. 2016, 56, 124–130. [CrossRef] 17. Palola, S.; Vuorinen, J.; Noordermeer, J.W.M.; Sarlin, E. Development in Additive Methods in Aramid Fiber Surface Modification
to Increase Fiber-Matrix Adhesion: A Review. Coatings 2020, 10, 556. [CrossRef] 18. Hazarika, A.; Deka, B.K.; Kim, D.; Kong, K.; Park, Y.; Park, H.W. Growth of aligned ZnO nanorods on woven Kevlar® fiber and
its performance in woven Kevlar® fiber/polyester composites. Compos. Part A Appl. Sci. Manuf. 2015, 78, 284–293. [CrossRef] 19. Malakooti, M.H.; Hwang, H.; Goulbourne, N.C.; Sodano, H.A. Role of ZnO nanowire arrays on the im
fabrics. Compos. Part B Eng. 2017, 127, 222–231. [CrossRef] H.; Hwang, H.; Goulbourne, N.C.; Sodano, H.A. Role of ZnO nanowire arrays on the impact response of aram
Part B Eng. 2017, 127, 222–231. [CrossRef] p
g
20. Ehlert, G.J.; Sodano, H.A. Zinc oxide nanowire interphase for enhanced interfacial strength in lightweight polymer fiber
composites. ACS Appl. Mater. Interfaces 2009, 1, 1827–1833. [CrossRef] [PubMed] pp
f
; Sodano, H.A. References Enhanced Interfacial Strength and UV Shielding of Aramid Fiber Composites through Zn
zing ACS Appl Mater Interfaces 2016 8 33963–33971 [CrossRef] [PubMed] p
pp
f
21. Patterson, B.A.; Sodano, H.A. Enhanced Interfacial Strength and UV Shielding of Aramid Fiber Composites through ZnO
Nanoparticle Sizing. ACS Appl. Mater. Interfaces 2016, 8, 33963–33971. [CrossRef] [PubMed] g
g
anoparticle Sizing. ACS Appl. Mater. Interfaces 2016, 8, 33963–33971. [CrossRef] [PubMed] 22. Cheng, Z.; Zhang, L.; Jiang, C.; Dai, Y.; Meng, C.; Luo, L.; Liu, X. Aramid fiber with excellent interfacial properties suitable for
resin composite in a wide polarity range. Chem. Eng. J. 2018, 347, 483–492. [CrossRef] 23. Wang, B.; Duan, Y.; Zhang, J. Titanium dioxide nanoparticles-coat
resistance properties. Mater. Des. 2016, 103, 330–338. [CrossRef] 23. Wang, B.; Duan, Y.; Zhang, J. Titanium dioxide nanoparticles-coated aramid fiber showing enhanced interfacial strength and UV
resistance properties. Mater. Des. 2016, 103, 330–338. [CrossRef] 24. Gonzalez-Chi, P.I.; Rodríguez-Uicab, O.; Martin-Barrera, C.; Uribe-Calderon, J.; Canché-Escamilla, G.; Yazdani-Pedram, M.;
May-Pat, A.; Avilés, F. Influence of aramid fiber treatment and carbon nanotubes on the interfacial strength of polypropylene
hierarchical composites. Compos. Part B Eng. 2017, 122, 16–22. [CrossRef] p
p
g
25. Hazarika, A.; Deka, B.K.; Kim, D.; Park, Y.; Park, H.W. Microwave-induced hierarchical iron-carbon nanotubes nanostructures
anchored on polypyrrole/graphene oxide-grafted woven Kevlar® fiber. Compos. Sci. Technol. 2016, 129, 137–145. [CrossRef] 89 Polymers 2021, 13, 3114 27. Nasser, J.; Lin, J.; Steinke, K.; Sodano, H.A. Enhanced interfacial strength of aramid fiber reinforced composites through adsorbed
aramid nanofiber coatings. Compos. Sci. Technol. 2019, 174, 125–133. [CrossRef] g
p
[
]
28. Yang, M.; Cao, K.; Sui, L.; Qi, Y.; Zhu, J.; Waas, A.; Arruda, E.M.; Kieffer, J.; Thouless, M.D.; Kotov, N.A. Dispersions of Aramid
Nanofibers: A New Nanoscale Building Block. ACS Nano 2011, 5, 6945–6954. [CrossRef] [PubMed] g
p
28. Yang, M.; Cao, K.; Sui, L.; Qi, Y.; Zhu, J.; Waas, A.; Arruda, E.M.; Kieffer, J.; Thouless, M.D.; Kotov, N.A. Dispersions of Aramid
N
fib
A N
N
l B ildi
Bl
k ACS N
2011 5 6945 6954 [C
R f] [P bM d] g
p
28. Yang, M.; Cao, K.; Sui, L.; Qi, Y.; Zhu, J.; Waas, A.; Arruda, E.M.; Kieffer, J.; Thouless, M.D.; Kotov, N.A g
Q
J
J
Nanofibers: A New Nanoscale Building Block. ACS Nano 2011, 5, 6945–6954. [CrossRef] [PubMed] g
29. Patterson, B.A.; Malakooti, M.H.; Lin, J.; Okorom, A.; Sodano, H.A. References Aramid nanofibers for multiscale fiber reinforcement of
polymer composites. Compos. Sci. Technol. 2018, 161, 92–99. [CrossRef] Patterson, B.A.; Malakooti, M.H.; Lin, J.; Okorom, A.; Sodano, H.A. Aramid nanofibers for multiscale fiber 29. Patterson, B.A.; Malakooti, M.H.; Lin, J.; Okorom, A.; Sodano, H.A. Aramid nanofibers for multisc
polymer composites. Compos. Sci. Technol. 2018, 161, 92–99. [CrossRef] 30. Nasser, J.; Groo, L.; Zhang, L.; Sodano, H. Laser induced graphene fibers for multifunctional aramid fiber reinforced composite. Carbon 2020, 158, 146–156. [CrossRef] 31. Li, H.; Xu, Y.; Zhang, T.; Niu, K.; Wang, Y.; Zhao, Y.; Zhang, B. Interfacial adhesion and shear behaviors of
6 composites under different thermal treatments. Polym. Test. 2020, 81, 106209. [CrossRef] ; Zhang, T.; Niu, K.; Wang, Y.; Zhao, Y.; Zhang, B. Interfacial adhesion and shear behaviors of aramid fiber/poly
s under different thermal treatments. Polym. Test. 2020, 81, 106209. [CrossRef] p
y
32. Cheng, Z.; Li, X.; Lv, J.; Liu, Y.; Liu, X. Constructing a new tear-resistant skin for aramid fiber to enhance composites interfacial
performance based on the interfacial shear stability. Appl. Surf. Sci. 2021, 544, 148935. [CrossRef] 32. Cheng, Z.; Li, X.; Lv, J.; Liu, Y.; Liu, X. Constructing a new tear-resistant skin for aramid fiber to enhan
performance based on the interfacial shear stability. Appl. Surf. Sci. 2021, 544, 148935. [CrossRef] Cheng, Z.; Li, X.; Lv, J.; Liu, Y.; Liu, X. Constructing a new tear resistant skin for aramid fiber to enhance
erformance based on the interfacial shear stability. Appl. Surf. Sci. 2021, 544, 148935. [CrossRef] 33. Palola, S.; Sarlin, E.; Azari, S.K.; Koutsos, V.; Vuorinen, J. Microwave induced hierarchical nanostructures on aramid fibers and
their influence on adhesion properties in a rubber matrix. Appl. Surf. Sci. 2017, 410, 145–153. [CrossRef] 34. Laurikainen, P.; Kakkonen, M.; von Essen, M.; Tanhuanpää, O.; Kallio, P.; Sarlin, E. Identification and compensation of error
sources in the microbond test utilising a reliable high-throughput device. Compos. Part A Appl. Sci. Manuf. 2020, 137, 105988. [CrossRef] 35. Miller, B.; Muri, P.; Rebenfel, L. A microbond method for determination of the shear strength of a fiber/resin interface. Compos. Sci. Technol. 1987, 28, 17–32. [CrossRef] 36. Neˇcas, D.; Klapetek, P. Gwyddion: An open-source software for SPM data analysis. Cent. Eur. J. Phys. 2012, 10, 181–188. [CrossRef] 37. EsB Detector—Make Sub-Surface Information and Nano-Scale Composition Visible; Carl Zeiss Microscopy GmbH: Oberkochen,
Germany, 2016. y
38. Bajpai, R.; Wagner, H.D. Citation: Rodes-Carbonell, A.M.;
Ferri, J.; Garcia-Breijo, E.; Montava, I.;
Bou-Belda, E. Influence of Structure
and Composition of Woven Fabrics
on the Conductivity of Flexography
Printed Electronics. Polymers 2021, 13,
3165. https://doi.org/10.3390/
polym13183165 Ana María Rodes-Carbonell 1,*, Josué Ferri 2, Eduardo Garcia-Breijo 3, Ignacio Montava 4 and Eva Bou-Belda 4 Carbonell 1,*, Josué Ferri 2, Eduardo Garcia-Breijo 3, Ignacio Montava 4 and Eva Bou-Belda 4 na María Rodes-Carbonell 1,*, Josué Ferri 2, Eduardo Garcia-Breijo 3, Ignacio Montava 4 and Eva Ana María Rodes-Carbonell 1,*, Josué Ferri 2, Eduardo Garcia-Breijo 3, Ignacio Montava 4 and Eva Bou-Belda 4 1
Textile Research Institut, Universitat Politècnica de València, 46022 Valencia, Spain
2
Instituto Tecnológico del Textil (AITEX), 03801 Alcoy, Spain; josue.ferri@aitex.es
3
Instituto Interuniversitario de Investigación de Reconocimiento Molecular y Desarrollo Tecnológico (IDM),
Universitat Politècnica de València, 46022 Valencia, Spain; egarciab@eln.upv.es
4
Department of Textile and Paper Engineering, Universitat Politècnica de València,
Plaza Ferrándiz y Carbonell s/n., 03801 Alcoy, Spain; imontava@txp.upv.es (I.M.);
evbobel@upvnet.upv.es (E.B.-B.) p
p
*
Correspondence: anarodescarbonell@gmail.com Abstract: The work is framed within Printed Electronics, an emerging technology for the manufacture
of electronic products. Among the different printing methods, the roll-to-roll flexography technique
is used because it allows continuous manufacturing and high productivity at low cost. Nevertheless,
the incorporation of the flexography printing technique in the textile field is still very recent due
to technical barriers such as the porosity of the surface, the durability and the ability to withstand
washing. By using the flexography printing technique and conductive inks, different printings were
performed onto woven fabrics. Specifically, the study is focused on investigating the influence of
the structure of the woven fabric with different weave construction, interlacing coefficient, yarn
number and fabric density on the conductivity of the printing. In the same way, the influence of
the weft composition was studied by a comparison of different materials (cotton, polyester, and
wool). Optical, SEM, color fastness to wash, color measurement using reflection spectrophotometer
and multi-meter analyses concluded that woven fabrics have a lower conductivity due to the ink
expansion through the inner part of the textile. Regarding weft composition, cotton performs worse
due to the moisture absorption capacity of cellulosic fiber. A solution for improving conductivity
on printed electronic textiles would be pre-treatment of the surface substrates by applying different
chemical compounds that increase the adhesion of the ink, avoiding its absorption. Citation: Rodes-Carbonell, A.M.;
Ferri, J.; Garcia-Breijo, E.; Montava, I.;
Bou-Belda, E. Influence of Structure
and Composition of Woven Fabrics
on the Conductivity of Flexography
Printed Electronics. Polymers 2021, 13,
3165. https://doi.org/10.3390/
polym13183165 Keywords: flexography; e-textiles; wearables; printed-electronics; textiles; electronic textiles Received: 28 August 2021
Accepted: 15 September 2021
Published: 18 September 2021 References Fast growth of carbon nanotubes using a microwave oven. Carbon 2015, 82, 32 y
R.; Wagner, H.D. Fast growth of carbon nanotubes using a microwave oven. Carbon 2015, 82, 327–336. [CrossR 39. He, S.; Sun, G.X.; Cheng, X.D.; Dai, H.M.; Chen, X.F. Nanoporous SiO2 grafted aramid fibers with low thermal conductivity. Compos. Sci. Technol. 2017, 146, 91–98. [CrossRef] 40. Li, Y.; Luo, Z.; Yang, L.; Li, X.; Xiang, K. Study on Surface Properties of Aramid Fiber Modified in Supercritical Carbon Dioxide by
Glycidyl-POSS. Polymers 2018, 11, 700. [CrossRef] 41. Shebanov, S.M.; Novikov, I.K.; Pavlikov, A.V.; Ana´nin, O.B.; Gerasimov, I.A. IR and Raman Spectra of Modern Aramid Fibers. Fibre Chem. 2016, 48, 158–164. [CrossRef] 42. Mosadegh, M.; Mahdavi, H. Chemical Functionalization of Graphene Oxide by Poly(styrene sulf
Radical and Free Radical Polymerization: A Comparative Study. Polym. Plast. Technol. Eng. 2017, 56 42. Mosadegh, M.; Mahdavi, H. Chemical Functionalization of Graphene Oxide by Poly(styrene sulfona
Radical and Free Radical Polymerization: A Comparative Study Polym Plast Technol Eng 2017 56 12 Mahdavi, H. Chemical Functionalization of Graphene Oxide by Poly(styrene sulfonate) Using Atom Trans
e Radical Polymerization: A Comparative Study. Polym. Plast. Technol. Eng. 2017, 56, 1247–1258. [CrossRef] adegh, M.; Mahdavi, H. Chemical Functionalization of Graphene Oxide by Poly(styrene sulfonate) Using A
cal and Free Radical Polymerization: A Comparative Study. Polym. Plast. Technol. Eng. 2017, 56, 1247–1258. [C g
p
y
y( y
)
Radical and Free Radical Polymerization: A Comparative Study. Polym. Plast. Technol. Eng. 2017, 56, 1247–125 43. Raja, P.M.V.; Barron, A.R. IR Spectroscopy. 2019. Available online: https://chem.libretexts.org/@go/page/167013 (accessed on
3 June 2021). J
)
44. Wang, C.; Liu, C.R. Transcrystallization of polypropylene composites: Nucleating ability of fibres. Polymer 1999, 40, 289–298. [CrossRef] 45. Beter, J.; Schrittesser, B.; Fuchs, P.F. Investigation of adhesion properties in load coupling applications for flexible composites. Mater. Today Proc. 2020, 34, 41–46. [CrossRef] 90 polymers 1. Introduction Printed electronics (PE) refers to the technology that allows electronic device man-
ufacturing through a printing process. PE is one of the fastest growing technologies in
the world as it provides different printing techniques for manufacturing low-cost and
large-area flexible electronic devices [1]. In recent years, flexible electronics technology
has attracted considerable attention since it can be applied to wearable devices includ-
ing flexible displays, flexible batteries and flexible sensors [2,3] in different areas such as
aerospace and automotive, biomedical, robotics, and health applications [4]. Among them,
wearable electronic textiles (e-textiles) are of great significance since they provide better
comfortability, durability and lighter weight as well as maintaining desirable electrical
properties [5]. Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article
distributed
under
the
terms
and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). The PE printing technique choice must be done according to the electronic application
(e.g., small, thin, lightweight, flexible, and disposable, etc.), the manufacturing cost and
volume. Additionally, the main materials (inks/pastes and substrates) must meet certain
requirements, depending on the printing technology selected and the final application. 91 https://www.mdpi.com/journal/polymers Polymers 2021, 13, 3165. https://doi.org/10.3390/polym13183165 Polymers 2021, 13, 3165 PE technologies are divided into contact techniques (e.g., flexography, gravure printing
and soft lithography techniques), where the printing plate is in direct contact with the
substrate and non-contact techniques (e.g., screen printing, aerosol printing, inkjet printing,
laser direct writing), where only the ink contacts the substrate [6]. Those techniques suitable
for roll-to-roll (R2R) processing, such as flexography, are especially attractive since they
offer continuous manufacturing and high productivity [7]. Flexographic printing is known for depositing a wide range of thicknesses with the
same resolution. The impression cylinder, plate cylinder, anilox roller, doctor blade and
inking unit are the main parts of the flexographic printing process [1], as illustrated in
Figure 1. Variables associated with the flexographic printing process include print speed,
print force/engagement, anilox cell volume, anilox force/engagement as well as the ink and
substrate properties [8]. Those variables have a direct impact on the print’s morphological
and electrical behavior. The print uniformity has a considerable influence on the final
functionality of the device [9]. 1. Introduction Topographical
characteristics of the fabrics strongly depend on their construction parameters such as
the type and fineness of filaments, yarn fineness, yarn density, and the type of weave. These characteristics have strong influence on, and in many cases, control the wetting
properties [18]. It is important that printing ink has good adhesion to the substrate [19]. This be-
comes particularly complex in printing electronics onto fabrics where the intrinsic porous
structures and texture characteristic of textiles affects the diffusion and penetration of
conductive ink, being able to deteriorate the printing precision and electrical performance
of conductive lines [20]. Related to the fabric structure, it was proved in other printing techniques, such as
screen-printing, that the smallest pore size and roughness shows a higher printing precision
and lower electrical resistance of printed conductive lines [20]. According to material composition, natural fibers such as cotton tend to absorb the
ink more readily than synthetic fibers such as polyester, due to hydrophilic and wettability
properties of cellulosic fibers [21]. Generally, synthetic textile materials have smooth, tight
surfaces that offer little texture for ink adhesion [19]. However, there are not systematic investigations about the relationship between the
electrical performance of a flexo printed textile with the structure of the woven fabric
(i.e., interlacing coefficient, yarn count and weft density) as well as with the material
composition of the weft. With the aim of establishing this relation, different textiles were specifically man-
ufactured varying their structural parameters and materials. After that, a controlled
flexographic printing process was performed by using a silver electrical ink. Finally, fabrics
were physically and electrically examined and compared. The research and the obtained results are presented from two different approaches. On the one hand, an approach is followed from a perspective focused on distinguishing the
impact of the structural parameters of the woven fabric on the conductivity of the printing. Structural parameters include, but are not limited to, weave construction, interlacing
coefficient, yarn count and fabric density. On the other hand, from a point of view based
on establishing the influence of the weft material composition on the conductivity, a
comparison between fabrics with different weft materials (cotton, polyester, and wool) was
also performed. Physical and electrical analyses were carried out for both approaches. Several method-
ologies were used including optical, FE-SEM, color fastness to wash, and color measure-
ment using reflection spectrophotometer or multi-meter analyses. 1. Introduction Within the context of printing electronics onto fabrics, it
must be highlighted the challenge of durability and withstanding bending, stretching,
abrasion and washing [10]. Figure 1. Significant parts of the flexography process: inking unit (1); anilox roller (2); doctor blade
(3); plate cylinder (4); impression cylinder (5); printing substrate (6). Figure 1. Significant parts of the flexography process: inking unit (1); anilox roller (2); doctor blade
(3); plate cylinder (4); impression cylinder (5); printing substrate (6). Numerous reviews and books were previously published, considering printed elec-
tronics on substrates such as glass, metal, paper or polymers [4,7]. Different studies were
focused on the application of printed electronics onto textiles to obtain electrical devices
such as capacitive sensors [11], perovskite solar cells [12] or RFID tags [13], but by using
other printing techniques, such as ink jet or screen printing which are more common. However, the incorporation of the flexography printing technique for printed electron-
ics in the textile field is still very recent and there are not enough studies for its application. Continuing our previous work [14], this research is focused on the influence of the textile
substrate parameters on the electrical performance of the printing. The most used textile type for garments and even for technical applications is woven
fabrics, where two sets of perpendicular yarns are crossed and interweave each other to
create a coherent and stable structure [15]. In the context of this study, the term ‘structure’
refers to binding patterns of interlacing threads in woven fabrics, also considering internal
structural features of the threads involved. Therefore, the structure of the woven fabric is
determined, among other factors, by the weave construction, the interlacing coefficient, the
density of threads in the fabric and the characteristics of warp and weft threads [16]. All the
other physical (crimp, thickness, physical density, mass of unit area, porosity, etc.), mechan-
ical (breaking strength and elongation, tensity, forces of rupture, resistance to abrasion, etc.)
and permeability properties (permeabilities of gasses, liquids, light, sound, energy, water
vapor, bacteria, etc.) are affected by previously mentioned selection [17]. Fabric texture and 92 Polymers 2021, 13, 3165 composition affect the porosity and strongly influence the textile characteristics such as
the fabric mass, thickness, draping ability, stress–strain behavior, or air permeability. The
surface topography of fabrics is responsible for their functionality—appearance and handle,
wettability, soiling behavior and cleanability, abrasion resistance and wear. 1. Introduction Significant results were
obtained and therefore studied. It is believed that these findings will provide some important support for printing
electronic devices on woven textiles by using the flexographic technique. 2. Materials and Methods
2.1. Materials 2.1. Materials 2.1. Materials With the aim of establishing the influence of structure and composition of woven
fabrics on the conductivity of flexographic printed electronics, eleven textile types were
specifically manufactured for the research (Textiles Joyper S.L., Cocentaina, Spain), to be
used as substrates for the printing. On the one hand, eight different textiles structures were defined by varying the
following constructional parameters: weave construction, weft yarn count, and weft
density. On the other hand, textiles alternatives of the material used for the weft were
added into the composition of three fabrics while maintaining the fabric structure. 93 Polymers 2021, 13, 3165 Table 1 shows the main characteristics of these fabrics, where test samples from T1–T8
correspond to textile structure variations and textiles from T9–T11 to alternative weft
materials. It should be noted that polyester yarn was used in the warp in all samples. Table 1. Textile substrate’s characterization (I): composition and ligament. Substrate Code
3D Modeling
Weft
Composition
Weave
Construction
Graphic
Representation
Interlacing
Coefficient (KL)
T1
Polyester
Plain
1
T2
Polyester
Plain
1
T3
Polyester
Plain
1
T4
Polyester
Plain
1
T5
Polyester
Twill
0.4
T6
Polyester
Twill
0.4
T7
Polyester
Twill
0.4
94 Table 1 shows the main characteristics of these fabrics, where test samples from T1–T8
correspond to textile structure variations and textiles from T9–T11 to alternative weft
materials. It should be noted that polyester yarn was used in the warp in all samples. Table 1 shows the main characteristics of these fabrics, where test samples from T1–T8
correspond to textile structure variations and textiles from T9–T11 to alternative weft
materials. It should be noted that polyester yarn was used in the warp in all samples. 94 94 Polymers 2021, 13, 3165 Table 1. Cont. Substrate Code
3D Modeling
Weft
Composition
Weave
Construction
Graphic
Representation
Interlacing
Coefficient (KL)
T8
Polyester
Twill
0.4
T9
Polyester
Twill
0.4
T10
Cotton
Twill
0.4
T11
Wool
Twill
0.4
The weave construction of textile materials is the regular structure produced by a
pattern (unit cell) of interlaced threads repeating at regular intervals in two transversal di-
rections [22]. The weave interlacing coefficient, KL, which depends on weave construction, The weave construction of textile materials is the regular structure produced by a
pattern (unit cell) of interlaced threads repeating at regular intervals in two transversal di-
rections [22]. 2.1. Materials Substrate Code
Warp Density
(ends/cm)
Weft Density
(picks/cm)
Weft Count
(dtex)
Cover Factor
(%)
Fabric Weight 1
(g/m2)
Thickness
(μm)
T1
58.0
10.5
333.3
84.8
154
515
T2
58.8
15.3
333.3
93.6
171
550
T3
58.4
10.7
666.7
92.9
191
622
T4
57.2
15.8
666.7
103.4
233
650
T5
59.2
10.6
333.3
64.5
160
705
T6
55.9
16.0
333.3
68.3
180
725
T7
57.3
11.6
666.7
70.0
222
744
T8
56.3
16.5
666.7
77.6
241
805
T9
59.4
15.9
666.7
79.3
235
800
T10
58.8
16.2
666.7
78.5
230
795
T11
60.4
16.8
666.7
82.1
225
790
1 Mass per unit area determined according to the standard ISO 3801 Table 2. Textile substrate’s characterization (II): size and weight characteristics. Therefore, the structure variations among samples from T1–T8 are the following: the
weave construction, as samples from T1–T4 are plain and T5–T8 are twill; the yarn count
used in for the weft, which is thicker in samples T4, T5, T7 and T8; and the weft density,
which is lower in samples T1, T3, T5 and T7. Meanwhile, while maintaining the structure parameters, the difference among samples
T9–T11 is the weft composition, which is made from polyester in T9, from cotton in T10
and from wool in T11. For the research, other structural parameters have been accordingly obtained to
complete the characterization of the woven fabric samples. Cover factor (the degree of
fabric fullness) is the proportion of the fabric area covered by warp and weft yarns. It
means that in practice, cover factor is calculated independently for warp and weft yarn by
the proportion of fabric area covered by those yarn, according to Equation (2): Cf = Cfwa + Cfwe −Cfwa × Cfw
(2) (2) Cf = Cfwa + Cfwe −Cfwa × Cfw Cf = Cfwa + Cfwe −Cfwa × Cfw as Cfwa = Dwa × dwa and Cfwe = Dwe × dwe, where Dwa and Dwe are densities of
warp and weft and dwa and dwe are the diameter of warp and weft yarns, respectively. The cover factor directly depends on the yarn density and the yarn count [26]. Finally, the
fabric weight was obtained according to the standard ISO 3801 by measuring the textile
mass per unit area. p
Regarding the ink, flexographic printing technology requires low-viscosity printing
inks, which allows regular ink flow in the printing unit. Viscosity is generally lower than
0.05–0.5 Pa·s [27]. 2.1. Materials The weave interlacing coefficient, KL, which depends on weave construction,
is calculated by Equation (1): The weave construction of textile materials is the regular structure produced by a
pattern (unit cell) of interlaced threads repeating at regular intervals in two transversal di-
rections [22]. The weave interlacing coefficient, KL, which depends on weave construction,
is calculated by Equation (1): KL =
i
w1 × w2
(1) (1) where i is the number of interlacing points in the weave repeat, w1 is the number of ends
in the weave repeat, and w2 is the number of picks in the weave repeat [23]. The research
was focused on the two most traditional and commonly used weaves—plain and twill. The plain weave is the basic weave where one warp yarn is lifted over one weft yarn. The
interlacing is opposite in all neighboring cells. Plain weave allows the highest possible
number of interlacing. The twill weave has a pattern of diagonal lines; each warp yarn lifts
over more than one weft, so the diagonal lines in fabric reach high densities [24]. Table 2 completes the main characteristics of the fabrics, adding the characteristics of
the textile yarns in a detailed way for a better understanding. Yarn number or yarn count refers to the thickness of a yarn and it is determined by
its mass per unit length [25]. The fabric samples used in this study were produced by
variation of the weft yarn count (167 dtex/333 dtex), without changing the warp count
(167 dtex). The density of the warp and weft is defined by the number of warps ends per cm and
the number of picks per cm. 95 Polymers 2021, 13, 3165 Table 2. Textile substrate’s characterization (II): size and weight characteristics. 1 Mass per unit area determined according to the standard ISO 3801. 2.2. Flexographic Electronic Printing 2.2. Flexographic Electronic Printing The manufacturing technology that is used is based on the flexographic printing
technique of thick film. Flexography is a roll-to-roll direct printing technology, where an
anilox roller, covered with micro-cavities on its surface, allows the collection of ink, and
then is transferred to the printing plate cylinder. The specification of the anilox determines
the volume of ink transferred to the printing plate. The ink is taken into these cells and the
excess ink is subsequently removed by a doctor blade assembly. For the research, one-layer flexographic prints were performed by using a printing
experimental plant (K Printing Proofer, RK Print Coat Instruments Ltd., Litlington, United
Kingdom). The equipment allows high quality proofs using flexography, among other
printing techniques, with variable printing speeds of up to 40 m/min. Printing plates for
use with the experimental plant are electronically engraved in exactly the same way as
production cylinders. Using the flexo head, ink is transferred from the printing plate to a
plain stereo roller and then onto the substrate. Adjustments can be made by micrometers. As developing a specific electronic device is out of the scope of this research, a plain design
has been used for the test; nevertheless the printing plate and the printing roller in the
industrial machine could be customized. The experimental phase of this research consisted of the flexo-printing of the woven
fabrics shown in Tables 1 and 2 using the silver ink described in Table 3. With the objective
of studying the influence of the textile substrates on the conductivity of the printing, the
equipment settings were kept fixed so that they did not interfere with the results. The
specific setup conditions are shown in Table 4. Printed layers were dry cured in a FED-
115 air oven from BINDER at 140 ◦C for one minute in order to use the same curing
characteristics for all the samples. Moreover, previous to the printing, a thermic treatment
was applied to all the fabrics to avoid variations of size due to the curing temperature of
inks. The thermic treatment applied consisted of introducing the fabrics in the same oven
at 130 ◦C for 15 min. Table 4. Printing parameters. Ink
Anilox Volume
Resolution
Printed Area
Speed
Curing
PFI-RSA6012—Silver ink
11 cm3/m2
150 LPI
150 × 95 mm2
12 m/min
60 s
140 ◦C Table 4. Printing parameters. 2.1. Materials Same aqueous flexo-printable conductive ink, PFI-RSA6012—silver ink
from Novacentrix (Austin, United States), was used in all prints to ensure comparable
results. Details can be consulted in Table 3. The selection was made considering high
conductivities and stretching properties for printing electronics on flexible substrates. It
should be highlighted that there is a low supply on the market due to the novelty of
the application of printed electronics in the textile through the flexography technique as
stated above. Table 3. PFI-RSA6012—Silver ink characteristics. Ink Code
Density
(g/mL)
Solids
(%)
Viscosity (Pas)
Volume Resistivity
(μΩ·cm)
Curing
Properties
PFI-RSA6012—
Silver ink
2.22
60 (±2)
0.05–0.15 @1000 s−1
8–10
10–60 s
140 ◦C
• Flexible
• Compatible with Polyester Table 3. PFI-RSA6012—Silver ink characteristics. 96 Polymers 2021, 13, 3165 The ink contains silver nanoparticles and was formulated for high conductivity, fast
curing, and improved levelling at lower printing speed. 2.3. Characterization Once dried, the printed textile samples were physically and electrically analyzed by
carrying out several studies. Regarding physical characterization, the following measurement methods were used:
optical, scanning electron microscopy (FE-SEM) (Oxford Instruments plc, Abingdon,
United Kingdom), and color fastness to wash and color measurement using reflection
spectrophotometer. The optical macroscopic images were taken with a LEICA MZ APO stereomicroscope. It was used to analyze the print uniformity of each layer of the electronic printed samples. High-resolution topographic images by SE (secondary electrons) and maps of crys-
talline and textural orientations by EBSD (electron backscatter diffraction) (Oxford Instru-
ments plc, Abingdon, United Kingdom) were taken with a ZEISS ULTRA 55 Scanning
Electron Microscope Field Emission Gun (field emission scanning electron microscopy
(FE-SEM))) (Oxford Instruments plc, Abingdon, UK). They were used to analyze the ink
penetration and the adhesion in each substrate. Color fastness to domestic and commercial laundering was evaluated with a Gy-
rowash according to the standard method EN ISO 105-C06:2010. The test conditions were: 97 Polymers 2021, 13, 3165 temperature of 40 ◦C, 10 steel balls and standardized ECE soap reference without optical
or chemical whitener. After the test, the printed woven textiles were dried in a forced-air
circulation dryer and treated samples were compared with untreated samples visually
using a grey scale, according to ISO 105-A02 standard. Color measurement was evaluated by the determination of CIELAB coordinates
according to the standard method ISO 105-J01:1997. The apparatus used was DATACOLOR
DC 650 (400–700 nm) (Datacolor, New Jersey, United States) with the following conditions:
illuminant D65/10◦, diffuse measuring geometry and 6.6 mm of observation area. With respect to electrical characterization, a usual two-terminal sensing unit was firstly
considered to measure the conductivity behavior. Nevertheless, a two-wire system does
not provide correct output due to variation in ambient temperature, as the resistance of
the lead wires (both sides) changes unpredictably. Meanwhile, 4-wire Kelvin resistance
measurement makes it possible to accurately measure resistance values less than 0.1 Ω
while eliminating the inherent resistance of the lead wires connecting the measurement
instrument to the component being measured [28]. For that reason, 4-wire system measure-
ments were made. Resistance measurements were made with a FLUKE 8845A multimeter
from FLUKE CORPORATION (Everett, WA, USA). 3.1. Electrical Characterization For the purpose of determining the electrical conductivity of the printings an approach
based on measuring the electrical resistance was considered. A low resistivity indicates
a material that readily allows electric current. Table 5 shows a summary of the printing
results for all the fabrics in terms of electrical resistivity. Four different samples of each
fabric were measured. The orientation of the measurements on the woven fabrics were
separately considered. In addition, results are graphically shown in Figure 2. Table 5. Electrical resistance of the printings. Measurements have been made both in warp and weft
directions. Table 5. Electrical resistance of the printings. Measurements have been made both in warp and weft
directions. Substrate Code
Resistance (Ω)
Warp Direction
Resistance (Ω)
Weft Direction
T1
0.4
0.3
T2
0.5
0.4
T3
0.4
0.5
T4
1.8
0.6
T5
0.4
1.1
T6
0.4
0.5
T7
0.5
0.4
T8
7
8.6
T9
7
8.6
T10
22.3
2.5
T11
0.3
0.4 Regarding the influence of the textile structure on the electrical behavior shown in
Figure 2a, both measures on warp and weft directions agree that the woven fabric that
presents the higher electrical resistance, and therefore worse electrical conductivity, is the
substrate coded as T8. T8 was characterized by a lower interlacing coefficient and the
highest mass per unit area. These results coincide with the conclusions about the surface properties of woven
fabric on electrical performance through a screen-printed technique. The fabric substrate
with the smallest pore size and roughness shows a higher printing precision and lower
electrical resistance of screen-printed conductive lines [20]. Therefore, the surface structure
of the fabric substrate determines to some degree, not only the printing precision of
conductive lines, but its electrical properties as well. 98 Polymers 2021, 13, 3165 Figure 2. Graphic representation of the electrical resistance of the printings (
) measured on warp direction and
(
)measured on weft direction. (a) Electrical resistance affected by the structural variations of the textiles; and (b) Electrical
resistance affected by the material composition of the textiles. Figure 2. Graphic representation of the electrical resistance of the printings (
) measured on warp direction and
(
)measured on weft direction. (a) Electrical resistance affected by the structural variations of the textiles; and (b) Electrical
resistance affected by the material composition of the textiles. 3.1. Electrical Characterization In order to address these challenges, surface pre-treatment onto rough and porous
substrates or coatings and lamination processes should be performed in order to produce a
continuous conductive pathway onto the textiles [5,10]. p
y
In order to deeply explore the relation found between woven fabric density and
conductivity, results were plotted, as shown in Figure 3. Figure 3. Graphic representation of the relation between electrical resistance of the printings mea-
sured on warp direction (
) and the mass per unit area (
). Figure 3. Graphic representation of the relation between electrical resistance of the printings mea-
sured on warp direction (
) and the mass per unit area (
). According to results shown in Figure 3, with regard to the influence of the textile
structure on the electrical behavior, it could be expected that the higher the mass per unit,
the lower conductivity due to the ink expansion through the inner part of the textile. Moreover, Figure 2b allows the analysis in terms of the impact of the textile composi-
tion on the electrical behavior of electronic printing through the flexographic technique. Again, resistance measures in both directions agree and show that the woven fabric T10
presents higher electrical resistance. Even though the measure on the warp direction shows 99 Polymers 2021, 13, 3165 better electrical conductivity, it is worse than the other textile samples made from other
materials. The weft of T10 was made from cotton. This makes sense, because of the mois-
ture absorption capacity of cellulosic fiber. In this way it can be demonstrated that cotton
performs worse as the weft material for printing electronics with the flexo technique. An alternative solution for improving conductivity on printed electronic textiles with
similar problems would be pre-treatment of the surface substrates by applying different
chemical compounds that increase the adhesion of the ink avoiding its absorption. 3.2. Physical Characterization Focusing on analyzing the print uniformity of each layer of the electronic flexo printed
woven textiles, optical macroscopic images were taken with 20 magnifications. In parallel,
in order to discuss the ink penetration and the adhesion in each substrate, high-resolution
topographic images were taken with Scanning Electron Microscope Field Emission Gun
(FE-SEM). Table 6 shows the images obtained for the Optical and FE-SEM characterization of
the woven substrates with different textile structures. The FE-SEM (×100) images show a
visual characterization of the fabric and ink, and the FE-SEM (×150) images on the right
show maps of crystalline and textural orientations by EBSD for a determination of the
position of the silver particles. Table 6. Optical and FE-SEM characterization (I). Substrate
Code
Optical
(×20)
FE-SEM
(×100)
FE-SEM
(×150)
T1
T2
T3 Table 6. Optical and FE-SEM characterization (I). Table 6. Optical and FE-SEM characterization (I). FE-SEM
(×100) T2 T2 T3 T3 100 Polymers 2021, 13, 3165 Table 6. Cont. Substrate
Code
Optical
(×20)
FE-SEM
(×100)
FE-SEM
(×150)
T4
T5
T6
T7
T8
Regarding optical results, images in Table 6 show a total color uniformity on the
substrates’ surface not depending on the textile structure. The silver shade obtained is
101 Substrate
Code
Optical
(×20)
T4 T5 T5 T6 T6 T7 T7 T8 T8 Regarding optical results, images in Table 6 show a total color uniformity on the
substrates’ surface not depending on the textile structure. The silver shade obtained is Regarding optical results, images in Table 6 show a total color uniformity on the
substrates’ surface not depending on the textile structure. The silver shade obtained is 101 Polymers 2021, 13, 3165 typical from conductive inks which contains silver nanoparticles. In addition, the images
reveal the porosity of the woven fabrics as they show their characteristic textiles holes,
being different for each structure. It should be highlighted the picture of the substrate T8
as it is the one with more holes between the yarns forming the woven fabric, in comparison
with the other samples. In respect of FE-SEM results, thanks to images of the cross section of the substrates
shown in Table 6, it can be observed how weft threads with different titles and densities
are intertwined with the warp threads. These pictures reveal the enclaves formed by
the interlacing of the threads in both directions, obtaining a roughness of the fabric and
presenting different heights. 3.2. Physical Characterization In order to address these challenges in woven textiles with natural fibers, surface pre-
treatment should be done in order to improve the ink adhesion and therefore the electrical
behavior [5]. It has been proved that special pretreatment processes on the fabric substrates
103 Table 8. Color fastness results (I). Substrate Code
Change in Color
Staining
Wool
Acrylic
Polyester
Polyamide
Cotton
Acetate
T1
4–5
4–5
4–5
4–5
4–5
4–5
4–5
T2
4–5
4–5
4–5
4–5
4–5
4–5
4–5
T3
4–5
4–5
4–5
4–5
4–5
4–5
4–5
T4
4
4–5
4–5
4–5
4–5
4–5
4–5
T5
4–5
4–5
4–5
4–5
4–5
4–5
4–5
T6
4–5
4–5
4–5
4–5
4–5
4–5
4–5
T7
4–5
4–5
4–5
4–5
4–5
4–5
4–5
T8
4
4–5
4–5
4–5
4–5
4–5
4–5
Table 9. Color fastness results (II). Substrate Code
Change in Color
Staining
Wool
Acrylic
Polyester
Polyamide
Cotton
Acetate
T9
4
4–5
4–5
4–5
4–5
4–5
4–5
T10
1
4
4
4
4
4
4
T11
1
4
4
4
4
4
4 Table 8. Color fastness results (I). Table 9. Color fastness results (II). For assessing the change in color, woven treated textiles were compared with a grey
scale complying with ISO 105-A02. Regarding assessing staining, the grey scale used
follows ISO 105-A03. Both scales consist of five pair of non-glossy grey color chips which
illustrate the perceived color differences corresponding to fastness rating 5, 4, 3, 2 and 1. A
range of 5 is given only when there is no perceived difference between the tested specimen
and the original material. According to results shown in Table 8, obtained values for change in color and staining
are between 4 (Very Good) and 5 (Excellent). Therefore, it can be concluded that structural
variations have not a relevant influence on the color fastness of the flexo electronic printings. p
g
However, results shown in Table 9 demonstrate that color fastness decreases with
natural fibers such as cotton and wool. The reason can be that ink adhesion improves in in
synthetic fibers such as polyester thanks to the curing time after the flexography printing
using 150 ◦C, as this type of fiber is a thermoplastic material and being its glass transition
temperature around 70 ◦C [30]. In order to address these challenges in woven textiles with natural fibers, surface pre-
treatment should be done in order to improve the ink adhesion and therefore the electrical
behavior [5]. 3.2. Physical Characterization Depending on the structure and yarn title, the enclave formed
is different and it directly affects to the fabric thickness. As already stated on the fabrics’
characterization in Table 2, the FE-SEM (×100) images in Table 6 confirm the thickness is
greater for fabrics whose ligament is sarge (substrates T5–T8). Furthermore FE-SEM (×150) results with more magnification allow to observe the ink
penetration and distribution in the inner part of the textile. Images demonstrate that there
are particles of the silver ink between the fibers of the weft threads. p
Meanwhile, Table 7 shows the images obtained for the Optical and FE-SEM characteri-
zation of the woven substrates with different materials on the weft. Table 7. Optical and FE-SEM characterization (II). Substrate
Code
Optical
(×20)
FE-SEM
(×100)
FE-SEM
(×150)
T9
T10
T11
Regarding ink uniformity, all the woven fabrics present a good visual level at the
print in spite of the porous surface. To ensure this uniformity in printing electronics with Table 7. Optical and FE-SEM characterization (II). T9 T9 T10 T10 T11 Regarding ink uniformity, all the woven fabrics present a good visual level at the
print in spite of the porous surface. To ensure this uniformity in printing electronics with 102 Polymers 2021, 13, 3165 low viscosity inks onto a rough and porous textile surface is a great challenge, due to the
orientation of fibers or yarns and the change of fiber morphology constantly [5]. y
g
gy
y
With respect to ink penetration, the images in Table 7 show that ink does not remain
at the surface, but penetrates until the lower side of the textile. Thus, results coincide
with the relation pointed out in Figure 3 about the influence of the mass per unit area on
the electrical behavior. Whereas conventional printing that uses thickeners to increase
the viscosity of the paste achieve deposition only on the surface, the low ink viscosity
used in flexo printing penetrates to the inner of the textile, the interior of the textile. Thus,
the topography of the sample plays an important role in achieving the homogeneity and
continuity of the ink. Therefore, it is demonstrated that the higher the mass per unit, the
lower conductivity due to the ink expansion through the inner part of the textile. 3.2. Physical Characterization y
p
g
p
In terms of the impact of the material of the weft, the images shown in Table 7 do not
show significant differences among them. The color loss and staining resulting from desorption and/or abrasive action of the
samples was evaluated according to EN ISO 105-C06:2010 tests for color fastness [29]. The
grade of color fastness to domestic and commercial laundering is presented in Tables 8 and 9. g
g
p
Table 8. Color fastness results (I). Substrate Code
Change in Color
Staining
Wool
Acrylic
Polyester
Polyamide
Cotton
Acetate
T1
4–5
4–5
4–5
4–5
4–5
4–5
4–5
T2
4–5
4–5
4–5
4–5
4–5
4–5
4–5
T3
4–5
4–5
4–5
4–5
4–5
4–5
4–5
T4
4
4–5
4–5
4–5
4–5
4–5
4–5
T5
4–5
4–5
4–5
4–5
4–5
4–5
4–5
T6
4–5
4–5
4–5
4–5
4–5
4–5
4–5
T7
4–5
4–5
4–5
4–5
4–5
4–5
4–5
T8
4
4–5
4–5
4–5
4–5
4–5
4–5
Table 9. Color fastness results (II). Substrate Code
Change in Color
Staining
Wool
Acrylic
Polyester
Polyamide
Cotton
Acetate
T9
4
4–5
4–5
4–5
4–5
4–5
4–5
T10
1
4
4
4
4
4
4
T11
1
4
4
4
4
4
4
For assessing the change in color, woven treated textiles were compared with a grey
scale complying with ISO 105-A02. Regarding assessing staining, the grey scale used
follows ISO 105-A03. Both scales consist of five pair of non-glossy grey color chips which
illustrate the perceived color differences corresponding to fastness rating 5, 4, 3, 2 and 1. A
range of 5 is given only when there is no perceived difference between the tested specimen
and the original material. According to results shown in Table 8, obtained values for change in color and staining
are between 4 (Very Good) and 5 (Excellent). Therefore, it can be concluded that structural
variations have not a relevant influence on the color fastness of the flexo electronic printings. However, results shown in Table 9 demonstrate that color fastness decreases with
natural fibers such as cotton and wool. The reason can be that ink adhesion improves in in
synthetic fibers such as polyester thanks to the curing time after the flexography printing
using 150 ◦C, as this type of fiber is a thermoplastic material and being its glass transition
temperature around 70 ◦C [30]. 3.2. Physical Characterization It has been proved that special pretreatment processes on the fabric substrates 103 Polymers 2021, 13, 3165 improve the wash fastness for other printing techniques such as digital printing [31] or
ink-jet [32]. According to a previous review [33], an increment of ink volume improves
the ink coverage, upgrading in this case the conductivity, nevertheless it enhances the ink
wash-out effect. For this reason, the ink volume transferred to the substrate should be
optimized when conductivity and color fastness to washing are the objectives. In addition,
coating and lamination processes could be done to ensure the continuous conductive
pathway on textiles [10]. Spectrophotometric methods are adequate and objective for determining the color
values of the fabric surface [34]. To assess variations found on the printings, measurements
have been performed according to ISO 105-J01:1997 General principles for measurement of
surface color [35]. The CIELAB, or CIE L* a* b*, color system represents quantitative relationship of
colors on three axes: L*value indicates lightness, and a* and b* are chromaticity coordinates. It is the most widely used method for measuring and ordering object color. The results
of the analysis of color of the printings onto the woven fabrics are shown in Table 10. For
a better understanding, results have been plotted and can be consulted at the Figure 4. On
the color space diagram, L* is represented on a vertical axis with values from 0 (black) to
100 (white). The a* value indicates red-green component of a color, where +a* (positive)
and −a* (negative) indicate red and green values, respectively. The yellow and blue
components are represented on the b* axis as +b* (positive) and −b* (negative) values,
respectively. At the centre of the plane is neutral or achromatic [36]. Figure 4. Two-dimensional CIELAB (+L*, +a *, +b*) coordinates representation. (a) CIELAB lightness (+L*) of textile
structure variations T1–T8; (b) CIELAB a* and b* coordinates of textile structure variations T1–T8; (c) CIELAB lightness
(+L*) of textile material variations T9–T11; (d) CIELAB a* and b* coordinates of textile material variations T9–T11. Figure 4. Two-dimensional CIELAB (+L*, +a *, +b*) coordinates representation. (a) CIELAB lightness (+L*) of textile
structure variations T1–T8; (b) CIELAB a* and b* coordinates of textile structure variations T1–T8; (c) CIELAB lightness
(+L*) of textile material variations T9–T11; (d) CIELAB a* and b* coordinates of textile material variations T9–T11. 104 Polymers 2021, 13, 3165 Table 10. CIELAB* coordinates (D65/10◦). Author Contributions: Conceptualization, J.F., E.G.-B., I.M., E.B.-B. and A.M.R.-C.; methodology,
E.G.-B., I.M., E.B.-B. and A.M.R.-C.; validation, J.F., E.G.-B., I.M., E.B.-B. and A.M.R.-C.; formal
analysis, A.M.R.-C.; investigation, J.F., E.G.-B., I.M., E.B.-B. and A.M.R.-C.; writing—original draft
preparation, E.G.-B., I.M., E.B.-B. and A.M.R.-C.; writing—review and editing, E.G.-B., I.M., E.B.-
B. and A.M.R.-C.; supervision, E.G.-B., I.M. and E.B.-B. All authors have read and agreed to the
published version of the manuscript. 3.2. Physical Characterization Substrate Code
L*
a*
b*
T1
50.19
1.56
11.04
T2
48.86
2.36
11.48
T3
58.07
1.21
12.36
T4
47.78
1.49
9.36
T5
51.88
0.83
8.49
T6
54.29
1.37
8.86
T7
51.58
0.87
7.71
T8
45.95
2
9.19
T9
45.95
2
9.19
T10
48.91
−3.55
5.53
T11
55.58
−4.24
6.19 Lower L* indicates that the sample becomes darker, and all printed samples show
low light (L*) values near to 50. It is observed that there is no correlation between the
conductivity and the tone obtained from the coordinates a* and b* in contrast to silver
coatings of polymeric substrates, where it is demonstrated that s shifting in the a* and b*
during sintering means best conductivity [37]. On the other hand, it is observed that the
sample T8/T9, the one with more weight and less conductivity, shows the lowest L* value,
being darker than the other samples. This fact corroborates firstly, that variations on the
fabric density have an influence on the color values [34], and secondly that the penetration
of the silver particles into the textile, when the silver particles remain on the surface, thanks
to emulsion resins, present values of L* between 60–70 [38]. 4. Conclusions It was proved that a direct relationship exists between the electrical performance of a
flexographic printed textile with the structure of the woven as well as with the material
composition of the weft. After performing controlled flexographic printing processes onto woven fabrics by
using a silver electrical ink, physical and electrical analysis led to the following conclusions. On the one hand, from a perspective focused on the structural parameters, the weave
construction, interlacing coefficient, yarn count and fabric density, there was an effect due
to the higher mass per unit of the fabric and lower conductivity due to the ink expansion
through the inner part of the textile. On the other hand, based on the weft material
composition (cotton, polyester, and wool), it was shown that cotton performs worse as weft
material for printing electronics with the flexo technique due to the moisture absorption
capacity of cellulosic fiber. It is believed that these findings will provide some important support for printing
electronic devices on woven textiles by using the flexographic technique. y
g
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p
q
The study’s next steps will consist of improving the conductivity of flexographic
printed textile by carrying out surface pre-treatment, coating and lamination processes that
increase the ink adhesion and therefore the electrical behavior. 5. Patents FERRI, J.; RODES-CARBONELL, A.M. and MORENO, J. (2020). Dispositivo NFC flex-
ible para la medición, almacenamiento y transmisión de datos (Spain U202130440) http://
www.oepm.es/pdf/ES/0000/000/01/26/48/ES-1264864_U.pdf (accessed on 12 April 2021). 105 Polymers 2021, 13, 3165 Funding: This work was supported by the Spanish Government/FEDER funds [Ministerio de
Economía y Empresa (MINECO)/Fondo Europeo de Desarrollo Regional (FEDER)] under Grant
RTI2018-100910-B-C43. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the
corresponding author. Data Availability Statement: The data presented in this study are available on request from the
corresponding author. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results. References Chemistry of solid metal-based inks and paste nchev, Z.Z.; Cruz, S.F.; Viana, J.C. Chemistry of solid metal-based inks and pastes for printed electronics—A
r. Today 2019, 15, 416–430. [CrossRef] ano-Raya, C.; Denchev, Z.Z.; Cruz, S.F.; Viana, J.C. Che 6. Cano-Raya, C.; Denchev, Z.Z.; Cruz, S.F.; Viana, J.C. Chem
review. Appl. Mater. Today 2019, 15, 416–430. [CrossRef] review. Appl. Mater. Today 2019, 15, 416–430. [CrossRef] 7. Søndergaard, R.R.; Hosel, M.; Krebs, F. Roll to roll fabrication of large area functional organic materials. J. Polym. Sci. 2012, 51,
16–34. [CrossRef] 8. Mogg, B.T.; Claypole, T.; Deganello, D.; Phillips, C. Flexographic printing of ultra-thin semiconductor polymer layers. Transl. Mater. Res. 2016, 3, 015001. [CrossRef] 9. Davide, D. Control of morphological and electrical properties of flexographic printed electronics through tailored ink rheology. Org. Electron. 2019, 73, 212–218. 10. Yang, K.; Torah, R.; Wei, Y.; Beeby, S.; Tudor, J. Waterproof and durable screen printed silver conductive tracks on textiles. Text. Res. J. 2013, 83, 2023–2031. [CrossRef] L.; Moreno, J.; Lidón-Roger, J.V.; Garcia-Breijo, E. An investigation into the fabrication parameters of screen- Llopis, R.L.; Moreno, J.; Lidón-Roger, J.V.; Garcia-Breijo, E. An investigation into the fabrication parameters
capacitive sensors on e textiles Text Res J 2020 90 1749 1769 [CrossRef] 11. Ferri, J.; Llopis, R.L.; Moreno, J.; Lidón-Roger, J.V.; Garcia-Breijo, E. An investigation into the
printed capacitive sensors on e-textiles. Text. Res. J. 2020, 90, 1749–1769. [CrossRef] 12. Quinto, C.; Linares, A.; Llarena, E.; Montes, C.; González, O.; Molina, D.; Pío, A.; Ocaña, L.; Friend, M.; Cendagorta, M. Screen
Printing for Perovskite Solar Cells Metallization. In Proceedings of the 31st European Photovoltaic Solar Energy Conference and
Exhibition, Hamburg, Germany, 14–18 September 2015; pp. 1144–1148. [CrossRef] Printing for Perovskite Solar Cells Metallization. In Proceedings of the 31st European
Exhibition, Hamburg, Germany, 14–18 September 2015; pp. 1144–1148. [CrossRef] g
y
p
pp
rret, E.; Tedjini, S. Implementation and Measurements of Chipless RFID Tags. In Chipless RFID Based on RF En
E Press—Elsevier: London, UK, 2016. [CrossRef] Vena, A.; Perret, E.; Tedjini, S. Implementation and Measurements of Chipless RFID Tags. In Chipless RFID
P
ti l
ISTE P
El
i
L
d
UK 2016 [C
R f] 13. Vena, A.; Perret, E.; Tedjini, S. Implementation and Measurements of Chipless RFID Tags. In Chiples
Particle; ISTE Press—Elsevier: London, UK, 2016. [CrossRef] 13. Vena, A.; Perret, E.; Tedjini, S. Implementation and Measureme
Particle; ISTE Press—Elsevier: London, UK, 2016. [CrossRef] 14. References 1. Avuthu, S.G.; Gill, M.; Ghalib, N.; Sussman, M.R.; Wable, G.; Richstein, J.; Circuit, J. An introduction to the process of printed
electronics. In Proceedings of the SMTA International, Rosemont, IL, USA, 25–29 September 2016. g
p
2. Kim, C.; Jeon, S.W.; Kim, C.H. Reduction of Linearly Varying Term of Register Errors Using a Dancer System in Roll-to-Roll
Printing Equipment for Printed Electronics Int J Precis Eng Manuf 2019 20 1485 1493 [CrossRef] g
p
2. Kim, C.; Jeon, S.W.; Kim, C.H. Reduction of Linearly Varying Term of Register Errors Using a Dancer System in Roll-to-Roll Printing Equipment for Printed Electronics. Int. J. Precis. Eng. Manuf. 2019, 20, 1485–1493. [CrossRef] 3. Wiklund, J.; Karakoç, A.; Palko, T.; Yi˘gitler, H.; Ruttik, K.; Jäntti, R.; Paltakari, J. A Review on Printed
M
h d
I k
S b
A
li
i
d E
i
l I
J M
f M
P
2021 5 8 und, J.; Karakoç, A.; Palko, T.; Yi˘gitler, H.; Ruttik, K.; Jäntti, R.; Paltakari, J. A Review on Printed Electronic 3. Wiklund, J.; Karakoç, A.; Palko, T.; Yi˘gitler, H.; Ruttik, K.; Jäntti, R.; Paltakari, J. A Review on Prin
Methods, Inks, Substrates, Applications and Environmental Impacts. J. Manuf. Mater. Process. 2021, 3. Wiklund, J.; Karakoç, A.; Palko, T.; Yi˘gitler, H.; Ruttik, K.; Jäntti, R.; Paltakari, J. A Review on Printed Electronics: Fabrication
Methods, Inks, Substrates, Applications and Environmental Impacts. J. Manuf. Mater. Process. 2021, 5, 89. [CrossRef] 3. Wiklund, J.; Karakoç, A.; Palko, T.; Yigitler, H.; Ruttik, K.; Jäntti, R.; Paltakari, J. A Review on Printed
Methods, Inks, Substrates, Applications and Environmental Impacts. J. Manuf. Mater. Process. 2021, 5, 8 strates, Applications and Environmental Impacts. J. Manuf. Mater. Process. 2021, 5, 89. [CrossRef] ethods, Inks, Substrates, Applications and Environmental Impacts. J. Manuf. Mater. Process. 2021, 5, 89. [Cro 4. Rocha, L.A.; Viana, J.C. Printing Technologies on Flexible Substrates for Printed Electronics. 2018. Available online: https:
//doi.org/10.5772/intechopen.76161 (accessed on 15 June 2021). [CrossRef] intechopen.76161 (accessed on 15 June 2021). [CrossRe 5. Karim, N.; Afroj, S.; Tan, S.; Novoselov, K.S.; Yeates, S.G. All Inkjet-Printed Graphene-Silver Composite Ink on Textiles for Highly
Conductive Wearable Electronics Applications. Sci. Rep. 2019, 9, 8035. [CrossRef] [PubMed] Cano-Raya, C.; Denchev, Z.Z.; Cruz, S.F.; Viana, J.C. Chemistry of solid metal-based inks and pastes for printe
eview. Appl. Mater. Today 2019, 15, 416–430. [CrossRef] 6. Cano-Raya, C.; Denchev, Z.Z.; Cruz, S.F.; Viana, J.C. References Rodes-Carbonell, A.M.; Ferri, J.; Garcia-Breijo, E.; Bou-Belda, E. A preliminary study of printed electronics through flexography
impression on flexible substrates. Ind. Textila 2021, 72, 133–137. [CrossRef] 15. Kumar, B.; Hu, J. Woven fabric structures and properties. In The Textile Institute Book Series; Woodhead Publishing: Sawston, UK,
2017; pp. 133–151. [CrossRef] aque, M. Effect of weave structure on fabric properties. Ann. Univ. Oradea Fascicle Text. 2011, 12, 161–165. 16. Amin, R.; Haque, M. Effect of weave structure on fabric properties. Ann. Univ. Oradea Fascicle Text. 201 17. Rogina-Car, B.; Kovaˇcevi´c, S.; Schwarz, I.; Dimitrovski, K. Microbial Barrier Properties of Cotton Fabric—Influence of Weave
Architecture. Polymers 2020, 12, 1570. [CrossRef] y
18. Hasan, M.; Calvimontes, A.; Synytska, A.; Dutschk, V. Effects of Topographic Structure on Wettability of Differently Woven
Fabrics. Text. Res. J. 2008, 78, 996–1003. [CrossRef] ki´c, N.; Erceg, T.; Budinski, N.; Lavicza, Z.; Lera, I.; Koji´c, D. The analysis of the substrate influence on the print 19. Meseldžija, M.; Vuki´c, N.; Erceg, T.; Budinski, N.; Lavicza, Z.; Lera, I.; Koji´c, D. The analysis of the substrate influence on the print
quality parameters of screen-printed textile. In Proceedings of the VIII International Conference on Social and Technological
Development, Bosnia and Herzegovina, Balkans, 8–9 November 2019. [CrossRef] p
g
20. Hong, H.; Hu, J.; Yan, X. Effect of the basic surface properties of woven lining fabric on printing precision and electrical
performance of screen-printed conductive lines. Text. Res. J. 2019, 90, 1212–1223. [CrossRef] 21. Agathian, K.; Kannammal, L.; Meenarathi, B.; Kailash, S.; Anbarasan, R. Synthesis, characterization and adsorption behavior of
cotton fiber based Schiff base. Int. J. Biol. Macromol. 2018, 107, 1102–1112. [CrossRef] [PubMed] 22. Grishanov, S.; Meshkov, V.; Omelchenko, A. A Topological Study of Textile Structures. Part I: An Introduction to Topological
Methods. Text. Res. J. 2009, 79, 702–713. [CrossRef] 106 Polymers 2021, 13, 3165 23. Özdemir, H.; Mert, E. The effects of fabric structural parameters on the tensile, bursting, and impact strengths of cellular woven
fabrics. J. Text. Inst. 2013, 104, 330–338. [CrossRef]
Š 24. Šajn Gorjanc, D. The Influence of Constructional Parameters on Deformability of Elastic Cotton Fabrics. J. Eng. Fibers Fabr. 2014,
9, 38–46. [CrossRef] 25. Vrljiˇcak, Z.; Dragana, K.; Skenderi, Z. Impact of yarn count, noil percentage and yarn tension on structure of jersey fabric. Tekstil
2015, 64, 158–168. 26. Kostajnšek, K.; Dimitrovski, K. Use of Extended Cover Factor Theory in UV Protection of Woven Fabric. References Polymers 2021, 13, 1188. [CrossRef] [PubMed] 27. ˙Zołek-Tryznowska, Z. Rheology of Printing Inks. In The Printing Ink Manual; Springer: Berlin, Germany, 2016; pp. 87–99. [CrossRef] Revuelta, P.S.; Litrán, S.P.; Thomas, J.P. Electrical Power Terms in the IEEE Std 1459 Framework. In Active Po 28. Revuelta, P.S.; Litrán, S.P.; Thomas, J.P. Electrical Power Terms in the IEEE Std 1459 Framework. In A
Elsevier: Amsterdam, The Netherlands, 2016; pp. 23–49. [CrossRef] a, P.S.; Litrán, S.P.; Thomas, J.P. Electrical Power Terms in the IEEE Std 1459 Framework. In Active Power Line Co
Amsterdam, The Netherlands, 2016; pp. 23–49. [CrossRef] 28. Revuelta, P.S.; Litrán, S.P.; Thomas, J.P. Electrical Power Terms in the IEEE Std 1459 Framework. In Activ
Elsevier: Amsterdam, The Netherlands, 2016; pp. 23–49. [CrossRef] pp
29. Textiles—Tests for Colour Fastness—Part C06: Colour Fastness to Domestic and Commercial Laundering (ISO 105-C06:2010). A
il bl
li
htt
//
/
t
t
/b
t
/
? N0045649 (
d
15 J
2021) iles—Tests for Colour Fastness—Part C06: Colour Fastness to Domestic and Commercial Laundering (ISO 1 29. Textiles—Tests for Colour Fastness—Part C06: Colour Fastness to Domestic and Commercial Launde
Available online: https://www.une.org/encuentra-tu-norma/busca-tu-norma/norma?c=N0045649 (a Available online: https://www.une.org/encuentra-tu-norma/busca-tu-norma/norma?c=N0045649 (accessed p
g
30. Deopura, B.L.; Padaki, N.V. Synthetic textile fibres: Polyamide, polyester and aramid fibres. In Textiles and Fashion; Woodhead
Publishing: Sawston, UK, 2015; pp. 97–114. g
pp
31. Jong, S.E.; Mok, L.Y.; Lee, J.S.; Chul, Y.S. Development of Cellulosic Woven Fabric for Digital Textile Printing. Text. Coloration
Finish. 2005, 17, 20–26. 32. Zhang, Y.; Westland, S.; Cheung, V.; Burkinshaw, S.M.; Blackburn, R.S. A custom ink-jet printing system using a novel pretreatment
method. Color. Technol. 2009, 125, 357–365. [CrossRef] 33. Kašikovi´c, N.; Vladi´c, G.; Mili´c, N.; Novakovi´c, D.; Miloševi´c, R.; Dedijer, S. Colour fastness to washing of multi-layered digital
prints on textile materials. J. Natl. Sci. Found. Sri Lanka 2018, 46, 381. [CrossRef] mitrovski, K. Influence of yarn count and warp and weft thread density on colour values of woven surface. r. 2004, 12, 32–39. 34. Gabrijelˇciˇc, H.; Dimitrovski, K. Influence of yarn count and warp and weft thread density on colour v
Fibres Text. East. Eur. 2004, 12, 32–39. 35. Available online: https://www.une.org/encuentra-tu-norma/busca-tu-norma/norma?c=N0023384 (accessed on 15 July 2021). 35. Available online: https://www.une.org/encuentra-tu-norma/busca-tu-norma/norma?c=N0023384 (accessed on 15 July 2021). 36. Ly, B.C.K.; Dyer, E.B.; Feig, J.L.; Chien, A.L.; Del Bino, S. Research Techniques Made Simple: Cutaneous Colorimetry: A Reliable 35. Available online: https://www.une.org/encuentra-tu-norma/busca-tu-norma/norma?c=N0023384 (accessed on 15 July 2021). Article
Preparation of Multifunctional Plasma Cured Cellulose Fibers
Coated with Photo-Induced Nanocomposite toward
Self-Cleaning and Antibacterial Textiles y 1,2,*, Mehrez E. El-Naggar 3,*, Tawfik A. Khattab 3 and Ayman El-Faham 1,4 1
Chemistry Department, College of Science, King Saud University, Riyadh 11451, Saudi Arabia;
aelfaham@ksu.edu.sa p
y,
y
,
y,
,
gyp
3
Textile Research Division, National Research Center (Affiliation ID: 60014618), Cairo 12622, Egypt;
ta.khattab@nrc.sci.eg 3
Textile Research Division, National Research Center (Affiliation ID: 60014618), Cairo 12622, Egypt;
ta.khattab@nrc.sci.eg 4
Department of Chemistry, Faculty of Science, Alexandria University, Ibrahimia, Alexandria 21321, Egypt
*
Correspondence: helhamshary@ksu.edu.sa (H.E.-H.); mehrez_chem@yahoo.com (M.E.E.-N.) 4
Department of Chemistry, Faculty of Science, Alexandria University, Ibrahimia, Alexandria 21321, Egypt
*
Correspondence: helhamshary@ksu.edu.sa (H.E.-H.); mehrez_chem@yahoo.com (M.E.E.-N.) Abstract: Multifunctional fibrous surfaces with ultraviolet protection, self-cleaning, or antibacterial
activity have been highly attractive. Nanocomposites consisting of silver (AgNPs) and titanium
dioxide (TiO2 NPs) nanoparticles (Ag/TiO2) were developed and coated onto the surface of viscose
fibers employing a straightforward pad–dry–cure procedure. The morphologies and elemental
compositions were evaluated by scan electron microscopy (SEM), infrared spectra (FTIR), and
energy-dispersion X-ray spectra (EDS). The resultant multifunctional textile materials displayed
antibacterial and photo-induced catalytic properties. The photocatalyzed self-cleaning properties
were investigated employing the photochemical decay of methylthioninium chloride, whereas
the antibacterial properties were studied versus E. coli. The viscose fibers coated with Ag/TiO2
nanocomposite demonstrated improved efficiency compared with viscose fibers coated with pure
anatase TiO2 nano-scaled particles. Citation: El-Hamshary, H.;
El-Naggar, M.E.; Khattab, T.A.;
El-Faham, A. Preparation of
Multifunctional Plasma Cured
Cellulose Fibers Coated with
Photo-Induced Nanocomposite
toward Self-Cleaning and
Antibacterial Textiles. Polymers 2021,
13, 3664. https://doi.org/10.3390/
polym13213664 Keywords: Ag/TiO2; nanocomposite; antibacterial; photocatalysis; viscose fibers Academic Editors: Antonio Pizzi,
Tarek M. Abou Elmaaty and Maria
Rosaria Plutino Academic Editors: Antonio Pizzi,
Tarek M. Abou Elmaaty and Maria
Rosaria Plutino Citation: El-Hamshary, H.;
El-Naggar, M.E.; Khattab, T.A.;
El-Faham, A. Preparation of
Multifunctional Plasma Cured
Cellulose Fibers Coated with
Photo-Induced Nanocomposite
toward Self-Cleaning and
Antibacterial Textiles. Polymers 2021,
13, 3664. https://doi.org/10.3390/
polym13213664 References 36
Ly B C K ; Dyer E B ; Feig J L ; Chien A L ; Del Bino S Research Techniques Made Simple: Cutaneous Colorimetry: A Reliable p //
g/
/
/
(
Ly, B.C.K.; Dyer, E.B.; Feig, J.L.; Chien, A.L.; Del Bino, S. Research Techniques Made Simple: Cutaneous Colori 36. Ly, B.C.K.; Dyer, E.B.; Feig, J.L.; Chien, A.L.; Del Bino, S. Research Techniques Made Simple: Cutaneous Colorimetry: A Reliable
Technique for Objective Skin Color Measurement. J. Investig. Dermatol. 2020, 140, 3–12.e1. [CrossRef] [PubMed] y,
;
y ,
;
g, J
;
,
;
,
q
p
y
Technique for Objective Skin Color Measurement. J. Investig. Dermatol. 2020, 140, 3–12.e1. [CrossRef] [PubMed] y
g
q
p
ue for Objective Skin Color Measurement. J. Investig. Dermatol. 2020, 140, 3–12.e1. [CrossRef] [PubMed] Cherrington, M.; Claypole, T.; Gethin, D.; Worsley, D.; Deganello, D. Non-contact assessment of electrical pe
sintered nanoparticle silver coatings through colorimetry. Thin Solid Films 2012, 522, 412–414. [CrossRef] gton, M.; Claypole, T.; Gethin, D.; Worsley, D.; Deganello, D. Non-contact assessment of electrical performa Cherrington, M.; Claypole, T.; Gethin, D.; Worsley, D.; Deganello, D. Non contact assessment of electrical perfor
intered nanoparticle silver coatings through colorimetry. Thin Solid Films 2012, 522, 412–414. [CrossRef] 38. Soleimani-gorgani, A.; Pishvaei, M. Water Fast Ink Jet Print Using an Acrylic /Nano-Silver Ink. Prog. Color Colorants Coat. 2021, 4,
79–83. [CrossRef] 107 polymers polymers 1. Introduction High-performance textiles have significant potential in marketing nano-based func-
tional commodities, such as antibacterial and self-cleaning textiles [1–5]. These nano-based
functional textiles can be accomplished by the immobilization of metal and/or metal oxide
nanoparticles onto the fabric surface during the finishing process. The great prospects of
metal nanoparticles can be effectively employed to provide multifunctional stimulation
without deteriorating the exterior properties or negatively affecting the native features
of the fibers [6]. Various studies were explored for the usage of Ag0, TiO2, and ZnO
nanoparticles as agents for textile surface modification to provide smart fibers with a
variety of distinctive properties, like ultraviolet blocking, self-cleaning, and antibacterial
activity [7–9]. Different techniques were described recently to tie TiO2 nanoparticles into
the fiber surface to present self-cleanable products [10]. The photocatalytic performance
of TiO2 nanoparticles upon irradiation with a UN supply was described. The exposure of
TiO2 nanoparticles to UV (λ < 388 nm) results in stimulating the electrons of the valence
band into the other conduction one to generate holes (h+) and electrons (e−). Those reactive
entities showed a major role in the commencement of a reduction-oxidation course [11]. TiO2 nanoparticles have been reported as a high-quality substance in photocatalysis under
irradiation with an ultraviolet supply owing to its satisfactory optical properties, chemi-
cal/physical stability, non-toxicity, and cheapness [12,13]. Nonetheless, some weakness
was linked to the use of TiO2 nanoparticles, such as an elevated band-gap (Eg = 3.2 eV). In addition, TiO2 nanoparticles can be excited only under irradiation with an ultraviolet Received: 16 September 2021
Accepted: 19 October 2021
Published: 24 October 2021 Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article
distributed
under
the
terms
and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). 109 https://www.mdpi.com/journal/polymers Polymers 2021, 13, 3664. https://doi.org/10.3390/polym13213664 Polymers 2021, 13, 3664 supply (λ < 388 nm) to release electrons to conduction band departing holes to the other
valence one, limiting their photocatalytic activity under visible or sunlight. Furthermore,
the high recombination rate between holes and electrons on TiO2 nanoparticles results in
less effective photocatalysis [2–5]. Silver nanoparticles (AgNPs) have been applied as an antimicrobial agent onto a
variety of textile substrates in the absence of UV light [14,15]. 2.1. Materials Viscose fabrics were supplied from Spin and Weaving Misr El-Mahalla Co. (El-Mahalla
City, Egypt) Silver(I) nitrate, titanium isopropoxide (TTIP; 97%), acetic acid (65%), silver
nitrate (≥99.0%), acetic acid (CH3COOH; 96%), nitric acid (HNO3; 65%), sodium carbonate
(Na2CO3), and oxalic acid were obtained from Aldrich (Cairo, Egypt). TiO2 nanoparticles
were synthesized according to the previously reported low temperature sol-gel method [18]. 2.2. Synthesis of TiO2 Nanoparticles TTIP (2% v-v) was added to a solution of HNO3 (1% v-v), CH3COOH (10% v-v), and
distilled water (DW). The mixture was subjected to stirring for an extra 16 h at 60 ◦C. After
cooling, TiO2 NPs were provided by continuously adding Na2CO3(aq) (5%) until reaching
a full sedimentation of TiO2 NPs. The generated dispersion was centrifuged (4000 rpm)
for 5 min, decanted, washed with DW, and dried at 100 ◦C over 3 h. The dispersion of
the generated TiO2 NPs in DW was transparent and stable for several weeks at room
temperature. 1. Introduction However, silver nanoparticles
can simply influence the colorimetric properties of the treated textile surface by oxidation
into the brownish AgO or by aggregation into bigger black microparticles. In addition,
silver is a costly metal, and small amounts are ineffective for a variety of realistic products. In order to accomplish the advantageous effects from both Ag0 and TiO2 nanoparticles
and reduce their weaknesses, Ag/TiO2 composites were developed by producing AgNPs
onto TiO2 nanoparticles, employing a variety of methods to enhance the photocatalytic
and antimicrobial properties. The deposition of AgNPs can significantly improve the
light-induced catalytic activity of TiO2 nanoparticles. This could be ascribed to the ability
of AgNPs to trap electrons at Schottky bar at each contact area of Ag/TiO2 [16–19]. This
results in a decrease in the recombination effect between electrons and holes on the surface
of TiO2 nanoparticles. Thus, separating the charge was stimulated and the transfer of
electrons took place to result in a higher life-time of hole/electron pairs [20–22]. p
g
p
Viscose is a significant material for textiles owing to its high resistance to radiation and
high stability to body fluids. The improvement of the antimicrobial properties of viscose
has been critical for a variety of healthcare purposes. Therefore, various techniques have
been reported to improve the antimicrobial properties of viscose fibers [23]. The weak
binding of the colloidal nanoparticles to viscose fibers has been a substantial problem that
can be overwhelmed by plasma treatment [24]. Plasma curing by etching was employed
to activate the fibrous surfaces to induce the creation of polar groups, such as carbonyl,
alcohol, carboxyl, and ether, facilitating better binding to nano-scaled particles [5,25]. Herein, we report the synthesis of TiO2 NPs and Ag/TiO2 nanocomposites as antibacterial
and photocatalytic agents and their immobilization onto the surface of viscose fibers via
a pad–dry–cure procedure to introduce multifunctional textiles. The morphologies and
elemental compositions were evaluated by different analytical techniques. The performance
of the Ag/TiO2-coated viscose fibers showed an improved efficiency compared with TiO2-
coated viscose fibers. 2.3. Synthesis of Ag/TiO2 2.7. Antibacterial Properties The antibacterial performance was examined against E. coli according to the earlier
procedure [29]. 2.6. Evaluation of Self-Cleaning The self-cleaning activity was assessed by the light-induced decay of methylthion-
inium chloride (MTC) under visible (410 nm) and ultraviolet irradiation (315–380 nm)
according to previous literature procedures [28]. 2.5. Characterization Methods TEM (JEOL-1230, Akishima, Japan) was applied to inspect the morphology of the
prepared TiO2 NPs. The morphologies of the coated viscose were explored by Quanta
SEM FEG 250 (Brno- ˇCernovice, Czech Republic) linked to EDS (TEAM) to investigate
the elemental contents of the viscose coated surface. FT-IR spectra were assessed by
Nexus 670 (Nicolet; Watertown, MA, USA). UV/Vis absorption spectra and CIE Lab of
the coated viscose were collected by UltraScanPro (Hunter Lab, Reston, VA, USA). The
optical band gap was assessed from the absorbance spectrum utilizing Tauc’s equation
[εhν = C (hν−Eg)n], where Eg is the average band gap, ε is molar extinction coefficient, C is
a constant, and n relies on the transition type. 2.4. Deposition of Ag/TiO2 onto Plasma-Activated Viscose 2.4. Deposition of Ag/TiO2 onto Plasma-Activated Viscose As demonstrated in Figure 1, the plasma tool was applied to viscose fibers for 3 min at
a power of 400 W and a constant pressure of 3 × 10−3 mbar [27]. The above-prepared solu-
tions were then applied to the plasma-activated viscose fibers by the pad–dry–cure process. Both TiO2 NPs (0.1 g) and Ag0/TiO2 (0.1 g) were stirred in DW (150 mL) and homogenized
for 45 min under ambient conditions. The plasma-activated fabric (15 cm × 15 cm) was
soaked in the prepared solutions for 60 min, and subjected to pad–dry–cure. The viscose
was then dried at 90 ◦C, subjected to curing at 120 ◦C, and finally rinsed with DW. The
binding stability of Ag0/TiO2 and TiO2 onto viscose can be attributed to the electrostatic
forces among Ti4+ existing on TiO2 or Ag0/TiO2, and the negative charges on the viscose
surface. The negative charges on viscose could be attributed to the negatively charged
substituents, such as O–O– and –COO– generated by plasma. Figure 1. Schematic diagram representing the deposition of Ag0/TiO2 onto plasma-cured viscose fabric. Ch
i
i
h d Figure 1. Schematic diagram representing the deposition of Ag0/TiO2 onto plasma-cured viscose fabric. 2.5. Characterization Methods 2.3. Synthesis of Ag/TiO2 Ag/TiO2 nanocomposite was synthesized utilizing UV technology [26]. Oxalic acid
(0.005 mol/L) and AgNO3 (0.0002 mol/L) in DW were mixed with a suspension of the
above-prepared TiO2 NPs (1 g). After stirring for 15 min, the mixture was added to 110 Polymers 2021, 13, 3664 DW (450 mL) with vigorous stirring. The pH was adjusted in the range of 6.8–7.0 using
NaOH(aq). The admixture was irradiated with UV supply for 60 min. The admixture was
then placed to settle down for 8 h to form a brownish precipitation of Ag0/TiO2, which
was filtered and dried at 120 ◦C for 3 h to give Ag/TiO2. 2.4. Deposition of Ag/TiO2 onto Plasma-Activated Viscose 3.1. Development of Composite Ag/TiO2 was synthesized under UV technology [19], starting from a mixture of
oxalic acid, AgNO3, and TiO2 NPs (Figure 2). The reaction color was found to change
from colorless to a brownish shade under irradiation with ultraviolet light to verify the
reduction of silver ions (Ag+) to silver metal (Ag0) and incorporating AgNPs onto TiO2
NPs. The color shift presented a visual verification for the photo-metallization process in
the reaction system. Silver ions were initially subjected to cationic adsorption onto the
surface of TiO2 NPs. When a suspension of TiO2 has a pH value <6, the main surface
entities become TiOH2+, while the main surface entities becomes TiOH−for a suspension
of TiO2 with a pH value higher than 6. Thus, NaOH(aq) was added for complete deposition
of the adsorbed Ag+ onto the surface of TiO2 NPs to result in the formation of silver(I)
oxide (Ag2O), which were then reduced to Ag0 by an ultraviolet supply. The ultraviolet
irradiation has the ability to induce the transfer of free electrons from valence of TiO2 NPs
to the other conduction band. TiO2 NPs comprises negative charges in the presence of
Ti-OH−, facilitating deposition of Ag+ onto its surface. Thus, the photo-induced generated
electrons function as reductive agents for Ag+ to provide Ag0. Production of tiny Ag0
crystals could occur by cathode-like reduction or by aggregation of Ag0. AgNPs has been
known to show an absorbance band attributed to Plasmon effect owing to the interaction
of the metallic NPs with UV, leading to oscillation of electrons. The color change of the
solution to brown was attributed to the improved absorption at low wavelength owing to
surface Plasmon. The reaction mechanism between AgNO3 and TiO2 is illustrated by the
equations described below [30]. Ag+ →Ag+ (adsorbed onto the surface of TiO2 NPs)
2Ag+ (adsorbed) + 2OH−→Ag2O + H2O
2Ag2O + hυ →4Ag0 + O2
TiO2 (e-/h+) + Ag+ →TiO2@Ag0 Figure 2. TEM graphs of TiO2 (a) and Ag0/TiO2 (b). Figure 2. TEM graphs of TiO2 (a) and Ag0/TiO2 (b). UV/Vis absorption spectra were studied to explore the influence of Ag0 on the TiO2
optical activity, as illustrated in Figure 3. The absorption spectra of TiO2 and Ag0/TiO2
showed broad absorbance bands with a wavelength maxima <400 nm. This can be at-
tributed to the electron transition in TiO2 depending on its energy band gap (~3.12 eV)
owing to a charge transfer. 2.8. Durability Test To study the durability of the treated viscose against washing, the coated samples
(15 cm × 15 cm) were subjected to washing for 10 laundry cycles under AATCC 61:1989
standard procedure. The coated samples were charged in a laundry-o-meter machine,
and subjected to washing with a detergent solution (200 mL) at 40 ◦C for 45 min. Both
antibacterial and self-cleaning were assessed as indicators to evaluate the durability of the
coated fibers. 111 Polymers 2021, 13, 3664 3. Result and Discussion 3.1. Development of Composite 3.1. Development of Composite The absorbance spectral curves of TiO2 were enhanced in
Ag0/TiO2. Obvious variations in absorbance activity of Ag0/TiO2 were detected in the
visible spectrum range as a result of the weak Plasmon effect owing to the low Ag0 content 112 Polymers 2021, 13, 3664 on TiO2 NPs. This could enhance both surface excitation and electron/hole separation. The
absorbance band of Ag0/TiO2 demonstrated that Ag0/TiO2 exhibits properties similar to
TiO2 NPs. The absorbance intensities were observed to exhibit a red shift for Ag0/TiO2,
representing a decrease in TiO2 gap. The absorbance spectra showed maximum absorbance
wavelengths at 383 and 388 nm for TiO2 and Ag0/TiO2, respectively. This monitored
shift in the wavelength and the reduced band gap led to the increase in the light-induced
catalytic activity of TiO2 NPs in the visible range.
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Figure 3. UV/Vis absorption spectral curves of the prepared composites coated onto viscose fibers. $EVRUEDQFH Figure 3. UV/Vis absorption spectral curves of the prepared composites coated onto viscose fibers. 3.2. Characterization of Viscose Fibers 3.2. Characterization of Viscose Fibers The morphology of the coated viscose before and after treatment with plasma, as well
as plasma-pretreated viscose before and after coating, were studied by SEM as depicted
in Figure 4. A surface of moderate smoothness was monitored for plasma-inactivated
viscose. Plasma-cured viscose displayed etches on the fiber surface. Irregular nanoparticles
were monitored on the surface of the plasma-treated fibers. Decreasing the thickness of
the surface layers resulted in improving the rough surface in comparison with pristine
fibers. Fibers loaded with TiO2 showed irregular and uneven clusters. Fibers coated with
Ag0/TiO2 displayed a skinny film of inconsistent Ag0/TiO2. No cracking was detected
and the small particles were monitored to cover the fibers. The changes in chemical
compositions of samples due to plasma-curing and deposition of nanoparticles onto the
surface of viscose were explored by EDX. The chemical compositions of blank and plasma-
untreated fibers loaded with nanoparticles are summarized in Table 1. Both carbon and
oxygen were detected as major contents due to the fabric, whereas Ti and/or Ag were
detected as minor contents due to the deposition of TiO2 or Ag0/TiO2 onto the fabric
surface. The plasma-cured sample showed a slight increase in the oxygen content due
to generating oxygen-containing substituents onto the fiber by oxygen plasma treatment. Plasma curing by etching and oxidation has been employed to activate the fiber surface
to induce the creation of substituents [31], such as carbonyl, alcohol, carboxyl, and ether,
facilitating strong binding to nano-scaled particles. 113 Polymers 2021, 13, 3664 Figure 4. SEM images of TiO2 NPs incorporated plasma-activated (a,b) and Ag0/TiO2 incorporated plasma-activated
(c,d) fibers. Figure 4. SEM images of TiO2 NPs incorporated plasma-activated (a,b) and Ag0/TiO2 incorporated plasma-activated
(c,d) fibers. Figure 4. SEM images of TiO2 NPs incorporated plasma-activated (a,b) and Ag0/TiO2 incorporated plasma-activated
(c,d) fibers. Table 1. Elemental contents of viscose fibers. Sample
C
O
Ti
Ag
Blank
62.12 ± 1.3
37.88 ± 1.2
0
0
Plasma-activated
61.71 ± 1.1
38.29 ± 1.0
0
0
Plasma-inactivated (TiO2)
59.44 ± 1.6
38.91 ± 1.1
1.65 ± 0.1
0
Plasma-activated (TiO2)
57.14 ± 1.4
39.12 ± 1.6
3.74 ± 0.3
0
Plasma-inactivated (Ag0/TiO2)
59.03 ± 1.0
38.73 ± 1.3
1.72 ± 0.1
0.52 ± 0.1
Plasma-activated (Ag0/TiO2)
56.11 ± 1.2
39.43 ± 1.2
3.34 ± 0.2
1.12 ± 0.1 FT-IR spectra were explored for the coated viscose with and without plasma treatment,
as shown in Figure 5. 3.2. Characterization of Viscose Fibers The main characteristic peaks were detected at 3339 cm−1 for the
hydroxyl group stretch vibration, as well as two peaks at 2932 and 1030 cm−1 for the
aliphatic C-H stretch and bend vibrations, respectively. No major shifts were detected in
the absorbance bands; however, the intensity of the hydroxyl group was found to increase
with the increasing deposition of the nanoparticles. 3.3. Self-Cleaning Properties The reduction potential of MTC is about 0.011 V, whereas the energy level of the
conduction band for TiO2 is about −0.5 V. Thus, MTC is a suitable model to investigate
the photo-induced catalysis process. The self-cleaning performance of TiO2 or Ag0/TiO2
deposited onto viscose fibers could be studied by testing the decomposition of MTC
underneath UV and visible lights, as shown in Figure 6. Ultraviolet/visible absorption
spectral curves of MTC were collected for the treated viscose under irradiation with
UV and visible light over 24 h. The absorbance peak at 665 nm decreased as a result
of the degradation of MTC. The self-cleaning activity was tested by exploring the total
content (C/C0) of MTC as a function of time. The degradation of MTC on the uncoated
fibers showed almost no variations under irradiation with either UV or visible daylight 114 Polymers 2021, 13, 3664 to prove that the uncoated fibers do not exhibit any light-induced decay ability. The
deposition of TiO2 NPs onto plasma-activated fibers proved an improvement in photo-
induced degradation of MTC under UV light. However, this photo-induced degradation
of MTC was incomplete. The deposition of TiO2 onto plasma-pretreated fibers displayed
a negligible photo-induced degradation under visible light owing to the adsorption and
diffusion of MTC within the coated viscose. The integration of the nanocomposite into
the plasma-pretreated fibers induced a total photoinduced degradation of MTC under
ultraviolet and visible light, as the blue shade was monitored neither on the coated fibers
nor in solution to prove a complete photo-induced degradation of MTC. The photo-induced
degradation of MTC for fibers coated with Ag0/TiO2 demonstrated improved activity in
comparison with TiO2 NPs, proposing that the inclusion of AgNPs onto the surface of
TiO2 is an efficient approach. The photo-induced degradation rate for TiO2 and Ag0/TiO2
coated onto viscose fibers decreased with washing. Nonetheless, they persisted higher
than the plasma-inactivated viscose fibers. This proposed higher adhesion of particles onto
plasma-activated viscose. After washing, the light-induced decay of methylthioninium
chloride (MTC) for Ag0/TiO2 deposited onto viscose fibers was lower than the case of the
viscose fibers coated with TiO2 under visible/UV light. Thus, the nanocomposite enhanced
the self-cleaning activity of viscose as a beneficial effect of silver on the light-induced
catalysis of TiO2. 3.3. Self-Cleaning Properties
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Figure 5. FT-IR spectra of coated viscose fibers. Figure 5. FT-IR spectra of coated viscose fibers. 115 Polymers 2021, 13, 3664 Figure 6. Degradation of MTC on fibers under UV (a) and visible (b) lights for pristine fibers (Vis0
TiO2/fibers after wash (Vis1), TiO2/fibers before wash (Vis2), Ag0/TiO2/fibers after wash (Vis3), an
Ag0/TiO2/fibers before wash (Vis4). Figure 6. Degradation of MTC on fibers under UV (a) and visible (b) lights for pristine fibers (Vis0),
TiO2/fibers after wash (Vis1), TiO2/fibers before wash (Vis2), Ag0/TiO2/fibers after wash (Vis3), and
Ag0/TiO2/fibers before wash (Vis4). 3.4. Antibacterial Activity The antibacterial properties of plasma-cured and coated viscose were examined
against E. coli by measuring optical density (OD) at 620 nm versus time, as shown in
Figure 7. OD was found to improve, reflecting the decrease in the quantity of growing
bacteria in the tested sample. Both blank and TiO2 coated viscose fibers displayed no
inhibition. The viscose fibers coated with Ag0/TiO2 showed antibacterial properties at all
contents, yet followed by washing to confirm the positive effect of loading Ag0 onto TiO2. AgNPs have been described to exhibit a broad of activity against a variety of pathogens. It has been recognized that the increase in surface area results in improved antibacterial
properties [32]. 116 Polymers 2021, 13, 3664 Figure 7. Activity of coated viscose fibers against E. coli; Vis0 is pristine viscose, Vis1 is 103 (a) or 10
(b) bacterial density, Vis2 is NPs/fibers, Vis3 is nanocomposite/fibers following washing, and Vis4
nanocomposite/fibers prior to washing. 4 C
l
i Figure 7. Activity of coated viscose fibers against E. coli; Vis0 is pristine viscose, Vis1 is 103 (a) or 106
(b) bacterial density, Vis2 is NPs/fibers, Vis3 is nanocomposite/fibers following washing, and Vis4 is
nanocomposite/fibers prior to washing. References Machine-washable smart textiles with photothermal and antibacterial activities
from nanocomposite fibers of conjugated polymer nanoparticles and polyacrylonitrile. Polymers 2019, 11, 16. [CrossRef] [PubMed] 8. Hiremath, L.; Kumar, S.N.; Sukanya, P. Development of antimicrobial smart textiles fabricated with magnetite nano particles
obtained through green synthesis. Mater. Today Proc. 2018, 5, 21030–21039. [CrossRef] 9. Moazzenchi, B.; Montazer, M. Click electroless plating and sonoplating of polyester with copper nanoparticles producing
conductive fabric. Fibers Polym. 2020, 21, 522–531. [CrossRef] 10. Dhineshbabu, N.R.; Bose, S. Smart textiles coated with eco-friendly UV-blocking nanoparticles derived from natural resources. ACS Omega 2018, 3, 7454–7465. [CrossRef] [PubMed] b, T.A.; Mashaly, H.M.; El-Halwagy, A.A.; Rehan, M. Plasma activation toward multi-stimuli responsive cotton
velopment of polyaniline derivatives and silver nanoparticles. Cellulose 2020, 27, 2913–2926. [CrossRef] g
11. Ahmed, H.; Khattab, T.A.; Mashaly, H.M.; El-Halwagy, A.A.; Rehan, M. Plasma activation toward multi-
fabric via in situ development of polyaniline derivatives and silver nanoparticles. Cellulose 2020, 27, 29 H.; Khattab, T.A.; Mashaly, H.M.; El-Halwagy, A.A.; Rehan, M. Plasma activation toward multi-stimuli respons
a in situ development of polyaniline derivatives and silver nanoparticles Cellulose 2020 27 2913–2926 [Cros H.; Khattab, T.A.; Mashaly, H.M.; El-Halwagy, A.A.; Rehan, M. Plasma activation toward multi-stimuli respons
a in situ development of polyaniline derivatives and silver nanoparticles. Cellulose 2020, 27, 2913–2926. [Cros 12. Elwakeel, K.Z.; Abd El-Ghaffar, M.A.; El-Kousy, S.M.; El-Shorbagy, H.G. Enhanced remediation of Reactive Black 5 from aqueous
media using new chitosan ion exchangers. J. Dispers. Sci. Technol. 2013, 34, 1008–1019. [CrossRef] g
g
p
13. Rabie, S.T.; Ahmed, A.E.; Sabaa, M.W.; Abd El-Ghaffar, M.A. Maleic diamides as photostabilizers for polystyrene. J. Ind. Eng. Chem. 2013, 19, 1869–1878. 14. Chen, H.; Huang, M.; Wang, Z.; Gao, P.; Cai, T.; Song, J.; Zhang, Y.; Meng, L. Enhancing rejection per
resistance genes by a TiO2/AgNPs-modified nanofiber forward osmosis membrane. Chem. Eng. J. 2020 14. Chen, H.; Huang, M.; Wang, Z.; Gao, P.; Cai, T.; Song, J.; Zhang, Y.; Meng, L. Enhancing rejection performance of tetracycline
resistance genes by a TiO2/AgNPs-modified nanofiber forward osmosis membrane. Chem. Eng. J. 2020, 382, 123052. [CrossRef]
15
Jatoi A W ; Kim I S ; Ni Q Q Cellulose acetate nanofibers embedded with AgNPs anchored TiO nanoparticles for long term 14. Chen, H.; Huang, M.; Wang, Z.; Gao, P.; Cai, T.; Song, J.; Zhang, Y.; Meng, L. Enhancing rejection performance of tetracycline
resistance genes by a TiO2/AgNPs-modified nanofiber forward osmosis membrane. Chem. Eng. J. 4. Conclusions Multifunctional viscose fibers coated with Ag/TiO2 nanocomposite were developed by
the simple pad–dry–cure technology. The synthesis, characterization, and use of nanocom-
posite as an antibacterial and light-induced self-cleaning agent were explored. Ag/TiO2
was prepared using a double-stage procedure of sol–gel TiO2 synthesis, followed by deposit-
ing of Ag0 onto the surface of TiO2 by ultraviolet irradiation. The deposition of Ag0/TiO2
onto plasma-pretreated viscose fibers was accomplished using the facile pad–dry–cure
technology. Ag0/TiO2 displayed better absorption in the visible spectrum and higher
antibacterial activity and light-induced catalysis in comparison with plasma-activated
viscose coated with TiO2. This considerable improvement in antibacterial and self-clean
properties could be attributed to AgNPs deposited onto the surface of TiO2. The current
study presented a good strategy to produce Ag/TiO2 composite with the ability to impart
antibacterial, self-cleaning photo-induced catalytic properties to plasma-cured fibers, un-
der irradiation with UV/visible lights to make this Ag0/TiO2 nanocomposite potentially
practical as a multifunctional agent for a variety of applications, such as medical clothing. 117 Polymers 2021, 13, 3664 Author Contributions: Conceptualization H.E.-H. and M.E.E.-N.; methodology, M.E.E.-N., T.A.K. and A.E.-F.; formal analysis and discussion, M.E.E.-N., A.E.-F. and T.A.K.; writing—original draft
preparation, H.E.-H. and M.E.E.-N.; writing—review and editing, H.E.-H., M.E.E.-N., T.A.K. and
A.E.-F.; supervision, H.E.-H. and M.E.E.-N. All authors have read and agreed to the published version
of the manuscript. Funding: Deanship of Scientific Research at King Saud University, Research Group Program (no. RGP-
201), King Saud University, Riyadh, Saudi Arabia. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the
corresponding author. Acknowledgments: The authors would like to extend their appreciation to the Deanship of Scientific
Research at King Saud University for funding this work through research group (no. RGP-201). Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References 1. Yasin, S.; Sun, D. Propelling textile waste to ascend the ladder of sustainability: EOL study on prob
technical textiles. J. Clean. Prod. 2019, 233, 1451–1464. [CrossRef] 1. Yasin, S.; Sun, D. Propelling textile waste to ascend the ladder of su
technical textiles. J. Clean. Prod. 2019, 233, 1451–1464. [CrossRef] ,
;
,
p
g
technical textiles. J. Clean. Prod. 2019, 233, 1451–1464. [CrossRef] 2. Sharaf, S.; El-Naggar, M.E. Wound dressing properties of cationized cotton fabric treated with carrageenan/cyclodextrin hydrogel
loaded with honey bee propolis extract. Int. J. Biol. Macromol. 2019, 133, 583–591. [CrossRef] y
p
p
3. Hebeish, A.; El-Naggar, M.E.; Tawfik, S.; Zaghloul, S.; Sharaf, S. Hyperbranched polymer–silver nanohybrid induce super
antibacterial activity and high performance to cotton fabric. Cellulose 2019, 26, 3543–3555. [CrossRef] y
p
p
3. Hebeish, A.; El-Naggar, M.E.; Tawfik, S.; Zaghloul, S.; Sharaf, S. Hyperbranched polymer–silver nanohybrid induce super
ib
i l
i i
d hi h
f
f b i
C ll l
2019 26 3543 3555 [C
R f] y
p
p
eish, A.; El-Naggar, M.E.; Tawfik, S.; Zaghloul, S.; Sharaf, S. Hyperbranched polymer–silver nanohybrid Hebeish, A.; El-Naggar, M.E.; Tawfik, S.; Zaghloul, S.; Sharaf, S. Hyperbranched polymer–silver na
antibacterial activity and high performance to cotton fabric. Cellulose 2019, 26, 3543–3555. [CrossRef] gg
g
yp
p
y
ntibacterial activity and high performance to cotton fabric. Cellulose 2019, 26, 3543–3555. [CrossRef] 4. El-Naggar, M.E.; Abdelgawad, A.M.; Elsherbiny, D.A.; El-shazly, W.A.; Ghazanfari, S.; Abdel-Aziz
Bioactive Wound Dressing Gauze Loaded with Silver Nanoparticles Mediated by Acacia Gum. J. C l-Naggar, M.E.; Abdelgawad, A.M.; Elsherbiny, D.A.; El-shazly, W.A.; Ghazanfari, S.; Abdel-Aziz, M.S.; Abd 4. El-Naggar, M.E.; Abdelgawad, A.M.; Elsherbiny, D.A.; El-shazly, W.A.; Ghazanfari, S.; Abdel-Aziz, M.S.; Abd-Elmoneam, Y.K. Bioactive Wound Dressing Gauze Loaded with Silver Nanoparticles Mediated by Acacia Gum. J. Clust. Sci. 2019. [CrossRef] 5. El-Naggar, M.E.; Khattab, T.A.; Abdelrahman, M.S.; Aldalbahi, A.; Hatshan, M.R. Development of antimicrobial, UV blocked
and photocatalytic self-cleanable cotton fibers decorated with silver nanoparticles using silver carbamate and plasma activation. Cellulose 2021, 28, 1105–1121. [CrossRef] 6. Chen, G.; Li, Y.; Bick, M.; Chen, J. Smart textiles for electricity generation. Chem. Rev. 2020, 120, 366 .; Li, Y.; Bick, M.; Chen, J. Smart textiles for electricity generation. Chem. Rev. 2020, 120, 3668–3720. [CrossRef] 7. Lee, D.; Sang, J.S.; Yoo, P.J.; Shin, T.J.; Oh, K.W.; Park, J. 19.
El-Naggar, M.E.; Hasanin, M.; Youssef, A.M.; Aldalbahi, A.; El-Newehy, M.H.; Abdelhameed, R.M. Hydroxyethyl cellu-
lose/bacterial cellulose cryogel dopped silver@titanium oxide nanoparticles: Antimicrobial activity and controlled release of
Tebuconazole fungicide. Int. J. Biol. Macromol. 2020, 165, 1010–1021. [CrossRef] [PubMed] References 2020, 382, 123052. [CrossRef]
15. Jatoi, A.W.; Kim, I.S.; Ni, Q.-Q. Cellulose acetate nanofibers embedded with AgNPs anchored TiO2 nanoparticles for long term
excellent antibacterial applications. Carbohydr. Polym. 2019, 207, 640–649. [CrossRef] [PubMed] resistance genes by a TiO2/AgNPs-modified nanofiber forward osmosis membrane. Chem. Eng. J. 2020, 382, 123052. [CrossRef]
15. Jatoi, A.W.; Kim, I.S.; Ni, Q.-Q. Cellulose acetate nanofibers embedded with AgNPs anchored TiO2 nanoparticles for long term
excellent antibacterial applications. Carbohydr. Polym. 2019, 207, 640–649. [CrossRef] [PubMed] 15. Jatoi, A.W.; Kim, I.S.; Ni, Q.-Q. Cellulose acetate nanofibers embedded with AgNPs anchored TiO2 na
excellent antibacterial applications. Carbohydr. Polym. 2019, 207, 640–649. [CrossRef] [PubMed] V.T.; Nguyen, T.A.; Tran, V.K.; Nguyen-Tri, P. Antibacterial activity of TiO2-and ZnO-decorated with silver
mpos. Sci. 2019, 3, 61. [CrossRef] 16. Nguyen, V.T.; Vu, V.T.; Nguyen, T.A.; Tran, V.K.; Nguyen-Tri, P. Antibacterial activity of TiO2-and Zn
nanoparticles. J. Compos. Sci. 2019, 3, 61. [CrossRef] 17. Djellabi, R.; Basilico, N.; Delbue, S.; D’Alessandro, S.; Parapini, S.; Cerrato, G.; Laurenti, E.; Falletta, E.; Bianchi, C.L. Oxidative
Inactivation of SARS-CoV-2 on Photoactive AgNPs@ TiO2 Ceramic Tiles. Int. J. Mol. Sci. 2021, 22, 8836. [CrossRef] [PubMed] 17. Djellabi, R.; Basilico, N.; Delbue, S.; D’Alessandro, S.; Parapini, S.; Cerrato, G.; Laurenti, E.; Falletta, E.; Bianchi, C.L. Oxidative
Inactivation of SARS-CoV-2 on Photoactive AgNPs@ TiO2 Ceramic Tiles. Int. J. Mol. Sci. 2021, 22, 8836. [CrossRef] [PubMed]
18. Chou, J.-C.; Lin, Y.-C.; Lai, C.-H.; Kuo, P.-Y.; Nien, Y.-H.; Syu, R.-H.; Yong, Z.-R.; Wu, Y.-T. AgNWs@ TiO2 and AgNPs@ TiO2
Double-Layer Photoanode Film Improving Light Capture and Application under Low Illumination. Chemosensors 2021, 9, 36. [CrossRef] g
18. Chou, J.-C.; Lin, Y.-C.; Lai, C.-H.; Kuo, P.-Y.; Nien, Y.-H.; Syu, R.-H.; Yong, Z.-R.; Wu, Y.-T. AgNWs@ TiO2 and AgNPs@ TiO2
Double-Layer Photoanode Film Improving Light Capture and Application under Low Illumination. Chemosensors 2021, 9, 36. [CrossRef] 19. El-Naggar, M.E.; Hasanin, M.; Youssef, A.M.; Aldalbahi, A.; El-Newehy, M.H.; Abdelhameed, R.M. Hydroxyethyl cellu-
lose/bacterial cellulose cryogel dopped silver@titanium oxide nanoparticles: Antimicrobial activity and controlled release of
Tebuconazole fungicide. Int. J. Biol. Macromol. 2020, 165, 1010–1021. [CrossRef] [PubMed] 118 Polymers 2021, 13, 3664 20. Kusiak-Nejman, E.; Czy˙zewski, A.; Wanag, A.; Dubicki, M.; Sadłowski, M.; Wróbel, R.J.; Morawski, A.W. of nitric oxide over AgNPs/TiO2-loaded carbon fiber cloths. J. Environ. Manag. 2020, 262, 110343. [Cro 21. Abdelwahab, N.A.; El-Nashar, D.E.; El-Ghaffar, M.A.A. Polyfuran, polythiophene and their blend as novel antioxidants for
styrene-butadiene rubber vulcanizates. Mater. Des. 2011, 32, 238–245. 32.
Binia´s, D.; Binia´s, W.; Machnicka, A.; Hanus, M. Preparation of antimicrobial fibres from the EVOH/EP
nanoparticles. Polymers 2020, 12, 1827. [CrossRef] [PubMed] Tarek Abou Elmaaty 1,2, Sally Raouf 3, Khaled Sayed-Ahmed 4 and Maria Rosaria Plutino 5,* Tarek Abou Elmaaty 1,2, Sally Raouf 3, Khaled Sayed-Ahmed 4 and Maria Rosaria Plutino 5,* 1
Department of Material Arts, Faculty of Art & Design, Galala University, Galala 43713, Egypt;
tasaid@gu.edu.eg 2
Department of Textile Printing, Dyeing & Finishing, Faculty of Applied Art, Damietta University
Damietta 34511, Egypt 2
Department of Textile Printing, Dyeing & Finishing, Faculty of Applied Art, Damietta University,
Damietta 34511, Egypt 3
Department of Textile Printing, Dyeing & Finishing, Faculty of Applied Art, Banha University,
Banha 13518, Egypt; soola.a@hotmail.com gyp
4
Department of Agricultural Chemistry, Faculty of Agriculture, Damietta University, Damietta 34511, Egypt;
dr_khaled@yahoo.com
5 5
Consiglio Nazionale Delle Ricerche, c/o Dipartment ChiBioFarAm, Istituto per lo Studio dei Materiali
Nanostrutturati, University of Messina, Viale F. D’Alcontres 31, Vill. S. Agata, 98166 Messina, Italy
*
Correspondence: mariarosaria.plutino@cnr.it; Tel.: +39-(09)-06765713 Abstract: This work aims to utilize selenium nanoparticles (Se-NPs) as a novel dyestuff, which
endows wool fibers with an orange color because of their localized surface plasmon resonance. The
color characteristics of dyed fibers were evaluated and analyzed. The color depth of the dyed fabrics
under study was increased with the increase in Se content and dyeing temperature. The colored wool
fabrics were characterized using scanning electron microscopy (SEM), energy dispersive spectroscopy
(EDX) and an X-ray diffraction (XRD) analysis. The results indicated that spherical Se-NPs with a
spherical shape were consistently deposited onto the surface of wool fibers with good distribution. In
addition, the influence of high temperature on the color characteristics and imparted functionalities
of the dyed fabrics were also investigated. The obtained results showed that the proposed dyeing
process is highly durable to washing after 10 cycles of washes, and the acquired functionalities,
mainly antimicrobial activity and UV-blocking properties, were only marginally affected, maintaining
an excellent fastness property. Keywords: multifunctional finishing; selenium nanoparticles; wool fabrics; UV-blocking properties;
antimicrobial activity Article
Multifunctional Dyeing of Wool Fabrics Using
Selenium Nanoparticles Tarek Abou Elmaaty 1,2, Sally Raouf 3, Khaled Sayed-Ahmed 4 and Maria Rosaria Plutino 5,* References [CrossRef] y
22. El-Enany, G.M.; Ghanem, M.A.; El-Ghaffar, M.A. Electrochemical deposition and characterization of poly (3,4-ethylene dioxythio-
phene), poly (aniline) and their copolymer onto glassy carbon electrodes for potential use in ascorbic acid oxidation. Port. Electrochim. Acta 2010, 28, 336–348. 23. Li, W.; Yu, Z.; Wu, Y.; Liu, Q. Preparation, characterization of feather protein-g-poly (sodium allyl sulfonate) and its application as
a low-temperature adhesive to cotton and viscose fibers for warp sizing. Eur. Polym. J. 2020, 136, 109945. [CrossRef] p
p
g
24. Revol, B.P.; Vauthier, M.; Thomassey, M.; Bouquey, M.; Ruch, F.; Nardin, M. Design of experience to evaluate the Interfacial
compatibility on high tenacity viscose fibers reinforced Polyamide-6 composites. Compos. Sci. Technol. 2021, 203, 108615. [CrossRef] 25. Katouah, H.; El-Metwaly, N.M. Plasma treatment toward electrically conductive and superhydrophobic cotton fibers by in situ
preparation of polypyrrole and silver nanoparticles. React. Funct. Polym. 2021, 159, 104810. [CrossRef]
á d
G
d ll
ll
d í
G
ál
h
f
h
l
h 26. Hernández-Gordillo, A.; Arroyo, M.; Zanella, R.; Rodríguez-González, V. Photoconversion of 4-nitrophenol in the presence
of hydrazine with AgNPs-TiO2 nanoparticles prepared by the sol–gel method. J. Hazard. Mater. 2014, 268, 84–91. [CrossRef]
[PubMed] [
]
27. Atta, A.M. Immobilization of silver and strontium oxide aluminate nanoparticles integrated into plasma-activated cotton fabric:
Luminescence, superhydrophobicity, and antimicrobial activity. Luminescence 2021, 36, 1078–1088. [CrossRef] [PubMed]
f
h b d
h l 28. Wu, S.; Huang, J.; Cui, H.; Ye, T.; Hao, F.; Xiong, W.; Liu, P.; Luo, H. Preparation of organic–inorganic hybrid methylene
blue polymerized organosilane/sepiolite pigments with superhydrophobic and self-cleaning properties. Text. Res. J. 2019, 89,
4220–4229. [CrossRef] [
]
29. Atta, A.M.; Abomelka, H.M. Multifunctional finishing of cotton fibers using silver nanoparticles via microwave-assisted reduction
of silver alkylcarbamate. Mater. Chem. Phys. 2021, 260, 124137. [CrossRef] y
30. Katouah, H.A.; El-Sayed, R.; El-Metwaly, N.M. Solution blowing spinning technology and plasma-assisted oxidation-reduction
process toward green development of electrically conductive cellulose nanofibers. Environ. Sci. Pollut. Res. 2021, 1–13. 31. Chiam, S.-L.; Soo, Q.-Y.; Pung, S.-Y.; Ahmadipour, M. Polycrystalline TiO2 particles synthesized via one-step rapid heating
method as electrons transfer intermediate for Rhodamine B removal. Mater. Chem. Phys. 2021, 257, 123784. [CrossRef] 32. Binia´s, D.; Binia´s, W.; Machnicka, A.; Hanus, M. Preparation of antimicrobial fibres from the EVOH/EPC blend containing silver
nanoparticles. Polymers 2020, 12, 1827. [CrossRef] [PubMed] 119 polymers 1. Introduction Recently, the usage of unique dyes and finishing agents has had a substantial influence
on textiles’ functionality. The dyeing of textiles with multi-functional characteristics has
attracted significant interest in recent years. Nanotechnologies and nanomaterials offer
a wider application potential for preparing functional textiles, such as flame retardant,
self-cleaning, wrinkle-resistant, antistatic, antimicrobial, and UV-protective, etc. [1–14] In
recent years, several approaches to metal and metal oxide NP fabrication and application
have been developed to impart functionalities to different types of textile substrates [15–23]. In addition, nanomaterials, such as gold and silver nanoparticles, stabilized by opportune
functional capping agents, have been used as a novel class of functionalizing nanofillers
either themselves [24–29] or for the functionalization of different types of materials (such
as concrete, glass, geopolymer and also embedded in coloring or antibacterial coatings),
especially for textile fabrics [30]. Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
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iations. Copyright:
© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article
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conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). p
y
The coloration of fabric/fiber can be achieved through a combination with metal
nanoparticles by means of the particular optical properties of plasmon nanomaterials,
called localized surface plasmon resonance (LSPR), producing brilliant and vivid colors, 121 https://www.mdpi.com/journal/polymers Polymers 2022, 14, 191. https://doi.org/10.3390/polym14010191 Polymers 2022, 14, 191 which are different from traditional dyes in that they are not the chromophore of traditional
dyes but the shape and size of nanoparticles that determine the colors [31,32]. This has
recently motivated research activities to directly employ metal nanoparticles in the dyeing
of different textile substrates to overcome color fading and high water and energy con-
sumption problems associated with conventional dyeing methods. Furthermore, excessive
exposure to dyes may cause respiratory problems and skin irritation and increase risk of
cancer [33]. Hence, several different approaches have been developed to impart colors
and functionalities to diverse fabrics/fibers, such as cotton, wool [34–41], silk [42–45],
bamboo [46], ramie [47], viscose and acrylic [48] using noble metal nanoparticles via a
self-assembly or in-situ synthesis methods. y
y
Owing to the localized Surface Plasmon Resonance (LSPR) property of selenium
nanoparticles (Se-NPs), they exhibit adaptable colors depending on the synthesizing mea-
sures. 2.1. Materials The fabrics used here were 100% scoured and bleached wool. Sodium hydrogen
selenite, vitamin C and polyvinylpyrrolidone (PVP) were purchased from Loba Chemie,
India. In addition, the other chemicals used in this study were of commercial grade. 2.3. Methods 2.3. Methods 2.3.1. Green Synthesis of Se-NPs 1. Introduction Besides, Se-NPs have strong cytotoxicity towards a broad range of microorganisms,
low toxicity to human cells, high selectivity, and long-term durability [49]. Se-NPs have
significant antimicrobial and antioxidant activity, which have been widely reported in
the literature [50–52], and their applications in textile fabrics have been reported once by
Joanne Yip et al. [53] as an antimicrobial agent for polyester fabric. In our previous study, cotton and polyester were colored with silver and gold NPs via
a printing technique to render different functions of fibrous materials [54]. Subsequently,
Se-NPs were used in the wool fabric printing process to fabricate antimicrobial textiles
with low cytotoxicity [55]. In the current study, wool fabrics were dyed with Se-NPs in a
different way to produce multifunctional fabrics with brilliant stable colors in one step and
compare between the results obtained from both dyeing and printing techniques. 2.2. Dyeing Apparatus Dyeing was carried out using an Infra Color Dyeing Machine, which consists of
12 beakers that are mounted in a rotating beaker-carrying wheel. Heating occurs through
infrared radiation, cooling through air and automation through the microprocessor pro-
grammer DC4 F/R. The maximum temperature was up to 140 ◦C, a maximum heating rate
was up to 5 ◦C/min and cooling had a maximum rate up to 3 ◦C/min. 2.3. Methods 2.3.1. Green Synthesis of Se-NPs Se-NPs were prepared according to the method reported by Abou Elmaaty et al. [55]
through a redox reaction. Sodium hydrogen selenite at different concentrations of 25 mM,
50 mM, 75 mM and 100 mM was added to vitamin C at the same concentration and
the volume ratio of 1:1 under magnetic stirring. PVP was used as a stabilizer at the
concentration of 0.75–3 g/100mL of vitamin C to enhance the stability of Se-NPs. The
change in color from colorless to orange to dark orange indicated the formation of Se-NPs. 2.6.2. Fastness Properties The color fastness properties were evaluated for all Se-dyed wool fabrics in accor-
dance with the standard test method. Washing, rubbing and light fastness were measured
according to the method of AATCC test methods (61–1972), (8–1972) and (16A–1972),
respectively. 2.5.1. Antimicrobial Activity The biological activity of the Se-dyed wool samples was evaluated qualitatively against
G+ve bacteria (Staphylococcus aureus and Bacillus cereus), G-ve bacteria (Escherichia coli)
and yeast (Candida utilis). The antimicrobial test was performed according to the AATCC
Test Method (147–1988). The antimicrobial activity was expressed as the growth inhibition
zone (mm). 2.6.1. Color Measurements 2.6.1. Color Measurements The color coordinates of CIE lab (L *, a *, b *, C *, and h*) and color strength (K/S) for
both blank and Se-dyed wool fabrics were measured in the wavelength range of 360–720 nm
using a Konica Minolta spectrophotometer (CM-3600 d, Minolta, Tokyo, Japan). All samples
were measured in three different areas, bearing in mind both sides of the fabrics, and the
mean values were recorded. 2.5. Functional Properties of Se-Dyed Wool Fabrics 2.5. Functional Properties of Se-Dyed Wool Fabrics The functionalities of wool fabrics dyed with Se-NPs were evaluated in terms of the
UV protection factor (UPF) and antimicrobial activity. 2.4. Characterization TEM images of the synthesized Se-NPs were obtained using a JEM-2100 Transmission
Electron Microscope (TEM) with an acceleration voltage of 200 kV to determine the size
and morphology of the NPs. Samples for the TEM analysis were prepared by dripping a
drop of Se-NP solution onto a carbon-coated copper grid and drying at room temperature. The surface morphology of wool fabrics, either blank or dyed fabrics, was examined
using high resolution scanning electron microscopy (JEOL JSM-6510LB with a field emission
gun, Tokyo, Japan). The chemical structure of the dyed samples was analyzed using a
surface energy dispersive spectroscopy (EDX) analysis unit (EDAX AMETEK analyzer,
Rigaku, Japan) attached to the electron microscope. X-ray diffraction was analyzed for both the prepared Se-NPs and Se-dyed wool fabrics
using an X-ray diffractometer system (XRD) (Bruker D8 ADVANCE, Karlsruhe, Germany). The Se-NP solution was dried at 130 ◦C until completely dried. 2.5. Functional Properties of Se-Dyed Wool Fabrics 2.3.2. Dyeing Procedure of Wool Fabrics with Selenium NPs 2.3.2. Dyeing Procedure of Wool Fabrics with Selenium NPs Wool fabrics were dyed with different concentrations of Se-NP solution using a wet
chemistry method through an immersion process. Briefly, the wool fabrics were immersed
into the Se-NP solutions (dyeing solutions) without any chemical additives. Dyeing was
performed in a laboratory-scale thermal HT dyeing machine with a liquor-to-goods ratio
of 100:1. The solutions containing Se-NPs and wool fabrics were shaken at different
temperatures, including room temperature, 40 ◦C, 70 ◦C and 100 ◦C for 60 min to obtain 122 Polymers 2022, 14, 191 the coloration of fabrics. At the end of the dyeing process, the dyed wool samples were
removed, rinsed with water and dried at room temperature. the coloration of fabrics. At the end of the dyeing process, the dyed wool samples were
removed, rinsed with water and dried at room temperature. 2.6. Testing 2.6.1. Color Measurements 2.5.2. UV-Protection Properties The ultraviolet protection factor expressed as UPF and transmission of ultraviolet
(UV) radiation through wool fabrics were evaluated based on the AS/NZS 4399:1996 test
method. The UV protection was rated as good, very good or excellent if the UPF values
were between 15–24, 25–39 or above 40, respectively. 2.6.4. Mechanical Properties 2.6.4. Mechanical Properties The tensile strength (maximum load) and elongation at break (maximum strain) of
blank and Se-dyed wool fabrics at high temperature were evaluated using the strip test
method on a multi-tester machine according to ASTM-D4850 with a load cell 500 N, preload
0.01 N, speed of 100 mm/min and gauge length of 100 mm. 2.7. Statistical Analysis Antimicrobial, UV-protection, and mechanical tests were performed by taking the
average of three readings (samples). The standard error of the mean was calculated
according to the equation given below and was found to be + (−) 0.1 SEX = S/√n Where S = sample standard deviation and n = the number of observations of
the samples. 2.6.3. Durability Test Durability to washing of the imparted functionalities (antimicrobial activity and UV-
protection properties) as well as color strength (K/S) of Se-dyed wool fabrics were evaluated
according to AATCC Test Method 61(2A)-1996 after 10 laundering cycles. 123 Polymers 2022, 14, 191 3.1. Characterization of Se-NPs and Dyed Wool Fabrics 3.1. Characterization of Se-NPs and Dyed Wool Fabrics 3. Results and Discussion The dyeing process of wool fabrics using selenium nanoparticles (Se-NPs) as a new
functional colorant was investigated to obtain multifunctional dyed fabrics based on
colloidal solutions of nanomaterials. The influences of different dyeing temperatures on the
adsorption of Se-NPs onto wool fabrics and alteration of the color characteristics of wool
samples were observed. The imparted functionalities were also determined. The results
obtained and appropriate discussions are presented below. 3.1.2. SEM and EDX Analysis 3.1.2. SEM and EDX Analysis In Figure 2, SEM images show the topographical characteristics of the blank and
Se-dyed wool fabrics. The SEM images of the blank wool show typically clear, clean scales
and a smooth longitudinal fibrous structure surface, as shown in Figure 2(0). The SEM
images of the surface of the Se-dyed wool fabrics revealed that the surfaces of wool fabrics
were covered by a sufficient layer of Se in the nano size, and Se-NPs on the wool fabric
surfaces had a wide range of size distribution. The surface chemical elements of the Se-dyed wool fabrics were determined by EDX
spectroscopy, as shown in Figure 2e–h. The peaks appearing at about 1 and 11 Kev in each
figure are attributed to selenium NPs. The SEM images and EDX results provide strong
evidence for the formation of Se-NPs on the surface of the wool fabrics. 3.1.1. Transmission Electron Microscopy (TEM) 3.1.1. Transmission Electron Microscopy (TEM) Figure 1 shows TEM images of colloidal Se-NPs prepared at different concentrations. The obtained TEM micrographs revealed that the Se-NPs prepared at a concentration of
50 mM had the lowest diameter range (25–90 nm) compared to the other prepared Se-NPs. ȱ
Figure 1. TEM images of synthesized Se-NPs on different concentrations: (a) 12.5 mM, (b) 25 mM,
(c) 37.5 mM and (d) 50 mM. ȱ Figure 1. TEM images of synthesized Se-NPs on different concentrations: (a) 12.5 mM, (b) 25 mM,
(c) 37.5 mM and (d) 50 mM. 124 Polymers 2022, 14, 191 The Se-NPs were well-dispersed and mostly spherical in shape when no agglomeration
or deformation of Se-NPs were observed. The Se-NPs prepared at a concentration of
12.5 mM showed the largest size up to 115 nm, resulting in the appearance of a block
structure, as shown in Figure 1a. While the concentration of the stabilizer PVP (3 g/100mL)
and sodium hydrogen selenite increased, the shape of the Se-NPs was observed to be
uniformly spherical instead of an aggregated form and had the lowest diameter, as shown
in Figure 1d. 3.2. Functional Properties of Se-NP Dyed Wool Fabrics
3.2.1. Biological Activity 3.2. Functional Properties of Se-NP Dyed Wool Fabrics 3.1.3. X-ray Diffraction (XRD) An XRD analysis was performed for further confirmation of the Se-NP formation. This
analytical method aids in the determination of crystallite materials and provides details
of the unit cell dimensions. As shown in Figure 3, the obtained Se-NPs for both prepared An XRD analysis was performed for further confirmation of the Se-NP formation. This
analytical method aids in the determination of crystallite materials and provides details
of the unit cell dimensions. As shown in Figure 3, the obtained Se-NPs for both prepared
solutions and Se-dyed wool fabrics were highly crystalline, and all diffraction peaks were
well indexed as 24.28◦, 29.24◦and 43.64◦, corresponding to 100, 101 and 102 crystal planes,
respectively, in accordance with the JCPDS 86-2246 international database. [56] The results
obtained from the SEM, EDX and XRD analyses confirmed the sufficient deposition of
Se-NPs on the wool fabric surface. solutions and Se-dyed wool fabrics were highly crystalline, and all diffraction peaks were
well indexed as 24.28◦, 29.24◦and 43.64◦, corresponding to 100, 101 and 102 crystal planes,
respectively, in accordance with the JCPDS 86-2246 international database. [56] The results
obtained from the SEM, EDX and XRD analyses confirmed the sufficient deposition of
Se-NPs on the wool fabric surface. 3.2.1. Biological Activity 2 0
3 0
4 0
5 0
6 0
7 0
102
100
101
Intensity (a.u.)
2 th e ta (d e g r e e s )
S e N P s
W o o lS e N P s
Figure 3. XRD patterns of the synthesized Se-NPs and Se-dyed wool fabric. Intensity (a.u.) Figure 3. XRD patterns of the synthesized Se-NPs and Se-dyed wool fabric. Figure 3. XRD patterns of the synthesized Se-NPs and Se-dyed wool fabric. Table 1. Antimicrobial activity of the Se-dyed wool fabrics at different temperatures. Se-NPs
Conc. Temp. ◦C
Antimicrobial Activity
ZI 1 (mm.)
Blank wool
G+ve
G−ve
Yeast
S. aureus
Bacillus cereus
E. coli
Candida utilis
0
0
0
0
12.5 mM
At room
temp. 13.4 (13) 2
10.6 (9)
10 (10)
14 (13.5)
25 mM
15.9 (15)
12.6 (12)
13.5 (12.4)
17.5 (16)
37.5 mM
17.7 (17)
14 (13.2)
14.3 (14)
19 (18)
50 mM
19 (18)
15.4 (14.8)
18.2 (18)
23 (22.3)
12.5 mM
At 40 ◦C
13 (13)
10.2 (10)
9.3 (9)
14 (12)
25 mM
14.7 (13.8)
12 (11)
12.5 (11)
16.6 (15)
37.5 mM
17 (15)
13.8 (13)
14 (12.3)
18.5 (15.3)
50 mM
18.2 (17.4)
15 (14.3)
17.4 (15.6)
20.8 (20)
12.5 mM
At 70 ◦C
10 (8)
8 (8)
8.6 (7.2)
12 (10)
25 mM
11.5 (11)
10 (8.5)
9.7 (9)
12.8 (11.2)
37.5 mM
13 (10.3)
10.5 (10)
10.5 (9.2)
14 (12.6)
50 mM
14.8 (14)
12 (10)
11.5 (11)
17.2 (15.4)
12.5 mM
At 100 ◦C
8 (7)
7.6 (6.3)
7 (6)
10.6 (10)
25 mM
9 (8.4)
8.2 (7)
8.5 (7)
11 (9.3)
37.5 mM
11 (10.5)
8.8 (8)
9 (8.2)
11.8 (11)
50 mM
11.8 (10)
10 (8.8)
10 (9)
13.5 (12.6)
1 ZI (zone of inhibitions). 2 Values in parentheses indicate durability of antimicrobial activity after 10 washing cycles. Table 1. Antimicrobial activity of the Se-dyed wool fabrics at different temperatures. On the other hand, the antimicrobial activity of the Se-dyed samples showed a slight
decrease with an increase in the dyeing temperatures, which may be attributed to the sig-
nificant impact of heat treatment on the size, structure and bioactivity of Se-NPs. 3.2.1. Biological Activity The large surface area of natural textile fabrics and their ability to retain moisture
provide an excellent environment for microorganism growth. Therefore, imparting the
antimicrobial properties for natural fabrics is of great interest. Thus, the antimicrobial
activity of the unwashed and washed Se-dyed wool fabrics was evaluated qualitatively
against G+ve (S. aureus and & Bacillus cereus), G-ve (E. coli) and yeast (Candida utilis). The
results are reported in Table 1 and are expressed as the zone of growth inhibition ZI (mm). From the results, it is quite clear that the blank wool fabric did not show any antimi-
crobial effect. However, the Se-dyed wool showed a notable antimicrobial activity that
increased with the increase in Se-NP concentration against the tested pathogens. In this
respect, SEM micrographs illustrated that the deposition of Se-NPs on the wool surface
increased with the increase in Se-NP concentration, confirming the results obtained from
the antimicrobial test. This enhancement may be due to the adsorption of Se-NPs, leading to
cell wall depolarization, which changes the typically negative charge of the wall to become
more permeable, and then, inhibition of cell membrane metabolisms causing the death
of bacteria, and/or the increase in NP concentrations leading to a concomitant increase
of highly reactive oxygen radicals causing the destruction of the molecular structure of
bacteria [57]. 125 Polymers 2022, 14, 191 Figure 2. SEM micrographs of (0) blank wool fabric, and (a–d) wool fabrics dyed with Se
different concentrations of: (a) 12.5 mM, (b) 25 mM, (c) 37.5 mM and (d) 50 mM, as well
spectra of Se-dyed wool fabrics with varied concentrations of: (e) 12.5 mM, (f) 25 mM, (g) 3
and (h) 50 mM. Figure 2. SEM micrographs of (0) blank wool fabric, and (a–d) wool fabrics dyed with Se-NPs at
different concentrations of: (a) 12.5 mM, (b) 25 mM, (c) 37.5 mM and (d) 50 mM, as well as EDX
spectra of Se-dyed wool fabrics with varied concentrations of: (e) 12.5 mM, (f) 25 mM, (g) 37.5 mM
and (h) 50 mM. 126 Polymers 2022, 14, 191 2 0
3 0
4 0
5 0
6 0
7 0
102
100
101
Intensity (a.u.)
2 th e ta (d e g r e e s )
S e N P s
W o o lS e N P s
Figure 3. XRD patterns of the synthesized Se-NPs and Se-dyed wool fabric. 3.2.1. Biological Activity Typically,
smaller NPs have higher antimicrobial activity; this phenomenon can be explained by
the small NPs having a larger surface area than larger NPs, which can extremely increase
the production of highly reactive oxygen species (ROS), which severely damaged and
inactivated the essential biomolecules, including proteins, DNA and lipids [57,58] Moreover, subjecting the Se-dyed samples to up to 10 cycles of consecutive laundry
cycles according to AATCC Test Method 61(2A)-1996 led to a non-sense decrement in their
antimicrobial properties. Colored wool fabrics with low amounts of Se-NPs were enough
to achieve excellent antimicrobial activity. On the other hand, Adomaviciute et al. (2016) revealed that no antimicrobial activity
was observed for the PVP solution (8%) against Staphylococcus aureus, Bacillus cereus, Es-
cherichia coli and Candida albicans [59]. However, the PVP concentration required for Se-NPs 127 Polymers 2022, 14, 191 preparation did not exceed 1.5% in this study, indicating that the antimicrobial activity
mainly corresponded to Se-NPs. These results demonstrated the activity of Se-NPs for ingrain dyed wool fabrics against
the microbial pathogens and confirmed the high efficiency of the current processing in the
acquisition of wool fabrics’ bio-functionality, in addition to the ingrain coloration of fabrics. 3.2.2. UV-Protection Properties Skin diseases, including allergies, premature skin aging, sunburn, and skin cancer, can
occur due to skin exposure to UV irradiation. A great number of approaches were done to
protect human skin against the harmful effects of UV irradiation using nanoparticles [60–62]. Therefore, the UV-blocking properties of the wool fabrics dyed with Se-NPs were evaluated. Table 2 showd the transmittance values of UV light in the range of UV-A (315–400 nm)
and UV-B (280–315 nm) ranges and the UV protection factor (UPF) values of the undyed
wool fabric and the wool fabrics dyed with Se-NPs. The average transmittance values
of the wool fabrics were decreased obviously after wool fabrics were dyed using Se-NPs,
which confirmed that the Se-NPs prominently enhanced the UV-blocking activity of wool
fabrics. Both transmittance values in the UV-A and UV-B regions decreased with increasing
concentrations of Se-NPs on the wool fabrics. Table 2. UV protection properties of Se-NPs dyed wool fabrics at different temperatures. Se-NPs conc. Temp. ◦C
UV Protection Properties
Blank wool
UV-A
Transmittance
UV-B
Transmittance
UPF
10.43
1.72
33.46
12.5 mM
At room temp. 2.33
1.58
62.5 (60.2) 1
25 mM
1.51
1.37
81.8 (80)
37.5 mM
1.46
0.83
120 (117)
50 mM
1.21
0.36
255 (250)
12.5 mM
At 40 ◦C
1.44
1.23
90 (87)
25 mM
1.32
0.96
130 (125.6)
37.5 mM
1.04
0.54
189.5 (180.7)
50 mM
0.76
0.21
274 (270)
12.5 mM
At 70 ◦C
0.71
0.88
133.5 (130.2)
25 mM
0.52
0.73
176 (170.5)
37.5 mM
0.35
0.61
258.7 (255)
50 mM
0.27
0.42
321 (320)
12.5 mM
At 100 ◦C
0.59
0.77
170.2 (166)
25 mM
0.5
0.65
210 (204.8)
37.5 mM
0.41
0.32
266.9 (260)
50 mM
0.08
0.06
452.6 (447.5)
1 Values in parentheses indicate durability of UV protection properties after 10 washing cycles. Table 2. UV protection properties of Se-NPs dyed wool fabrics at different temperatures. The UPF refers to the fabric efficiency to block out UV irradiation from passing through
and reaching the skin. The UPF value of the undyed wool fabrics was also measured to
be 33.46 (Table 2). From the results obtained, it was obvious that the dyeing process with
Se-NPs increased the UPF value of wool fabrics, as shown in Figure 4 and Table 2. The
results confirmed that the Se-NP dyed wool fabrics provide excellent UV protection, which
increased by increasing the Se concentrations. 3.2.2. UV-Protection Properties 128 Polymers 2022, 14, 191 ȱ
62.5
81.8
120
255
0
90
130
189.5
274
0
133.5
176
258.7
321
0
170.2
210
266.9
452.6
0
50
100
150
200
250
300
350
400
450
500
atroomtemp. at40°C
at70°C
at100°C
UPF
SeͲNPsconc.
Figure 4. UV protection properties of Se-dyed wool fabrics at different temperatures. Figure 4. UV protection properties of Se-dyed wool fabrics at different temperatures. Additionally, the influence of dyeing temperature on the UV protection properties of
wool fabrics was examined. The UPF value of the wool fabrics were increased dramatically,
as shown in Figure 4, as well as the UV light transmittance values were decreased with an
increase in dyeing temperature. This may be due to the larger size of Se particles because
of high temperature coloration; it could be considered that enlarged particles have a better
chance to reflect more radiation [45]. Furthermore, the results in Table 2 also showed that repeated laundering for up to 10
cycles of the Se-dyed samples, evaluated according to AATCC Test Method 61(2A)-1996,
caused a slight decrease in the imparted UV-protection functionality, expressed as UPF
values; however, it was still rated as excellent values. 3.3. Color Characteristics and Color Fastness of Se-Dyed Wool Fabrics This indicates that the Se-NPs
were still tightly loaded and fixed onto the simultaneous functional dyeing of the wool
fabric surface. This might be due to the electrostatic interaction between wool fibers, which
protonated to carry positive charges and Se-NPs carrying negative charges, leading to the
coloration of fibers. The washing, rubbing and light color fastness of dyed wool fabrics with Se-NPs at
different dyeing temperatures were evaluated according to AATCC standard test methods
(61–1972), (8–1972) and (16A–1972), respectively. The results represented in Table 4 showed washing, rubbing and light fastness prop-
erties for all Se-dyed samples. Both Se-dyed wool fabrics at room temperature and 40◦C
exhibited excellent results for washing and rubbing fastness, which agreed with the K/S
data after the washing process, while the light fastness showed a very good rating of 4/5. Table 3. Color characteristics for Se-dyed wool fabrics at different temperatures. Se-NPs Conc. Temp. ◦C
L *
a *
b *
c *
H *
K/S
Blank wool
Room temp. 86.87
0.96
12.33
12.37
85.56
1.1
12.5 mM
79.77
7.83
20.2
21.67
68.81
2.21 (2)1
25 mM
67.81
18.84
26.05
32.15
60.12
3.71 (3.5)
37.5 mM
68.77
17.97
28.98
34.1
58.19
4.68 (4.3)
50 mM
59.14
24.52
29.26
37.41
49.05
6.63 (6.4)
12.5 mM
40 ◦C
75.56
11.47
21.36
24.24
61.77
2.53 (2.5)
25 mM
64.14
22.06
27.21
32.75
57.66
3.34 (3.2)
37.5 mM
66.9
23.85
29.28
32.15
51.86
4.23 (4)
50 mM
42.32
27.34
30.12
39.77
44.24
6.31 (6)
12.5 mM
70 ◦C
42.16
12.82
15.1
19.81
49.67
9.8 (9.5)
25 mM
42.76
18.03
21.51
28.07
50.02
12.7 (12.3)
37.5 mM
42.26
24.63
28.37
37.57
49.04
15.3 (15)
50 mM
36.03
34.3
27.04
42.25
49.99
16.3 (16)
12.5 mM
100 ◦C
33.21
5.76
9.09
10.76
57.63
13.8 (13.3)
25 mM
29.27
13.56
13.56
18.44
42.63
18.2 (18)
37.5 mM
28.42
16.96
16.96
23.01
42.53
21.7 (21.4)
50 mM
28.43
17.06
17.06
23.15
42.53
22.1 (22)
* Values in parentheses indicate color strength (K/S) properties after 10 washing cycles. Table 3. Color characteristics for Se-dyed wool fabrics at different temperatures. By increasing the dyeing temperature from 40 ◦C to 70 ◦C and 100 ◦C, the wool fabrics
became red or brown, as indicated by the positive a* and b* values. 3.3. Color Characteristics and Color Fastness of Se-Dyed Wool Fabrics 3.3. Color Characteristics and Color Fastness of Se-Dyed Wool Fabrics At the end of the dyeing process, the color of the wool fabrics was changed, and the
fabric color reflected the color of the impregnated Se-NPs, as shown in Figures 5 and 6. Color characteristics were quantified by estimating the color space in terms of L *, a *, b *
and color strength (K/S). In this system, L * refers to lightness/darkness values from 100 to
0 representing white to black, a * values run from negative (green) to positive (red) and b *
values run from negative (blue) to positive (yellow). The detected data are represented in
Table 3. An illustration of the color data can be pointed in the following: dyeing at room
temperature and 40 ◦C resulted in linear decrement in lightness values with increment in
Se content, which was reflected in the color acquired by the incorporation of Se-NPs on
wool fabrics. The lightness results were reduced from 79.77 to 59.14 at room temperature
and from 75.56 to 42.32 for dyeing at 40 ◦C by increment in Se content from 12.5 to 50 mM. Red/green values (a *) were found to be raised in a positive direction by the increment
in Se content, which might have been attributed to acquiring the red color by ingrain
clustering of Se-NPs, and the yellow/blue ratio (b *) was slightly increased by increasing
the content of Se, reflecting the redness/yellowness color of the fabrics, as shown in Figure 5,
which increased with Se content. 129 Polymers 2022, 14, 191 Figure 5. Photographs of Se-dyed wool fabrics with different concentrations of synthesized Se-NPs
at room temperature and 40 ◦C. Figure 5. Photographs of Se-dyed wool fabrics with different concentrations of synthesized Se-NPs
at room temperature and 40 ◦C. Figure 6. Photographs of Se-dyed wool fabrics with different concentrations of synthesized Se-NPs
at 70 ◦C and 100 ◦C. Figure 6. Photographs of Se-dyed wool fabrics with different concentrations of synthesized Se-NPs
at 70 ◦C and 100 ◦C. 130 Polymers 2022, 14, 191 Table 3. Color characteristics for Se-dyed wool fabrics at different temperatures. Se-NPs Conc. Temp. ◦C
L *
a *
b *
c *
H *
K/S
Blank wool
Room temp. 3.3. Color Characteristics and Color Fastness of Se-Dyed Wool Fabrics 86.87
0.96
12.33
12.37
85.56
1.1
12.5 mM
79.77
7.83
20.2
21.67
68.81
2.21 (2)1
25 mM
67.81
18.84
26.05
32.15
60.12
3.71 (3.5)
37.5 mM
68.77
17.97
28.98
34.1
58.19
4.68 (4.3)
50 mM
59.14
24.52
29.26
37.41
49.05
6.63 (6.4)
12.5 mM
40 ◦C
75.56
11.47
21.36
24.24
61.77
2.53 (2.5)
25 mM
64.14
22.06
27.21
32.75
57.66
3.34 (3.2)
37.5 mM
66.9
23.85
29.28
32.15
51.86
4.23 (4)
50 mM
42.32
27.34
30.12
39.77
44.24
6.31 (6)
12.5 mM
70 ◦C
42.16
12.82
15.1
19.81
49.67
9.8 (9.5)
25 mM
42.76
18.03
21.51
28.07
50.02
12.7 (12.3)
37.5 mM
42.26
24.63
28.37
37.57
49.04
15.3 (15)
50 mM
36.03
34.3
27.04
42.25
49.99
16.3 (16)
12.5 mM
100 ◦C
33.21
5.76
9.09
10.76
57.63
13.8 (13.3)
25 mM
29.27
13.56
13.56
18.44
42.63
18.2 (18)
37.5 mM
28.42
16.96
16.96
23.01
42.53
21.7 (21.4)
50 mM
28.43
17.06
17.06
23.15
42.53
22.1 (22)
* Values in parentheses indicate color strength (K/S) properties after 10 washing cycles. By increasing the dyeing temperature from 40 ◦C to 70 ◦C and 100 ◦C, the wool fabrics
became red or brown, as indicated by the positive a* and b* values. The color of the wool
fabrics darkened and changed to brown as the Se content and dyeing temperature increased,
as shown in Figure 6, which indicated that temperature had a visible influence on the colors
of Se-NP dyed wool fabrics, and this might be due to the increase on the sizes of Se-NPs
when the solution was subjected to heat treatment [58]. The color strength (K/S) increased
with the increase of dyeing temperature and Se-NP concentration. The maximum intensity
of K/S was recorded in the wavelength range of 360–390 nm. From the observations, it could be concluded that the dyeing process of wool fabric
using synthesized Se-NPs resulted in the change of the fabric’s color from the original
creamy-white color to a yellowish red (orange) color, varying to a brownish color, according
to the increment in dyeing temperature and the concentration of Se-NPs. The significant
increase in color depth with increasing Se content confirmed the main responsibility of
Se-NPs in the ingrain coloration of wool fabrics due to the SPR effects of Se-NPs. Moreover, Table 3 revealed that the color depth (K/S) of the Se-dyed samples were still
high after 10 washing cycles of successive laundry cycles. 3.3. Color Characteristics and Color Fastness of Se-Dyed Wool Fabrics The color of the wool
fabrics darkened and changed to brown as the Se content and dyeing temperature increased,
as shown in Figure 6, which indicated that temperature had a visible influence on the colors
of Se-NP dyed wool fabrics, and this might be due to the increase on the sizes of Se-NPs
when the solution was subjected to heat treatment [58]. The color strength (K/S) increased
with the increase of dyeing temperature and Se-NP concentration. The maximum intensity
of K/S was recorded in the wavelength range of 360–390 nm. From the observations, it could be concluded that the dyeing process of wool fabric
using synthesized Se-NPs resulted in the change of the fabric’s color from the original
creamy-white color to a yellowish red (orange) color, varying to a brownish color, according
to the increment in dyeing temperature and the concentration of Se-NPs. The significant
increase in color depth with increasing Se content confirmed the main responsibility of
Se-NPs in the ingrain coloration of wool fabrics due to the SPR effects of Se-NPs. Moreover, Table 3 revealed that the color depth (K/S) of the Se-dyed samples were still
high after 10 washing cycles of successive laundry cycles. This indicates that the Se-NPs
were still tightly loaded and fixed onto the simultaneous functional dyeing of the wool
fabric surface. This might be due to the electrostatic interaction between wool fibers, which
protonated to carry positive charges and Se-NPs carrying negative charges, leading to the
coloration of fibers. The washing, rubbing and light color fastness of dyed wool fabrics with Se-NPs at
different dyeing temperatures were evaluated according to AATCC standard test methods
(61–1972), (8–1972) and (16A–1972), respectively. The results represented in Table 4 showed washing, rubbing and light fastness prop-
erties for all Se-dyed samples. Both Se-dyed wool fabrics at room temperature and 40◦C
exhibited excellent results for washing and rubbing fastness, which agreed with the K/S
data after the washing process, while the light fastness showed a very good rating of 4/5. 131 131 Polymers 2022, 14, 191 Table 4. Fastness properties of Se-dyed wool fabrics at different temperatures. Se-NPs
Conc. Temp. ◦C
WF 1
RF 2
LF 3
Alt. St. Wet
Dry
12.5 mM
Room
temp. 3.3. Color Characteristics and Color Fastness of Se-Dyed Wool Fabrics 5 (5) 4
5 (5)
5 (5)
5 (5)
4/5 (4/5)
25 mM
5 (5)
5 (5)
5 (5)
5 (5)
4/5 (4/5)
37.5 mM
5 (5)
5 (5)
5 (5)
5 (5)
4/5 (4/5)
50 mM
5 (5)
5 (5)
5 (5)
5 (5)
4/5 (4/5)
12.5 mM
40 ◦C
5 (5)
5 (5)
5 (5)
5 (5)
4/5 (4/5)
25 mM
5 (5)
5 (5)
5 (5)
5 (5)
4/5 (4/5)
37.5 mM
5 (5)
5 (5)
5 (5)
5 (5)
4/5 (4/5)
50 mM
5 (5)
5 (5)
5 (5)
5 (5)
4/5 (4/5)
12.5 mM
70 ◦C
4/5 (4/5)
4/5 (4/5)
4/5 (4)
4/5 (4/5)
4 (4)
25 mM
4/5 (4/5)
4/5 (4/5)
4 (4)
4/5 (4/5)
4 (4)
37.5 mM
4/5 (4/5)
4/5 (4/5)
4 (4)
4/5 (4/5)
4 (4)
50 mM
4/5 (4/5)
4/5 (4/5)
4 (4)
4/5 (4/5)
4 (4)
12.5 mM
100 ◦C
4/5 (4)
4/5 (4)
3/4 (3)
4 (4)
4 (4)
25 mM
4/5 (4)
4/5 (4)
3/4 (3)
4 (4)
4 (4)
37.5 mM
4/5 (4)
4/5 (4)
3/4 (3)
4 (4)
4 (4)
50 mM
4/5 (4)
4/5 (4)
3/4 (3)
4 (4)
4 (4)
1 wash fastness. 2 rubbing fastness. 3 light fastness. 4 Values in parentheses indicate durability of fastness
properties after 10 washing cycles. Table 4. Fastness properties of Se-dyed wool fabrics at different temperatures. On the other hand, wool samples dyed at high temperatures (70 and 100 ◦C) exhibited
lower color fastness properties. While washing fastness had a very good rating in both
color alteration and staining, the rubbing fastness showed good results, and the wet one
had a rate of 4 at 70 ◦C to 3/4 at 100 ◦C. In addition, the light fastness exhibited a very
slight color fading rate of 4. It is well known that the larger the colorant (in size), the poorer
the fastness for the colored fabrics. Therefore, the moderate fastness properties of dyed
wool fabrics at a high temperature could be attributed to the enlarged size of Se-NPs onto
the fabric surface, caused by heat treatment and, consequently, easy leaching [45,58] y
q
y
y
g
Moreover, a washing durability test was conducted according to AATCC Test Method
61(2A)-1996, and the results indicated that the fastness properties of all Se-dyed wool
fabrics were still high, even after 10 cycles of consecutive laundry cycles. 3.3. Color Characteristics and Color Fastness of Se-Dyed Wool Fabrics These results
demonstrated that the wool fabrics dyed with synthesized Se-NPs have very good color
fastness properties. 3.4. Mechanical Properties of Se-Dyed Wool Fabrics 3.4. Mechanical Properties of Se-Dyed Wool Fabrics Tensile strength (maximum load N/cm2) and elongation at break (maximum strain %)
were both evaluated for blank and dyed wool fabrics to give an indication for the change
in mechanical properties of wool fabrics after dyeing with Se-NPs at a high temperature
(100 ◦C). The results are reported in Table 5. The tensile strength as well as the elongation
at break slightly decreased after the dyeing process. Table 5. Mechanical properties of blank and Se-dyed wool fabrics at different temperatures. Samples
Mechanical Properties
Tensile Strength (N/cm2)
Elongation (%)
Blank wool
356.8
26.95
Se-dyed wool at RT. 330.8
25.54
40 ◦C
314.5
23.22
70 ◦C
284.4
20.22
100 ◦C
276
19.94
120 ◦C
176.6
13.88 ble 5. Mechanical properties of blank and Se-dyed wool fabrics at different temperatures. Tensile strength decreased from 356.8 N/cm2 for the blank wool to 176.6 N/cm2 and
elongation at break for the blank sample also decreased from 26.95% to 13.88% for the 132 Polymers 2022, 14, 191 applied concentration of 50 mM Se-NPs at 120 ◦C. This decrease in mechanical properties is
probably related to high temperature dyeing processes, which could damage the chemical
structure of the wool fiber, which means that 100 ◦C is the maximum temperature for
dyeing wool fabrics with Se-NPs. All experimental findings clearly show the great efficiency of the proposed dyeing
process in terms of color fastness combined with improved functionalities, such as an-
timicrobial activity and UV-blocking properties. These results lay the groundwork for a
durable dyeing of different kinds of fabrics by use of the simple impregnation with colloidal
solutions of metallic nanoparticles prepared with a sustainable method. This represents an
important result, considering that, today, the main worldwide pollution of white waters is
produced by textile treatments and dyes. Furthermore, the pollution produced by the textile
industry, employing a large consumption of water and the use of harmful dyes (often azo
dyes), is harmful for the environment and for human health, with a huge negative impact
on the planet. Often, wastewater is not adequately treated to remove pollutants before
being discharged into the environment. Very recently, specific, and targeted treatments
have been developed as a suitable method for the removal of azo dyes from textile effluents. 3.4. Mechanical Properties of Se-Dyed Wool Fabrics Many recent studies refer to the use of photocatalytic degradation under visible light, the
employment of non-living cells of marine microalgae (i.e., Nannochloropsis oceanica) and
other waste materials, with a careful optimization of their response surface methodology,
as a suitable and quite valuable wastewater treatment [63–65]. Funding: The authors are grateful to the P.O. FESR SICILIA 2014/2020 n. 08CL4120000131-SETI
(Sicilia Eco Tecnologie Innovative- n. 08CL4120000131) project for funding. Institutional Review Board Statement: Not applicable. References 1. Yetisen, A.K.; Qu, H.; Manbachi, A.; Butt, H.; Dokmeci, M.R.; Hinestroza, J.P.; Skorobogatiy, M.; Khademhosseini, A.; Yun, S.H. Nanotechnology in textiles. ACS Nano 2016, 10, 3042–3068. [CrossRef] [PubMed] 1. Yetisen, A.K.; Qu, H.; Manbachi, A.; Butt, H.; Dokmeci, M.R.; Hinestroza, J.P.; Skorobogatiy, M.; Khademhosseini, A.; Yun, S.H. Nanotechnology in textiles. ACS Nano 2016, 10, 3042–3068. [CrossRef] [PubMed] Abou Elmaaty, T.; Elsisi, H.G.; Elsayad, G.M.; Elhadad, H.H.; Sayed-Ahmed, K.; Plutino, M.R. Fabrication of New
Cotton/Lycra Composites Protective Textiles through Deposition of Nano Silica Coating. Polymers 2021, 13, 28 2. Abou Elmaaty, T.; Elsisi, H.G.; Elsayad, G.M.; Elhadad, H.H.; Sayed-Ahmed, K.; Plutino, M.R. Fabrication of New Multifunctional
Cotton/Lycra Composites Protective Textiles through Deposition of Nano Silica Coating. Polymers 2021, 13, 2888. [CrossRef] 2. Abou Elmaaty, T.; Elsisi, H.G.; Elsayad, G.M.; Elhadad, H.H.; Sayed-Ahmed, K.; Plutino, M.R. Fabrication
Cotton/Lycra Composites Protective Textiles through Deposition of Nano Silica Coating. Polymers 202 3. Castellano, A.; Colleoni, C.; Iacono, G.; Mezzi, A.; Plutino, M.R.; Malucelli, G.; Rosace, G. Synthesis and characterization of a
phosphorous/nitrogen based sol-gel coating as a novel halogen- and formaldehyde-free flame retardant finishing for cotton
fabric. Polym. Degrad. Stab. 2019, 162, 148–159. [CrossRef] 4. Plutino, M.R.; Colleoni, C.; Donelli, I.; Freddi, G.; Guido, E.; Maschi, O.; Mezzi, A.; Rosace, G. Sol-gel 3-glycidoxypropyltriethoxy-
silane finishing on different fabrics: The role of precursor concentration and catalyst on the textile performances and cytotoxic
activity. J. Colloid Interface Sci. 2017, 506, 504–517. [CrossRef] [PubMed] y
f
5. Rosace, G.; Guido, E.; Colleoni, C.; Brucale, M.; Piperopoulos, E.; Milone, C.; Plutino, M.R. Halochromic resorufin-GPTMS hybrid
sol-gel: Chemical-physical properties and use as pH sensor fabric coating. Sens. Actuators B Chem. 2017, 241, 85–95. [CrossRef] 6. Dong, J.; Luo, S.; Ning, S.; Yang, G.; Pan, D.; Ji, Y.; Liu, C. MXene-Coated Wrinkled Fabrics for Stretchable and Multifunctional
Electromagnetic Interference Shielding and Electro/Photo-Thermal Conversion Applications. ACS Appl. Mater. Interf. 2021, 13,
60478–60488. [CrossRef] [PubMed] 7. Plutino, M.R.; Guido, E.; Colleoni, C.; Rosace, G. Effect of GPTMS functionalization on the improvement of the pH-sensitive
methyl red photostability. Sens. Actuators B 2017, 238, 281–291. [CrossRef] V.; Rosace, G.; Colleoni, C.; Sfameni, S.; Migani, V.; Plutino, M.R. Sol-gel based coatings for the protection o
f
f ace, G.; Colleoni, C.; Sfameni, S.; Migani, V.; Plutino, M.R. Sol-gel based coatings for the protection of cultu
IOP Conf Ser Mater Sci Eng 2020 777 012007 [CrossRef] 8. 4. Conclusions Data Availability Statement: The data presented in this study are available on request from the
corresponding author. Data Availability Statement: The data presented in this study are available on request from the
corresponding author. Acknowledgments: MURST: CNR and MIUR are gratefully acknowledged for financial support. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References Trovato, V.; Rosace, G.; Colleoni, C.; Sfameni, S.; Migani, V.; Plutino, M.R. Sol-gel based coatings for the protection of cultural
heritage textiles. IOP Conf. Ser. Mater. Sci. Eng. 2020, 777, 012007. [CrossRef] ato, V.; Rosace, G.; Colleoni, C.; Sfameni, S.; Migani, V.; Plutino, M.R. Sol-gel based coatings for the protect
age textiles. IOP Conf. Ser. Mater. Sci. Eng. 2020, 777, 012007. [CrossRef] 9. Ielo, I.; Galletta, M.; Rando, G.; Sfameni, S.; Cardiano, P.; Sabatino, G.; Drommi, D.; Rosace, G.; Plutino, M.R. Design, synthesis,
and characterization of hybrid coatings suitable for geopolymeric-based supports for the restoration of cultural heritage. IOP
Conf. Ser. Mater. Sci. Eng. 2020, 777, 012003. [CrossRef] f
g
10. Rosace, G.; Cardiano, P.; Urzì, C.; De Leo, F.; Galletta, M.; Ielo, I.; Plutino, M.R. Potential roles of fluorine-containing sol-gel
coatings against adhesion to control microbial biofilm. IOP Conf. Ser. Mater. Sci. Eng. 2018, 459, 012021. [CrossRef] g
ce, G.; Cardiano, P.; Urzì, C.; De Leo, F.; Galletta, M.; Ielo, I.; Plutino, M.R. Potential roles of fluorine-cont
ngs against adhesion to control microbial biofilm. IOP Conf. Ser. Mater. Sci. Eng. 2018, 459, 012021. [CrossRef Cardiano, P.; Urzì, C.; De Leo, F.; Galletta, M.; Ielo, I.; Plutino, M.R. Potential roles of fluorine-containing ce, G.; Cardiano, P.; Urzì, C.; De Leo, F.; Galletta, M.; Ielo, I.; Plutino, M.R. Potential roles of fluorine-cont
ngs against adhesion to control microbial biofilm. IOP Conf. Ser. Mater. Sci. Eng. 2018, 459, 012021. [CrossRef against adhesion to control microbial biofilm. IOP Conf. Ser. Mater. Sci. Eng. 2018, 459, 012021. [CrossRef] 11. Grancari´c, A.M.; Tarbuk, A.; Sutlovi´c, A.; Castellano, A.; Colleoni, C.; Rosace, G.; Plutino, M.R. Enhancement of acid dyestuff
salt-free fixation by a cationizing sol-gel based coating for cotton fabric. Colloids Surf. A Physicochem. Eng. Asp. 2021, 612, 125984. [CrossRef] nino, C.; Trovato, V.; Iacopetta, D.; Piperopoulos, E.; Triolo, C.; Bonomo, M.G.; Drommi, D.; Parisi, O.I.; Milo 12. Puoci, F.; Saturnino, C.; Trovato, V.; Iacopetta, D.; Piperopoulos, E.; Triolo, C.; Bonomo, M.G.; Drommi, D.; Parisi, O.I.; Milone,
C.; et al. Sol–Gel Treatment of Textiles for the Entrapping of an Antioxidant/Anti-Inflammatory Molecule: Functional Coating
Morphological Characterization and Drug Release Evaluation. Appl. Sci. 2020, 10, 2287. [CrossRef] p
g
g
pp
13. Ielo, I.; Giacobello, F.; Sfameni, S.; Rando, G.; Galletta, M.; Trovato, V.; Rosace, G.; Plutino, M.R. Nanostructured Surface Finishing
and Coatings: Functional Properties and Applications. Materials 2021, 14, 2733. [CrossRef] [PubMed] p
g
g
pp
13. 4. Conclusions The present study has reported a one step process for the multi-functionalization of
dyeing wool fabrics using a colloidal solution of Se-NPs. All Se-NP concentrations were
adsorbed onto wool fabrics even though the dyeing temperatures were different, which was
confirmed by the color fastness properties and color strength (K/S). The obtained colored
wool fabrics had a stable bright color ranging from light orange to a dark orange color
according to the concentration of the prepared Se-NPs as well as the dyeing temperatures. Moreover, the wool fabrics with Se-NPs exhibited significant antimicrobial activity and
excellent UV protection properties with excellent washing durability up to 10 laundering
cycles. XRD, SEM and EDX analyses revealed that the Se-NP solution was effectively
absorbed on wool fabric surfaces. Furthermore, a high dyeing temperature led to a change
in the color characteristics from a dark orange color to dark brownish color, especially
at 100 ◦C. However, the UV-protection properties were not affected by high temperature
dyeing; the antimicrobial properties slightly decreased, and the mechanical properties of the
dyed wool fabrics also decreased when the dyeing temperature was increased. The study
concluded that the Se-wool dyeing process at a low temperature (at room temperature or
40 ◦C) was better than that of high temperature dyeing. The proposed dyeing process of
wool fabrics with Se-NPs demonstrated that the dyeing of fibers based on nanoparticles
may facilitate the functionalization of fibrous materials better than the traditional dyestuffs
that require energy and an increased temperature for dyeing. This work will open the way
to the use of opportune nanoparticles for the functionalization of different kinds of surfaces,
to obtain implemented mechanical properties (i.e., color, sensing and antibacterial). Author Contributions: T.A.E. conceived the original idea and participated in the manuscript draft-
ing. S.R. was responsible for the dyeing process using the prepared Se-NPs and evaluation of the
characteristics of the dyed wool fabrics. She discussed the obtained results with T.A.E. and M.R.P. as
well as participated in the manuscript writing. K.S.-A. prepared Se-NPs and interpreted the obtained
results from SEM and XRD analyses and antimicrobial activity test as well as participated in the
manuscript writing. M.R.P. put a lot of effort into reviewing the manuscript and took charge of all
correspondence. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement: Not applicable. 133 Polymers 2022, 14, 191 Informed Consent Statement: Not applicable. References Ibrahim, N.A.; Nada, A.A.; Eid, B.M.; Al-Moghazy, M.; Hassabo, A.G.; Abou-Zeid, N.Y. Nano-structured metal oxides: Synthesis,
characterization and application for multifunctional cotton fabric. Adv. Nat. Sci. Nanosci. Nanotechnol. 2018, 9, 35014. [CrossRef]
23. Eid, B.M.; Ibrahim, N.A. Recent developments in sustainable finishing of cellulosic textiles employing biotechnology. J. Clean. Prod. 2021, 284, 124701–124722. [CrossRef] 23. Eid, B.M.; Ibrahim, N.A. Recent developments in sustainable finishing of cellulosic textiles employing biotechnology. J. Clean. Prod. 2021, 284, 124701–124722. [CrossRef]
l
l
d
l
d l
l
( ) 24. Barattucci, A.; Plutino, M.R.; Faggi, C.; Bonaccorsi, P.; Monsù Scolaro, L.; Aversa, M.C. Mono- and trinuclear tripodal platinum(II)
chelated complexes containing a pyridine/sulfoxide based anchoring framework. Eur. J. Inorg. Chem. 2013, 19, 3412–3420. [CrossRef] 25. Ibrahim, H.M.; Zaghloul, S.; Hashem, M.; El-Shafei, A. A green approach to improve the antibacterial properties of cellulose
based fabrics using Moringa oleifera extract in presence of silver nanoparticles. Cellulose 2021, 28, 549–564. [CrossRef]
l
l g
g
p
p
26. De Luca, G.; Bonaccorsi, P.; Trovato, V.; Mancuso, A.; Papalia, T.; Pistone, A.; Casaletto, M.P.; Mezzi, A.; Brunetti, B.; Minuti,
L ; et al Tripodal tris disulfides as capping agents for a controlled mixed functionalization of gold nanoparticles New J Chem 26. De Luca, G.; Bonaccorsi, P.; Trovato, V.; Mancuso, A.; Papalia, T.; Pistone, A.; Casaletto, M.P.; Mezzi, A.; Brunetti, B.; Minuti,
L.; et al. Tripodal tris-disulfides as capping agents for a controlled mixed functionalization of gold nanoparticles. New J. Chem. 2018, 42, 16436–16440. [CrossRef] 27. Mehravani, B.; Ribeiro, A.I.; Zille, A. Gold Nanoparticles Synthesis and Antimicrobial Effect on Fibrous Materials. Nanomaterials
2021, 11, 1067. [CrossRef] 28. Ielo, I.; Iacopetta, D.; Saturnino, C.; Longo, P.; Galletta, M.; Drommi, D.; Rosace, G.; Sinicropi, M.S.; Plutino, M.R. Gold Derivatives
Development as Prospective Anticancer Drugs for Breast Cancer Treatment. Appl. Sci. 2021, 11, 2089. [CrossRef] 29. Ielo, I.; Rando, G.; Giacobello, F.; Sfameni, S.; Castellano, A.; Galletta, M.; Drommi, D.; Rosace, G.; Plutino, M.R. Synthesis,
Chemical–Physical Characterization, and Biomedical Applications of Functional Gold Nanoparticles: A Review. Molecules 2021,
26, 5823. [CrossRef] 30. Riaz, S.; Ashraf, M.; Hussain, T.; Hussain, M.T.; Rehman, A.; Javid, A.; Iqbal, K.; Basit, A.; Aziz, H. Functional finishing, and
coloration of textiles with nanomaterials. Color. Technol. 2018, 134, 327–346. [CrossRef] 31. Rivero, P.J.; Goicoechea, J.; Arregui, F.J. Localized Surface Plasmon Resonance for Optical Fiber-Sensing Applications. In
Nanoplasmonics—Fundamentals and Applications; Intech Open: London, UK, 2017; Chapter 17. 32. References Phytochemicals and volatile compounds of peanut red skin
extract: Simultaneous coloration and in situ synthesis of silver nanoparticles for multifunctional viscose fibers. Cellulose 2020, 27, 39. Rehan, M.; Elshemy, N.S.; Haggag, K.; Montaser, A.S.; Ibrahim, G.E. Phytochemicals and volatile compounds of peanut red skin
extract: Simultaneous coloration and in situ synthesis of silver nanoparticles for multifunctional viscose fibers. Cellulose 2020, 27,
9893–9912. [CrossRef] 40. Tang, B.; Yao, Y.; Chen, W.; Chen, X.; Zou, F.; Wang, X. Kinetics of dyeing natural protein fibers with silver nanoparticles. Dye. Pigment. 2018, 148, 224–235. [CrossRef] g
41. Jafari-Kiyan, A.; Karimi, L.; Davodiroknabadi, A. Producing colored cotton fabrics with functional properties by combining silver
nanoparticles with nano titanium dioxide. Cellulose 2017, 24, 3083–3094. [CrossRef] Jafari-Kiyan, A.; Karimi, L.; Davodiroknabadi, A. Producing colored cotton fabrics with func
nanoparticles with nano titanium dioxide. Cellulose 2017, 24, 3083–3094. [CrossRef] 42. Tang, B.; Li, J.; Hou, X.; Afrin, T.; Sun, L.; Wang, X. Colorful and antibacterial silk fiber from anisotropic silver nanoparticles. Ind. Eng. Chem. Res 2013, 52, 4556–4563. [CrossRef] 43. Tang, B.; Sun, L.; Kaur, J.; Yu, Y.; Wang, X. In-situ synthesis of gold nanoparticles for multifunctionalization of silk fabrics. Dye. Pigment. 2014, 103, 183–190. [CrossRef] hen, W.; Sun, L.; Wang, X. Sunlight-induced coloration of silk. Nanoscale Res. Lett. 2016, 11, 293. [CrossRef] 44. Yao, Y.; Tang, B.; Chen, W.; Sun, L.; Wang, X. Sunlight-induced coloration of silk. Nanoscale Res. Lett. [PubMed] 45. Emam, H.E.; Rehan, M.; Mashaly, H.M.; Ahmed, H.B. Large scaled strategy for natural/synthetic fabrics functionalization via
immediate assembly of AgNPs. Dye. Pigment. 2016, 133, 173–183. [CrossRef] y
g
y
g
46. Tang, B.; Sun, L.; Li, J.; Kaur, J.; Zhu, H.; Qin, S.; Yao, Y.; Chen, W.; Wang, X. Functionalization of bamboo pulp fabrics with noble
metal nanoparticles. Dye. Pigment. 2015, 113, 289–298. [CrossRef] Yao, Y.; Li, J.; Qin, S.; Zhu, H.; Kaur, J.; Chen, W.; Sun, L.; Wang, X. Functional application of noble metal nanop
thesized on ramie fibers Nanoscale Res Lett 2015 10 366 [CrossRef] [PubMed] 47. Tang, B.; Yao, Y.; Li, J.; Qin, S.; Zhu, H.; Kaur, J.; Chen, W.; Sun, L.; Wang, X. Functional application of noble metal nanoparticles in
situ synthesized on ramie fibers. Nanoscale Res. Lett. 2015, 10, 366. [CrossRef] [PubMed] 48. Mowafi, S.; Rehan, M.; Mashaly, H.M.; Abou El-Kheir, A.; Emam, H.E. Influence of silver nanoparticles on the fabrics functions
prepared by in-situ technique. J. Text. Inst. References Wang, X.; Xu, D.; Jaquet, B.; Yang, Y.; Wang, J.; Huang, H.; Chen, Y.; Gerhard, C.; Zhang, K. Structural Colors by Synergistic
Birefringence and Surface Plasmon Resonance. ACS Nano 2020, 14, 16832–16839. [CrossRef] [PubMed] g
33. Tan, K.B.; Vakili, M.; Horri, B.A.; Poh, P.E.; Abdullah, A.Z.; Salamatinia, B. Adsorption of dyes
developments and adsorption mechanisms. Sep. Purif. Technol. 2015, 150, 229–242. [CrossRef] 34. Kafafy, H.; Helmy, H.; Zaher, A. Treatment of cotton and wool fabrics with different nanoparticles for m
Egyptian J. Chem. 2021, 64, 5255–5267. [CrossRef] gyp
J
[
]
35. Wu, M.; Ma, B.; Pan, T.; Chen, S.; Sun, J. Silver-nanoparticle-colored cotton fabrics with tunable colors and durable antibacterial
and self-healing superhydrophobic properties. Adv. Funct. Mater. 2016, 26, 569–576. [CrossRef] g
p
y
p
p
p
36. Razmkhah, M.; Montazer, M.; Rezaie, A.B.; Rad, M.M. Facile technique for wool coloration via locally forming of nano selenium
photocatalyst imparting antibacterial and UV protection properties. J. Ind. Eng. Chem. 2021, 101, 153–164. [CrossRef] g
p
y
p
p
p
36. Razmkhah, M.; Montazer, M.; Rezaie, A.B.; Rad, M.M. Facile technique for wool coloration via locally forming of nano selenium
h
l
i
i
ib
i l
d UV
i
i
J I d E
Ch
101 153 164 [C
R f] g
p
y
p
p
p
ah, M.; Montazer, M.; Rezaie, A.B.; Rad, M.M. Facile technique for wool coloration via locally forming of nano ocatalyst imparting antibacterial and UV protection properties. J. Ind. Eng. Chem. 2021, 101, 153–164. [CrossR 37. Abdelrahman, M.S.; Nassar, S.H.; Mashaly, H.; Mahmoud, S.; Maamoun, D.; El-Sakhawy, M.; Khattab, T.; Kamel, S. Studies of
polylactic acid and metal oxide nanoparticles-based composites for multifunctional textile prints. Coatings 2020, 10, 58. [CrossRef] hman, M.S.; Nassar, S.H.; Mashaly, H.; Mahmoud, S.; Maamoun, D.; El-Sakhawy, M.; Khattab, T.; Kamel, S. id
d
l
id
i l
b
d
i
f
l if
i
l
il
i
C
i
10 58 ,
;
,
;
y,
;
,
;
,
;
y,
;
,
;
,
polylactic acid and metal oxide nanoparticles-based composites for multifunctional textile prints. Coatings 2020, 10, 58. [CrossRef] p
y
p
p
p
g
38. Johnston, J.H.; Lucas, K.A. Nanogold synthesis in wool fibres: Novel colourants. Gold Bull. 2011, 44, 85 H.; Lucas, K.A. Nanogold synthesis in wool fibres: Nove 39. Rehan, M.; Elshemy, N.S.; Haggag, K.; Montaser, A.S.; Ibrahim, G.E. 22.
Ibrahim, N.A.; Nada, A.A.; Eid, B.M.; Al-Moghazy, M.; Hassabo, A.G.; Abou-Zeid, N.Y. Nano-structured metal oxides: Synthesis,
characterization and application for multifunctional cotton fabric. Adv. Nat. Sci. Nanosci. Nanotechnol. 2018, 9, 35014. [CrossRef]
23.
Eid, B.M.; Ibrahim, N.A. Recent developments in sustainable finishing of cellulosic textiles employing biotechnology. J. Clean.
Prod. 2021, 284, 124701–124722. [CrossRef] References Ielo, I.; Giacobello, F.; Sfameni, S.; Rando, G.; Galletta, M.; Trovato, V.; Rosace, G.; Plutino, M.R. Nanostructured Surface Finishing
d C
ti
F
ti
l P
ti
d A
li
ti
M t i l 2021 14 2733 [C
R f] [P bM d] g
p
pp
14. AbouElmaaty, T.; Abdeldayem, S.A.; Ramadan, S.M.; Sayed-Ahmed, K.; Plutino, M.R. Coloration and M
Polypropylene Fabrics with Selenium Nanoparticles. Polymers 2021, 13, 2483. [CrossRef] [PubMed] AbouElmaaty, T.; Abdeldayem, S.A.; Ramadan, S.M.; Sayed-Ahmed, K.; Plutino, M.R. Coloration and M 14. AbouElmaaty, T.; Abdeldayem, S.A.; Ramadan, S.M.; Sayed-Ahmed, K.; Plutino, M.R. Coloration and Multi-Functionalization of
Polypropylene Fabrics with Selenium Nanoparticles. Polymers 2021, 13, 2483. [CrossRef] [PubMed] y,
;
y
,
;
,
;
y
,
;
,
Polypropylene Fabrics with Selenium Nanoparticles. Polymers 2021, 13, 2483. [CrossRef] [PubMed] 15. Ibrahim, N.A.; Eid, B.M.; El-Aziz, E.A.; Elmaaty, T.M.A.; Ramadan, S.M. Loading of chitosan–Nano metal oxide hybrids onto
cotton/polyester fabrics to impart permanent and effective multifunctions. Int. J. Biol. Macromol. 2017, 105, 769–776. [CrossRef]
[PubMed] [
]
16. Ibrahim, N.A.; Eid, B.M.; El-Aziz, E.A.; Elmaaty, T.M.A.; Ramadan, S.M. Multifunctional cellulose-containing fabrics using
modified finishing formulations. RSC Adv. 2017, 7, 33219–33230. [CrossRef] 17. Ibrahim, N.A.; Eid, B.M.; El-Aziz, E.A.; Elmaaty, T.M.A. Functionalization of linen/cotton pigment prints using inorganic nano
structure materials. Carbohydr. Polym. 2013, 97, 537–545. [CrossRef] [PubMed] 18. Ibrahim, N.A.; Eid, B.M.; Elmaaty, T.M.A.; El-Aziz, E.A. A smart approach to add antibacterial function
prints. Carbohydr. Polym. 2013, 94, 612–618. [CrossRef] [PubMed] 18. Ibrahim, N.A.; Eid, B.M.; Elmaaty, T.M.A.; El-Aziz, E.A. A smart ap
prints. Carbohydr. Polym. 2013, 94, 612–618. [CrossRef] [PubMed] y
y
si, M.; Othman, R.; Yassin, F.A. Improving antibacterial and ultraviolet properties of cotton fabrics via p
y
y
19. El-bisi, M.; Othman, R.; Yassin, F.A. Improving antibacterial and ultraviolet properties of cotton f
nano-metal oxide and Moringa oleifera extract. Egypt. J. Chem. 2020, 63, 3441–3451. [CrossRef] El-bisi, M.; Othman, R.; Yassin, F.A. Improving antibacterial and ultraviolet properties of cotton
nano-metal oxide and Moringa oleifera extract. Egypt. J. Chem. 2020, 63, 3441–3451. [CrossRef] 20. Ibrahim, N.A.; El-Aziz, E.A.; Eid, B.M.; Elmaaty, T.M.A. Single-stage process for bifunctionalization and eco-friendly pigment
coloration of cellulosic fabrics. J. Text. Inst. 2016, 107, 1022–1029. 21. Ibrahim, N.A.; Eid, B.M.; Abdel-Aziz, M.S. Effect of plasma superficial treatments on antibacterial functionalization and coloration
of cellulosic fabrics. Appl. Surf. Sci. 2017, 392, 1126–1133. [CrossRef] 134 Polymers 2022, 14, 191 22. References 2017, 108, 1828–1839. [CrossRef] 49. Hong, Z. Preparation and antioxidant properties of selenium nanoparticles-loaded chitosan microspheres. Int. J. Nanomed. 2017,
12, 4527–4539. 135 Polymers 2022, 14, 191 50. Zhai, X.; Zhang, C.; Zhao, G.; Stoll, S.; Ren, F.; Leng, X. Antioxidant capacities of the selenium nanoparticles stabilized by chitosan. J. Nanobiotechnology 2017, 15, 4. [CrossRef] 51. Nastulyavichus, A.; Kudryashov, S.; Smirnov, N.; Saraeva, I.; Rudenko, A.; Tolordava, E.; Ionin, A.; Romanova, Y.; Zayarny, D. Antibacterial coatings of Se and Si nanoparticles. Appl. Surf. Sci 2019, 469, 220–225. [CrossRef] g
p
pp
f
52. Huang, X.; Chen, X.; Chen, Q.; Yu, Q.; Sun, D.; Liu, J. Investigation of functional selenium nanoparticles as potent antimicrobial g
p
pp
f
52. Huang, X.; Chen, X.; Chen, Q.; Yu, Q.; Sun, D.; Liu, J. Investigation of functional selenium nanoparticles as potent antimicrobial
agents against superbugs. Acta Biomater. 2016, 30, 397–407. [CrossRef] [PubMed] 52. Huang, X.; Chen, X.; Chen, Q.; Yu, Q.; Sun, D.; Liu, J. Investigation of functional s
agents against superbugs. Acta Biomater. 2016, 30, 397–407. [CrossRef] [PubMed] 53. Yip, J.; Liu, L.; Wong, K.H.; Leung, P.H.M.; Yuen, C.W.M.; Cheung, M.C. Investigation of antifungal and antibacterial effects of
fabric padded with highly stable selenium nanoparticles. J. Appl. Polym. Sci. 2014, 131, 8886–8893. [CrossRef] 54. Elmaaty, T.A.; El-Nagare, K.; Raouf, S.; Abdelfattah, K.; El-Kadi, S.; Abdelaziz, E. One-step green approach for functional printing
and finishing of textiles using silver and gold NPs. RSC Adv. 2018, 8, 25546–25557. [CrossRef] 55. Elmaaty, T.A.; Raouf, S.; Sayed-Ahmed, K. Novel one step printing and functional finishing of
nanoparticles. Fiber Polym. 2019, 21, 1983–1991. [CrossRef] 56. Vieira, A.P.; Stein, E.M.; Andreguetti, D.X.; Cebrián-Torrejón, G.; Doménech-Carbó, A.; Colepicolo, P.; Ferreira, A.M.D.C. “Sweet
Chemistry”: A Green Way for Obtaining Selenium Nanoparticles Active against Cancer Cells. J. Braz. Chem. Soc. 2017, 28,
2021–2027. [CrossRef] nis, J.; Häfeli, U.O.; Bach, H. Metal nanoparticles: Understanding the mechanisms behind antibacterial activity
gy 2017, 15, 65. [CrossRef] [PubMed] 57. Slavin, Y.N.; Asnis, J.; Häfeli, U.O.; Bach, H. Metal nanoparticles: Understanding the mechanisms behin
Nanobiotechnology 2017, 15, 65. [CrossRef] [PubMed] gy
58. Zhang, J.; Taylor, E.W.; Wan, X.; Peng, D. Impact of heat treatment on size, structure, and bioactivity of elemental selenium
nanoparticles. Int. J. Nanomed. 2012, 7, 815. [CrossRef] p
59. Adomaviciute, E.; Stanys, S.; Žilius, M.; Juškaite, V.; Pavilonis, A.; Briedis, V. 51.
Nastulyavichus, A.; Kudryashov, S.; Smirnov, N.; Saraeva, I.; Rudenko, A.; Tolordava, E.; Ionin, A.; Romanova, Y.; Zayarny, D.
Antibacterial coatings of Se and Si nanoparticles. Appl. Surf. Sci 2019, 469, 220–225. [CrossRef] 50.
Zhai, X.; Zhang, C.; Zhao, G.; Stoll, S.; Ren, F.; Leng, X. Antioxidant capacities of the selenium nanoparticles stabilized by chitosan.
J. Nanobiotechnology 2017, 15, 4. [CrossRef] anusa, A.; Zuorro, A.; Lavecchia, R.; Marrosu, G.; Petrucci, R. Recovery of Natural Antioxidants from Spent C
Agric. Food Chem. 2013, 61, 4162–4168. [CrossRef] 64.
Zuorro, A.; Maffei, G.; Lavecchia, R. Kinetic modeling of azo dye adsorption on non-living cells of Nannochloropsis oceanica. J.
Environ. Chem. Eng. 2017, 5, 4121–4127. [CrossRef] Article
Soybean Oil-Based Biopolymers Induced by Nonthermal
Plasma to Enhance the Dyeing of Para-Aramids with a
Cationic Dye Received: 18 January 2022
Accepted: 4 February 2022
Published: 6 February 2022 Keywords: para-aramid; nonthermal plasma; ambient air; soybean oil; acrylic acid; acrylated
epoxidized soybean oil; cationic dye Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
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iations. Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. References Formation and biopharmaceutical characterization
of electrospun PVP mats with propolis and silver nanoparticles for fast releasing wound dressing. Biomed. Res. Int. 2016, 2016,
4648287. [CrossRef] [
]
60. Lu, Z.; Mao, C.; Meng, M.; Liu, S.; Tian, Y.; Yu, L.; Sun, B.; Li, C.M. Fabrication of CeO2 nanoparticle-modified silk for UV
protection and antibacterial applications. J. Colloid Interface Sci. 2014, 435, 8–14. [CrossRef] [PubMed] 60. Lu, Z.; Mao, C.; Meng, M.; Liu, S.; Tian, Y.; Yu, L.; Sun, B.; Li, C.M. Fabrication of CeO2 nanopartic
protection and antibacterial applications. J. Colloid Interface Sci. 2014, 435, 8–14. [CrossRef] [PubMed] 61. Khan, M.Z.; Baheti, V.; Ashraf, M.; Hussain, T.; Ali, A.; Javid, A.; Rehman, A. Development of UV protective, superhydrophobic
and antibacterial textiles using ZnO and TiO2 nanoparticles. Fibers Polym. 2018, 19, 1647–1654. [CrossRef] g
p
y
62. Chitichotpanya, P.; Pisitsak, P.; Chitichotpanya, C. Sericin—Copper-functionalized silk fabrics for enhanced ultraviolet protection
and antibacterial properties using response surface methodology. Text. Res. J. 2019, 89, 1166–1179. [CrossRef] 62. Chitichotpanya, P.; Pisitsak, P.; Chitichotpanya, C. Sericin—Copper-functionalized silk fabrics for enhanced ultraviolet protection
and antibacterial properties using response surface methodolog
Text Res J 2019 89 1166 1179 [CrossRef] Chitichotpanya, P.; Pisitsak, P.; Chitichotpanya, C. Sericin—Copper-functionalized silk fabrics for enhanced ul p
y
p
y
pp
nd antibacterial properties using response surface methodology. Text. Res. J. 2019, 89, 1166–1179. [CrossRef] 63. Zuorro, A.; Lavecchia, R.; Monaco, M.M.; Iervolino, G.; Vaiano, V. Photocatalytic Degradation of Azo
Fe-Doped Titania Catalysts under Visible Light Irradiation. Catalysts 2019, 9, 645. [CrossRef]
64
Z
A M ff i G L
hi
R Ki
ti
d li
f
d
d
ti
li i
ll
f N 63. Zuorro, A.; Lavecchia, R.; Monaco, M.M.; Iervolino, G.; Vaiano, V. Photocatalytic Degradation of Azo Dye Reactive Violet 5 on
Fe-Doped Titania Catalysts under Visible Light Irradiation. Catalysts 2019, 9, 645. [CrossRef] 64. Zuorro, A.; Maffei, G.; Lavecchia, R. Kinetic modeling of azo dye adsorption on non-living cells of Nannochloropsis oceanica. J. Environ. Chem. Eng. 2017, 5, 4121–4127. [CrossRef] 136 polymers Citation: Metzcar, C.; Ye, X.P.; Wang,
T.; Doona, C.J. Soybean Oil-Based
Biopolymers Induced by Nonthermal
Plasma to Enhance the Dyeing of
Para-Aramids with a Cationic Dye.
Polymers 2022, 14, 628. https://
doi.org/10.3390/polym14030628 Article
Soybean Oil-Based Biopolymers Induced by Nonthermal
Plasma to Enhance the Dyeing of Para-Aramids with a
Cationic Dye Caleb Metzcar 1, Xiaofei Philip Ye 2,*, Toni Wang 1 and Christopher J. Doona 3,4 1
Department of Food Science, The University of Tennessee, Knoxville, TN 37996, USA;
cmetzcar@vols.utk.edu (C.M.); twang46@utk.edu (T.W.) p
y
cmetzcar@vols.utk.edu (C.M.); twang46@utk.edu (T.W. g
2
Department of Biosystems Engineering and Soil Science, The University of Tennessee,
Knoxville, TN 37996, USA 2
Department of Biosystems Engineering and Soil Science, The University of Tennessee,
Knoxville, TN 37996, USA 3
U.S. Army Combat Capabilities Development Command—Soldier Center, Natick, MA 01760, USA;
doonac@mit.edu 4
Massachusetts Institute of Technology—Institute for Soldier Nanotechnologies, 77 Massachusetts Ave
NE47-4F, Cambridge, MA 02139, USA 4
Massachusetts Institute of Technology—Institute for Soldier Nanotechnologies, 77 Massachusetts Ave
NE47-4F, Cambridge, MA 02139, USA g
*
Correspondence: xye2@utk.edu; Tel.: +1-865-974-7129; Fax: +1-865-974-4514 g
*
Correspondence: xye2@utk.edu; Tel.: +1-865-974-7129; Fax: +1-865-974-4514 Abstract: To overcome the recalcitrance of para-aramid textiles against dyeing, this study demon-
strated that increasing the functionalities of soybean oil applied to the surface of para-aramids
followed by a nonthermal plasma (NTP) treatment improved the dyeing color strength compared
with the use of soybean oil alone, and that dyeing occurred through covalent bonding. Particularly,
compared with the pretreatment using soybean oil that obtained the highest color strength of 3.89 (as
K/S value determined from spectral analysis of the sample reflectance in the visible range), the
present pretreatments with either acrylated epoxidized soybean oil (AESO) or a mixture of acrylic
acid and soybean oil (AA/Soy) achieved K/S values higher than nine (>9.00). The NTP treatment,
after the AESO or AA/Soy pretreatment, was essential in inducing the formation of a polymerized
network on the surface of para-aramids that bonded the dye molecules and generating covalent
bonds that anchored the polymerized network to the para-aramids, which is difficult to achieve given
the high crystallinity and chemical inertness of para-aramids. As an important economic considera-
tion, the sequential experimentation method demonstrated that a simple mixture of AA/Soy could
replace the expensive AESO reagent and render a comparable performance in dyeing para-aramids. Among the auxiliary additives tested with the AESO and AA/Soy pretreatments followed by NPT
treatment in this study, Polysorbate 80 as a surfactant negatively affected the dyeing, benzyl alcohol
as a swelling agent had minimal effect, and NaCl as an electrolyte showed a positive effect. The
dyeing method developed in this study did not compromise the strength of para-aramids. 1. Introduction Typically, aramid filaments/yarns are dyed to a single color using solu-
tion dyeing methods, a process in which acid-tolerant colorant is added to the polymer
dope at the time of aramid filament production, and the dyed filaments are subsequently
woven into usable fabrics with only a limited number of color choices available by these
methods [8]. While it is highly desirable to incorporate aramids into new applications for
protective clothing or outerwear, for example, the difficulty in durably dyeing or print-
ing aramids is a barrier to using aramids in these applications. New methods of dyeing
para-aramids that avoid the use of harsh or environmentally unsafe chemicals would be
advantageous for these applications. g
pp
Our previous studies [9,10] demonstrated that pretreatment with soybean oil followed
by nonthermal plasma (NTP) treatment enabled dyeing para-aramids to significantly high
color strength without other chemical additives; and this new method is compatible with
both a disperse dye and a basic dye, showing the potential of this method replacing
the environmentally unfriendly chemicals in current dyeing practices with renewable,
environmentally friendly materials to improve the dyeing of para-aramid fabrics. The
proposed mechanism for improved dyeing is that the soybean oil diffuses onto woven
fabric and adsorbs onto the surfaces of yarns and fibers, and the subsequent NTP treatment
induces the formation of a polymerized network in situ, enabling dyeing to a higher color
strength. The color strength in term of K/S value, determined from spectral analysis of the
sample reflectance in the visible range, increased to 3.89 from ~1 of the untreated samples. Along this line of sustainable dyeing method, in this study, we aim at further improving
the dyeing color strength and colorfastness by deriving more functionalities of soybean oil
on the surface of para-aramids. p
Acrylated epoxidized soybean oil (AESO) synthesized from soybean oil has already
occupied a significant share of market as a “green” alternative to petroleum-based epoxy
resins, plasticizers, and pre-polymers [11]. It contains three highly reactive functionalities
of double (C=C) bonds, –OH groups, and epoxy rings. The C=C bond in AESO is capable
of self-polymerizing and copolymerizing with other components via a free radical initiation
(including UV and NTP, which generate both UV and free radical species), forming a
network with ample functionalities to bind with dyes [12]. 1. Introduction The invention of aramids with commercial names such as Kevlar® (para-aramid)
and Nomex® (meta-aramid) brought about a synthetic fiber with high strength-to-weight
ratio, low elongation to break, superior heat and flame resistance, high cut resistance,
and excellent ballistic properties [1]. Since the first commercial use of Kevlar® in the
early 1970s as a replacement for steel in racing tires, aramid materials have been used in
an increasing number of diverse applications. Examples include textiles for protective
clothing (e.g., flame-resistant apparel) and body armor (e.g., bullet-proof vests, helmets, and
puncture-resistant correctional wear), sportswear, and reinforced composites (e.g., brake
pads, gaskets, hot-air filters, industrial belts and ropes, and strength member in fiber Copyright:
© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article
distributed
under
the
terms
and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). 137 https://www.mdpi.com/journal/polymers Polymers 2022, 14, 628. https://doi.org/10.3390/polym14030628 Polymers 2022, 14, 628 optics) [2]. It is highly desirable to incorporate aramids into additional applications for
protective clothing, but these efforts are complicated by the difficulty associated with
durably dyeing or printing aramids. Between the two major types of aramids, meta-aramids and para-aramids, the meta-
aramid fibers consist of poly(m-phenylene isophthalamide) that binds via meta-linked
aromatic rings to result in a semi-crystalline fiber with the molecular chain oriented along
the fiber axis, while the building-blocks of para-aramids are poly(p-phenylene terephthala-
mide) with stiff para-linked aromatic rings and densely arranged hydrogen bond donors
and acceptors throughout their backbones [3]. This inherent molecular rigidity of para-
aramids, combined with strong intermolecular hydrogen bonding interactions, enables
the molecules to achieve excellent alignment with their neighbors, resulting in a highly
anisotropic unit cell consisting of covalent bonds, hydrogen bonds, and van der Waals in-
teractions along each fundamental axis, forming a highly crystalline structure [4]. The high
degree of crystallinity and chemical inertness makes it difficult to dye para-aramids with
conventional dyeing methods, because para-aramids cannot entrap or bind dye molecules. There are a number of methods reported for dyeing meta-aramids, but few methods
reported for dyeing para-aramids (especially continuous filament para-aramids) with-
out using harsh chemicals that can damage the dyed materials and sacrifice mechanical
strength [5–7]. 2.1. Materials The para-aramid fabrics used in this study are made of tightly woven 300 Denier con-
tinuous filament and were provided by the U.S. Army Development Command—Soldier
Center (Natick, MA, USA). This highly crystalline material has been proven undyeable by
conventional methods using either a cationic dye or a disperse dye, even with nonthermal
plasma surface treatment, but were dyed using soybean oil/NTP pretreatments [9,10]. Blue cationic dye (Victoria Blue R, CAS Number 2185-86-6), Disperse Red 1 acrylate dye
(CAS Number 13695-46-0), AESO (CAS Number 91722-14-4), acrylic acid (CAS Number
79-10-7), and ethyl acetate (CAS Number 141-78-6) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Polysorbate 80 (a nonionic surfactant with the common name TWEEN
80 and the IUPAC name polyoxyethylene (20) sorbitan monooleate) and benzyl alcohol (a
common swelling agent) were purchased from Chem Center @ Amazon.com. Commercial
food-grade refined soybean oil (typically consisting of about 23% monounsaturated fat,
58% polyunsaturated fat, and 15% saturated fat) and laundry detergent (ECOS® plus stain-
fighting enzymes) were purchased from a local supermarket. All materials in this study
were used as-is and without further purification. 1. Introduction However, the current method of
soybean oil epoxidation followed by acrylate addition for AESO production is a tedious
process, rendering a high price for AESO. We hypothesized that pretreating para-aramids
with a simple mixture of soybean oil and acrylic acid followed by NTP treatment would
induce a similar polymerized network in situ to enhance the dyeing. Importantly, acrylic
acid can also be produced from renewable glycerol, which is a co-product of massive
biodiesel production, mainly from soybean oil in the U.S. Acrylic acid can be produced 138 Polymers 2022, 14, 628 via the intermediate of glycerol dehydration to acrolein, and this strategy has received
much attention because it appears to be one of the most promising ways to valorize
glycerol [13–15]. Furthermore, because the dyeing industry often uses auxiliary chemical additives
of surfactants, electrolytes, and swelling agents to improve dyeing performance [16–18],
the effects of Polysorbate 80 as a surfactant, sodium chloride (NaCl) as an electrolyte, and
benzyl alcohol as a swelling agent, were also examined in our new method. 2. Materials and Methods The following section describes all materials and methods used in this study. Overall,
a series of experiments, designed as sequential experimentation, directed this study and
demonstrated the potential for this new method to improve the dyeing of para-aramids. The
experimental design and analysis were conducted using Design-Expert software (version 6,
Stat-Ease, Inc., Minneapolis, MN, USA). Because many experimental factors were involved,
our design of experiments emphasized the sequential use of two-level factorial designs to
identify critical factors for improving dyeing, and the sequential assembly of second-order
designs to elucidate the nature of the response surface in the improved formulations and
process conditions. 2.3. Dyeing Experimental Procedure and Washing The dye bath was prepared by dissolving 0.1 wt.% of Victoria Blue R dye in distilled
water. Depending on the experimental design, auxiliary additives of a surfactant, elec-
trolyte, and/or swelling agent were added to the dye bath, expressed as a mass percentage
based on the solvent. Then, the dye bath liquid was transferred into vials of a combined
heating/stirring system (Reacti-Therm, ThermoFisher Scientific, Waltham, MA, USA) to
conduct the dyeing experiments. The Reacti-Therm system can hold up to eight vials
with different dye-bath formulations and precisely control the dyeing temperature. The
Reacti-Therm system was initially set at 60 ◦C with stirring while the samples were loaded
into their respective vials, then the temperature was raised to T = 90 ◦C in about 30 min
and held there for 1 h. Subsequently, the Reacti-Therm system was turned off, and samples
were left in the vials to cool for 20 min. After dyeing, all the samples were removed from the vials and rinsed under flowing
warm tap water for 2 min followed by a cold-water rinse for another 2 min. Then, the
samples were dried in a programmable convective oven starting at T = 30 ◦C, raising
the temperature to T = 150 ◦C at a rate of 30 ◦C/min, and holding at T = 150 ◦C for
2 min, to fix the dye to the fabric samples. To prepare the samples for color strength
analysis, the samples were washed with detergent (ECOS® plus stain-fighting enzymes) to
remove the oily pretreatment materials using a home-made tumbler to simulate laundering,
in accordance with the protocol described in ISO standard 105-C10:2006 [19]. After the
detergent washing, the samples were rinsed and dried again in the same way as described
above. 2.2. Pretreatment and NTP Treatment Each para-aramid fabric sample was cut into approximately one square inch swatch. Depending on the experimental design, the pretreatment was conducted by submerging
the samples in a solution of AESO or a mixture of acrylic acid and soybean oil (denoted
as AA/Soy hereafter) in ethyl acetate in a sealed beaker for a designated time. Then, the
samples were taken out, placed on a paper towel, and pressed with a roller two times
to remove excessive pretreatment liquid, followed by NTP treatment if required by the
experimental design. The NTP treatment of the fabric samples was carried out using an in-house made
surface dielectric barrier discharge (SDBD) apparatus consisting of two electrodes separated
by a 108 × 95 mm alumina dielectric plate with a thickness of 1 mm (Figure 1). The alumina
plate has an induction electrode made of a rectangular copper tape embedded in insulation
tape on its top and a discharge electrode made of 17 interconnected tungsten strips on its
lower surface. Teflon-coated aramids have been made wettable after a 30 s exposure to
this SDBD. Compared with volume dielectric barrier discharge (VDBD), SDBD generates a
higher density of micro-discharges that are limited to the surface of the sample, and thereby
avoids pin-holing that VDBD caused in para-aramids by the hot electron bombardment, 139 Polymers 2022, 14, 628 weakening the fibers. The feedgas for the SDBD is ambient air, and the power for the SDBD
was a 9.2 kV sinusoidal high voltage source tuned to a resonance frequency of 23.2 kHz. Para-aramid samples were placed on top of a Plexiglas platform mounted on a rotating
stage, and the SDBD plate was lowered via an adjustable stand to 1 mm above and parallel
to the sample to ensure uniform treatment of the sample. Surface emissions, reactive
oxygen and nitrogen species (RONS), and other radicals generated in the plasma interacted
with the para-aramid samples. At completion of the NTP treatment of a specified time, the
power to the SBSD was turned off and the sample was removed immediately thereafter. Figure 1. (A) Schematic of NTP treatment and (B) Photo image showing glowing surface discharge
on top of samples in dark background. Figure 1. (A) Schematic of NTP treatment and (B) Photo image showing glowing surface discharge
on top of samples in dark background. 2.3. Dyeing Experimental Procedure and Washing 2.4. Color Strength Analysis 2.4. Color Strength Analysis The color strength of each dyed sample was quantified by measuring its spectral
reflectance (R in %) in the visible range using a spectrophotometer (SPECTRO 1, Variable 140 Polymers 2022, 14, 628 Inc., Chattanooga, TN, USA). The color strength was calculated at the wavelength of
maximum absorbance for Victoria Blue R (λ = 615 nm) using the K/S value defined by
the Kubelka–Munk equation (Equation (1)) that relates R with sample absorption (K) and
scattering characteristics (S) [20]. K/S = (1 −0.01R)2
2(0.01R)
(1) (1) The Kubelka–Munk equation is used in formulating colors for the textile, paper, and
coatings industries. For these applications, it is assumed that the absorption (K) of light
depends on the properties of the colorant, and the scattering (S) of a dye or pigment
depends on the properties of the substrate or opacifier. The K/S value is roughly linear
with respect to colorant concentration [21]. 2.5. FTIR Analysis To provide insight into the chemical changes occurring over the entire dyeing process,
FTIR analysis was carried out on the para-aramid samples at the different stages of treat-
ments. Attenuated total reflection Fourier transform infrared spectroscopy (ATR–FTIR)
spectra were recorded using an FTIR spectrometer (Excalibur 3100, Varian Inc., Palo Alto,
CA, USA) equipped with an overhead attenuated total reflection (ATR) accessory with
germanium crystal (UMA 400, Varian Inc.) and a liquid nitrogen cooled mercury cadmium
telluride detector. A sample was placed on a potassium bromide (KBr) plate and pressed
under the germanium crystal for scanning. Each spectrum was collected within the mid-IR
region from 50–4000 cm−1 at a resolution of 4 cm−1 after averaging 128 scans. The ATR
spectra of samples were presented in absorbance units after taking into account the back-
ground spectrum acquired using a blank KBr plate. Between successive measurements, the
germanium ATR crystal was carefully cleaned with ethanol, rinsed with distilled water,
and dried to prevent cross contamination. All the spectra were ATR-corrected using Varian
Resolutions Pro software (Varian Inc., Palo Alto, CA, USA). 2.6. SEM, Extraction Test, Tensile Strength 3.1. Analysis of Dyeing Experiments We designed Experiment A to investigate the impact of AESO concentration, the use
of benzyl alcohol (Benzyl-OH) as a swelling agent, and NaCl as an electrolyte on dyeing. Furthermore, we tested the NTP treatment prior to or post AESO pretreatment in order to
understand the function of NTP in improving the dyeing color strength. The design of this
4-factor, 2-level, full factorial experiment and results are presented in Table 1. Table 1. Experiment A: factorial design and the results *. Sample
AESO conc. (wt.%)
Benzyl-OH
(wt.%)
NaCl
(wt.%)
NTP
Order
K/S **
Std **
A1
30
None
None
Prior
4.23
0.023
A2
30
None
None
Post
9.81
0.121
A3
30
None
8
Prior
6.14
0.075
A4
30
None
8
Post
10.53
0.505
A5
30
2
None
Prior
5.96
0.059
A6
30
2
None
Post
8.49
0.283
A7
30
2
8
Prior
7.56
0.013
A8
30
2
8
Post
10.04
0.081
A9
39
None
None
Prior
3.94
0.122
A10
39
None
None
Post
10.58
0.724
A11
39
None
8
Prior
6.48
0.191
A12
39
None
8
Post
9.22
0.318
A13
39
2
None
Prior
5.91
0.324
A14
39
2
None
Post
9.90
0.350
A15
39
2
8
Prior
7.78
0.202
A16
39
2
8
Post
8.79
0.311
* Experiment conducted at soaking time in AESO in ethyl acetate = 1 h, dye conc. = 0.1 wt.%, swelling
agent = 2 wt.%, benzyl alcohol, electrolyte = 8 wt.%, NaCl, NTP treatment time = 90 s, dyeing temperature
T = 90 ◦C, dyeing time = 1 h. ** Mean and standard error of three repeated measurements. Table 1. Experiment A: factorial design and the results *. For the analysis of variance (ANOVA) of the factorial design, model term selection (in-
cluding factorial interactions) was first conducted based on the half-normal probability plot,
showing the factorial impact in the ascending order of (AESO conc.) < (Benzyl-OH) < (NaCl)
< (NTP order). This resulted in an overall significant model (F-test, p < 0.0001). T-tests
for each model term coefficients showed that all model terms were significant (p < 0.002),
except for the main effect of (AESO conc.) (p = 0.89). This was not surprising because we
did not know a suitable AESO concentration in this first experiment and randomly selected
the two AESO concentrations. 2.6. SEM, Extraction Test, Tensile Strength Scanning Electron Micrographs (SEM) of original para-aramid fibers and dyed sam-
ples were acquired on a Zeiss Auriga scanning electron microscope (Carl Zeiss SMT Inc.,
Oberkochen, Germany). Complementary to the FTIR analysis, an extraction test was conducted on a set of
samples dyed with AA/Soy pretreatment followed by NTP treatment, to investigate how
the polymerized AA/Soy binds with the dye and para-aramids. Each sample was extracted
with 40 mL of either hexane, ethanol, or 2:1 (v/v) chloroform:methanol of varying polarities
at room temperature for 15 h. It was assumed that dye molecules covalently bonded to
the fabric and highly polymerized AA/Soy network could not be extracted. The K/S
value of each sample before and after the extraction was compared to evaluate the extent
of the extraction that reduced the color strength. Furthermore, we replicated a reported
novel method [22] for the fabrication of colored materials with significantly reduced dye
leaching through covalent immobilization of the desired dye using plasma-generated
surface radicals; this plasma dye coating procedure immobilizes a pre-adsorbed layer of a
dye functionalized with a radical sensitive group on the surface through radical addition
caused by a short NTP treatment. We need to point out that this study demonstrated
successful dyeing of some hard-to-dye materials such as inert plastics of polyethylene and
polytetrafluoroethylene but dyeing para-aramids was not attempted. We followed the same
procedure of dyeing described in this reference [22] using a Disperse Red 1 acrylate dye
(CAS Number 13695-46-0, Sigma-Aldrich, St. Louis, MO, USA), which is the only one used
in this study that is commercially available, to dye our para-aramids. However, because
our NTP source is different from that used in the referenced study, we optimized the NTP
treatment time and found that a 30 s NTP time resulted in the highest K/S value (longer 141 Polymers 2022, 14, 628 time would degrade the dye). Para-aramid samples dyed with the Disperse Red 1 acrylate
were subjected to the same extraction procedure for comparison. To evaluate if our pretreatment and dyeing process would affect the strength of the
para-aramids, tensile testing of the para-aramid yarns was performed using a TA.XT plus
texture analyzer (Stable Micro Systems, Godalming, UK), following ASTM standard [23]. 2.6. SEM, Extraction Test, Tensile Strength Yarns of 300 Denier continuous filaments were subjected to the same pretreatment and
dyeing process developed in this study, and then placed in a sealed bag to be conditioned
for three days together with undyed yarns for comparison. The test was completed in
triplicates and peak load at breakpoint was recorded as an indicator of the tensile strength. 3. Results and Discussion 3.1. Analysis of Dyeing Experiments 3.1. Analysis of Dyeing Experiments Since all factors were involved in significant interactions,
the statistical inferences are based on the interaction plots as shown in Figure 2. 142 Polymers 2022, 14, 628 Figure 2. Statistical inferences of Experiment A: significant factorial interactions. Figure 2. Statistical inferences of Experiment A: significant factorial interactions. The difference between the two selected AESO concentrations was insignificant. Ad-
dition of NaCl in the dye bath slightly improved color strength, apparently when the
NTP was applied prior to AESO pretreatment. The order of NTP application played an
important role; NTP application post AESO pretreatment greatly increased the dyed color
strength, indicating that the chemical reactions induced by NTP on AESO were the key. It
appeared that the effect of NTP was confounded by the addition of benzyl alcohol and NaCl
in the dye bath, so the two did not show an obvious effect if the fabrics were NTP-treated
post AESO pretreatment. A reasonable explanation would be that NTP application prior to
AESO pretreatment facilitated the attraction of polar NaCl and benzyl alcohol to the fabrics,
while in the case of NTP application post AESO pretreatment of the fabrics was covered by
AESO, and NTP was mainly used to induce reactions on the AESO. y
Because the interaction between NTP and AESO was important, in Experiment B, we
used a central composite design to find optimal AESO concentration and NTP treatment
time post AESO pretreatment, without the addition of benzyl alcohol and NaCl in the dye
bath. The detailed design and results are presented in Table 2. Table 2. Experiment B: central composite design and the results *. Table 2. Experiment B: central composite design and the results *. Sample
AESO conc. (wt.%)
NTP Time (s)
K/S **
Std **
B1
0.85
75
2.51
0.086
B2
0.85
139
2.54
0.049
B3
5
30
3.63
0.032
B4
5
120
3.98
0.008
B5
15
11
3.80
0.072
B6
15
75
5.25
0.065
B7
15
75
4.65
0.127
B8
15
75
4.73
0.116
B9
15
75
5.25
0.065
B10
15
75
5.54
0.017
B11
15
75
5.77
0.104
B12
15
139
5.45
0.125
B13
25
30
5.05
0.008
B14
25
120
7.16
0.090
B15
29
11
3.55
0.016
B16
29
75
6.84
0.126
* Experiment conducted at soaking time in AESO in ethyl acetate = 15 h, dye conc. * Experiment conducted at soaking time in AESO in ethyl acetate = 15 h, dye conc. = 0.1 wt.%, dyeing temperature
T = 90 ◦C, dyeing time = 1 h. ** Mean and standard error of three repeated measurements. 3.1. Analysis of Dyeing Experiments = 0.1 wt.%, dyeing temperature
T = 90 ◦C dyeing time = 1 h ** Mean and standard error of three repeated measurements 143 Polymers 2022, 14, 628 Stepwise regression resulted in the selection of a reduced quadratic model as the best
fit (F-test, p < 0.001), including only three terms of the main effect of NTP time, main effect
of AESO concentration, and the interaction of the two. The quadratic terms of both NTP
time and AESO concentration were not significant. The response surface plot for Experiment B is shown in Figure 3. Increasing both
NTP time and AESO concentration increased color strength. At low AESO concentration,
increasing NTP time only slightly increased color strength. However, at higher AESO con-
centration, increasing NTP time prominently improved color strength. This is reasonable
because higher concentration of AESO and longer NTP time induced more polymerized
AESO on the fabric surface that attracted more dye molecules, again corroborating the
importance of AESO pretreatment followed by NTP treatment. Figure 3. Statistical inference of Experiment B: Response surface of the central composite design. Figure 3. Statistical inference of Experiment B: Response surface of the central composite design. We also observed that AESO concentration higher than 25 wt.% resulted in an unnec-
essarily thick coating on the para-aramid fabrics. Therefore, we considered 25 wt.% as an
optimal concentration for AESO pretreatment because the highest K/S in Experiment B
was achieved at NTP time = 120 s. and AESO conc. = 25 wt.%. In Experiment C, based on the results of Experiment A and B, we fixed AESO con-
centration at 25 wt.% and tested if there is any difference between just applying NTP post
AESO pretreatment and applying NTP both before and after AESO pretreatment. We also
slightly extended the NTP post AESO pretreatment time to 150 s, based on our observation
in Figure 3. Experiment C focused on the functions of auxiliary additives of a swelling
agent (benzyl alcohol), an electrolyte (NaCl), and a surfactant of Polysorbate 80 (denoted
as TWEEN hereafter). The factorial design and results are shown in Table 3. Table 3. Experiment C: factorial design and the results *. 3.1. Analysis of Dyeing Experiments Sample
Benzyl-OH
(wt.%)
NaCl (wt.%)
TWEEN
(wt.%)
NTP
Order
K/S **
Std **
C1
None
None
None
Both
7.93
0.063
C2
None
None
None
Post
7.94
0.074
C3
None
None
3
Both
7.68
0.019
C4
None
None
3
Post
8.25
0.044
C5
None
8
None
Both
7.47
0.096
C6
None
8
None
Post
7.10
0.164
C7
None
8
3
Both
6.20
0.222 Table 3. Experiment C: factorial design and the results *. 144 Polymers 2022, 14, 628 Table 3. Cont. Sample
Benzyl-OH
(wt.%)
NaCl (wt.%)
TWEEN
(wt.%)
NTP
Order
K/S **
Std **
C8
None
8
3
Post
6.72
0.162
C9
2
None
None
Both
8.46
0.059
C10
2
None
None
Post
7.39
0.092
C11
2
None
3
Both
7.26
0.099
C12
2
None
3
Post
7.58
0.232
C13
2
8
None
Both
8.29
0.171
C14
2
8
None
Post
8.41
0.097
C15
2
8
3
Both
9.23
0.038
C16
2
8
3
Post
6.23
0.491
* Experiment conducted at soaking time in 25 wt.% AESO in ethyl acetate = 15 h, dye conc. = 0.1 wt.%, NTP
treatment time = 150 s post AESO soaking (designated Post in Table 3) or 90 s before AESO soaking and 150 s
post AESO soaking (designated Both in Table 3), dyeing temperature T = 90 ◦C, dyeing time = 1 h. ** Mean and
standard error of three repeated measurements. * Experiment conducted at soaking time in 25 wt.% AESO in ethyl acetate = 15 h, dye conc. = 0.1 wt.%, NTP
treatment time = 150 s post AESO soaking (designated Post in Table 3) or 90 s before AESO soaking and 150 s
post AESO soaking (designated Both in Table 3), dyeing temperature T = 90 ◦C, dyeing time = 1 h. ** Mean and
standard error of three repeated measurements. We determined an ANOVA model as the best fit (F-test, p < 0.0001) according to the
half-normal probability plot, including all the four main factorial effects and only two
interaction terms, which were statistically significant based on the t-test of the model
coefficients (p < 0.05). Statistical inference is presented in Figure 4. Figure 4. Statistical inference of Experiment C: main effect of a factor not involved in any interaction
and significant interactions. Figure 4. Statistical inference of Experiment C: main effect of a factor not involved in any interaction
and significant interactions. 3.1. Analysis of Dyeing Experiments TWEEN was the only factor not involved in any significant interactions; the addition
of the surfactant negatively affected dyeing performance, consistent with our previous
observations [9,10]. The surfactant, although helping to form a dye dispersion, might
also hinder the diffusion of the dye onto the para-aramid fiber fabrics. The positive effect
of benzyl alcohol was significant only when the NTP was applied both before and after
AESO pretreatment. Furthermore, benzyl alcohol and NaCl were confounding factors. In
the absence of benzyl alcohol, the addition of NaCl lowered color strength, but with the
presence of benzyl alcohol, the NaCl effect was insignificant. We may speculate that both
NaCl and benzyl alcohol participated in the NTP-induced reactions with AESO, leading
to different reaction pathways, and therefore, different dyeing effects. Overall, all the
samples were dyed to high color strength with small variations because we fixed AESO
concentration and NTP treatment time at optimal conditions. The effects of auxiliary 145 Polymers 2022, 14, 628 additives were limited, compared with the major improvement of color strength caused by
AESO pretreatment followed by NTP treatment. additives were limited, compared with the major improvement of color strength caused by
AESO pretreatment followed by NTP treatment. Experiment D was conducted as a starting point to investigate the possibility of replac-
ing AESO with a simple mixture of soybean oil and acrylic acid, because hypothetically a
cross-linked network could be formed with the application of NTP to help dyeing. How-
ever, longer NTP treatment time is needed based on our preliminary trials. Furthermore,
because one soybean oil molecule has an average of 4.6 double bonds and NTP could
trigger self-polymerization of soybean oil [24], we tested two molar ratios of acrylic acid to
soybean oil (denoted as AA/Soy hereafter) of 1.6 and 4. We also tested if the application of
NTP again after dyeing would help fixing the dye. The design and results of this 3-factor,
2-level factorial experiment are shown in Table 4. Table 4. Experiment D: factorial design and the results *. Table 4. Experiment D: factorial design and the results *. 3.1. Analysis of Dyeing Experiments Sample
AA/Soy
NTP Time (s)
Post-Dye NTP
Time (s)
K/S **
Std **
D1
1.6
240
0
7.67
0.087
D2
1.6
240
30
3.99
0.054
D3
1.6
360
0
9.6
0.175
D4
1.6
360
30
6.85
0.012
D5
4
240
0
8.84
0.056
D6
4
240
30
7.66
0.039
D7
4
360
0
9.59
0.113
D8
4
360
30
8.26
0.023
* Experiment conducted at soaking time in AA/Soy mixture = 15 h, dye conc. = 0.1 wt.%, dyeing temperature
T = 90 ◦C, dyeing time = 1 h. ** Mean and standard error of three repeated measurements. Based on the half-normal probability plot, we selected an ANOVA model (F-test,
p < 0.0001), which included all three main factors and two interaction terms (t-test, p < 0.001). Because all the factors were involved in the interactions, the statistical inference was based
on the significant interaction plots as shown in Figure 5. Figure 5. Statistical inference of Experiment D: significant factorial interactions. Figure 5. Statistical inference of Experiment D: significant factorial interactions. Overall, the color strength achieved in Experiment D is comparable with that in
Experiment C using AESO, showing the potential of using AA/Soy to replace AESO. Generally, longer NTP treatment time and higher AA/Soy resulted in higher K/S values. It
is interesting to observe that at AA/Soy = 1.6, a 30 s NTP treatment after dyeing greatly Overall, the color strength achieved in Experiment D is comparable with that in
Experiment C using AESO, showing the potential of using AA/Soy to replace AESO. Generally, longer NTP treatment time and higher AA/Soy resulted in higher K/S values. It
is interesting to observe that at AA/Soy = 1.6, a 30 s NTP treatment after dyeing greatly 146 Polymers 2022, 14, 628 decreased color strength, whereas at AA/Soy = 4 there was no change. A plausible
explanation would be that NTP could quickly degrade the dye molecules [10]; however, at
higher AA/Soy, more chemical functionalities could absorb most NTP energy and reactive
species, thus protecting the dye from degradation. p
p
g
y
g
In order to evaluate if the use of auxiliary agents would help improve dyeing with
the AA/Soy pretreatment, Experiment E focused on the effects of NaCl and benzyl alco-
hol. 3.1. Analysis of Dyeing Experiments Furthermore, because we observed unevenly dyed samples in Experiment D due to
inadequate mixing of the soybean oil and acrylic acid, the AA/Soy well dissolved in ethyl
acetate (50 wt.% of AA/Soy in ethyl acetate) was used as the pretreatment solution. The
design and results of this 4-factor full factorial experiment are presented in Table 5. Table 5. Experiment E: factorial design and the results *. Sample
AA/Soy
NTP Time (s)
NaCl
(wt.%)
Benzyl-OH
(wt.%)
K/S
Std **
E1
1.6
240
None
None
6.18
0.321
E2
1.6
240
None
2
4.6
0.059
E3
4
240
None
None
10.21
0.327
E4
4
240
None
2
9.82
0.589
E5
1.6
240
5
None
8.93
0.42
E6
1.6
240
5
2
7.12
0.034
E7
4
240
5
None
8.69
0.037
E8
4
240
5
2
6.51
0.056
E9
1.6
360
None
None
6.76
0.049
E10
1.6
360
None
2
7.44
0.655
E11
4
360
None
None
7.92
0.051
E12
4
360
None
2
6.74
0.017
E13
1.6
360
5
None
9.01
0.032
E14
1.6
360
5
2
7.18
0.048
E15
4
360
5
None
8.41
0.066
E16
4
360
5
2
7.36
0.031
* Experiment conducted at soaking time in AA/Soy in ethyl acetate = 15 h, dye conc. = 0.1 wt.%, swelling
agent = 2 wt.%, benzyl alcohol, electrolyte = 5 wt.%, NaCl, dyeing temperature T = 90 ◦C, dyeing time = 1 h. ** Mean and standard error of three repeated measurements. Table 5. Experiment E: factorial design and the results *. Table 5. Experiment E: factorial design and the results *. Table 5. Experiment E: factorial design and the results *. Based on the half-normal probability plot, we selected the best fit of ANOVA model
(F-test, p < 0.0001). The statistical inference is shown in Figure 6. Only two interaction
terms were significant (t-test, p < 0.01). However, the main effect of NTP treatment time
was not significant (t-test, p = 0.53) due to the confounding effect of NaCl. Figure 6. Statistical inference of Experiment E: main effect of a factor not involved in any interaction
and significant interactions. Figure 6. Statistical inference of Experiment E: main effect of a factor not involved in any interaction
and significant interactions. 147 Polymers 2022, 14, 628 The swelling agent of benzyl alcohol was the only significant main factor not involved
in any interactions; it negatively affected the dyeing color strength. 3.1. Analysis of Dyeing Experiments In comparison with
Experiment D, Experiment E showed similar interactions between the NTP treatment time
and the AA/Soy ratio. Specifically, fixing the NTP time = 360 s, increasing AA/Soy from
1.6 to 4, did not improve color strength, while fixing NTP time = 240 s, increasing AA/Soy,
significantly increased color strength. g
y
g
A confounding effect of NaCl was observed. Without NaCl, higher AA/Soy yielded
higher color strength; with the addition of NaCl in the dye bath, AA/Soy = 4 and
AA/Soy = 1.6 resulted in statistically the same K/S. A plausible explanation would be that
both NTP and NaCl would induce reactions in AA/Soy (as revealed in our FTIR analysis
in Section 3.2), and the higher dosage of both would result in overreactions that decreased
surface functionality for binding with the dye molecules. y
g
y
Because of the kinetic nature of the reactions catalyzed by NTP and influenced by
the presence of NaCl, we speculated that there exists an optimal combination of NTP
treatment time, AA/Soy, and NaCl concentration. Therefore, in Experiment F, we utilized a
Box–Behnken response surface design in an effort to find this optimal combination. The
detailed experimental design and results are presented in Table 6. Table 6. Experiment F: Box–Behnken design and the results *. Sample
AA/Soy
NTP Time (s)
NaCl (wt.%)
K/S
Std **
F1
1.5
180
5
10.59
0.019
F2
1.5
270
2
5.56
0.209
F3
1.5
270
8
7.44
0.573
F4
1.5
360
5
10.50
0.387
F5
3.25
180
2
7.96
0.190
F6
3.25
180
8
9.61
0.339
F7
3.25
270
5
13.18
1.157
F8
3.25
270
5
8.94
0.052
F9
3.25
270
5
13.37
0.220
F10
3.25
270
5
12.30
0.259
F11
3.25
270
5
12.16
1.564
F12
3.25
360
2
10.97
0.126
F13
3.25
360
8
10.72
0.063
F14
5
180
5
9.62
0.044
F15
5
270
2
10.94
0.183
F16
5
270
8
9.84
0.125
F17
5
360
5
10.86
0.756
* Experiment conducted at soaking time in AA/Soy (50 wt.%) in ethyl acetate = 15 h, dye conc. = 0.1 wt.%, dyeing
temperature T = 90 ◦C, dyeing time = 1 h. ** Mean and standard error of three repeated measurements. Table 6. Experiment F: Box–Behnken design and the results *. 3.1. Analysis of Dyeing Experiments A reduced quadratic model of response surface was selected as the best fit (F-test,
p < 0.05) after performing stepwise regression, including the three main factor effects and
the quadratic terms of only AA/Soy and NaCl concentration. Representative response
surfaces are presented in Figure 7. 148 Polymers 2022, 14, 628 Figure 7. Statistical inference of Experiment F: Response surface of the Box–Behnken design. Figure 7. Statistical inference of Experiment F: Response surface of the Box–Behnken design. Within the selected ranges for the main factors, increasing NTP treatment time linearly
increased color strength, so the longest NTP time = 360 s gave the best results. However,
there existed an optimal point for AA/Soy and NaCl concentration. Based on the above
plots, we determined an optimal condition as NTP time = 360 s, NaCl = 5% wt.%, and
AA/Soy = 3.25. Experiment G was conducted at the determined optimal conditions, and we varied
the pretreatment soaking time for practical applications, making it a one-factor factorial
design with three replicates. Furthermore, all the samples in Experiment G were washed a
second time with detergent by following the same protocol described in Section 2.3, and
the change of K/S values (denoted as ΔK/S) was used to evaluate the colorfastness against
wash. The results are presented in Figure 8, showing each data point, the means, and the
least significant difference (LSD) lines. Figure 8. Effect of pretreatment soaking time on color strength and colorfastness (ΔK/S is defined as
subtracting K/S after 2nd detergent wash from that after 1st detergent wash). ANOVA for the one-factor model did not detect any significant difference among the
four pretreatment soaking times (F-test, p > 0.2), because of a large variation of K/S when Figure 8. Effect of pretreatment soaking time on color strength and colorfastness (ΔK/S is defined as
subtracting K/S after 2nd detergent wash from that after 1st detergent wash). ANOVA for the one-factor model did not detect any significant difference among the
four pretreatment soaking times (F-test, p > 0.2), because of a large variation of K/S when
the soaking time was short. There was an outlier with low K/S = 6.2, occurring with 1 min 149 Polymers 2022, 14, 628 of soaking time. Generally, the soaking time appeared to affect the consistency of color
strength. The ΔK/S indicated slight reduction in color strength after second detergent wash. 3.1. Analysis of Dyeing Experiments Similarly, shorter soaking time resulted in larger variation of ΔK/S. It is recommended
that a soaking time ≥60 min be used, or we need to develop a better method to evenly
distribute AA/Soy into the fabrics in a shorter time (e.g., continuous soaking and pressing
with rollers). To provide a visual demonstration comparing the dyeing strengths of different treat-
ments, images of selected samples acquired using a flatbed scanner are presented in the
Supplementary File (Figure S1). 3.2. FTIR Analysis To understand chemical changes during the pretreatment and dyeing processes, ATR–
FTIR spectroscopy was performed at different stages of the processes. As a foundation for
interpreting the FTIR spectra, Table S1 in the Supplementary File summarizes major FTIR
bands of AESO, soybean oil, acrylic acid, and para-aramids. Figure 9 presents ATR–FTIR spectra of materials at different stages of our processes
using AESO for pretreatment. The AESO signal is dominated by the major peaks of
soybean oil backbone (line I). A strong peak for ester carbonyl groups should appear at
1742 cm−1; however, due to the formation of acrylate esters, a peak should appear at nearby
1723 cm−1 [25]. Therefore, the peak with a deformed shape around 1737 cm−1 should be the
overlapping of 1742 and 1723 cm−1. Compared with the dominant signal from soybean oil
backbone, characteristics of AESO/acrylate can be found as small peaks. There are residual
epoxides in the AESO that were not fully acrylated as revealed by the oxirane peaks at
1270 and 822 cm−1. Although major acrylate peaks (such as 1638 and 1619 cm−1) overlap
with a strong signal from para-aramids, two distinguishable acrylate peaks at 1406 and
810 cm−1 can be tracked along the way of our processes to indicate the consumption of the
acrylates. Figure 9. FTIR spectra at different stages of materials of (I) AESO; (II) Para-aramids; (III) Para-aramid
after soaking in AESO; (IV) Para-aramid after soaking in AESO and subsequent NTP treatment;
(V) dyed sample A3 pretreated with AESO without subsequent NTP treatment; (VI) dyed sample C9
pretreated with AESO with subsequent NTP treatment; (VII) Victoria Blue R. Figure 9. FTIR spectra at different stages of materials of (I) AESO; (II) Para-aramids; (III) Para-aramid
after soaking in AESO; (IV) Para-aramid after soaking in AESO and subsequent NTP treatment;
(V) dyed sample A3 pretreated with AESO without subsequent NTP treatment; (VI) dyed sample C9
pretreated with AESO with subsequent NTP treatment; (VII) Victoria Blue R. 150 Polymers 2022, 14, 628 For para-aramids, the bands shown in Table S1 and correspondingly in Figure 9 (line
II) can be considered as an internal standard [26]. The FTIR spectrum of AESO-soaked para-
aramids apparently show the addition of signals from both AESO and para-aramids (line
III). After subsequent NTP treatment, changes can be seen (line IV): the acrylate peaks at
1406 and 810 cm−1 disappeared or significantly decreased. 3.2. FTIR Analysis Although this observation was
somewhat obscured by a nearby peak of para-aramids, the shapes of the two peaks more
closely resemble those of the para-aramid peaks nearby, indicating the consumption of the
acrylate functional groups. A sobservation can be found around 1737 cm−1, showing the
consumption of 1723 cm−1 acrylate esters. The oxirane peak at 1270 cm−1 also disappeared
(line IV). However, the signal of vinyl functional group at 986 cm−1 remained, albeit
decreased, indicating that the AESO was not fully polymerized. The 1190 cm−1 peak of
C–O stretching in the esters of soybean oil backbone also remained, revealing that the NTP
treatment did not fully break the ester bonds. y
Figure 9 also shows the FTIR spectrum of a dyed sample which was pretreated with
AESO without subsequent NTP treatment (line V, sample A3, K/S = 6.14), and another
dyed sample pretreated with AESO with subsequent NTP treatment (line VI, sample C9,
K/S = 8.46). Other than differences at a few minor peaks, lines V and VI are very similar. The –C=O carbonyl stretching and C–O stretching in the esters at 1737 cm−1 and 1190 cm−1,
respectively remained strong in lines V and VI, indicating that the NTP treatment and the
dyeing process did not break much of the ester bonds, which remained after dyeing and
detergent wash. However, the 1190 cm−1 peak in line VI shifted to the right, probably due
to the influence of the dye peak at 1170 cm−1, because the sample of line VI was dyed to
a higher K/S value. The three spectra (lines IV, V, and VI) all have a new peak emerging
at 786 cm−1, which is in the typical region of 1,3-disubstituted or 1,2,3-trisubstituted
C–H bending, meaning a possible new attachment of AESO to the para-aromatic rings. Interestingly, this new peak at 786 cm−1 could be generated by the NTP treatment or the
thermal dyeing process, because the sample of line V was not treated with NTP after AESO
soaking. In line VI, the weak absorption band at 1585 cm−1 was assigned to aromatic
rings conjugated with α-carbonyl group, indicating that some AESO covalently bonded to
para-aramid rings after NTP treatment [27]. Additionally, lines IV, V, and VI obviously have stronger FTIR signal because the
AESO pretreatment, NTP treatment, and dyeing process changed the reflective index of the
treated samples, enabling deeper ATR–FTIR penetration. 3.2. FTIR Analysis The FTIR spectrum of the Victoria Blue R dye is also shown in Figure 9 (line VII). Because of low uptake of the dye by the fabrics and that most of the dye peaks were
obscured by the characteristic peaks of the para-aramids and AESO, no significant dye
signal was detected in the dyed samples, except for that at 1355 and 1170 cm−1 that
rendered a small shoulder in the spectra of dyed samples (lines V and VI), especially clear
for the sample of line VI, which was treated with NTP and consequently dyed to a higher
color strength. Because the strong signal of para-aramids obscured many meaningful peaks that reveal
chemical changes, we used ATR–FTIR to monitor reactions between soybean oil and acrylic
acid on KBr plates (instead of on para-aramids) in contrast to those of AESO. It is important
to note that the reagents acrylic acid and AESO contain 200 or 4000 ppm of monomethyl
ether of hydroquinone (MEHQ), respectively, as an inhibitor of self-polymerization. Fig-
ure 10 shows the results; line I is the spectrum of soybean oil; line II is that of acrylic acid;
and line III is that of the mixture of acrylic acid and soybean oil in a molar ratio of 4:1,
apparently showing the signal addition of soybean oil and acrylic acid. 151 151 Polymers 2022, 14, 628 Figure 10. FTIR spectra at different stages of materials on KBr of (I) soybean oil; (II) acrylic acid;
(III) mixture of soybean oil and acrylic acid; (IV) mixture of soybean oil and acrylic acid after 6 min
NTP treatment; (V) mixture of soybean oil, acrylic acid, and NaCl; (VI) AESO; (VII) AESO after
2.5 min NTP treatment. Figure 10. FTIR spectra at different stages of materials on KBr of (I) soybean oil; (II) acrylic acid;
(III) mixture of soybean oil and acrylic acid; (IV) mixture of soybean oil and acrylic acid after 6 min
NTP treatment; (V) mixture of soybean oil, acrylic acid, and NaCl; (VI) AESO; (VII) AESO after
2.5 min NTP treatment. After a 6 min NTP treatment of the mixture of soybean oil and acrylic acid, significant
changes could be observed (line IV). A small but obvious broad peak appeared around
3650 cm−1, which is assigned to O–H stretching usually from alcohols or phenols. 3.2. FTIR Analysis Because
the NTP generated in ambient air has ozone, singlet oxygen atoms, and hydrogen peroxide
as highly reactive oxidants, the unstable C=C double bonds in soybean oil could be first
attacked to form epoxidized soybean oil, which could be followed by ensuing reaction with
acrylic acid catalyzed by UV and/or radical species in NTP to form AESO. However, a
competitive reaction could also lead to the production of polyols [28], which is evident in
line IV, and the polyols could react with acrylic acid to form acrylate polymers [29,30]. p
y
y
y
p
y
After NTP treatment (line IV), the 3009 cm−1 peak (=C–H in soybean oil) disappeared
and some new peaks appeared between 3009 and 2922 cm−1. The peak at 2980 cm−1 can be
assigned to C–H (sp3 stretching), indicating hydrogen abstraction from the –CH3 or –CH2
of the soybean oil. However, if the FTIR spectrum was acquired on para-aramids pretreated
with AA/Soy followed by NTP treatment, (see line A in Figure S2 of Supplementary File),
this peak at 2980 cm−1 was not observed. Instead, a small peak appeared at 786 cm−1,
which is the typical region of 1,3-disubstituted or 1,2,3-trisubstituted C–H bending and
similar to the case in Figure 9 for samples pretreated with AESO. Therefore, we reasoned
that the AESO or AA/Soy network was “anchored” to para-aramids at the C–H point. The 1695 cm−1 peak (C=O stretching) in acrylic acid was significantly consumed with
the application of NTP, and a new peak at 1556 cm−1 indicated nitro groups caused by the
reactive nitrogen and oxygen species (RONS) in NTP; a small peak at the same wavelength
can also be observed in NTP-treated AESO (line VII). Interestingly, by simply adding NaCl to the mixture of soybean oil and acrylic acid,
similar changes can be observed around 3650 and 2980 cm−1 (line V), indicating that NaCl
triggered reactions between the soybean oil and acrylic acid at room temperature, which
explains the confounding effect of NaCl we observed in Experiment E, in that the reactions
with or without NaCl might lead to different kinetics and pathways. With NTP application
on the mixture of soybean oil and acrylic acid (line III vs. VI), the 1637 and 1617 cm−1 of 152 Polymers 2022, 14, 628 C=C double bonds in the acrylic acid were consumed, indicating that the NTP treatment did
induce radical polymerization through the C=C double bonds. 3.2. FTIR Analysis A similar observation could
be found in NTP-treated AESO (line VI vs. VII). However, for the NTP-treated mixture of
soybean oil and acrylic acid, the 984 cm−1 peak (=CH2) was almost completely consumed
after a 6 min NTP treatment (line IV), whereas the 986 cm−1 peak of vinyl functional groups
in AESO was still visible (line VII), indicating that a 2.5 min NTP treatment did not totally
consume the vinyl groups, and longer NTP time could be applied on the AESO. The FTIR
spectra of dyed samples pretreated with AESO or AA/Soy were very similar, except for
some small peaks (see line B vs. C in Figure S2 of Supplementary File). The NTP-induced chemical reactions in AESO and AA/Soy are complex, and the
precise details of reaction pathways on the surface of para-aramids deserve further study. Nonetheless, based on the FTIR results, we can propose the following mechanism of im-
proved dyeing with the pretreatment of AESO or AA/Soy. Because of the high crystallinity
and inertness of para-aramids, few dye molecules can be directly adsorbed on the para-
aramid surface. With the pretreatment of AESO or AA/Soy and subsequent NTP treatment,
a cross-linked network (yet flexible due to the preserved long acyl chains) is formed, “an-
choring” to the para-aramids with covalent bonds, and also providing functional groups
that bind the dye molecules. Although the pretreatment with AESO vs. AA/Soy generated
differences in the polymerized network on para-aramids, comparable dyeing effects were
achieved, as evident in Section 3.1. 3.3. Supporting Evidence: SEM, Extraction Test, and Tensile Strength 3.3. Supporting Evidence: SEM, Extraction Test, and Tensile Strength Scanning electron micrographs (SEM) reveal the smooth surface of original para-
aramid fibers, and a 4 min NTP treatment did not show any visible changes on the surface
(Figure 11), explaining why the NTP treatment alone would not significantly improve
dyeing. The samples dyed with AESO or AA/Soy pretreatment have a resin-like coating on
the para-aramid fibers, in which dye particles are embedded. It is notable that the coating
survived two washes with detergent, indicating a polymerized network. Agglomeration of
dye particles can also be observed, especially in the AA/Soy pretreated sample, indicating
that our pretreatment procedure needs further improvement in order to evenly distribute
the AESO or AA/Soy and deliver the NTP treatment. After a 15 h extraction with three solvents of different polarity, the color strength of all
the tested samples decreased. Because the K/S values were measured at different wave-
lengths for the blue cationic dye vs. the red disperse dye, we report here the percentage
decrease in K/S value after extraction for comparison. For the samples dyed with AA/Soy
pretreatment followed by NTP treatment, the K/S value decreased by 20%, 64%, and 63%
after the extraction using hexane, ethanol, or 2:1 (v/v) chloroform:methanol, respectively. For the sample dyed with Disperse Red 1 acrylate, the K/S value decreased by 6%, 54%, and
84% after the extraction using hexane, ethanol, or 2:1 (v/v) chloroform:methanol, respec-
tively. This indicates that not all the AA/Soy were polymerized (and the unpolymerized
soybean oil could be easily extracted by hexane), or not all the AA/Soy were covalently
bonded to the fabric. However, there was evidence that almost all the dye molecules were
covalently bonded to the NTP-induced polymerized network. When the solvents were
evaporated from the extractable matters and the dry lipid-dye matrices were re-dissolved
in hexane, then water was added to study the partition of the dye between the hexane and
water, no blue color was observed in the aqueous layer, indicating that this water-soluble
dye was chemically linked to the soybean oil network. 153 Polymers 2022, 14, 628 Figure 11. SEM micrograph of samples. Figure 11. SEM micrograph of samples. 3.3. Supporting Evidence: SEM, Extraction Test, and Tensile Strength In addition, significant color strength remained after extraction with the three solvents
of different polarities, indicating a covalently bonded network formed on the surface of
para-aramid fabrics, with one exception of the 84% reduction in K/S value for the sample
dyed with Disperse Red 1 acrylate and extracted with chloroform:methanol, which indicates
the lack of bonding between the polymerized dye and the para-aramids; the extracted
sample exhibited a yellow color close to that of the undyed fabric. In this respect, the
method developed in this study performed better in binding with the inert para-aramids. Furthermore, the samples dyed with Disperse Red 1 acrylate became more rigid because of
the short chain of the starting monomers, while the samples dyed with AA/Soy remained
flexible due to the preserved long chains of soybean oil. It is worthwhile to mention that in dyeing textiles, dyes are usually fixed on textiles
through physical entrapment or the relatively weak hydrogen bonds and van der Waals
force of attraction, or stronger ionic bonds in the case of acid dye or basic dye; few dye
molecules binded to textiles via covalent bonds, except for reactive dyes with cotton, wool,
or polyamides such as nylon [31,32]. Tensile test results show that our developed method of dyeing para-aramids did not
reduce the strength of para-aramid fibers measured as breakpoint peak load. 4. Conclusions We demonstrated that deriving more functionalities of soybean oil on the surface of
para-aramids further improved the dyeing color strength and colorfastness. Compared
with the pretreatment using soybean oil that obtained the highest K/S value up to 3.89,
pretreatment with either AESO or AA/Soy achieved K/S values higher than nine in
this study. Importantly, we demonstrated through sequential experimentation that a
simple mixture of AA/Soy could replace the pricy AESO to render comparable dyeing
performance. NTP treatment after the AESO or AA/Soy pretreatment was essential for
our developed method, because the NTP not only induced the formation of a polymerized 154 Polymers 2022, 14, 628 network on the surface of para-aramids that bonded with dye molecules, but also generated
covalent bonds anchoring the polymerized network to the para-aramids, which is difficult
given the high crystallinity and chemical inertness of the para-aramids. Among the auxiliary additives tested in this study, Polysorbate 80 (TWEEN) as a
surfactant negatively affected the dyeing, and the effect of benzyl alcohol as a swelling
agent was minimal. However, NaCl as an electrolyte showed positive effect. Therefore, we
recommend an optimal formulation and condition as NTP time = 360 s (specific for the NTP
source used in this study), NaCl = 5% wt.%, and AA/Soy = 3.25 for dyeing para-aramids
without other chemical additives. The dyeing method developed in this study did not
sacrifice the strength of para-aramids, showing the potential for this method to replace
hazardous chemicals currently used in dyeing practices with renewable materials and
environmentally friendly technologies, while achieving improved dyeing of para-aramid
textiles for new applications. Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/polym14030628/s1, Table S1: Characteristic FTIR bands of materials
and the references; Figure S1: Scanned images of dyed samples in contrast with original undyed
fabric (labels correspond to the sample numbers in Tables 3–5 and Figure 8; G4 (W2) indicates sample
G4 after 2nd detergent wash); Figure S2: FTIR spectra of (A) para-aramid sample after soaking in
AA/Soy and subsequent NTP treatment; (B) dyed para-aramid sample pretreated with AA/Soy with
subsequent NTP treatment; (C) dyed para-aramid sample pretreated with AESO with subsequent
NTP treatment. Author Contributions: Conceptualization, C.J.D. and X.P.Y.; methodology, X.P.Y. and C.J.D.; valida-
tion, X.P.Y., T.W. and C.M.; formal analysis, C.J.D., C.M. and X.P.Y.; investigation, C.M., T.W. and
X.P.Y.; resources, C.J.D. and X.P.Y.; data curation, C.M. y
,
,
[
]
6.
Xi, M.; Li, Y.-L.; Shang, S.-Y.; Li, D.-H.; Yin, Y.-X.; Dai, X.-Y. Surface modification of aramid fiber by air DBD plasma at atmospheric
pressure with continuous on-line processing. Surf. Coat. Technol. 2008, 202, 6029–6033. [CrossRef] 4. Conclusions and X.P.Y.; writing—original draft preparation,
X.P.Y.; writing—review and editing, X.P.Y., T.W. and C.J.D.; visualization, C.M. and X.P.Y.; supervision,
X.P.Y. and T.W.; project administration, C.J.D. and X.P.Y.; funding acquisition, C.J.D. and X.P.Y. All
authors have read and agreed to the published version of the manuscript. Funding: This work was partially supported by the Department of Agriculture HATCH project No. TEN00521. Funding: This work was partially supported by the Department of Agriculture HATCH project No. TEN00521. Funding: This work was partially supported by the Department of Agriculture HATCH project No. TEN00521. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the
corresponding author. Acknowledgments: We thank the support of the U.S. Department of Agriculture HATCH project No. TEN00521. Conflicts of Interest: The authors declare no conflict of interest. 4.
Roenbeck, M.R.; Sandoz-Rosado, E.J.; Cline, J.; Wu, V.; Moy, P.; Afshari, M.; Reichert, D.; Lustig, S.R.; Strawhecker, K.E. Probing
the internal structures of Kevlar® fibers and their impacts on mechanical performance. Polymer 2017, 128, 200–210. [CrossRef]
5.
Sun, Y.; Liang, Q.; Chi, H.; Zhang, Y.; Shi, Y.; Fang, D.; Li, F. The application of gas plasma technologies in surface modification of
aramid fiber. Fibers Polym. 2014, 15, 1–7. [CrossRef] g
pp
3.
Roenbeck, M.R.; Cline, J.; Wu, V.; Afshari, M.; Kellner, S.; Martin, P.; Londono, J.D.; Clinger, L.E.; Reichert, D.; Lustig, S.R.;
et al. Structure–property relationships of aramid fibers via X-ray scattering and atomic force microscopy. J. Mater. Sci. 2019, 54,
6668–6683. [CrossRef] References Used for High-Performance Apparel. In High-Performance Apparel; McLoughlin, J., Sabir, T., Eds.; Woodhead
n, UK, 2018; pp. 7–32. 1. Sabir, T. 2—Fibers Used for High-Performance Apparel. In High-Performance Apparel; McLoughlin, J., S
Publishing: Sawston, UK, 2018; pp. 7–32. g
pp
g
f
pp
g
J
Publishing: Sawston, UK, 2018; pp. 7–32. 2. Gong, R.H.; Chen, X. 3—Technical Yarns. In Handbook of Technical Textiles, 2nd ed.; Horrocks, A.R., Anand, S.C., Eds.; Woodhead
P bli hi
S
t
UK 2016
43 62 g
,
,
; pp
2. Gong, R.H.; Chen, X. 3—Technical Yarns. In Handbook of Technical Textiles, 2nd ed.; Horrocks, A.R., Anand, S.C., Eds.; Woodhead
Publishing: Sawston, UK, 2016; pp. 43–62. g
pp
2. Gong, R.H.; Chen, X. 3—Technical Yarns. In Handbook of Technical Textiles, 2nd ed.; Horrocks, A.R., A
Publishing: Sawston, UK, 2016; pp. 43–62. 3. Roenbeck, M.R.; Cline, J.; Wu, V.; Afshari, M.; Kellner, S.; Martin, P.; Londono, J.D.; Clinger, L.E.; Reichert, D.; Lustig, S.R.;
et al. Structure–property relationships of aramid fibers via X-ray scattering and atomic force microscopy. J. Mater. Sci. 2019, 54,
6668–6683. [CrossRef] 4. Roenbeck, M.R.; Sandoz-Rosado, E.J.; Cline, J.; Wu, V.; Moy, P.; Afshari, M.; Reichert, D.; Lustig, S.R.; Strawhecker, K.E. Probing
the internal structures of Kevlar® fibers and their impacts on mechanical performance. Polymer 2017, 128, 200–210. [CrossRef]
5. Sun, Y.; Liang, Q.; Chi, H.; Zhang, Y.; Shi, Y.; Fang, D.; Li, F. The application of gas plasma technologies in surface modification of
aramid fiber. Fibers Polym. 2014, 15, 1–7. [CrossRef] y
,
,
[
]
6. Xi, M.; Li, Y.-L.; Shang, S.-Y.; Li, D.-H.; Yin, Y.-X.; Dai, X.-Y. Surface modification of aramid fiber by air DBD plasma at atmospheric
pressure with continuous on-line processing. Surf. Coat. Technol. 2008, 202, 6029–6033. [CrossRef] 155 Polymers 2022, 14, 628 7. Su, M.; Gu, A.; Liang, G.; Yuan, L. The effect of oxygen-plasma treatment on Kevlar fibers and the properties of Kevlar
fibers/bismaleimide composites. Appl. Surf. Sci. 2011, 257, 3158–3167. [CrossRef] p
pp
f
8. Kaul, B.L. Synthetic Fibre Dyeing; Hawkyard, C., Ed.; Society of Dyers and Colourists: Bradford, 8. Kaul, B.L. Synthetic Fibre Dyeing; Hawkyard, C., Ed.; Society of Dyers and Colourists: Bradford, UK, 2004; p. 230. Doona, C.J. Soybean Oil and Nonthermal Plasma Pretr 9. Morris, M.; Ye, X.P.; Doona, C.J. Soybean Oil and Nonthermal Plasma Pretreatment to Dye Para-Aramid Woven Fabrics with
Disperse Dye Using a Glycerol-Based Dye Bath. J. Am. Oil Chem. References In Soy Based Chemicals and Materials; Brentin, R
Chemical Society: Washington, DC, USA, 2014; pp. 43–80. Chemical Society: Washington, DC, USA, 2014; pp. 43–80. y
g
pp
ngh, S.; Jena, J.; Das, T.; Das, N.B. Role of cationic and anionic surfactants in textile dyeing with natural dyes y
g
pp
16. Baliarsingh, S.; Jena, J.; Das, T.; Das, N.B. Role of cationic and anionic surfactants in textile dyeing with natural dyes extracted
from waste plant materials and their potential antimicrobial properties. Ind. Crops Prod. 2013, 50, 618–624. [CrossRef] waste plant materials and their potential antimicrobial properties. Ind. Crops Prod. 2013, 50, 618–624. [CrossR 17. Arif, M.F.; Butt, M.T.; Khan, M.N.; Ali, M. Effect of electrolyte concentration on dyeing process of cotton. Pak. J. Sci. Ind. Res. 2007,
50, 159–164. 18. Shao, D.; Xu, C.; Wang, H.; Du, J. Enhancing the Dyeability of Polyimide Fibers with the Assistance of Swelling Agents. Materials
2019, 12, 347. [CrossRef] 19. Standard ISO 105-C10:2006; Colour Fastness to Washing with Soap or Soap and Soda. Textiles—Tests for Colour Fastness. ISO: London, UK, 2006. Etters, J.N.; Hurwitz, M.D. Opaque reflectance of translucent fabric. Text. Chem. Colorist 1986, 18, 19–26. 21. Hunter, R.S.; Harold, R.W. The Measurement of Appearance, 2nd ed.; Wiley: New York, NY, USA, 1987. 21. Hunter, R.S.; Harold, R.W. The Measurement of Appearance, 2nd ed.; Wiley: New York, NY, USA, 1987. 22. De Smet, L.; Vancoillie, G.; Minshall, P.; Lava, K.; Steyaert, I.; Schoolaert, E.; Van De Walle, E.; Dubruel, P.; De Clerck, K.;
Hoogenboom, R. Plasma dye coating as straightforward and widely applicable procedure for dye immobilization on polymeric
materials. Nat. Commun. 2018, 9, 1123. [CrossRef] materials. Nat. Commun. 2018, 9, 1123. [CrossRef] 23. ASTM. Standard Test Methods for Tensile Testing of Aramid Yarns; D7269/D7269M−17; ASTM International: West Conshohocken,
PA, USA, 2017. cterization and Analysis of High Voltage Atmospheric Cold Plasma Treatment of Soilbean Oil. Ph.D. Thesis,
West Lafayette, IN, USA, 2020. 24. Ximena, V.Y. Characterization and Analysis of High Voltage Atmospheric Cold Plasma Treatment of So
Purdue University, West Lafayette, IN, USA, 2020. 25. Esen, H.; Çayli, G. Epoxidation and polymerization of acrylated castor oil. Eur. J. Lipid Sci. Technol. 2016, 118, 959–966. [CrossRef] 25. Esen, H.; Çayli, G. Epoxidation and polymerization of acrylated castor oil. Eur. J. Lipid Sci. Techno 26. Mukherjee, M.; Kumar, S.; Bose, S.; Das, C.; Kharitonov, A. References Soc. 2021, 98, 463–473. [CrossRef] P.; Doona, C.J. Dyeing Para-Aramid Textiles Pretreated with Soybean Oil and Nonthermal Plasma Using
mers 2021, 13, 1492. [CrossRef] Morris, M.; Ye, X.P.; Doona, C.J. Dyeing Para-Aramid Textiles Pretreated with Soybean Oil and Nonth
Cationic Dye. Polymers 2021, 13, 1492. [CrossRef] Cationic Dye. Polymers 2021, 13, 1492. [CrossRef] y
y
11. Omonov, T.S.; Curtis, J.M. 7—Plant Oil-Based Epoxy Intermediates for Polymers. In Bio-Based Plant
M db
l
S A Zh
C K
l
M R Ed
Willi
A d
P bli hi
N
Y
k NY USA 2 y
y
11. Omonov, T.S.; Curtis, J.M. 7—Plant Oil-Based Epoxy Intermediates for Polymers. In Bio-Based Plant Oil Polymers and Composites; 11. Omonov, T.S.; Curtis, J.M. 7—Plant Oil-Based Epoxy Intermediates for Polymers. In Bio-Based Plant Oil Polymers and Composites;
Madbouly, S.A., Zhang, C., Kessler, M.R., Eds.; William Andrew Publishing: New York, NY, USA, 2016; pp. 99–125. ly, S.A., Zhang, C., Kessler, M.R., Eds.; William Andrew Publishing: New York, NY, USA, 2016; pp. 99–125. y
g
g
pp
12. Behera, D.; Banthia, A.K. Synthesis, characterization, and kinetics study of thermal decomposition of epoxidized soybean oil
acrylate. J. Appl. Polym. Sci. 2008, 109, 2583–2590. [CrossRef] 12. Behera, D.; Banthia, A.K. Synthesis, characterization, and kinetics study of
acrylate. J. Appl. Polym. Sci. 2008, 109, 2583–2590. [CrossRef] a, D.; Banthia, A.K. Synthesis, characterization, and k 12. Behera, D.; Banthia, A.K. Synthesis, characterization, and kinetics study of thermal decompositio
acrylate. J. Appl. Polym. Sci. 2008, 109, 2583–2590. [CrossRef] y
pp
y
13. Zou, B.; Ren, S.; Ye, X.P. Glycerol Dehydration to Acrolein Catalyzed by ZSM-5 Zeolite in Supercritical Carbon Dioxide Medium. ChemSusChem 2016, 9, 3268–3271. [CrossRef] ChemSusChem 2016, 9, 3268–3271. [CrossRef] 14. Liu, L.; Ye, X.P.; Bozell, J.J. A Comparative Review of Petroleum-Based and Bio-Based Acrolein Production. ChemSusChem 2012, 5,
1162–1180. [CrossRef] 15. Ye, X.P.; Ren, S. 3—Value-Added Chemicals from Glycerol. In Soy-Based Chemicals and M
Chemical Society: Washington, DC, USA, 2014; pp. 43–80. X.P.; Ren, S. 3—Value-Added Chemicals from Glycerol. In Soy-Based Chemicals and Materials; Brentin, R., E
i
l S
i t
W
hi
t
DC USA 2014
43 80 15. Ye, X.P.; Ren, S. 3—Value-Added Chemicals from Glycerol. In Soy-Based Chemicals and Materials; Br Ren, S. 3—Value-Added Chemicals from Glycerol. In Soy-Based Chemicals and Materials; Brentin, R., Ed.;
al Society: Washington, DC, USA, 2014; pp. 43–80. Ye, X.P.; Ren, S. 3
Value Added Chemicals from Glycerol. Article
Simultaneous Sonochemical Coloration and Antibacterial
Functionalization of Leather with Selenium Nanoparticles
(SeNPs) Tarek Abou Elmaaty 1,2,*, Khaled Sayed-Ahmed 3, Radwan Mohamed Ali 4, Kholoud El-Khodary 2
and Shereen A. Abdeldayem 2 1
Department of Material Art, Galala University, Galala 43713, Egypt 2
Department of Textile Printing, Dyeing and Finishing, Faculty of Applied Arts, Damietta University,
Damietta 34512, Egypt; khloud.el.khodary@gmail.com (K.E.-K.); shereen.abdeldayem.82@gmail.com (S.A.A.)
3
Department of Agricultural Chemistry, Faculty of Agriculture, Damietta University, Damietta 34512, Egypt;
drkhaled_1@du.edu.eg 2
Department of Textile Printing, Dyeing and Finishing, Faculty of Applied Arts, Damietta University,
Damietta 34512 Egypt; khloud el khodary@gmail com (K E -K ); shereen abdeldayem 82@gmail com (S A A ) Damietta 34512, Egypt; khloud.el.khodary@gmail.com (K.E.-K.); shereen.abdeldayem.82@gmail.com (S.A.A.)
3
Department of Agricultural Chemistry, Faculty of Agriculture, Damietta University, Damietta 34512, Egypt;
drkhaled_1@du.edu.eg 3
Department of Agricultural Chemistry, Faculty of Agriculture, Damietta University, Damietta 34512, Eg
drkhaled_1@du.edu.eg g
4
Department of Biochemistry, Faculty of Agriculture, Al-Azhar University, Cairo 11651, Egypt;
yaseenjjar@gmail.com 4
Department of Biochemistry, Faculty of Agriculture, Al-Azhar University, Cairo 11651, Egypt;
jj
@
il yaseenjjar@gmail.com *
Correspondence: tasaid@gu.edu.eg Abstract: The development of antibacterial coatings for footwear components is of great interest
both from an industry and consumer point of view. In this work, the leather material was developed
taking advantage of the intrinsic antibacterial activity and coloring ability of selenium nanoparticles
(SeNPs). The SeNPs were synthesized and implemented into the leather surface by using ultrasonic
techniques to obtain simultaneous coloring and functionalization. The formation of SeNPs in the
solutions was evaluated using UV/Vis spectroscopy and the morphology of the NPs was deter-
mined by transmission electron microscopy (TEM). The treated leather material (leather/SeNPs)
was characterized by scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy
(EDX). The effects of SeNPs on the coloration and antibacterial properties of the leather material were
evaluated. The results revealed that the NPs were mostly spherical in shape, regularly distributed,
and closely anchored to the leather surface. The particle size distribution of SeNPs at concentrations
of 25 mM and 50 mM was in the range of 36–77 nm and 41–149 nm, respectively. It was observed
that leather/SeNPs exhibited a higher depth of shade compared to untreated ones, as well as ex-
cellent fastness properties. The results showed that leather/SeNPs can significantly enhance the
antibacterial activity against model of bacteria, including Gram-positive bacteria (Bacillus cereus) and
Gram-negative bacteria (Pseudomonas aeruginosa, Salmonella typhi and Escherichia coli). Moreover, the
resulting leather exhibited low cytotoxicity against HFB4 cell lines. polymers polymers Article
Simultaneous Sonochemical Coloration and Antibacterial
Functionalization of Leather with Selenium Nanoparticles
(SeNPs) This achievement should be quite
appealing to the footwear industry as a way to prevent the spread of bacterial infection promoted
by humidity, poor breathability and temperature which promote the expansion of the microflora of
the skin. Citation: Abou Elmaaty, T.;
Sayed-Ahmed, K.; Mohamed Ali, R.;
El-Khodary, K.; Abdeldayem, S.A. Simultaneous Sonochemical
Coloration and Antibacterial
Functionalization of Leather with
Selenium Nanoparticles (SeNPs). Polymers 2022, 14, 74. https://
doi.org/10.3390/polym14010074
Academic Editor: Sándor Kéki
Received: 10 November 2021
Accepted: 6 December 2021
Published: 25 December 2021 Citation: Abou Elmaaty, T.;
Sayed-Ahmed, K.; Mohamed Ali, R.;
El-Khodary, K.; Abdeldayem, S.A. Simultaneous Sonochemical
Coloration and Antibacterial
Functionalization of Leather with
Selenium Nanoparticles (SeNPs). Polymers 2022, 14, 74. https://
doi.org/10.3390/polym14010074 Academic Editor: Sándor Kéki Keywords: selenium nanoparticles; leather; sonochemical; antibacterial; toxicity; footwear Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. References Study on the Mechanical, Rheological, and Morphological Properties
of Short Kevlar™Fiber/s-PS Composites. Polym. Plast. Technol. Eng. 2008, 47, 623–629. [CrossRef] J.; van Loon, A.; Boon, J.J. FTIR Studies of the Effects of Pigments on the Aging of Oil. Stud. Conserv. 2005,
] 27. Van der Weerd, J.; van Loon, A.; Boon, J.J. FTIR Studies of the Effects of Pigments on the Aging of
3–22. [CrossRef] 28. Santacesaria, E.; Turco, R.; Russo, V.; Tesser, R.; Di Serio, M. Soybean Oil Epoxidation: Kinetics of the Epoxide Ring Opening
Reactions. Processes 2020, 8, 1134. [CrossRef] 29. Cheong, M.; Ooi, T.; Ahmad, S.; Yunus, W.; Kuang, D. Synthesis and Characterization of Palm-Based Resin for UV Coating. J. Appl. Polym. Sci. 2008, 111, 2353–2361. [CrossRef] 29. Cheong, M.; Ooi, T.; Ahmad, S.; Yunus, W.; Kuan
Appl. Polym. Sci. 2008, 111, 2353–2361. [CrossRef] 29. Cheong, M.; Ooi, T.; Ahmad, S.; Yunus, W.; Ku g
Appl. Polym. Sci. 2008, 111, 2353–2361. [CrossRef] 30. Li, Y.; Sun, S. Polyols from epoxidized soybean oil and alpha hydroxyl acids and their adhesion properties from UV polymerization. Int. J. Adhes. Adhes. 2015, 63, 1–8. [CrossRef] 31. Nicolai, S.; Tralau, T.; Luch, A.; Pirow, R. A scientific review of colorful textiles. J. Consum. Prot. Food Saf. 2021, 16, 5–17. [CrossRef]
32. Fleischmann, C.; Lievenbrück, M.; Ritter, H. Polymers and Dyes: Developments and Applications. Polymers 2015, 7, 717–746. [CrossRef] 31. Nicolai, S.; Tralau, T.; Luch, A.; Pirow, R. A scien f
32. Fleischmann, C.; Lievenbrück, M.; Ritter, H. Polymers and Dyes: Developments and Applications. Polymers 2015, 7, 717–746. [CrossRef] 156 Citation: Abou Elmaaty, T.;
Sayed-Ahmed, K.; Mohamed Ali, R.;
El-Khodary, K.; Abdeldayem, S.A.
Simultaneous Sonochemical
Coloration and Antibacterial
Functionalization of Leather with
Selenium Nanoparticles (SeNPs).
Polymers 2022, 14, 74. https://
doi.org/10.3390/polym14010074 1. Introduction Leather is a durable and flexible material made by tanning animal rawhide and
skin, most commonly cattle hide [1]. It has unique characteristics, including high tensile
strength, elasticity, tear resistance, high porosity, and air/water permeability [2,3]. In
addition, collagen is the main component of natural leather and amino acids are the main
units of collagen. It contains functional groups such as –NH4+, –COO−as well as OH
groups [4]. Collagen of leather can provide ideal conditions such as moisture, temperature,
oxygen, and nutrients required for the rapid growth of bacteria and fungi [5]. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article
distributed
under
the
terms
and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). Leathers have undergone many developments concerning their color and acquired
functionalization such as antibacterial, self-cleaning, and UV protection [6,7]. In addition, 157 https://www.mdpi.com/journal/polymers Polymers 2022, 14, 74. https://doi.org/10.3390/polym14010074 Polymers 2022, 14, 74 diverse dyes and dyeing methods have been utilized for sustainable development in the
leather industry [8]. The most important of these developments is the use of nanotechnol-
ogy, which has an eco-friendly effect on the environment [9,10]. Nowadays, coloration and functionalization of leather surfaces with organic nanopar-
ticles have received considerable attention [7,11–16]. The multifunctional features of leather,
such as antimicrobial and UV radiation protection, as well as self-cleaning capabilities,
have improved leather’s adaptability [1,6,9,17–19]. Recently, many studies have reported the use of AgNPs to functionalize leather sur-
faces due to their antibacterial properties [5,20–23]. In addition, silicon nanoparticles were
used with polyurethanes and polyacrylates to produce leather with high water vapor per-
meability, good mechanical properties as well as good thermal properties [24,25]. Addition-
ally, Ag-TiO2 nanoparticles were deposited on the leather surface to obtain antimicrobial
properties and improve photocatalytic activity [26,27]. As well, TiO2-SiO2 nanocomposite
was used to enhance the performance such as colorfastness and adhesion [28]. From what was mentioned previously, we found that the multifunctional properties of
leathers improved by different kinds of nanoparticles, which were investigated to evaluate
their antimicrobial, mechanical, and thermal properties, as well as UV protection and
photocatalytic activity. However, the use of NPs as colorants and finishing agents in leather
processing has not been sufficiently investigated. Moreover, leather finishing with NPs
requires multiple steps, a long reaction time, and the use of more hazardous chemical
agents. 2.1. Materials Salted wet cowhide leathers were obtained from the Timex Tannery Company, Cairo,
Egypt, and then tanned, as reported by Ali et al. [39]. As for chemicals; sodium hydrogen
selenite, ascorbic acid, and polyvinylpyrrolidone (PVP) were purchased from Loba Chemie,
Mumbai, India. Other chemicals were of commercial grade. 1. Introduction As a result, there is a great demand for a simple and environmentally safe process
for the incorporation of nanoparticles into leather surfaces without the use of hazardous
chemical ingredients. One of the most effective nanoparticles in this context is selenium
nanoparticles (SeNPs) which have excellent coloring capability [29–32], antimicrobial,
antitumor, antioxidant, and antibiofilm properties [33–37]. Moreover, SeNPs exhibit low
cytotoxicity [29,38]. In this work we have developed a simple technique to obtain simultaneous coloration
and functionalization of leather via the incorporation of SeNPs into the leather under study
utilizing an ultrasonic technique. The SeNPs was synthesized through a green process and
used as a colorant without any harmful additives, achieving outstanding color fastness
and antibacterial properties. To the best of our knowledge, the utilization of SeNPs for coloration and functional-
ization of leather has not been reported elsewhere. 2. Materials and Methods 2.1. Materials 2.5.4. Exhaustion of SeNPs onto Leather The UV/Vis spectrum of the SeNPs colloidal solutions before and after leather treat-
ment was measured by UV/Vis spectrophotometer (Alpha-1860, Noble, IN, USA) to
evaluate the SeNPs exhaustion. 2.5.1. SEM and EDX Analysis The surface morphology of the leather samples, including blank samples and those
treated with SeNPs, was observed using a scanning electron microscope (JEOL JSM-6510LB,
Tokyo, Japan) with attached energy dispersive X-ray spectrum (EDX) unit. 2.5.3. Colorimetric Study The color uptake, expressed as the color strength (K/S) of blank leather and leather/SeNPs,
was determined using a spectrophotometer (CM3600A; Konica Minolta, Japan). K/S values
were evaluated at the wavelength of maximum absorption (λmax) of the color’s reflectance
curve at 360 nm. The total color difference (ΔE) was represented in terms of CIE LAB color
space data. It was calculated using (Equation (1)), ΔE =
(L ∗2 −L∗1)2 + (a ∗2 −a∗1)2 + (b ∗2 −b∗1) 2
(1) (1) where, ΔE is the total difference between blank leather and leather/SeNPs, L* is the
lightness from black (0) to white (100), a* is the red (+)/green (−) ratio and b* is the yellow
(+)/blue (−) ratio. 2.5.2. Raman Spectroscopy Analysis The types of bonds found in the blank or treated leather were detected using a confocal
Raman microscope (Jasco NRS-4500, Tokyo, Japan) at the range of 200–1600 cm−1. For both
Raman data acquisition and processing, Jasco spectroscopy suite software was used. 2.2. Green Synthesis of SeNPs 2.2. Green Synthesis of SeNPs SeNPs were prepared via redox reaction as reported by Abou Elmaaty et al. [32] with
an improved modification. Sodium hydrogen selenite was used at different concentrations
(50 mM and 100 mM) as a precursor for SeNPs. Polyvinylpyrrolidone (PVP) was dissolved
in sodium hydrogen selenite solution at a concentration of 12 g/100 mL to maintain the
nanoparticles stability. Then, ascorbic acid at varied concentrations of 100 mM and 200 mM
was mixed with this mixture at a volume ratio of 1:1 and molar ratio of 2:1 (ascorbic
acid: NaHSeO3). The color change from colorless to dark orange indicated the formation
of SeNPs. 2.3. Implementation of Selenium Nanoparticles (SeNPs) into Leather Before treatment, the leather samples were scoured with nonionic detergent (3%) on a
weight of leather at 50 ◦C for 15 min at a liquor ratio of 1:50 to remove impurities. Then, 158 Polymers 2022, 14, 74 the leather sample was rinsed with distilled water, followed by treatment into a solution of
SeNPs at a liquor ratio (LR) of 1:50 by ultrasonic water bath. The treatment was carried
out at varied temperatures (at room, 40, 50, 60, 65 and 70 ◦C), different periods (30, 60, 90
and 120 min) as well as different pH values (3,4,5,6,7,8) and two concentrations of SeNPs of
(25 and 50) mmol/l. Subsequently, the leather sample was removed, rinsed with nonionic
detergent (2%) on weight of leather at 40 ◦C for 15 min at a liquor ratio of 1:50, rinsed with
distilled water, and dried in an oven at 50 ◦C for 25 min. Finally, the leather samples were
placed in a desiccator to remove traces of water [4]. 2.4. Transmission Electron Microscopy (TEM) Analysis SeNPs characteristics included morphology and size were characterized by transmis-
sion electron microscope (JEOL, JEM 2100F) at 200 kV. A drop of the NPs colloidal solution
was loaded onto a 400-mesh copper grid with an amorphous carbon film, and water was
evaporated in air at room temperature. 2.5. Leather Characterization 2.5.6. Cytotoxicity Test of Leather/SeNPs Cytotoxicity was evaluated on human normal melanocyte cell line (HFB4) using MTT
assay [45]. The leather/SeNPs dyed under the optimum conditions were sterilized and
cut. Then it was plated on the six-well tissue culture plate. This plate was inoculated with
cells and incubated for 24 h at 37 ◦C. The cell monolayer was washed twice using washing
media after growth medium decantation. The cytotoxicity physical signs were checked
in the tested cells. The tissue was picked up, and then 20 μL of 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide dye (MTT) dissolved in phosphate buffer saline at a
concentration of 5 mg/mL was added to each well with shaking for 5 min and incubated at
37 ◦C and 5% CO2 for 5 h. The formazan was formed as MTT metabolite and resuspended
in 200 μL dimethyl sulfoxide with shaking for 5 min. The absorbance was measured at
560 nm and the background was read at 620 nm, and then subtracted. 2.5.7. Antibacterial Activity The antibacterial activity test for leather/SeNPs was conducted according to AATCC
(147-2004) [46]. The bacterial strains were spread on the media using a sterile cotton swab. Antibacterial activity was evaluated against G+ve bacteria (Bacillus cereus) and G-ve bacte-
ria (Escherichia coli, Salmonella typhi and Pseudomonas aeruginosa). Leather/SeNPs samples
were cut to be square in shape and measuring 10 × 10 mm, while standard drugs such as
tetracycline and ciprofloxacin were loaded in the disks to compare between their antibacte-
rial activity and that of the tested leather/SeNPs. The petri dishes were then incubated at
37 ◦C for 24 h. while the growth inhibition zone diameters (mm) were determined. 2.5.5. Physical Properties of Leather The color fastness of blank leather and leather/SeNPs was tested according to the
following standard methods. They were determined using the AATCC (61-1996) [40],
(8-1996) [41], and (16-2004) [42] tests for washing, rubbing, and light fastness, respectively. The tensile strength tests of blank leather and leather/SeNPs were performed accord-
ing to ASTM D638-14 [43], using a tensile testing machine (Zwick Z010, Staufenberg,
Germany). Additionally, the durability to washing was evaluated according to AATCC
61(2A)-1996 [44] after five washing cycles. 159 Polymers 2022, 14, 74 2.5.6. Cytotoxicity Test of Leather/SeNPs 2.5.8. Statistical Analysis All tests have been performed by taking the average of three (samples) readings. The
standard error of the mean was calculated according to the equation given below and
found to be + (−) 0.1 SEX = S/√n where S = sample standard deviation, and n = the number of observations of the sample 3.1. Transmission Electron Microscopy (TEM) Analysis 3.1. Transmission Electron Microscopy (TEM) Analysis 3.1. Transmission Electron Microscopy (TEM) Analysis The morphology and size of the synthesized SeNPs at different concentrations (25,
50 mM) were characterized using a transmission electron microscope. SeNPs prepared at
the concentrations of 25 and 50 mM ranged from 36–77 nm and 41 to 149 nm, indicating
the increase in average size with the increase in SeNPs concentration. The obtained TEM
micrographs revealed that SeNPs were spherical in shape and well dispersed in the colloidal
solution, especially that prepared at the low concentration. Furthermore, no agglomeration
or deformation of SeNPs was observed, as displayed in Figure 1a,b. The width of bins in
SeNPs histogram was 20 nm, as illustrated in Figure 1c, and these bins were centered at 40,
60, 80, 100, 120 and 140 nm. For example, all particles with diameters between 70 nm and
90 nm were considered together as particles with a diameter of 80 nm. In the case of low
concentration (25 mM), SeNPs around 60 nm in size exhibited the highest percentage value
of 76.06. On the other hand, the majority of SeNPs at the high concentration of 50 mM were
from 70 to 110 nm, which confirmed the increase in SeNPs diameter and polydispersity as
the increase in NPs concentration. 160 Polymers 2022, 14, 74 Figure 1. TEM images of SeNPs synthesized at different concentrations of (a) 25 mM and (b) 50 mM
in addition to (c) the size distribution histogram of the prepared SeNPs. Figure 1. TEM images of SeNPs synthesized at different concentrations of (a) 25 mM and (b) 50 mM
in addition to (c) the size distribution histogram of the prepared SeNPs. Over and above, many SeNPs at the low concentration had a hollow shape (ring-
shaped), while SeNPs at the concentration of 50 mM were ordinary solid spherical particles,
illustrating that SeNPs specific surface area increased with the decrease in SeNPs concen-
tration due to the reduction in NPs average size and the change in their morphology as
shown in Figure 1. 3.2. Leather Characterization before and after Functionalization 3.2.1. Scanning Electron Microscopy (SEM) Analysis 3.2.1. Scanning Electron Microscopy (SEM) Analysis The micrographs obtained from SEM analysis showed that the surface of untreated
leather is typically clear with clean scales and smooth longitudinal collagen fibers as shown
in Figure 2a. On the other hand, the SEM micrographs of leather/SeNPs revealed that the
leather surface was coated sufficiently with a layer of SeNPs without aggregation and the
SeNPs were distributed well on the leather surface as displayed in Figure 2b,c. Moreover,
the chemical elements analysis of leather/SeNPs confirmed the presence of SeNPs on the
surface after the dyeing process as shown in Figure 2d. 161 Polymers 2022, 14, 74 Figure 2. SEM micrographs of (a) blank leather and (b,c) leather dyed with SeNPs under the optimum
conditions at different scales as well as (d) EDX spectrum of the dyed leather. Figure 2. SEM micrographs of (a) blank leather and (b,c) leather dyed with SeNPs under the optimum
conditions at different scales as well as (d) EDX spectrum of the dyed leather. 3.2.3. Colorimetric Study Table 1 showed the (L*, a*, b*, C*, h, K/S) values of blank leather and leather/SeNPs. The results revealed that the K/S value of leather/SeNPs was significantly higher than the
blank leather. The (L*, a*, b*) values for leather/SeNPs were varied considerably from blank
leather. The color of blank leather is cream-white with a relatively high L* value (74.9),
low a* value (8.6), and low b* value (17.7). The lightness (L*) value of leather/SeNPs was
decreased due to the presence of SeNPs, resulting in the coloration of leather. The a* values
of leather/SeNPs was increased, the increment in a* value indicated more redness color for
the treated leather. The rise in b* value of leather/SeNPs was observed as yellow/brownish,
which confirming the coverage of leather with SeNPs. It can be concluded from the equation
1 and Table 1 that the total color difference (ΔE) between the two leathers is ~42. Table 1. Color strength and colorimetric data of blank leather and leather/SeNPs. Table 1. Color strength and colorimetric data of blank leather and leather/SeNPs. Type
Sample
Color Parameters
L*
a*
b*
C*
h
K/S
Blank leather
74.9
8.6
17.7
22.5
64.0
1.7
Leather/SeNPs
37.1
26.1
24.8
19.6
43.5
18.7
Effect of Treatment pH on Color Strength (K/S) Table 1. Color strength and colorimetric data of blank leather and leather/SeNPs. Type
Sample
Color Parameters
L*
a*
b*
C*
h
K/S
Blank leather
74.9
8.6
17.7
22.5
64.0
1.7
Leather/SeNPs
37.1
26.1
24.8
19.6
43.5
18.7
Effect of Treatment pH on Color Strength (K/S) Table 1. Color strength and colorimetric data of blank leather and leather/SeNPs. Table 1. Color strength and colorimetric data of blank leather and leather/SeNPs. Type
Sample
Color Parameters
L*
a*
b*
C*
h
K/S
Blank leather
74.9
8.6
17.7
22.5
64.0
1.7
Leather/SeNPs
37.1
26.1
24.8
19.6
43.5
18.7
Effect of Treatment pH on Color Strength (K/S) Effect of Treatment pH on Color Strength (K/S) To investigate the color changes of leather/SeNPs at different pH values under an
ultrasonic water bath, Figure 4 showed the effect of pH on K/S of leather/SeNPs. The pH
was adjusted from 3 to 8 to reflect the different colors expressed in leather/SeNPs. As
shown, the maximum K/S value appeared at pH 6; this might be caused by overcrowding of
SeNPs molecules into the leather matrix in this pH value due to the ultrasonic power [48]. Effect of Treatment Temperature on Color Strength (K/S) Figure 5 demonstrated the K/S values of leathers/SeNPs at different temperatures. The results showed that the maximum K/S value was found to be 18.7 at 65 ◦C. In addition,
when the temperature was increased above 65 ◦C, the K/S value was simultaneously
decreased. This may be attributed to the tough muscle fiber of leather which did not allow
the nanoparticles to enter the leather matrix, causing a lower SeNPs uptake. Moreover,
high temperatures can cause the leather to shrink, harden, stiffen, and become brittle. It
can be concluded that the temperature of 65 ◦C was set to be the optimum temperature for
binding SeNPs into the leather [48]. 3.2.2. Raman Analysis Raman analysis was conducted to confirm the deposition of SeNPs on the dyed leather
surface as shown in Figure 3. The comparison between the spectra of leather surface before
and after treatment with SeNPs was investigated in the range of 200–1600 cm−1. After
treatment two intense peaks at 230 and 295 cm−1 appeared as a result of SeNPs deposition. Similar results were obtained by Abou Elmaaty et al. [29] who treated the polypropylene
fabric with SeNPs and found a peak around 236 cm−1 after SeNPs treatment, while Lukács
et al. [47] found a pronounced peak at 292 cm−1 as an indicator for the presence of SeNPs
that confirms the successful deposition of SeNPs on the treated leather surface.
,QWHQVLW\
:DYHQXPEHU FP
8QWUHDWHG
/HDWKHU6H13V
? ? ? Figure 3. Raman spectra of untreated leather and leather/SeNPs−. 162 Figure 3. Raman spectra of untreated leather and leather/SeNPs−. Figure 3. Raman spectra of untreated leather and leather/SeNPs−. Polymers 2022, 14, 74 3.2.3. Colorimetric Study The results indicated that the K/S of leather/SeNPs can be fine-tuned by controlling the pH. According to Lee and Kim et al. [49], the correlation between SeNPs and the leather can be
attributed to pH of treatment bath [48]. Effect of Treatment Time on Color Strength (K/S) Figure 6 exhibited the effect of treatment time on SeNPs uptake by leather. As depicted,
the K/S was increased upon increasing time of treatment. The optimum K/S value of 18.7
was achieved at 60 min after which, K/S started to decrease with increasing time. The
decrease in K/S as the increase in time from 90 to 120 min may be due to the aggregation of
SeNPs as a result of SeNPs accumulation upon the leather surface [48]. 163 Polymers 2022, 14, 74 1.7
7.6
8.7
10.3
11.7
9.7
8.2
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
Blank
3
4
5
6
7
8
K/S
pH
K/S
Figure 4. Effect of pH on color strength (K/S) of leather/SeNPs. 6.4
11.2
11.5
13.1
18.7
12.2
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
18.0
20.0
At
room
40
50
60
65
70
K/S
Temperature (ȤC)
K/S
Figure 5. Effect of treatment temperature on color strength (K/S) of leather/SeNPs. 1.7
7.6
8.7
10.3
11.7
9.7
8.2
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
Blank
3
4
5
6
7
8
K/S
pH
K/S
Figure 4. Effect of pH on color strength (K/S) of leather/SeNPs. 1.7
7.6
8.7
10.3
11.7
9.7
8.2
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
Blank
3
4
5
6
7
8
K/S
pH
K/S
Figure 4. Effect of pH on color strength (K/S) of leather/SeNPs. Figure 4. Effect of pH on color strength (K/S) of leather/SeNPs. 6.4
11.2
11.5
13.1
18.7
12.2
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
18.0
20.0
At
room
40
50
60
65
70
K/S
Temperature (ȤC)
K/S
Figure 5. Effect of treatment temperature on color strength (K/S) of leather/SeNPs. 6.4
11.2
11.5
13.1
18.7
12.2
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
18.0
20.0
At
room
40
50
60
65
70
K/S
Temperature (ȤC)
K/S Figure 5. Effect of treatment temperature on color strength (K/S) of leather/SeNPs. 15.4
18.7
17.7
17.4
0.0
5.0
10.0
15.0
20.0
30 min
60 min
90 min
120 min
K/S
Time (min.)
K/S
Figure 6. Effect of treatment time on color strength (K/S) of leather/SeNPs. Effect of SeNPs Concentration on Color Strength (K/S)
The color strength of the leather/SeNPs was found to be dependent on S Figure 6. Effect of treatment time on color strength (K/S) of leather/SeNPs. 3.2.4. Exhaustion of SeNPs onto Leather The UV/Visible spectrum of SeNPs colloidal solution was measured before and after
leather treatment to evaluate the SeNPs exhaustion by treated leather as shown in Figure 8.
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Figure 8. UV/Visible spectrum of SeNPs before and after leather treatment. Figure 8. UV/Visible spectrum of SeNPs before and after leather treatment. The absorbance of SeNPs was measured in the range of 235 to 700 nm. The absorption
spectrum of SeNPs showed a sharp absorption band at 290 nm before the exhaustion, while
SeNPs spectrum after leather treatment decreased obviously after exhaustion, confirming
the deposition of SeNPs on the treated leather. Effect of Treatment Time on Color Strength (K/S) Effect of SeNPs Concentration on Color Strength (K/S) Effect of SeNPs Concentration on Color Strength (K/S) The color strength of the leather/SeNPs was found to be dependent on SeNPs con-
centration. Upon increasing the concentration of SeNPs, the K/S value was decreased as The color strength of the leather/SeNPs was found to be dependent on SeNPs con-
centration. Upon increasing the concentration of SeNPs, the K/S value was decreased as 164 Polymers 2022, 14, 74 shown in Figure 7. The highest K/S value for the treated leather was monitored at the
lowest concentration. This proved that treating leather with lower concentration of SeNPs
(25 mM) was adequate to achieve the optimum K/S. The aforementioned behavior could be
attributed to the thermal stability of the leather, as it increased at low SeNPs concentration
and decreased at high SeNPs concentration. The improvement in thermal stability could
be recognized as the crosslinking between SeNPs and collagen of leather [4]. 18.7
14.4
0.0
5.0
10.0
15.0
20.0
25
50
K/S
SeNPs Conc. (mM)
K/S
Figure 7. Effect of SeNPs concentrations on color strength (K/S) of leather/SeNPs. 18.7
14.4
0.0
5.0
10.0
15.0
20.0
25
50
K/S
SeNPs Conc. (mM)
K/S Figure 7. Effect of SeNPs concentrations on color strength (K/S) of leather/SeNPs. 3.2.5. Physical Properties of Leather/SeNPs The tensile modulus and elongation at break % values were listed in Table 2. The
obtained results indicated that there were no significant differences about the mechanical
properties of leathers, mainly between the leather/SeNPs compared to the blank leather. 165 Polymers 2022, 14, 74 The color fastness properties of leather/SeNPs were evaluated and the results were tabu-
lated in Table 2. Leather/SeNPs revealed excellent fastness results referring to the chemical
stability of the SeNPs onto the leather surfaces along with long-term durable interactions
between the SeNPs and the leather surface [4]. Table 2. Properties of the Leather and Leather/SeNPs under optimum conditions. Sample
Wash Fastness
Rubbing Fastness
Light
Fastness
Tensile Strength
St. Alt. Dry
Wet
Tensile Modulus,
a MPa
Elongation, b %
Blank leather
-
-
-
-
-
8
36
Leather covered
with SeNPs
5
5
5
4/5
4/5
7
32
Leather covered
with SeNPs after 5
washing cycles
(durability test)
5
5
5
4/5
4/5
7
32
Treatment conditions: SeNPs, (25 mM); Time (60) min; Temp. (65) ◦C; pH 6. a Standard deviation for untreated
leather = 0.5, standard deviation for treated leather = 0.5. b Standard deviation for untreated leather = 0.5, standard
deviation for treated leather = 0.5. The color fastness properties of leather/SeNPs were evaluated and the results were tabu-
lated in Table 2. Leather/SeNPs revealed excellent fastness results referring to the chemical
stability of the SeNPs onto the leather surfaces along with long-term durable interactions
between the SeNPs and the leather surface [4]. The color fastness properties of leather/SeNPs were evaluated and the results were tabu-
lated in Table 2. Leather/SeNPs revealed excellent fastness results referring to the chemical
stability of the SeNPs onto the leather surfaces along with long-term durable interactions
between the SeNPs and the leather surface [4]. Table 2. Properties of the Leather and Leather/SeNPs under optimum conditions. Treatment conditions: SeNPs, (25 mM); Time (60) min; Temp. (65) ◦C; pH 6. a Standard deviation for untreated
leather = 0.5, standard deviation for treated leather = 0.5. b Standard deviation for untreated leather = 0.5, standard
deviation for treated leather = 0.5. 3.2.6. Cytotoxicity of Leather/SeNPs Leather/SeNPs cytotoxicity was evaluated against healthy human melanocyte cell
line (HFB4) using MTT assay. The viability of cells in the presence of leather/SeNPs was
90.67% compared to that of negative control. The average relative cell viability is over
70% [50], indicating the low toxicity of leather/SeNPs toward human skin. 3.2.7. Antibacterial Activity The antibacterial activity of leather/SeNPs was evaluated against four bacterial strains,
including Bacillus cereus as Gram-positive bacteria in addition to Escherichia coli, Pseu-
domonas aeruginosa and salmonella typhi as Gram-negative bacteria. The results revealed
that the leather/SeNPs showed outstanding antibacterial activity against the tested strains
in comparison with standard drugs as listed in Table 3. Leather treated with 25 mM SeNPs
colloidal solution was more effective against Escherichia coli, Pseudomonas aeruginosa and
Bacillus cereus than that treated with high concentration (50 mM) due to the difference in
average size. SeNPs (25 mM) with small average size exhibited higher specific area and in
contact with bacterial cells more than that at the high concentration (50 mM) [51]. There
was no obvious variation in inhibition zone diameters for leather/SeNPs before and after
five washing cycles that confirms the durability of the leather/SeNPs samples at the tested
concentrations. Moreover, the cross-linking between leather and SeNPs as antimicrobial
agents protects leather against laundering and mechanical abrasion that makes the leather
products more durable [22]. Table 3. The inhibition zone (mm) values of leathers/SeNPs treated with different SeNPs concentrations. Substrate
Bacillus
cereus
(G+)
Escherichia
coli
(G−)
Pseudomonas
aeruginosa
(G−)
Salmonella
typhi
(G−)
Leather/SeNPs (25 mM)
20
15
19
16
Leather/SeNPs (50 mM)
18
9
15
18
Leather/SeNPs (25 mM);
after 5 washing cycles
20
14
18
14
Leather/SeNPs (50 mM);
after 5 washing cycles
17
8
15
18
Tetracycline (30 μg)
15
19
16
13
Ciprofloxacin (10 μg)
18
21
17
15 Table 3. The inhibition zone (mm) values of leathers/SeNPs treated with different SeNPs concentrations. 166 Polymers 2022, 14, 74 The antibacterial effect on bacteria may be due to the release of ions or the formation
of reactive oxygen species that lead to DNA damage. Furthermore, the deposited SeNPs
can contact bacteria or fungi cells much easier than colloidal form [22]. 4. Conclusions Leather material was successfully colored and functionalized utilizing selenium
nanoparticles (SeNPs) which were synthesized and deposited simultaneously onto the
leather surface. The SeNPs decorated the leather surface with shining colors, which can
be controlled by adjusting the pH at 6, treatment time for 60 min., treatment temperature
at 65 ◦C and SeNPs concentration of 25 mM. The results showed that a yellow/brown
color was imparted to the leather after the implementation with SeNPs by ultrasound
technique. Moreover, the colored leather samples acquired good color fastness to rub,
wash, and light. Leathers/SeNPs exhibited excellent antibacterial activities against models
of bacteria, including Gram-positive bacteria (Bacillus cereus) and Gram-negative bacteria
(Pseudomonas aeruginosa, Escherichia coli and Salmonella typhi). The results of coloration,
cytotoxicity and antibacterial properties clarified that the SeNPs can be used to impart color
and antibacterial properties to leather material. The proposed methodology emphasized
the effectiveness and applicability of this simple approach to the footwear industry to color
the leather as well as prevent the spread of bacterial infection promoted by humidity, poor
breathability and temperature. Author Contributions: T.A.E. conceived the original idea of this study and participated in design
and coordination as well as manuscript drafting. S.A.A. participated in the manuscript writing and
interpreted the obtained results from colorimetric and mechanical properties study. K.S.-A. prepared
the SeNPs and interpreted the obtained results obtained from TEM, SEM analysis, antimicrobial
and cytotoxicity tests as well as participation in manuscript writing. K.E.-K. was responsible for the
dyeing experiments and leather finishing steps in addition to participation in SeNPs preparation. R.M.A. provided the tanned leather required for this study. All authors have read and agreed to the
published version of the manuscript. Funding: This paper is based upon work supported by Science, Technology & Innovation Funding
Authority (STDF); Egypt under grant (POST GRADUATE SUPPORT GRANT, CALL-1). Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the
corresponding author. Conflicts of Interest: The authors declare no conflict of interest. p
g
5.
Sportelli, M.C.; Picca, R.A.; Paladini, F.; Mangone, A.; Giannossa, L.C.; Franco, C.D.; Gallo, A.L.; Valentini, A.; Sannino, A.;
Pollini, M.; et al. Spectroscopic Characterization and Nanosafety of Ag-Modified Antibacterial Leather and Leatherette. Nanoma-
terials 2017, 7, 203. [CrossRef] [PubMed] 6.
Gurera, D.; Bhushan, B. Fabrication of bioinspired superliquiphobic synthetic leather with self-clea
Colloids Surf. A Physicochem. Eng. Asp. 2018, 545, 130–137. [CrossRef] 7.
Bao, Y.; Feng, C.; Wang, C.; Ma, J.; Tian, C. Hygienic, antibacterial, UV-shielding performance of polyacrylate/ZnO composite
coatings on a leather matrix. Colloids Surf. A Physicochem. Eng. Asp. 2017, 518, 232–240. [CrossRef] y
y
y
p
,
,
[
]
4.
Elsayed, H.; Hasanin, M.; Rehan, M. Enhancement of multifunctional properties of leather surface decorated with silver
nanoparticles (Ag NPs). J. Mol. Struct. 2021, 1234, 130130. [CrossRef] an, B. Fabrication of bioinspired superliquiphobic synthetic leather with self-cleaning and low adhesion.
sicochem. Eng. Asp. 2018, 545, 130–137. [CrossRef] 3.
Su, Y.; Li, P.; Gao, D.; Lyu, B.; Ma, J.; Zhang, J.; Lyu, L. High-efficiency antibacterial and anti-mildew properties under self-
assembly: An environmentally friendly nanocomposite. Adv. Powder Technol. 2021, 32, 2433–2440. [CrossRef] References [CrossRef] 13. Solangi, B.; Nawaz, H.; Solangi, B.; Zehra, B.; Nadeem, U. Preparation of Nano Zinc Oxide and its Application in Leather
as a Retanning and Antibacterial Agent Preparation of Nano Zinc Oxide and its Application in Leather as a Retanning and
Antibacterial Agent. Can. J. Sci. Ind. Res. 2011, 2, 164–170. g
J
14. Liu, J.; Ma, J.; Bao, Y.; Wang, J.; Tang, H.; Zhang, L. Polyacrylate/Surface-Modified ZnO Nanocomposite as Film-Forming Agent
for Leather Finishing. Int. J. Polym. Mater. Polym. Biomater. 2014, 63, 809–814. [CrossRef] 15. Nidhin, D.M.; Rathinam, A.; Sreeram, K.J. GREEN synthesis of monodispersed iron oxide nanoparticles for leather finishing. J. Am. Leather Chem. Assoc. 2014, 109, 184–188. 16. Chen, Y.; Fan, H.; Shi, B. Nanotechnologies for leather manufacturing: A review. J. Am. Leather Chem. A g
g
J
17. Yorgancioglu, A.; Bayramoglu, E.; Renner, M. Preparation of Antibacterial Fatliquoring Agents Containing Zinc Oxide Nanoparti-
cles for Leather Industry. J. Am. Leather Chem. Assoc. 2019, 114, 171–179. y J
,
,
18. Petica, A.; Gaidau, C.; Ignat, M.; Sendrea, C.; Anicai, L. Doped TiO2 nanophotocatalysts for leather surface finishing with
self-cleaning properties. J. Coat. Technol. Res. 2015, 12, 1153–1163. [CrossRef] 19. Ma, J.; Zhang, X.; Bao, Y.; Liu, J. A facile spraying method for fabricating superhydrophobic leather coating. Colloids Surf. A
Physicochem. Eng. Asp. 2015, 472, 21–25. [CrossRef] y
g
p
20. Velmurugan, P.; Lee, S.-M.; Cho, M.; Park, J.-H.; Seo, S.-K.; Myung, H.; Bang, K.-S.; Oh, B.-T. Antibacterial activity of silver
nanoparticle-coated fabric and leather against odor and skin infection causing bacteria. Appl. Microbiol. Biotechnol. 2014,
98, 8179–8189. [CrossRef] [PubMed] 21. Velmurugan, P.; Cho, M.; Lee, S.-M.; Park, J.-H.; Bae, S.; Oh, B.-T. Antimicrobial fabrication of cotton fabric and leather using
green-synthesized nanosilver. Carbohydr. Polym. 2014, 106, 319–325. [CrossRef] [PubMed] 22. Xiang, J.; Ma, L.; Su, H.; Xiong, J.; Li, K.; Xia, Q.; Liu, G. Layer-by-layer assembly of antibacterial composite coating for leather
with cross-link enhanced durability against laundry and abrasion. Appl. Surf. Sci. 2018, 458, 978–987. [CrossRef] 23. Zohreh, M.; Ani, I.; Peiman, V. Antibacterial improvement of leather by surface modification using
nanoparticles application. J. Sci. Technol. 2014, 5, 1–15. p
pp
24. Elsayed, H.; Attia, R.; Mohamed, O.; Haroun, A.; El-Sayed, N. Preparation of Polyurethane Silicon Oxide Nanomaterials as a
Binder in Leather Finishing. Fibers Polym. 2018, 19, 832–842. [CrossRef] g
y
25. Mohamed, O.; Elsayed, H.; Attia, R.; Haroun, A.; El-Sayed, N. References 1. Velmurugan, P.; Shim, J.; Bang, K.-S.; Oh, B.-T. Gold nanoparticles mediated coloring of fabrics and leather for antibacterial
activity. J. Photochem. Photobiol. B Biol. 2016, 160, 102–109. [CrossRef] 3. Su, Y.; Li, P.; Gao, D.; Lyu, B.; Ma, J.; Zhang, J.; Lyu, L. High-efficiency antibacterial and anti-milde
assembly: An environmentally friendly nanocomposite. Adv. Powder Technol. 2021, 32, 2433–2440. [Cro u, Y.; Li, P.; Gao, D.; Lyu, B.; Ma, J.; Zhang, J.; Lyu, L. High efficiency antibacterial and anti mildew prop
ssembly: An environmentally friendly nanocomposite. Adv. Powder Technol. 2021, 32, 2433–2440. [CrossRef] 4. Elsayed, H.; Hasanin, M.; Rehan, M. Enhancement of multifunctional properties of leather surface decorated with silver
nanoparticles (Ag NPs). J. Mol. Struct. 2021, 1234, 130130. [CrossRef] p
g
5. Sportelli, M.C.; Picca, R.A.; Paladini, F.; Mangone, A.; Giannossa, L.C.; Franco, C.D.; Gallo, A.L.; Valentini, A.; Sannino, A.;
Pollini, M.; et al. Spectroscopic Characterization and Nanosafety of Ag-Modified Antibacterial Leather and Leatherette. Nanoma-
terials 2017, 7, 203. [CrossRef] [PubMed] 6. Gurera, D.; Bhushan, B. Fabrication of bioinspired superliquiphobic synthetic leather with self-cleaning and low adhesion. Colloids Surf. A Physicochem. Eng. Asp. 2018, 545, 130–137. [CrossRef] 167 Polymers 2022, 14, 74 8. Khan, S.A.; Shahid, S.; Kanwal, S.; Rizwan, K.; Mahmood, T.; Ayub, K. Synthesis of novel metal complexes of 2-((phenyl (2-(4-
sulfophenyl) hydrazono) methyl) diazenyl) benzoic acid formazan dyes: Characterization, antimicrobial and optical properties
studies on leather. J. Mol. Struct. 2019, 1175, 73–89. [CrossRef] J
9. Liu, G.; Li, K.; Luo, Q.; Wang, H.; Zhang, Z. PEGylated chitosan protected silver nanoparticles as water-borne coating for leather
with antibacterial property. J. Colloid Interface Sci. 2017, 490, 642–651. [CrossRef] [PubMed] p
p
y
f
10. Tamil Selvi, A.; Aravindhan, R.; Madhan, B.; Raghava Rao, J. Studies on the application of natura
seeds for dyeing and finishing of leather. Ind. Crop. Prod. 2013, 43, 84–86. [CrossRef] l Selvi, A.; Aravindhan, R.; Madhan, B.; Raghava Rao, J. Studies on the application of natural dye
s for dyeing and finishing of leather. Ind. Crop. Prod. 2013, 43, 84–86. [CrossRef] 11. Gaidau, C.; Ignat, M.; Iordache, O.; Madalina, L.; Piticescu, R.; Ditu, L.-M.; Ionescu, M. ZnO Nanoparticles for Antimicrobial
Treatment of Leather Surface. Revista De Chimie 2018, 69, 767–771. [CrossRef] ,
,
[
]
12. Erciyes, A.; Ocak, B. Physico-mechanical, thermal, and ultraviolet light barrier properties of collagen hydrolysate films from
leather solid wastes incorporated with nano TiO2. Polym. Compos. 2019, 40, 4716–4725. References Preparation of acrylic silicon dioxide nanoparticles as a binder for
leather finishing. Adv. Polym. Technol. 2018, 37, 3276–3286. [CrossRef] 26. Carvalho, I.; Ferdov, S.; Mansilla, C.; Marques, S.M.; Cerqueira, M.A.; Pastrana, L.M.; Henriques, M.; Gaidau, C.; Ferreira, P.;
Carvalho, S. Development of antimicrobial leather modified with Ag–TiO2 nanoparticles for footwear industry. Sci. Technol. Mater. 2018, 30, 60–68. [CrossRef] 27. Gaidau, C.; Petica, A.; Ignat, M.; Iordache, O.; Ditu, L.-M.; Ionescu, M. Enhanced photocatalysts based on Ag-TiO2 and Ag-N-TiO2
nanoparticles for multifunctional leather surface coating. Open Chem. 2016, 14, 383–392. [CrossRef] 28. Kaygusuz, M.; Meyer, M.; Aslan, A. The Effect of TiO2-SiO2 Nanocomposite on the Performance Characteristics of Leather. Mater. Res. Ibero-Am. J. Mater. 2017, 20, 1103–1110. [CrossRef] 29. AbouElmaaty, T.; Abdeldayem, S.A.; Ramadan, S.M.; Sayed-Ahmed, K.; Plutino, M.R. Coloration and Multi-Functionalization of
Polypropylene Fabrics with Selenium Nanoparticles. Polymers 2021, 13, 2483. [CrossRef] 30. Abou Elmaaty, T.M.; Abdeldayem, S.A.; Elshafai, N. Simultaneous Thermochromic Pigment Printin
Finishing of Cotton Fabrics for Smart Childrenswear. Cloth. Text. Res. J. 2020, 38, 182–195. [CrossRe 30. Abou Elmaaty, T.M.; Abdeldayem, S.A.; Elshafai, N. Simultaneous Thermochromic Pigment Printing an y
y
g
g
Finishing of Cotton Fabrics for Smart Childrenswear. Cloth. Text. Res. J. 2020, 38, 182–195. [CrossRef] 31. Razmkhah, M.; Montazer, M.; Bashiri Rezaie, A.; Rad, M.M. Facile technique for wool coloration via locally forming of nano
selenium photocatalyst imparting antibacterial and UV protection properties. J. Ind. Eng. Chem. 2021, 101, 153–164. [CrossRef] 31. Razmkhah, M.; Montazer, M.; Bashiri Rezaie, A.; Rad, M.M. Facile technique for wool coloration via locally forming of nano
selenium photocatalyst imparting antibacterial and UV protection properties. J. Ind. Eng. Chem. 2021, 101, 153–164. [CrossRef]
32. Elmaaty, T.M.A.; Raouf, S.; Sayed-Ahmed, K. Novel One Step Printing and Functional Finishing of Wool Fabric Using Selenium 31. Razmkhah, M.; Montazer, M.; Bashiri Rezaie, A.; Rad, M.M. Facile technique for wool coloration via locally forming of nano
selenium photocatalyst imparting antibacterial and UV protection properties. J. Ind. Eng. Chem. 2021, 101, 153–164. [CrossRef]
32. Elmaaty, T.M.A.; Raouf, S.; Sayed-Ahmed, K. Novel One Step Printing and Functional Finishing of Wool Fabric Using Selenium
Nanoparticles. Fibers Polym. 2020, 21, 1983–1991. [CrossRef] p
y
p
g
p
p
p
g
32. Elmaaty, T.M.A.; Raouf, S.; Sayed-Ahmed, K. Novel One Step Printing and Functional Finishing of Wool Fabric Using Selenium
Nanoparticles. Fibers Polym. 2020, 21, 1983–1991. [CrossRef] 32. Elmaaty, T.M.A.; Raouf, S.; Sayed-Ahmed, K. Novel One Step Prin
Nanoparticles. p
y
y
35.
Kong, H.; Yang, J.; Zhang, Y.; Fang, Y.; Nishinari, K.; Phillips, G.O. Synthesis and antioxidant propertie
selenium nanoparticles. Int. J. Biol. Macromol. 2014, 65, 155–162. [CrossRef] References Fibers Polym. 2020, 21, 1983–1991. [CrossRef] 33. Jia, X.; Liu, Q.; Zou, S.; Xu, X.; Zhang, L. Construction of selenium nanoparticles/β-glucan composites for enhancement of the
antitumor activity. Carbohydr. Polym. 2015, 117, 434–442. [CrossRef] 34. Chen, W.; Li, Y.; Yang, S.; Yue, L.; Jiang, Q.; Xia, W. Synthesis and antioxidant properties of chitosan and carboxymethyl
chitosan-stabilized selenium nanoparticles. Carbohydr. Polym. 2015, 132, 574–581. [CrossRef] p
y
y
35. Kong, H.; Yang, J.; Zhang, Y.; Fang, Y.; Nishinari, K.; Phillips, G.O. Synthesis and antioxidant properties of gum arabic-stabilized
selenium nanoparticles. Int. J. Biol. Macromol. 2014, 65, 155–162. [CrossRef] 35. Kong, H.; Yang, J.; Zhang, Y.; Fang, Y.; Nishinari, K.; Phillips, G.O. Synthesis a
selenium nanoparticles. Int. J. Biol. Macromol. 2014, 65, 155–162. [CrossRef] 168 Polymers 2022, 14, 74 36. Wang, Q.; Barnes, L.-M.; Maslakov, K.I.; Howell, C.A.; Illsley, M.J.; Dyer, P.; Savina, I.N. In situ synthe
nanoparticles on cationized cellulose fabrics for antimicrobial application. Mater. Sci. Eng. C 2021, 121, es, L.-M.; Maslakov, K.I.; Howell, C.A.; Illsley, M.J.; Dyer, P.; Savina, I.N. In situ synthesis of silver or seleniu
n cationized cellulose fabrics for antimicrobial application. Mater. Sci. Eng. C 2021, 121, 111859. [CrossRef] 37. Shakibaie, M.; Forootanfar, H.; Golkari, Y.; Mohammadi-Khorsand, T.; Shakibaie, M.R. Anti-biofilm activity of biogenic selenium
nanoparticles and selenium dioxide against clinical isolates of Staphylococcus aureus, Pseudomonas aeruginosa, and Proteus
mirabilis. J. Trace Elem. Med. Biol. 2015, 29, 235–241. [CrossRef] 37. Shakibaie, M.; Forootanfar, H.; Golkari, Y.; Mohammadi-Khorsand, T.; Shakibaie, M.R. Anti-biofilm activity of biogenic selenium nanoparticles and selenium dioxide against clinical isolates of Staphylococcus aureus, Pseudomonas aeruginosa, and Proteus
mirabilis. J. Trace Elem. Med. Biol. 2015, 29, 235–241. [CrossRef] 38. Biswas, D.P.; O’Brien-Simpson, N.M.; Reynolds, E.C.; O’Connor, A.J.; Tran, P.A. Comparative study of novel in situ decorated
porous chitosan-selenium scaffolds and porous chitosan-silver scaffolds towards antimicrobial wound dressing application. J. Colloid Interface Sci. 2018, 515, 78–91. [CrossRef] [PubMed] f
39. Nagar, R. Replacement of Lime with Sodium Hydroxide in Leather Tanning. Environ. Sci. 2020, 15, 40. American Association of Textile Chemists and Colorists. AATTCC 61-1996: Colorfastness to Laundering: Accelerated; American
Association of Textile Chemists and Colorists: Research Triangle Park, NC, USA, 1996. ation of Textile Chemists and Colorists. AATTCC 61-1 40. American Association of Textile Chemists and Colorists. AATTCC 61-1996: Colorfastness to Laund
Association of Textile Chemists and Colorists: Research Triangle Park, NC, USA, 1996. g
41. American Association of Textile Chemists and Colorists. References AATCC Test Method 8-1996, Colorfastness to Crocking; American Association
of Textile Chemists and Colorists: Research Triangle Park, NC, USA, 1996. g
42. American Association of Textile Chemists and Colorists. AATCC16-2004: Colorfastness to Light; American Association of Textile
Chemists and Colorists: Research Triangle Park, NC, USA, 2004. g
national. ASTM D638-14, Standard Test Method for Tensile Properties of Plastics; ASTM International: West Conshoh
15. 43. ASTM International. ASTM D638-14, Standard Test Method for Tensile Properties of Plastics; ASTM Internatio
PA, USA, 2015. ,
,
44. American Association of Textile Chemists and Colorists. AATCC 61(2A): Colorfastness to Laundering; American Association of
Textile Chemists and Colorists: Research Triangle Park, NC, USA, 1996. 45. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods 1983, 65, 55–63. [CrossRef] [
]
46. American Association of Textile Chemists and Colorists. AATTCC 147-2004: Antibacterial Activity Assessment of Textile Materials;
American Association of Textile Chemists and Colorists: Research Triangle Park, NC, USA, 2010. American Association of Textile Chemists and Colorists. AATTCC 147-2004: Antibacterial Activity Assessment o
A
i
A
i
i
f T
il Ch
i
d C l
i
R
h T i
l P
k NC USA 2010 46. American Association of Textile Chemists and Colorists. AATTCC 147-2004: Antibacterial Activity Asses rican Association of Textile Chemists and Colorists. AATTCC 147-2004: Antibacterial Activity Assessment of Te
rican Association of Textile Chemists and Colorists: Research Triangle Park, NC, USA, 2010. American Association of Textile Chemists and Colorists: Research Triangle Park, NC, USA, 2010. 47. Lukács, R.; Veres, M.; Shimakawa, K.; Kugler, S. On photoinduced volume change in amorphous selenium: Quantum chemical
calculation and Raman spectroscopy. J. Appl. Phys. 2010, 107, 073517. [CrossRef] 48. Velmurugan, P.; Vedhanayakisri, K.A.; Park, Y.-J.; Jin, J.-S.; Oh, B.-T. Use of Aronia melanocarpa Fruit Dye Combined with Silver
Nanoparticles to Dye Fabrics and Leather and Assessment of Its Antibacterial Potential Against Skin Bacteria. Fibers Polym. 2019,
20, 302–311. [CrossRef] 49. Lee, Y.-H.; Kim, H.-D. Dyeing properties and colour fastness of cotton and silk fabrics dyed with Cassia tora L. extract. Fibers
Polym. 2004, 5, 303–308. [CrossRef] 50. Kangwansupamonkon, W.; Lauruengtana, V.; Surassmo, S.; Ruktanonchai, U. Antibacterial effect of apatite-coated titanium
dioxide for textiles applications. Nanomed. Nanotechnol. Biol. Med. 2009, 5, 240–249. [CrossRef] Kangwansupamonkon, W.; Lauruengtana, V.; Surassmo, S.; Ruktanonchai, U. Antibacterial eff
dioxide for textiles applications. Nanomed. Nanotechnol. Biol. Med. 2009, 5, 240–249. References [CrossRef] g
p
,
;
g
,
;
,
;
,
p
dioxide for textiles applications. Nanomed. Nanotechnol. Biol. Med. 2009, 5, 240–249. [CrossRef] g
p
g
ide for textiles applications. Nanomed. Nanotechnol. Biol. Med. 2009, 5, 240–249. [CrossRef] 51. Inam, M.; Foster, J.C.; Gao, J.; Hong, Y.; Du, J.; Dove, A.P.; O’Reilly, R.K. Size and shape affects the
quaternized nanoparticles. J. Polym. Sci. Part A Polym. Chem. 2019, 57, 255–259. [CrossRef] 169 MDPI
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https://openalex.org/W3095680431
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https://www.nature.com/articles/s41598-020-75543-2.pdf
|
English
| null |
A novel classification regression method for gridded electric power consumption estimation in China
|
Scientific reports
| 2,020
|
cc-by
| 7,777
|
A novel classification regression
method for gridded electric power
consumption estimation in China
OPEN In contrast, gridded EPC data are
more efficient in fine scales research. Therefore, developing a convenient and reliable approach for fine scale
estimation of EPC is urgently needed. Remotely sensed nighttime light (NTL) has long been considered as a direct measure of socio-economic
development, and has served as primary data for a wide range of human-based research1,2, such as population
modelling3,4, GDP estimation5,6, urban expansion mapping7,8, and EPC estimation9,10. Many studies have dem-
onstrated a strong correlation between NTL and EPC at multiple levels, and consequently regression models
for such estimation have been built1,9,11–15. However, the relationship between NTL and EPC varies across areas,
owing to the local socioeconomic diversity16. In other words, the relationship between NTL and EPC is spatial
non-stationary17, which tends to be overlooked in existing literature, where global models are often used10,18–20. Thus, from the spatial non-stationary perspective, local regression would be more suitable for EPC estimation. As pointed out by Shi et al.21, a single regression model for all administrative units is susceptible to incorrect
estimation and an appropriate subdivision of administrative units could improve the estimation accuracy22. Some researchers have divided their study area into several sub-regions based on factors, such as geographic
location12,23, income consideration24, and city function17. Yet, such classifications are limited in terms of accuracy,
as they do not fully consider the complexity of interactions between NTL and EPC24. For instance, a classifica-
tion based on economic factors may not sufficiently distinct the relationship between NTL and EPC, given that
cities at the same level of economic development can have different energy consumption patterns and NTL levels
depending on their key industries17. Hence, exploring an appropriate classification basis from the perspective
of the relationship between EPC and NTL would provide a better understanding and a new insight for gridded
EPC estimation. 1State Key Laboratory of Resources and Environmental Information System, Institute of Geographic Sciences and
Natural Resources Research, Chinese Academy of Sciences, 11A, Datun Road, Chaoyang District, Beijing 100101,
China. 2University of Chinese Academy of Sciences, Beijing, China. 3College of Urban and Environment, Liaoning
Normal University, Dalian, China. *email: caihy@igsnrr.ac.cn g
y
Remotely sensed nighttime light (NTL) has long been considered as a direct measure of socio-economic
development, and has served as primary data for a wide range of human-based research1,2, such as population
modelling3,4, GDP estimation5,6, urban expansion mapping7,8, and EPC estimation9,10. www.nature.com/scientificreports www.nature.com/scientificreports A novel classification regression
method for gridded electric power
consumption estimation in China
OPEN Mulin Chen1,2, Hongyan Cai1*, Xiaohuan Yang1 & Cui Jin3 Spatially explicit information on electric power consumption (EPC) is crucial for effective electricity
allocation and utilization. Many studies have estimated fine-scale spatial EPC based on remotely
sensed nighttime light (NTL). However, the spatial non-stationary relationship between EPC and
NTL at prefectural level tends to be overlooked in existing literature. In this study, a classification
regression method to estimate the gridded EPC in China based on imaging NTL via a Visible Infrared
Imaging Radiometer Suite (VIIRS) was described. In addition, owing to some inherent omissions
in the VIIRS NTL data, the study has employed the cubic Hermite interpolation to produce a more
appropriate NTL dataset for estimation. The proposed method was compared with ordinary least
squares (OLS) and geographically weighted regression (GWR) approaches. The results showed that our
proposed method outperformed OLS and GWR in relative error (RE) and mean absolute percentage
error (MAPE). The desirable results benefited mainly from a reasonable classification scheme that
fully considered the spatial non-stationary relationship between EPC and NTL. Thus, the analysis
suggested that the proposed classification regression method would enhance the accuracy of the
gridded EPC estimation and provide a valuable reference predictive model for electricity consumption. Electric power is essential for modern life and society. Having a clear understanding of electric power consump-
tion (EPC) is important for infrastructure planning, energy allocation and environmental protection. However,
given the commercial and confidential nature of electricity supply, detailed EPC information is difficult to obtain. Current EPC data are available mainly at administrative units, however, such data rarely meet the needs for fine
scale application because of the coarse resolution. Furthermore, the data are also difficult to integrate with spatial
data at other scales, and this can limit interdisciplinary applications of EPC. In contrast, gridded EPC data are
more efficient in fine scales research. Therefore, developing a convenient and reliable approach for fine scale
estimation of EPC is urgently needed. 1
V l (0123
Scientific Reports | (2020) 10:18558
| https://doi.org/10.1038/s41598-020-75543-2
given the commercial and confidential nature of electricity supply, detailed EPC information is difficult to obtain. Current EPC data are available mainly at administrative units, however, such data rarely meet the needs for fine
scale application because of the coarse resolution. Furthermore, the data are also difficult to integrate with spatial
data at other scales, and this can limit interdisciplinary applications of EPC. A novel classification regression
method for gridded electric power
consumption estimation in China
OPEN Many studies have dem-
onstrated a strong correlation between NTL and EPC at multiple levels, and consequently regression models
for such estimation have been built1,9,11–15. However, the relationship between NTL and EPC varies across areas,
owing to the local socioeconomic diversity16. In other words, the relationship between NTL and EPC is spatial
non-stationary17, which tends to be overlooked in existing literature, where global models are often used10,18–20. Thus, from the spatial non-stationary perspective, local regression would be more suitable for EPC estimation. As pointed out by Shi et al.21, a single regression model for all administrative units is susceptible to incorrect
estimation and an appropriate subdivision of administrative units could improve the estimation accuracy22. Some researchers have divided their study area into several sub-regions based on factors, such as geographic
location12,23, income consideration24, and city function17. Yet, such classifications are limited in terms of accuracy,
as they do not fully consider the complexity of interactions between NTL and EPC24. For instance, a classifica-
tion based on economic factors may not sufficiently distinct the relationship between NTL and EPC, given that
cities at the same level of economic development can have different energy consumption patterns and NTL levels
depending on their key industries17. Hence, exploring an appropriate classification basis from the perspective
of the relationship between EPC and NTL would provide a better understanding and a new insight for gridded
EPC estimation. 1State Key Laboratory of Resources and Environmental Information System, Institute of Geographic Sciences and
Natural Resources Research, Chinese Academy of Sciences, 11A, Datun Road, Chaoyang District, Beijing 100101,
China. 2University of Chinese Academy of Sciences, Beijing, China. 3College of Urban and Environment, Liaoning
Normal University, Dalian, China. *email: caihy@igsnrr.ac.cn | https://doi.org/10.1038/s41598-020-75543-2 Scientific Reports | (2020) 10:18558 www.nature.com/scientificreports/ In previous literature, the NTL used for EPC estimation was collected mainly from several sources including
the Defense Meteorological Satellite Program Operational Linescan System (DMSP/OLS)23,25–27 and the day/
night band (DNB) on the Visible Infrared Imaging Radiometer Suite (VIIRS) on-board the Suomi National
Polar-orbiting Partnership satellite21. Many studies have documented that estimation accuracy would be reduced
when using DMSP/OLS NTL, due to the saturation and coarse resolution21,28,29. In contrast, VIIRS/DNB NTL has
finer resolution and better data quality than DMSP/OLS29–32, and consequently would provide a more reliable
gridded EPC estimation21,33. Even so, some imperfections in the current VIIRS/DNB NTL (details given in the
“Materials” section) are undesirable for gridded estimation34. Materialsh The VIIRS/DNB NTL data provided by the National Oceanic and Atmospheric Administration’s National Cent-
ers for Environmental Information (NOAA/NCEI, https://ngdc.noaa.gov/eog/download.html) were used in this
study. This dataset has widely used for socioeconomic indicators spatialization owing to its fine resolution21,31,35. However, the data are vulnerable to stray light, resulting in emergence of distorted pixels in the mid and high
latitude areas, especially in the summer32,34,36,37. To address this problem, two versions of the NTL are provided
by NOAA, denoted by the filenames VCM (VIIRS Cloud Mask) and VCMSL (VIIRS Cloud Mask with Stray
Light), respectively. The VCM version eliminates all the pixels that were contaminated by stray light, resulting
in numerous missing pixels in the dataset. The VCMSL version corrects the distorted pixels through implemen-
tation of a stray light correction procedure38, but the data quality may be reduced in these regions29,37,39. Thus,
both the VCM and the VCMSL data have some limitations in respect of data quality. In the present study, a more
appropriate NTL dataset was produced through interpolation, which was then used for estimation purposes. pp
p
p
g
p
p
p
Statistical EPC data of 2015 at the prefectural level were obtained from the China City Statistical Yearbook
released by the National Statistics Bureau of China. The statistical data include EPC, industrial electricity con-
sumption (IEC) and household electricity consumption.h p
y
p
The land use and cover change (LUCC) data of 2015 were obtained from the land use database at a scale of
1:100,000, and collected from the Resources and Environmental Scientific Data Center (RESDC) of the Chinese
Academy of Sciences (CAS). These data were interpreted by a human–computer interactive method from the
Landsat TM/ ETM + images with an accuracy greater than 90%40,41. A novel classification regression
method for gridded electric power
consumption estimation in China
OPEN Given these problems, more data processing steps
are needed to remove the imperfections before estimation.i p
Against this background, the aim of this study was to develop a classification regression approach for high
accuracy gridded EPC estimation. To achieve this goal, a more suitable NTL dataset for EPC estimation was
first produced, based on the original VIIRS/DNB NTL. Secondly, a classification estimation scheme for the grid-
ded EPC estimation was proposed. At the same time, apart from the proposed approach, the gridded EPC was
also estimated using ordinary least squares (OLS) and geographically weighted regression (GWR). Finally, the
accuracy in estimation of these three methods were compared. Methodsh The methodology flow chart is displayed in Fig. 1. Given that the EPC activities occur mainly on built-up land,
the EPC estimation was only performed for built-up land. First, the built-up land was extracted from the LUCC
data. Second, a new NTL dataset (hereafter called the interpolated NTL) was obtained by interpolation. The
annual total nighttime light (TNL) data were then composited by the interpolated NTL. Third, the gridded EPC
was estimated by OLS, classification regression, and GWR, respectively. Finally, the accuracy was evaluated and
compared at the prefectural level. Improved VIIRS/DNB NTL data production. The original monthly VIIRS/DNB NTL data are subject
to stray light, and do not have background noise or ephemeral light filtered out, causing emergence of outliers
such as negative pixels and abnormally high-valued pixels in the imagery42. Such negative effects will reduce the
accuracy of estimation21. Therefore, an improved VIIRS/DNB NTL data, which has the outliers excluded and the
missing data interpolated, was produced prior to estimation.hhi The threshold method was used to eliminate outliers. Theoretically, the highest level of artificial light should
be emitted from the most developed areas43. Thus, the maximal radiation in the most developed cities in China
(Beijing, Shanghai, Guangzhou and Shenzhen), which was 264.47 nW cm−2 sr−1, was determined as the thresh-
old, and pixels with a radiation value larger than 264.47 nW cm−2 sr−1 were reassigned to that value. Meanwhile,
negative-valued pixels were reassigned to zero21.ti After filtering the outliers, the missing values were interpolated by a temporal interpolation method, as the
missing pixels tend to exist over a large area. According to the study of Chen et al.44, the cubic Hermite method
was used for interpolation. The monthly VCM data from August 2014 to April 2015, and from September 2015 to
April 2016 were served as the initial data in interpolation proceeding at the pixel level. Subsequently, the annual
TNL was calculated based on the interpolated NTL data at the pixel level (Eq. (1)), that is: (1)
TNLi =
12
j=1
NTLij (1) where NTLij is the monthly radiation value of the ith pixel in the jth month, and TNLi is the annual total radia-
tion value of the ith pixel. where NTLij is the monthly radiation value of the ith pixel in the jth month, and TNLi is the annual total radia-
tion value of the ith pixel. Methodsh https://doi.org/10.1038/s41598-020-75543-2 Scientific Reports | (2020) 10:18558 | www.nature.com/scientificreports/ Figure 1. Flow chart for production and evaluation of the gridded EPC. Figure 1. Flow chart for production and evaluation of the gridded EPC. Regression by OLS, GWR, classification regression. Statistical EPC and TNL were regressed by OLS,
GWR, and classification regression, respectively. Due to the lack of EPC statistical data in some cities, 259 cities
were used for regression. OLS was given in Eq. (2). (2)
EPCs = a
TNLi + b + e (2) where EPCs was the statistical data of a certain city, ∑TNLi was the amount of TNL for all the pixels in the cor-
responding areas, a was the regression coefficient, b was the intercept, and e was the error term. In previous
studies, the intercept was assumed to be zero10,18,24,45. However, since NTL is not fully indicative of EPC, the
intercept accounts for non-light emitting EPC. Hence, the intercept was presumed to be non-zero in this study
and the F-test was applied to achieve statistical significance.f
6 pp
gi
In consideration of the spatial spillover effect on EPC46, GWR was also adopted. GWR is an extension of lin-
ear regression model47. It uses some distance-based weighting function to permit the coefficients to vary locally
within the bandwidth48. GWR was performed by ArcGIS 10.2 and the bandwidth was determined by AICc49.hi The classification regression was built based on OLS. In order to maximize the stationary aspect of the rela-
tionship between EPC and NTL, the classification regression grouped the cities into three patterns based on
the 95% confidence interval of the OLS regression coefficients, and then each pattern was regressed by Eq. (2),
respectively. The three patterns were: the upper pattern, which consists of cities that were over the 95% confi-
dence interval (72 cities, blue points in Fig. 2b); the middle pattern, which consists of cities that were within the
confidence interval (78 cities, green points in Fig. 2b); and the down pattern, which consists of cities that were
under the confidence interval (109 cities, red points in Fig. 2b). In general, the upper pattern displayed a low
TNL with a relative high EPC, the down pattern featured a high TNL with a relative low EPC, and the middle pat-
tern was in between these two. EPC and TNL spatial distributions of the three patterns were displayed in Fig. 3. Gridded EPC estimation. Results
l
i Evaluation of the interpolated NTL data. The monthly TNL time series for the original VCM, VCMSL
and the interpolated NTL data at the national scale were compared and shown in Fig. 4. The TNL data for
the VCM version in the summer months were much lower than those in other months, resulting in dramatic
fluctuations in the time series curve. In contrast, the VCMSL and interpolated versions were smoother. If a cer-
tain region did not suffered from any severe events, such as natural disasters or power outages, but its TNL fluc-
tuated dramatically, it could be inferred that its NTL data did not reflect the real socio-economic conditions50,51,
and, therefore, were inappropriate for robust socio-economic research. From this standpoint, the interpolated
NTL data are more suitable for estimation of the EPC than the VCM data.f As shown in Fig. 4, there was only a slight difference between the VCMSL version and the interpolation
version at the national level. Thus, a further comparison of these two versions was performed at the prefectural
level. The results demonstrated that the interpolated NTL data performed better than the VCMSL product in the
mid and high latitude areas. The TNL values for some cities in the VCMSL version appeared to be susceptible
to sudden sharp drops in certain months, but this feature did not show up in the interpolated NTL data. For
reporting purposes, we defined cities where the TNL values suddenly became zero as being atypical cities. A
latitudinal statistic was used to calculate the frequency of the atypical cities (Fig. 5). Since a high ratio of 50%
came from the 45–50° N (13 cities) and 50–55° N (66.7%, 2 cities) regions, the atypical cities tended to occur in
the mid-high latitude regions where stray light shows severe contamination. In comparison, the atypical cities
were not evident in the interpolated NTL dataset. This difference in the respective NTL datasets most likely
results from the different processes used to produce the two NTL versions. The VCMSL NTL was produced by a
stray light correction procedure52, and, consequently, the mid-high latitude areas, where there was severe stray
light contamination, most likely was susceptible to this problem34, whereas the missing pixels were corrected
based on the long, historical, time series data in the interpolated NTL, which could effectively avoid dramatic
TNL time series changes. Evaluation of the gridded EPC estimation. www.nature.com/scientificreports/ www.nature.com/scientificreports/ more, IEC varies significantly between the industrial and non-industrial regions within a city. Thus, this differ-
ence was taken into consideration in the estimation. (3)
EPCGi =
aTNLi + b1
TNLi
TNLi industrial region
aTNLi + b2
TNLi
TNLi non −industrial region (3) (4)
b1 = b IECs
EPCs (4)
b1 = b IECs
EPCs
(5)
b2 = b−b1 b1 = b IECs
EPCs (4) b2 = b−b1 (5)
b2 = b−b1 (5) where EPCGi was the estimated EPC of the ith grid in a certain city. The values of a and b were obtained from
the three regression methods. IECs and EPCs represented the statistical IEC and EPC of the city. TNLi was the
annual total nighttime light radiation of the ith grid, and ∑TNLi was the city’s aggregated TNL. where EPCGi was the estimated EPC of the ith grid in a certain city. The values of a and b were obtained from
the three regression methods. IECs and EPCs represented the statistical IEC and EPC of the city. TNLi was the
annual total nighttime light radiation of the ith grid, and ∑TNLi was the city’s aggregated TNL. Accuracy evaluation. The accuracy at the prefectural level was evaluated using relative error (RE) (Eq. (6))
and mean absolute percentage error (MAPE) (Eq. (7)). (6)
RE = EPCe −EPCs
EPCs
× 100%
(7)
MAPE = 1
n
n
i=1
EPCei −EPCsi
EPCsi
× 100% (6)
RE = EPCe −EPCs
EPCs
× 100% (6) (7)
MAPE = 1
n
n
i=1
EPCei −EPCsi
EPCsi
× 100% (7) where EPCe represented the estimated EPC and EPCs represented the statistical EPC. i represented the ith city. n
represented the total number of cities and equaled to 259 in present study. The closer RE and MAPE are to zero,
the more accurate the estimation is. where EPCe represented the estimated EPC and EPCs represented the statistical EPC. i represented the ith city. n
represented the total number of cities and equaled to 259 in present study. The closer RE and MAPE are to zero,
the more accurate the estimation is. Methodsh The gridded EPC for the three assessing methods was calculated using Eqs. (3)–
(5). It was assumed that all the grids in a certain city had a segmented intercept based on their weight. Further- https://doi.org/10.1038/s41598-020-75543-2 Scientific Reports | (2020) 10:18558 | www.nature.com/scientificreports/ 0) 10:18558 |
https://doi.org/10.1038/s41598-020-75543-2
Figure 2. Regression of TNL and EPC by OLS and the proposed classification regression. (a) OLS. (b) Classification regression. 4
S i
ifi R
|
(
)
8
8 |
h
//d i
/10 1038/ 41598 020 75543 2
Figure 2. Regression of TNL and EPC by OLS and the proposed classification regression. (a) OLS. (b) Classification r Figure 2. Regression of TNL and EPC by OLS and the proposed classification reg https://doi.org/10.1038/s41598-020-75543-2 Scientific Reports | (2020) 10:18558 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ 0
200000
400000
600000
800000
1000000
1200000
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Dec
Original VCM
Original VCMSL
Interpolated NTL
Radiation/nWcm-2sr-1
Month
Figure 4. Monthly total nighttime light (TNL) time series at the national level for the original visible infrared
imaging radiometer suite cloud mask (VCM) version, the original visible infrared imaging radiometer suite
cloud mask with stray light (VCMSL) version and the interpolated nighttime light (NTL) data. 0
200000
400000
600000
800000
1000000
1200000
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Dec
Original VCM
Original VCMSL
Interpolated NTL
Radiation/nWcm-2sr-1
Month Figure 4. Monthly total nighttime light (TNL) time series at the national level for the original visible infrared
imaging radiometer suite cloud mask (VCM) version, the original visible infrared imaging radiometer suite
cloud mask with stray light (VCMSL) version and the interpolated nighttime light (NTL) data. 0%
20%
40%
60%
80%
100%
0
20
40
60
80
100
120
140
20~25
25~30
30~35
35~40
40~45
45~50
50~55
Proportion of atypical cities
Proportion of normal cities
Number of all cities
Number of atypical cities
Percentage
Number of cities
Latitude/ °N
Figure 5. Frequency of occurrence of the sudden TNL drop for the visible infrared imaging radiometer suite
cloud mask with stray light (VCMSL) product. Cities that had the sudden drop were called the atypical cities. The “number of all cities” is the total number of cities within the corresponding latitude zone. Figure 5. Frequency of occurrence of the sudden TNL drop for the visible infrared imaging radiometer suite
cloud mask with stray light (VCMSL) product. Cities that had the sudden drop were called the atypical cities. The “number of all cities” is the total number of cities within the corresponding latitude zone. exceeded 50% decreased from over a hundred to 62 after classification. The lowest MAPE values for the proposed
classification estimation also demonstrated that the approach resulted in a significant improvement in accuracy
compared to OLS and GWR. exceeded 50% decreased from over a hundred to 62 after classification. The lowest MAPE values for the proposed
classification estimation also demonstrated that the approach resulted in a significant improvement in accuracy
compared to OLS and GWR. Results
l
i The gridded EPC based on classification regression was
shown in Fig. 6. The EPC was concentrated in eastern China. The high EPC areas were centered on megacities
and provincial capitals, especially Shanghai, Guangzhou and Beijing. The low TNL areas were mostly distributed
in rural regions and less developed satellite cities. Within a city, the highest EPC tended to occur mainly at air-
ports, railway stations and business districts.hh p
y
The accuracy of the evaluated results at the prefectural level is shown in Table 1. There were 145 cities whose
RE values fell into the [− 25%, 25%] bin when adopting the classification estimation, while there were only 72
cities in that bin for OLS and 86 for GWR. Furthermore, the number of cities for which the absolute RE values Scientific Reports | (2020) 10:18558 | https://doi.org/10.1038/s41598-020-75543-2 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 3. Distributions of the three observed patterns. (a) EPC in the three observed patterns. (b) TNL in the three observed patterns. The map was generated by ArcGIS 10.2 (https://www.arcgi
s.com/index.html). 3. Distributions of the three observed patterns. (a) EPC in the three observed patterns. (b) TNL in the three observed patterns. The map was generated by ArcGIS 10.2 (https://www.arcgi
ndex.html). Figure 3. Distributions of the three observed patterns. (a) EPC in the three observed patterns. (b) TNL in the three observed patterns. The map was generated by ArcGIS 10.2 (https://www.arcgi
s.com/index.html). b) TNL in the three observed patterns. The map was generated by ArcGIS 10.2 (https://ww igure 3. Distributions of the three observed patterns. (a) EPC in the three observed patterns. (b) TNL in the three observed patterns. The map was generated by ArcGIS 10.2 (https://w
com/index.html). https://doi.org/10.1038/s41598-020-75543-2 Scientific Reports | (2020) 10:18558 | Scientific Reports | (2020) 10:18558 | Discussion
ff
f Different from most previous literature, a novel classification regression method that fully considered the spa-
tial non-stationary relationship between the EPC and NTL was described. The results indicated that the pro-
posed method performed best among the three assessing methods (Table 1). Compared to OLS, the relationship
between the EPC and NTL was stronger for the classification regression, as revealed by the R2 values (0.900,
0.994 and 0.934 vs. 0.805). Moreover, a previous study by Shi et al.21 also confirmed this finding. In that study,
the VIIRS/DNB NTL data were also applied to gridded EPC estimation based on OLS. The RE values in the
present work were significantly reduced compared to that study, notably in Anhui, Hebei, Heilongjiang and
Shaanxi provinces. In addition, GWR is powerful for spatial non-stationary parameters regression53. However,
the Moran’s bivariate spatial autocorrelation index of statistical EPC and TNL was only 0.070. It was evident
that the spatial autocorrelation between EPC and TNL was weak, in other words, the distance between cities has
little influence on the regression. Thereby, the distance-based weighting scheme was not likely to render a better
performance than the proposed method.hiihi The favorable results benefited mainly from a reasonable classification scheme being realized. The classifica-
tion basis is important to high accuracy estimation. In a study of Li et al.17, cities were grouped into technology
and education cities, industrial cities, and service-oriented cities. However, the classification basis based on city
function still have some limitations on ensuring the spatial stationary relationship between EPC and TNL. For
example, Changchun, Shenyang, Baotou, Dongguan were the famous industrial cities in China, but differed
greatly in the relationship between EPC and TNL (Fig. 2). In part because EPC used for different purposes would
emit different levels of light. Industrial electricity consumed for electrolysis, heating and other energy-intensive
processes, has a little light emitted22, whereas industrial electricity consumed for oil, natural gas, and coal mineral
resources mining, has bright light emitted due to the mining lights and industrial fires22. It leads to different
key industries playing a role in the relationship between EPC and TNL17. The classification basis based on city Scientific Reports | (2020) 10:18558 | https://doi.org/10.1038/s41598-020-75543-2 www.nature.com/scientificreports/ Figure 6. Distribution of the gridded EPC based on the classification regression method. (a) Spatial
distribution of gridded EPC in China. Gridded EPC distribution in Beijing, Shanghai and Guangzhou is
displayed in (b–d), respectively. Discussion
ff
f The map was generated by ArcGIS 10.2 (https://www.arcgis.com/index.html). Figure 6. Distribution of the gridded EPC based on the classification regression method. (a) Spatial
distribution of gridded EPC in China. Gridded EPC distribution in Beijing, Shanghai and Guangzhou is
displayed in (b–d), respectively. The map was generated by ArcGIS 10.2 (https://www.arcgis.com/index.html Table 1. Number of cities in the different relative error (RE) bins and mean absolute percentage error (MAPE)
values for each of the three estimation methods. OLS
GWR
Classification regression
|RE|≤ 25%
72
86
145
25% <|RE|≤ 50%
71
70
52
50% <|RE|
116
103
62
MAPE
57.1%
73.9%
38.0% Table 1. Number of cities in the different relative error (RE) bins and mean absolute percentage error (MAPE)
values for each of the three estimation methods. Table 1. Number of cities in the different relative error (RE) bins and mean absolute percentage error (MAPE)
values for each of the three estimation methods. function, thus, was insufficient for spatial stationary classification of EPC and TNL; instead, the classification
scheme we proposed effectively avoided ambiguous classification.hiih function, thus, was insufficient for spatial stationary classification of EPC and TNL; instead, the classification
scheme we proposed effectively avoided ambiguous classification.hiih f
y
gi
The threshold of confidence interval may cause uncertainty of the classification method. Thus, apart from
95% confidence interval, 90% and 99% confidence intervals were also served as the threshold, respectively. As the
comparison results shown, whether the regression coefficients, or the MAPE values, the differences were slight
(Fig. 7). It was because for the most cities, their patterns did not change as the confidence intervals changed, and,
consequently, the classification regression method was seldom affected by the threshold of confidence interval. Thus, from this perspective, the classification regression method was robust. ,
p
p
,i
g
Inevitably, some limitations existed when estimating the gridded EPC based on the VIIRS/DNB NTL. First
he overpass time of the SNPP satellite is after midnight and near 01:30 am54. The outdoor artificial light is low at Scientific Reports | (2020) 10:18558 | https://doi.org/10.1038/s41598-020-75543-2 www.nature.com/scientificreports/ Figure 7. Classification regression based on different confidence intervals. (a) 90% confidence interval. (b) 95% confidence interval. (c) 99% confidence interval. Figure 7. Classification regression based on different confidence intervals. (a) 90% confidence interval. (b) 95% confidence interval. (c) 99% confidence interval. erval. (c) 99% confidence interval. igure 7. Classification regression based on different confidence intervals. (a) 90% confidence interval. Conclusionsi Artificial light is closely related to the EPC, therefore the NTL is a superior indicator of the EPC and is widely
used for gridded EPC estimation9,10. However, the current VIIRS/DNB monthly NTL data did not meet high
estimation accuracy requirements due to the impact of missing pixels and ephemeral light57. Thus, in this study,
a more suitable interpolated NTL dataset was first proposed based on the cubic Hermite method, then the data
were subjected to regression analysis. Moreover, few studies took the spatial non-stationary relationship between
EPC and NTL into account in the estimation. In contrast to previous research12,58, this study has developed a
classification regression method that based on the relationship between EPC and NTL.ih i
Generally, the proposed approach resulted in a significant improvement in accuracy over OLS and GWR. The
R2 values for the proposed method were greater than the OLS (0.900, 0.994 and 0.934 vs. 0.805). Furthermore,
with regard to the RE values, the proposed method had more cities (145 vs. 72 and 86) that fell into the [− 25%,
25%] bin than OLS and GWR. In addition, by comparing estimation results with previous literature, results for
this study showed a higher accuracy in many provinces. In conclusion, the comparative results for this study
indicate that the approach used gives new insights into the explicit spatial distribution estimation of EPC based
on the NTL. In addition to gridded EPC estimation, the proposed method also has a great potential for other
socio-economic indicators estimation (e.g., GDP and carbon dioxide emission). Received: 6 January 2020; Accepted: 16 October 2020 Received: 6 January 2020; Accepted: 16 October 2020 Discussion
ff
f (b) 95% confidence interval. (c) 99% confidence interval. t confidence intervals. (a) 90% confidence interval. (b) 95% confidence interval. (c) Figure 7. Classification regression based on different confidence intervals. (a) 90% confiden igure 7. Classification regression based on different confidence interv https://doi.org/10.1038/s41598-020-75543-2 Scientific Reports | (2020) 10:18558 | www.nature.com/scientificreports/ this time, leading to much EPC that is not reflected in the NTL dataset. Second, monthly changes in vegetation
and snow cover are also contained in the current VIIRS/DNB NTL dataset. These physical changes are known
to be undesirable for socio-economic studies55. Despite this, given that China is mainly located in the low-mid
latitude regions, most parts of the country would not be impacted by that seasonal noise56, and, consequently,
the EPC estimation result is considered reliable. However, for high latitude areas, researchers should give close
attention to the physical effects of using the VIIRS/DNB NTL data for EPC estimation and other socio-economic
studies. Besides, NTL is just one of components of EPC, other factors such as GDP, population, and urbaniza-
tion also impact on EPC46. However, coarse statistical GDP and population data are unfeasible for fine scale
estimation. Furthermore, using estimated gridded GDP and population product would introduce inevitable
errors. Thus, GDP, population, and urbanization were not considered when EPC estimation in the present study. References 1. Proville, J., Zavala-Araiza, D. & Wagner, G. Night-time lights: A global, long term look at links to socio-economic trends. PLoS
ONE 12, e0174610 (2017). 2. Kyba, C. et al. High-resolution imagery of earth at night: New sources, opportunities and challenges. Remote Sens. 7, 1–23 (2
3 T
M
l M d li
l i
d
i
b
d
i h i
li h i
d l
d
d
i Chi
A
l G
90 239 2. Kyba, C. et al. High-resolution imagery of earth at night: New sources, opportunities and challenges. Remote Sens. 7, 1–23 (2015)
3. Tan, M. et al. Modeling population density based on nighttime light images and land use data in China. Appl. Geogr. 90, 239–247 2. Kyba, C. et al. High-resolution imagery of earth at night: New sources, opportunities and challenges. Remote Sens. 7, 1–23 (2015). 3. Tan, M. et al. Modeling population density based on nighttime light images and land use data in China. Appl. Geogr. 90, 239–247
(2018). y
,
g
g
y
g
, pp
g
,
(
)
3. Tan, M. et al. Modeling population density based on nighttime light images and land use data in China. Appl. Geogr. 90, 239–247
(2018). 4. Wang, L. et al. Mapping population density in China between 1990 and 2010 using remote sensing. Remote Sens. Environ. 210,
269–281 (2018).f 5. Zhao, M. et al. GDP spatialization and economic differences in south China based on NPP-VIIRS nighttime light imagery. Remote
Sens. 9, 673 (2017). 6. Zhu, X., Ma, M., Yang, H. & Ge, W. Modeling the spatiotemporal dynamics of gross domestic product in China using extended
temporal coverage nighttime light data. Remote Sens. 9, 626 (2017). g
g
g
7. Huang, X., Schneider, A. & Friedl, M. A. Mapping sub-pixel urban expansion in China using MODIS and DMSP/OLS nighttime
lights. Remote Sens. Environ. 175, 92–108 (2016). 7. Huang, X., Schneider, A. & Friedl, M. A. Mapping lights. Remote Sens. Environ. 175, 92–108 (2016). g
8. Xie, Y. & Weng, Q. Updating urban extents with nighttime light imagery by using an object-based thresholding method. Remote
Sens. Environ. 187, 1–13 (2016).i 9. Dugoua, E., Kennedy, R. & Urpelainen, J. Satellite data for the social sciences: Measuring rural electrification with night-time
lights. Int. J. Remote Sens. 39, 2690–2701 (2018). g
10. Pan, J. & Li, J. References Spatiotemporal dynamics of electricity consum p
p
y
y
p
pp
p
y
1. Shi, K. et al. Detecting spatiotemporal dynamics of global electric power consumption using DMSP-OLS nighttime stable ligh
data. Appl. Energy 184, 450–463 (2016). 2. Xie, Y. & Weng, Q. Detecting urban-scale dynamics of electricity consumption at Chinese cities using time-series DMSP-OLS
(Defense Meteorological Satellite Program-Operational Linescan System) nighttime light imageries. Energy 100, 177–189 (2016) 12. Xie, Y. & Weng, Q. Detecting urban-scale dynamics of electricity consumption at Chinese cities using time-series DMSP-OLS
(Defense Meteorological Satellite Program-Operational Linescan System) nighttime light imageries. Energy 100, 177–189 (2016). 13. Shi, K., Yu, B., Huang, C., Wu, J. & Sun, X. Exploring spatiotemporal patterns of electric power consumption in countries along
the Belt and Road. Energy 150, 847–859 (2018). 3. Shi, K., Yu, B., Huang, C., Wu, J. & Sun, X. Exploring spatiotemporal patterns of electric power consumption in countries along
the Belt and Road. Energy 150, 847–859 (2018). gy
4. Fobi, S., Deshpande, V., Ondiek, S., Modi, V. & Taneja, J. A longitudinal study of electricity consumption growth in Kenya. Energy
Policy 123, 569–578 (2018). y
5. Xiao, H. et al. Spatio-temporal simulation of energy consumption in China’s provinces based on satellite night-time light data
Appl. Energy 231, 1070–1078 (2018). pp
gy
(
)
16. Hu, T. & Huang, X. A novel locally adaptive method for modeling the spatiotemporal dynamics of global electric power consump-
tion based on DMSP-OLS nighttime stable light data. Appl. Energy 240, 778–792 (2019). g
g
pp
gy
7. Li, S. et al. City type-oriented modeling electric power consumption in China using NPP-VIIRS nighttime stable light data. Energy
189, 116040 (2019). 8. Cao, X., Wang, J., Chen, J. & Shi, F. Spatialization of electricity consumption of China using saturation-corrected DMSP-OLS data
Int. J. Appl. Earth Obs. Geoinf. 28, 193–200 (2014). pp
f
19. Chi, Z., Zhou, K., Yang, S. & Zhen, S. On electricity consumption and economic growth in China. Renew. Sustain. Energy Rev. 76,
353–368 (2017). 20. Elvidge, C. D. et al. Relation between satellite observed visible-near infrared emissions, population, economic activity and electric
power consumption. Int. J. Remote Sens. 18, 1373–1379 (1997). p
p
J
(
)
1. Shi, K. et al. Evaluating the ability of NPP-VIIRS nighttime light data to estimate the gross domestic product and the electric power
consumption of China at multiple scales: A comparison with DMSP-OLS data. Remote Sens. www.nature.com/scientificreports/ 31, 292–302 (2011). g
g
p
y
g
g
pp
g
4. Miller, S. D. et al. Illuminating the capabilities of the suomi national polar-orbiting partnership (NPP) visible infrared imaging
radiometer suite (VIIRS) day/night band. Remote Sens. 5, 6717–6766 (2013). D. et al. Illuminating the capabilities of the suomi national polar y
g
55. Levin, N. & Zhang, Q. A global analysis of factors controlling VIIRS nighttime light levels from densely populated areas. Remote
Sens. Environ. 190, 366–382 (2017).h 6. Levin, N. The impact of seasonal changes on observed nighttime brightness from 2014 to 2015 monthly VIIRS DNB composites
Remote Sens. Environ. 193, 150–164 (2017). 7. Wu, R., Yang, D., Dong, J., Zhang, L. & Xia, F. Regional inequality in China based on NPP-VIIRS night-time light imagery. Remote
Sens. 10, 240 (2018). 58. Tripathy, B. R. et al. Modeling of electric demand for sustainable energy and management in India using spatio-temporal DMSP-
OLS night-time data. Environ. Manage. 61, 615–623 (2018). www.nature.com/scientificreports/ www.nature.com/scientificreports/ 23. He, C., Ma, Q., Li, T., Yang, Y. & Liu, Z. Spatiotemporal dynamics of electric power consumption in Chinese mainland from 1995
to 2008 modeled using DMSP/OLS stable nighttime lights data. J. Geogr. Sci. 22, 125–136 (2012). g
g
g
g
4. Xie, Y. & Weng, Q. World energy consumption pattern as revealed by DMSP-OLS nighttime light imagery. GISci. Remote Sens. 53
265–282 (2016). 5. Liu, Z., He, C., Zhang, Q., Huang, Q. & Yang, Y. Extracting the dynamics of urban expansion in China using DMSP-OLS nighttime
light data from 1992 to 2008. Landsc. Urban Plann. 106, 62–72 (2012). g
6. Zhao, N., Ghosh, T. & Samson, E. L. Mapping spatio-temporal changes of Chinese electric power consumption using night-time
imagery. Int. J. Remote Sens. 33, 6304–6320 (2012).h imagery. Int. J. Remote Sens. 33, 6304–6320 (2012). 27. Townsend, A. C. & Bruce, D. A. The use of night-time lights satellite imagery as a measure of Australia’s regional electricity con-
sumption and population distribution Int J Remote Sens 31 4459–4480 (2010) g
y
7. Townsend, A. C. & Bruce, D. A. The use of night-time lights satellite imagery as a measure of Australia’s regional electricity con-
sumption and population distribution. Int. J. Remote Sens. 31, 4459–4480 (2010).f p
p p
8. Shi, K. et al. Evaluating spatiotemporal patterns of urban electricity consumption within different spatial boundaries: A case study
of Chongqing, China. Energy 167, 641–653 (2019). of Chongqing, China. Energy 167, 641–653 (2019). gq
g
gy
9. Zhao, M. et al. Applications of satellite remote sensing of nighttime light observations: Advances, challenges, and perspectives
Remote Sens. 11, 1971 (2019). 0. Baugh, K. E., Hsu, F. C., Elvidge, C. D. & Zhizhin, M. Nighttime lights compositing using the VIIRS day-night band: Preliminary
results. Proc. Asia-Pac. Adv. Netw. 35, 70–86 (2013). 1. Elvidge, C. D., Baugh, K. E., Zhizhin, M. N. & Hsu, F. C. Why VIIRS data are superior to DMSP for mapping nighttime lights
Proc. Asia-Pac. Adv. Netw. 35, 62–69 (2013). Proc. Asia Pac. Adv. Netw. 35, 62 69 (2013). 2. Elvidge, C. D., Baugh, K. E., Zhizhin, M. N., Hsu, F. C. & Ghosh, T. VIIRS night-time lights. Int. J. Remote Sens. 38, 5860–5879
(2017) 2. Elvidge, C. D., Baugh, K. E., Zhizhin, M. N., Hsu, F. C. & Ghosh, T. VIIRS night-time lights. Int. J. Remote Sens. 38, 5860–5879
(2017). (
)
33. www.nature.com/scientificreports/ & Nishio, F. Regional-scale estimation of electric power and power plant CO2 emissions using defense
meteorological satellite program operational linescan system nighttime satellite data. Environ. Sci. Technol. Lett. 1, 259–265 (2014). 46 H
Y & H
H E
i
i
i
G
d
f Chi
(2006 2015) A
i l d
i
l
ilib i
i 6. He, Y. & Huang, H. Energy intensity in Guangdong of China (2006–2015): A spatial dynamic general equilibrium econometric
model. Nat. Resour. Modell. 22, 1–14 (2019). 7. Fotheringham, A. S., Charlton, M. E. & Brunsdon, C. Geographically weighted regression: A natural evolution of the expansion
method for spatial data analysis. Environ. Plan. A 30, 1905–1927 (1998).fii p
y
8. Griffith, D. A. Spatial-filtering-based contributions to a critique of geographically weighted regression (GWR). Environ. Plan. A
40, 2751–2769 (2008). 48. Griffith, D. A. Spatial-filtering-based contributions to a critique of geographically weighted regression (GWR). Environ. Plan. A
40, 2751–2769 (2008). 49 Geniaux, G & Martinetti, D A new method for dealing simultaneously with spatial autocorrelation and spatial heterogeneity in 40, 2751–2769 (2008). 49. Geniaux, G. & Martinetti, D. A new method for dealing simultaneously with spatial autocorrelation and spatial heterogeneity in
i
d l
R
i
U b
E
(
) 9. Geniaux, G. & Martinetti, D. A new method for dealing simultaneously with spatial autocorrelation and spatial heterogeneity in
regression models. Region. Urban Econ. 72, 74–85 (2017). g
g
0. Li, X., Zhang, R., Huang, C. & Li, D. Detecting 2014 Northern Iraq insurgency using night-time light imagery. Int. J. Remote Sens
36, 3446–3458 (2015). 1. Li, X., Li, D., Xu, H. & Wu, C. Intercalibration between DMSP/OLS and VIIRS night-time light images to evaluate city light dynam-
ics of Syria’s major human settlement during Syrian Civil War. Int. J. Remote Sens. 38, 5934–5951 (2017). ics of Syria’s major human settlement during Syrian Civil War. Int. J. Remote Sens. 38, 5934–5951 (2017). 52. Krikigianni, E., Tsiakos, C. & Chalkias, C. Estimating the relationship between touristic activities and night light emissions. Eur. J Remote Sens 52 233–246 (2019) y
j
g y
J
,
(
)
52. Krikigianni, E., Tsiakos, C. & Chalkias, C. Estimating the relationship between touristic activities and night light emissions. Eur. J. Remote Sens. 52, 233–246 (2019). 3. Gao, J. & Li, S. Detecting spatially non-stationary and scale-dependent relationships between urban landscape fragmentation and
related factors using Geographically Weighted Regression. Appl. Geogr. www.nature.com/scientificreports/ Jing, X., Shao, X., Cao, C., Fu, X. & Yan, L. Comparison between the Suomi-NPP day-night band and DMSP-OLS for correlating
socio-economic variables at the provincial level in China. Remote Sens. 8, 17 (2016). 34. Lee, S. & Cao, C. Soumi NPP VIIRS day/night band stray light characterization and correction using calibration view data. Remote
Sens. 8, 138 (2016). 5. Cao, J., Chen, Y., Wilson, J. P., Tan, H. & Xu, Z. Modeling China’s prefecture-level economy using VIIRS imagery and spatia
methods. Remote Sens. 12, 839 (2020). 36. Liang, C. K., Mills, S., Hauss, B. I. & Miller, S. D. Improved VIIRS day/night band imagery with near-constant contrast. IEEE Trans. Geosci. Remote Sens. 52, 6964–6971 (2014). 37. Zhao, X., Li, D., Li, X., Zhao, L. & Wu, C. Spatial and seasonal patterns of night-time lights in global ocean derived from VIIRS
DNB images. Int. J. Remote Sens. 39, 8151–8181 (2018). g
8. Chen, H., Xiong, X., Sun, C., Chen, X. & Chiang, K. Suomi-NPP VIIRS day–night band on-orbit calibration and performance. J
Appl. Remote Sens. 11, 036019 (2017). 39. Chen, M. L. & Cai, H. Y. VIIRS/DNB monthly and yearly nighttime light dataset in Beijing-Tianjin-Hebei region (2013–2018). J. Glob. Change Data Discov. 3, 376–381 (2019).l 40. Liu, W., Liu, J., Kuang, W. & Ning, J. Examining the influence of the implementation of major function-oriented zones on built-up
area expansion in China. J. Geogr. Sci. 27, 643–660 (2017).h p
g
41. Jiyuan, L. et al. The land use and land cover change database and its relative studies in China. J. Geogr. Sci. 12, 275–282 (200
h
f
d
d
h
l h 41. Jiyuan, L. et al. The land use and land cover change database and its relative studies in China. J. Geogr. Sci. 12, 275–282 (2002). 42. Ma, T., Zhou, C., Pei, T., Haynie, S. & Fan, J. Responses of Suomi-NPP VIIRS-derived nighttime lights to socioeconomic activity
in China’s cities. Remote Sens. Lett. 5, 165–174 (2014). 3. Ma, W. & Li, P. An object similarity-based thresholding method for urban area mapping from visible infrared imaging radiometer
suite day/night band (VIIRS DNB) data. Remote Sens. 10, 263 (2018). y
g
(
)
(
)
44. Chen, M. L. & Cai, H. Y. Interpolation methods comparison of VIIRS/DNB nighttime light monthly composites: A case study of
Beijing. Prog. Geogr. 38, 126–138 (2019). j
g
g
g
45. Letu, H., Nakajima, T. Y. References 6, 1705–1724 (2014).hff p
p
p
,
(
)
22. Dai, Z., Hu, Y. & Zhao, G. The suitability of different nighttime light data for GDP estimation at different spatial scales and regional
levels. Sustainability 9, 305 (2017). Scientific Reports | (2020) 10:18558 | https://doi.org/10.1038/s41598-020-75543-2 The authors declare no competing interests. The authors declare no competing interests. Acknowledgements We would like to thank the State Key Laboratory of Resources and Environmental Information System, Institute
of Geographic Sciences and Natural Resources Research, Chinese Academy of Sciences and University of Chinese
Academy of Sciences. This work is supported by the Strategic Priority Research Program of Chinese Academy of
Sciences [No. XDA20010203]; the National Key R&D Program of China [No.2018YFC1800103]; the National
Natural Science Foundation of China [No. 41771460]. https://doi.org/10.1038/s41598-020-75543-2 Scientific Reports | (2020) 10:18558 | www.nature.com/scientificreports/ Author contributions
M C
i
d h
d M.C. conceived the proposed method, generated all the GIS maps and figures, and wrote the manuscript, under
the guidance of H.C. and X.Y., C.J. polished the manuscript. All authors discussed the results and commented
on the manuscript at all stages. Additional information Correspondence and requests for materials should be addressed to H.C. Correspondence and requests for materials should be addressed to H.C. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
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English
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Metabolic and clinical responses to Bunium Persicum (black caraway) supplementation in overweight and obese patients with type 2 diabetes: a double-blind, randomized placebo-controlled clinical trial
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Nutrition & metabolism
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Research Version of Record: A version of this preprint was published on August 26th, 2020. See the published
version at https://doi.org/10.1186/s12986-020-00494-4. Page 1/19 Abstract Background: Diabetes mellitus is the most common metabolic disorder worldwide. We aimed to
determine the effects of Bunium Persicum (BP) on serum glucose indices, lipid profile, and nesfatin-1
levels in overweight or obese T2DM patients. Methods: Participant recruitment took place in the diabetic clinic of Bu-Ali hospital in Zahedan. Based on
the eligibility criteria, 60 participants were randomly divided into two groups, namely BP (n=30) and
placebo (n=30). The supplementation was one 1000 mg capsule 2 times /day BP with meals (lunch and
dinner) for 8 weeks. Bodyweight, waist circumference, serum nesfatin-1, fasting blood sugar (FBS) and
insulin (FBI), total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-
density lipoprotein cholesterol (HDL-C) were measured. Quantitative insulin sensitivity checks index
(QUICKI), homeostasis model assessment-insulin resistance (HOMA-IR), and Body Mass Index (BMI) were
also calculated. Results: In comparison with placebo, BP significantly decreased FBS, HOMA-IR, and BMI (P<0.05). The
differences in the FBI, QUICKI, TG, TC, LDL, HDL, WC, and Nesfatin-1 were not significant (P>0.05). Results: In comparison with placebo, BP significantly decreased FBS, HOMA-IR, and BMI (P<0.05). The
differences in the FBI, QUICKI, TG, TC, LDL, HDL, WC, and Nesfatin-1 were not significant (P>0.05). Conclusion: BP supplement improved serum glucose indices and decreased BMI among overweight or
obese T2DM patients; though, further trials are suggested to confirm results. Trial Registration: Iranian Registry of Clinical Trials (IRCT), IRCT20181207041876N1, Registered
18/01/2019, https://irct.ir/trial/35752 Background Diabetes is a multifactorial autoimmune disorder, which is determined by a high blood glucose level. The
prevalence of diabetes mellitus type 2 (T2DM) is increasing globally and its complications are a major
health problem (1, 2). In 2014, over 422 million had diabetes and a continuous rise in DM prevalence is
expected (3). Being overweight, obesity, impaired blood glucose, and hyperlipidemia are some risk factors of T2DM (4,
5). Some metabolic perturbations such as B-cell dysfunction, impaired insulin secretion, insulin resistance,
and dyslipidemia contribute to the pathogenesis of diabetes (6). Adipose tissue is an active endocrine organ which produces adipokines that control many physiological
functions such as immunity, inflammation, and energy homeostasis (7). Nesfatin-1 is one of these
adipokines that is involved in appetite regulation and body fat storage by participating in important
functions in metabolizing glucose, phosphorylation specific signaling proteins, and increasing liver Page 2/19 insulin sensitivity, specifically by AMP-activated protein kinase (8, 9). This adipokine is expressed in parts
of the brain that interfere with metabolism regulation and dietary behavior (10). Nesfatin gene expression
is activated by peroxisome proliferator-activated receptors (PPARs), especially PPARγ (11, 12). Nesfatin-1
can decrease food intake when administered into the third cerebral ventricle of rats (13). Nesfatin-1 can
decrease food intake when administered into the third cerebral ventricle of rats. Fasting blood nesfatin-1
was significantly decreased in T2DM patients compared to healthy subjects and might be one of the
appetite-related hormones involved in diabetic Polyphagia(14). Management of diabetes to prevent complexity include changes in dietary patterns, regular physical
activity, and anti-diabetic medications (15, 16). Various types of traditional, complementary, or alternative therapies have been increasingly used for the
treatment of diabetes in human and animal models of type 2 diabetes (17). Herbal medicines are a major
part of these therapies (18). One of the medicinal plants is Bunium Persicum (Black Caraway). Bunium
Persicum, which belongs to the Apiaceae family and is generally known in Iran as ‘Zireh Siah’ has always
been used in traditional Iranian medicine for certain disorders (19, 20). The main constituents of BP, that
have been demonstrated in recent studies, are cumin aldehyde, caffeic acid, p-coumaric acid, gamma-
terpinene, p-cymene, cuminal, flavonoids (such as quercetin and kaempferol) and a high level of
polyphenols compound (21-25). Ethical Considerations The ethics committee of Zahedan University of Medical Sciences approved this double-blind randomized
placebo-controlled clinical trial under the code; IR.ZAUMS.REC.1397.332. The present study was
registered in the Iranian Registry of Clinical Trials; IRCT20181207041876N1 on 18/01/2019. The
participants were overweight or obese T2DM patients who had referred to the diabetic clinic of Bu-Ali
Hospital of Zahedan. This trial lasted from 23 June 2019 until 22 October 2019. An informed written consent form was obtained from the subjects before data collection. Participants
were informed at the outset of the study about risks and possible side effects of BP, blood sampling, the
confidentiality of participants' personal information, right to withdraw from the study at any point, free-of-
charge participation in the trial, weekly contact by the researcher to participants, and the approval section. -Study Design and Subjects: This double-blind, randomized, placebo-controlled clinical trial was carried out on 60 overweight or obese
patients with T2DM who were randomly divided into two groups (Placebo group [n=30] and Bunuim
Persicum Group [n=30]) by the block randomization method. Selected patients took Bunuim Persicum and Placebo capsules for 8 weeks. The packaging of
supplements and capsules were similar in appearance. The researchers and subjects were blinded to
group assignment and capsule content until the end of the analysis. Inclusion criteria were T2DM diagnosed, use of oral hypoglycemic drugs, age 30–65 years, and
25≤BMI<40 kg/m2. Exclusion criteria were suffering from cognitive disease or other psychotic illnesses,
depression, acute systemic disease, cystic fibrosis disease, muscular dystrophy, protein malnutrition, a
history of gastrointestinal surgery, neurological disorders, structural abnormalities of the gastrointestinal
tract, disability, uncontrolled hypertension (>140/90 mmHg), antihypertensive, probiotics, multivitamin-
mineral and antioxidant supplements during the three past months, pregnancy or lactation, expert athlete,
intake of statins, caraway interacting drugs, alcohol, cigarette, and drug abuse, losing weight during the
past 3-months, changing the type and dose of T2DM medications, and not taking more than 10% of the
intervention supplement. This trial adhered to CONSORT guidelines and included a completed CONSORT checklist as an additional
file. Background The reported effects of cumin aldehyde one of the most studied agent,
include the inhibition of aldose reductase and r-Glucosidase, the insulinotropic and b cell-protective
action, anti-obesity, and liver protective effect (26-29). According to the data available, the results of previous studies on the effect of Bunium Persicum (black
caraway) on T2DM patients and normal subjects were inconsistent. Some previous animal and human
studies found that caraway intake could improve lipid profiles and decrease blood glucose
concentrations (30, 31). However, Ghorbani et al. reported that caraway supplementation had no beneficial
effects on fasting blood glucose concentrations and some other researchers revealed no significant
effect of caraway intake on lipid profiles and fasting blood glucose (31, 32). Furthermore, evidence has suggested that Bunium Persicum and Carum Carvi are plant members of the
Apiaceae family and have similar active ingredients (33, 34). In addition, slimming, appetite-suppressing,
hypoglycemic, and antihyperlipidemic effects of Carum Carvi have been shown in recent studies (35, 36). With regard to the food intake decreasing effects of nesfatin-1 shown in previous studies (37), we
hypothesized that Bunium Persicum can suppress appetite and reduce food intake by increasing levels of
nesfatin-1 that can, in turn, lead to the decrease of blood sugar and lipid profiles. However, no human
studies have been carried out to determine the effect of BP on T2DM and nesfatin-1 in overweight or
obese T2DM patients and requires further investigation. This trial was designed to assess the metabolic
and clinical responses to Bunium Persicum (Black Caraway) supplementation in overweight and obese
patients with type 2 diabetes. Page 3/19 Page 3/19 Methods Ethical Considerations -Randomization and Intervention: According to the block randomization method, participants were divided into two equal groups by an
assistant (BP [n=30] or placebo [n=30] groups). The stratified randomization method was used for age
and gender. The ratio of the two groups was 1:1. Three patients from the BP group and 2 patients from Page 4/19 Page 4/19 the placebo group declined to participate after randomization and before the beginning of the study
(Figure 1). The blinding of intervention allocation was done for both the participants and researchers as A and B
packages. The BP and placebo capsules were made by the Herbarium center of the Kerman Faculty of
Pharmacy, Kerman University of Medical Sciences, Kerman, Iran. The shape, size, and color of the
capsules were similar. The contents of capsules were 1 gr of whole BP or Starch powder. Before
supplementation, the capsules were placed alongside each other for one week for their smell to rub off on
each other. The absorbed Bunium Persicum volatile oil in the placebo capsules was very low to be able to
change health parameters. According to a similar previous study that investigated the effect of cumin
(one of the Apiaceae family plants) on overweight and obese women, the dose of supplements was
determined at 2 grams per day or 2 capsules per day, taken with meals (twice a day with lunch and
dinner) (38). However, the intake and absorption of the BP either with or without food should be
investigated further. The supplements were distributed monthly and the checking of compliance status
was also done monthly face to face and weekly over the telephone. The duration of the intervention
period was 8 weeks. The BP voucher number was Bunium Persicum (Boiss) B. Fedtch, Family: Apiaceae, KF1141-1. The
analysis of the whole BP was performed by the Herbarium center of the Kerman Faculty of Pharmacy,
Kerman University of Medical Sciences, Kerman, Iran. The contents of Bunium Persicum essential oil
according to the gas chromatography-mass spectrometry (GC-MS) was 46.10 % γ-Terpinene and 15.50 %
cumin aldehyde. -Assessments and Measurements: Anthropometric measurements: Weight (at the beginning and end), height (at the beginning), and waist circumference (at the beginning
and end) were determined by using a digital scale and stadiometer (Seca® Germany, Model: 755
1021994). They were measured as such: Weight without shoes, with minimal clothing, and with 100-gram
accuracy; height without shoes, standing, heels sticking to the wall, flat and forward head, and with 0.5-
centimeter accuracy; and waist circumference with minimal clothing, at the middle of the last rib and the
iliac crest. Body mass index (BMI) was calculated via dividing weight in kilograms by squared height in
meters. General Characteristics, Dietary Intakes, and Physical Activity: The T2DM patients were identified, the eligibility criteria checked, the study details were clarified, and
informed consent was obtained by the main investigator (BSC student). The questionnaires including the
general questionnaire, 24-hour food recall (at the beginning and end), and short-form IPAQ (SF-IPAQ)
questionnaire (at the beginning and end) were administered through interviewing. The dietary status was determined using gram per day of values from 24-hour food recall (valid in Iran
(39)) by the Nutritionist 4 software (39, 40). The IPAQ questionnaire provides information on physical activity that people do as part of their everyday
lives. The questions are about the time when the person has been active during the last 7 days. This
questionnaire addresses the activities in the workplace or as part of the homework and the garden, place
to place movement, exercises, and leisure activities. It also considers all the intense activities over the
past 7 days. Intense activities require a great deal of physical power and more intense breathing. The
IPAQ addresses only continuous activities for at least 10 minutes. Its short-form has 7 classified Page 5/19 Page 5/19 questions determining the three activity levels (1-3 or low-to-high levels). This questionnaire had been
validated in Iran (41, 42). HOMA-IR = FBI [µiu/ml] × FBS [mg/dl] / 405 HOMA-IR = FBI [µiu/ml] × FBS [mg/dl] / 405 Blood biomarkers measurement: At the beginning and the end of the study, 10 ml of blood (at the beginning and end) was taken from the
peripheral vein after 12-hour fasting during the night and centrifuged for 20 min (3000 g). Serum glucose
was determined on the same day of blood withdrawal. The remaining serums (5 ml) were frozen and
stored at -80°C up to the analysis. Serum nesfatin was determined using the sandwich ELISA and kit as Shanghai Crystal Day Biotech Co. Ltd®; Intra-assay CV<8 %, Inter-assay CV<10 % by an automatic device (Elisys Uno Human®). Similarly,
the ELISA kit for insulin was diametra® Co of Italy, DCM076-8; Intra-assay CV≤5 %, Inter-assay CV≤10 %. Serum glucose was measured according to the glucose oxidase method using the Hitachi analyzer device
(q17®) and the specific kit as Bionik®, Liquid Stable, Glucose oxidase GOD-POD, Mono-reagent; Intra-
assay CV≤2.10 %, Inter-assay CV≤3.09 %. Also, the serum levels of TC, TG, LDL-C, and HDL-C were
measured using Hitachi analyzer device (q17®) and the specific kits as 1-Bionik®, Liquid Stable,
Enzymatic Colorimetric CHOD-POD, 2-Bionik®, Liquid Stable, GPO-POD, Mono-reagent, 3-Bionik®, Liquid
Stable, Direct. Enzymatic Colorimetric, and 4-Bionik®, Liquid Stable, Direct. Enzymatic Colorimetric,
respectively. The intra- and inter-assay coefficients of variation for TC, TG, LDL-C, and HDL-C were
≤1.22% and ≤6.90%, ≤1.57% and ≤7.70%, ≤1.76% and ≤0.65 %, and ≤0.70% and ≤1.50%, respectively. HOMA-IR and QUICKI indices were calculated by the following formulas: -Participants’ characteristics According to Figure 1, overall, 680 people were screened based on medical history. Specifically, 82
subjects had the eligibility criteria, of whom 12 declined and 4 could not participate. Also, 66 subjects
were randomized, with 3 subjects in the BP group and 3 subjects in the placebo group refusing to
participate and as such did not receive the intervention. Thus, the first visit was completed with 60
subjects (bunuim persicum n = 30; placebo n = 30). Also, 6 subjects could not continue the follow-up
stage (for personal reasons, travel and did not take more than 10% of the intervention supplement;
bunuim persicum n = 3; placebo n = 3). Further, the baseline serum sample of two subjects in the placebo
group was not available. Eventually, data analysis was performed on 52 subjects. The general characteristics and physical activity level of the patients are presented in Table 1. Most of the
participants had a similar education, high economic, and low physical activity level. Both groups used
more than 95% of the prescribed supplements. Sample Size: The sample size was calculated using the ’two mean comparison formula‘ and one of the similar
previous studies (43), considering the cholesterol as the main variable, and errors I and II, the mean Page 6/19 difference of cholesterol between the groups, the standard deviation of group 1 and 2 were 0.05 and 0.20,
17.38, 29.34 and 4.77 respectively. difference of cholesterol between the groups, the standard deviation of group 1 and 2 were 0.05 and 0.20,
17.38, 29.34 and 4.77 respectively. n=(Z1-α/2+Z1-β)2(S1
2+S2
2)/(µ1-µ2)2 The calculated sample size was 23 participants in each group. Considering the anticipation of a 30%
drop‐out, the sample size was estimated at 30 participants in each group. -Data Analysis and Accessibility: Data management including entry, security, coding, and storage was performed at this stage. The
Kolmogorov-Smirnov, Chi-square, and t-test assessed the normality of continuous variables as well as
categorical and continuous baseline characteristics, respectively. Two-way repeated-measures analysis
of variance (TWRM-ANOVA) was used to determine time effects and time by treatment interaction effects
on all dependent variables. Moreover, TWRM-ANOVA was adjusted for dietary intake of vitamin B12. Also,
95% confidence interval (CI) and a P-value<0.05 were considered for reporting the measurements. Data
analysis was conducted using SPSS16 (statistical package for the social sciences). The main researcher
had access to the final dataset and the results were presented by the publication. -Safety Any side effects related to the treatment were reported, and only vomiting was observed in one patient in
the BP group in one of his follow-ups. Any side effects related to the treatment were reported, and only vomiting was observed in one patient in
the BP group in one of his follow-ups. -Changes in dietary intake and blood biomarkers The dietary intake of carbohydrates, protein percent, and iron in the baseline were higher in the BP group,
while the other baseline features were similar in the two groups (Tables 3). The dietary intake of vitamin B12 during the study was higher in the BP group (P<0.05, Table 3), while the
other dietary intakes were almost similar in the two groups. This significant intake was considered as a Page 7/19 confounder in the final analysis model. Within the BP group, the mean difference of TC, TG, HDL, and LDL
were not significant (P>0.05). On the other hand, FBS, FBI, BMI, WC, and HOMA-IR reduced, while QUICKI
and nesfatin-1 increased significantly (P<0.05). Within the placebo group, the mean differences of FBS,
FBI, HOMA-IR, QUICKI, TC, TG, LDL-C, HDL-C, and nesfatin-1 were not significant (P>0.05) (Table 4). According to the time by treatment interaction effect in the final analysis model, FBS, HOMA-IR, BMI, and
WC declined while QUICKI increased significantly in the BP group in comparison with the placebo group
(P<0.05) (Table 4). In other words, BP in comparison with placebo significantly elevated QUICKI and
reduced FBS and HOMA-IR (P<0.05). After adjustment for confounders, the significant changes were
similar (P<0.05) with an exception of WC (P=0.12) (Table 4). Discussion This may be attributed to the sample size and form of the
supplementation in our intervention. A hypothesis for the possible mechanism of hypoglycemic activity
of this plant may be through its main bioactive compounds. The hypoglycemic effect of limonene has
been previously reported in diabetic rats by decreasing the activities of gluconeogenic enzymes, In several animal studies, caraway (Bunuim Persicum and Carum Carvi) significantly decreased FBS
levels (36, 56, 57). In contrast with our study, the caraway in obese and overweight patients over 12 weeks
had no significant effects on FBS (31). This may be attributed to the sample size and form of the
supplementation in our intervention. A hypothesis for the possible mechanism of hypoglycemic activity
of this plant may be through its main bioactive compounds. The hypoglycemic effect of limonene has
been previously reported in diabetic rats by decreasing the activities of gluconeogenic enzymes,
increasing the glycolytic enzymes, and stimulating insulin secretion in pancreatic β-cells (58, 59). Cumin
aldehyde in the Bunium Persicum oilseeds has significant inhibitory activity against the α-glucosidase
enzyme that catalyzes the final step in the digestive process of carbohydrates. The inhibitory effect of
cumin aldehyde can postpone glucose uptake and reduce hyperglycemia (26). Also, nesfatin-1 can
improve glucose indices through many mechanisms such as the enhancement of glucose-induced insulin
secretion by promoting ca2+ influx through L-type channels (60, 61). Thus, the present improvements may
be attributed to increased nesfatin-1 levels. Even though in our study lipid profile was not changed significantly, two human studies demonstrated the
improvement of lipid profile by using caraway. The beneficial effects of caraway on lipid profile including
LDL, HDL, TG, and cholesterol had been observed in most of the animal models (19, 62). In our study, black caraway significantly reduced body weight, body mass index compared to the placebo
group although waist circumference reduction was not significant. This may be attributed to the short
time of the intervention in our study. Results in our study are almost consistent with previous studies. Caraway extract supplementation in overweight and obese women for 90 days decreased weight, body
mass index, body fat percentage, and waist-to-hip ratio (43). Another human study on the effects of taking
the caraway supplement and eight weeks of aerobic exercise showed a significant alteration in the weight
and BMI in the supplement-exercise group compared to the other groups (63). Discussion This was the first time the effects of Bunium Persicum (black caraway) on blood glucose indices, lipid
profile, and serum levels of nesfatin-1 in overweight or obese patients with type 2 diabetes mellitus
(T2DM) had been assessed. The different clinical usages and the lack of awareness concerning the
advantages and disadvantages of Bunium Persicum in patients with T2DM have made it very relevant. According to both unadjusted and adjusted analysis models, consumption of 2,000 mg of BP for 8 weeks
caused a significant reduction in FBG, HOMA-IR, and BMI and increased QUICKI in the intervention group
compared with the placebo group. Furthermore, the decrease in WC was significant in the unadjusted
model but not significant in the adjusted model. Limited human studies have investigated the effect of Bunium Persicum on glucose indices and lipid
profiles and according to our knowledge, no study has examined the effects of Bunium Persicum on
serum levels of nesfatin-1 in these patients. Some results of different studies on the effects of Bunium
Persicum or other plants of the Apiaceae family have shown some contradictions are presented below. The flavonoids, especially quercetin, can increase nesfatin-1 gene expression (44). In our study, Bunium
Persicum increased the serum nesfatin-1 significantly in the BP group, however, the increase of nesfatin-1
in the BP group in comparison with the placebo group was not significant. The reasons are likely to be the
short time of intervention and the lower sample size in the study. Furthermore, Kaempferol is a natural
flavonol, a type of flavonoid that can up-regulate the satiety gene NUCB-2/nesfatin-1 which plays an
important role in controlling energy balance, reducing body weight and increasing satiety (13, 45-47). Nesfatin-1 can produce several activities including suppression of inflammation, lipid mobilization
activity, enhancement of antioxidant defense, and a regulatory role in glucose homeostasis that may
prevent and reduce the complications, development, and progression of T2DM (48-50). Nesfatin-1 mRNA is
expressed in white adipose tissue, gastric mucosa, and central nervous system (CNS) such as the Page 8/19 hypothalamus and brainstem (51-54). However, nesfatin-1 was identified in human plasma and negatively
correlated with body mass index (55). In several animal studies, caraway (Bunuim Persicum and Carum Carvi) significantly decreased FBS
levels (36, 56, 57). In contrast with our study, the caraway in obese and overweight patients over 12 weeks
had no significant effects on FBS (31). Conclusion BP supplementation in overweight or obese T2DM patients showed a significant beneficial effect on
serum glucose indices and BMI. Further trials are required to use BP in clinical practice. Discussion The explained mechanisms
in the improvement of weight, BMI, and WC may be related to the increased levels of serum nesfatin-1 by
BP. Nesfatin-1 can affect energy homeostasis, decrease appetite, and reduce body weight (64-66). The observed metabolic and clinical response to BP in T2DM patients would make this study relevant. Although BP has some effects on some clinical and metabolic indicators in our study, these effects are
small and require further studies with larger sample size and longer duration and in different diseases. This study has several strengths in its procedure: To start, there was the first assessment of BP effects in
overweight or obese T2DM patients especially by assessing nesfatin-1 levels; second, the double-blinded
stratified blocked randomization design; third, assessing dietary intake and physical activity status and
adjustment in them and other potential confounders; fourth, considering the control group and fifth,
considering multiple eligibility criteria. Page 9/19 However, our study had some limitations. First, the sample size was small, second, the intervention
duration was short to understand the real effects of bunuim persicum (black caraway) supplementation,
third, self-reporting of diet and physical activity, fourth, failure to check the bioavailability of BP and
measure serum levels of its components, fifth, 24-h food recall is not a good index for assessing the
usual food intake sixth, failure to measure body composition and blood pressure, seventh, no assessment
of the dietary intake status of the participants during the intervention period, and eighth, the effect size
was small. Abbreviations T2DM: type 2 diabetes mellitus, BP: Bunium Persicum, BMI: body mass index, IPAQ: international physical
activity questionnaire, I.C.V injection: Intracerebroventricular injection, TG: triglyceride, ELISA: enzyme-
linked immunosorbent assay, TWRM-ANOVA: two-way repeated-measures analysis of covariance Declarations -Ethics approval and consent to participate: This trial was approved by the Ethics Committee of Zahedan
University of Medical Sciences (Ethical Code: IR.ZAUMS.REC.1397.332). A written informed consent form
(in Persian) and informed assent (in Persian) obtained from all patients. Participation was free, and a
patient could withdraw at whatever point the person feels he/she was unable to continue. The lifestyle
advice was presented free to the patients and there was no bar to receiving the other health care services
of the center. Side-effects of Bunium Persicum (Black Caraway) supplement (up to 2 gram/day) have not
been reported previously. The personal information of patients was kept secret before and after the study. -Consent to publish: Not applicable. -Consent to publish: Not applicable. -Availability of data and materials: The datasets used and/or analyzed during the current study are
available from the corresponding author on a reasonable request. -Competing Interests: There is no potential conflict of interest to research, authorship, and publication. -Funding: Funding was supported by the Zahedan University of Medical Sciences. The funding body did
not have a role in the design of the study and collection, analysis, interpretation of data and writing the
manuscript. -Authors Contributions: MS and MDM contributed equally to this work. SJM, MS, MDM, and ZM
conceived and developed the idea for the paper and revised the manuscript. ASG wrote numerous drafts. Page 10/19 Page 10/19 ADP contributed to statistical interpretations. All authors reviewed and revised the manuscript. All authors
read and approved the final manuscript. -Acknowledgments: The support of Zahedan University of Medical Sciences and the cooperation of the
Bu-Ali hospital of Zahedan and participants are acknowledged. -Acknowledgments: The support of Zahedan University of Medical Sciences and the cooperation of the
Bu-Ali hospital of Zahedan and participants are acknowledged. References 1. Control CfD, Prevention. National diabetes statistics report, 2017. Atlanta, GA: Centers for Disease
Control and Prevention, US Department of Health and Human Services. 2017. 2. Yazdanpanah L, Shahbazian H, Aleali AM, Jahanshahi A, Ghanbari S, Latifi S. Prevalence, awareness
and risk factors of diabetes in Ahvaz (South West of Iran). Diabetes & Metabolic Syndrome: Clinical
Research & Reviews. 2016;10(2):S114-S8. 3. Lovic D, Piperidou A, Zografou I, Grassos H, Pittaras A, Manolis A. The Growing Epidemic of Diabetes
Mellitus. Current Vascular Pharmacology. 2020;18(2):104-9. 4. Zheng M, Yang S, Liu Y, He Q, Hu P, editors. A Model for Physical Activity Behavioural Change in
Middle Aged and Older People with Type 2 Diabetes. E3S Web of Conferences; 2019: EDP Sciences. 4. Zheng M, Yang S, Liu Y, He Q, Hu P, editors. A Model for Physical Activity Behavioural Change in
Middle Aged and Older People with Type 2 Diabetes. E3S Web of Conferences; 2019: EDP Sciences. 5. Chen G-Y, Li L, Dai F, Li X-J, Xu X-X, Fan J-G. Prevalence of and risk factors for type 2 diabetes
mellitus in hyperlipidemia in China. Medical science monitor: international medical journal of
experimental and clinical research. 2015;21:2476. 5. Chen G-Y, Li L, Dai F, Li X-J, Xu X-X, Fan J-G. Prevalence of and risk factors for type 2 diabetes
mellitus in hyperlipidemia in China. Medical science monitor: international medical journal of
experimental and clinical research. 2015;21:2476. 6. Thévenod F. Pathophysiology of diabetes mellitus type 2: roles of obesity, insulin resistance and β-
cell dysfunction. Diabetes and Cancer. 19: Karger Publishers; 2008. p. 1-18. 6. Thévenod F. Pathophysiology of diabetes mellitus type 2: roles of obesity, insulin resistance and β-
cell dysfunction. Diabetes and Cancer. 19: Karger Publishers; 2008. p. 1-18. 7. Smitka K, Marešová D. Adipose tissue as an endocrine organ: an update on pro-inflammatory and
anti-inflammatory microenvironment. Prague Med Rep. 2015;116(2):87-111. 7. Smitka K, Marešová D. Adipose tissue as an endocrine organ: an update on pro-inflammatory and
anti-inflammatory microenvironment. Prague Med Rep. 2015;116(2):87-111. 8. Saldanha J, Carrero J, Lobo J, Stockler-Pinto M, Leal V, Calixto A, et al. The newly identified
anorexigenic adipokine nesfatin-1 in hemodialysis patients: are there associations with food intake,
body composition and inflammation? Regulatory peptides. 2012;173(1-3):82-5. 9. Ayada C, Toru Ü, Korkut Y. Nesfatin-1 and its effects on different systems. Hippokratia. 2015;19(1):4. 9. Ayada C, Toru Ü, Korkut Y. Nesfatin-1 and its effects on different systems. Hippokratia. 2015;19(1):4. References 10. Gonzalez R, Kerbel B, Chun A, Unniappan S. Molecular, cellular and physiological evidences for the
anorexigenic actions of nesfatin-1 in goldfish. PLoS One. 2010;5(12). 10. Gonzalez R, Kerbel B, Chun A, Unniappan S. Molecular, cellular and physiological evidences for the
anorexigenic actions of nesfatin-1 in goldfish. PLoS One. 2010;5(12). 11. Jazayeri-Tehrani SA, Rezayat SM, Mansouri S, Qorbani M, Alavian SM, Daneshi-Maskooni M, et al. Nano-curcumin improves glucose indices, lipids, inflammation, and Nesfatin in overweight and obese
patients with non-alcoholic fatty liver disease (NAFLD): a double-blind randomized placebo-
controlled clinical trial. Nutrition & metabolism. 2019;16(1):8. 12. Başar Ö, Akbal E, Köklü S, Koçak E, Tuna Y, Ekiz F, et al. A novel appetite peptide, nesfatin-1 in
patients with non-alcoholic fatty liver disease. Scandinavian journal of clinical and laboratory
investigation. 2012;72(6):479-83. Page 11/19 13. Oh S, Shimizu H, Satoh T, Okada S, Adachi S, Inoue K, et al. Identification of nesfatin-1 as a satiety
molecule in the hypothalamus. Nature. 2006;443(7112):709-12. 14. Li Q-C, Wang H-Y, Chen X, Guan H-Z, Jiang Z-Y. Fasting plasma levels of nesfatin-1 in patients with
type 1 and type 2 diabetes mellitus and the nutrient-related fluctuation of nesfatin-1 level in normal
humans. Regulatory peptides. 2010;159(1-3):72-7. 15. Malaguti-Boyle M. Evidence for the effectiveness of clinical nutrition therapy in diabetes mellitus
type 2: management in primary care. Journal of the Australian Traditional-Medicine Society. 2016;22(2):74. 16. Odegard PS, Capoccia K. Medication taking and diabetes. The Diabetes Educator. 2007;33(6):1014-
29. 17. Chang Hy, Wallis M, Tiralongo E. Use of complementary and alternative medicine among people
living with diabetes: literature review. Journal of advanced nursing. 2007;58(4):307-19. 18. Jabbari M, Daneshfard B, Emtiazy M, Khiveh A, Hashempur MH. Biological effects and clinical
applications of dwarf elder (Sambucus ebulus L): A review. Journal of evidence-based
complementary & alternative medicine. 2017;22(4):996-1001. 19. Haidari F, Seyed-Sadjadi N, Taha-Jalali M, Mohammed-Shahi M. The effect of oral administration of
Carum carvi on weight, serum glucose, and lipid profile in streptozotocin-induced diabetic rats. Saudi
Med J. 2011;32(7):695-700. 19. Haidari F, Seyed-Sadjadi N, Taha-Jalali M, Mohammed-Shahi M. The effect of oral administration of
Carum carvi on weight, serum glucose, and lipid profile in streptozotocin-induced diabetic rats. Saudi
Med J. 2011;32(7):695-700. 20. Eddouks M, Maghrani M, Lemhadri A, Ouahidi M-L, Jouad H. Ethnopharmacological survey of
medicinal plants used for the treatment of diabetes mellitus, hypertension and cardiac diseases in
the south-east region of Morocco (Tafilalet). Journal of ethnopharmacology. 2002;82(2-3):97-103. 20. References Eddouks M, Maghrani M, Lemhadri A, Ouahidi M-L, Jouad H. Ethnopharmacological survey of
medicinal plants used for the treatment of diabetes mellitus, hypertension and cardiac diseases in
the south-east region of Morocco (Tafilalet). Journal of ethnopharmacology. 2002;82(2-3):97-103. 21. Hajhashemi V, Sajjadi SE, Zomorodkia M. Antinociceptive and anti-inflammatory activities of Bunium
persicum essential oil, hydroalcoholic and polyphenolic extracts in animal models. Pharmaceutical
biology. 2011;49(2):146-51. 21. Hajhashemi V, Sajjadi SE, Zomorodkia M. Antinociceptive and anti-inflammatory activities of Bunium
persicum essential oil, hydroalcoholic and polyphenolic extracts in animal models. Pharmaceutical
biology. 2011;49(2):146-51. 22. Nickavar B, Adeli A, Nickavar A. Analyses of the essential oil from Bunium persicum fruit and its
antioxidant constituents. Journal of oleo science. 2014;63(7):741-6. 22. Nickavar B, Adeli A, Nickavar A. Analyses of the essential oil from Bunium persicum fruit and its
antioxidant constituents. Journal of oleo science. 2014;63(7):741-6. 23. Sharififar F, Yassa N, Mozaffarian V. Bioactivity of major components from the seeds of Bunium
persicum (Boiss.) Fedtch. Pakistan journal of pharmaceutical sciences. 2010;23(3). 23. Sharififar F, Yassa N, Mozaffarian V. Bioactivity of major components from the seeds of Bunium
persicum (Boiss.) Fedtch. Pakistan journal of pharmaceutical sciences. 2010;23(3). 24. Azizi M, Davareehejad G, Bos R, Woerdenbag HJ, Kayser O, Davareenejad G. The Essential Oil
Content and Constituents of Black Zira (Bunium persicum [Boiss.] B. Fedtsch.) during Field
Cultivation. 한국원예학회 기타간행물. 2006:89-. 25. Seri A, Khorsand M, Rezaei Z, Hamedi A, Takhshid MA. Inhibitory effect of bunium persicum
hydroalcoholic extract on glucose-induced albumin glycation, oxidation, and aggregation in vitro. Iranian journal of medical sciences. 2017;42(4):369. 25. Seri A, Khorsand M, Rezaei Z, Hamedi A, Takhshid MA. Inhibitory effect of bunium persicum
hydroalcoholic extract on glucose-induced albumin glycation, oxidation, and aggregation in vitro. Iranian journal of medical sciences. 2017;42(4):369. 26. Lee H-S. Cuminaldehyde: aldose reductase and α-glucosidase inhibitor derived from Cuminum
cyminum L. seeds. Journal of agricultural and food chemistry. 2005;53(7):2446-50. 26. Lee H-S. Cuminaldehyde: aldose reductase and α-glucosidase inhibitor derived from Cuminum
cyminum L. seeds. Journal of agricultural and food chemistry. 2005;53(7):2446-50. Page 12/19
27. Patil SB, Takalikar SS, Joglekar MM, Haldavnekar VS, Arvindekar AU. Insulinotropic and β-cell
protective action of cuminaldehyde, cuminol and an inhibitor isolated from Cuminum cyminum in Page 12/19
27. Patil SB, Takalikar SS, Joglekar MM, Haldavnekar VS, Arvindekar AU. Insulinotropic and β-cell
protective action of cuminaldehyde, cuminol and an inhibitor isolated from Cuminum cyminum in streptozotocin-induced diabetic rats. British journal of nutrition. 2013;110(8):1434-43. 28. References Haque MR, Ansari HS. Anti-obesity effect of Arq Zeera and Its main components thymol and
cuminaldehyde in high fat diet induced obese rats. Drug research. 2018;68(11):637-47. 29. Haque M, Ansari S. Aromatic aldehyde compound cuminaldehyde protects nonalcoholic fatty liver
disease in rats feeding high fat diet. Human & experimental toxicology. 2019;38(7):823-32. 30. Khaksari M, Ahmadi M, Najafipour H, Shahrokhi N. Effect of Bunium persicum aqueous extract plus
endurance exercise on cardiorespiratory capacity and serum lipid profile. Avicenna journal of
phytomedicine. 2014;4(2):118. 31. Kazemipoor M, Cordell GA. Clinical effects of caraway, a traditional medicine for weight loss. Evidence-Based Validation of Herbal Medicine: Elsevier; 2015. p. 339-62. 32. Ghorbani S, Alizadeh R, Moradi L. The effect of high intensity interval training along with
consumption of caraway seeds (Carum carvi L.) on liver enzymes, lipid profile, and blood glucose in
obese and overweight women. Ebnesina. 2017;19(2):12-20. 32. Ghorbani S, Alizadeh R, Moradi L. The effect of high intensity interval training along with
consumption of caraway seeds (Carum carvi L.) on liver enzymes, lipid profile, and blood glucose in
obese and overweight women. Ebnesina. 2017;19(2):12-20. 33. Hassanzad Azar H, Taami B, Aminzare M, Daneshamooz S. Bunium persicum (Boiss.) B. Fedtsch: An
overview on Phytochemistry, Therapeutic uses and its application in the food industry. Journal of
Applied Pharmaceutical Science. 2018;8(10):150-8. 33. Hassanzad Azar H, Taami B, Aminzare M, Daneshamooz S. Bunium persicum (Boiss.) B. Fedtsch: An
overview on Phytochemistry, Therapeutic uses and its application in the food industry. Journal of
Applied Pharmaceutical Science. 2018;8(10):150-8. 34. Moubarz G, Taha MM, Mahdy-Abdallah H. Antioxidant effect of Carum carvi on the immune status of
streptozotocin-induced diabetic rats infected with Staphylococcus aureus. World Applied Sciences
Journal. 2014;30(1):63-9. 34. Moubarz G, Taha MM, Mahdy-Abdallah H. Antioxidant effect of Carum carvi on the immune status of
streptozotocin-induced diabetic rats infected with Staphylococcus aureus. World Applied Sciences
Journal. 2014;30(1):63-9. 35. Kazemipoor M, Hamzah S, Hajifaraji M, Radzi CWJBWM, Cordell GA. Slimming and Appetite‐
Suppressing Effects of Caraway Aqueous Extract as a Natural Therapy in Physically Active Women. Phytotherapy Research. 2016;30(6):981-7. 35. Kazemipoor M, Hamzah S, Hajifaraji M, Radzi CWJBWM, Cordell GA. Slimming and Appetite‐
Suppressing Effects of Caraway Aqueous Extract as a Natural Therapy in Physically Active Women. Phytotherapy Research. 2016;30(6):981-7. 36. Sadjadi NS, Shahi MM, Jalali M-T, Haidari F. Short-Term Caraway Extract Administration Improves
Cardiovascular Disease Risk Markers in Streptozotocin-Induced Diabetic Rats: A Dose-Response
Study. Journal of Dietary Supplements. 2014;11(1):30-9. 36. References Sadjadi NS, Shahi MM, Jalali M-T, Haidari F. Short-Term Caraway Extract Administration Improves
Cardiovascular Disease Risk Markers in Streptozotocin-Induced Diabetic Rats: A Dose-Response
Study. Journal of Dietary Supplements. 2014;11(1):30-9. 37. Mirzaei K, Hossein-nezhad A, Keshavarz SA, Koohdani F, Eshraghian MR, Saboor-Yaraghi AA, et al. Association of nesfatin-1 level with body composition, dietary intake and resting metabolic rate in
obese and morbid obese subjects. Diabetes & Metabolic Syndrome: Clinical Research & Reviews. 2015;9(4):292-8. 38. Zare R, Heshmati F, Fallahzadeh H, Nadjarzadeh A. Effect of cumin powder on body composition and
lipid profile in overweight and obese women. Complementary therapies in clinical practice. 2014;20(4):297-301. 39. JAZAYERI S, NOURI M, POUREBRAHIM R, FAKHRZADEH H, ARDESHIR LARIJANI MB. FOOD AND
NUTRIENT INTAKES AMONG 20-60 AGED INHABITANTS OF TEHRAN UNIVERSITY OF MEDICAL
SCIENCES POPULATION LAB REGION. JOURNAL OF DIABETES AND METABOLIC DISORDERS. 2004;3(Supplement 1 (MONICA PROJRCT)):-. 40. Ghafarpour M, Houshiar-Rad A, Kianfar H, Ghaffarpour M. The manual for household measures,
cooking yields factors and edible portion of food. 1999. Page 13/19 41. Baghiani-Moghaddam M, Bakhtari-Aghdam F, Asghari-Jafarabadi M, Allahverdipour H, Saeed
Dabagh-Nikookheslat S, Nourizadeh R. Comparing the results of pedometer-based data and
International Physical Activity Questionnaire (IPAQ). J Health Syst Res. 2013;9(6):605-12. 42. Vahedian-Shahroodi M, Tehrani H, Dogonchi M, Lael-monfared E. The Effectiveness of Physical
Activity Training on Depersonalization and lack of accomplishment of Employees. Iranian Journal of
Health Education and Health Promotion. 2015;3(2):116-24. 43. Kazemipoor M, Hajifaraji M, Haerian BS, Mosaddegh MH, Cordell GA. Antiobesity effect of caraway
extract on overweight and obese women: a randomized, triple-blind, placebo-controlled clinical trial. Evidence-Based Complementary and Alternative Medicine. 2013;2013. 44. Khorchani MJ, Zal F, Neisy A. The phytoestrogen, quercetin, in serum, uterus and ovary as a potential
treatment for dehydroepiandrosterone-induced polycystic ovary syndrome in the rat. Reproduction,
Fertility and Development. 2020;32(3):313-21. 45. Hossain AM, Brennan MA, Guo X, Zeng X-A, Brennan CS. Cellular biological activity and regulation of
gene expression of antioxidant dietary fibre fraction isolated from blackcurrant incorporated in the
wholemeal cereals cookies. Food Chemistry. 2020;312:125829. 46. García-Galiano D, Pineda R, Ilhan T, Castellano J, Ruiz-Pino F, Sánchez-Garrido M, et al. Cellular
distribution, regulated expression, and functional role of the anorexigenic peptide, NUCB2/nesfatin-1,
in the testis. Endocrinology. 2012;153(4):1959-71. 46. García-Galiano D, Pineda R, Ilhan T, Castellano J, Ruiz-Pino F, Sánchez-Garrido M, et al. Cellular
distribution, regulated expression, and functional role of the anorexigenic peptide, NUCB2/nesfatin-1,
in the testis. Endocrinology. 2012;153(4):1959-71. 47. References Holland TM, Agarwal P, Wang Y, Leurgans SE, Bennett DA, Booth SL, et al. Dietary flavonols and risk
of Alzheimer dementia. Neurology. 2020. 47. Holland TM, Agarwal P, Wang Y, Leurgans SE, Bennett DA, Booth SL, et al. Dietary flavonols and risk
of Alzheimer dementia. Neurology. 2020. 48. Jiang L, Xu K, Li J, Zhou X, Xu L, Wu Z, et al. Nesfatin-1 suppresses interleukin-1β-induced
inflammation, apoptosis, and cartilage matrix destruction in chondrocytes and ameliorates
osteoarthritis in rats. Aging (Albany NY). 2020;12(2):1760. 49. Liu Y, Chen X, Qu Y, Song L, Lin Q, Li M, et al. Central nesfatin‐1 activates lipid mobilization in
adipose tissue and fatty acid oxidation in muscle via the sympathetic nervous system. BioFactors. 2020. 50. Li Z, Gao L, Tang H, Yin Y, Xiang X, Li Y, et al. Peripheral effects of nesfatin-1 on glucose
homeostasis. PloS one. 2013;8(8). 51. SHimizu H, OH S, Okada S, Mori M. Nesfatin-1: an overview and future clinical application. Endocrine
journal. 2009;56(4):537-43. 52. Stengel A, Goebel M, Yakubov I, Wang L, Witcher D, Coskun T, et al. Identification and characterization
of nesfatin-1 immunoreactivity in endocrine cell types of the rat gastric oxyntic mucosa. Endocrinology. 2009;150(1):232-8. 53. Ramanjaneya M, Chen J, Brown JE, Tripathi G, Hallschmid M, Patel S, et al. Identification of nesfatin-
1 in human and murine adipose tissue: a novel depot-specific adipokine with increased levels in
obesity. Endocrinology. 2010;151(7):3169-80. 54. Kim J, Chung Y, Kim H, Im E, Lee H, Yang H. The tissue distribution of nesfatin-1/NUCB2 in mouse. Development & reproduction. 2014;18(4):301. Page 14/19 55. Tsuchiya T, Shimizu H, Yamada M, Osaki A, Oh-I S, Ariyama Y, et al. Fasting concentrations of
nesfatin-1 are negatively correlated with body mass index in non-obese males. Clinical
Endocrinology. 2010;73(4):484-90. 56. Eidi A, Eidi M, Haeri Rohani A, Basati F. Hypoglycemic effect of ethanolic extract of Carum carvi L. seeds in normal and streptozotocin-induced diabetic rats. Journal of Medicinal Plants. 2010;3(35):106-13. 57. Giancarlo § S, Rosa § LM, Nadjafi F, Francesco M. Hypoglycaemic activity of two spices extracts:
Rhus coriaria L. and Bunium persicum Boiss. Natural product research. 2006;20(9):882-6. 58. Murali R, Saravanan R. Antidiabetic effect of d-limonene, a monoterpene in streptozotocin-induced
diabetic rats. Biomedicine & Preventive Nutrition. 2012;2(4):269-75. 59. Conforti F, Statti GA, Tundis R, Loizzo MR, Menichini F. In vitro activities of Citrus medica L. cv. Diamante (Diamante citron) relevant to treatment of diabetes and Alzheimer's disease. References Phytotherapy
Research: An International Journal Devoted to Pharmacological and Toxicological Evaluation of
Natural Product Derivatives. 2007;21(5):427-33. 60. Nakata M, Manaka K, Yamamoto S, Mori M, Yada T. Nesfatin-1 enhances glucose-induced insulin
secretion by promoting Ca2+ influx through L-type channels in mouse islet β-cells. Endocrine journal. 2011:1102080532-. 61. Öztürk Özkan G. Effects of Nesfatin-1 on Food Intake and Hyperglycemia. Journal of the American
College of Nutrition. 2019:1-7. 62. Saghir MR, Sadiq S, Nayak S, Tahir MU. Hypolipidemic effect of aqueous extract of Carum carvi
(black Zeera) seeds in diet induced hyperlipidemic rats. Pakistan Journal of Pharmaceutical
Sciences. 2012;25(2). 63. Mohammadkhani PG, Irandoust K, Taheri M, Mirmoezzi M, Baić M. Effects of eight weeks of aerobic
exercise and taking caraway supplement on C-reactive protein and sleep quality in obese women. Biological Rhythm Research. 2019:1-9. 64. Tsuchiya T, Shimizu H, Yamada M, Osaki A, Oh‐I S, Ariyama Y, et al. Fasting concentrations of
nesfatin‐1 are negatively correlated with body mass index in non‐obese males. Clinical
endocrinology. 2010;73(4):484-90. 65. Abaci A, Catli G, Anik A, Kume T, Bober E. The relation of serum nesfatin‐1 level with metabolic and
clinical parameters in obese and healthy children. Pediatric diabetes. 2013;14(3):189-95. 66. Dong J, Xu H, Wang P-f, Cai G-j, Song H-f, Wang C-c, et al. Nesfatin-1 stimulates fatty-acid oxidation
by activating AMP-activated protein kinase in STZ-induced type 2 diabetic mice. PLoS One. 2013;8(12):e83397. *t-test; BMI: body mass index, WC: waist circumference, HOMA-IR: homeostasis model assessment-insulin
resistance, QUICKI: quantitative insulin sensitivity check index, FBS: fasting blood sugar, FBI: fasting blood Tables Table 1. General characteristics and physical activity of overweight or obese patients with
type 2 diabetes mellitus (T2DM) Page 15/19 Page 15/19 General variables and physical activity
Caraway
(n=27)
n(%) or
Mean(SD)
Placebo
(n=25)
n(%) or
Mean(SD)
P-
value
Age (yrs)
52.4(10.8)
50.1(10.9)
0.5*
Gender
male
8(30.0)
9(36.0)
0.6**
female
19(70.0)
16(64.0)
Blood
sugar‐lowering
agents
Metformin
18(66.7)
16(64.0)
0.8**
Glibenclamide
4(14.8)
5(20.0)
Metformin+Glibenclamide
4(14.8)
4(16)
other
1(3.7)
0(0.0)
Education level
up to associate degree
11(42.3)
10(47.6)
0.6**
Bachelor and higher
15(57.7)
11(52.4)
Economic level
Low/moderate (≤6 living
items)
7(26.9)
6(28.6)
0.2**
High (≥7 living items)
19(73.1)
15(71.4)
Physical activity level (Baseline)
1.4(0.6)
1.3(0.5)
0.6*
Physical activity level (End)
1.3(0.6)
1.4(0.5)
0.6* seline mean for BMI and serum nesfatin-1, glucose indices, and
r obese patients with type 2 diabetes mellitus (T2DM) . Comparison of baseline mean for BMI and serum nesfatin-1, glucose in
ofile in overweight or obese patients with type 2 diabetes mellitus (T2DM)
Baseline Dependent Variables
Caraway
(n=27)
n(%) or Mean(SD)
Placebo
(n=25)
n(%) or Mean(SD)
P-value*
BMI (kg/m2)
28.8(3.8)
29.2(3.3)
0.7
WC (cm)
100.32(7.06)
100.35(8.45)
0.9
FBS (mg/dl)
175.3(17.6)
174.0(17.9)
0.9
FBI (µIU/ml)
9.4(1.6)
11.6(1.9)
0.3
HOMA-IR (score)
4.2(3.0)
5.0(3.7)
0.5
QUICKI (score)
0.450(0.004)
0.445(0.002)
0.6
Nesfatin-1 (ng/ml)
4.4(1.6)
5.5(2.1)
0.07
TC (mg/dl)
159.0(36.0)
152.6(48)
0.6
TG (mg/dl)
163.3(117.1)
164.2(82.4)
0.9
LDL-C (mg/dl)
81.3(27.6)
83.3(29.6)
0.8
HDL-C (mg/dl)
45.0(12.0)
43.0(7.6)
0.5 Table 2. Comparison of baseline mean for BMI and serum nesfatin-1, glucose indices, and
lipid profile in overweight or obese patients with type 2 diabetes mellitus (T2DM) Page 16/19 Page 16/19 insulin, TC: total cholesterol, TG: triglyceride, HDL-C: high-density lipoprotein cholesterol, LDL-C: low-density
lipoprotein-cholesterol insulin, TC: total cholesterol, TG: triglyceride, HDL-C: high-density lipoprotein cholesterol, LDL-C: low-density
lipoprotein-cholesterol able 3. Tables Mean of dietary intakes during the study on overweight or obese patients with t
diabetes mellitus (T2DM)
Dietary intakes during the study
Bunium persicum (n=27)
Mean(95% CI)
Placebo (n=25)
Mean(95% CI)
P-value*
Energy (kcal)
1982.6(1951, 2014.2)
1799.3(1793.4, 1805.3)
0.45
Protein (g)
72.1(71.6, 72.6)
77.8(78.9, 76.8)
0.24
Protein (%)
14.4(14.5, 14.3)
17.3(17.5, 17.2)
0.76
Carbohydrate (g)
274.6(268.6, 280.7)
227.0(230.7, 223.3)
0.27
Carbohydrate (%)
54.8(54.5, 55.1)
49.6(50.4, 48.8)
0.25
Fat (g)
69.1(69.0, 69.3)
66.5(65.3, 67.8)
0.55
Fat (%)
30.8(31, 30.6)
33.0(32.0, 34.1)
0.17
Cholesterol (mg)
133.4(123.5, 143.3)
130.9(132.3, 129.5)
0.11
Saturated fat (g)
20.3(19.6, 21.1)
16.1(15.9, 16.3)
0.69
Monounsaturated fatty acid (g)
25.3(26.9, 23.7)
24.4(25.0, 23.9)
0.42
Polyunsaturated fatty acid (g)
17.0(16.4, 17.6)
17.8(17.7, 18.0)
0.63
Vitamin A [RAE] (µg)
806.2(884.3, 728.2)
676(752.7, 599.3)
0.99
Vitamin C (mg)
136.4(123.3, 149.5)
125.4(118.2, 132.6)
0.59
Potassium
2708.6(2625.0, 2792.3)
2588.1(2540.4, 2635.8)
0.81
Calcium (mg)
823.4(837.1, 809.8)
911.4(934.2, 889.4)
0.88
Iron (mg)
19.4(19.1, 19.7)
15.2(15.2, 15.3)
0.40
Vitamin D (µg)
0.6(0.5, 0.7)
0.5(0.4, 0.6)
0.81
Vitamin E (mg)
17.5(19.8, 15.3)
16.0(16.6, 15.4)
0.18
Vitamin B1 (mg)
1.9(2.1, 1.7)
1.8(1.9, 1.8)
0.20
Vitamin B2 (mg)
1.8(1.9, 1.7)
1.8(1.9, 1.7)
0.88
Vitamin B3 (mg)
23.3(23.9, 21.7)
25.7(25.6, 25.9)
0.33
Vitamin B6 (mg)
1.7(2.5, 2.4)
2.8(3.3, 2.4)
0.37
Folate (DFE) (µg)
143.7(138.3, 149.2)
144.7(147.5, 141.9)
0.51
Vitamin B12 (µg)
2.4(2.3, 2.5)
2.2(2.4, 2.0)
0.03
Vitamin K (µg)
182.0(129.3, 234.7)
154.9(106.8, 203.0)
0.91
Zinc (mg)
9.5(9.5, 9.6)
8.6(8.4, 8.8)
0.70
Selenium (µg)
33.1(32.2, 34.1)
39.2(39.0, 39.4)
0.82
Total fiber (g)
4.7(4.4, 5)
4.1(3.8, 4.5)
0.42 Table 3. Mean of dietary intakes during the study on overweight or obese patients with type
2 diabetes mellitus (T2DM) *Two way repeated measures-ANOVA (TWRM-ANOVA) Table 4. Tables The changes in BMI, WC, serum nesfatin-1, glucose indices, and lipid profile in
overweight or obese patients with type 2 diabetes mellitus (T2DM) Page 17/19 Page 17/19 Variables
Supplement
Baseline
Mean
End
Mean
P-
value$
Mean
Changes
(95 % CI)
P-value#
Time
Treatment
Interaction
BMI
Caraway
(n=27)
28.8(3.8)
28.3(4.0)
0.003
-0.5
(0.2, 0.7)
0.4
0.5
0.02
Placebo
(n=25)
29.2(3.3)
29.4(3.3)
0.4
0.2
(-.8, 0.3)
0.8
0.6
0.04
WC (cm)
Caraway
(n=27)
100.3(7.0)
99.0(7.4)
0.002
-1.3
(0.5, 2.0)
0.013
0.8
0.04
Placebo
(n=25)
100.3(8.4)
100.2(8.3)
0.7
-0.1
(-0.7, 0.9)
0.1
0.6
0.2
FBS (mg/dl)
Caraway
(n=27)
175.4(69.9)
142.6(53.8)
0.002
-32.8
(11.9,
51.2)
0.036
0.3
0.005
Placebo
(n=25)
174.0(52.3)
178.9(73.5)
0.6
4.9
(-22.7,
12.9)
0.2
0.8
0.02
FBI (µIU/ml)
Caraway
(n=27)
9.4(6.8)
5.6(2.5)
0.03
-3.8
(0.3, 7.3)
0.245
0.006
0.15
Placebo
(n=25)
11.6(7.5)
12.0(9.0)
0.9
0.4
(-5.1, 5.3)
0.140
0.01
0.05
HOMA-IR
Caraway
(n=27)
4.2(3.7)
2.3(1.3)
0.041
-1.9
(0.9, 3.8)
0.181
0.009
0.047
Placebo
(n=25)
5.0(3.1)
5.2(4.7)
0.87
0.2
(-2.5, 2.1)
0.173
0.034
0.018
QUICKI
Caraway
(n=27)
0.450(0.03)
0.47(0.03)
0.006
0.02
(-.03, 0.0)
0.014
0.128
0.035
Placebo
(n=25)
0.445(0.02)
0.446(0.03)
0.761
0.01
(-.04, 0.1)
0.050
0.4
0.054
TC (mg/dl)
Caraway
(n=27)
159.0(36.8)
159.5(37.1)
0.942
0.5
(-13.3,
12.4)
0.4
0.8
0.5
Placebo
(n=25)
152.6(49.0)
159.9(45.2)
0.395
7.3
(-24.9,
10.3)
0.8
0.6
0.9
TG (mg/dl)
Caraway
(n=27)
163.3(117.2)
175.3(121.4)
0.389
-12.0
(-39.9,
16.1)
0.1
0.9
0.8
Placebo
(n=25)
164.2(82.4)
179.3(100.4)
0.189
15.1
(-38.1, 8.0)
0.3
0.9
0.8
LDL-C
(mg/dl)
Caraway
(n=27)
81.3(27.6)
82.6(27.7)
0.842
1.3
(-13.7,
11.3)
0.6
0.9
0.4
Placebo
(n=25)
83.3(29.6)
77.8(39.3)
0.422
-5.5
(-8.5, 19.5)
0.9
0.4
0.1
HDL-C
(mg/dl)
Caraway
(n=27)
45.0(12.0)
44.2(11.2)
0.520
-0.8
(-1.6, 3.2)
0.9
0.7
0.3
Placebo
(n 25)
43.0(7.6)
44.0(9.2)
0.518
1.0
( 4 3 2 3)
0.7
0.8
0.3 Page 18/19 Page 18/19 Nesfatin-1
(ng/ml)
Caraway
(n=27)
4.4(1.6)
6.1(3.4)
0.021
1.7
(-3.2, 0.3)
0.01
0.3
0.59
Placebo
(n=25)
5.5(2.1)
6.6(3.9)
0.168
1.1
(-2.9, 0.5)
0.2
0.7
0.8 $Paired t-test; #Two way repeated measures-ANOVA (TWRM-ANOVA), top row P-value: unadjusted; bottom row $Paired t-test; #Two way repeated measures-ANOVA (TWRM-ANOVA), top row P-value: unadjusted; bottom row $Paired t-test; #Two way repeated measures-ANOVA (TWRM-ANOVA), top row P-value: unadjusted; bottom row
P-value: adjusted for vitamins B12 dietary intake $Paired t-test; #Two way repeated measures-ANOVA (TWRM-ANOVA), top row P P-value: adjusted for vitamins B12 dietary intake BMI: body mass index, WC: waist circumference, HOMA-IR: homeostasis model assessment-insulin resistance,
QUICKI: quantitative insulin sensitivity check index, FBS: fasting blood sugar, FBI: fasting blood insulin, TC:
total cholesterol, TG: triglyceride, HDL-C: high-density lipoprotein cholesterol, LDL-C: low-density lipoprotein-
cholesterol Figures Figures Figure 1 Figure 1 Flow chart of the study participants Page 19/19
|
https://openalex.org/W2095370893
|
https://zenodo.org/records/1622895/files/article.pdf
|
English
| null |
CXXXIX.—The bromination of silver cyanate
|
Journal of the Chemical Society. Transactions
| 1,904
|
public-domain
| 2,665
|
Published on 01 January 1904. Downloaded by University of Exeter on 19/07/2016 15:10:28. Published on 01 January 1904. Downloaded by University of Exeter on 19 View Article Online / Journal Homepage / Table of Contents for this issue View Article Online / Journal Homepage / Table of Contents for this issue DEAN: THE BROMINATION OF SILVER CYANATE. 1390 DEAN: THE BROMINATION OF SILVER CYANATE. Published on 01 January 1904. Downloaded by University of Exeter on 19/07/2016 15:10:28. 904. Downloaded by University of Exeter on 19/07/2016 15:10:28. blished on 01 January 1904. Downloaded by University of Exeter on 19/07/2016 15:10: The greater portion of the excess of bromine present mas drawn off
with the aid of the water-pump, but some difficulty was experienced
in getting rid of the last traces without having to raise the tempera-
ture. On gently heating, a few minute rosettes of needle-shaped
crystals were seen to have condensed from the vapour. These
crystals, which volatilised during the course of
the experiment,
probably consisted of cyanogen bromide. Published on 01 January 1904. Downloaded by University of E The final weight of the brominated cyanate was 15.065 grams, cor-
responding with an addition of 76 units per molecule of the silver salt. The presenceof cyanogen bromide in the vapour would be explained
if the reaction on bromination had followed the course represented by
the equation : 2AgCNO + 2Br, = 2AgBr + 2CNBr + 0,. In that case, it should have been accompanied by free oxygen. To
determine if this were so, several grams of silver cyanate were treated
with bromine in an exhausted flask and the gas pumped out by means
of a Sprengel pump. About 80 C.C. were obtained and found on
analysis to be a mixture of carbon dioxide and nitrogen, roughly in
the proportions 1 to 2-5, but no oxygen was present. The bromine
used had not in this instance been specially dried, and I suspected that
the gas produced might be due to the oxidising action of the moist
halogen, especially as the increase in weight after bromination was
much lower than usual, The experiment was accordingly repeated
with fresh material, which was prepared as follows. The silver cyanate
was obtained by evaporating a solution containing silver nitrate and
urea in molecular proportions, The mass of fine needle-shaped crystals
which separated was repeatedly washed with hot water, drained, dried
in a vacuum, heated for several hours at 1004 and finally left for some
weeks over concentrated sulphuric acid in a vacuum. Commercial
bromine was well washed with water, and then left with successive
solutions of potassium bromide which removed chlorine and cyanogen
chloride. Traces of iodine were eliminated by means of zinc oxide,
and water was removed by fused zinc bromide. cxxx1x.- The
of
Silver Cyan ate.
By GEORGE DEAN, M.A. By GEORGE DEAN, M.A. DURING
some experiments on the preparation of certain cyanogen
compounds, equivalent weights of silver cyanate and bromine were
taken in a sealed tube and gently heated, the bromine being intro-
duced in a thin glass bulb. On opening the tube, most of the halogen
bad disappeared, only a trace of vapour remaining in the enclosed air. The solid residue appeared to be an additive compound of silver
cyanate and bromine. When heated, it gave off bromine, and on the
addition of
water decomposed with effervescence, bromine being
evolved. Other specimens, which had been freed from uncombined
bromine by being kept for several weeks over caustic soda, irop
filings, or in a vacuum, behaved similarly. The substance was of a brittle, friable nature, and had a deep
yellow colour quite distinct from that of silver bromide ; when exposed
to light in a tightly-stgppered bottle, it preserved its colour indefinitely,
ID the presence of traces of moisture, silver bromide was produced, the
colour fading t o a pale yellow which slowly blackened. When a tube
containing the substance was plunged into a bath of sulphur at
300-400°,
the bromine evolved was accompanied by a thick white
sublimate of cyanogen bromide. At that temperature, a n interaction
had evidently taken place between part of the liberated halogen and
the silver cyanate. The following experiment was made in order to find out the reacting
proportions. Ten grams of cyanate were placed in a small, stoppered View Article Online DEAN: THE BROMINATION OF SILVER CYANATE. 1371 distilling flask and bromine added until the whole was liquid. During
the reaction, the heat evolved was sufficient to produce an appreciable
rise in temperature. Next morning, the dark brown mass had
solidified, and showed signs of crystalline structure, the surface being
crossed by several fine long needles. Published on 01 January 1904. Downloaded by University of Exeter on 19/07/2016 15:10:28. 904. Downloaded by University of Exeter on 19/07/2016 15:10:28. blished on 01 January 1904. Downloaded by University of Exeter on 19/07/2016 15:10: In order to determine the weight of bromine retained by the
cyanate when the conditions of temperature and pressure were varied,
the apparatus used was modified somewhat ; brominstion was effected
in a Wurtz flask, provided with a well-ground stopper, the side-tube
being connected with a large glass bulb, which also had two other
outlets. One of these communicated with a water-pump, through a
wash-bottle containing strong sulphuric acid, and the other could
be quickly opened to the air. The glass bulb thus employed served
as a trap between the flask and the drying bottle, and the latter pre-
vented the passage of water vapour from the pump to the substance. Published on 01 January 1904. Downloaded by University of E Much heat was evolved on bromination, and the product was then
left until the next day, more bromine being added on successive days
until the mass was quite liquid, Excess of bromine was then removed
by heating the flask to about 60' in a water-bath and gradually
diminishing the pressure to 300-400
mm. of mercury. As soon as
the space above the solid appeared to be free from bromine vapour,
the flask was disconnected and weighed. This gave the first deter-
mination of the added bromine. The flask was then replaced,
exhausted, and weighed. The details of the separate treatments,
together with the results obtained, are given below. Bromine. 1st exhaustion-continued
until no bromine vapour could be
2nd
3rd
4th
5th
6th
7th
8th
9th
10th
seen at 60' under 300-400 mm. pressure. In-
crease in weight per molecule of cyanate.. .... continued for 30 mins. at same temperature and
pressure as before .................................... 82.6
continued for 30 mins. at same temperature and
pressure as before .................................... 81.7
continued for 30 mins. at same temperature and
pressure as before.. .................................. 81 *3
continued for 30 mins. at same temperature and
pressure as before .................................... 81.0
af cer long exhaustion at 100" (300-400 mm.). .. after long exhaustion in the cold (300-400
mm.)
after long exhaustion to 10-20 mm. in the cold
on heating at 100' for 15 mins., bromine was
seen to be coming off, and therefore no weigh-
ing was made. on heating at the temperature OF a saturated
calcium
chloride
solution, bromine
was
evolved. Published on 01 January 1904. Downloaded by University of Exeter on 19/07/2016 15:10:28. The bromine thus
purified was finally distilled from fresh zinc bromide and the first and
last portions of the distillate rejected. On repeating the bromination in a vacuum with the dried substances,
several grams of cyanate evolved only 5 C.C. of gag, consisting approxi-
mately of 1 vol. of carbon dioxide and 2 of nitrogen. In no instance View Article Online View Article Online DEAN: TEE BROMINATION OF SILVER CYANATE. 1372 could any appreciable amount of oxygen have been produced, and the
gas evolved is apparently due to a secondary reaction caused by the
presence of water. Published on 01 January 1904. Downloaded by University of Exeter on 19/07/2016 15:10:28. tion of bromine vapour, which was still being tion of bromine vapour, which was still being given off when the apparatus was disconnected
66.92 The conclusions drawn from the foregoing experiment are as
follows : 1. The weight remains fairly steady when the composition of the
product corresponds with the formula (AgCNOBr),. 2. Dissociation does not take place below 70', but begins below 100'
p
p
g
and is not complete at 200'. 3. A t the ordinary temperature, dissociation can be produced by
p
lowering the pressure to 10-20
mm. of mercury. 3. A t the ordinary temperature, dissociation can be produced by
lowering the pressure to 10-20
mm. of mercury. Published on 01 January 1904. Downloaded by University of Exeter on 19/07/2016 15:10:28. 90.56
79.74
79.74
79.66 View Article Online View Article Online DEAN: THE BROMINATION OF SILVER CYANATE. 1373 Cromiiie . 11th exhaustion-on heating at 220', there was n steady evolu- 904. Downloaded by University of Exeter on 19/07/2016 15:10:28. Published on 01 January 1904. Downloaded by University of Exeter on 19/07/2016 15:10:28. shed on 01 January 1904. Downloaded by University of Exeter on 19/07/2016 15:10:28. Published on 01 January 1904. Downloaded by University of Exeter on 19/07/2016 15:10:28. Published on 01 January 1904. Downloaded by University of Exeter on 19/07/2016 15:10:28. y 1904. Downloaded by University of Exeter on 19/07/2016 15:10:28. Published on 01 January 1904. Downloaded by University of Exeter on 19/07/2016 15:1 Hydrogen Cldo&le.-On
passing the dried gas over the substance,
bromine was at first given off, the amount^ increasing on gently heating. At the same time, a few drops of clear liquid distilled which gradually
turned milky and finally solidified ; this product was probably cyanogen
chloride, the residue being a mixture of silver chloride and bromide : 6AgCNOBr + 8HCl= 2AgCl+ 4AgBr + 6CNCl+ 4H,O + 0, + Br,. Action of Diferent Reagents on the 8ubstcmce. Water.-Treatment
with water causes an effervescence due to a mix-
ture of gas and bromine vapour. In order to test the gas produced, the
substance was introduced into a small flask provided with a dropping
funnel and connected with a Sprengel pump by means of a tube packed
with iron wire, to absorb the bromine. The flask was exhausted and
the gas which was evolved on the addition of water was pumped out
and tested ; it proved to be a mixture of carbon dioxide and nitrogen in
the proportions by volume of 2.5 of the former to 1 of the latter. The
solid residue was chiefly silver bromide, but contained in addition a
white constituent. To obtain a specimen of this, 10 grams of cyanate
were brominated and the product treated with water after removing
the excess of halogen. The dry residue weighed 13.6 grams instead of
12.53, as calculated for silver bromide. By careful levigation, a little
of the white portion was removed from the bromide and tested. It
answered the tests for cyanuric acid, and on combustion the percentages
of carbon and hydrogen agreed fairly well with the calculated values. The action of water on the substance is approximately represented by
the following equation : 18AgCNOBr + 6H,O = 18AgBr + 4H,C30,N3 + 6C0, + 3N2. Alcohol.-Absolute
alcohol was heated with some of the substance,
when a slight evolution of gas took place and a distinct odour of
aldehyde was observed. The liquid turned yellow and reduced
ammoniacal silver solution. A white deposit settled on the surface of
the silver bromide, but was not further examined. The reaction
probably took the following course : 2AgCNOBr + C,H,O = C2H40 + 2AgBr + 2HCNO. Ammonia.-When
well-dried ammonia was passed over about
10 grams of the substance enclosed in a tube, white fumes were formed View Article Online View Article Online DEAN: THE EROMINATION OF SILVER CYANATE. 1.374 and a considerable evolution of heat was noticed. The yellow residue,
unlike the original substance, then rapidly turned dark, and on fwther
heating gave, in addition to a little ammonium bromide, a white
sublimate insoluble in water. WEST HAM TECHNICAL INSTITUTE
CHEMICAL DEPARTMENT, A Higher Additive Compound. Evidence was not wanting of the existence of a more unstable pro-
duct containing a larger proportion of bromine. In the second experi-
ment, the dark brown solid product had the appearance of such a
compound, and as the excess of bromine was removed it lost its super-
ficial crystalline structure. This fact is supported by the following
experiments, which were made in order to determine whether bromina-
tion at a lower temperature would have any effect on the reacting
proportions. To bring about interaction a t a low temperature, bromine vapour
was allowed to diffuse gradually in through the silver cyanate. The
apparatus used consisted of a small retort, tightly stoppered, to the
neck of which a flask was ground. Into the latter, the cyanate was
introduced, whilst the bromine was poured into the retort. The
progress of the reaction could be traced from the movement of the
encroaching ring of additive compound in the mass of cyanate. From
time to time, the apparatus was disconnected, the vapour in the flask
removed by a current of air, and the increase in weight determined. The rate at which the halogen was taken up diminished gradually, and
after about thirty days there was no further addition, the increase
then corresponding with 73 parts per molecule of cyanate. After
that, the proportion of bromine diminished, a slight decomposition
taking place, due probably to the presence of moisture in the aspirated
air. The following curve shows the rate of bromination. Bromine and cyanate from the same samples as those used in the
foregoing experiments were next allowed to interact in exactly the same
manner, but for a longer period. No disturbance of the apparatus was
made for intermediate weighing. On attempting to remove the free
vapour from the flask before weighing, by passing air through as
before, a copious and prolonged evolution of bromine took place. On
weighing the added bromine at this stage, there were still 120 parts View Article Online View Article Online DEAN: THE BROMINATION OF SILVER CYANATIL 1375 per molecule of cyannte. On again passing air through the flask,
more bromine was expelled, and the ordinary yellow product remained. per molecule of cyannte. On again passing air through the flask,
more bromine was expelled, and the ordinary yellow product remained. A Higher Additive Compound. In order to see whether this additional absorption of bromine could
b
ib
d
l
h
i
l
i
f
h
fi
l
di id d n 01 January 1904. Downloaded by University of Exeter on 19/07/2016 15:10:28. per molecule of cyannte. On again passing air through the flask,
more bromine was expelled, and the ordinary yellow product remained. In order to see whether this additional absorption of bromine could
be attributed to any purely mechanical action of the finely-divided
componnd, an experiment was performed, precisely similar in method,
but in which the cyanate was replaced by dry precipitated barium
sulphate. No absorption whatever took place, the siilphate remaining p
m
b
c
b
s
Published on 01 January 1904. Downloaded by University of Exeter on 19/07/2016 15:10:28. Published on 01 January 1904. Downloaded by University of Exeter on 19/07/2016 15:10:28. y 1904. Downloaded by University of Exeter on 19/07/2016 15:10:28. Published on 01 January 1904. Downloaded by University of Exeter on 19/07/2016 15:10:28. aded by University of Exeter on 19/07/2016 15:10:28. In order to see whether this additional absorption of bromine could
be attributed to any purely mechanical action of the finely-divided
componnd, an experiment was performed, precisely similar in method,
but in which the cyanate was replaced by dry precipitated barium
sulphate. No absorption whatever took place, the siilphate remaining Y .-
I
I
I
I
I
l
a
3
12
19
26
33
38
41
Days. absolutely constant in weight after several months’ immersion in the
vapour . absolutely constant in weight after several months’ immersion in the
vapour . I have much pleasure in thanking the Research Committee of the
Chemical Society for a grant, which has defrayed part of the cost of
the materials used in the foregoing experiments.
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https://openalex.org/W3022444072
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https://cris.unibo.it/bitstream/11585/760931/1/animals-10-00828-v2.pdf
|
English
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Early Renal Involvement in Cats with Natural Feline Morbillivirus Infection
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Animals
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cc-by
| 11,535
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animals animals animals animals Early Renal Involvement in Cats with Natural Feline
Morbillivirus Infection Paolo Emidio Crisi 1,*, Francesco Dondi 2
, Eliana De Luca 1,3
, Morena Di Tommaso 1,
Kateryna Vasylyeva 2, Enea Ferlizza 2
, Giovanni Savini 3, Alessia Luciani 1, Daniela Malatesta 3,
Alessio Lorusso 3 and Andrea Boari 1 1
Faculty of Veterinary Medicine, Veterinary University Hospital, University of Teramo,
Località Piano d’Accio, 64100 Teramo, Italy; edeluca@unite.it (E.D.L.); mditommaso@unite.it (M.D.T.);
aluciani@unite.it (A.L.); aboari@unite.it (A.B.) 2
Department of Veterinary Medical Sciences, Alma Mater Studiorum-University of Bologna,
Via Tolara di Sopra 50, 40064 Ozzano dell’Emilia (BO), Italy; f.dondi@unibo.it (F.D);
kateryna.vasylyeva2@unibo.it (K.V.); enea.ferlizza2@unibo.it (E.F.) y
y y
(
);
(
)
3
Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise (IZSAM), Campo Boario, 64100 Teramo, Italy
g.savini@izs.it (G.S.); d.malatesta@izs.it (D.M.); a.lorusso@izs.it (A.L.) g
*
Correspondence: pecrisi@unite.it *
Correspondence: pecrisi@unite.it 1. Introduction Feline morbillivirus (FeMV), family Paramyxoviridae, was first discovered and isolated in stray
and domestic cats in Hong Kong in 2009 [1]. Later, the presence of FeMV was reported in cats from
Japan [2,3], Italy [4–6], Germany [7], USA [8], Turkey [9], Brazil [10] and the United Kingdom [11]. Some of these studies suggested a possible association between FeMV infection and tubulointerstitial
nephritis (TIN), which represents the most frequent histopathological finding in feline chronic kidney
disease (CKD) [1–3,9,12], even though a clear association between FeMV infection and the development
of CKD is lacking. CKD is a common feline disease with a reported prevalence ranging from 1 to 3% in the general
population and reaching 30% in geriatric cats [13]. Therefore, an early diagnosis of CKD is needed to
prevent further damage and to reduce the progressive decline of renal function. The determination
of the glomerular filtration rate (GFR) is still considered the most sensitive index for the evaluation
of renal function, but it is impractical for routine clinical use, particularly in cats [14]. At an early
stage, histopathological examination of renal tissue can help to identify the presence of kidney disease
and to obtain a pathological diagnosis of renal damage, including TIN [15]. However, the renal
biopsy is an invasive procedure with some potential adverse effects [16] and in most cases is unable to
recognize the etiology of the renal injury [12,15]. Currently, in feline clinical practice, CKD diagnosis
and staging are mainly based on serum creatinine concentration (sCr), urine concentrating ability,
quantitative proteinuria measured by urine protein:creatinine ratio (UPC) and imaging results [17]. In addition, these variables are late markers and are more useful to detect advanced stages of CKD. Serum symmetric dimethylarginine (SDMA) has been proposed as an early indicator of CKD, as reported
by the International Renal Interest Society guidelines (IRIS) [18], although its early diagnostic role
needs further evidence [19]. For these reasons, other sensitive and specific biomarkers, measurable
in non- or minimally invasive biological samples, are required in clinical practice to identify early
renal damage. Urine represents a readily available source of biomarkers for the evaluation of renal function
or damage. In particular, qualitative proteinuria, by means of proteomic techniques, and urinary
fractional excretion (FE) of electrolytes have been studied in the evaluation of renal damage in cats and
dogs [20–22]. 1. Introduction Regarding qualitative proteinuria and proteomics, sodium-dodecyl-sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis have been applied in cats,
leading to the identification of putative biomarkers of tubulointerstitial damage in feline CKD including
uromodulin, cauxin and retinol binding protein (RBP) [21–23]. Previously reported studies have suggested a correlation between viral infection and the presence
of TIN in affected cats, hypothesizing the involvement of FeMV in the pathogenesis of feline CKD. Notwithstanding, knowledge on the clinical implications and role of FeMV in feline nephrology is
still lacking. Therefore, the aim of the study was to identify and characterize renal disease in cats
naturally infected with FeMV using clinical and clinicopathological data including SDS-PAGE and
urine chemistry. Received: 12 March 2020; Accepted: 8 May 2020; Published: 10 May 2020 Simple Summary: Feline morbillivirus is a newly discovered paramyxovirus infecting domestic cats. Its pathogenetic role in domestic cats is still debated, however some evidences suggest a potential
involvement of this novel feline virus in the pathogenesis of feline chronic kidney disease. In this
study, clinical data of cats infected by morbillivirus were retrospectively reviewed and compared
with data obtained from healthy cats and cats affected by chronic kidney disease. The results of the
present study suggest that this infection can be associated with the presence of a sub-clinical kidney
damage and with different grades of renal dysfunction in cats younger than those typically affected
by chronic kidney disease. Abstract: Feline morbillivirus (FeMV) is a newly discovered paramyxovirus infecting domestic cats
and its role in the pathogenesis of feline chronic kidney disease (CKD) has been suggested, however
not confirmed. The primary aim of the study was to evaluate the renal damage associated with FeMV
infection in cats. In this retrospective study, clinical and clinicopathological data were compared
among 14 FeMV naturally infected, 21 CKD and 22 healthy cats. FeMV positive cats had serum
chemistry analytes and main urine chemistry results similar to the healthy subjects. FeMV positive
cats had significantly decreased urine specific gravity (median 1054, range 1022–1065) and urine
creatinine (median 227.23 mg/dL, range 83.02–489.75) when compared with healthy cats (median
1067, range 1040–1080, p < 0.001; median 406.50 mg/dL, range 195.32–575.58; p < 0.001, respectively). Urine protein:creatinine ratio (UPC) results of FeMV and CKD were not different (median 0.20,
range 0.08–1.03; median 0.23, range 0.10–0.80, respectively), however UPC results were significantly
increased in both groups, if compared with healthy cats (median 0.1, range 0.04–0.250, p < 0.01). Based on clinical data, serum creatinine concentration, urine specific gravity and UPC results, CKD
was suspected by clinicians in 3/14 FeMV cats. Urine protein sodium-dodecyl-sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) in 10/13 (77%) FeMV cats indicated a tubular pattern, with a decrease
of uromodulin and an increase in the number and intensity of low molecular weight proteins. FeMV
infection can be associated with different grades of renal dysfunction ranging from mild tubular
proteinuria with less concentrated urine to azotemia in cats younger than those typically affected
by CKD. Keywords: feline morbillivirus; urine protein electrophoresis; urine chemistry; chronic kidney disease Animals 2020, 10, 828; doi:10.3390/ani10050828 www.mdpi.com/journal/animals www.mdpi.com/journal/animals 2 of 15 Animals 2020, 10, 828 2. Materials and Methods FeMV Infection Diagnosis and Serological Evaluation 2. Materials and Methods This was a retrospective, cross-sectional study, performed between July 2015 and March 2017 at
two Veterinary University Hospitals (VUH) (Faculty of Veterinary Medicine, University of Teramo,
and Department of Veterinary Medical Science, University of Bologna, Italy). Cats included in the
study were divided into three groups: FeMV positive cats, CKD cats and healthy cats. The FeMV group
was composed of client-owned cats, from different sites in the Abruzzo and Emilia Romagna regions,
naturally infected with FeMV and with evidence of urinary shedding of the viral genome [24]. These cats
were diagnosed during a wider epidemiological evaluation regarding the diffusion of FeMV in a cat
population [6]. The CKD group was composed of cats with naturally occurring CKD; in particular,
cats were diagnosed with CKD based on the presence of consistent history, clinical, laboratory and 3 of 15 Animals 2020, 10, 828 imaging findings. Included cats must have evidence of abnormal renal size and architecture as
determined by abdominal ultrasonography (Logiq S8; GE Healthcare, Solingen, Germany) and either
(a) persistent azotemia (serum creatinine concentration [sCr] >1.6 mg/dL), or (b) persistently inadequate
urine specific gravity (USG < 1035), both assessed and confirmed over 1-month [17]. Only cats that
were re-evaluated on at least two consecutive occasions 2–4 weeks apart were eligible for inclusion in
the study. CKD cats were staged according to International Renal Interest Society (IRIS) guidelines for
feline CKD staging, and as previously reported [17], as follows: stage 1: sCr < 1.6 mg/dL; stage 2: sCr
1.6–2.8 mg/dL; stage 3: 2.9–5.0 mg/dL; stage 4: > 5.0 mg/dL. Cats were excluded from the CKD group if they had acute kidney injury (AKI) or decompensated
CKD, or if they were affected by non-renal diseases or treated with therapies known to influence
USG and proteinuria, regardless of the presence of renal disease itself (e.g., hypercalcemia, diuretics,
fluid therapy; pre-renal, post-renal or extra-urinary proteinuria, hyperthyroidism, hypertension non
related to CKD, respectively). p
y
Healthy group: during the same study period, client-, student- and hospital staff-owned as well
as blood donor cats were enrolled as a control group. These cats underwent to the same evaluation of
FeMV and CKD cats and were defined healthy based on history, physical examination including blood
pressure measurement, imaging results and blood and urine screening tests. Healthy and CKD cats
must result FeMV-negative using the same diagnostic protocol used in FeMV cats. 2.1. FeMV Infection Diagnosis and Serological Evaluation 2.1. 2.1. FeMV Infection Diagnosis and Serological Evaluation FeMV infection was diagnosed by the demonstration of FeMV RNA in the urine, as described in
a previous paper [24]. Immunofluorescence (IF) was also performed to detect antibodies (Ab) to FeMV-N protein in
cat sera [6]. The Ab titers of tested cat sera were recorded as the reciprocals of the highest dilutions
producing positive staining. Ab titers of less than 1:40 were considered negative. 2.2. Clinical and Clinicopathological Evaluation 2.2. Clinical and Clinicopathological Evaluation 2.2. Clinical and Clinicopathological Evaluation Samples with severe hematuria (gross hematuria or ≥100 red blood cells at ×400), pyuria or both,
as defined previously, were excluded from urine chemistry and SDS-PAGE [26]. Urine chemistry evaluation included urine creatinine (uCr), urine total protein (uTP) and other
urinary analytes concentration measurement (urea, uUrea; calcium, uCa; phosphate, uP; sodium, uNa;
potassium, uK; magnesium, uMg; chloride, uCl). The uTP:uCr ratio (UPC) was calculated as follows:
UPC = uTP (mg/dL)/uCr (mg/dL). Proteinuria and borderline proteinuria were considered when UPC
value were above 0.4, and between 0.2 and 0.4, respectively [18]. Fractional excretion of urinary analytes was calculated by the following formula: (1) FEe = ue × sCr/uCr × se × 100 (based on spot urine sample)
(1) FEe = ue × sCr/uCr × se × 100 (based on spot urine sample) where “e” indicated the analyte, and “u” and “s” indicated urine and serum values, respectively. Urinalysis was performed within 2 h from the collection and then urine supernatants, after centrifugation
for 10 min at 1500× g, were stored at −80 ◦C for the further evaluations. Urine chemistry and SDS-PAGE
were performed on stored supernatants within 1 month from the collection; in particular, urine
proteins were separated using an electrophoretic system (NuPage; Thermo Fisher Scientific; Waltham,
MA, USA) on precast 12% polyacrylamide gel in reducing conditions with a MOPS (3–(N–morpholino)
propanesulfonic acid) buffer containing sodium-dodecyl-sulfate (Thermo Fisher Scientific; Waltham,
MA, USA). Five micrograms of proteins for each sample were loaded. Gels were stained with
Coomassie blue staining (PageBlu protein staining solution; Thermo Fisher Scientific; Waltham,
MA, USA). After staining, each gel was digitalized by a semiautomated system (ChemiDoc MP;
Bio-Rad Laboratories, Hercules, CA, USA) and pherograms were obtained using a commercial software
(ImageLab 5.2.1 Software; Bio-Rad Laboratories, Hercules, CA, USA). To avoid manual correction of
band, the same optimized default settings (background subtraction disk size 5.00 mm, sensitivity 20.00,
size scale 5, noise filter 4, shoulder 1) were applied to each gel and used to detect and count protein
bands. Each band was verified by two of the authors (E.F., F.D.). Protein bands were classified as high
molecular mass (HMM) proteins when presenting a molecular mass equal or above to albumin, and low
molecular mass (LMM) proteins when below to albumin. Electrophoretic patterns were evaluated
qualitatively and quantitatively by the enumeration of total, HMM and LMM number of bands. 2.2. Clinical and Clinicopathological Evaluation All cats underwent to a complete physical examination, including blood pressure measurement,
and abdominal ultrasound. Body weight and 9-point body condition score (BCS) were recorded. Doppler (Minidop ES-100VX; Hadeco, Kawasaki, Japan) or oscillometric (petMAP graphic;
Ramsey Medical, Tampa, FL, USA) systolic blood pressure (SBP) measurements were performed as
recommended by IRIS guidelines and as reported by Taylor and colleagues [25]. Cats were considered
hypertensive if their SBP was persistently above 160 mmHg. Blood samples were obtained for all cats using blood vacuum systems (Vacutest Kima;
Arzegrande, Italy), after at least a 12-h fasting period. Concurrent urine samples were obtained
by spontaneous voiding or cystocentesis. The following analyses were performed: venous blood
gas analysis (ABL 800 Flex; Radiometer Medical ApS, Brønshøj-Husum, Denmark) including pH,
base excess (BE), anion gap, lactate and ionized calcium (iCa) concentration; complete blood count (CBC)
(ADVIA 2120; Siemens Healthcare Diagnostics, Tarrytown, NY, USA) and blood smear examination;
serum chemistry profile (OLYMPUS AU 400; Olympus-Beckman Coulter, Brea, CA, USA) including sCr,
albumin, total proteins, glucose, alanine transaminase (ALT), aspartate transaminase (AST), alkaline
phosphatase (ALP), total bilirubin, gamma(γ)-glutamyltransferase (GGT), total cholesterol, sodium,
potassium, magnesium, chloride, total calcium and phosphate. The serum SDMA concentration was
also determined from serum stored at −20 ◦C (IDEXX SDMA; IDEXX Laboratories, Milano, Italy). Feline Immunodeficiency Virus (FIV) antibody and Feline Leukaemia Virus (FeLV) antigen were
evaluated with a commercial assay kit following manufacturer instruction (SNAP FIV/FeLV Combo Test;
IDEXX Laboratories, Westbrook, ME, USA). Urinalysis including refractometric urine specific gravity
(USG) (American Optical, Buffalo, NY, USA), dipstick examination (Combur10Test; Roche Diagnostic,
Basel, Switzerland) and microscopic evaluation of the sediment, were performed. In cats with
active sediment, bacteriuria or suspected urinary tract infection (UTI), urine culture was performed, 4 of 15 Animals 2020, 10, 828 when required by attending clinicians. Standardized technique was used for bacterial culture: urine
samples were plated onto 5% sheep blood agar and incubated at 37 ◦C for 48 h. when required by attending clinicians. Standardized technique was used for bacterial culture: urine
samples were plated onto 5% sheep blood agar and incubated at 37 ◦C for 48 h. Samples with severe hematuria (gross hematuria or ≥100 red blood cells at ×400), pyuria or both,
as defined previously, were excluded from urine chemistry and SDS-PAGE [26]. 2.3. Renal Histopathology and Immunohistochemistry When available, kidney tissue samples of deceased cats belonging to the FeMV group were
submitted for histopathological and immunohistochemical (IHC) analyses, as reported previously [6,24]. Briefly, all renal tissues, resulted FeMV positive by quantitative polymerase chain reaction (qPCR),
were fixed in 10%neutral buffered formalin, embedded in paraffin and cut into 3-µm-thick sections,
mounted on positive charged glass slides, dewaxed and rehydrated using standard procedures and
stained by Haematoxylin and eosin (HE). For IHC, 3-µm-thick tissue sections were dewaxed and
rehydrated and then incubated with 3% hydrogen peroxide diluted in absolute methanol for 30 min to
inhibit endogenous peroxidase activity and then rinsed in 0.05 M Tris-buffered saline (TBS), pH 7.6,
for 5 min. Antigen retrieval was performed by heat treatment in citrate buffer 0.01 M pH 6.0 at 121 ◦C
for 5 min. To reduce nonspecific binding, slides were incubated with 20% normal swine serum (Dako,
Copenhagen, Denmark) diluted in TBS for 20 min, followed by a second incubation with 10% skimmed
milk diluted in TBS for 20 min at room temperature. Then tissue sections were incubated overnight
in a humidified chamber at 4 ◦C with the rabbit polyclonal antibody against the N protein of FeMV. After three washing in TBS, tissues sections were incubated with an horseradish peroxidase-labeled
polymer conjugated with the secondary anti-rabbit antibody (REAL™Envision detection system
peroxidase Dako, Copenhagen, Denmark) for 30 min. Immune reactions were detected by means of
a peroxidase technique (REAL™Envision detection system peroxidase Dako, Copenhagen, Denmark) 5 of 15 Animals 2020, 10, 828 and visualized using 3-3′-diaminobenzi-dine as chromogen. Positive and negative controls were
included in each IHC run. In this respect, the specificity of immunolabeling was verified by testing the
same IHC protocol on qPCR FeMV negative renal sections, with and without inflammatory lesions. In addition, all samples were tested with an irrelevant antibody directed against an unrelated antigen
(anti-Von Willebrand factor rabbit polyclonal Ab 1:800, DakoCytomation, Denmark). 2.4. Statistical Analysis Statistical analysis was performed using a statistical software (MedCalc version 17.6; MedCalc
Software Ltd, Ostend, Belgium). All data were evaluated using standard descriptive statistics and
reported as mean ± standard deviation (SD) or as median and range (minimum–maximum), based
on their distribution. Normality was checked graphically or using the D’Agostino Pearson test. Variables collected were compared among the three groups using the ANOVA or a Kruskall–Wallis test
and post-hoc tests (Student–Newman–Keuls test or Dunn test). Categorical variables were analyzed
using the χ2 test. A p-value < 0.05 was considered significant. 3. Results A total number of 57 cats were included in the study. Of these, 14/57 cats were FeMV PCR positive
and included in the FeMV group, 21/57 in the CKD group and 22/57 were enrolled as healthy control
group. Cats belonging to CKD and Healthy groups were FeMV PCR and IF negative. In the FeMV group, 8/14 (57%) infected cats were neutered males and 6/14 (43%) were females
(5 spayed). The median age was 35 months (range 14–101), and all of the animals were European
domestic cats. In PCR positive cats, the median Cycle threshold value was 33.5 and ranged between 32
and 39. Within this group, sera of 12/14 (86%) cats underwent IF in order to detect antibodies against
specific FeMV–N protein. Median IF titer was 1:2560 and ranged between 1:160 and 1:10,240; one cat
was negative to serology. In the CKD group, 14/21 (67%) cats were males (12 neutered), and 7/21 (33%) were spayed females. The CKD cats were significantly older than FeMV and healthy cats with a median age of 174 months
(range 40–205; p < 0.01). Breeds included in the CKD group were European domestic cats (18/21),
Persian (1/21), Chartreaux (1/21) and Siamese (1/21). Of the CKD cases, 2/21 (10%) were classified as
IRIS stage 1, 7/21 (33%) stage 2, 8/21 (38%) stage 3 and 4/21 (19%) stage 4. The healthy control group included 11/22 (50%) males (8 neutered) and 11/22 (50%) females
(9 spayed) cats with a median age of 55 months (range 12–109). Breeds represented in this group
included European domestic cats (15/22), Main Coon (4/22), Sphynx (1/22), Chartreaux (1/22) and
Devon Rex (1/22). FeMV positive cats and healthy subjects had similar age (p = 0.15), while cats of
both groups were significantly younger than CKD cats (p < 0.001). There was no significant difference
in sex among the three groups. Body weight and BCS were not different between FeMV and CKD cats, however they were
significantly lower if compared with healthy ones (p = 0.005 and p = 0.001, respectively) (Table 1). Body weight and BCS were not different between FeMV and CKD cats, however they were
significantly lower if compared with healthy ones (p = 0.005 and p = 0.001, respectively) (Table 1). Among the FeMV positive cats, 2 tested positive for FeLV while no cats tested positive for FIV. 3. Results The main clinical obser ations and imaging features of cats belonging to the health
FeMV and y
g
y
antly lower if compared with healthy ones (p = 0.005 and p = 0.001, respectively) (Table 1). mong the FeMV positive cats, 2 tested positive for FeLV while no cats tested positive for FIV. h
i
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Among the FeMV positive cats, 2 tested positive for FeLV while no cats tested positive for FI g
p
p
p
The main clinical observations and imaging features of cats belonging to the healthy, FeMV and
CKD groups are summarized in Table 1. 6 of 15 Animals 2020, 10, 828 Table 1. Main complaints, physical examination and ultrasound findings and retroviral status of
healthy cats (n = 22) and cats belonging to the FeMV (n = 14) and the CKD (n = 21) groups. 3. Results Variable
Healthy (n = 22)
FeMV (n = 14)
CKD (n = 21)
p Value
Median body weight (kg)
5.0 (2.6–7.6)
3.8 (3.0–6.0)
4.0 (2.1–7.4)
0.005 *
Median BCS
6 (5–8)
5 (2–7)
5 (3–6)
0.001 *
Poor coat conditions n (%)
–
8 (57%)
4 (19%)
0.003
Gingivostomatitis n (%)
–
4 (29%)
8 (38%)
0.721
Diarrhea n (%)
–
3 (21%)
2 (9%)
0.369
Hyporexia/Anorexia n (%)
–
2 (14%)
12 (57%)
0.016
FeLV n (%)
–
2 (14%)
0
0.153
FIV n (%)
–
0
0
1.000
Weight loss n (%)
–
1 (7%)
11 (52%)
0.009
Vomiting n (%)
–
1 (7%)
6 (29%)
0.203
Pale mucous n (%)
–
1 (7%)
6 (29%)
0.203
Lethargy n (%)
–
1 (7%)
3 (14%)
0.635
Enlarged abdomen n (%)
–
1 (7%)
0
0.400
Dehydration n (%)
–
1 (7%)
7 (33%)
0.108
PU/PD n (%)
–
0
10 (48%)
0.002
Heart murmur n (%)
–
0
3 (14%)
0.259
Constipation n (%)
–
0
2 (9%)
0.506
Abnormal renal palpation n (%)
–
1 (7%)
7 (33%)
0.108
Hypertension
(SBP > 160 mmHg) n (%)
–
0
9 (43%)
0.005
Ultrasound abnormalities
n (%)
3 (14%)
2 (14%)
21 (100%)
<0.001
Hyperechoic cortex
3 (14%)
2 (7%)
21 (100%)
<0.001
Decreased corticomedullar
distinction
–
0
13 (62%)
<0.001
Small kidneys
–
0
5 (24%)
0.063
Irregular kidney shape
–
0
10 (48%)
0.002
Data were reported as median and range (in brackets) or as number of cases and percentage (%). FeMV =
Feline morbillivirus; CKD = chronic kidney disease; BCS = body condition score; FeLV = Feline Leukaemia Virus;
FIV = Feline Immunodeficiency Virus; PU/PD = polyuria and polydipsia; SBP = Systolic Blood Pressure; * Statistically
significant difference in Healthy compared with CKD and FeMV cats. Data were reported as median and range (in brackets) or as number of cases and percentage (%). FeMV =
Feline morbillivirus; CKD = chronic kidney disease; BCS = body condition score; FeLV = Feline Leukaemia Virus;
FIV = Feline Immunodeficiency Virus; PU/PD = polyuria and polydipsia; SBP = Systolic Blood Pressure; * Statistically
significant difference in Healthy compared with CKD and FeMV cats. 3.1. Hematology, Chemistry and Urinalysis Results The results of CBC and serum chemistry, and their comparison between the three groups are
reported in Table 2. The results of CBC and serum chemistry, and their comparison between the three groups are
reported in Table 2. Table 2. Complete blood count and serum chemistry values of cats belonging to the three study groups:
Healthy (n = 22), FeMV (n = 14) and CKD (n = 21). Variable
RI
Healthy (n = 22)
FeMV (n = 14)
CKD (n = 21)
p Value
Hct %
32–48
38.3 (33.3–48.7)
32.0 (19.9–41.4)
27.0 (17.7–40.1)
<0.001 *
WBCs cells/mm3
4800–14,900
8600
(4980–18,830)
16,345
(7990–23,340)
9660 (5480–27,970)
<0.001 ¶
Neutrophils cells/mm3
1600–10,000
4710
(1570–13,520)
9320
(4080–16,870)
6270 (2740–24,790)
<0.001 *
Lymphocytes
cells/mm3
900–5600
3050 (1150–4610)
4530
(1660–9700)
1760 (800– 7770)
<0.001 *
Monocytes cells/mm3
0–650
135.0 (50.0–490.0)
470.0
(0.0–920.0)
240.0 (70.0–870.0)
<0.001 *
Eosinophils cells/mm3
60–1470
645 (60–1260)
955 (330–3910)
490 (200–1060)
0.009 ¶
Platelets cells/mm3
150,000–500,000
208,000
(45,000–563,000)
362,000
(58,000–615,000)
332,000
(95,000–618,000)
0.033 †
Basophils cells/mm3
0–60
20 (0–40)
20 (10–60)
10 (0–30)
<0.001 ‡
Creatinine mg/dL
0.8–1.6
1.46 (0.78–1.90)
0.82 (0.67–2.13)
2.98 (1.52–9.49)
<0.001 *
Creatinine > 1.6
mg/dL, n (%)
–
–
1/14 (7%)
19/21 (90%)
<0.001 Table 2. Complete blood count and serum chemistry values of cats belonging to the three study groups:
Healthy (n = 22), FeMV (n = 14) and CKD (n = 21). 7 of 15 Animals 2020, 10, 828 Table 2. Cont. 3.1. Hematology, Chemistry and Urinalysis Results Variable
RI
Healthy (n = 22)
FeMV (n = 14)
CKD (n = 21)
p Value
Urea mg/dL
30–65
46.48
(31.47–73.01)
45.35
(30.20–63.30)
99.4 (41.51–318.82)
<0.001 ‡
SDMA µg/dL
<18
12.5 (7.0–18.0)
10.0 (6.0–15.0)
21.5 (7.0–50.0)
<0.001 ‡
SDMA ≥18 µg/dL, n
(%)
–
–
0/14
12/18 (67%)
<0.001 *
Total calcium mg/dL
8.5–10.5
9.75 (8.80–10.80)
9.20
(8.24–10.38)
10.10 (8.30–11.60)
0.004 ¶
Ionized calcium
mmol/L
1.19–1.38
1.27 (1.07–1.34)
1.25 (0.80–1.40)
1.32 (1.07–1.50)
0.001 ‡
Phosphate mg/dL
2.5–6.2
4.4 (3.0–6.1)
5.43 (3.79–6.50)
4.9 (4.0–12.3)
0.010 †
Sodium mEq/L
145–155
151.0
(147.0–155.0)
149.0
(147.0–154.0)
151.0 (143.0–157.0)
0.299
Chloride mEq/L
110–123
119.0
(113.0–129.0)
120.5
(117.0–125.0)
116.6 (108.4–125.0)
0.022 †
Potassium mEq/L
3.4–5.1
4.2 (3.4–4.9)
4.93 (4.08–5.38)
4.2 (3.1–4.8)
<0.001 ¶
Magnesium mg/dL
1.9–2.8
2.30 (2.12–2.48)
2.32 (2.04–2.72)
2.27 (1.80–3.66)
0.952
Total proteins g/dL
6.5–8.8
7.72 (7.06–8.89)
7.87 (7.05–9.12)
7.51 (5.36–9.97)
0.790
Albumin g/dL
2.6–4.0
3.6 (2.0–4.0)
3.1 (2.5–3.7)
3.0 (2.3–3.4)
<0.001 †
A:G
0.52–1.20
0.85 (0.58–1.28)
0.63 (0.37–0.87)
0.67 (0.37–0.90)
0.001 †
Glucose mg/dL
65–148
99.5 (71.0–240.0)
85.0
(62.0–193.0)
106.0 (72.0–148.0)
0.055
AST U/L
9–40
25.5 (16.0–157.0)
23.5 (5.0–65.0)
28.0 (17.0–79.0)
0.419
ALT U/L
20–72
47.5 (33.0–92.0)
55.0
(20.0–117.0)
58.0 (20.0–224.0)
0.309
GGT U/L
0–4
0.1 (0.1–0.6)
0.1 (0.0–1.3)
0.1 (0.1–2.3)
0.590
ALP U/L
20–140
69.0 (22.0–163.0)
26.5
(13.0–120.0)
45.0 (16.0–128.0)
<0.001 *
Total bilirubin mg/dL
0–0.35
0.18 (0.10–0.31)
0.08 (0.05–0.20)
0.19 (0.14–0.32)
<0.001 ¶
Total cholesterol
mg/dL
59–230
130.5 (45.0–370.0)
86.0
(55.0–142.0)
195.0 (81.0–478.0)
<0.001 *
Data are reported as median and range (in brackets); FeMV = Feline morbillivirus; CKD = chronic kidney
disease; RI = reference interval; Hct = haematocrit value; WBCs = white blood cells; SDMA = symmetric
dimethylarginine; A:G = albumin:globulin ratio; AST = aspartate transaminase; ALT = alanine transaminase; GGT
= gamma(γ)–glutamyltransferase; ALP = alkaline phosphatase; * Statistically significant difference among the three
study groups; † Statistically significant difference in healthy compared with CKD and FeMV cats; ‡ Statistically
significant difference in CKD compared with healthy and FeMV cats; ¶ Statistically significant difference in FeMV
compared with CKD and healthy cats. Healthy (n = 22)
FeMV (n = 14) Serum creatinine was above the study cut-offvalues in 1/14 (7%) FeMV and 19/21 (90%) CKD
cats and USG was persistently below to 1035 in 1/14 (7%) FeMV and 19/21 (90%) CKD cats; the both
abnormalities were simultaneously present in none of the FeMV and in 18/21 (86%) CKD cats,
respectively. 3.2. Quantitative and Qualitative Urine Analysis 3.2. Quantitative and Qualitative Urine Analysis Urine culture was performed in 13/14 (93%) FeMV and 2/21 (9%) CKD, and yielded negative
results in all these cats. One cat included in the FeMV group was excluded from urine chemistry
(including UPC evaluation) and SDS-PAGE due to the presence of relevant hematuria, as defined
above. Pyuria was never detected in the cats included in the study. When compared with the healthy ones, FeMV cats had significantly decreased USG, uCr and
uUrea and significantly increased UPC. Conversely, FeMV cats had significantly increased USG, uCr
and uUrea when compared with the CKD ones (Table 3). UPC values were not different between these
two groups. Proteinuria, as previously defined, was detected in 1/13 (8%) FeMV and 6/21 (29%) CKD
cats, respectively. Frequency of proteinuria was not significantly different between CKD and FeMV
cats (p = 0.149). Table 3. Urinalysis and urine chemistry values of cats belonging to the three study groups: Healthy (n = 22),
FeMV (n = 14) and CKD (n = 21). 3.2. Quantitative and Qualitative Urine Analysis Variable
RI
Healthy (n = 22)
FeMV (n = 14)
CKD (n = 21)
p Value
USG
>1035
1067 (1040–1080)
1054 (1022–1060)
1017 (1008–1040)
<0.001 *
USG < 1035, n (%)
–
–
1/14 (7 %)
19/21 (90 %)
<0.001
uCreatinine mg/dL
120–593
406.5 (117.8–575.6)
227.2 (83.0–489.8)
112.7 (48.4–489.6)
<0.001 *
uUrea mg/dL
1500–10700
7597.8
(3650.6–9235.1)
5174.9
(1625.2–6508.3)
2225.8
(967.6–6478.8)
<0.001 *
uTP mg/dL
21–134
41.14 (22.09–151.24)
41.97 (16.44–172.74)
33.42 (5.96–337.90)
0.244
UPC
0.01–0.4
0.1 (0.04–0.25)
0.20 (0.08–1.03)
0.23 (0.10–0.80)
<0.001 †
UPC 0.2–0.4, n (%)
–
–
5/13 (38%)
9/21 (43%)
0.803
UPC > 0.4, n (%)
–
–
1/13 (8%)
6/21 (29%)
0.149
uGlucose mg/dL
4.0–14.0
8.0 (5.0–16.1)
9.9 (3.3–139.0)
2 (0–1408.0)
<0.001 ‡
uNa mEq/L
49.0–375.0
210.40 (20.40–296.0)
83.10 (16.20–217.10)
78.80 (13.10–298.20)
<0.001 †
uNa/uCrea
0.15–1.0
0.51 (0.10–0.72)
0.31 (0.07–1.23)
0.76 (0.11–1.20)
0.040 ‡
FE Na%
0–0.97
0.49 (0.08–0.88)
0.19 (0.04–0.78)
1.13 (0.21–6.11)
<0.001 ‡
uK mEq/L
60–231
134.30
(62.50–230.40)
149.95 (85.0–200.40)
53.50 (19.0–169.30)
<0.001 ‡
uK/uCrea
0.16–0.66
0.34 (0.15–0.64)
0.68 (0.22–1.30)
0.47 (0.16–0.76)
<0.001 *
FE K%
7.0–22.0
11.87 (6.20–22.79)
12.69 (8.63–27.12)
38.96 (7.91–128.76)
<0.001 ‡
uCa mg/dL
1.0–10.0
2.40 (1.0–11.60)
2.05 (1.10–3.50)
2.40 (0.80–9.40)
0.586
uCa/uCrea
0–0.03
0.01 (0–0.03)
0.01 (0–0.03)
0.02 (0–0.14)
<0.001 ‡
FE Ca%
0–0.26
0.09 (0.05–0.45)
0.10 (0.04–0.26)
0.72 (0.10–11.15)
<0.001 ‡
uP mg/dL
90.0–385.0
187.70
(105.20–381.30)
230.0
(103.50–377.30)
58.10 (9.7–268.20)
<0.001 ‡
uP/uCrea
0.25–1.13
0.51 (0–0.89)
1.16 (0.32–2.26)
0.55 (0.04–0.87)
<0.001 ¶
FE P%
0–40
16.51 (8.28–37.32)
17.20 (9.54–38.15)
31.55 (3.02–54.99)
0.002 ‡
uCl mEq/L
51–366
179.40 (85.0–330.0)
87.0 (10.50–133.0)
190.50 (56.0–386.0)
<0.001 ‡
uCl/uCrea
0.13–1.10
0.43 (0.15–1.08)
1.02 (0.14–2.47)
0.64 (0.09–1.29)
0.004 †
FE Cl%
0.18–1.26
0.55 (0.20–1.08)
0.80 (0.26–1.97)
1.42 (0.21–9.15)
<0.001 ‡
uMg mg/dL
0.76–17
11.06 (3.0–19.40)
8.07(1.04–14.64)
4.15 (0.76–9.70)
0.001 ‡
uMg/uCrea
0–0.05
0.03 (0.01–0.06)
0.05 (0–0.09)
0.04 (0–0.11)
0.051 †
FE Mg%
0.05–3.23
1.92 (0.41–3.22)
1.72 (0.21–3.87)
6.38 (0.15–31.61)
<0.001 ‡
Total bands
number
–
13 (9–15)
17 (5–24)
15 (6–21)
0.016 ∞
HMM number
–
6 (5–7)
6 (2–8)
4 (1–9)
0.049 ‡
LMM number
–
7 (3–10)
11 (3–17)
9 (5–16)
0.024 †
Data are reported as median and range (in brackets). 3.1. Hematology, Chemistry and Urinalysis Results Based on these results, CKD was suspected by clinicians in 2/14 (14%) FeMV cats (IRIS
stage 1, n = 1; IRIS stage 2, n = 1). SDMA results were available for 14/14 FeMV and 18/21 CKD
cats. None of the FeMV and 12/18 CKD cats had SDMA above the IRIS CKD suggested threshold
(≥18 µg/dL) [18]. FeMV cats had significantly lower sCr, urea, SDMA, total and ionized calcium
concentrations as compared with CKD cats. Serum phosphate of FeMV cats was significantly higher
than in healthy cats but no difference was detected by the comparison with the CKD group. Albumin
concentration and albumin:globulin ratio (A:G) of FeMV and CKD groups were not different, however
both were significantly lower as compared with healthy cats. In FeMV cats, total cholesterol and total
bilirubin concentrations were significantly lower than in CKD and healthy cats. A reduced activity of
ALP was observed in both FeMV and CKD groups as compared with healthy cats. The FeMV cats
had significantly higher serum potassium concentrations than healthy and CKD groups, and higher
chloride concentration than healthy cats. There were no differences in serum sodium and magnesium
concentration among the three groups. No differences were observed for other serum chemistry analytes among the three group Animals 2020, 10, 828 8 of 15 3.2. Quantitative and Qualitative Urine Analysis FeMV = Feline morbillivirus; CKD = chronic kidney disease;
RI = reference interval; USG = urine specific gravity; uCreatinine = urinary creatinine; uUrea = urinary urea;
uTP = urinary total proteins; UPC = urine protein:creatinine ratio; uGlucose = urinary glucose; uNa = urinary
sodium; FE Na = fractional excretion of sodium; uK = urinary potassium; FE K = fractional excretion of potassium;
uCa = urinary calcium; FE Ca = fractional excretion of calcium; uP = urinary phosphorus; FE P = fractional excretion
of phosphorus; uCl = urinary chloride; FE Cl = fractional excretion of chloride; uMg = urinary magnesium;
FE Mg = fractional excretion of magnesium; HMM = high molecular mass protein; LMM = low molecular mass
protein; * Statistically significant difference among the three study groups; † Statistically significant difference in
healthy compared with CKD and FeMV cats; ‡ Statistically significant difference in CKD compared with healthy
and FeMV cats; ¶ Statistically significant difference in FeMV compared with CKD and healthy cats; ∞Statistically
significant difference between FeMV and healthy cats. Table 3. Urinalysis and urine chemistry values of cats belonging to the three study groups: Healthy (n = 22),
FeMV (n = 14) and CKD (n = 21). Data are reported as median and range (in brackets). FeMV = Feline morbillivirus; CKD = chronic kidney disease;
RI = reference interval; USG = urine specific gravity; uCreatinine = urinary creatinine; uUrea = urinary urea;
uTP = urinary total proteins; UPC = urine protein:creatinine ratio; uGlucose = urinary glucose; uNa = urinary
sodium; FE Na = fractional excretion of sodium; uK = urinary potassium; FE K = fractional excretion of potassium;
uCa = urinary calcium; FE Ca = fractional excretion of calcium; uP = urinary phosphorus; FE P = fractional excretion
of phosphorus; uCl = urinary chloride; FE Cl = fractional excretion of chloride; uMg = urinary magnesium;
FE Mg = fractional excretion of magnesium; HMM = high molecular mass protein; LMM = low molecular mass
protein; * Statistically significant difference among the three study groups; † Statistically significant difference in
healthy compared with CKD and FeMV cats; ‡ Statistically significant difference in CKD compared with healthy
and FeMV cats; ¶ Statistically significant difference in FeMV compared with CKD and healthy cats; ∞Statistically
significant difference between FeMV and healthy cats. Data are reported as median and range (in brackets). 3.2. Quantitative and Qualitative Urine Analysis FeMV = Feline morbillivirus; CKD = chronic kidney disease;
RI = reference interval; USG = urine specific gravity; uCreatinine = urinary creatinine; uUrea = urinary urea;
uTP = urinary total proteins; UPC = urine protein:creatinine ratio; uGlucose = urinary glucose; uNa = urinary
sodium; FE Na = fractional excretion of sodium; uK = urinary potassium; FE K = fractional excretion of potassium;
uCa = urinary calcium; FE Ca = fractional excretion of calcium; uP = urinary phosphorus; FE P = fractional excretion
of phosphorus; uCl = urinary chloride; FE Cl = fractional excretion of chloride; uMg = urinary magnesium;
FE Mg = fractional excretion of magnesium; HMM = high molecular mass protein; LMM = low molecular mass
protein; * Statistically significant difference among the three study groups; † Statistically significant difference in
healthy compared with CKD and FeMV cats; ‡ Statistically significant difference in CKD compared with healthy
and FeMV cats; ¶ Statistically significant difference in FeMV compared with CKD and healthy cats; ∞Statistically
significant difference between FeMV and healthy cats. 9 of 15 Animals 2020, 10, 828
CKD and healt
Statistically sign FE of sodium, potassium, calcium, phosphorous, chloride and magnesium of the FeMV group were
not different from the healthy group but were significantly lower than those of the CKD group (Table 3). FE of sodium, potassium, calcium, phosphorous, chloride and magnesium of the FeMV group
were not different from the healthy group but were significantly lower than those of the CKD group
Table 3) FE of sodium, potassium, calcium, phosphorous, chloride and magnesium of the FeMV group were
not different from the healthy group but were significantly lower than those of the CKD group (Table 3). FE of sodium, potassium, calcium, phosphorous, chloride and magnesium of the FeMV group
were not different from the healthy group but were significantly lower than those of the CKD group
Table 3) SDS-PAGE of urine samples was conducted for each group (Figure 1) and the pherograms acquired
(Figure 2). In healthy cats, a typical electrophoretic profile was found, common to all the examined
samples. This pattern was characterized by the presence of HMM proteins, including uromodulin
(94 kDa), transferrin (73 kDa), cauxin (65 kDa) and albumin (62 kDa), and a limited number of
LMM proteins. In most samples, the bands of cauxin and albumin were distinguishable, though not
quantifiable separately. (Table 3). 3.2. Quantitative and Qualitative Urine Analysis SDS‐PAGE of urine samples was conducted for each group (Figure 1) and the pherograms
acquired (Figure 2). In healthy cats, a typical electrophoretic profile was found, common to all the
examined samples. This pattern was characterized by the presence of HMM proteins, including
uromodulin (94 kDa), transferrin (73 kDa), cauxin (65 kDa) and albumin (62 kDa), and a limited
number of LMM proteins. In most samples, the bands of cauxin and albumin were distinguishable,
though not quantifiable separately g
q
p
y
Figure 1. Representative sodium‐dodecyl‐sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) of
cat urine samples: lane 1 (L1), molecular mass marker; lanes 2–4 (L2–L4), urine samples from healthy
cats; lanes 5–7 (L5–L7), urine samples from FeMV cats; lanes 8–10 (L8–L10), urine samples from CKD
cats. White continuous line indicates uromodulin (100‐92 kDa); white dashed line indicates transferrin
(76 kDa); white dotted line indicates cauxin and albumin (67‐62 kDa); black dotted and black
continuous lines indicate immunoglobulin heavy (54 kDa) and light (26 kDa) chains, respectively;
black dashed line indicates haptoglobin (36 kDa); black dotted and dashed line indicates retinol
binding protein (20 kDa). LMM, low molecular mass. Figure 1. Representative sodium-dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of
cat urine samples: lane 1 (L1), molecular mass marker; lanes 2–4 (L2–L4), urine samples from healthy
cats; lanes 5–7 (L5–L7), urine samples from FeMV cats; lanes 8–10 (L8–L10), urine samples from CKD
cats. White continuous line indicates uromodulin (100-92 kDa); white dashed line indicates transferrin
(76 kDa); white dotted line indicates cauxin and albumin (67-62 kDa); black dotted and black continuous
lines indicate immunoglobulin heavy (54 kDa) and light (26 kDa) chains, respectively; black dashed
line indicates haptoglobin (36 kDa); black dotted and dashed line indicates retinol binding protein
(20 kDa). LMM, low molecular mass. Figure 1. Representative sodium‐dodecyl‐sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) of
cat urine samples: lane 1 (L1), molecular mass marker; lanes 2–4 (L2–L4), urine samples from healthy
cats; lanes 5–7 (L5–L7), urine samples from FeMV cats; lanes 8–10 (L8–L10), urine samples from CKD
cats. White continuous line indicates uromodulin (100‐92 kDa); white dashed line indicates transferrin
(76 kDa); white dotted line indicates cauxin and albumin (67‐62 kDa); black dotted and black
continuous lines indicate immunoglobulin heavy (54 kDa) and light (26 kDa) chains, respectively;
black dashed line indicates haptoglobin (36 kDa); black dotted and dashed line indicates retinol
binding protein (20 kDa). LMM, low molecular mass. Figure 1. 3.2. Quantitative and Qualitative Urine Analysis Representative sodium-dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of
cat urine samples: lane 1 (L1), molecular mass marker; lanes 2–4 (L2–L4), urine samples from healthy
cats; lanes 5–7 (L5–L7), urine samples from FeMV cats; lanes 8–10 (L8–L10), urine samples from CKD
cats. White continuous line indicates uromodulin (100-92 kDa); white dashed line indicates transferrin
(76 kDa); white dotted line indicates cauxin and albumin (67-62 kDa); black dotted and black continuous
lines indicate immunoglobulin heavy (54 kDa) and light (26 kDa) chains, respectively; black dashed
line indicates haptoglobin (36 kDa); black dotted and dashed line indicates retinol binding protein
(20 kDa). LMM, low molecular mass. In CKD cats, a decrease or disappearance of uromodulin and cauxin was accompanied by
an increase appearance of albumin, transferrin and LMM proteins as immunoglobulin light (26 kDa)
and heavy chains (54 kDa) and haptoglobin (36 kDa); in most samples the presence of the RBP (20 kDa)
was recorded. In CKD cats, a decrease or disappearance of uromodulin and cauxin was accompanied by
an increase appearance of albumin, transferrin and LMM proteins as immunoglobulin light (26 kDa)
and heavy chains (54 kDa) and haptoglobin (36 kDa); in most samples the presence of the RBP (20 kDa)
was recorded. FeMV positive cats presented variable profiles, which were in part similar to CKD profiles
(Figure 2). Uromodulin (92 kDa) and cauxin (65 kDa) were still present, although in lesser amounts
than in healthy cats. The pattern was characterized by an increased number of LMM proteins and
few samples presented the RBP band. This profile was detected in 10/13 (77%) FeMV-positive cats. The HMM, LMM and total number of bands detected in the study groups are reported in Table 3. CKD
and FeMV cats had an increased number of LMM bands if compared with the healthy ones (p = 0.024);
HMM bands were significantly decreased in CKD cats if compared to the others (p = 0.049). Moreover,
FeMV cats had a significantly increased total number of bands if compared with the healthy ones
(p = 0.016). 10 of 15
15 Animals 2020, 10, 828
Animals 2020 10 x Figure 2. Representative pherograms of cat urine samples: (a) Healthy cat (same sample of lane 3
Figure 1); (b) FeMV cat (same sample of lane 5 Figure 1); (c) CKD cat (same sample of lane 9 Figure
1). The number above each peak correspond to the Molecular Mass of the band in kD. intensity; Rf, ratio o
I
CKD
d
3.3. Renal Histopathology In CKD cats, a decrease or disappearance of uromodulin and cauxin was accompanied by an
increase appearance of albumin, transferrin and LMM proteins as immunoglobulin light (26 kDa)
and heavy chains (54 kDa) and haptoglobin (36 kDa); in most samples the presence of the RBP (20
kDa) was recorded. FeMV positive cats presented variable profiles, which were in part similar to CKD profiles
(Fi
2) U
d li
(92 kD )
d
i
(65 kD )
till
t
lth
h i
l
t
Three FeMV positive cats deceased within two months from the clinical visit because hit by car and
renal histopathology showed moderate to severe chronic TIN. In all of the cases, immunohistochemical
staining revealed FeMV–N positive cells mostly involving the cortical kidney tubules with an extensive
inflammatory infiltrate surrounding the FeMV–N positive cells (Figure 3)
Animals 2020, 10, x
11 of 15 In CKD cats, a decrease or disappearance of uromodulin and cauxin was accompanied by an
increase appearance of albumin, transferrin and LMM proteins as immunoglobulin light (26 kDa)
and heavy chains (54 kDa) and haptoglobin (36 kDa); in most samples the presence of the RBP (20
kDa) was recorded. FeMV positive cats presented variable profiles, which were in part similar to CKD profiles
(Fi
2) U
d li
(92 kD )
d
i
(65 kD )
ill
l h
h i
l
Three FeMV positive cats deceased within two months from the clinical visit because hit by car and
renal histopathology showed moderate to severe chronic TIN. In all of the cases, immunohistochemical
staining revealed FeMV–N positive cells mostly involving the cortical kidney tubules with an extensive
inflammatory infiltrate surrounding the FeMV–N positive cells (Figure 3)
Animals 2020, 10, x
11 of 15 (
g
)
(
)
(
)
p
,
g
than in healthy cats. The pattern was characterized by an increased number of LMM proteins and
few samples presented the RBP band. This profile was detected in 10/13 (77%) FeMV‐positive cats. The HMM, LMM and total number of bands detected in the study groups are reported in Table 3. CKD and FeMV cats had an increased number of LMM bands if compared with the healthy ones (P
= 0.024); HMM bands were significantly decreased in CKD cats if compared to the others (P = 0.049). 3.2. Quantitative and Qualitative Urine Analysis Arrows indicates the
peaks corresponding to the bands of uromodulin (100-92 kDa), cauxin (67-65 kDa), albumin (65-62 kDa),
transferrin (76 kDa), immunoglobulin heavy chains (HC, 54 kDa) and light chains (LC, 26 kDa), haptoglogin
(36 kDa) and retinol binding protein (RBP, 20 kDa). Int, pixel intensity; Rf, ratio of the front. 3.2. Quantitative and Qualitative Urine Analysis Representative pherograms of cat urine samples: (a) Healthy cat (same sample of lane 3
Figure 1); (b) FeMV cat (same sample of lane 5 Figure 1); (c) CKD cat (same sample of lane 9 Figure
1). The number above each peak correspond to the Molecular Mass of the band in kD. Arrows
indicates the peaks corresponding to the bands of uromodulin (100‐92 kDa), cauxin (67‐65 kDa),
albumin (65‐62 kDa), transferrin (76 kDa), immunoglobulin heavy chains (HC, 54 kDa) and light
chains (LC, 26 kDa), haptoglogin (36 kDa) and retinol binding protein (RBP, 20 kDa). Int, pixel
i
i
Rf
i
f h f
Figure 2. Representative pherograms of cat urine samples: (a) Healthy cat (same sample of lane 3
Figure 1); (b) FeMV cat (same sample of lane 5 Figure 1); (c) CKD cat (same sample of lane 9 Figure 1). The number above each peak correspond to the Molecular Mass of the band in kD. Arrows indicates the
peaks corresponding to the bands of uromodulin (100-92 kDa), cauxin (67-65 kDa), albumin (65-62 kDa),
transferrin (76 kDa), immunoglobulin heavy chains (HC, 54 kDa) and light chains (LC, 26 kDa), haptoglogin
(36 kDa) and retinol binding protein (RBP, 20 kDa). Int, pixel intensity; Rf, ratio of the front. Figure 2. Representative pherograms of cat urine samples: (a) Healthy cat (same sample of lane 3
Figure 2. Representative pherograms of cat urine samples: (a) Healthy cat (same sample of lane 3
Figure 1); (b) FeMV cat (same sample of lane 5 Figure 1); (c) CKD cat (same sample of lane 9 Figure 1) Figure 1); (b) FeMV cat (same sample of lane 5 Figure 1); (c) CKD cat (same sample of lane 9 Figure
1). The number above each peak correspond to the Molecular Mass of the band in kD. Arrows
indicates the peaks corresponding to the bands of uromodulin (100‐92 kDa), cauxin (67‐65 kDa),
albumin (65‐62 kDa), transferrin (76 kDa), immunoglobulin heavy chains (HC, 54 kDa) and light
chains (LC, 26 kDa), haptoglogin (36 kDa) and retinol binding protein (RBP, 20 kDa). Int, pixel
i
i
Rf
i
f h f
g
p
g
p
g
The number above each peak correspond to the Molecular Mass of the band in kD. 3.2. Quantitative and Qualitative Urine Analysis Arrows
indicates the peaks corresponding to the bands of uromodulin (100‐92 kDa), cauxin (67‐65 kDa),
albumin (65‐62 kDa), transferrin (76 kDa), immunoglobulin heavy chains (HC, 54 kDa) and light
chains (LC, 26 kDa), haptoglogin (36 kDa) and retinol binding protein (RBP, 20 kDa). Int, pixel
intensity; Rf, ratio of the front. Figure 2. Representative pherograms of cat urine samples: (a) Healthy cat (same sample of lane 3
Figure 1); (b) FeMV cat (same sample of lane 5 Figure 1); (c) CKD cat (same sample of lane 9 Figure 1). The number above each peak correspond to the Molecular Mass of the band in kD. Arrows indicates the
peaks corresponding to the bands of uromodulin (100-92 kDa), cauxin (67-65 kDa), albumin (65-62 kDa),
transferrin (76 kDa), immunoglobulin heavy chains (HC, 54 kDa) and light chains (LC, 26 kDa), haptoglogin
(36 kDa) and retinol binding protein (RBP, 20 kDa). Int, pixel intensity; Rf, ratio of the front. Figure 2. Representative pherograms of cat urine samples: (a) Healthy cat (same sample of lane 3
Figure 1); (b) FeMV cat (same sample of lane 5 Figure 1); (c) CKD cat (same sample of lane 9 Figure
1). The number above each peak correspond to the Molecular Mass of the band in kD. Arrows
indicates the peaks corresponding to the bands of uromodulin (100‐92 kDa), cauxin (67‐65 kDa),
albumin (65‐62 kDa), transferrin (76 kDa), immunoglobulin heavy chains (HC, 54 kDa) and light
chains (LC, 26 kDa), haptoglogin (36 kDa) and retinol binding protein (RBP, 20 kDa). Int, pixel
i te
ity Rf
atio of the f o t
Figure 2. Representative pherograms of cat urine samples: (a) Healthy cat (same sample of lane 3
Figure 1); (b) FeMV cat (same sample of lane 5 Figure 1); (c) CKD cat (same sample of lane 9 Figure 1). The number above each peak correspond to the Molecular Mass of the band in kD. Arrows indicates the
peaks corresponding to the bands of uromodulin (100-92 kDa), cauxin (67-65 kDa), albumin (65-62 kDa),
transferrin (76 kDa), immunoglobulin heavy chains (HC, 54 kDa) and light chains (LC, 26 kDa), haptoglogin
(36 kDa) and retinol binding protein (RBP, 20 kDa). Int, pixel intensity; Rf, ratio of the front. Figure 2. 4. Discussion The present study aimed to identify and characterize renal dysfunction in cats infected by
FeMV using non-invasive methods. To achieve these goals, clinical and clinicopathological findings,
including urinalysis and urine chemistry results, of a population of cats naturally infected with
FeMV were retrospectively compared with those obtained from clinically healthy and CKD cats. Since urine SDS-PAGE represents a highly sensitive way to evaluate renal damage in cats, as reported
in previous studies [21,23], qualitative proteinuria was included in the assessment of these animals. The retrospective nature of the study and the inclusion of cats affected by spontaneous disease in
different stages may not allow to prove a clear cause-effect relationship between FeMV infection and
development of renal damage in FeMV infected cats. Nonetheless, in contrast with a previous study
that not detected a clear association between urinary excretion or past exposure to FeMV and azotemic
CKD [11], the findings of our study suggest that FeMV infected cats have some degree of renal damage
while they are shedding virus in their urine. To date, knowledge on epidemiology of FeMV infection in cats is lacking, and sex, age or breed
predisposition is still unknown. In the current study, adult (i.e., >1-year-old) European domestic cats
were over-represented in the FeMV group while no differences were highlighted regarding sex. CKD is
the most common cause of death in cats, affects 30%–40% of older cats [17] and aging is an important
factor implicated in the initiation of the disease [27]. Interestingly, in this study, healthy cats and FeMV
cats had similar age and FeMV cats were significantly younger than CKD cats, suggesting that the renal
involvement, observed in the cohort of FeMV infected cats, might not be related to the aging process. The majority of FeMV infected cats had hematological and biochemical results within the reference
interval. However, slight differences were observed when they were compared with the healthy cats. In particular, CBC in FeMV positive cats showed lower hematocrit (Hct) values, and higher platelet and
white blood cell (WBC) counts. These findings, in association with FeMV infection, are non-specific
and potentially related to inflammation experienced by the FeMV cats, although not necessarily related
to FeMV infection [28,29]. Creatinine and, most recently, SDMA are useful serum markers of decreased kidney function
in cats and dogs [30]. intensity; Rf, ratio o
I
CKD
d
3.3. Renal Histopathology Moreover, FeMV cats had a significantly increased total number of bands if compared with the
healthy ones (P = 0.016). 3.2. Renal Histopathology
Three FeMV positive cats deceased within two months from the clinical visit because hit by car
and renal histopathology showed moderate to severe chronic TIN. In all of the cases,
immunohistochemical staining revealed FeMV–N positive cells mostly involving the cortical kidney
tubules with an extensive inflammatory infiltrate surrounding the FeMV–N positive cells (Figure 3)
Figure 3. Cat, Kidney, Immunohistochemestry for FeMV N protein. Severe tubulointerstitial nephritis
(TIN) with mononuclear cells infiltration. Epithelial cells of some cortical distal tubules showed
diffuse and granular immunoreactivity for viral antigen. Mayer’s hematoxylin counterstain, final
magnification × 200. Scale bar: 100 μm. 4. Discussion
The present study aimed to identify and characterize renal dysfunction in cats infected by FeMV
Figure 3. Cat, Kidney, Immunohistochemestry for FeMV N protein. Severe tubulointerstitial nephritis
(TIN) with mononuclear cells infiltration. Epithelial cells of some cortical distal tubules showed diffuse
and granular immunoreactivity for viral antigen. Mayer’s hematoxylin counterstain, final magnification
× 200. Scale bar: 100 µm. Figure 3. Cat, Kidney, Immunohistochemestry for FeMV N protein. Severe tubulointerstitial nephritis
(TIN) with mononuclear cells infiltration. Epithelial cells of some cortical distal tubules showed
diffuse and granular immunoreactivity for viral antigen. Mayer’s hematoxylin counterstain, final
magnification × 200. Scale bar: 100 μm. 4. Discussion
The present study aimed to identify and characterize renal dysfunction in cats infected by FeMV
Figure 3. Cat, Kidney, Immunohistochemestry for FeMV N protein. Severe tubulointerstitial nephritis
(TIN) with mononuclear cells infiltration. Epithelial cells of some cortical distal tubules showed diffuse
and granular immunoreactivity for viral antigen. Mayer’s hematoxylin counterstain, final magnification
× 200. Scale bar: 100 µm. Animals 2020, 10, 828 11 of 15 11 of 15 4. Discussion In our study, 13/14 (93%) and 14/14 (100%) FeMV-positive cats had sCr and
SDMA concentration, respectively, below the suggested IRIS CKD staging cut-offvalues for stage 2. Nevertheless, based on the criteria used in our study for the diagnosis of CKD and on the follow-up
evaluation, CKD IRIS stage 1 and IRIS stage 2 were suspected in 2/14 FeMV cats. Furthermore, CKD
IRIS stage 1 was a consistent diagnosis, suspected by clinicians, in an additional FeMV-positive cat
presenting proteinuria (UPC > 0.4) that remained persistently proteinuric during the follow-up period
(data not shown). These findings suggest that FeMV might be associated with CKD cases also in cats
younger than the senior ones. When compared with CKD, FeMV cats had a significantly lower sCr concentration. Although lower
than the healthy ones, FeMV cats had similar body weight and BCS, if compared with CKD, and for
this reason a poorer body condition in FeMV cats could not be considered as a possible explanation
of creatinine underestimation. Hyperfiltration experienced by FeMV cats could represent another
hypothesis raised to explain this finding; however, it remains difficult to be demonstrated without the
evaluation of a measured GFR, and should be confirmed in further studies. In the present study, although 13/14 (93%) FeMV cats had appropriately concentrated urine
(i.e., USG >1035) upon admission and in the follow up period (data not shown), USG results were
significantly lower if compared with the healthy group. This finding, together with the lower values of
uCr, may suggest an early renal dysfunction in cats infected by FeMV characterized by an impaired
ability to concentrate urine despite a normal urine handling of electrolytes, as suggested by the values
of FE of electrolytes. Nevertheless, comparing USG in the appropriately elevated range could not be
clinically relevant and additional studies using more accurate indicators of renal concentrating ability
should be performed to confirm these results. 12 of 15 Animals 2020, 10, 828 The UPC values of FeMV and CKD cats were similar, the frequency of proteinuria was not
different between these groups and, furthermore, 6/13 (46%) FeMV positive cats were proteinuric or
borderline proteinuric, suggesting a potential kidney damage. Indeed, Jepson and colleagues [20]
have identified UPC and USG, among others, as early predictors for the onset of CKD in 118 cats. These findings seemed to be further corroborated when SDS-PAGE results were combined with
quantitative proteinuria. 4. Discussion SDS-PAGE is helpful for the early diagnosis of renal damage or dysfunction
in cats and dogs, and can aid to distinguish between glomerular and tubular proteinuria [21,26,31]. Healthy cats included in this study had a typical pattern characterized by the constant presence of
uromodulin and albumin or cauxin bands. Uromodulin and cauxin are known markers of tubular
damage and their decrease was already demonstrated in cats affected by kidney disease [21,23,26]. In the CKD group, a lack or decrease of uromodulin and cauxin and an increase of albumin and
transferrin were observed. Moreover, the presence of a significantly higher mean number of bands with
an LMM compared with healthy cats confirmed the prevalent tubular involvement that characterize
CKD in cats, as reported previously [21]. In cats infected by FeMV, SDS-PAGE showed differences
when compared with the healthy cats. In particular, FeMV cats had frequently a tubular pattern
characterized by an increase in the number and intensity of LMM proteins (not different to CKD
cats) and a decrease of uromodulin. Moreover, the appearance of RBP, another known marker of
tubular damage [32], in urines of CKD cats and in some samples of FeMV cats, let us confirm the renal
involvement also in FeMV cats, although less severe than in the CKD ones. All tested cats, except one, had detectable antibodies against specific FeMV-N protein. As previously
suggested, it is important to interpret with caution serological results. Moreover, combining the results of
RT-PCR with serology could be useful to understand the length of exposure in infected cats [33]. Indeed,
the seronegativity herein observed in a PCR-positive cat may suggests an acute infection that could have
been confirmed by serology only using a convalescent titer in this case. The three FeMV cats that underwent kidney histopathology revealed tubulointerstitial changes. These lesions are common in cats regardless the presence of renal disease [34] thus, it is hard to interpret
and discuss the clinical implication of tubular lesions in positive cats. However, several studies
reported a higher incidence of TIN in cats with a FeMV infection compared with negative cats [1,3,9]. In particular, in a recent study, the detection of FeMV antigens was significantly associated with
some morphological abnormalities in feline kidney tissues, particularly those in renal tubular and
interstitial areas [35]. References 1. Woo, P.C.Y.; Lau, S.K.P.; Wong, B.H.L.; Fan, R.Y.Y.; Wong, A.Y.P.; Zhang, A.J.X.; Wu, Y.; Choi, G.K.Y.; Li, K.S.M.;
Hui, J.; et al. Feline morbillivirus, a previously undescribed paramyxovirus associated with tubulointerstitial
nephritis in domestic cats. Proc. Natl. Acad. Sci. USA 2012, 109, 5435–5440. [CrossRef] 2. Furuya, T.; Sassa, Y.; Omatsu, T.; Nagai, M.; Fukushima, R.; Shibutani, M.; Yamaguchi, T.; Uematsu, Y.;
Shirota, K.; Mizutani, T. Existence of feline morbillivirus infection in Japanese cat populations. Arch. Virol. 2014, 159, 371–373. [CrossRef] 2. Furuya, T.; Sassa, Y.; Omatsu, T.; Nagai, M.; Fukushima, R.; Shibutani, M.; Yamaguchi, T.; Uematsu, Y.;
Shirota, K.; Mizutani, T. Existence of feline morbillivirus infection in Japanese cat populations. Arch. Virol. 2014, 159, 371–373. [CrossRef] 3. Park, E.-S.; Suzuki, M.; Kimura, M.; Mizutani, H.; Saito, R.; Kubota, N.; Hasuike, Y.; Okajima, J.; Kasai, H.;
Sato, Y.; et al. Epidemiological and pathological study of feline morbillivirus infection in domestic cats in
Japan. BMC Vet. Res. 2016, 12, 228. [CrossRef] [PubMed] 3. Park, E.-S.; Suzuki, M.; Kimura, M.; Mizutani, H.; Saito, R.; Kubota, N.; Hasuike, Y.; Okajima, J.; Kasai, H.;
Sato, Y.; et al. Epidemiological and pathological study of feline morbillivirus infection in domestic cats in
Japan. BMC Vet. Res. 2016, 12, 228. [CrossRef] [PubMed] 4. Lorusso, A.; Di Tommaso, M.; Di Felice, E.; Zaccaria, G.; Luciani, A.; Marcacci, M.; Aste, G.; Bo
Savini, G. First report of feline morbillivirus in Europe. Vet. Ital. 2015, 51, 235–237. [CrossRef] [Pub 5. Marcacci, M.; De Luca, E.; Zaccaria, G.; Di Tommaso, M.; Mangone, I.; Aste, G.; Savini, G.; Boari, A.;
Lorusso, A. Genome characterization of feline morbillivirus from Italy. J. Virol. Methods 2016, 234, 160–163. [CrossRef] [PubMed] 6. De Luca, E.; Crisi, P.E.; Marcacci, M.; Malatesta, D.; Di Sabatino, D.; Cito, F.; D’Alterio, N.; Puglia, I.;
Berjaoui, S.; Colaianni, M.L.; et al. Epidemiology, pathological aspects and genome heterogeneity of feline
morbillivirus in Italy. Vet. Microbiol. 2019, 108484. [CrossRef] y
7. Sieg, M.; Heenemann, K.; Rückner, A.; Burgener, I.; Oechtering, G.; Vahlenkamp, T.W. Discovery of new feline
paramyxoviruses in domestic cats with chronic kidney disease. Virus Genes 2015, 51, 294–297. [CrossRef] 8. Sharp, C.R.; Nambulli, S.; Acciardo, A.S.; Rennick, L.J.; Drexler, J.F.; Rima, B.K.; Williams, T.; Duprex, W.P. Chronic Infection of Domestic Cats with Feline Morbillivirus, United States. Emerg. Infect. Dis. 2016, 22,
760–762. [CrossRef] 9. 4. Discussion This study had some limitations, first of all its retrospective nature that makes difficult to establish
a clear association of FeMV-positivity with changes in renal function or CKD and that shall be overcome
in further studies using experimental infection models. The limited cohort size and the fact that the study
groups were not age-matched could be a source of bias in the statistical comparison for some variables. Additionally, despite the use of same instrumentations, analytical methods, calibration and quality
control material in the two institutions that performed the clinicopathological evaluation, a potential
degree of variation between the laboratories results cannot be completely excluded. Moreover, even
though FeMV was detected in cats using a specific molecular assay targeting the viral genome, it is
difficult to assess whether infectious virus was still present at the time of urine collection. In this regard,
indeed, we were not able to provide clear indications regarding the phase of the infection. To circumvent
this issue, an experimental study is therefore highly recommended. Furthermore, the potential role of
comorbidities on clinical and clinicopathological findings (e.g., body weight and BCS) in FeMV cats
should be taken into account when interpreting our results. However, no concomitant diseases were
clearly diagnosed by clinicians at the time of inclusion in the study with the exception of FeLV infection
in two cases. Retroviral infections are known to be associated with clinicopathological abnormalities
including proteinuria in cats and could have partially biased our results. Nevertheless, the two
FeLV-positive cats were non-proteinuric, had no significant laboratory abnormalities and presented
a lower number of bands in their urines at qualitative proteinuria evaluation if compared with the
other FeMV cats (data not shown). For these reasons, these cats were not excluded from the study. 13 of 15 Animals 2020, 10, 828 Finally, histopathology and immunohistochemistry would have been necessary in order to characterize
the FeMV-associated kidney damage, however, in this study, were available only in three cases. Finally, histopathology and immunohistochemistry would have been necessary in order to characterize
the FeMV-associated kidney damage, however, in this study, were available only in three cases. 5. Conclusions Cats affected by FeMV had less concentrated urines if compared with healthy cats; furthermore,
they were proteinuric or borderline proteinuric in half of the cases. These findings, associated with
the results of urine electrophoresis, suggest a tubular damage or dysfunction in FeMV positive cats,
and most of these cases could be classified as CKD IRIS stage 1. FeMV infection could represent a potential renal insult leading to an early renal dysfunction in
the infected cats included in the present study; however, a clear pathogenetic role of the FeMV cannot
be determined in a spontaneous disease setting due to other potential confounders. Finally, further prospective studies combining long-term patient follow-up, renal pathology and
urine tests are warranted to obtain a better characterization of potential FeMV-associated renal damage
in the clinical setting. Author Contributions: Conceptualization, P.E.C., A.B., F.D., A.L. and G.S.; methodology, P.E.C. and F.D.; formal
analysis, P.E.C.; investigation, P.E.C., A.L., E.D.L., M.D.T., D.M., E.F., K.V. and F.D.; writing—original draft
preparation, P.E.C. and K.V.; writing—review and editing, A.B. and F.D.; supervision, A.B., F.D., A.L. and G.S. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Funding: This research received no external funding. Funding: This research received no external funding. Acknowledgments: The authors would like to acknowledge Park Eun-Sil and Shigeru Morikawa, Department of
Veterinary Science National Institute of Infectious Diseases (Tokyo, Japan) for providing anti-FeMV/N protein
rabbit polyclonal antibody. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. Author Contributions: Conceptualization, P.E.C., A.B., F.D., A.L. and G.S.; methodology, P.E.C. and F.D.; formal
analysis, P.E.C.; investigation, P.E.C., A.L., E.D.L., M.D.T., D.M., E.F., K.V. and F.D.; writing—original draft
preparation, P.E.C. and K.V.; writing—review and editing, A.B. and F.D.; supervision, A.B., F.D., A.L. and G.S.
All authors have read and agreed to the published version of the manuscript. References Yilmaz, H.; Tekelioglu, B.K.; Gurel, A.; Bamac, O.E.; Ozturk, G.Y.; Cizmecigil, U.Y.; Altan, E.; Aydin, O.;
Yilmaz, A.; Berriatua, E.; et al. Frequency, clinicopathological features and phylogenetic analysis of feline
morbillivirus in cats in Istanbul, Turkey. J. Feline Med. Surg. 2017, 19, 1206–1214. [CrossRef] 14 of 15 Animals 2020, 10, 828 10. Darold, G.M.; Alfieri, A.A.; Muraro, L.S.; Amude, A.M.; Zanatta, R.; Yamauchi, K.C.I.; Alfieri, A.F.; Lunardi, M. First report of feline morbillivirus in South America. Arch. Virol. 2017, 162, 469–475. [CrossRef] 11. McCallum, K.E.; Stubbs, S.; Hope, N.; Mickleburgh, I.; Dight, D.; Tiley, L.; Williams, T.L. Detection and
seroprevalence of morbillivirus and other paramyxoviruses in geriatric cats with and without evidence of 11. McCallum, K.E.; Stubbs, S.; Hope, N.; Mickleburgh, I.; Dight, D.; Tiley, L.; Williams, T.L. Detection and
seroprevalence of morbillivirus and other paramyxoviruses in geriatric cats with and without evidence of
azotemic chronic kidney disease. J. Vet. Intern. Med. 2018, 32, 1100–1108. [CrossRef] [PubMed] 12. Chakrabarti, S.; Syme, H.M.; Brown, C.A.; Elliott, J. Histomorphometry of feline chronic kidney disease and
correlation with markers of renal dysfunction. Vet. Pathol. 2013, 50, 147–155. [CrossRef] [PubMed] 13. Bartges, J.W. Chronic kidney disease in dogs and cats. Vet. Clin. N. Am. Small Anim. Pract. 2012, 42, 669–692. [CrossRef] [PubMed] 14. Finch, N. Measurement of glomerular filtration rate in cats: Methods and advantages over routine markers
of renal function. J. Feline Med. Surg. 2014, 16, 736–748. [CrossRef] 15. Polzin, D.J. Chronic kidney disease. In Textbook of Veterinary Internal Medicine: Disease of the Dog and the Cat;
Elsevier: St. Louis, MO, USA, 2017; pp. 1938–1959. 16. Vaden, S.L.; Levine, J.F.; Lees, G.E.; Groman, R.P.; Grauer, G.F.; Forrester, S.D. Renal biopsy: A retrospective
study of methods and complications in 283 dogs and 65 cats. J. Vet. Intern. Med. 2005, 19, 794–801. [CrossRef] 17. Sparkes, A.H.; Caney, S.; Chalhoub, S.; Elliott, J.; Finch, N.; Gajanayake, I.; Langston, C.; Lefebvre, H.P.;
White, J.; Quimby, J. ISFM Consensus Guidelines on the Diagnosis and Management of Feline Chronic
Kidney Disease. J. Feline Med. Surg. 2016, 18, 219–239. [CrossRef] 18. IRIS Staging of CKD. Available online:
http://www.xn--iriskidney-ru6e.com/guidelines/staging.html
(accessed on 26 January 2020). 19. Hall, J.A.; Yerramilli, M.; Obare, E.; Yerramilli, M.; Jewell, D.E. Comparison of serum concentrations of
symmetric dimethylarginine and creatinine as kidney function biomarkers in cats with chronic kidney
disease. J. Vet. Intern. Med. 2014, 28, 1676–1683. [CrossRef] 20. References Jepson, R.E.; Coulton, G.R.; Cowan, M.L.; Markwell, P.; Syme, H.M.; Elliott, J. Evaluation of mass spectrometry
of urinary proteins and peptides as biomarkers for cats at risk of developing azotemia. Am. J. Vet. Res. 2013,
74, 333–342. [CrossRef] 21. Ferlizza, E.; Campos, A.; Neagu, A.; Cuoghi, A.; Bellei, E.; Monari, E.; Dondi, F.; Almeida, A.M.; Isani, G. The effect of chronic kidney disease on the urine proteome in the domestic cat (Felis catus). Vet. J. 2015, 204,
73–81. [CrossRef] 22. Martorelli, C.R.; Kogika, M.M.; Chacar, F.C.; Caragelasco, D.S.; de Campos Fonseca Pinto, A.C.B.;
Lorigados, C.A.B.; Andrade, L.C. Urinary Fractional Excretion of Phosphorus in Dogs with Spontaneous
Chronic Kidney Disease. Vet. Sci. 2017, 4, 67. [CrossRef] 23. Miyazaki, M.; Soeta, S.; Yamagishi, N.; Taira, H.; Suzuki, A.; Yamashita, T. Tubulointerstitial nephritis causes
decreased renal expression and urinary excretion of cauxin, a major urinary protein of the domestic cat. Res. Vet. Sci. 2007, 82, 76–79. [CrossRef] [PubMed] 24. De Luca, E.; Crisi, P.E.; Di Domenico, M.; Malatesta, D.; Vincifori, G.; Di Tommaso, M.; Di Guardo, G.;
Di Francesco, G.; Petrini, A.; Savini, G.; et al. A real-time RT-PCR assay for molecular identification and
quantitation of feline morbillivirus RNA from biological specimens. J. Virol. Methods 2018, 258, 24–28. [CrossRef] [PubMed] 25. Taylor, S.S.; Sparkes, A.H.; Briscoe, K.; Carter, J.; Sala, S.C.; Jepson, R.E.; Reynolds, B.S.; Scansen, B.A. ISFM
Consensus Guidelines on the Diagnosis and Management of Hypertension in Cats. J. Feline Med. Surg. 2017,
19, 288–303. [CrossRef] [PubMed] 26. Hokamp, J.A.; Leidy, S.A.; Gaynanova, I.; Cianciolo, R.E.; Nabity, M.B. Correlation of electrophoretic urine
protein banding patterns with severity of renal damage in dogs with proteinuric chronic kidney disease. Vet. Clin. Pathol. 2018, 47, 425–434. [CrossRef] [PubMed] 27. Brown, C.A.; Elliott, J.; Schmiedt, C.W.; Brown, S.A. Chronic Kidney Disease in Aged Cats: Clinical Features,
Morphology, and Proposed Pathogeneses. Vet. Pathol. 2016, 53, 309–326. [CrossRef] 28. Rizzo, F.; Tappin, S.W.; Tasker, S. Thrombocytosis in cats: A retrospective study of 51 cases (2000–2005). J. Feline Med. Surg. 2007, 9, 319–325. [CrossRef] 29. Chikazawa, S.; Dunning, M.D. A review of anaemia of inflammatory disease in dogs and cats. J. Small Anim. Pract. 2016, 57, 348–353. [CrossRef] 30. Relford, R.; Robertson, J.; Clements, C. Symmetric Dimethylarginine: Improving the Diagnosis and Staging of
Chronic Kidney Disease in Small Animals. Vet. Clin. N. Am. Small Anim. Pract. 2016, 46, 941–960. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/). References [CrossRef] 15 of 15 Animals 2020, 10, 828 15 of 15 31. Littman, M.P. Protein-losing nephropathy in small animals. Vet. Clin. N. Am. Small Anim. Pract. 2011, 41,
31–62. [CrossRef] 32. Hokamp, J.A.; Nabity, M.B. Renal biomarkers in domestic species. Vet. Clin. Pathol. 2016, 45, 28–56. [CrossRef] 33. Beatty, J.A.; Sharp, C.R.; Duprex, W.P.; Munday, J.S. Novel feline viruses: Emerging significance of
gammaherpesvirus and morbillivirus infections. J. Feline Med. Surg. 2019, 21, 5–11. [CrossRef] [PubMed] 33. Beatty, J.A.; Sharp, C.R.; Duprex, W.P.; Munday, J.S. Novel feline viruses: Emerging significance of
gammaherpesvirus and morbillivirus infections. J. Feline Med. Surg. 2019, 21, 5–11. [CrossRef] [PubMed]
34. Lawler, D.F.; Evans, R.H.; Chase, K.; Ellersieck, M.; Li, Q.; Larson, B.T.; Satyaraj, E.; Heininger, K. The aging
feline kidney: A model mortality antagonist? J. Feline Med. Surg. 2006, 8, 363–371. [CrossRef] [PubMed]
35. Sutummaporn, K.; Suzuki, K.; Machida, N.; Mizutani, T.; Park, E.-S.; Morikawa, S.; Furuya, T. Association of
feline morbillivirus infection with defined pathological changes in cat kidney tissues. Vet. Microbiol. 2019,
228, 12–19. [CrossRef] [PubMed] gammaherpesvirus and morbillivirus infections. J. Feline Med. Surg. 2019, 21, 5–11. [CrossRef] [PubMed]
34. Lawler, D.F.; Evans, R.H.; Chase, K.; Ellersieck, M.; Li, Q.; Larson, B.T.; Satyaraj, E.; Heininger, K. The aging
feline kidney: A model mortality antagonist? J. Feline Med. Surg. 2006, 8, 363–371. [CrossRef] [PubMed]
35
Sutummaporn K ; Suzuki K ; Machida N ; Mizutani T ; Park E S ; Morikawa S ; Furuya T Association of 34. Lawler, D.F.; Evans, R.H.; Chase, K.; Ellersieck, M.; Li, Q.; Larson, B.T.; Satyaraj, E.; Heininger, K. The aging
feline kidney: A model mortality antagonist? J. Feline Med. Surg. 2006, 8, 363–371. [CrossRef] [PubMed] 35. Sutummaporn, K.; Suzuki, K.; Machida, N.; Mizutani, T.; Park, E.-S.; Morikawa, S.; Furuya, T. Association of
feline morbillivirus infection with defined pathological changes in cat kidney tissues. Vet. Microbiol. 2019,
228, 12–19. [CrossRef] [PubMed] © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/). © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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COLD CHAIN MANAGEMENT IN MEAT SUPPLY: «OLD» AND NOVEL STRATEGIES
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Teoriâ i praktika pererabotki mâsa
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Theory and Practice of Meat Processing №4 | 2017 УДК/UDC 637.5.037 DOI 10.21323/2414–438X–2017–2–4–20–34 Обзор
DOI 10.21323/2414–438X–2017–2–4–20–34 Аннотация ц
Мясо это скоропортящийся продукт с коротким сроком годности и, следовательно, с коротким сроком реализации. Поэтому, управление холодильной цепью при поставке имеет первостепенное значение для поддержания качества
и безопасности мяса и мясных продуктов. Сырое мясо и мясные продукты могут поддерживать рост патогенных
микроорганизмов или микроорганизмов, вызывающих порчу, и, таким образом, должны храниться при темпера-
турах, которые исключают возникновение риска здоровью. Холодильная цепь должна соблюдаться на всех этапах
реализации. В связи со сложностью глобальной цепи поставок мяса и мясных продуктов, которая нередко бывает
достаточно протяженной и связанной с транспортировкой продукта, как в внутри страны, так и при поставках
из одной страны в другую, или c одного континента на другой, требуется необходимость соблюдать режимы охла-
ждения и замораживания, отслеживать время и температуру, чтобы потребитель был уверен в свежести данных
продуктов и их безопасности. В настоящее время существует несколько доступных вариантов контроля и управле-
ния холодильной цепью, таких как комбинации хранения в охлажденном и замороженном виде, суперохлаждение, иони-
зирующее излучение, биоконсервация, высокое гидростатическое давление (HHP), активная упаковка, беспроводные
датчики поддерживаемые компьютерной базой данных холодильной цепи (CCD). Review paper Abstract Meat is a perishable product with a short shelf life and therefore short selling times. Therefore, cold chain management in meat
supply is of utmost importance for the maintenance of quality and safety of meat/meat products. Raw meat/meat products are
likely to support the growth of pathogenic microorganisms and/or spoilage bacteria, and should be kept at temperatures that
do not result in a risk to health. The cold chain should not be interrupted at all times along the meat distribution chain. The
complexity of global meat supply chain, with frequently long distribution chains associated with transportation of the product
within one country, from one to another country and from one to another continent, makes the solutions for the chilling and
freezing regimes, as well as monitoring of time-temperature profiles, very important for the overall success in delivery of prod-
uct which will be accepted by consumer for its freshness and safety levels. From recently, there are several available options for
control and management of the cold chain, such as chilled and frozen storage combinations, superchilling, ionizing radiation,
biopreservation, high hydrostatic pressure (HHP), active packaging, wireless sensors, supported with the software-based cold
chain database (CCD). УПРАВЛЕНИЕ ХОЛОДИЛЬНОЙ ЦЕПЬЮ
ПРИ ПОСТАВКАХ МЯСА: «СТАРЫЕ» И НОВЫЕ СТРАТЕГИИ Настасиевич И.,*Лакицевич Б., Петрович З. Институт гигиены мяса и технологии, Белград, Сербия вые слова: поставка мяса, порча, холодильнаяцепь, хранение, дистрибуция, розничная торговля Для цитирования: Настасиевич И., Лакицевич Б., Петрович З. Управление холодильной цепью при поставках мяса: «старые» и новые
стратегии. Теория и практика переработки мяса. 2017; 2(4):20-34. DOI:10.21323/2414–438X‑2017–2–4-20-34 Для цитирования: Настасиевич И., Лакицевич Б., Петрович З. Управление холодильной цепью при поставках мяса: «старые» и новые
стратегии. Теория и практика переработки мяса. 2017; 2(4):20-34. DOI:10.21323/2414–438X‑2017–2–4-20-34
For citation:
Nastasijevic I., Lakicevic B., Petrovic Z. Cold chain management in meat supply: «old» and novel strategies.Theory and practice of
i
2017 2(4) 20 34 (I R
) DOI 10 21323/2414 438X 2017 2 4 20 34 Theory and Practice of Meat Processing №4 | 2017 Theory and Practice of Meat Processing №4 | 2017 c I., Lakicevic B., Petrovic Z. Cold chain management in meat supply: «old» and novel strategies.Theory and practice of
essing. 2017; 2(4):20-34. (In Russ.) DOI:10.21323/2414–438X‑2017–2–4-20-34 COLD CHAIN MANAGEMENT
IN MEAT SUPPLY: «OLD» AND NOVEL STRATEGIES Ivan Nastasijevic*, Brankica Lakicevic and Zoran Petrovic
Institute of Meat Hygiene and Technology, Belgrade, Serbia Keywords: meat supply, spoilage, cold chain, storage, distribution, retail 1. Введение ингредиенты, полуфабрикаты и готовые продукты,
которые могут поддерживать рост патогенных ми-
кроорганизмов или микроорганизмов, вызывающих
порчу, должны храниться при температурах, кото-
рые исключают возникновение риска здоровью. Хо-
лодильная цепь должна соблюдаться на всех этапах
реализации [3]. Мясо это скоропортящийся продукт с коротким
сроком годности и следовательно с коротким сроком
реализации. В отличие от свежих фруктов и ово-
щей, упакованное мясо должно иметь на этикетке
обозначенный срок годности [1]. Поддержание хо-
лодильной цепи является одним из основных прин-
ципов и базовым требованием законодательства Ев-
ропейского союза (EU) по пищевой гигиене. Сырье, Общеизвестно, что срок годности охлажденного
мяса может быть увеличен с помощью различных ре- For citation: 20 2017 | №4 теория и практика переработки мяса
имеет управление холодильной цепью. Рис. 1. Схема холодильной цепи при поставках мяса
Потре-
битель
Нацио-
нальный
Между-
народный
Транспор-
тировка
Дистрибуция
Оптовая
торговля
Скотобойня
Холодильная камера
Помещение для
обвалки
Камера
холодильного
хранения
Транспор-
тировка
Национальная
Между-
народная
Дистрибуция
Ферма
Рознич-
ная тор-
говля Потре-
битель
Нацио-
нальный
Между-
народный Транспор-
тировка
Дистрибуция
Оптовая
торговля
Дистрибуция Транспор-
тировка Рознич-
ная тор-
говля Ферма Рис. 1. Схема холодильной цепи при поставках мяса шений в области упаковки, такими как вакуумная упа-
ковка или упаковка с модифицированной атмосферой
(MAP) [4, 5, 6]. Однако на сроки гожности охлажден-
ного мяса большое влияние оказывает температура. Неадекватная температура при хранении, транспор-
тировании и в точках розничной торговли может
привести к значительному сокращению срока годно-
сти и продолжительности хранения, и как следствие
к порче мяса и мясных продуктов [6]. Рис.1. Схема холодильной цепи при по шений в области упаковки, такими как вакуумная упа-
ковка или упаковка с модифицированной атмосферой
(MAP) [4, 5, 6]. Однако на сроки гожности охлажден-
ного мяса большое влияние оказывает температура. Неадекватная температура при хранении, транспор-
тировании и в точках розничной торговли может
привести к значительному сокращению срока годно-
сти и продолжительности хранения, и как следствие
к порче мяса и мясных продуктов [6]. Рис.1. Схема холодильной цепи при по спортировка/поставка-розничная торговля-потреби-
тель является постоянным потоком), например, для
поставки мяса из одной страны в другую или с одного
континента на другой (Рис. 1). тавках мяса Поддержание безопасности и свежести охлажден-
ного мяса до тех пор, пока оно не достигнет конечного
потребителя является постоянной проблемой, поэто-
му первостепенное значение имеет управление холо-
дильной цепью. Здоровые животные, подвергнутые убою с соблю-
дением правил гигиены после надлежащего отдыха
и голодной выдержки, дают практические асептиче-
ское мясо. 1. Введение Однако во время операций убоя, нутровки
и разделки обычно происходит перекрестная конта-
минация микроорганизмами, особенно на поверхно-
сти мяса в результате контакта с оборудованием, ин-
струментами, руками, одеждой, другими объектами
и т. п. Мясо — это особенно благоприятная среда для
роста микроорганизмов из-за его химического соста-
ва, например, большого количества белков, липидов
и воды. Содержание липидов в мясе делает его очень
чувствительным к окислению (реакции кислорода
с жирными кислотами) и последующему производст-
ву пероксидов. Продукты распада пероксидов имеют
характерный неприятный запах и вкус прогорклого
мяса. 2. Порча мяса Туши свиней, крупного рогатого скота (КРС),
мелкого рогатого скота (МРС), а также тушки птицы
сразу же охлаждаются по окончании послеубойной
экспертизы на линии убоя и/или в холодильных ка-
мерах скотобойни. Процесс охлаждения — аэроб-
ный, так как туши обычно подвергаются воздей-
ствию циркулирующего воздуха. Наиболее часто,
после 24–96 ч охлаждения, туши обычно переме-
щаются в помещение для обвалки/разделки, где они
дополнительно разделываются на сортовые отруба. Сортовые отруба обычно хранят до 6 недель в ва-
куумных упаковках при анаэробных условиях [3]. Продукты из измельченного мяса могут быть приго-
товлены из тримминга от обвалки и/или триммин-
га от сортовых отрубов после 6 недель анаэробного
хранения; они могут храниться аэробно или ана
эробно (Рис. 2). Существует несколько слабых точек в мясной
холодильной цепи, как например, охлаждение про-
дуктов во время хранения (перед отправкой), пре-
вышение температуры во время транспортировки
и передаче продуктов от одного участника холодиль-
ной цепи другому, а также время ожидания во время
комплектования и расформирования партий продук-
та в розничной торговле [7, 8, 9]. Нарушения темпе-
ратурных режимов приводит к изменениям качества
продукции во время дистрибуции и в конце срока
хранения, и может привести к порче до истечения
срока годности, вызывая дополнительные затраты
на ее утилизацию и, как следствие, к экономическим
потерям. Известно, что охлаждение красного мяса и туш
домашней птицы необходимо для задержки роста
бактерий. Охлаждение также требуется для сохра-
нения внешнего вида и потребительского качест-
ва мяса. Наиболее часто туши КРС, свиней и МРС
охлаждают, используя принудительную конвекцию
охлажденного воздуха [10], хотя также может ис-
пользоваться охлаждение распылением (примене-
ние тонкого распыления), так как оно более быст-
рое по сравнению с воздушным охлаждением, и оно
в основном используется для птицы, но также может
применяться на предприятиях по переработке КРС,
свиней и МРС. На сегодняшний день были выявлены слабые ме-
ста в холодильной цепи поставки, где распределение
по цепи порой трудоемко и длительно (бойня-тран- В соответствии с Регламентом (EC) 853/2004/EC
туши после убоя должны сразу же охлаждаться по 21 Theory and Practice of Meat Processing №4 | 2017 Рис. 2. 2. Порча мяса Условия охлаждения и холодильного хранения туш КРС, свиней и МРС, и их сортовых отрубов и тримминга (адаптировано из [3])
Туша
Помещение
для обвалки
Камера для
холодильного
хранения
Аэробное хранение
24-96 ч
Температура туши
снижается до
температуры
в центре ниже 7°C
Подготовка
сортовых отрубов
Комнатная
температура
ниже 12 °C
Анаэробное хранение
Вакуум-упакованные
сортовые отруба
хранившиеся при 0-2 °C,
в течении 6 недель
максимум
Охлаждающая
камера
Тримминг
Тримминг
Отрубы хранятся аэробно или анаэробно
(вакуумная упаковка) при 0-2 °C
Камера для
холодильного хранения Камера для
холодильного
хранения Помещение
для обвалки Охлаждающая
камера Вакуум-упакованные
сортовые отруба
хранившиеся при 0-2 °C,
в течении 6 недель
максимум Туша Туша Тримминг Камера для
холодильного хранения Отрубы хранятся аэробно или анаэробно
(вакуумная упаковка) при 0-2 °C Рис. 2. Условия охлаждения и холодильного хранения туш КРС, свиней и МРС, и их сортовых отрубов и тримминга (адаптировано из [3]) холодильного хранения туш КРС, свиней и МРС, и их сортовых отрубов и тримминга (адаптировано из [3]) РС, свиней и МРС, и их сортовых отрубов и тримминга (адаптировано из [3]) например, меняется цвет, возникает посторонний
запах или слизь, что обычно обусловлено вызываю-
щими порчу бактериями (Табл. 1), хотя к этому мо-
гут быть причастны эндогенные ферменты [11]. На-
пример, Pseudomonads, Lactobacillus и Enterococcus
влияют на образование слизи на мясе, в то время
как Enterococcus продуцирует пероксид водорода,
приводящий к образованию зеленых пятен, анало-
гичных позеленению, вызванному Clostridium spp. Рост бактерий на поверхности мяса вызван влия-
нием температуры, показателем рН, активностью
воды, доступность нутриентов, атмосферой хране-
ния (аэробная или анаэробная) и взаимодействием
с другими микроорганизмами, присутствующими
на мясе [12]. например, меняется цвет, возникает посторонний
запах или слизь, что обычно обусловлено вызываю-
щими порчу бактериями (Табл. 1), хотя к этому мо-
гут быть причастны эндогенные ферменты [11]. На-
пример, Pseudomonads, Lactobacillus и Enterococcus
влияют на образование слизи на мясе, в то время
как Enterococcus продуцирует пероксид водорода,
приводящий к образованию зеленых пятен, анало-
гичных позеленению, вызванному Clostridium spp. Рост бактерий на поверхности мяса вызван влия-
нием температуры, показателем рН, активностью
воды, доступность нутриентов, атмосферой хране-
ния (аэробная или анаэробная) и взаимодействием
с другими микроорганизмами, присутствующими
на мясе [12]. окончании послеубойной экспертизы (рекомендо-
ванная температура для мяса ниже 7 °C и субпро-
дуктов ниже 3 °C). Следует отметить, что временные
рамки, в течение которых должна быть достигнута
эта температура, не определены. В скотобойне холодильная цепь начинается с двух
основных этапов: В скотобойне холодильная цепь начинается с двух
основных этапов: (a) первичное охлаждение (быстрое охлаждение туш
после убоя), таким образом, чтобы в наиболее те-
плой точке туши (центр задней ноги) достигалась
температура около 7 °C и 3 °C (для пищевых суб-
продуктов) и приблизительно 4 °C для тушек пти-
цы для предотвращения роста микроорганизмов
и увеличения продолжительности хранения. При
современных технологиях эти температуры мож-
но получить спустя 16–24 часа в маленьких тушках
(МРС), в течении 48 часов в больших тушах (говя-
жья, свиная) и менее чем за 2 ч для тушек птицы
(глубоко внутри грудок). Средние и поверхност-
ные температуры намного ниже, достигая 0 °C
в течение 4 часов. Это очень важно для замедления
пролиферации микроорганизмов; 3.1.2. Иммерсионное охлаждение. Это наиболее
старый и наименее дорогой способ охлаждения, ко-
торый обеспечивает очень быстрое охлаждение без
риска замораживания. Иммерсионное охлаждение —
это система вне линии, которая не позволяет авто-
матизировать взвешивание до и после охлаждения,
и обычно используется в США для охлаждения ту-
шек птицы. Аппараты для иммерсионного охлажде-
ния занимают небольшую площадь и обычно очень
энергоэффективные. Процесс может быть осуществ-
лен, используя водопроводную воду с добавлением
или без добавления чешуйчатого льда или предвари-
тельно охлажденную воду для создания более низких
температур иммерсионных ванн. Такая система спо-
собна понизить температуру водопроводной воды до
1 °C. Данный процесс может привести к увеличению
массы продукта до 12 % в результате контролируемой
абсорбции воды. 3.1.2. Иммерсионное охлаждение. Это наиболее
старый и наименее дорогой способ охлаждения, ко-
торый обеспечивает очень быстрое охлаждение без
риска замораживания. Иммерсионное охлаждение —
это система вне линии, которая не позволяет авто-
матизировать взвешивание до и после охлаждения,
и обычно используется в США для охлаждения ту-
шек птицы. Аппараты для иммерсионного охлажде-
ния занимают небольшую площадь и обычно очень
энергоэффективные. Процесс может быть осуществ-
лен, используя водопроводную воду с добавлением
или без добавления чешуйчатого льда или предвари-
тельно охлажденную воду для создания более низких
температур иммерсионных ванн. Такая система спо-
собна понизить температуру водопроводной воды до
1 °C. Данный процесс может привести к увеличению
массы продукта до 12 % в результате контролируемой
абсорбции воды. (b) (b) вторичное охлаждение (поддержание температуры
мяса ниже 7 °C (красное мясо) и ниже 4 °C (мясо
птицы) в течение всего времени во время холо-
дильного хранения, разделки, обвалки, измельче-
ния перед транспортировкой. 2. Порча мяса Например, туши
КРС и МРС обычно не охлаждают до ниже 10 °C
(температура в центре) в течение первых 10 ч, для
того, чтобы избежать холодового сокращения и при-
обретения жесткой структуры мяса. Однако такие
условия благоприятны для роста бактерий на по-
верхности туши, до тех пор пока температура не бу-
дет снижена достаточно, чтобы остановить жизнеде-
ятельность бактерий [3]. окончании послеубойной экспертизы (рекомендо-
ванная температура для мяса ниже 7 °C и субпро-
дуктов ниже 3 °C). Следует отметить, что временные
рамки, в течение которых должна быть достигнута
эта температура, не определены. Например, туши
КРС и МРС обычно не охлаждают до ниже 10 °C
(температура в центре) в течение первых 10 ч, для
того, чтобы избежать холодового сокращения и при-
обретения жесткой структуры мяса. Однако такие
условия благоприятны для роста бактерий на по-
верхности туши, до тех пор пока температура не бу-
дет снижена достаточно, чтобы остановить жизнеде-
ятельность бактерий [3]. Мясо считается испорченным, когда проявля-
ются изменения органолептических показателей, Таблица 1. Обзор основных причин возникновения порчи мяса/мясных продуктов и связанные с этим бактерии
(адаптированный из [8]) ор основных причин возникновения порчи мяса/мясных продуктов и связанные с этим бактерии
й з [8]) Таблица 1. Обзор основных причин возникновения порчи мяса/мясных продуктов и связанные с этим бактерии
(адаптированный из [8])
Дефект
Мясо/мясные продукты
Вызывающие порчу бактерии
Слизь
Свежее мясо
Pseudomonads, Lactobacillus, Enterococcus, Weissella, Brochothrix
Позеленение, вызванное
перекисью водорода
Свежее мясо
Weissella, Leuconostoc, Enterococcus, Lactobacillus
Образование сероводорода
Посоленные мясопродукты
Vibrio, Enterobacteriaceae
Запах серы
Вакуум-упакованное
охлажденное мясо
Clostridium, Hafnia
Запах капусты
Бекон
Providencia
Сырный или молочный аромат
Вакуум-упакованное
охлажденное мясо
Brochothrix thermosphacta
Гниение
Окорок
Enterobacteriaceae, Proteus
Порча мяса у кости
Цельномышечные
мясные продукты
Clostridium, Enterococcus
Закисание
Вакуум-упакованные
мясные продукты
Lactic acid bacteria, Enterococcus, Micrococcus, Bacillus, Clostridium р
у
р
Вызывающие порчу бактерии
Pseudomonads, Lactobacillus, Enterococcus, Weissella, Brochothrix
Weissella, Leuconostoc, Enterococcus, Lactobacillus
Vibrio, Enterobacteriaceae
Clostridium, Hafnia
Providencia
Brochothrix thermosphacta
Enterobacteriaceae, Proteus
Clostridium, Enterococcus
Lactic acid bacteria, Enterococcus, Micrococcus, Bacillus, Clostridium 22 2017 | №4 теория и практика переработки мяса поставок мяса Для успешной внутренней и международной тор-
говли и при экспорте очень важно вакуумное хранение
красного мяса и мяса птицы в виде герметично упако-
ванного продукта в охлажденном или в заморожен-
ном состоянии при достаточно низких температурах
[13]. Ранее были проведены полные исследования по
влиянию длительного холодильного хранения и хра-
нения при замораживании мяса и мясных продуктов
для целей экспорта. [12, 14]. Было доказано, что при
заморозке продолжительность хранения может быть
увеличена на срок более одного года [15]. Несмотря на
эти преимущества, улучшение в технологиях консер-
вирования в современной экспортной холодильной
цепи остается актуальным вопросом, особенно на
промышленном уровне, включая потенциал развития
суперохлаждения, ионизирующего излучения, био-
консервирования и обработку высоким гидростати-
ческим давлением или обработку высоким давлением
(ННР) [16]. 3. Управление холодильной цепью
поставок мяса лодильной камеры. Температура воздуха должна быть
около 0 °C, не снижаясь ниже минус 1 °C, что может
привести к замораживанию поверхности мяса и ухуд-
шению его внешнего вида. Скорость воздуха может
колебаться в диапазоне от 0, 25 до 3, 0 м/с. Однако по
экономическим причинам, наиболее широко приме-
няемые скорости находятся в диапазоне от 0, 75 до 1,
5 м/с. в пустой холодильной камере. Во время проце-
дуры охлаждения должен поддерживаться высокий
уровень влажности чтобы предотвратить чрезмерную
потерю массы. Рекомендуемый уровень 90 % — 95 %,
хотя это наиболее трудный для контроля параметр. Стоит отметить, что иногда быстрое охлаждение пред-
ставляет собой проблемы из-за «холодового сокраще-
ния». Холодовое сокращение может часто наблюдать-
ся в говядине и баранине, когда мясо, все еще в фазе
посмертного окоченения, достигает температур 10 °C
и ниже. Эти условия вызывают необратимые сокраще-
ния мышечной ткани, что увеличивает жесткость мяса
даже после продолжительного созревания. Быстрое
первичное охлаждение приводит к увеличению стои-
мости продукции и более высоким эксплуатационным
затратам. Период охлаждения может быть уменьшен,
снижением температуры воздуха (риски заморажива-
ния поверхности) или увеличением скорости воздуха
(более высокие эксплуатационные затраты), или од-
новременным применением двух способов. Чтобы по-
лучить более низкие температуры, чем в предыдущих
процедурах, иногда холодильные камеры охлаждают
заранее (до температуры минус 5–6 °C для говядины
и минус 10–12 °C для свинины), получая преимущества
термической инерции, чтобы компенсировать влия-
ние теплого мяса [8]. 3.3. Холодильная цепь в розничной торговле тов» или «птиц интенсивной шпарки», а также «све-
жих продуктов» или «птиц мягкой шпарки». Этот
метод охлаждения основан на комбинации прерыви-
стого распыления холодной воды и потока холодного
воздуха по всей поверхности продукта (начальный
этап охлаждения) и использование только потока
воздуха (финишная часть охлаждения). При его ис-
пользовании не происходит высыхание поверхности
туш, ускоряется процесс охлаждения, сохраняется
цвет мяса. Как правило, камеры (туннели) охлажде-
ния с распылителями разработаны специально для
обработки тушек птиц расположенных в один слой,
для предотвращения перекрестной контаминации. Возможно расположение и в два слоя, но в этом случае
нижний слой должен быть размещен таким образом,
чтобы предотвратить попадание загрязненных капель
от тушек верхнего слоя, и для этого требуются допол-
нительные емкости под сток. тов» или «птиц интенсивной шпарки», а также «све-
жих продуктов» или «птиц мягкой шпарки». Этот
метод охлаждения основан на комбинации прерыви-
стого распыления холодной воды и потока холодного
воздуха по всей поверхности продукта (начальный
этап охлаждения) и использование только потока
воздуха (финишная часть охлаждения). При его ис-
пользовании не происходит высыхание поверхности
туш, ускоряется процесс охлаждения, сохраняется
цвет мяса. Как правило, камеры (туннели) охлажде-
ния с распылителями разработаны специально для
обработки тушек птиц расположенных в один слой,
для предотвращения перекрестной контаминации. Возможно расположение и в два слоя, но в этом случае
нижний слой должен быть размещен таким образом,
чтобы предотвратить попадание загрязненных капель
от тушек верхнего слоя, и для этого требуются допол-
нительные емкости под сток. Поддержание холодильной цепи во время разме-
щения в холодильной витрине розничной торговли
имеет огромное значение для предотвращения микро-
биологической порчи, а также для поддержания све-
жести и безопасности мяса. Размер и производитель-
ность холодильных камер на предприятиях розничной
торговли, размер помещений, исходная температура
поступающего мяса, процедура обработки мяса (раз-
делка, измельчение), температура окружающей сре-
ды, расположение холодильного оборудования, вен-
тиляция и свет — это все возможные слабые точки,
на которые необходимо обращать внимание. Особое
внимание необходимо уделять температуре при ком-
плектовании/расформировании партий во время пе-
реработки мяса (например, ежедневное перемещение
мясных отрубов из холодильных камер до витрины
розничной торговли и наоборот (при этом, темпе-
ратура внутри кусков мяса должна быть постоянно
меньше чем 7 °C). Управленческий подход, который
доминирует на мясном рынке связан с принципом
«первый поступил- первый отправлен». Однако такой
подход также должен применяться на всех стадиях хо-
лодильной цепи (Рис. 3.3. Холодильная цепь в розничной торговле 1), и он может быть достигнут
с помощью правильно разработанных регламентов пе-
ремещения продукции в холодильных камерах хране-
ния мяса и мясных продуктов и витринах розничной
торговли. В целом, все точки в цепи транспортировки
от холодильного хранения на скотобойне до предпри-
ятия розничной торговли, а затем до холодильника
потребителя являются критическими точками для об-
щего качества и безопасности мяса. 3.1.4. Вакуумное охлаждение. Это быстрый пери-
одический процесс, при которой сырые продукты,
содержащие свободную воду, охлаждаются путем ис-
парения воды в условиях вакуума. Преимуществом
данной технологии является существенное снижение
количеств психрофильных и мезофильных бактерий
после хранения мяса в течение нескольких дней. У ох-
лажденного мяса в вакуумной упаковке срок годности
значительно дольше, чем у охлажденного мяса в тра-
диционной упаковке. Например, вакуум-упакованные
говяжьи отруба могут храниться до 12 недель, в то вре-
мя как отруба баранины и свинины имеют продолжи-
тельность хранения до 5 и 8 недель при хранении при
температуре 0 °C, соответственно. Недостатком явля-
ется большие потери массы мяса. Theory and Practice of Meat Processing №4 | 2017 Theory and Practice of Meat Processing №4 | 2017 В скотобойне холодильная цепь начинается с двух
основных этапов: Для охлаждения мяса перед транспортировкой
наиболее распространенны такие технологии, как:
(i) воздушное охлаждение, (ii) иммерсионное охла-
ждение, (iii) охлаждение распылением, и (iv) вакуум-
ное охлаждение [8]. 3.1.1. Воздушное охлаждение. Эффективность при-
менения воздушного охлаждения зависит от ряда
факторов включая температуру воздуха, скорость
воздушного потока, относительную влажность среды,
массу туши, толщину шпика туш, а также загрузки хо- 3.1.1. Воздушное охлаждение. Эффективность при-
менения воздушного охлаждения зависит от ряда
факторов включая температуру воздуха, скорость
воздушного потока, относительную влажность среды,
массу туши, толщину шпика туш, а также загрузки хо- 3.1.3. Охлаждение орошением. Это альтернативный
иммерсионному охлаждению метод, который все чаще
используется, в США для охлаждения тушек птицы. Охлаждение орошением может быть применено при
переработке и производстве «замороженных продук- 3.1.3. Охлаждение орошением. Это альтернативный
иммерсионному охлаждению метод, который все чаще
используется, в США для охлаждения тушек птицы. Охлаждение орошением может быть применено при
переработке и производстве «замороженных продук- 23 3.2. Холодильная цепь
при транспортировке мяса Как было сказано, если холодильная цепь наруша-
ется, то возникает ряд проблем, связанных с качест-
вом мясных продуктов и продуктов из мяса птицы,
как например, усушка, гниение, потери при зачистке,
неприятный запах, изменения цвета и текстуры, а так-
же усугубленные риски для здоровья со стороны таких
патогенов как Salmonella, Campylobacter, вырабатываю-
щие шига-токсин E. coli (STEC) и Listeriamonocytogenes. Несмотря на многочисленные исследования, наце-
ленные на лучшее понимание микробной экологии
микроорганизмов в мясе (микроорганизмы, вызыва-
ющие порчу и пищевые патогены) и ее взаимоотноше-
ния с температурой/временем в сложной цепи поста-
вок мяса (например, хранение мяса, транспортировка,
дистрибуция и розничная торговля), поддержание хо-
лодильной цепи, а также качество и безопасность мяса
все еще остаются проблемой. На сегодняшний день,
существует несколько вариантов/мер контроля, кото-
рые могут быть эффективно и синергетически приме-
нены для улучшения контроля в «классической» холо-
дильной цепи поставок мяса. Для обеспечения стабильного качества и безопас-
ности продукции холодильная цепь должна стабильно
поддерживаться о всей цепи транспортировки мяса
до конечного потребителя — оптового холодильно-
го хранения или холодильной витрины предприятия
розничной торговли. При этом холодильные камеры,
оборудованные в специальном транспорте или в ку-
зове грузовика, имеют различные особенности и про-
изводительность. Их размер, начальная температура
отгружаемого мяса, время достижения рекоменду-
емой температуры во время транспортировки, кон-
структивные особенности (например, мощность ком-
прессоров, вентиляции и изоляции), а также вопросы
энергозатрат — являются приоритетными при тран-
спортировке мяса [8]. В целом, транспортные средства
должны быть оснащены необходимым холодильным
оборудованием и системами охлаждения, способных
поддерживать нужный температурный режим для
мяса и субпродуктов в течение всего времени тран-
спортирования (3. 1.). 24 2017 | №4 теория и практика переработки мяса 4.1. Комбинации хранения в охлажденном
и замороженном виде живания/размораживания и, таким образом, увели-
чить выход, снизить энергию, трудовые затраты и за-
траты на транспортировку [21]. В течение предыдущих двух десятилетий были
проведены многочисленные исследования для оцен-
ки увеличения продолжительности хранения, а также
улучшения качества и безопасности мяса, достигаемых
с помощью режимов охлаждения и замораживания. Однако, следует отметить, что было проведено огра-
ниченное количество исследований по оценке комби-
нированных практик холодильного и морозильного
хранения, особенно в отношении длительного холо-
дильного хранения, а затем морозильного хранения
с общей продолжительностью свыше 3–4 мес. 4.3. Ионизирующее излучение Обработка пищевых при помощи ионизирующего
излучения обладает рядом положительных эффектов,
имеет благоприятное воздействие, которые не может
быть достигнуто другими способами и, в частности,
традиционными методами, такими как например ох-
лаждение. Облучение пищевых продуктов, приме-
няемое при низкой дозе (до 1 кГр) может быть очень
эффективным против паразитов, присутствующих
в красном мясе, а также для инактивации патогенных
и вызывающих порчу бактерий в охлажденном и/или
замороженном мясе и птице. По всей вероятности, продолжительное холо-
дильное хранение в вакуум-упакованном виде (две
недели для говядины и баранины при 4 °C и даже до
30 недель для говядины при –0,5 °C и 7 недель для ба-
ранины при –1,5 °C) в комбинации с последующим
морозильным хранением (до одного года при –18 °C)
оказывает благоприятное влияние на параметры ка-
чества (усилие среза, нежность, влагоудерживающую
способность, флейвор и цвет) мяса; улучшенная неж-
ность была подтверждена как основное достижение
[15, 19, 20]. Очевидно, что пролонгированное комби-
нированное холодильное/морозильное хранение мо-
жет обладать благоприятным эффектом для экспор-
та мяса. Необходимы дальнейшие и более глубокие
исследования для изучения цветовых характеристик
и микробиологического профиля охлажденного, а за-
тем замороженного мяса, так как эти исследования
тесно связаны с доверием потребителя и вопросами
здоровья [20]. С другой стороны, с самого начала несколько об-
щественных организаций по защите прав потреби-
телей выражали обеспокоенность в отношении без-
опасности облученных продуктов для потребления. Однако после более 100 лет исследований, возникшие
вопросы разрешены, и потребители принимают об-
лученные пищевые продукты там, где они доступны
с понятным объяснением этой новой технологии. На-
пример, в Китае, имеют свою долю рынка фирменные
блюда — маринованные куриные ножки (обработан-
ные излучением). В ЕС количество облученных пи-
щевых продуктов на рынке и существенно отличает-
ся от США. Последние исследования в ЕС по опросу
потребителей в отношении употребления облученных
пищевых продуктов на данный момент не доступны. Использование ионизирующего излучения при пере-
работке пищевых продуктов все еще остается недоо-
цененной и недостаточно используемой технологией,
несмотря на большой потенциал [22]. 4.2. Суперохлаждение Суперохлаждение используется для обеспечения
сохранности пищевых продуктов путем их обработки,
при которой температура пищевого продукта снижена
на 1–2 °C ниже исходной точки замерзания (для боль-
шинства продуктов между 0,5 °C и 2,8 °C). Технология
суперохлаждения является комбинацией благопри-
ятного эффекта низких температур с превращением
некоторого количества воды в лед, что является недо-
статком из-за ухудшения качества. Суперохлаждение
обеспечивает пищевой продукт внутренним льдом,
так что нет необходимости во внешнем льде вокруг
продукта во время транспортировки или хранения
в течение более коротких периодов времени. В общем,
суперохлаждение позиционируется между заморажи-
ванием и охлаждением (традиционное охлаждение),
где температура окружающей среды устанавливается
ниже исходной точки замерзания. Хранение пищевого
продукта при температурах суперохлаждения имеет
три основных преимущества: поддержание свежести
и обеспечение высокого качества пищевого продукта,
а также подавление роста вредных микроорганизмов. Кроме того, оно может снизить применение замора- 4.6. Активная упаковка Активная упаковка — это инновационная техно-
логия, которая позволяет взаимодействовать продук-
ту и его окружающей средой для увеличения срока
хранения продукта и обеспечения его микробиоло-
гической безопасности, при поддержании качества
упакованного пищевого продукта. В соответствии,
с законодательством ЕС, активная упаковка — это
тип упаковки с дополнительной функцией, помимо
обеспечения защитного барьера против внешнего
влияния. Это означает, что химические вещества,
полученные из пищевого продукта и окружающей
среды могут абсорбироваться в упаковку, или сама
упаковка выделяет вещества в пищевой продукт или
окружающую среду, например, консерванты, анти-
оксиданты и вкусо-ароматические вещества [25]. Существует три основных типа активной упаковки,
которые были разработаны к настоящему времени:
антимикробная активная упаковка, антиоксидант-
ная активная упаковка, выделяющая/генерирующая
диоксид углерода упаковка. 4.6.3. Упаковка, выделяющая диоксид углерода. Ди-
оксид углерода обладает доказанным ингибирующим
эффектом в отношении ряда аэробных бактерий
и грибков при пониженном содержании кислорода
и прямого антимикробного эффекта (путем удлине-
ния лаг-фазы и времени генерации во время логариф-
мической фазы роста микроорганизмов). Таким обра-
зом, генерирующая CO2 упаковочная система — это
метод, дополняющий поглощение кислорода. Уров-
ни CO2, применяемые для сохранения красного мяса
и мяса птицы, относительно высокие (10–80 %). На-
пример, для ингибирования Pseudomonas необходимо
10–20 % CO2, в то время как для контроля пролифера-
ции C. perfringens, C. botulinum и Listeriamonocytogenes
нужно 50 % CO2. 4.6.1. Антимикробная активная упаковка. Это
одна из наиболее важных концепций активной упа-
ковки мяса. Существуют четыре основных категории
антимикробной упаковки: (i) внесение антимикроб-
ных веществ в мягкую подложку внутри упаковки
для обеспечения медленного высвобождения анти-
микробных веществ, (ii) прямое введение антими-
кробных агентов в упаковочную пленку путем тепло-
вой обработки (ко-экструзия упаковочных пленок
с антимикробными веществами), таким образом, что
антимикробные вещества могут постепенно высво-
бождаться из упаковочных пленок в свободное от 4.4. Биоконсервирование Натуральные компоненты, такие как эфирные ма-
сла, низин, лизоцим, а также естественная или контр-
олируемая микробиота, например, молочнокислые
бактерии (МКБ) и их антимикробные продукты, такие
как молочная кислота и бактериоцины, также были ис-
следованы для увеличения срока хранения мяса и мяс-
ных продуктов, и для получения продуктов с «зеленой
этикеткой». Например, низин является единственным
коммерчески доступным бактериоцином и показал
хорошее антибактериальное действие в искусственно
контаминированной свинине, а в комбинации с 2 %
хлоридом натрия превосходный противолистериоз-
ный эффект в говяжьем фарше; пентоцин 31–1 (вы-
рабатываемый Lactobacilluspentosus 31–1, который был
выделен из традиционного китайского ферментиро-
ванного окорока Xuanwei) эффективно ингибировал
азот летучих оснований (АЛО) и подавлял рост инди-
генной микробиоты, особенно Listeria и Pseudomonas
в свинине при холодильном хранении [16]. 25 Theory and Practice of Meat Processing №4 | 2017 4.5. Высокое гидростатическое давление (HHP) продукта пространство в упаковке или на поверх-
ность продукта, (iii) покрытие упаковки матрицей,
которая действует как носитель для антимикробных
агентов, так чтобы выделить антимикробных веществ
на поверхность пищевого продукта через испарение
в свободное от продукта пространство (летучие ве-
щества) или миграцию в пищевой продукт через
диффузию (нелетучие вещества) и (iv) использование
полимеров, которые обладают антимикробной актив-
ностью (например, хитозан, поли-L-лизин). Кроме
того, были проведены исследования большого коли-
чество антимикробных веществ для ингибирования
роста микроорганизмов в пищевых продуктах, на-
пример, этанол, диоксид углерода, ионы серебра, ди-
оксид хлора, антибиотики, бактериоцины, органиче-
ские кислоты, эфирные масла, специи, растительные
экстракты (розмарин), пептиды и т. д. [26]. Процесс сохранения мяса с использованием высо-
кого гидростатического давления (HHP) — нетерми-
ческой технологии, может инактивировать вызыва-
ющие порчу продукта микроорганизмы и ферменты
при низких температурах без существенного измене-
ния органолептических или пищевых характеристик
продукта; нежность даже улучшается, в то время как
цвет сырого мяса несколько изменяется после обра-
ботки HHP. HHP также является мощным средст-
вом контроля рисков, ассоциированных с Salmonella
и Listeriamonocytogenes в сырых или маринованных
мясных продуктах [23]. Обработка давлением обыч-
но производится в цилиндрах из нержавеющей стали,
которые содержат передающую давление среду, на-
пример, воду, в то время как продукт защищен от пря-
мого контакта с помощью применения герметичной
пластичной упаковки [16]. Рекомендуемая комбина-
ция давления/времени для мяса и мясных продуктов
(600–700 MПa в течение 2–5 мин. ) показала быструю
инактивацию L. monocytogenes, а также другой инди-
генной микробиоты [24]. 4.6.2. Антиоксидантная активная упаковка. Высо-
кие уровни кислорода в упаковке мяса могут способст-
вовать росту микроорганизмов, окислению липидов,
образованию постороннего флейвора и запаха, изме-
нению цвета и потерям питательных веществ. Таким
образом, контроль уровня кислорода в упаковке мяса
очень важен для предотвращения ухудшения качест-
ва и порчи мяса. Системы антиоксидантной актив-
ной упаковки могут быть подразделены на 2 группы:
(i) независимые антиоксидантные приспособления
(саше, мягкие подложки или этикетки с поглотителя-
ми кислорода — мелкий порошок железа и оксид же-
леза) и (ii) антиоксидантные упаковочные материалы
(антиоксидантные активные агенты вводятся в стенки
упаковочной пленки (например, терпеноиды из про-
полиса). 4.7. Беспроводные сенсоры Производители в пищевой промышленности часто
сталкиваются с дилеммой относительно типа управ-
ления холодильной цепью при поставках пищевых
продуктов до розничной торговли или конечных по-
требителей, стараясь выбрать между хранением в за-
мороженном состоянии или холодильным хранением 26 2017 | №4 теория и практика переработки мяса (охлаждение, замораживание, поставка и хранение). Температура — это основной параметр после процес-
са переработки в определении продолжительности
хранения в холодильной цепи охлажденных или замо-
роженных пищевых продуктов. Хранение в заморо-
женном состоянии требует больших энергетических
затрат для сохранения пищевых продуктов, в то время
при холодильном хранении существует постоянная
угроза порчи, вызванной бактериями. Современное
управление холодильной цепью включает контроль
и регулирование температуры и сфокусирован на
простой логистической цепи, а не на обработке не-
скольких каналов. Для преодоления вышеуказанных
недостатков разработана система холодильной цепи
на основе температурно-временного индикатора
(TTI), в которой используются беспроводные сенсо-
ры для сбора температурных данных на протяжении
цепи поставок мяса (от холодильного хранения на
скотобойне до розничной торговли) и внедряются по-
ложения критериев критических контрольных точек
(ККТ) на протяжении всего процесса поставок. При
строгом контроле температуры, возможен перевод
ряда пищевых продуктов с замороженного хранения
на охлажденное, для преодоления недостатков, свя-
занных с замороженным хранением (высокоэнергети-
ческое потребление, ухудшение вкуса, ограниченное
число продаж). Создаются контрольные диаграммы
для контроля каждой точки процесса. Этот подход
основан на архитектуре «Интернет вещей» Internet
of Things (IoT) и Международном стандарте по без-
опасности и качестве пищевых продуктов (ISO 22000). IoT является растущей тенденцией из-за сильного
влияния в результате развития информационно-ком-
муникационных технологий (ICT), например, бирки
радиочастотной идентификации животных и пище-
вой продукции (RFID), датчики, исполнительные ме-
ханизмы и даже интеллектуальные устройства такие
как мобильные телефоны [28]. На практике, ожида-
ется, что IoT будет развиваться в таких областях, как
беспроводные сенсорные сети с целью сбора контекс-
туальных данных. В последнее время идет разработка
программного обеспечения для расширения сетевых
услуг, используя возможности IoT (сеть вещей — Web
of Things, WoT). Наконец, также были разработаны
новые бизнес модели в пищевой промышленности:
(i) холодильная цепь в службах доставки продуктов на
дом; (ii) непрямая доставка из магазинов «шаговой до-
ступности»; (iii) прямая доставка из магазинов «шаго-
вой доступности»; (iv) служба комбинатов бортового
питания [28]. 4 8 База данных холодильной цепи (CCD) требителя. 4.7. Беспроводные сенсоры На практике, ожида-
ется, что IoT будет развиваться в таких областях, как
беспроводные сенсорные сети с целью сбора контекс-
туальных данных. В последнее время идет разработка
программного обеспечения для расширения сетевых
услуг, используя возможности IoT (сеть вещей — Web
of Things, WoT). Наконец, также были разработаны
новые бизнес модели в пищевой промышленности:
(i) холодильная цепь в службах доставки продуктов на
дом; (ii) непрямая доставка из магазинов «шаговой до-
ступности»; (iii) прямая доставка из магазинов «шаго-
вой доступности»; (iv) служба комбинатов бортового
питания [28]. 4.8. База данных холодильной цепи (CCD) 5. Заключение Управление холодильной цепью при поставках
мяса имеет высокое значение для сохранения безопас-
ности и качества мяса/мясных продуктов. В связи со
сложностью глобальной цепи поставок мяса и мясных
продуктов, которая нередко бывает достаточно протя-
женной и связанной с транспортировкой продукта, как
в внутри страны, так и при поставках из одной стра-
ны в другую, или c одного континента на другой, тре-
бует необходимости соблюдать режимы охлаждения
и замораживания, отслеживать время и температуру,
чтобы потребитель был уверен в свежести данных
продуктов и их безопасности. Хотя режимы охлажде-
ния и замораживания эффективны, как и контроль
температуры в течении всего периода поставок мяса,
это остается постоянной проблемой для промышлен-
ности, дистрибьюторов, ритейлеров и потребителей. В настоящее время существует несколько доступных
вариантов контроля и управления холодильной це-
пью, таких как комбинации хранения в охлажденном
и замороженном виде, суперохлаждение, ионизирую-
щее излучение, биоконсервирование, высокое гидро-
статическое давление (HHP), активная упаковка, бес-
проводные датчики поддерживаемые компьютерной
базой данных холодильной цепи (CCD). 4.7. Беспроводные сенсоры Целенаправленный сбор данных профи-
лей температуры-времени (t – Т) в системе поста-
вок мяса, а также в пределах определенного модуля
стадии холодильной цепи (например, холодильное
хранение на скотобойне, транспортировка/достав-
ка, розничная торговля) может служить ценным
вкладом для прогнозирования статуса срока годно-
сти пищевого продукта. Недавно была разработана
сетевая платформа, в рамках европейского проекта
ФРИСБИ (http://frisbee-project. eu), для сбора данных
температурных условий по всей системе поставок
охлажденных и замороженных пищевых продуктов. Были собраны данные всех холодильных модулей
цепи (промышленность, дистрибьюторы, розничная
торговля и потребители), для создания обширной
базы данных, включающей более чем 14000 профи-
лей времени/температуры (t/T). Такая платформа
может служить ценным инструментом управления
холодильной цепью. Компьютерная программа по
прогнозированию в холодильной цепи (CCP) на ос-
нове базы данных холодильной цепи (CCD) позволя-
ет рассчитывать статус срока хранения на различных
этапах холодильной цепи на основе существующих
или определенных пользователем программы кине-
тических данных. Разработанные средства обладают
потенциалом проверки модельных сценариев на ос-
нове реальных данных холодильной цепи и дают воз-
можность совершенствовать систему и управление
холодильной цепью [29]. (охлаждение, замораживание, поставка и хранение). Температура — это основной параметр после процес-
са переработки в определении продолжительности
хранения в холодильной цепи охлажденных или замо-
роженных пищевых продуктов. Хранение в заморо-
женном состоянии требует больших энергетических
затрат для сохранения пищевых продуктов, в то время
при холодильном хранении существует постоянная
угроза порчи, вызванной бактериями. Современное
управление холодильной цепью включает контроль
и регулирование температуры и сфокусирован на
простой логистической цепи, а не на обработке не-
скольких каналов. Для преодоления вышеуказанных
недостатков разработана система холодильной цепи
на основе температурно-временного индикатора
(TTI), в которой используются беспроводные сенсо-
ры для сбора температурных данных на протяжении
цепи поставок мяса (от холодильного хранения на
скотобойне до розничной торговли) и внедряются по-
ложения критериев критических контрольных точек
(ККТ) на протяжении всего процесса поставок. При
строгом контроле температуры, возможен перевод
ряда пищевых продуктов с замороженного хранения
на охлажденное, для преодоления недостатков, свя-
занных с замороженным хранением (высокоэнергети-
ческое потребление, ухудшение вкуса, ограниченное
число продаж). Создаются контрольные диаграммы
для контроля каждой точки процесса. Этот подход
основан на архитектуре «Интернет вещей» Internet
of Things (IoT) и Международном стандарте по без-
опасности и качестве пищевых продуктов (ISO 22000). IoT является растущей тенденцией из-за сильного
влияния в результате развития информационно-ком-
муникационных технологий (ICT), например, бирки
радиочастотной идентификации животных и пище-
вой продукции (RFID), датчики, исполнительные ме-
ханизмы и даже интеллектуальные устройства такие
как мобильные телефоны [28]. 2. Meat spoilage Pork, beef, lamb and poultry carcasses are chilled im-
mediately after post-mortem inspection at slaughter line
and/or in slaughterhouse chilling rooms. The chilling pro-
cess is aerobic as the carcasses are exposed to air circula-
tion. Most often, after 24–96h of chilling, the carcasses are
usually moved to a boning/cutting room where they are
further cut into primary cuts (primals). The primals can be
typically stored up to 6 weeks in vacuum packs under an-
aerobic conditions [3]. Ground meat products may be pre-
pared from trimmings from deboning and/or trimmings
from primals after 6 weeks of anaerobic storage; these may
be stored aerobically or anaerobically (Figure 2). usually occurs, especially on the surface of It is known that shelf life of chilled fresh meat can be
extended by various packaging solutions, such as vacuum
or modified atmosphere packaging (MAP) [4, 5, 6]. How-
ever, a freshness of chilled meat is strongly influenced by
temperature. Inadequate storage, distribution and retail
temperatures can lead to a significant reduction in shelf life
and early spoilage of meat and meat products [6]. operations the microbialcross-contaminatio
i
i h
i
l It is well known that chilling of red meat and poultry
carcasses is essential to retard bacterial growth. Chill-
ing is also required for appearance and eating quality of
meat. Most frequently bovine, pork and lamb carcasses
are chilled using a forced convection of chilled air [10], al-
though spray chilling (application of a fine spray) may also
be used since it is faster than air chilling and it is primarily
used in poultry, but may be also used in beef, pork and
lamb processing plants. hands, clothes, objects, etc. Meat is a
wth of microorganisms due to its chemical
water. The lipid content of meat also makes
of oxygen with fatty acids) andsubsequent
products of the peroxides produce the
of rancid meat. ld chain, such as the chilling of products
b
d
i
t
t
d t
f
i Healthy animals that are hygienically slaughtered af-
ter proper resting and fasting provide a practically aseptic
meat. However, during slaughter, evisceration and dress-
ing operations the microbialcross-contamination usually
occurs, especially on the surface of meat, via contact with
equipment, tools, hands, clothes, objects, etc. Meat is a par-
ticularly favorable substrate for the growth of microorgan-
isms due to its chemical composition, e. g. rich in proteins,
lipids and water. 2. Meat spoilage The lipid content of meat also makes it
very sensitive to oxidation (the reaction of oxygen with
fatty acids) and subsequent production of peroxides. The
breakdown products of the peroxides produce the charac-
teristic objectionable odor and flavor of rancid meat. meat, via contact with equipment,tools,
particularly favorable substrate for the gro
composition, e.g. rich in proteins, lipids and
it very sensitive to oxidation (the reaction
production of peroxides. The breakdow
characteristic objectionable odor and flavor
Several weak points exist in meat c
during storage - before shipping, tempera
products from one actor to another an
deconsolidation at retail [7, 8, 9]. Temper
quality during distribution and at the end of According to Regulation (EC) 853/2004/EC the car-
casses should be immediately chilled after post-mortem
inspection to ensure that the temperature throughout the
meat(carcass)is lower than 7 °C, and lower than 3 °C for of-
fal. Interestingly, the provision on time limit by when this
temperature must be achieved, is not defined. For example,
beef and lamb carcasses are usually not chilled to the tem-
perature lower than 10 °C (core temperature) within the first
10 h, to avoid cold shortening and toughening of the meat. Therefore, such conditions are favorable for the bacterial
growth on the surface of the carcass, until the temperature
is sufficiently reduced to retard bacterial activity [3]. ure abuse during transport and transferring
waiting times during consolidation and
ture abuses result in variations of product
shelf life and may cause spoilage before the
e and economical losses. The vulnerability
ant in modern, global meat trade where
complex
and
long
(slaughterhouse-
ontinuum), where meat has to be shipped
another continent (Figure 1). Therefore, the
unt importance and presents a permanent l
Several weak points exist in meat cold chain, such as
the chilling of products during storage — before shipping,
temperature abuse during transport and transferring prod-
ucts from one actor to another and waiting times during
consolidation and deconsolidation at retail [7, 8, 9]. Tem-
perature abuses result in variations of product quality dur-
ing distribution and at the end of shelf life and may cause
spoilage before the use by date is reached, leading to food
waste and economical losses. 1. Introduction 1. Introduction Meat is a perishable product with a short shelf life and
therefore short selling times. In contrast to fresh fruit and
vegetables, packaged meat has to be declared with a «use
by» date [1]. The maintenance of the cold chain is also one
of the main principles and basic requirements of European
Union (EU) legislation on food hygiene [2]. Raw materials,
ingredients, intermediate products and finished products
that are likely to support the growth of pathogenic micro-
organisms and/or spoilage bacteria, are to be kept at tem-
peratures that do not result in a risk to health. The cold
chain should not be interrupted at all times along the meat
distribution chain [3]. Theory and Practice of Meat Processing №4 | 2017 tail-consumer continuum), where meat has to be shipped
from one to another country or from one to another conti-
nent (Figure 1). Therefore, the management of cold chain is
of paramount importance and presents a permanent chal-
lenge to maintain the safety and freshness of chilled fresh
meat until it reaches the final consumer. 4.8. База данных холодильной цепи (CCD) Эффективное управление холодильной цепью
может оптимизировать свежесть и безопасность
продукта от фермы/скотобойни до конечного по- 27 Theory and Practice of Meat Processing №4 | 2017 2. Meat spoilage The chilling and chilled storage conditions for beef, pork and lamb carcasses and their primals and trimmings (adapted from [3])
Figure 2. The chilling and chilled storage conditions for beef, pork and lamb also be involved [11]. For instance Pseudomonads, Lacto-
bacillus and Enterococcus produce slime on meat, while
Enterococcus produce hydrogen peroxide greening spots,
similar to greening caused by Clostridium spp. The growth
of bacteria on meat surface is influenced by temperature,
pH, water activity, nutrient availability, storage atmosphere
(aerobic or anaerobic) and competition with other micro-
biota present on meat [12]. p
g (
It is well known that chilling of red m
retard bacterial growth. Chilling is also requi
meat. Most frequently bovine, pork and lam
convection of chilled air [10], although spray
also be used since it is faster than air chilling an
be also used in beef, pork and lamb processing
According to Regulation (EC) 853/2004 ments encompassing superchilling, ionizing radiation,
biopreservation and high hydrostatic pressure or high
pressure processing (HPP) [16]. [ ])
at and poultry carcasses is essential to
d for appearance and eating quality of 3.1. Cold chain in slaughterhouse
arcasses are chilled using a forc
ng (application of a fine spray) m In slaughterhouse the meat chill chain starts with two
main steps: (a) the primary chilling (rapid cooling of meat
carcasses after slaughter) so that the warmest point of the
carcass (center of the hind leg) has to reach a temperature
lower than 7 °C and 3 °C for edible offal and lower than
4 °C for poultry carcasses, to prevent microbial growth
and extend the shelf life; with current technology these
temperatures can be arrived at in 16–24 hours in small
carcasses (lamb), in less than 48 hours in large carcasses
(beef, pork) and less than 2h for poultry carcasses (internal
deep breast); average and surface temperatures are obvi-
ously much lower, reaching 0 °C on the surface within four
hours; this is very important to slow microbial prolifera-
tion; and (b) secondary chilling (maintenance of the meat
temperature below 7 °C (red meat) and below 4 °C (poultry
g ( pp
p y)
y
d it is primarily used in poultry, but may
lants. C the carcasses should be immediately
e that the temperature throughout the
°C for offal. Interestingly, the provision
achieved, is not defined. For example,
o the temperature lowerthan 10°C (core
shortening and toughening of the meat. bacterial growth on the surface of the
ced to retard bacterial activity [3]. certain sensory changes occur, e.g. nt and is usually primarily triggered by
enzymes may also be involved [11]. For
rococcus produce slime on meat, while
ng spots, similar to greening caused by 3. Meat supply cold chain management
chilled after post-mortem inspe
meat(carcass)is lowerthan 7°C, a Preservation of red and poultry meat for trade and
export purposes as anaerobic vacuum-packed product in
either chilled or frozen form at sufficiently cold tempera-
tures is essential for successful national and international
trade [13]. The impact of long-term chilled storage, as well
as frozen storage for export purposes was thoroughly in-
vestigated [12, 14]. It was proved that frozen storage dura-
tions were extended for more than one year [15]. In spite of
these advances, improvement of preservation technologies
within the current export cold chain remains an issue, in
particular at industrial level, with potential for advance-
meat(carcass)is lowerthan 7 C, and lowerthan
on time limit by when this temperature must b
beef and lamb carcasses are usually not chilled
temperature) within the first 10h, to avoid cold
Therefore, such conditions are favorable for th
carcass, until the temperature is sufficiently red
Meat is considered to be spoiled wh
discoloration, off-odor and/or slime developm
spoilage bacteria (Table 1) although indigenou
instance Pseudomonads, Lactobacillus and En
Enterococcus produce hydrogen peroxide gree
Clostridium spp. The growth of bacteria on m
pH water activity nutrient availability storag
Table 1. The overview of the main meat/meat products spoilage defe eat surface is influenced by temperature,
ge atmosphere (aerobic or anaerobic) and
meat [12]. ects and causal bacteria (adapted from [8]). Causal bacteria
Pseudomonads, Lactobacillus, Enterococcus, Weissella, Brochothrix
Weissella, Leuconostoc, Enterococcus, Lactobacillus
Vibrio, Enterobacteriaceae
Clostridium, Hafnia
Providencia
Brochothrixthermosphacta
Enterobacteriaceae, Proteus
Clostridium, Enterococcus
Lactic acid bacteria, Enterococcus, Micrococcus, Bacillus, Clostridium pH, water activity, nutrient availability, storage atmosphere (aerobic or anaerobic) and
competition with other microbiota present on meat [12]. Table 1. The overview of the main meat/meat products spoilage defects and causal bacteria (adapted from [8]). 3. Meat supply cold chain management
chilled after post-mortem inspe
meat(carcass)is lowerthan 7°C, a Defect
Meat/Meat product
Causal bacteria
Slime
Fresh meat
Pseudomonads, Lactobacillus, Enterococcus, Weissella, Brochothrix
Hydrogen peroxide greening
Fresh meat
Weissella, Leuconostoc, Enterococcus, Lactobacillus
Hydrogen sulfide production
Cured meats
Vibrio, Enterobacteriaceae
Sulfide odour
Vacuum-packed fresh meat
Clostridium, Hafnia
Cabbage odour
Bacon
Providencia
Cheesy or dairy odour
Vacuum-packed fresh meat
Brochothrixthermosphacta
Putrefaction
Ham
Enterobacteriaceae, Proteus
Bone taint
Whole meats
Clostridium, Enterococcus
Souring
Vacuum-packed meats
Lactic acid bacteria, Enterococcus, Micrococcus, Bacillus, Clostridium pH, water activity, nutrient availability, storage atmosphere (aerobic or anaerobic) and
competition with other microbiota present on meat [12].h
p
p
g
p
Defect
Meat/Meat product
Causal bacteria
Slime
Fresh meat
Pseudomonads, Lactobacillus, Enterococcus, Weissella, Brochothrix
Hydrogen peroxide greening
Fresh meat
Weissella, Leuconostoc, Enterococcus, Lactobacillus
Hydrogen sulfide production
Cured meats
Vibrio, Enterobacteriaceae
Sulfide odour
Vacuum-packed fresh meat
Clostridium, Hafnia
Cabbage odour
Bacon
Providencia
Cheesy or dairy odour
Vacuum-packed fresh meat
Brochothrixthermosphacta
Putrefaction
Ham
Enterobacteriaceae, Proteus
Bone taint
Whole meats
Clostridium, Enterococcus
Souring
Vacuum-packed meats
Lactic acid bacteria, Enterococcus, Micrococcus, Bacillus, Clostridium )
Causal bacteria Pseudomonads, Lactobacillus, Enterococcus, Weissella, Brochothrix
W i
ll
L
t
E t
L
t b
ill 2. Meat spoilage The vulnerability of meat
cold chain became very important in modern, global meat
trade where distribution chain is sometimes very complex
and long (slaughterhouse-transportation/distribution-re-
quality during distribution and at the end of
use by date is reached, leading to food wa
of meat cold chain became very impor
distribution
chain
is
sometimes
ver
transportation/distribution-retail-consumer
from one to another country or from one to
management of cold chain is of paramo
challenge to maintain the safety and freshn
final consumer. fi
Meat is considered to be spoiled when certain sensory
changes occur, e. g. discoloration, off-odor and/or slime
development and is usually primarily triggered by spoil-
age bacteria (Table 1) although indigenous enzymes may
ss of chilled fresh meat until it reaches the Figure 1. The schematic layout of meat supply cold chain
Figure 1. The schematic layout of meat supply cold chain Figure 1. The schematic layout of meat supply cold chain
e 1. The schematic layout of meat suppl 28 2017 | №4 теория и практика переработки мяса 2017 | №4 теория и практика переработки мяса 2017 | №4 теория и практика переработки мяса
Figure 2. The chilling and chilled storage conditions for beef, pork and lamb carcasses and their primals and trimmings (adapted from [3])
Carcass
Figure 2. The chilling and chilled storage conditions for beef, pork and lamb
d h i
i
l
d
i
i
( d
d f
[3])
Deboning room
Storage chiller
Aerobic storage
24-96 h
Carcass
temperature
decreased to a core
temperature lower
7°C
Primals prepared
Room temperature
lower 12°C
Anaerobic storage
Vacuum-packed
primals stored at
temperature 0-2°С,
for up to 6 weeks
Chilling room
Trimmings
Trimmings
Trimmings stored aerobically or
anaerobically (vacuum-packed) at 0-2°C
Storage chiller Aerobic storage
24-96 h Vacuum-packed
primals stored at
temperature 0-2°С,
for up to 6 weeks Carcass Trimmings Storage chiller Trimmings stored aerobically or g
y
anaerobically (vacuum-packed) at 0-2°C gure 2. The chilling and chilled storage conditions for beef, pork and lamb carcasses and their primals and trimmings (adapted from [3])
Figure 2. The chilling and chilled storage conditions for beef, pork and lamb Figure 2. The chilling and chilled storage conditions for beef, pork and lamb carcasses and their primals and trimmings (adapted from [3])
Figure 2. The chilling and chilled storage conditions for beef, pork and lamb Figure 2. Slime Weissella, Leuconostoc, Enterococcus, Lactobacillus Vibrio, Enterobacteriaceae Clostridium, Hafnia 29 Theory and Practice of Meat Processing №4 | 2017 of the product and intermittent spraying with cold water. This system prevents the skin from drying out and speeds
up the chilling process. With this technique, the discolou-
ration of the skin, which can occur with hard scalded birds,
is prevented. Usually, spray chilling tunnels are designed
to take one layer of birds in order to prevent cross con-
tamination. Two layers are possible, but the bottom layer
must be positioned in between the lines of the top layer
to prevent drip contamination and additional drip troughs
are required. h meat) at all times during chill storage, cutting, deboning,
mincing), before transportation. h The most common technologies to chill meat before
transportation are: (i) air chilling, (ii) immersion chilling,
(iii) spray chilling, and (iv) vacuum chilling [8]. hf 3.1.1. Air chilling. The effectiveness of air chilling appli-
cations depends on a number of factors including air tem-
perature and velocity, relative humidity, weight, fat cover of
carcasses and loading of chilling chamber. Air temperature
must be within the region of 0 °C, with no decrease below
minus 1 °C, which could freeze the meat surface and impair
its appearance. Air speed can range from 0. 25 to 3. 0 m/s. However, for economic reasons the most common speeds
in use are from 0. 75 to 1. 5 m/s in the empty section of the
cold chamber. Relative humidity during the chilling opera-
tion should be kept high to prevent excessive weight loss. The recommended rate is between 90 and 95 %, though
this is the most difficult factor to control. It is worth of note
that sometimes quick chilling has its problems because of
the «cold shortening». Cold shortening can often be seen
in beef and mutton, when the meat, still in its pre-rigor
phase, reaches temperatures of 10 °C or lower. These condi-
tions cause irreversible contractions of the muscle tissue
which toughen the meat even after prolonged ripening. Quick primary chilling also signifies an increase in invest-
ment and higher operational costs. The chilling period can
be reduced by lowering the air temperature (surface freez-
ing risks) or increasing air speed (higher operational costs)
or both. 3.2. Cold chain in meat distribution During meat distribution (transportation) route to the
final user — wholesale cold storage and/or display at retail,
the cold chain must be maintained vigorously. Industrial
and/or truck chambers have different characteristics and
performances. Its size, initial temperature of incoming
meat, targeted temperature during transportation, me-
chanical characteristics (e. g. power of compressors, ven-
tilation and insulation), as well as energy/cost matters are
issues of first priority when considering the meat distribu-
tion/transportation [8]. In general, the vehicle must be pro-
vided with a good refrigerated system, capable to maintain
the required temperature of meat/offal at all times during
distribution (see 3.1.). 3.1.2. Immersion chilling. This is the oldest chilling meth-
od and also the least expensive method and provides very
rapid cooling with no risk of freezing. Immersion chilling
is an off-line system that does not allow automatic weigh-
ing before and after chilling and is commonly used for the
chilling of poultry carcasses, predominantly in USA. Im-
mersion chillers use little floor space and are very energy
efficient. The process can be accomplished using just tap
water with or without flaked ice or pre-chilled water to cre-
ate lower temperatures in the immersion bath. Such sys-
tem is capable of lowering the temperature of the tap water
to 1 °C. This process can result in product weight increase
by means of a controllable water pick-up. An increase of up
to 12 % can be achieved. h Slime Occasionally cold chambers are refrigerated in ad-
vance to reach lower temperatures than those in operation
(minus 5–6 °C for beef; minus 10–12 °C for pork), taking
advantage of thermal inertia to offset the effect of warm
meat loads [8]. h q
3.1.4. Vacuum chilling. This is a rapid batch process
whereby moist products containing free water are cooled
by evaporation of moisture under vacuum. The advantage
of this technology is that it significantly reduces bacterial
counts of phychrophiles and mesophiles after the meat was
stored for several days. Vacuum packed chilled meat has
significantly longer shelf life compared with conventional-
ly chilled wrapped meat. For instance, vacuum packed beef
cuts can be stored for up to 12 weeks, while lamb and pork
cuts can have a shelf life for up to 5 and 8 weeks, stored at
0 °C, respectively. The disadvantage is the large weight loss
of meats. 3.3. Cold chain in meat retailh The maintenance of cold chain during display at re-
tail is of crucial importance for prevention of microbial
spoilage, as well as maintenance of meat freshness and
safety. The size and capacity of chilling chambers at re-
tail establishment, the size of cabinets, initial tempera-
ture of incoming meat, meat handling procedures (cut-
ting, mincing), temperatures of surroundings, location
of refrigeration machinery, ventilation and light are the
possible weak points to be addressed. The special focus
should be put on temperature consolidation/decon-
solidation during meat handling (e. g. daily transfer of
meat cuts from the chilling chamber to the retail cabinet
and vice versa, where internal temperature of meat cuts
should be at all times lower than 7 °C). The management
approach that dominates in the meat market is related to
the principle «First In — First Out». However, such ap-
proach should also adhere to all stages of the cold chain 3.1.3. Spray chilling. This is an alternative method to im-
mersion chilling which has been increasingly used espe-
cially in the USA for the chilling of poultry carcasses. Spray
chilling can be applied in the processing and production of
«frozen products» or «hard scalded birds», as well as «fresh
products» or «soft scalded birds». This chilling method is
based on combination of sprays and air during the initial
stage of the chilling cycle and the use of air for the rest of
the chilling period; it uses a cold airflow across the surface 30 2017 | №4 теория и практика переработки мяса (Figure 1) and has to be achieved through properly de-
signed handling procedures in the chill storage rooms
and retail cabinets. In all, different points of transport,
from cold storage in slaughterhouse to retail outlet, and
then to consumer refrigerator, are critical points for the
overall meat quality and safety. the conversion of some water into ice, which makes it less
available for deteriorative processes. Superchilling gives
the food product an internal ice reservoir so that there is
no need for external ice around the product during trans-
portation or storage for shorter periods. Generally, super-
chilling is positioned between freezing and refrigeration
(conventional chilling), where the surrounding tempera-
ture is set below the initial freezing point. Storing food
at superchilling temperature has three major advantages:
maintaining food freshness, retaining high food quality
and suppressing growth of harmful microbes. 3.3. Cold chain in meat retailh It can also
reduce the use of freezing/thawing for production and
thereby increase yield, reduce energy, labor and transport
costs [21]. 4. Available options in meat supply cold chain
management As said, if the cold supply chain is breached, meat and
poultry products will suffer from a range of quality prob-
lems such as shrinkage, rotting, trim loss, unpleasant odor,
color and texture changes, as well as exacerbated health
risks from pathogens such as Salmonella, Campylobacter,
Shiga toxin-producing E. coli (STEC) and Listeria mono-
cytogenes. Despite a numerous studies targeted to better
understanding of meat microbial ecology (spoilage micro-
organisms and food borne pathogens) and its relation to
temperature/time within the complex meat supply chain,
e. g. meat storage, transportation, distribution and retail,
the maintenance of cold chain, as well as meat quality and
safety remains a challenge. From recently, there are sev-
eral options/control measures which can be effectively
and synergistically applied to improve the control over the
«classic» meat supply cold chain. 4.4. Biopreservation Natural compounds, such as essential oils, nisin, lyso-
zyme, as well as natural or controlled microbiota, e. g. lactic
acid bacteria (LAB) and their antimicrobial products such
as lactic acid and bacteriocins, were investigated to extend
the shelf life of meat/meat products and to obtain «green
label» products. For example, nisin is only commercially
available bacteriocin and showed good antibacterial ac-
tion in artificially contaminated pork and in combination
with 2 % sodium chloride an excellent anti-listerial effect
in minced beef; pentocin 31–1 (produced by Lactobacillus
pentosus 31–1 isolated from the traditional Chinese fer-
mented Xuanwei ham) effectively inhibited volatile basic
nitrogen (VBN) and suppressed the growth of indigenous
microbiota, especially Listeria and Pseudomonas, in chilled
pork storage [16]. 4.1. Chilled and frozen storage combinations Over the previous two decades a numerous studies
had been carried out to assess the enhancement of shelf
life, as well as quality and safety of meat achieved by
chilling and freezing regimes. However, a limited num-
ber of studies were conducted to evaluate combined
chilled and frozen storage practices, particularly regard-
ing long-term chilled-than-frozen beyond 3–4 months
of total duration. It appeared that prolonged vacuum-packed chilled
storage (two weeks for beef and lamb, at 4 °C and even up
to 30 weeks for beef, at minus 0.5 °C and 7 weeks for lamb,
at minus 1.5 °C)combined with subsequent frozen storage
(up to one year, at minus 18 °C) had the beneficial effect
to quality parameters (shear force, tenderness, fluid levels,
flavor and color) of meat; the improved tenderness was
confirmed as a major achievement [15, 19, 20]. Obviously,
the prolonged chilled-frozen storage combinations can
have a beneficial effect for export of fresh meat. Further
and deeper studies are needed to investigate instrumental
color and microbial profile of chilled-then-frozen meat,
as these studies are highly relevant for consumer approval
and health issues [20]. 4.3. Ionizing radiation Processing of food by ionizing radiation offers arrange
of beneficial effects, which can»t be achieved by other and,
in particular, traditional techniques such as chilling alone. Food radiation, if applied at low dose (up to1kGy) can be
very effective against parasites presented in red meat, as
well as for inactivation of spoilage and pathogen bacteria
in chilled and/or frozen meat and poultry. On the other hand, several consumer organizations
raised concerns from the beginning whether it would be
«safe» to consume irradiated food? However, after more
than 100 years of research, the questions raised are resolved
and the consumers accept irradiated food where it has be-
come available together with an understandable explana-
tion of the new technology. For example, in China very
special items, as pickled chicken feet (irradiated), appear
to have a certain market share. In the European Union the
amount of irradiated food on the market place is marginal;
however, varying drastically between Member States. More
recent consumer studies are not available for the EU. The
use of ionizing radiation in food processing remains still to
be an under-estimated and under-exploited technology, in
spite of its great potential [22]. 4.7. Wireless sensorsh The manufacturers in the food industry frequently face
the dilemma regarding the type of cold chain manage-
ment in delivering products to retailers or end consumers,
having to choose between frozen storage and cool storage
(cooling, freezing, delivering and storage). Temperature
is the main post-processing parameter in the determina-
tion of shelf-life in a cold chain of chilled and frozen food
products. Frozen storage includes high-energy consump-
tion for the preservation of food products, whereas cool
storage involves the constant threat of bacterial-induced
spoilage. Contemporary cold chain management encom-
passes temperature control and is focused on single logistic
chain rather than serving multiple channels. In order to
overcome the aforementioned deficiency, a time-temper-
ature indicator (TTI) based cold-chain system is devel-
oped, which uses wireless sensors for collecting tempera-
ture data along the meat supply chain (from cold storage
in slaughterhouse to retail) and implements the formu-
lation of Critical Control Point (CCP) criteria through-
out the entire delivery process. Under strict temperature
monitoring, switching a number of products from frozen
storage to cool storage seems to be feasible to improve the
shortcomings associated with frozen storage (high-energy
consumption, deterioration of taste, limited number of
sale channels). Control charts are formulated for moni-
toring each point in the process. This approach is based
on Internet of Things (IoT) architecture and international
food standard (ISO 22000). IoT is a growing trend with a
powerful influence in shaping the development of the in- g g
g p
g
g
4.6.1. Antimicrobial active packaging. This is one of the
most important concepts of active packaging of meat. There are four basic categories of antimicrobial packag-
ing: (i) incorporation of antimicrobial substances into a
pad inside the package, with aim to provide a slow release
of antimicrobials, (ii) direct incorporation of the antimi-
crobial agents into the packaging film, by heat treatment
(co-extrusion of packaging films with antimicrobials), so
that antimicrobials can be gradually released from the
packaging films to the packaging head space or food sur-
face, (iii) coating of packaging with a matrix that acts as
a carrier for antimicrobial agents, so to allow the release
of antimicrobials onto the food surface through evapora-
tion into the headspace (volatile substances) or migration
into the food through diffusion (non-volatile substances),
and (iv) use of polymers that have antimicrobial activity
(e. g. chitosan, poly-l-lysine). 4.5. High hydrostatic pressure (HHP)h 4.6.2. Antioxidant active packaging. High levels of oxy-
gen in meat packaging can facilitate microbial growth,
lipid oxidation, development of off-flavors and off-odors,
color changes and nutritional losses. Therefore, control of
oxygen level in meat packaging is important to prevent/
retard the deterioration and spoilage of meat. Antioxidant
active packaging systems can be classified into 2 groups:
(i) independent antioxidant devices (sachets, pads or labels
with oxygen scavengers — fine iron powder and ferrous
oxide), and (ii) antioxidant packaging materials (antioxi-
dant active agent is incorporated into the walls of the pack-
aging film (e. g. terpenoids from the propolis). The meat preservation process using HHP, a non-ther-
mal technology, can effectively inactivate product-spoiling
microorganisms and enzymes at low temperatures without
changing dramatically the sensory or nutritional charac-
teristics of the product; the tenderness is even improved,
while the fresh meat color is slightly changed, after the
HHP treatment. HHP is also a powerful tool to control
risks associated with Salmonella and Listeria monocyto-
genes in raw or marinated meats [23]. Pressure processing
is usually carried out in a steel cylinder containing a liquid
pressure-transmitting medium, e. g. water, while the prod-
uct is protected from direct contact by using sealed flexible
packaging [16]. The recommended pressure/time combi-
nation for meat/meat products of 600–700 MPa for 2–5
minutes showed quick inactivation of L. monocytogenes, as
well as other indigenous microbiota [24]. g
gi
g
4.6.3. Carbon-dioxide emitting/generating packaging. Carbon dioxide has proven inhibitory effect for a range
of aerobic bacteria and fungi via reduced oxygen level
and through direct antimicrobial effect (by prolonging
a lag phase and generation time during the logarithmic
phase of microbial growth). Therefore, a CO2 generating
packaging system is a technique complementary to oxy-
gen scavenging. The levels of CO2 applied for meat and
poultry preservation are relatively high (10–80 %). For
example 10–20 % of CO2 is needed for the inhibition of
Pseudomonas, while 50 % of CO2 is necessary for control
of proliferation of C. perfringens, C. botulinum and Liste-
ria monocytogenes. 4.6. Active packaging Active packaging is an innovative technology that al-
lows the product and its environment to interact to extend
the product shelf life and to ensure its microbial safety,
while maintaining the quality of packed food. According to
EU legislation, active packaging is a type of food packaging
with an extra function, in addition to that of providing a
protective barrier against external influence. It means that
the packaging can absorb food- and environment-derived
chemicals within the packaging surrounding the food or
its releases substances into the food or the environment
surrounding the food, e. g. preservatives, antioxidants, and
flavorings [25]. There are three main types of active pack-
aging which were developed so far: antimicrobial active
packaging, antioxidant active packaging, carbon-dioxide
emitting/generating packaging. h Theory and Practice of Meat Processing №4 | 2017 Theory and Practice of Meat Processing №4 | 2017 4.2. Superchilling Superchillingis used for preserving foods by process
in which the temperature of a food product is lowered
to 1–2 °C below the initial freezing point (for most foods
between 0.5 °C and 2.8 °C). The superchilling technology
combines the favorable effect of low temperatures with 31 4.7. Wireless sensorsh In addition, a large number
of antimicrobials were tested for inhibiting the growth of
microorganisms in food, e. g. ethanol, carbon dioxide,
silver ions, chlorine dioxide, antibiotics, bacteriocins,
organic acids, essential oils, spices, plant extracts (rose-
mary), peptides, etc [26]. g g
g p
g
g
4.6.1. Antimicrobial active packaging. This is one of the
most important concepts of active packaging of meat. There are four basic categories of antimicrobial packag-
ing: (i) incorporation of antimicrobial substances into a
pad inside the package, with aim to provide a slow release
of antimicrobials, (ii) direct incorporation of the antimi-
crobial agents into the packaging film, by heat treatment
(co-extrusion of packaging films with antimicrobials), so
that antimicrobials can be gradually released from the
packaging films to the packaging head space or food sur-
face, (iii) coating of packaging with a matrix that acts as
a carrier for antimicrobial agents, so to allow the release
of antimicrobials onto the food surface through evapora-
tion into the headspace (volatile substances) or migration
into the food through diffusion (non-volatile substances),
and (iv) use of polymers that have antimicrobial activity
(e. g. chitosan, poly-l-lysine). In addition, a large number
of antimicrobials were tested for inhibiting the growth of
microorganisms in food, e. g. ethanol, carbon dioxide,
silver ions, chlorine dioxide, antibiotics, bacteriocins,
organic acids, essential oils, spices, plant extracts (rose-
mary), peptides, etc [26]. 32 2017 | №4 теория и практика переработки мяса The Cold Chain Predictor (CCP) software based on the
Cold Chain Database (CCD) allows calculation of prod-
uct shelf-life status at different cold chain stages based on
existing or user defined kinetic data. The developed tools
offer the potential to run simulation scenarios based on
real cold chain data and contribute to effective cold chain
improvement and management [29]. formation and communication technology (ICT) sector,
e. g. radio frequency identification (RFID) tags, sensors,
actuators, and even smart devices like mobile phones [27]. In practice, the IoTis expected to develop in areas such as
wireless sensor networks with the aim of collecting con-
textual data. Further, a software approach to expanding
web-based services using the capabilities of IoT (Web of
Things, WoT) were recently developed. Lastly, a new busi-
ness models in the food industry have been also developed:
(i) cold chain home delivery service; (ii) convenience store
(CVS) indirect delivery; (iii) CVS direct delivery; (iv) flight
kitchen service [28]. 5. Conclusionh The cold chain management in meat supply is of utmost
importance for the maintenance of quality and safety of
meat/meat products. The complexity of global meat supply
chain, with frequently long distribution chains associated
with transportation of the product within one country,
from one to another country, and from one to another con-
tinent, makes the solutions for the chilling and freezing re-
gimes, as well as monitoring of time-temperature profiles,
very important for the overall success in delivery of prod-
uct. These products shouldbe than accepted by consumer
for its freshness and safety levels. Although the importance
of chilling and freezing regime effectiveness and temper-
ature monitoring along the meat supply chain are well
known, it remains a permanent challenge for the industry,
distributors, retailers and consumers. From recently, there
are several available options for control and management
of the cold chain, such as chilled and frozen storage combi-
nations, superchilling, ionizing radiation, biopreservation,
high hydrostatic pressure (HHP), active packaging, wire-
less sensors, supported with the software-based cold chain
database (CCD). 4.8. Cold chain database (CCD)hf The effective cold chain management can optimize
freshness and safety of the product from farm/slaughter-
house up to the final consumer. The targeted data acqui-
sition of time-temperature (t - T) profiles along the meat
supply chain, as well as within the specific module of the
cold chain stage (e. g. cold storage in slaughterhouse, trans-
portation/delivery, retail) can serve as a valuable input for
prediction of a product shelf-life status. From recently, a
web-based platform was developed, within FRISBEE Eu-
ropean project (http://frisbee-project. eu), for temperature
conditions data collection throughout the chilled and fro-
zen food supply chain. Data including all cold chain mod-
ules (industry, distributors, retailers and consumers), were
collected to create the extensive database comprising more
than 14, 000 time/Temperature (t/T) profiles. Such plat-
form can serve as a valuable Cold Chain Management tool. БИБЛИОГРАФИЧЕСКИЙ СПИСОК/ REFERENCES M. (eds. ). Encyclopedia of Meat Sciences. Academic Press, Else-
vier Science, Ltd. 696–702. 1. The European Parliament and the Council. (2011). Regula-
tion (EU) No 1169/2011 of 25 October 2011 on the provision of
food information to consumers. Official Journal of the European
Union, OJ (L 304), 18–63. 11. Tsigarida, E., Nychas, G. — J. E. (2001). Ecophysiological attri-
butes of a Lactobacillus spp. and a Pseudomonas spp. on sterile
beef fillets in relation to storage temperature and film permeabil-
ity. Journal of Applied Microbiology, 90 (5), 696–705. ,
(
),
2. Regulation (EC) 853/2004 of the European Parliament and
of the Council of 29 April 2004, laying down specific hygiene
rules for on the hygiene of foodstuffs. Official Journal of the Euro-
pean Union, OJ (L 304), L139/55. 12. Sumner, J., Jenson, I. (2011). The effect of storage tempera-
ture on shelf life of vacuum-packed lamb shoulders. Food Austra-
lia, 63 (6), 249–251. 3. EFSA (2016). Growth of spoilage bacteria during storage and
transport of meat. Official Journal of the European Union, 14 (6),
4523. 13. Deards, B., Leith. R., Mifsud, C., Murray, C., Martin. P., Glee-
son, T. (2014). Live export trade assessment. Canberra: Department
of Agriculture. [Electronic resource: http://www. herefordsaus-
tralia. com. au/Portals/0/PDF/ABARES%20Live%20Export%20
Trade%20Assessment. pdf. Access date 30. 06. 2017] 4. Lambert, A. D., Smith. J. P., Dodds, K. L. (1991). Shelf life ex-
tension and microbiological safety of freshmeat — a review. Food
Microbiology, 8 (4), 267–297. 14. Kiermeier, A., Tamplin, M., May, D., Holds, G., Williams, M.,
Dann, A. (2013). Microbial growth, communities, and sensory
characteristics of vacuum and modified atmosphere packaged
lamb shoulders. Food Microbiology, 36 (2), 305–315. i 5. Kiermeier, A., Tamplin, M., May, D., Holds, G., Williams, M.,
Dann, A. (2013). Microbial growth, communities and sensory
characteristics of vacuum andmodified atmosphere packaged
lambshoulders. Food Microbiology, 36 (2), 305–315. l 15. Muela, E., Monge, P., Safiudo, C., Campo, M. M., Beltran, J. A. (2015). Meat quality of lamb frozen stored up to 21 months:
instrumental analysis on thawed meat during display. Meat
Science, 102, 35–40. 6. Berruga, M. I., Vergara, H., Gallego, L. (2005). Influence of
packaging conditions on microbial andlipid oxidation in lamb
meat. Small Ruminant Research, 57 (2–3), 257–264. 7. Raab, V., Petersen, B., Kreyenschmidt, J. (2011). Tempera-
ture monitoring in meat supply chains. British Food Journal, 113
(10), 1267–1289. 16. Zhou, G. H., Xu, X. L., Liu, Y. (2010). Theory and Practice of Meat Processing №4 | 2 Cold chain database development and application as a
tool for the cold chain management and food quality evaluation. International Journal of Refrigeration, 52, 109–21. 29. Gogou, E., Katsaros, G., Derens, E., Alvarez, G., Taoukis, P. S. (2015). Cold chain database development and application as a
tool for the cold chain management and food quality evaluation. International Journal of Refrigeration, 52, 109–21. 24. Lucore, L. A., Shellhammer, T. H., Yousef, A. E. (2000). Inac-
tivation of Listeria monocytogenes on artificially contaminated
frankfurters by high-pressure processing. Journal of Food Protec-
tion, 63 (5), 662–664. Theory and Practice of Meat Processing №4 | 2 Theory and Practice of Meat Processing №4 | 2017 2017 2017 20. Coombs, C. E. O., Holman, B. W. B., Friend, M. A., Hopkins, D. L. 20. Coombs, C. E. O., Holman, B. W. B., Friend, M. A., Hopkins, D. L. (2017). Long-term red meat preservation using chilled and frozen
storage combinations: A review. Meat Science, 125, 84–94. 25. EU. (2009). Guidance to the Commission Regulation (EC)
450/2009 on active and intelligent materials and articles intend-
ed to come into contact with food. Version 10. European Com-
mission Health and Consumers Directorate, General Directorate
E — Safety of the Food Chain, E6 — Innovation and sustainability. 26. Fang, Z., Zhao, Y., Warner, R. D., Johnson, S. K. (2017). Active
and intelligent packaging in meat industry. Trends in Food Sci-
ence and Technology, 61, 60–71. 25. EU. (2009). Guidance to the Commission Regulation (EC)
450/2009 on active and intelligent materials and articles intend-
ed to come into contact with food. Version 10. European Com-
mission Health and Consumers Directorate, General Directorate
E — Safety of the Food Chain, E6 — Innovation and sustainability. 26. Fang, Z., Zhao, Y., Warner, R. D., Johnson, S. K. (2017). Active
and intelligent packaging in meat industry. Trends in Food Sci-
ence and Technology, 61, 60–71. 21. Kaale, L. D., Eikevik, T. M., Rustad, T., Kolsake, r K. (2011). Superchilling of food: A review. Journal of Food Engineering, 107
(2), 141–146. 22. Ehlermann, D. A. E. (2016). Particular applications of food
irradiation: Meat, fish and others. Radiation Physics and Chem-
istry, 129, 53–57. 27. Kang, Y. S., Lee, Y. H. (2013). Development of generic RFID
traceability services. Computers in Industry, 64 (5), 609–623. 28. Shih, C. W., Wang, C. H., (2016). Integrating wireless sensor
networks with statistical quality control to develop a cold chain
system in food industries. Computer Standards & Interfaces, 45,
62–78. 23. Garriga, M., Grebol, N., Aymerich, M. T., Monfort, J. M., Hu-
gas, M. (2004. )Microbial inactivation after high-pressure pro-
cessing at 600 MPa in commercial meat products over its shelf
life. Innovative Food Science and Emerging Technologies, 5 (4),
451–457. 24. Lucore, L. A., Shellhammer, T. H., Yousef, A. E. (2000). Inac-
tivation of Listeria monocytogenes on artificially contaminated
frankfurters by high-pressure processing. Journal of Food Protec-
tion, 63 (5), 662–664. 29. Gogou, E., Katsaros, G., Derens, E., Alvarez, G., Taoukis, P. S. (2015). Принадлежность к организации Настасиевич Иван — адъюнкт-директор, Институт гигиены и
технологии мяса. Настасиевич Иван — адъюнкт-директор, Институт гигиены и
технологии мяса. Nastasijevic Ivan — Associate Director, Institute of Meat Hygiene
and Technology. Nastasijevic Ivan — Associate Director, Institute of Meat Hygiene
and Technology. Kaćanskog 13, 11000, Belgrade, Serbia
Tel. : +381-11-2650-722
E-mail: ivan.nastasijevic@inmes.rs
*corresponding author
Lakicevic Brankica—Research Associate, Institute of meat hygiene
and technology. Kaćanskog 13, 11000, Belgrade, Serbia
Tel. : +381-11-2650-722
E-mail: brankica.lakicevic@inmes.rs
Petrović Zoran — Research Associate, Institute of meat hygiene and
technology. Kaćanskog 13, 11000, Belgrade, Serbia
Tel. : +381-11-2650-722
E-mail: zoran.petrovic@inmes.rs
Contribution
The authors equally contributed to the writing of the manuscript and
are equally responsible for plagiarism. Conflict of interest
The authors declare no conflict of interest. Received 20.10.2017 Настасиевич Иван — адъюнкт-директор, Институт гигиены и
технологии мяса. Kaćanskog 13, 11000, Belgrade, Serbia
Teл. : +381-11-2650-722
E-mail: ivan.nastasijevic@inmes.rs
*автор для контактов
Лакицевич Бранкика — Научный сотрудник, Институт гигиены
и технологии мяса. Kaćanskog 13, 11000, Belgrade, Serbia
Teл. : +381-11-2650-722
E-mail: brankica.lakicevic@inmes.rs
Петрович Зоран — Научный сотрудник, Институт гигиены и
технологии мяса. Kaćanskog 13, 11000, Belgrade, Serbia
Teл. : +381-11-2650-722
E-mail: zoran.petrovic@inmes.rs
Критерии авторства
Авторы в равных долях имеют отношение к написанию рукописи
и одинаково несут ответственность за плагиат. Конфликт интересов
Авторы заявляют об отсутствии конфликта интересов. Поступила 20.10.2017 E-mail: ivan.nastasijevic@inmes.rs j
*автор для контактов БИБЛИОГРАФИЧЕСКИЙ СПИСОК/ REFERENCES Preservation technologies
for fresh meat — A review. Meat Science, 86(1), 119–28. 16. Zhou, G. H., Xu, X. L., Liu, Y. (2010). Preservation technologies
for fresh meat — A review. Meat Science, 86(1), 119–28. 17. UN FAO, (1991). Manual on meat cold storage operation
and management. [Electronic resource: http://www. fao. org/
docrep/004/T0098E/T0098E02. htm. Access date 29.06.2017]. 18. Hagyard, C. J, Keiller, A. H., Cummings, T. L., Chrystall, B. B. (1993). Frozen storage conditions and rancid flavor development
in lamb. Meat Science, 35 (2), 305–312. 8. Nychas, G. E., Skandamis, P. N, , Tassou, C. C., Koutsoumanis,
K. P. (2008). Meat spoilage duringdistribution. Meat Science, 78
(1–2), 77–89. 9. Olsson, A. (2004). Temperature controlled supply chains
call for improved knowledge andshared responsibilities. In
Conf. Proc. NOFOMA 2004 (ed. H. Aronsson), 569–582. Swe-
den: Linköping. 19. Muela, E., Monge, P., Sanudo, C., Campo, M. M., Medel, I.,
Beltran, J. A. (2016). Sensory quality of lamb following long-term
frozen storage. Meat Science, 114, 32–37. 10. James, S. J., James, C. (2004). Meat marketing (d) transport
of meat and meat products. In: Jensen, W. K., Devine, C. Dikeman, 33 Конфликт интересов 34
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English
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Excimer laser ablation combined with drug-coated balloon versus drug-coated balloon in the treatment of de novo atherosclerotic lesions in lower extremities (ELABORATE): study protocol for a real-world clinical trial
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BMC cardiovascular disorders
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cc-by
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Jiang et al. BMC Cardiovascular Disorders (2022) 22:317
https://doi.org/10.1186/s12872-022-02751-1 STUDY PROTOCOL
Excimer laser ablation combined
with drug‑coated balloon versus drug‑coated
balloon in the treatment of de novo
atherosclerotic lesions in lower extremities
(ELABORATE): study protocol for a real‑world
clinical trial
Xiaolang Jiang1†, Longhua Fan2†, Bin Chen1, Junhao Jiang1, Jianjun Liu2, Guanyu Qiao1, Shuai Ju3, Yun Shi1,
Tao Ma1, Changpo Lin1, Gang Fang1, Daqiao Guo1, Xin Xu1, Zhihui Dong1* and Weiguo Fu1
Open Access Open Access Excimer laser ablation combined
with drug‑coated balloon versus drug‑coated
balloon in the treatment of de novo
atherosclerotic lesions in lower extremities
(ELABORATE): study protocol for a real‑world
clinical trial Xiaolang Jiang1†, Longhua Fan2†, Bin Chen1, Junhao Jiang1, Jianjun Liu2, Guanyu Qiao1, Shuai Ju3, Yun Shi1,
Tao Ma1, Changpo Lin1, Gang Fang1, Daqiao Guo1, Xin Xu1, Zhihui Dong1* and Weiguo Fu1 Abstract Background: The efficacy and validity of excimer laser ablation (ELA) in the in-stent restenosis (ISR) has been con-
firmed. However, its application in de novo atherosclerotic lesions of lower extremity artery disease (LEAD) has not
been clearly defined and its procedure has not been standardized. Methods: ELABORATE is a prospective, multicenter, real-world study designed to evaluate the efficacy and safety
between ELA combined with drug-coated balloon (DCB) and DCB alone in de novo atherosclerotic lesions of LEAD. Discussion: ELABORATE is a prospective, multicenter, real-world study designed to assess the efficacy and safety
between ELA combined with drug-coated balloon (DCB) and DCB alone in patients with de novo atherosclerotic
lesions of LEAD. According to the real-world situation, eligible patients will be allocated to ELA + DCB group (group E)
and DCB group (group C). Baseline and follow-up information (at 3, 6, and 12 months) will be collected. The primary
efficacy point is primary patency at 12-months, and the secondary efficacy points include clinically driven target
lesion reintervention (CD-TLR), change of Rutherford class, ankle-brachial index and ulcer healing rate. These indexes
will be assessed and recorded at 3, 6, and 12-month follow-up. Also, safety evaluation, including major adverse event,
all-cause mortality through 30-day follow-up, unplanned major amputation, bailout stent and distal embolization,
will also be evaluated by an independent core laboratory. All the data will be collected and recorded by the electric
data capture system. This study will be finished in 3 years and the 12-month results will be available in 2023. All the
patients will be followed for 5 years. †Xiaolang Jiang and Longhua Fan have contributed equally to this work. *Correspondence: dzh926@126.com 1 Department of Vascular Surgery, Institute of Vascular Surgery, Zhongshan
Hospital, Fudan University, National Clinical Research Center for Interventional
Medicine, 180 Fenglin Road, Shanghai 200032, China
Full list of author information is available at the end of the article 1 Department of Vascular Surgery, Institute of Vascular Surgery, Zhongshan
Hospital, Fudan University, National Clinical Research Center for Interventional
Medicine, 180 Fenglin Road, Shanghai 200032, China
Full list of author information is available at the end of the article Background Furthermore, calcification in the intima or media could
interfere with the absorption and distribution of pacli-
taxel and reduce the effect of DCB, which has been
proven to be an independent risk factor of DCB [19]. In
addition, the patency of stent implantation in non-stent
zones is poor due to the special anatomy. The incidence
rate of ISR at 3-year reaches 45.8% [20]. Meanwhile,
the clinical outcome of stents implantation in below-
the-knee (BTK) arteries is unclear. It is well known that
patients with CLI mainly involved BTK arteries, espe-
cially in those with diabetes mellitus or chronic kid-
ney dysfunction [21]. Also the efficacy of PTA in such
patients need to be clearly defined, especially given the
high incidence of re-stenosis due to the diffused calci-
fication and plaque [22]. Excimer laser ablation (ELA)
could minimize the dissection compared to PTA without
debulking, owing to removal of plaque and calcification. Its efficacy in ISR lesions has been demonstrated and also
been approved by U.S. Food and Drug Administration
(FDA) [23]. However, its efficacy in de novo atheroscle-
rotic lesions has not been clearly defined. ELABORATE
is initiated as the first trial to compare the safety and
efficacy of ELA combined with DCB in patients with de
novo atherosclerotic obliterans (ASO) in a real-world
setting. With the increase of aging population, the incidence rate
of peripheral artery disease (PAD) is getting higher [1]. In Germany, the prevalence of PAD has reached 10% [2]. The prevalence is up to 20% among 70-year-old Ger-
man patient population [1]. More than 20 million peo-
ple worldwide were diagnosed with PAD in the year of
2009 [3, 4]. And it cost 4 billion in the treatment of PAD
patients per year [5]. Of note is the fact that the symp-
toms are not classic in the early stage in many patients. However, approximately 10–15% of them would progress
to critical limb ischemia (CLI) within 1 year [6]. Mean-
while, they are also more likely to suffer from other cardi-
ovascular diseases [7]. Hence, the aim of treating patients
with PAD is to improve the long-term results and to
improve the quality of life (QoL) and thus to reduce the
national medical burden. Lower extremity artery disease (LEAD) is the most
commonly involved location in patients with PAD. In
the past decades, endovascular therapy has become the
main method to treat these patients. Trial designh This trial is a prospective, multicenter, real-world study,
and it has been approved by the Ethics Committee of
Zhongshan Hospital, Fudan University (Approval num-
ber: B2021-578). The overall design is demonstrated in
Fig. 1. The data collection is shown in Table 1. The design
of this trial is accordance with the Standard Protocol
Items [24]. Objectivesh The primary objective is to demonstrate whether the
application of ELA in the de novo LEAD is associated
with an increased rate of efficacy outcomes. The superiority of drug-coated balloon (DCB) in de
novo atherosclerotic lesions and ISR has been demon-
strated in previous studies [14–17]. However, flow-lim-
iting dissection and recoil are still observed up to 20%
of de novo lesion and 16% of ISR after the application of
DCB, which is needed to be treated by stenting [16–18]. © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecom-
mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Jiang et al. BMC Cardiovascular Disorders (2022) 22:317 Jiang et al. BMC Cardiovascular Disorders Page 2 of 8 Trial registration number Chinese Clinical Trial Registry (ChiCTR2100051263). Registered 17 September 2019. http://
www.chictr.org.cn/listbycreater.aspx. Keywords: Excimer laser ablation, Drug-coated balloon, De novo lesions, Atherosclerotic oblitera Background Meanwhile, more
centers prefer the endovascular approach compared
with open surgery. Even in patients with long and com-
plex LEAD, endovascular therapy also demonstrates a
relatively high technical success and low risks. Currently,
endovascular therapy mainly included percutaneous
transluminal angioplasty (PTA) and stent implantation;
however, the drawbacks of these procedures are increas-
ingly appeared. Studies have reported that the vessel
patency was significantly higher in stents compared to
PTA alone when the length of femoropopliteal lesions
was < 5 cm [8]. The implantation of bare-mental stent
could prevent the occurrence of extensive recoil and dis-
section thus to improve clinical outcomes in long seg-
ment lesions. However, in-stent restenosis (ISR) with an
incidence rate of 14–50%, is the main factor affecting the
long-term clinical outcomes [9–12]. In order to prevent
ISR, drug-eluting stents has been applied recently, and no
significant difference has been identified compared with
bare-mental stents regarding freedom from restenosis at
24-months [13].h Interventions
Th
bl h The establishment of access was as our previous study
[25]. And the extend of the calcification of the targeted
lesions will be assessed according to Peripheral Arterial
Calcium Scoring System (PACSS) [26]. The length, run-
off vessels situations, minimal luminal diameter (MLD),
and the reference vessel diameter (RVD) will be visu-
ally assessed under the digital subtraction angiography
(DSA). 1. Aged ≥ 18 years. 1. Aged ≥ 18 years. 2. Target lesion length ≤ 10 cm of the femoropopliteal
and BTK arteries. 3. Significant stenosis ≥ 70% or chronic total occlusion
(CTO) lesions assessed by the angiography. 4. Rutherford class 2–5 with ankle-brachial index
(ABI) < 0.9 in the target limb. After passage of the lesion, the necessity of the usage
of the distal protective device is at the surgeon’s discre-
tion. One runoff vessel existed and contaminant (1) long-
segment CTO or ISR lesions; (2) severe calcified lesions;
(3) subacute thrombosis, is recommended the applica-
tion of the distal protective device. For patients in the
treatment group, the laser guide (Spectranetics Corp.,
Colorado Springs, Colorado) will be used to achieve
maximum debulking with starting parameter: 45 mJ/
mm2, 45 Hz, followed by 50–60 mJ/mm2 and 50–70 Hz. Recruitmenth This study will be carried out from September 1, 2021 to
30 June, 2024. Each participant will be fully notified by
the trial leaders from each center and signs the informed
consent. Inclusion criteria. Jiang et al. BMC Cardiovascular Disorders (2022) 22:317 Page 3 of 8 Fig. 1 The flow-chart of the ELABORATE trial Fig. 1 The flow-chart of the ELABORATE trial The inclusion criteria of this trial are as follows: Fig. 1 The flow-chart of the ELABORATE trial Exclusion criteria are as follows: 1. ISR lesions. 1. ISR lesions. 2. Acute limb ischemia. 3. Simultaneously usage of other debulking devices. 4. Serum creatinine > 250 umol/L unless dialysis-
dependent, contrast agent allergy, coagulation dys-
function, and other severe comorbidities that is
impossible to tolerate the procedure. Jiang et al. BMC Cardiovascular Disorders (2022) 22:317 Page 4 of 8 Table 1 Schedule of enrollment and follow-up points
ABI ankle-brachial index, QoL quality of life, PACSS peripheral arterial calcium scoring system [25], DSA digital substract angiography, IVUS intravascular ultrasound,
NRS numerical rating scale, DUS Doppler ultrasound
(a) (b): evaluated by DSA in both groups before and after intervention; (c) (d): evaluated by IVUS in study group before and after laser
Follow-up visits
Long-term follow-up
Measuring
Outcome variables
Baseline
Intervention
1 month
3, 6, 12 months
(Every 12 months until
60 months)
Instrument
Allowed visit window
4 weeks to 1 week
± 5 days
± 10 days
± 30 days
Before intervention
Informed consent
X
Inclusion and exclusion criteria
X
Calcification class
X
PACSS
Target lesion length
X
Location of lesions
X
Runoff vessels
X
Minimal luminal diameter (a)
X
DSA
Reference vessel diameter (b)
X
DSA
Minimal luminal diameter (c)
X
IVUS
Reference vessel diameter (d)
X
IVUS
ABI
X
X
X
Rutherford class
X
X
X
Ulcer size
X
X
X
Pain
X
X
X
NRS
QoL
X
X
X
SF-36V2
Primary patency
X
X
DUS
All-cause mortality
X
Major amputation
X
X
Bailout stent
X
Distal embolization
X
Study completion/termination
record
At study exit Table 1 Schedule of enrollment and follow-up points ABI ankle-brachial index, QoL quality of life, PACSS peripheral arterial calcium scoring system [25], DSA digital substract angiography, IVUS intravascular ultrasound,
NRS numerical rating scale, DUS Doppler ultrasound (a) (b): evaluated by DSA in both groups before and after intervention; (c) (d): evaluated by IVUS in study group before and after laser Sample size A minimum sample size of 136 patients is determined to
be required for this study, assuming 1-sided α = 0.025,
β = 80%, assumed 76.1% and 84.9% primary patency at
12 months in the DCB and ELA + PTA groups, respec-
tively, and 25% attrition due to the nature of the real-
world study [23, 29]. Composition, roles, and responsibilities of the coordinating
center, steering committeeh The steering committee, including the principal investi-
gator Zhihui Dong, has full responsibility for the design
and implementation of this trial. Local primary investiga-
tors at each site are responsible for study administration,
coordination, and monitoring of this trial at their own
centers. Discussion Guideline on treatment of ASO by ELA are primarily
based on retrospective studies or randomized controlled
trials (RCT) [23, 31]. However, retrospective study com-
plies with its heterogeneity nature and RCT does not
represent the real clinical practice due to its registration
purpose. Only a small proportion of patients are eligible
for RCT due to the strict inclusion and exclusion criteria
[23]. Efficacy outcomeshfi The primary efficacy outcome is primary patency at
12-month, which is defined as no clinically driven target
lesion reintervention (CD-TLR) and peak systolic velocity
ratio (PSVR) ≤ 2.5 at the target lesion assessed by duplex
ultrasound. The secondary efficacy outcomes are: (1)
CD-TLR, (2) technical success, defined as residual steno-
sis ≤ 30% and flow-limiting dissection ≤ 40%, (3) ABI, (4)
change of Rutherford class, (5) pain evaluated by NRS, (6)
QoL evaluated by SF-36V2, and (7) ulcer healing. The strategy of monitoring will be carried in the mid-
dle and at the end of the trial just as Chen et al. [30]. No
audit is planned for the study. Meanwhile, the measures
that used to keep the confidentiality and safety of data are
consistent with those took by Chen et al. [30]. And the
paper copies of the CRFs will be kept by the trial leaders
for at least 5 years.h Safety evaluation includes major adverse event (MAE),
all-cause mortality through 30-day follow-up, unplanned
major amputation, bailout stent, and distal embolization.hi The definition of adverse event (AE) and adverse device
effect (ADE) are according to Zeller et al. [28]. Any AEs
and ADE will be recorded. The results of this trial will be present at vascular con-
ferences or be published in vascular journals. Data can be
provided to researchers upon request, subject to a review
of privacy. Requests for data can be sent to the investiga-
tor, Zhihui Dong, by email. Ethics approvalh This trial is approved by Ethics Community of Zhong-
shan Hospital, Fudan University (B2021-578) on August
30, 2021. Also it has been registered in the Chinese Clini-
cal Trial (ChiCTR2100051263). Reassessment will be
made by ethics communities once there is any changes of
the plan. Assignment of interventions According to the real-world situation, the group par-
ticipants will be allocated completely depends on the
patients’ request. Hence the sequence generation is not
applicable. And the double-blind method is also impos-
sible in this trial. ELABORATE is the first trial comparing the efficacy
of ELA combined with DCB and DCB alone in the treat-
ment of de novo atherosclerotic lesions in a real-world
setting. Compared with RCT, real-world study more
conforms to clinical practice and provides high exter-
nal validity [32]. The inclusion criteria in ELABORATE
represent the broad definition of a patient eligible for Statistical analysis Data will be analyzed by SPSS 20.0 (IBM Co., Armonk,
NY, USA). The distribution of continuous variables will
be checked firstly. Continuous variables were presented
as mean ± standard deviation (SD) with range values for
normally distributed data, or as the medians with the
interquartile ranges (IQRs) for non-normally distributed
data, respectively. Categorical variables were presented
as frequencies and percentages. The differences between
continuous variables are identified by using 2-sided Stu-
dent t tests or the Wilcoxon rank test. While chi-square
test or Fisher exact test is applied to compare the differ-
ence between categorical variables. All of the tests are
2-sided, and a p value < 0.05 was considered statistically
significant. Follow‑up strategyh The angiography will be performed to evaluate the MLD,
distal embolization, the extend of dissection. ELA could
be continued if the lumen acquired is not satisfactory. The angiography will be performed to evaluate the MLD,
distal embolization, the extend of dissection. ELA could
be continued if the lumen acquired is not satisfactory. These patients will be given aspirin (100 mg/d) and
clopidogrel (75 mg/d) for at least 6 months after inter-
vention. And they will be followed at 3, 6 12 months. ABI and Doppler ultrasound of the targeted lesions will
be evaluated at each follow-up point, and SF-36QoF
is used to assess the QoL. The pain score during the
follow-up is evaluated by the numerical rating scale
(NRS). Meanwhile, the ulcers (if existed) will be photo-
graphed at two different angles. All participants will be
followed for at least 5 years. After ELA in the treatment group, the following pro-
cedure is similar in two groups. A standard balloon, 0.5-
mm undersized to the RVD, is used before the DCB. And
DCB is selected with 1:1 ratio to RVD for 2 min. Angiog-
raphy is performed at least two different angles to record
the MLD and dissection (if existed). In the treatment
group, intravascular ultrasound (IVUS) (Volcano Corpo-
ration, Rancho Cordova, CA, USA) is applied to confirm
the type of the lesions, MLD, and RVD before ELA, and
MLD, RVD, and extend of dissection after ELA. The use
of other debulking devices is prohibited. The bailout stent
is implanted if flow-limiting dissection > 40% or residual
stenosis > 30% occurs. The distal protective device would
be retrieved and the debris > 2 mm will be recorded sig-
nificant ones [27]. Participant can withdrawal from the trial any time,
and their routine treatment would not be affected. The
allocated interventions can be modified when (1) par-
ticipant request, (2) the allocated intervention does
no good to participant’s disease. Dr Zhihui Dong is
responsible for any changes. i
Any special balloon, such as Chocolate balloon, cutting
balloon or Shockwave balloon, will be prohibited. Page 5 of 8 Jiang et al. BMC Cardiovascular Disorders (2022) 22:317 Outcomesfi size will be recorded and performed by one special tech-
nician using the same apparatuses. Participant retention
and follow-up engagement is enhanced by communi-
cating verbally and via WeChat app. A case report form
(CRF) is used to collect data for each participant. Mean-
while, the collected data will be stored in an electronic
data capture (EDC) system to ensure data authenticity. Data collection, monitoring and confidentiality Each patient’s ID and baseline information will be col-
lected. The occurrence and timing of clinical outcomes
will be documented. The ABI and measurements of ulcer Jiang et al. BMC Cardiovascular Disorders (2022) 22:317 Jiang et al. BMC Cardiovascular Disorders (2022) 22:317 Page 6 of 8 endovascular therapy in the real world. Meanwhile, study
accessibility is maximized by defining minimal exclu-
sion criteria. Observational studies and RCTs all have
their own nature limitations and any of them alone may
not fully represent the true value of certain treatment
for LEAD [33]. As such, well-designed real-world study
may offer complementary data to these standard types of
studies, representing true real-world effectiveness. The
application of ELA in ISR has been approved by U.S. FDA
[23]. And it also has been approved in our nation in the
treatment of PAD. However, its validity in PAD, especially
in de novo atherosclerotic lesions, has not been fully
demonstrated, and the operation is still not standardized. Although been approved by Chinese FDA, perform-
ing a trial of relatively new device in real-world setting
still poses many challenges. Participant’s safety is the
priority of the study. We have several steps to keep the
enrolled participant’s safety. Firstly, before initiation,
surgeons who participates in the trial will be trained by
professional engineer about the principles of excimer
laser, also they will be trained by an experienced vascular
surgeon who performed ≥ 50 ELA procedures to ensure
the standardization of ELA operation. The chief surgeon
participating in this study has performed at least 50 PAD
cases/year and accumulated ≥ 10 cases of ELA. Secondly,
participant’s safety will be monitored in real-time by a
combination of surveillance of EDC system and clinical
monitoring. than RCT, and we enrolled more than 25% of the sample
size to ensure the valid results. endovascular therapy in the real world. Meanwhile, study
accessibility is maximized by defining minimal exclu-
sion criteria. Observational studies and RCTs all have
their own nature limitations and any of them alone may
not fully represent the true value of certain treatment
for LEAD [33]. As such, well-designed real-world study
may offer complementary data to these standard types of
studies, representing true real-world effectiveness. The
application of ELA in ISR has been approved by U.S. FDA
[23]. And it also has been approved in our nation in the
treatment of PAD. Data collection, monitoring and confidentiality However, its validity in PAD, especially
in de novo atherosclerotic lesions, has not been fully
demonstrated, and the operation is still not standardized. Acknowledgements
None. Acknowledgements
None. Availability of data and materials Data can be provided to researchers upon request, subject to a review of
privacy. Requests for data can be sent to the corresponding author by email. Funding g
This work was supported externally by the Key Clinical Research Projects of
National Clinical Research Center for Interventional Medicine (2021–006). It
has no role in in study design, collection, management, analysis, or interpreta-
tion of data, writing of the report, or the decision to submit the report for
publication. Ethics approval and consent to participate The study will be conducted in accordance with the ethical principles of the
Declaration of Helsinki. The study is approved by Ethical Review Board of
Zhongshan Hospital, Fudan University (B2021-578). All patients will provide
written consent for participation and publication of trial data. i
The ELABORATE has limitations need to be demon-
strated. Participants and surgeons are not blinded due to
the real-world nature, which induces some selection bias
cannot be precluded. Also, due to the real-world setting
of the study, the lost follow-up rate is inevitably higher Author contributionsi XJ and LF are co-first authors. XJ, LF, BC, JJ, JL, GQ and SJ obtained the ethical
approval, registered and drafted the manuscript. XJ, YS, TM, CL, GF, DG, and XX
conceived the study and participated in the design of the study. XJ, LF, WF and
ZD participated in the study coordination. BC, JJ, JL, GQ and SJ contributed
to the statistical analysis plan. XJ, LF, YS, TM, CL, GF, DG and XX acquired and
analysed the data of the work. ZD is the corresponding author. All authors
read and approved the final manuscript and agreed the submission. The main purpose of this trial is to demonstrate the
efficacy of ELA in de novo atherosclerotic lesions in
LEAD compared with PTA, especially in popliteal and
BTK arteries. The concept of “leave nothing behind”
means more in the P2 segment. The laser could ablate
the calcification of the vessel and the hyperplastic intima
to reduce the occurrence of flow-limiting dissection
after PTA. Then the stent implantation, which shows
poor short- to long-term patency in P2 segment, could
more likely be avoided. BTK artery is another challenge
of endovascular treatment. Lesions involves BTK arter-
ies are usually severely calcified and diffused and have a
high incidence of restenosis after PTA [21]. Moreover,
these patients have a higher proportion of diabetic mel-
litus or chronic kidney insufficiency, which makes them
have a lower threshold of tolerance to surgery. Although
minimal invasive, repeated endovascular procedures are
impracticable. The longer working distance and smaller
diameter of the catheter show good compatibility with
BTK lesions and could significantly improve the patency.h Current trial statush This trial has been registered in the Chinese Clinical
Trial (ChiCTR2100051263) on September 17, 2021 after
approval of the Ethics Community of Zhongshan Hos-
pital, Fudan University (B2021-578) on August 30, 2021. The inclusion began on October 1, 2021, and 2 patients
in the ELA group and 2 patients in the controlled group
have been included. The other two centers are prepared
to begin or are awaiting approval of the Ethics Com-
mittee. Inclusion will be continued until 2024, and the
follow-up will be finished till 2024. The current trial pro-
tocol version is 1.0, and the informed consent version is
2.0. Although been approved by Chinese FDA, perform-
ing a trial of relatively new device in real-world setting
still poses many challenges. Participant’s safety is the
priority of the study. We have several steps to keep the
enrolled participant’s safety. Firstly, before initiation,
surgeons who participates in the trial will be trained by
professional engineer about the principles of excimer
laser, also they will be trained by an experienced vascular
surgeon who performed ≥ 50 ELA procedures to ensure
the standardization of ELA operation. The chief surgeon
participating in this study has performed at least 50 PAD
cases/year and accumulated ≥ 10 cases of ELA. Secondly,
participant’s safety will be monitored in real-time by a
combination of surveillance of EDC system and clinical
monitoring. ELABORATE is a multicenter, prospective study aim-
ing at evaluating the safety and effectiveness of ELA
combined with DCB in the treatment of de novo ath-
erosclerotic lesions in a real-world setting. Data from
ELABORATE will provide evidence for the efficacy of
ELA in ASO. Meanwhile, the trial will make a reference
for future evaluation of effectiveness of new therapies. References 1. Lawall H, Huppert P, Espinola-Klein C, Zemmrich CS, Ruemenapf G. German guideline on the diagnosis and treatment of peripheral artery
disease—a comprehensive update 2016. Vasa. 2017;46(2):79–86. 1. Lawall H, Huppert P, Espinola-Klein C, Zemmrich CS, Ruemenapf G. German guideline on the diagnosis and treatment of peripheral artery
disease—a comprehensive update 2016. Vasa. 2017;46(2):79–86. 19. Tepe G, Beschorner U, Ruether C, Fischer I, Pfaffinger P, Noory E, et al. Drug-eluting balloon therapy for femoropopliteal occlusive disease:
predictors of outcome with a special emphasis on calcium. J Endovasc
Ther. 2015;22(5):727–33. 2. Diehm C, Schuster A, Allenberg JR, Darius H, Haberl R, Lange S, et al. High prevalence of peripheral arterial disease and co-morbidity in
6880 primary care patients: cross-sectional study. Atherosclerosis. 2004;172(1):95–105. 2. Diehm C, Schuster A, Allenberg JR, Darius H, Haberl R, Lange S, et al. High prevalence of peripheral arterial disease and co-morbidity in
6880 primary care patients: cross-sectional study. Atherosclerosis. 2004;172(1):95–105. 20. San Norberto EM, Flota CM, Fidalgo-Domingos L, Taylor JH, Vaquero C. Real-world results of supera stent implantation for popliteal artery ath-
erosclerotic lesions: 3-year outcome. Ann Vasc Surg. 2020;62:397–405. 3. Fowkes FG, Rudan D, Rudan I, Aboyans V, Denenberg JO, McDermott
MM, et al. Comparison of global estimates of prevalence and risk factors
for peripheral artery disease in 2000 and 2010: a systematic review and
analysis. Lancet. 2013;382(9901):1329–40. 3. Fowkes FG, Rudan D, Rudan I, Aboyans V, Denenberg JO, McDermott
MM, et al. Comparison of global estimates of prevalence and risk factors
for peripheral artery disease in 2000 and 2010: a systematic review and
analysis. Lancet. 2013;382(9901):1329–40. 21. Diehm N, Shan A, Silvestro A, Do DD, Dick F, Schmidli J, et al. Associa-
tion of cardiovascular risk factors with pattern of lower limb athero-
sclerosis in 2659 patients undergoing angioplasty. Eur J Vasc Endovasc
Surg. 2006;31(1):59–63. 4. Adam DJ, Bradbury AW. TASC II document on the management of
peripheral arterial disease. Eur J Vasc Endovasc Surg. 2007;33(1):1–2. g
22. Capek P, McLean GK, Berkowitz HD. Femoropopliteal angioplasty. Fac-
tors influencing long-term success. Circulation. 1991;83(2 suppl):I70-80. 5. Hirsch AT, Hartman L, Town RJ, Virnig BA. National health care costs
of peripheral arterial disease in the medicare population. Vasc Med. 2008;13(3):209–15. 5. Hirsch AT, Hartman L, Town RJ, Virnig BA. National health care costs
of peripheral arterial disease in the medicare population. Vasc Med. 2008;13(3):209–15. 23. References Dippel EJ, Makam P, Kovach R, George JC, Patlola R, Metzger DC, et al. Randomized controlled study of excimer laser atherectomy for treat-
ment of femoropopliteal in-stent restenosis: initial results from the
EXCITE ISR trial (EXCImer Laser Randomized Controlled Study for Treat-
ment of FemoropopliTEal In-Stent Restenosis). JACC Cardiovasc Interv. 2015;8(1 Pt A):92–101. 6. Gerhard-Herman MD, Gornik HL, Barrett C, Barshes NR, Corriere MA,
Drachman DE, et al. 2016 AHA/ACC guideline on the management
of patients with lower extremity peripheral artery disease: executive
summary: a report of the American College of Cardiology/American
Heart Association task force on clinical practice guidelines. Circulation. 2017;135(12):e686–725. 24. Chan A-W, Tetzlaff JM, Gøtzsche PC, Altman DG, Mann H, Berlin JA,
et al. Spirit 2013 explanation and elaboration: guidance for protocols of
clinical trials. BMJ. 2013;346:e7586. 7. Criqui MH, Langer RD, Fronek A, Feigelson HS, Klauber MR, McCann TJ,
et al. Mortality over a period of 10 years in patients with peripheral arte-
rial disease. N Engl J Med. 1992;326(6):381–6. 7. Criqui MH, Langer RD, Fronek A, Feigelson HS, Klauber MR, McCann TJ,
et al. Mortality over a period of 10 years in patients with peripheral arte-
rial disease. N Engl J Med. 1992;326(6):381–6. 25. Jiang X, Ju S, Chen B, Jiang J, Shi Y, Ma T, et al. Safety and effectiveness
of excimer laser ablation combined with drug-coated balloon for ath-
erosclerotic obliterans in the lower extremity. J Endovasc Ther. 2022. https://doi.org/10.1177/15266028221092979. 8. Laird JR, Katzen BT, Scheinert D, Lammer J, Carpenter J, Buchbinder M,
et al. Nitinol stent implantation vs. balloon angioplasty for lesions in
the superficial femoral and proximal popliteal arteries of patients with
claudication: three-year follow-up from the RESILIENT randomized trial. J
Endovasc Ther. 2012;19(1):1–9. 26. Rocha-Singh KJ, Zeller T, Jaff MR. Peripheral arterial calcification:
prevalence, mechanism, detection, and clinical implications. Catheter
Cardiovasc Interv. 2014;83(6):E212–20. 9. Schillinger M, Sabeti S, Loewe C, Dick P, Amighi J, Mlekusch W, et al. Bal-
loon angioplasty versus implantation of nitinol stents in the superficial
femoral artery. N Engl J Med. 2006;354(18):1879–88. 27. Shammas NW, Coiner D, Shammas GA, Christensen L, Dippel EJ, Jerin
M. Distal embolic event protection using excimer laser ablation in
peripheral vascular interventions: results of the DEEP EMBOLI registry. J
Endovasc Ther. 2009;16(2):197–202. 10. Krankenberg H, Schluter M, Steinkamp HJ, Bürgelin K, Scheinert D,
Schulte KL, et al. Competing interests The authors declare that they have no competing interests. Meanwhile,
the study funders have no role in in study design, collection, management, Page 7 of 8 Jiang et al. BMC Cardiovascular Disorders (2022) 22:317 Jiang et al. BMC Cardiovascular Disorders (2022) 22:317 Jiang et al. BMC Cardiovascular Disorders (2022) 22:317 analysis, or interpretation of data, writing of the report, or the decision to
submit the report for publication. 14. Werk M, Albrecht T, Meyer DR, Ahmed MN, Behne A, Dietz U, et al. Paclitaxel-coated balloons reduce restenosis after femoro-popliteal
angioplasty: evidence from the randomized PACIFIER trial. Circ Cardio-
vasc Interv. 2012;5(6):831–40. Author details 1 Department of Vascular Surgery, Institute of Vascular Surgery, Zhongshan
Hospital, Fudan University, National Clinical Research Center for Interven-
tional Medicine, 180 Fenglin Road, Shanghai 200032, China. 2 Department
of Vascular Surgery, Qingpu Branch of Zhongshan Hospital, Fudan University,
1158 East Gongyuan Road, Shanghai 201715, China. 3 Department of Vascular
and Wound Treatment Center, Jinshan Hospital, Fudan University, Shang-
hai 200540, China. 15. Tepe G, Zeller T, Albrecht T, Heller S, Schwarzwälder U, Beregi JP, et al. Local delivery of paclitaxel to inhibit restenosis during angioplasty of
the leg. N Engl J Med. 2008;358(7):689–99. 16. Scheinert D, Duda S, Zeller T, Krankenberg H, Ricke J, Bosiers M, et al. The LEVANT I (Lutonix paclitaxel-coated balloon for the prevention
of femoropopliteal restenosis) trial for femoropopliteal revasculari-
zation: first-in-human randomized trial of low-dose drug-coated
balloon versus uncoated balloon angioplasty. J Am Coll Cardiol Intv. 2014;7(1):10–9. Received: 20 April 2022 Accepted: 7 July 2022 Received: 20 April 2022 Accepted: 7 July 2022 17. Stabile E, Virga V, Salemme L, Cioppa A, Ambrosini V, Sorropago G,
et al. Drug-eluting balloon for treatment of superficial femoral artery
in-stent restenosis. J Am Coll Cardiol. 2012;60(18):1739–42. 18. Liistro F, Angioli P, Porto I, Ricci L, Ducci K, Grotti S, et al. Paclitaxel-
eluting balloon vs. standard angioplasty to reduce recurrent restenosis
in diabetic patients with in-stent restenosis of the superficial femoral
and proximal popliteal arteries: the DEBATE-ISR study. J Endovasc Ther. 2014;21(1):1–8. References Nitinol stent implantation versus percutaneous
transluminal angioplasty in superficial femoral artery lesions up to
10 cm in length: the femoral artery stenting trial (FAST). Circulation. 2007;116(3):285–92. 28. Zeller T, Baumgartner I, Scheinert D, Brodmann M, Bosiers M, Micari
A, et al. IN.PACT Amphirion paclitaxel eluting balloon versus standard
percutaneous transluminal angioplasty for infrapopliteal revasculariza-
tion of critical limb ischemia: rationale and protocol for an ongoing
randomized controlled trial. Trials. 2014;15:63. 11. Schillinger M, Sabeti S, Dick P, Amighi J, Mlekusch W, Schlager O, et al. Sustained benefit at 2 years of primary femoropopliteal stenting
compared with balloon angioplasty with optional stenting. Circulation. 2007;115(12):2745–9. 29. Jia X, Zhang J, Zhuang B, Fu W, Wu D, Wang F, et al. Acotec drug-coated
balloon catheter: randomized, multicenter, controlled clinical study in
femoropopliteal arteries: evidence from the AcoArt I trial. JACC Cardio-
vasc Interv. 2016;9(18):1941–9. 12. Laird JR, Katzen BT, Scheinert D, Lammer J, Carpenter J, Buchbinder M,
et al. Nitinol stent implantation versus balloon angioplasty for lesions
in the superficial femoral artery and proximal popliteal artery: twelve-
month results from the RESILIENT randomized trial. Circ Cardiovasc Interv. 2010;3(3):267–76. 30. Chen S, Xu Z, Liu H, Zhang Y, Zhang J, Chen Y, et al. Perioperative
patient-controlled regional analgesia versus patient-controlled intrave-
nous analgesia for patients with critical limb ischaemia: a study proto-
col for a randomised controlled trial. BMJ Open. 2020;10(10):e037879. 13. Duda SH, Bosiers M, Lammer J, Scheinert D, Zeller T, Oliva V, et al. Drug-
eluting and bare nitinol stents for the treatment of atherosclerotic lesions
in the superficial femoral artery: long-term results from the SIROCCO trial. J Endovasc Ther. 2006;13(6):701–10. 31. Wissgott C, Kamusella P, Ludtke C, Andresen R. Excimer laser atherec-
tomy after unsuccessful angioplasty of TASC C and D lesions in femoro-
popliteal arteries. J Cardiovasc Surg (Torino). 2013;54(3):359–65. Page 8 of 8 Jiang et al. BMC Cardiovascular Disorders (2022) 22:317 Jiang et al. BMC Cardiovascular Disorders (2022) 22:317 Jiang et al. BMC Cardiovascular Disorders (2022) 22:317 Jiang et al. BMC Cardiovascular Disorders (2022) 22:317 32. Price D, Chisholm A, van der Molen T, Roche N, Hillyer EV, Bousquet J.
Reassessing the evidence hierarchy in asthma: evaluating comparative
effectiveness. Curr Allergy Asthma Rep. 2011;11(6):526–38.
33. Roche N, Reddel H, Martin R, Brusselle G, Papi A, Thomas M, et al. Quality
standards for real-world research. Focus on observational database stud-
ies of comparative effectiveness. Ann Am Thorac Soc. 2014;11:S99-104. Publisher’s Note
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ADAR1 overexpression is associated with cervical cancer progression and angiogenesis
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© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Analysis also showed that disease-free survival time of ADAR1 positive patients was shorter than
that of ADAR1 negative patients, and the difference was significant (P < 0.01). Finally, COX risk assessment showed that
parametrical invasion had independent prognostic factors for overall survival of squamous cell carcinoma. expression (P < 0.05). Analysis also showed that disease-free survival time of ADAR1 positive patients was shorter than
that of ADAR1 negative patients, and the difference was significant (P < 0.01). Finally, COX risk assessment showed that
parametrical invasion had independent prognostic factors for overall survival of squamous cell carcinoma. Conclusions: Results indicated that ADAR1 might play an important role in the occurrence, progression and prognosis Conclusions: Results indicated that ADAR1 might play an important role in the occurrence, progression and prognosis
of cervical squamous cancer. Keywords: Adenosine deaminase acting on RNA1, Cervical squamous cancer, Risk factor the mortality rate is 14%, which is higher compared with
that obtained in other developing countries. For a long
time, the studies of cancer mechanism had been greatly
focused on the mechanism of DNA imbalance. Recently,
we found that tumorigenesis might be caused by the
process of RNA transcription, in which RNA editing en-
zyme played an important role. RNA editing is wide-
spread in the evolutionary process [2]. It refers to RNA
modifications and processing after the transcription of
DNA into RNA. The edited RNA molecules may be
translated to completely different proteins compared to
their transcribed DNA sequences, hereby changing the
genetic information [3–7]. Studies have shown that ADAR1 ADAR1 overexpression is associated
with cervical cancer progression and
angiogenesis Ying Chen1,2, He Wang1*, Wenyi Lin3 and Ping Shuai4 Ying Chen1,2, He Wang1*, Wenyi Lin3 and Ping Shuai4 Abstract Background: This study aimed to assess the role of RNA-dependent adenosine deaminase (ADAR1) in cervical
squamous cell carcinoma occurrence and progression. Methods: ADAR1 expression levels in stage IA and stage IIA cervical squamous cell carcinoma (group A), cervical
intraepithelial neoplasia (CIN) specimens (group B), as well as normal and inflamed cervical tissue samples (group C)
were assessed by immunohistochemistry. Clinical and pathological data of cervical squamous cell carcinoma
patients undergoing surgery were retrospectively evaluated. Chi-square test, comparative analysis of survival curve,
disease-free survival and COX risk assessment method were used to understand the association of ADAR1 with the
occurrence and progression and prognostic significance of cervical squamous cell carcinoma. Results: ADAR1 is expressed in the cytoplasm and nuclei. The expression level was high in squamous cell carcinoma
tissues (81.18%), while relatively low in the CIN group (21.56%). And there was no expression in non-cancerous tissues. The differences between them were statistically significant using P < 0.05 as criterion. One-factor analysis revealed that
ADAR1 was significantly correlated with tumor diameter, horizontal diffusion diameter, vascular invasion, parametrial
invasion, vaginal involvement, and pathologically diagnostic criteria for perineural invasion (PNI). Meanwhile, the overall
survival rate of ADAR1 positive patients was significantly lower compared with that of patients with no ADAR1
expression (P < 0.05). Analysis also showed that disease-free survival time of ADAR1 positive patients was shorter than
that of ADAR1 negative patients, and the difference was significant (P < 0.01). Finally, COX risk assessment showed that
parametrical invasion had independent prognostic factors for overall survival of squamous cell carcinoma. Conclusions: Results indicated that ADAR1 might play an important role in the occurrence, progression and prognosis
of cervical squamous cancer. Results: ADAR1 is expressed in the cytoplasm and nuclei. The expression level was high in squamous cell carcinoma
tissues (81.18%), while relatively low in the CIN group (21.56%). And there was no expression in non-cancerous tissues. The differences between them were statistically significant using P < 0.05 as criterion. One-factor analysis revealed that
ADAR1 was significantly correlated with tumor diameter, horizontal diffusion diameter, vascular invasion, parametrial
invasion, vaginal involvement, and pathologically diagnostic criteria for perineural invasion (PNI). Meanwhile, the overall
survival rate of ADAR1 positive patients was significantly lower compared with that of patients with no ADAR1 expression (P < 0.05). Chen et al. Diagnostic Pathology (2017) 12:12
DOI 10.1186/s13000-017-0600-0 Chen et al. Diagnostic Pathology (2017) 12:12
DOI 10.1186/s13000-017-0600-0 Background Cervical carcinoma is the second most common malig-
nant tumor in women around the world, and the first
malignancy affecting females in developing countries. In-
deed, about half a million women are diagnosed yearly
with cervical cancer worldwide [1], with a mortality rate
of 9%. In China, the number of new cases of cervical
squamous cell carcinoma each year is 130,000, which ac-
counts for 28% of newly diagnosed cases globally. And * Correspondence: Wanghe_cd@126.com
1Department of Obstetrics and Gynecology, West China Second University
Hospital, Sichuan University, Chengdu 610041, Sichuan, China
Full list of author information is available at the end of the article Page 2 of 8 Chen et al. Diagnostic Pathology (2017) 12:12 Page 2 of 8 anti-mouse/anti-rabbit secondary antibody reagents were
purchased from Guangzhou Shenda Biological Products
Technology Co., Ltd (Guangzhou, China). Procedures
exactly followed the manufactures’ instructions:a) Par-
affin section was baked at 65 °C for 3 h, immersed in
xylene to dewax for l0 min for two times, and then
dehydrated using gradient alcohol; b) Paraffin section
was soaked in 3% hydrogen peroxide for l0 min, and
then washed three times using PBS buffer, 3 min for
each time; c) Microwave antigen repair was performed
using 0.4 g citric acid/3 g sodium citrate mixed solution
(1 L.pH 6.0). After 30 min of natural cooling, paraffin
section was washed two times using PBS buffer, 3 min
each time. d) The first antibody (ADAR1 rabbit anti-
human monoclonal antibody) was added and then paraffin
was placed in a humid chamber incubator at 37 °C for
60 min, following with 3 times of wash using PBS buffer; e)
The secondary antibody, i.e. goat anti-mouse/anti-rabbit,
was added and then paraffin was incubated at room
temperature for 30 min before PBS buffer washing for 3
times; f) DAB staining at room temperature for 5 min, and
then washed using tap water for 15–20 min; nuclei staining
using hematoxylin for 2–3 min, and then washed using tap
water for half an hour; g) 85% alcohol, 90% alcohol, 95%
Ethanol, pure ethanol wash for 5 min × 1 times, xylene
wash for 5 min × 2 times. After all of these steps, check
the paraffin using microscope. By the way, we set positive
control and negative control: normal cervical tissue as
a negative control with PBS solution instead of primary
antibody; known esophageal squamous cell carcinoma
as ADAR1 positive control. Measurement and comparison of the expressions of
ADAR1 in cervical squamous cell carcinoma in A, B, and C
groups Sample collection methods were the same as above,
with the only difference was to use S-100 rabbit anti-
human monoclonal antibody instead (purchased from
Guangzhou Shenda Biological Products Technology Co.,
Ltd, Guangzhou, China). Immunohistochemical paraffin sections came from the
Chengdu Women and Children’s Central Hospital, De-
partment of Pathology. All samples were fixed by 10%
formaldehyde, embedded in paraffin, and treated with
HE staining. And pathological results confirmed that to
be cervical squamous cell carcinoma. All patients did not
receive preoperative radiotherapy, chemotherapy and
biotherapy. Patients and clinical data Tissue samples of patients were collected in Gynecology
Department of Chengdu Women and Children’s Central
Hospital. Between February 2012 and December 2015, 170
tissue samples from patients with stage IA-IIA cervical
squamous cell carcinoma (Group A), 102 tissue specimens
from patients with CIN (51 cases of CINI and and 51 cases
of CIN II-III, respectively, Group B), as well as 31 normal
and inflamed cervical tissue samples (Group C) were col-
lected. Patients with cervical squamous cell carcinoma were
selected according to the following critieria: (1) diagnosed
with stage IA-IIA based on the FIGO (International Feder-
ation of Gynecology and Obstetrics) classification; (2)
treated with radical hysterectomy and pelvic lymphadenec-
tomy, with complete postoperative pathological data; (3)
had complete postoperative follow-up data. All th patients
were between 21–68 years old, with an average age of
48.79 years old. According to the FIGO classification, pa-
tients with stage I and stage IIA squamous cell carcinoma
accounted for 68.24 and 31.76%, respectively. Clinical and
pathological data of hospitalized patients were retrospect-
ively analyzed. Immunohistochemistry was used to detect whether
Pathologically diagnostic criteria for perineural invasion
(PNI) was expressed in cervical squamous cell carcin-
oma. Pathologically diagnostic criteria for perineural in-
vasion (PNI) refers to a focal invasion and metastasis
phenomenon, with tumor cells entering any layer of epi-
neurium, perineurium or endoneurium along nerves fi-
bers or around the nerves, or aggregating and wrapping
the nerves (≥33% of nerve circumference) as well as ex-
tending along them [12]. Background is a RNA-dependent adenosine deaminase, with highest
expression in tumor cells. It mainly plays a role of RNA
editing to change A into G in chronic myelocytic
leukemia [8–10], thereby changing the normal physio-
logical structures and functions of proteins. In addition,
ADAR1 plays an important role in laryngeal squamous
cell carcinoma, and tends to promote its occurrence
and progression [11]. However, relevant studies on cer-
vical squamous cell carcinoma have not been reported
previously. In this study, The mRNA and protein expres-
sion levels of ADAR1 in cervical squamous cell carcinoma
tissues, as well as their associations with patient prognosis,
were assessed, in order to have a preliminary investigation
of ADAR1’s role in cervical squamous cell carcinoma oc-
currence and progression. Follow-up Mainly, patients with cervical squamous cell carcinoma
were followed-up by telephone, mails or postoperative
visits, from pathological diagnosis to December 2015. Follow-up lasted for 1–47 months, with an average
follow-up time of 30.4 months. Immunohistochemical assessment Positive signals appeared as brown staining in the nuclei
and cytoplasm and percentage of positive cells/total cell
count is used to measure the expression level of ADAR1. With the normal squamous epithelium as a reference,
positive ADAR1 expression was indicated by a higher
expression, and negative expression, refers to weaker or
comparable expression levels, compared with the amounts
obtained for the corresponding squamous epithelium
specimens. The following scales were used for staining de-
grees: (−), only basal cells stained, with positive cell rate
<5%; (+), focal lesion stained, with positive cell rate ran-
ging from 5 to 30%; (++), extensive staining with positive
cell rate of 31- 60%; (+++), diffused staining with positive
cell rate greater than 60%. Escaping slices or those with
very small numbers of tumor cells (cell count < 200) were
considered ineffective (18 slices). Statistical analysis of association of ADAR1 with clinical
pathology of cervical squamous cell carcinoma According to FIGO staging of cervical pathology indica-
tors in combination with clinical practice, we chose 15
factors to describe the progression of cervical squamous
cancer, including menopause, motherhood, clinical stage,
keratosis, tumor size, horizontal diffusion distance, stromal ADAR1 rabbit anti-human monoclonal antibody was
bought from abcom Company (Shanghai, China). Goat Chen et al. Diagnostic Pathology (2017) 12:12 Page 3 of 8 Page 3 of 8 Page 3 of 8 invasion, histological grade, vascular invasion, parametrial
margin, parametrial invasion, vaginal involvement, lymph
node metastasis, HPV infection, and perineural invasion. They were compared with ADAR1 expression to explore
the association of ADAR1 with progress of cervical squa-
mous cancer. Clinical and pathological data of group A
undergoing surgery were retrospectively evaluated, using
chi-square test to verify the association of ADAR1 with the
occurrence and progression of cervical squamous cell car-
cinoma. Comparative analysis of survival curve, disease-free
survival and COX risk assessment method were performed
to verify the prognostic significance of ADAR1 in cervical
squamous cell carcinoma. were considered to be high expression, i.e. positive expres-
sion. Interestingly, the positive rate was found to be
81.18% (140/170) for Group A and 21.56% (22/102) for
Group B, while all Group C cases showed no expression. In addition, the signal intensity gradually increased with
the lesion severity. The differences among these groups
were statistically significant (p < 0.05). In ADAR1 positive tissue of cervical squamous cell
carcinoma, the PNI was found around the foci, account-
ing for 14.71% (25/170), which was mainly for the nerve
fibers distributing in the cancer stroma irregularly, and
perineural invasion around the foci was visible with ir-
regular growth of nerve and messy. However, the PNI
expression in ADAR1 negative tissue was negative. The
difference was statistically significant (p < 0.05) (Fig. 1). Associations of ADAR1 and cervical squamous cell
carcinoma related factors One-factor analysis data of ADAR1 are shown in Table 1. A total of 15 factors were analyzed, including menopause,
gravidity, clinical stage, keratinization, tumor diameter,
horizontal diffusion diameter, stroma invasion depth,
histological grade, vascular invasion, parametrial margin,
uterine invasion, vagina involvement, lymph node me-
tastasis, HPV infection, and PNI. Interestingly, tumor
diameter, horizontal diffusion diameter, vagina involve-
ment, vascular invasion, uterine invasion and PNI were
significantly associated with ADAR1 (p < 0.05), as high-risk
factors affecting ADAR1 expression. Besides uterine inva-
sion which had a p < 0.05, the remaining factors showed
p < 0.01, indicating statistical significance (Table 1). Effect of ADAR1expression in cervical carcinoma
prognosis p
g
Overall survival times of ADAR1 positive and negative
patients were 38.25 ± 1.3 and 45.46 ± 0.86 months, respect-
ively, with a significant difference of p < 0.05 (χ2 = 5.101,
p = 0.024) (Fig. 2). The overall disease-free survival times
were 39.048 ± 1.118 months. The disease-free survival
times of ADAR1 negative and positive patients were
45.458 ± 0.863 and 34.877 ± 1.408 months, respectively,
with a significant difference (χ2 = 12.925, p = 0.000) (Fig. 3). From overall survival curve, we could see that the 1-year
and 2 year survival rates for ADAR1 negative group were
100 and 93.8%, respectively; while the 1 year and two sur-
vival rates for ADAR1 positive group were 82.1 and 81.2%,
respectively. From disease-free survival curve, we could
see that 1-year and 2 year survival rates for ADAR1 nega-
tive group were both 100%; the 1-year and 2 year disease-
free survival rates for ADAR1 positive group were 80.5
and 70.6%, respectively. Taken together, our data suggest
that ADAR1 positive patients have worse prognosis. To
further study the effect of ADAR1 positive expression on
overall survival rate of cervical squamous cell carcinoma, Statistical analysis Statistical analyses were performed using the SPSS 19.0
software. One-factor analysis of categorical variables was
conducted with chi-square test; survival curves were
generated by the Kaplan-Meier method. The parameters
of statistical significance from univariate chi-square test
were included in the COX risk assessment to test
whether ADAR1 was an independent prognostic factor
for overall survival in cervical squamous cell carcinoma. Immunohistochemical data analysis ADAR1 is expressed in the cytoplasm and nuclei. Negative
and weakly positive signals were considered to be normal
expression, while moderately and strongly positive signals Chen et al. Diagnostic Pathology (2017) 12:12 Page 4 of 8 Fig. 1 Immunohistochemistry (IHC) data showing ADAR1 expression levels in normal cervical, CIN, and cervical squamous cancer tissue specimens. a Normal squamous epithelium (IHC × 200); b Uterine neck CIN III (IHC × 200); c Cervical keratinizing squamous cell carcinoma (IHC × 200);
d Cervicalnon-keratinizing squamous cell carcinoma (IHC × 200). e Perineural invasion exhibited messy and irregular next to cervical squamous
cell carcinoma lesion (IHC × 200) Fig. 1 Immunohistochemistry (IHC) data showing ADAR1 expression levels in normal cervical, CIN, and cervical squamous cancer tissue specimens. a Normal squamous epithelium (IHC × 200); b Uterine neck CIN III (IHC × 200); c Cervical keratinizing squamous cell carcinoma (IHC × 200);
d Cervicalnon-keratinizing squamous cell carcinoma (IHC × 200). e Perineural invasion exhibited messy and irregular next to cervical squamou
cell carcinoma lesion (IHC × 200) and progression. Leilei et al. [14] found by transcriptome
sequencing that ADAR1 with A-to-I RNA editing might be
a potential driver in the pathogenesis of human cancer, es-
pecially liver cancer. Jochen et al. [15] found that ADAR1
editing for nuclear adenosine of nerve tissue was crucial for
embryonic development of mouse liver. They generated
inducible ADAR1 interference in mice and found that
ADAR1 played an important role in the maintenance of
adult hematopoietic stem cells (HSC) and the inhib-
ition of interferon signaling pathway. The interferon
signaling pathway can protect many pathological pro-
cesses of the body mainly by downregulating the activation
of harmful effect on interferon, avoiding chronic inflam-
mation, autoimmune diseases and cancer [16, 17]. It is
also known that ADAR1 shows different expression levels
in cancer tissues such as laryngeal cancer, bladder cancer,
and hematologic malignancies, as well as different stages
of tumor progression. However, the molecular mecha-
nisms underlying its effects are largely unclear, with
no report linking ADAR1 to cervical squamous cell
carcinoma. we used multivariate COX proportional hazard model,
and it is showed that parametrial invasion for overall sur-
vival of ADAR1 positive cervical squamous cell carcinoma
had an independent prognostic factor with statistical sig-
nificance (Table 2). Discussion ADAR1, also known as RNA editase, has attracted in-
creasing attention in recent years. Athanasiadis et al. [13] found that ADAR1 had anti-viral and anti-tumor
effect, which was due to the fact that ADAR has a Z-
DNA-binding domain, zalpha, differing with other
members of the ADAR family. The Z-DNA-binding do-
main could bind to the CPG sequence with left-handed
helical structure with a high affinity and specificity. Once bound, it is associated with interferon response,
leading to the anti-tumor effect. On the other hand, its
erroneous editing or absence of editing may be closely
associated with the occurrence of tumors. The possible
mechanism may be the alteration of proteins involved in
important pathways, thereby leading to tumor occurrence Chen et al. Discussion Diagnostic Pathology (2017) 12:12 Page 5 of 8 Table 1 One-factor analysis of ADAR1
Characteristics
Case (n = 170)
Negative
Positive
P
Menopause
0.144
Yes
68
16(38.23%)
52(76.47%)
No
102
15(14.71%)
87(85.29%)
Gravidity
0.212
< 3
36
4(11.11%)
32(88.89%)
≥3
134
27(20.15%)
107(79.85%)
Clinical stage
0.224
I
116
24(20.69%)
92(79.31%)
IIA
54
7(12.96%)
47(87.04%)
Tumor diameter
0.000**
≤4 cm
96
27(28.13%)
69(71.88%)
> 4 cm
74
4(5.41%)
70(94.59%)
Keratinization
0.079
Yes
90
12(13.33%)
78(86.67%)
No
80
19(23.75%)
61(76.25%)
Horizontal diffusion diameter
0.000**
≤4 cm
102
28(27.45%)
74(72.55%)
> 4 cm
68
3(4.41%)
65(95.59%)
Stroma invasion
0.612
Superficial stroma
8
2(25.00%)
6(75.00%)
Deep stroma
162
29(17.90%)
133(82.10%)
Vascular invasion
0.008*
Negative
36
12(33.33%)
24(66.67%)
Positive
134
19(14.18%)
115(85.82%)
Lymph node metastasis
0.076
Negative
54
14(25.93%)
40(74.07%)
Positive
116
17(88.73%)
99(85.34%)
Parametrial invasion
0.023*
Negative
126
28(22.22%)
98(77.78%)
Positive
44
3(6.82%)
41(93.18%)
Parametrial margin
0.855
Negative
152
28(18.42%)
124(81.58%)
Positive
18
3(16.67%)
15(83.33%)
Vagina margin
0.001**
Negative
80
23(28.75%)
57(71.25%)
Positive
90
8(88.89%)
82(91.11%)
Histological grade
0.318
G1
30
6(20.00%)
24(80.00%)
G2
52
6(11.54%)
46(88.46%)
G3
88
19(21.59%)
69(78.41%)
HPV infection
0.955
Negative
16
3(18.75%)
13(81.25%) of ADAR1
Case (n = 170)
Negative
Positive
P
0.144
68
16(38.23%)
52(76.47%)
102
15(14.71%)
87(85.29%)
0.212
36
4(11.11%)
32(88.89%)
134
27(20.15%)
107(79.85%)
0.224
116
24(20.69%)
92(79.31%)
54
7(12.96%)
47(87.04%)
0.000**
96
27(28.13%)
69(71.88%)
74
4(5.41%)
70(94.59%)
0.079
90
12(13.33%)
78(86.67%)
80
19(23.75%)
61(76.25%)
0.000**
102
28(27.45%)
74(72.55%)
68
3(4.41%)
65(95.59%)
0.612
8
2(25.00%)
6(75.00%)
162
29(17.90%)
133(82.10%)
0.008*
36
12(33.33%)
24(66.67%)
134
19(14.18%)
115(85.82%)
0.076
54
14(25.93%)
40(74.07%)
116
17(88.73%)
99(85.34%)
0.023*
126
28(22.22%)
98(77.78%)
44
3(6.82%)
41(93.18%)
0.855
152
28(18.42%)
124(81.58%)
18
3(16.67%)
15(83.33%)
0.001**
80
23(28.75%)
57(71.25%)
90
8(88.89%)
82(91.11%)
0.318
30
6(20.00%)
24(80.00%)
52
6(11.54%)
46(88.46%)
88
19(21.59%)
69(78.41%)
0.955
16
3(18.75%)
13(81.25%)
154
28(18.18%)
126(81.82%)
0.011*
31
31(100.00%)
0(0.00%)
139
114(82.01%)
25(17.99%) Case (n = 170)
Negative
Positive
P
0.144
68
16(38.23%)
52(76.47%)
102
15(14.71%)
87(85.29%)
0.212
36
4(11.11%)
32(88.89%)
134
27(20.15%)
107(79.85%)
0.224
116
24(20.69%)
92(79.31%)
54
7(12.96%)
47(87.04%)
0.000**
96
27(28.13%)
69(71.88%)
74
4(5.41%)
70(94.59%)
0.079
90
12(13.33%)
78(86.67%)
80
19(23.75%)
61(76.25%)
0.000**
102
28(27.45%)
74(72.55%)
68
3(4.41%)
65(95.59%)
0.612
8
2(25.00%)
6(75.00%)
162
29(17.90%)
133(82.10%)
0.008*
36
12(33.33%)
24(66.67%)
134
19(14.18%)
115(85.82%)
0.076
54
14(25.93%)
40(74.07%)
116
17(88.73%)
99(85.34%)
0.023*
126
28(22.22%)
98(77.78%)
44
3(6.82%)
41(93.18%)
0.855
152
28(18.42%)
124(81.58%)
18
3(16.67%)
15(83.33%)
0.001**
80
23(28.75%)
57(71.25%)
90
8(88.89%)
82(91.11%)
0.318
30
6(20.00%)
24(80.00%)
52
6(11.54%)
46(88.46%)
88
19(21.59%)
69(78.41%)
0.955
16
3(18.75%)
13(81.25%)
154
28(18.18%)
126(81.82%)
0.011*
31
31(100.00%)
0(0.00%)
139
114(82.01%)
25(17.99%) Chen et al. Discussion Diagnostic Pathology (2017) 12:12 Page 6 of 8 Fig. 2 Overall survival curve of patients with ADAR1 positive and ADAR1 negative cervical squamous cell carcinoma. Survival time of ADAR1
positive group was 38.252 ± 1. 301 month, survival time of ADAR1 negative group was 45.458 ± 0. 863 months. The difference was statistically
significant (χ2 = 5.101, p = 0.024). 0 = ADAR1 negative group; 1 = ADAR1 positive group Fig. 2 Overall survival curve of patients with ADAR1 positive and ADAR1 negative cervical squamous cell carcinoma. Survival time of ADAR1
positive group was 38.252 ± 1. 301 month, survival time of ADAR1 negative group was 45.458 ± 0. 863 months. The difference was statistically
significant (χ2 = 5.101, p = 0.024). 0 = ADAR1 negative group; 1 = ADAR1 positive group Fig. 3 Disease-free survival curve of patients with ADAR1 positive and ADAR1 negative cervical squamous cell carcinoma. Overall disease-free
survival time was 39.048 ± 1.118 months. Disease-free survival time of ADAR1 negative group was 45.458 ± 0.863 months, disease-free survival time
of ADAR1 positive group was 34.877 ± 1.408. The difference was statistically significant (χ2 = 12.925, p < 0.001). 0 = ADAR1 negative group;
1 = ADAR1positive group Fig. 3 Disease-free survival curve of patients with ADAR1 positive and ADAR1 negative cervical squamous cell carcinoma. Overall disease-free
survival time was 39.048 ± 1.118 months. Disease-free survival time of ADAR1 negative group was 45.458 ± 0.863 months, disease-free survival time
of ADAR1 positive group was 34.877 ± 1.408. The difference was statistically significant (χ2 = 12.925, p < 0.001). 0 = ADAR1 negative group;
1 = ADAR1positive group Chen et al. Diagnostic Pathology (2017) 12:12 Page 7 of 8 Page 7 of 8 Table 2 Multivariate analysis of the predictive value of cervical squamous cell carcinoma overall survival for each variable clinical case
Variables
B
S.E. Wald
df
Sig. Associations of ADAR1 with different clinicopathologic
features of cervical squamous cell carcinoma A prospective study on extensive hysterectomy for initial
treatment of stage 1B cervical carcinoma conducted by
the [gynecologic oncology group] GOG revealed that
tumor diameter, invasion depth and vascular invasion
are independent prognostic factors [18]. Except this,as
shown above, we found that horizontal diffusion diam-
eter, parametrial invasion, and vagina involvement were
also significantly associated with ADAR1 expression. Surgery for phase Ib–IIa cervical carcinoma with a
diameter greater than 4 cm is very difficult and prone to
postoperative focal recurrence and distant metastasis
[19]. This is especially true when a large tumor size is
combined with deep myometrial invasion. Based on the
GOG study, it indicates that tumor diameter, invasion
depth and vascular invasion are related to horizontal dif-
fusion diameter, parametrial invasion, and vagina in-
volvement, affecting prognosis. Our findings indicated
an association of ADAR1 with the metastasis, invasion,
and malignancy of cervical squamous cell carcinoma. However, the mechanism is not clear, which needs for
further study. ADAR1 expression in different cervical tissues Previous studies reported that PNI is a risk factor pre-
dicting tumor recurrence and death, and tumor invasion,
whether to nerve trunks or endings, could increase the
risk of recurrence and decrease survival [20–22]. As shown above, we found that ADAR1 was highly
expressed in the cytoplasm and nuclei and its expression
level gradually increased with cervical disease stage. The
close association of ADAR1 with cervical squamous cell
carcinoma, as well as its progress indicates it may play
an oncogenic role in the occurrence and progression of
cervical squamous cell carcinoma. Thus, ADAR1 might
be considered as an oncogene in cervical squamous cell
carcinoma. In this study, we also found that PNI existed in the
foci of ADAR1 positive cervical squamous cell carcin-
oma, indicating the presence of the nerve fibers might
play a role in regulating the progress of cervical squa-
mous cell carcinoma. Furthermore, it was found that
the ADAR1 positive cases were concurrent with PNI
expression, indicating that PNI was associated with
ADAR1 positively. These data suggest that ADAR1 is
an important indicator of prognosis in cervical squamous
cell carcinoma. Discussion Exp(B)
95% CIfor EXP (B)
Lower
Upper
PNI
-.295
.634
.217
1
.641
.744
.215
2.579
Tumor diameter
.407
.454
0820
1
.370
.666
.212
2.969
Horizontal diffusion
-.231
.673
.118
1
.732
.794
1.183
15.581
Vessel invasion
.016
.572
.001
1
.978
1.016
.331
3.115
Parametrial invasion
1.090
.401
7.411
1
.006*
2.976
1.357
6.525
Vagina involvement
-.173
.574
.091
1
.763
.841
.273
2.590
Constant
-.277
1.039
.071
1
.790
.758
*p < 0.05
PNI pathologically diagnostic criteria for perineural invasion
B coefficient of regression
S.E standard error
Wald Wald Chi-Square
df degree of freedom
sig statistically significant
EXP(B) B coefficient index
95% CI for EXP (B) 95% confidence interval
OR odds ratio Author details
1 1Department of Obstetrics and Gynecology, West China Second University
Hospital, Sichuan University, Chengdu 610041, Sichuan, China. 2Department
of Obstetrics and Gynecology, Sichuan Province, Chengdu Women’s and
Children’s Central Hospital, Chengdu 610091, Sichuan, China. 3Department of
Pathology, Sichuan Province, Chengdu Women’s and Children’s Central
Hospital, Chengdu 610091, China. 4Department Physical Examination,
Sichuan People’s Hospital, Chengdu 610072, Sichuan, China. Conclusion 5. Valente L, Nishikura K. ADAR gene family and A-to-I RNA editing: diverse
roles in posttranscriptional gene regulation. Prog Nucleic Acid Res Mol Biol. 2005;79:299–338. Our preliminary study demonstrates the increased ex-
pression of ADAR1 is associated with development and
progress of cervical squamous cell carcinoma. However,
further studies are warranted to investigate its under-
lying molecular mechanisms. 6. Barlati S, Barbon A. RNA editing: a molecular mechanism for the fine
modulation of neuronal transmission. Acta Neurochir Suppl. 2005;93:53–7. 7. Hoopengardner B, Bhalla T, Staber C, Reenan R. Nervous system targets of
RNA editing identified by comparative genomics. Science. 2003;301:832–6. 7. Hoopengardner B, Bhalla T, Staber C, Reenan R. Nervous system targets of
RNA editing identified by comparative genomics. Science. 2003;301:832–6. 8. Bahn JH, Lee JH, Li G, Greer C, Peng G, Xiao X. Accurate identification of
A-to-I RNA editing in human by transcriptome sequencing. Genome Res. 2012;22:142–50. Funding 15. Hartner JC, Walkley CR, Lu J, Orkin SH. ADAR1 is essential for the
maintenance of hematopoiesis and suppression of interferon signaling. Nat
Immunol. 2009;10:109–15. 15. Hartner JC, Walkley CR, Lu J, Orkin SH. ADAR1 is essential for the
maintenance of hematopoiesis and suppression of interferon signaling. Nat
Immunol. 2009;10:109–15. Relationship between ADAR1 and cervical squamous cell
carcinoma prognosis From the overall survival curve, there was difference in
survival rate within 24 months between the ADAR1
positive group and the ADAR1 negative groups, and the
two sets of data tended to balance in 30 months. The
similar results were also found in disease-free survival
curve. From the figure, the recurrence and mortality of
the stage I-IIA patients with cervix squamous cell car-
cinoma in 30 months after surgery were greatly reduced
with stabilized condition. Combined with result of Cox
proportional hazards assessment and the PNI expression
in squamous cell carcinoma, it suggested that ADAR1
expression was not only associated with the metastasis,
invasion, and malignancy of cervical squamous cell
carcinoma but also closely related to the prognosis,
which further confirmed that ADAR1 expression had
guiding significance for prognosis of cervical squamous
cell carcinoma. Chen et al. Diagnostic Pathology (2017) 12:12 Page 8 of 8 Page 8 of 8 Page 8 of 8 Consent for publication
Not applicable. Consent for publication
Not applicable. 20. Memarzadeh S, Natarajan S, Dandade DP, Ostrzega N, Saber PA, Busuttil A, et
al. Lymphovascular and perineural invasion in the parametria: a prognostic
factor for early-stage cervical cancer. Obstet Gynecol. 2003;102:612–9. 20. Memarzadeh S, Natarajan S, Dandade DP, Ostrzega N, Saber PA, Busuttil A, et
al. Lymphovascular and perineural invasion in the parametria: a prognostic
factor for early-stage cervical cancer. Obstet Gynecol. 2003;102:612–9. Availability of data and materials All data generated or analysed during this study are included in the
Additional file 1. 16. Martinez HD, Jasavala RJ, Hinkson I, Fitzgerald LD, Trimmer JS, Kung HJ, et
al. RNA editing of androgen receptor gene transcripts in prostate cancer
cells. J Biol Chem. 2008;283:29938–49. Ethics approval and consent to participate
Th
d
l
d b
h E hi The study protocol was approved by the Ethics Committees of the Sichuan
Province, Chengdu Women’s and Children’s Central Hospital (No. 2015120018). All participants provided written informed consent. 21. Horn LC, Meinel A, Fischer U, Bilek K, Hentschel B. Perineural invasion in
carcinoma of the cervix uteri–prognostic impact. J Cancer Res Clin Oncol. 2010;136:1557–62. 21. Horn LC, Meinel A, Fischer U, Bilek K, Hentschel B. Perineural invasion in
carcinoma of the cervix uteri–prognostic impact. J Cancer Res Clin Oncol. 2010;136:1557–62. 22. Zhang G, Yang Y, Zhu Y, Cui L, Jia S, Shi Y, et al. Evidence of perineural
invasion on early-stage cervical cancer and prognostic significance. Zhonghua Fu Chan Ke Za Zhi. 2015;50:673–8. 22. Zhang G, Yang Y, Zhu Y, Cui L, Jia S, Shi Y, et al. Evidence of perineural
invasion on early-stage cervical cancer and prognostic significance. Zhonghua Fu Chan Ke Za Zhi. 2015;50:673–8. Additional file 9. Shah SP, Morin RD, Khattra J, Prentice L, Pugh T, Burleigh A, et al. Mutational evolution in a lobular breast tumour profiled at single
nucleotide resolution. Nature. 2009;461:809–13. Additional file 1: Original data of baseline, clinical pathological
diagnosis, prognosis and follow-up for ADAR1 patients. (DOC 825 kb) 10. Jiang Q, Crews LA, Barrett CL, Chun HJ, Court AC, Isquith JM, et al. ADAR1
promotes malignant progenitor reprogramming in chronic myeloid
leukemia. Proc Natl Acad Sci U S A. 2013;110:1041–6. Acknowledgements Acknowledgements
None. Acknowledgements
None. 13. Athanasiadis A. Zalpha-domains: at the intersection between RNA editing
and innate immunity. Semin Cell Dev Biol. 2012;23:275–80. 14. Chen L, Li Y, Lin CH, Chan TH, Chow RK, Song Y, et al. Recoding RNA
editing of AZIN1 predisposes to hepatocellular carcinoma. Nat Med. 2013;19:209–16. Received: 23 September 2016 Accepted: 12 January 2017 Received: 23 September 2016 Accepted: 12 January 2017 • We accept pre-submission inquiries
• Our selector tool helps you to find the most relevant journal
• We provide round the clock customer support
• Convenient online submission
• Thorough peer review
• Inclusion in PubMed and all major indexing services
• Maximum visibility for your research
Submit your manuscript at
www.biomedcentral.com/submit
p y
y
p • We accept pre-submission inquiries References
1. Schiffman M, Castle PE, Jeronimo J, Rodriguez AC, Wacholder S. Human
papillomavirus and cervical cancer. Lancet. 2007;370:890–907. 2. Reenan RA. Molecular determinants and guided evolution of species-specific
RNA editing. Nature. 2005;434:409–13. 3. Keller W, Wolf J, Gerber A. Editing of messenger RNA precursors and of
tRNAs by adenosine to inosine conversion. FEBS Lett. 1999;452:71–6. 4. Maas S, Rich A. Changing genetic information through RNA editing. Bioessays. 2000;22:790–802. Authors’ contributions YC carried out the molecular genetic studies, participated in the sequence
alignment and drafted the manuscript. WL carried out the immunoassays
and participated in the sequence alignment. PS participated in the design of
the study and performed the statistical analysis. HW conceived of the study,
and participated in its design and coordination and helped to draft the
manuscript. All authors have read and approved the final manuscript. 17. Galeano F, Leroy A, Rossetti C, Gromova I, Gautier P, Keegan LP, et al. Human BLCAP transcript: new editing events in normal and cancerous
tissues. Int J Cancer. 2010;127:127–37. 18. Delgado G, Bundy B, Zaino R, Sevin BU, Creasman WT, Major F. Prospective
surgical-pathological study of disease-free interval in patients with stage IB
squamous cell carcinoma of the cervix: a Gynecologic Oncology Group
study. Gynecol Oncol. 1990;38:352–7. Competing interests 19. Sedlis A, Bundy BN, Rotman MZ, Lentz SS, Muderspach LI, Zaino RJ. A
randomized trial of pelvic radiation therapy versus no further therapy in
selected patients with stage IB carcinoma of the cervix after radical
hysterectomy and pelvic lymphadenectomy: A Gynecologic Oncology
Group Study. Gynecol Oncol. 1999;73:177–83. Competing interests
The authors declare that they have no competing interests. The authors declare that they have no competing interests. Submit your next manuscript to BioMed Central
and we will help you at every step: • We accept pre-submission inquiries
• Our selector tool helps you to find the most relevant journal
• We provide round the clock customer support
• Convenient online submission
• Thorough peer review
• Inclusion in PubMed and all major indexing services
• Maximum visibility for your research
Submit your manuscript at
www.biomedcentral.com/submit
Submit your next manuscript to BioMed Central
and we will help you at every step: References
1.
Schiffman M, Castle PE, Jeronimo J, Rodriguez AC, Wacholder S. Human
papillomavirus and cervical cancer. Lancet. 2007;370:890–907.
2.
Reenan RA. Molecular determinants and guided evolution of species-specific
RNA editing. Nature. 2005;434:409–13.
3.
Keller W, Wolf J, Gerber A. Editing of messenger RNA precursors and of
tRNAs by adenosine to inosine conversion. FEBS Lett. 1999;452:71–6.
4.
Maas S, Rich A. Changing genetic information through RNA editing.
Bioessays. 2000;22:790–802. Abbreviations 11. Yu WF, Zhao YL, Wang K. Expression and significance of RNA editase 1
mRNA in laryngeal carcinoma and paracancerous tissues. J Clin Otorhinolar
Head Neck Sur. 2008;22:73–5. ADAR1: Adenosine deaminase; CIN: Cervical intraepithelial neoplasia;
HSC: Hematopoietic stem cells 12. Liebig C, Ayala G, Wilks JA, Berger DH, Albo D. Perineural invasion in cancer:
a review of the literature. Cancer. 2009;115:3379–91.
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Professor Dr. Erhard Kaliweit 65 Jahre
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Professor Dr. Erhard Kaliweit
65 Jahre
Erhard Kallweit hat seine Wurzeln in Ostpreußen, wo er am 4. Mai 1936 in Neu Lappienen
geboren wurde. Die Kriegsereignisse brachten ihn als Neunjährigen nach Niedersachsen, wo
er bis heute zu Hause ist. Das „Pferdeland" hat sicherlich die Ausübung seiner Hobbies, Pferdezucht und Reiten, die er in seinem Domizil in Evensen (Neustadt am Rübenberge) pflegt,
beflügelt. Nicht alle Menschen können ihr Hobby so intensiv in die beruflichen Aktivitäten
einbringen, wie dies Professor Kallweit vor allem in den letzten Jahren getan hat. In seiner
langjährigen Wirkungsstätte, dem Institut für Tierzucht und Tierverhalten Mariensee, konnte
er seine Liebe zum Pferd mit wissenschaftlichen Untersuchungen zu Problemen der Trainings- und Turnierbelastung und zum Trainingsmanagement bei Sportpferden sehr erfolgreich
verknüpfen. Hierzu kann man, in Verbindung mit dem 65. Geburtstag, herzlichst gratulieren.
Damit ist aber nur ein Teil der wissenschaftlichen Verdienste von Erhard Kallweit beleuchtet.
Nach landwirtschaftlicher Lehre in den Kreisen Rotenburg und Verden / Aller und dem Studium der Landwirtschaft an der Universität Göttingen legte er dort 1962 die Diplomprüfung
ab. Als Mitarbeiter von Prof. Fritz Haring und unter Betreuung von Prof. Dr. J. Weniger fertigte er im Institut für Tierzucht und Haustiergenetik eine Dissertation an zum Thema:
„Untersuchungen über Beziehungen zwischen Körperzusammensetzung und
Fleischbeschaffenheit an wachsenden Schweinen".
Dem darin zum Ausdruck kommenden Bemühen, Grundlagen für die wirtschaftliche Erzeugung qualitativ hochwertiger Produkte für die Verbraucher zu erarbeiten, ist Professor Kallweit bis heute verbunden geblieben. Die mit der Promotion zum Dr.sc.agr. im Jahre 1964 gelegte Basis für sein wissenschaftliches Engagement wurde zunächst vertieft durch die Arbeit
als Kellogg - Stipendiat in Madison an der University of Wisconsin, USA, in den Jahren
1964/65. Im Anschluss daran konnte Prof. Max Witt ihn für das damalige Max - Planck - Institut für Tierzucht und Tieremährung Mariensee, gewinnen, in dem er vielfältige Arbeiten
zur Schlachtkörper- und Fleischbeschaffenheit bei landwirtschaftlichen Nutztieren durchführte. Immer in der vordersten Linie wissenschaftlicher Entwicklungen, hat der Jubilar maßgeblichen Anteil an der Bewertung, Verbesserung und Sicherung der Fleischqualität in ihren
vielseitigen Beurteilungsfacetten. Dieses Gebiet war daher auch Inhalt seiner Habilitationsschrift „Beziehungen zwischen Mast- und Schlachtleistung und Merkmalen der Fleischbeschaffenheit bei verschiedenen Tierarten". Dort sind die Ergebnisse eingehender vergleichender Untersuchungen dargelegt. .
118
Prof. Dr. Erhard Kallweit 65 Jahre
Es war für die wissenschaftliche Kontinuität sehr wichtig, dass Erhard Kallweit seine Arbeiten auch nach der Überführung des Max - Planck - Instituts in das Institut für Tierzucht und
Tierverhalten der Bundesforschungsanstalt für Landwirtschaft in gleicher Richtung weiter
entwickeln konnte. Unter Erschließung neuer apparativer Messmöglichkeiten einschließlich
der Magnet - Resonanz - Tomographie wurden wesentliche Erkenntnisse gewonnen zu Phänomenen des Wachstums, der Körperzusammensetzung und der Gewebebeschaffenheit bei
Nutztieren in vivo. Besondere Aufmerksamkeit fanden dabei die Einflussfaktoren während
des Erzeugungsprozesses. Stets wurde den praktischen Auswirkungen aller Maßnahmen im
Rahmen von Zuchtwertschätzung und Schlachtkörperbewertung besonderes Gewicht beigemessen.
Ein wesentliches Kennzeichen des wissenschaftlichen Engagements von Professor Kallweit
war stets die institutionsübergreifende und internationale Kooperation. Als besonders nachhaltig wirksames Beispiel ist seine langjährige Zusammenarbeit mit dem Max - Planck - Institut für experimentelle Endokrinologie in Hannover und dessen Direktor, Professor Peter
Jungblut, zu nennen.
Die Ergebnisse seiner wissenschaftlichen Arbeit sind in vielen einschlägigen Veröffentlichungen im deutschen und internationalen Schrifttum sowie in verschiedenen Buchbeiträgen
dokumentiert. Besonders hervorzuheben ist hier das 1988 gemeinsam mit weiteren Autoren
herausgegebene Buch „Qualität tierischer Nahrungsmittel".
Aufgrund seines wissenschaftlichen Engagements war der Jubilar in einer Reihe wichtiger
Gremien tätig, die sich mit Fragen der Qualitätserzeugung von Fleisch sowie dessen Verarbeitung und Vermarktung befassen. So war er z.B. von 1982 bis 1999 Vorsitzender des Arbeitsausschusses „Fleischerzeugung" der Deutschen Gesellschaft für Züchtungskunde und ist
seit 1989 Mitglied einer CMA - Arbeitsgruppe zur Erarbeitung eines Kriterienkatalogs für
„Qualitätsfleisch". Als Mitglied des ALZ (Ausschuss für Leistungsprüfung und Zuchtwertschätzung beim Schwein) hielt und hält er nach wie vor Kontakte zur züchterischen Praxis.
Genannt sei auch sein langjähriges Wirken im Redaktionskollegium des „Archiv für Tierzucht".
Professor Kallweit hat stets das von ihm erarbeitete Wissen der Öffentlichkeit zugänglich
gemacht, unter anderem in umfangreicher Vortragstätigkeit und in langjährigem Engagement
in der akademischen Lehre an der Universität Göttingen. Er habilitierte sich dort 1972 an der
Landwirtschaftlichen Fakultät und wurde 1975 zum apl. Professor ernannt. 1978 nahm er eine
Lehrstuhlvertretung am Institut für Tierzucht und Haustiergenetik an der Universität Göttingen wahr. Er war stellvertretender Institutsleiter am Institut für Tierzucht und Tierverhalten
Mariensee und Mitglied des Senats der Bundesforschungsanstalt für Landwirtschaft (FAL).
1977 wurde er zum wissenschaftlichen Direktor ernannt. Die von Professor Kallweit im Institut für Tierzucht und Tierverhalten wahrgenommenen wissenschaftlichen und administrativen Aufgaben, z.B. die Leitung der Arbeitsgruppen Schaf- und Schweineproduktion, In vivo Analyse biologischer Systeme, Prozessqualität, haben wesentlich zum Ansehen des Instituts
beigetragen und solide Fundamente für eine erfolgreiche Weiterführung der Aufgabenbereiche gelegt.
Für den beginnenden Ruhestand wünschen wir dem Jubilar weiter eine schöne Zeit an der
Seite seiner Ehefrau Lore, eine bekömmliche Mischung aus Muße und verbleibender beruflicher Aktivität und weiterhin viel Freude im Kreis seiner Familie und Freunde und an seinem
Hobby namens Pferd.
Ad multos annos!
Diedrich Smidt, Frielingen und Martina Henning, Mariensee
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УДК 640.4(477.81)
DOI: 10.31866/2616-7468.3.1.2020.205574 УДК 640.4(477.81)
DOI: 10.31866/2616-7468.3.1.2020.205574 УДК 640.4(477.81)
DOI: 10.31866/2616-7468.3.1.2020.205574 СУЧАСНІ ТЕНДЕНЦІЇ
РОЗВИТКУ
ГОТЕЛЬНО-
РЕСТОРАННОГО
БІЗНЕСУ
РЕКРЕАЦІЙНОГО
РАЙОНУ Микола Ляшук,
старший викладач,
Східноєвропейський національний
університет імені Лесі Українки,
Луцьк, Україна,
nicklyashuk@i.ua
https://orcid.org/0000-0002-4993-0191
© Ляшук М. М., 2020 Ірина Єрко,
кандидат географічних наук,
Східноєвропейський національний
університет імені Лесі Українки,
Луцьк, Україна,
ierko@i.ua
https://orcid.org/0000-0002-5395-7557
© Єрко І. В., 2020 Актуальність. Дослідження готельно-ресторанного господарства (ГРГ), складової ча-
стини сфери рекреації, є актуальним, оскільки наявність існуючої мережі закладів ГРГ на
певній території є важливим фактором, необхідним для визначення шляхів подальшого
розвитку, удосконалення організації та функціонування. Мета статті – аналіз функціону-
вання закладів готельно-ресторанного господарства Дубенського рекреаційного району
України. Методи дослідження. При проведенні дослідження використовувались загаль-
нонаукові методи: аналізу, синтезу, порівняння та статистичний. Результати досліджен-
ня. Проаналізовано сучасний стан готельно-ресторанного господарства Дубенського ре-
креаційного району. Виявлено місця найбільшої та найменшої дислокації підприємств
готельно-ресторанного господарства у районі. Досліджено територіальну концентрацію
та рівень розвитку підприємств готельно-ресторанного господарства Дубенського рекре-
аційного району шляхом розрахунку індексу територіальної концентрації та індексу ло-
калізації. Охарактеризовано перспективи розвитку закладів ГРГ досліджуваного району. Висновки та обговорення. Дослідження ГРГ Дубенського рекреаційного району мало на
меті показати територіальні відмінності у розміщенні та рівні розвитку ГРГ району. Най-
кращими показниками розвитку ГРГ характеризуються Демидівський адміністративний
район та м. Дубно, найгіршими – Млинівський адміністративний район. Ключові слова: готельно-ресторанне господарство, рекреаційний район, готель, рес-
торан, індекс територіальної концентрації, індекс локалізації. Ресторанний і готельний консалтинг. Інновації. 2020 Том 3 № 1
Restaurant and hotel consulting. Innovations. 2020 Vol. 3 No 1 Ресторанний і готельний консалтинг. Інновації. 2020 Том 3 № 1
Restaurant and hotel consulting. Innovations. 2020 Vol. 3 No 1 Actual problems of hotel and restaurant business development
Актуальні проблеми розвитку готельно-ресторанного бізнесу (ГРГ), складової частини сфери рекреації, є актуальним, оскільки наявність існуючої
мережі закладів ГРГ на певній території є важливим фактором, необхідним для ви-
значення шляхів подальшого розвитку, удосконалення організації та функціонуван-
ня рекреаційної сфери. Сучасний стан ГРГ слід аналізувати з огляду його просторово-
го розміщення, враховуючи типи підприємств та ряд інших факторів. Це дає підстави
розглядати ГРГ з огляду економіки та рекреації. Аналіз ГРГ Дубенського рекреацій-
ного району дозволить детально розкрити напрями подальшого розвитку та функ-
ціонування ГРГ як компонента туристсько-рекреаційної інфраструктури Поліського
рекреаційного регіону. р
р
р
у
Стан вивчення проблеми. Сучасним тенденціям розвитку готельно-ресторанно-
го господарства України присвятили праці вітчизняні науковці П Курмаєв (2015),
С. Мазур, А. Прилуцький (2019), І. Мельник (2016). Аналізу стану розвитку ГРГ Полісь-
кого рекреаційного регіону з боку територіальної диференціації в межах адміністра-
тивних областей присвятили праці Н. Алєшугіна, О. Зеленська (2012), І. Єрко (2016),
Н. Кравченко (2007), М. Ляшук (2014), О. Романів, Т. Трусова, Р. Бричка (2015). Невирішені питання. Систематизований аналіз сучасного стану ГРГ у межах
Дубенського рекреаційного району Поліського рекреаційного регіону авторами
здійснюється уперше, саме це і визначає актуальність дослідження. Актуальність проблеми Постановка проблеми. Рекреаційні ресурси можна використати з метою відпо-
чинку, туризму та оздоровлення. Адже споживачами їх є не тільки туристи, а й місце-
ве населення. у структурі рекреаційних ресурсів досліджуваної території чітко виді-
ляються природні та соціально-економічні. Вони зосереджені у зонах транспортної
доступності відносно міських поселень. Для кращого використання рекреаційних
ресурсів необхідно мати розвинену інфраструктуру, одними зі складових якої є за-
клади розміщення та харчування. Дослідження готельно-ресторанного господарства 109 Результати дослідження Готельно-ресторанне господарство є складовою частиною рекреаційно-ту-
ристичної галузі України. Готельні підприємства забезпечують подорожуючих
житлом, а ресторанні заклади – поєднують різні форми організації виробництва,
продажу та споживання кулінарної продукції, послуг. Тобто ГРГ притаманні усі
характеристики рекреаційних послуг. У 2007 році Н. О. Кравченко (2007) запропонувала районування території По-
лісся за рівнем розвитку рекреаційного господарства. До складу Поліського регі-
ону входять Волинська, Житомирська, Рівненська, Київська, Чернігівська області
та м. Київ. Поліський рекреаційний регіон поділяється на чотири мезорегіони: Півден-
но-Поліський, Північно-Поліський, Східно-Поліський та Центрально-Поліський
(Кравченко, 2007). Загальна площа регіону становить 130,858 тис. км², населення–
9,08 млн чол. (21,7 % території та 19 % населення України). Дубенський рекреаційний район входить до складу Південно-Поліського ре-
креаційного мезорегіону. Загальна площа району становить 3,3 тис. км², а насе-
лення – 171 тис. чол. Факторами для розвитку ГРГ у районі є: природний потенціал; історико-куль-
турний потенціал; економічний потенціал. Факторами для розвитку ГРГ у районі є: природний потенціал; історико-куль-
турний потенціал; економічний потенціал. У 2019 р. на території Дубенського рекреаційного району функціонувало 243
заклади ГРГ (тоді як у 2018 р. – 245 од.), із них 208 – заклади ресторанного госпо-
дарства (ЗРГ) (у 2018 р. – 210 од.), 35 – заклади готельного господарства (ЗГГ) (табл. 1) ("Дубенська районна державна адміністрація", 2019). З наведених даних видно, що найбільша кількість ЗРГ зосереджена у м. Дубно
(32 %) та Дубенському районі (24 %), найменша – у Млинівському (13 %). Готельне
господарство краще розвинуте у Демидівському (51 %) та Дубенському (29 %) ад-
міністративних районах, гірше – у Млинівському (3 %). З наведених даних видно, що найбільша кількість ЗРГ зосереджена у м. Дубно
(32 %) та Дубенському районі (24 %), найменша – у Млинівському (13 %). Готельне
господарство краще розвинуте у Демидівському (51 %) та Дубенському (29 %) ад-
міністративних районах, гірше – у Млинівському (3 %). У 2018–2019 рр. змін зазнали ЗРГ, оскільки їх значно більше, ніж ЗГГ. Готельно-ресторанне господарство Дубенського рекреаційного району, 2019 р. Табл. 1. Готельно-ресторанне господарство Дубенського рекреаційного району, 2019 р. Tabl. 1. The hotel and restaurant industry of the Dubensky recreation district, 2019
№
з/п
Адміністративно-
територіальна одиниця
ГРГ
к-ть,
од. %
у тому числі
ЗРГ
ЗГГ
к-ть,
од. %
к-ть,
од. %
1
м. Ресторанний і готельний консалтинг. Інновації. 2020 Том 3 № 1
Restaurant and hotel consulting. Innovations. 2020 Vol. 3 No 1 Ресторанний і готельний консалтинг. Інновації. 2020 Том 3 № 1
Restaurant and hotel consulting. Innovations. 2020 Vol. 3 No 1 Мета і методи дослідження Метою статті є аналіз та функціонування закладів готельно-ресторанного
господарства Дубенського рекреаційного району України. Для досягнення цієї
мети розв’язувались такі завдання: аналіз діяльності закладів ГРГ рекреаційного
району; дослідження територіального розміщення закладів ГРГ; дослідження те-
риторіальної концентрації та рівня розвитку підприємств готельно-ресторанно-
го господарства Дубенського рекреаційного району шляхом розрахунку індексу
територіальної концентрації та індексу локалізації. Об’єктом дослідження є діяльність закладів готельно-ресторанного господар-
ства Дубенського рекреаційного району. Предметом дослідження є компонентний та територіальний аспекти дослі-
дження закладів готельно-ресторанного господарства Дубенського рекреаційно-
го району. Наукова новизна дослідження полягає у наступному: вперше на основі струк-
турного аналізу досліджені компонентний та територіальний аспекти ГРГ Дубен-
ського рекреаційного району; удосконалено визначення ролі закладів ГРГ рекреа-
ційного району; набули подальшого розвитку рекомендації щодо вдосконалення
розвитку закладів ГРГ Дубенського рекреаційного району. Методи дослідження. При проведенні дослідження використовувались загаль-
нонаукові методи: аналізу – передбачає аналіз чинників формування та терито-
ріальну організацію закладів ГРГ; синтезу – об’єднання окремих характеристик в
одне ціле; порівняння – зіставлення отриманих результатів дослідження діяльно-
сті закладів ГРГ; статистичний – вивчення та узагальнення тенденцій функціону-
вання закладів ГРГ Дубенського рекреаційного району. Ці методи ми застосовува-
ли на всіх стадіях дослідження. Інформаційною базою дослідження є монографії та наукові статті вітчизняних
науковців. Для виконання поставлених завдань здійснено комплексний аналіз
даних статистичної звітності Головного управління статистики у Рівненській об-
ласті та Рівненської облдержадміністрації. 110 Результати дослідження Дубно
70
29
66
32
4
11
2
Дубенський р-н
59
24
49
24
10
29
3
Демидівський р-н
47
19
29
14
18
51
4
Млинівський р-н
29
12
28
13
1
3
5
Радивилівський р-н
38
16
36
17
2
6
Разом по району
243
100
208
100
35
100
Джерело: Рівненська облдержадміністрація
Source: Rivne Regional State Administration 1. The hotel and restaurant industry of the Dubensky recreation district, 2019 111 Actual problems of hotel and restaurant business development
Актуальні проблеми розвитку готельно-ресторанного бізнесу Так, зростання кількості ЗРГ зафіксовано у м. Дубно та Дубенському адміні-
стративному районі (збільшились на 10 % та 26 % відповідно). у решті адміністра-
тивних районів зафіксована негативна динаміка (Млинівський та Радивилівський
адміністративні райони зменшились на 32 % та 10 % відповідно). Причиною та-
ких змін є відтік сільського населення на роботу за кордон або у міську місцевість. У загальній структурі рекреаційного району ГРГ найбільше зосереджено у м. Дубно (29 %), найменше – у Млинівському районі (12 %) (рис. 1). На решті терито-
рії ГРГ зосереджено майже порівну. Рис. 1. Структура готельно-ресторанного господарства
Дубенського рекреаційного району, 2019 р. Джерело: власна розробка
Fig. 1. The hotel and restaurant industry structure of the Dubensky recreation district, 2019
Source: own development Рис. 1. Структура готельно-ресторанного господарства
Дубенського рекреаційного району, 2019 р. Джерело: власна розробка Результати аналізу показали диспропорцію у дислокації підприємств ГРГ
у сільських і міських поселеннях. Готелі, мотелі, ресторани, бари, кафе зосередже-
ні у міських поселеннях. у сільській місцевості переважають мотелі, санаторії, ту-
ристичні бази та бази відпочинку, дитячі табори, їдальні, бари, кафе, закусочні. Саме тому у Дубенському рекреаційному районі виділяються чітко сформовані
Дубенський, Радивилівський, Млинівський, Смизький, Хрінниківський рекреа-
ційні вузли. Диференціація соціально-економічних рівнів життя міського та сільського
населення рекреаційного району загрожує відпливом молодого працездатного
сільського населення до міст, що веде до занепаду сільських населених пунктів. Цей чинник впливає на ЗРГ, розвиток яких істотно відрізняється у сільській та
міській місцевостях. у міській місцевості рівень забезпеченості ЗРГ вищий, ніж
у сільській. Аналіз статистичних даних свідчить про деформовану структуру роз-
міщення закладів ГРГ, наприклад: ресторани та готелі найбільш зосереджені у мі-
стах, у селах – кафе, бари, їдальні, туристичні бази. у
у
Серед загальної структури закладів ресторанного господарства у Дубенському
рекреаційному районі переважають кафе (38 %) та бари (28 %) (рис. 2). 112 Ресторанний і готельний консалтинг. Інновації. 2020 Том 3 № 1
Restaurant and hotel consulting. Innovations. 2020 Vol. 3 No 1 Ресторанний і готельний консалтинг. Інновації. 2020 Том 3 № 1
Restaurant and hotel consulting. Innovations. 2020 Vol. 3 No 1 Ресторанний і готельний консалтинг. Інновації. 2020 Том 3 № 1
Restaurant and hotel consulting. Innovations. 2020 Vol. 3 No 1 Рис. 2. Структура закладів ресторанного господарства
Дубенського рекреаційного району за типами, 2019 р. Джерело: власна розробка
Fig. 2. The restaurant industry structure of the Dubensky recreation district by type, 2019
Source: own development Рис. 2. Структура закладів ресторанного господарства
Дубенського рекреаційного району за типами, 2019 р. Джерело: власна розробка Рис. 2. Структура закладів ресторанного господарства
Дубенського рекреаційного району за типами, 2019 р. Джерело: власна розробка Fig. 2. The restaurant industry structure of the Dubensky recreation district by type, 2019
Source: own development Найменша кількість припадає на ресторани, їдальні та буфети (по 3 % відпо-
відно). Найбільша кількість кафе зосереджена у сільській місцевості, тоді як бари
– у міській. Серед закладів готельного господарства найбільшу частку у рекреацій-
ному районі посідають туристичні бази та бази відпочинку (49 %), які зосереджено
у Хрінниківському рекреаційному вузлі, та мотелі (34 %). Дитячі табори (3 %) та
санаторії, профілакторії, пансіонати (3 %) мають слабкорозвинуту мережу (рис. 3). Рис. 3. Структура закладів готельного господарства
Дубенського рекреаційного району за типами у 2019 р. Джерело: власна розробка
Fig. 3. The hotel industry structure of the Dubensky recreation district by type, 2019
Source: own development Рис. 3. Структура закладів готельного господарства
Дубенського рекреаційного району за типами у 2019 р. Джерело: власна розробка Fig. 3. The hotel industry structure of the Dubensky recreation district by type, 2019
Source: own development 113 Ресторанний і готельний консалтинг. Інновації. 2020 Том 3 № 1
Restaurant and hotel consulting. Innovations. 2020 Vol. 3 No 1 Ресторанний і готельний консалтинг. Інновації. 2020 Том 3 №
Restaurant and hotel consulting. Innovations. 2020 Vol. 3 No 1 Restaurant and hotel consulting. Innovations. 2020 Vol. 3 No 1 Про достатнє використання потенціалу ГРГ району може свідчити Іл > 1, коли
на адміністративний район припадає частка, більше пропорційної частки даного
рекреаційного району. Про недостатнє використання потенціалу ГРГ буде свідчи-
ти Іл < 1. л
Іл дозволив виявити відмінності рівня розвитку ГРГ в адміністративних райо-
нах рекреаційного району. Дослідження Іл показало найбільший рівень розвитку
ГРГ у м. Дубно (1,3) та Демидівському адміністративному районі (2,31), наймен-
ший – у Млинівському адміністративному районі (0,55). Рівень розвитку ГРГ по
досліджуваному рекреаційному району є достатнім. Результати дослідження ГРГ Дубенського рекреаційного району дають підстави
запропонувати шляхи подальшого розвитку з метою мінімізації впливу негатив-
них факторів на розвиток рекреаційної сфери («Стратегія сталого розвитку», 2017): них факторів на розвиток рекреаційної сфери («Стратегія сталого розвитку», 2017):
−−
активізація використання інструментів адміністративного менеджменту
та маркетингу; −−
активізація використання інструментів адміністративного менеджменту
та маркетингу; −−
залучення інвестицій для розвитку місцевої промисловості, окультурення
та створення зон відпочинку; р
у
−−
збільшення робочих місць у сфері малого та середнього бізнесу; −−
створення закладів дозвілля та відпочинку у досліджуваному рекреацій-
ному районі для популяризації здорового способу життя; −−
сприяння залученню іноземних інвестицій для розвитку Дубенського ре-
креаційного району, в тому числі через активізацію інформаційної присутності
інвестиційних можливостей м. Дубно та м. Рівне на ресурсах мережі інтернет; −−
розвиток туризму на основі історико-культурної спадщини та маркетин-
гові кампанії щодо залучення інвестицій у туристичну та супутню інфраструктуру
рекреаційного району; −−
використання енергозберігаючих технологій, а також перехід на альтер-
нативні та відновлювальні джерела енергії; −−
оптимізація системи підготовки кадрів та системи працевлаштування,
зокрема, через соціальні угоди для зменшення рівня зовнішньої міграції насе-
лення, в тому числі відтоку молодого працездатного населення за кордон; −−
активізація пошуку інвестиційних партнерів для створення нових робо-
чих місць у рекреаційній сфері, зокрема, туризму та готельно-ресторанної інду-
стрії. Саме тому реалізація перерахованих вище заходів допоможе уникнути на-
гальних проблем, використати можливості та якомога цілісніше розвивати
Дубенський рекреаційний район. Actual problems of hotel and restaurant business development
Актуальні проблеми розвитку готельно-ресторанного бізнесу В результаті аналізу встановлено, що на території досліджуваного рекреа-
ційного району з’явилися і комбіновані заклади ресторанного господарства, що
зумовлено відповідними пріоритетами споживчого попиту: удень – кафе, увече-
рі – дискоклуб, що дає можливість подовжити робочий час підприємства. Серед
закладів готельного господарства переважають сезонні підприємства. Для подальшого розвитку, зважаючи на сучасні економічні реалії, закладам
ГРГ району необхідно: враховувати потреби споживачів; систематично аналізу-
вати діяльність конкурентів; враховувати зміни в законодавстві та інфляційні
процеси; проводити активну рекламно-інформаційну діяльність; розробити єди-
ний перелік закладів ГРГ та ін. Крім того, досліджено, як спеціалізуються певні адміністративні райони на
наданні послуг розміщення та харчування, визначено кількісну територіальну
диференціацію, що є основою для подальшого аналізу територіальної органі-
зації ГРГ. Для цього було розраховано індекси територіальної концентрації (Ітк)
та локалізації (Іл) закладів ГРГ Дубенського рекреаційного району (табл. 2). Дані
індекси дозволяють з’ясувати як загальні (розташування, можливість збільшен-
ня та зменшення кількості), так і специфічні (локальні) характеристики закладів
ГРГ. Для дослідження територіальної концентрації закладів обслуговування було
обраховано індекс територіальної концентрації (Ітк) та індекс локалізації (Іл) ГРГ
у розрізі адміністративних районів Дубенського рекреаційного району. Це дасть
можливість дослідити повноту і збалансованість розміщення закладів ГРГ на те-
риторії досліджуваного району (Нємець та ін., 2008). Табл. 2. Індекси територіальної концентрації (Ітк) та локалізації (Іл) підприємств ГРГ
Дубенського рекреаційного району, 2019 р. Табл. 2. Індекси територіальної концентрації (Ітк) та локалізації (Іл) підприємств ГРГ
Дубенського рекреаційного району, 2019 р. №
з/п
Адміністративно-територіальна одиниця
Індекси
територіальної
концентрації
(Ітк)
локалізації (Іл)
1
м. Дубно
35,2
1,3
2
Дубенський р-н
0,67
0,93
3
Демидівський р-н
1,68
2,31
4
Млинівський р-н
0,42
0,55
5
Радивилівський р-н
0,69
0,73
Джерело: власна розробка
Source: own development Ітк показує концентрацію закладів ГРГ на певній території. Якщо Ітк > 1, це
свідчить про оптимальну концентрацію закладів ГРГ на досліджуваній території. Якщо навпаки, Ітк < 1, це недостатнє розташування закладів ГРГ у адміністратив-
ному районі. Аналіз Ітк показав високу концентрацію ГРГ у м. Дубно (35,2) і Де-
мидівському адміністративному районі (1,68) та низьку у Млинівському адміні-
стративному районі (0,42). Загалом по Дубенському рекреаційному району Ітк ГРГ
є високим. 114 Ресторанний і готельний консалтинг. Інновації. 2020 Том 3 № 1
Restaurant and hotel consulting. Innovations. 2020 Vol. 3 No 1 Висновки та обговорення результатів Дослідження ГРГ Дубенського рекреаційного району мало на меті показати те-
риторіальні відмінності у розміщенні та рівні розвитку ГРГ району. Використан-
ня різних методик дослідження ГРГ дало можливість виявити території з різною
концентрацією та рівнем розвитку ГРГ. Найкращими показниками розвитку ГРГ
характеризуються Демидівський адміністративний район та м. Дубно, найгірши-
ми – Млинівський адміністративний район. Досліджено, що у 2019 р. на території
Дубенського рекреаційного району функціонувало 243 заклади ГРГ, із них 208 –
заклади ресторанного господарства (ЗРГ), 35 – заклади готельного господарства 115 Actual problems of hotel and restaurant business development (ЗГГ). Серед загальної структури закладів ресторанного господарства у Дубен-
ському рекреаційному районі переважають кафе (37,5 %) та бари (28,4 %). (ЗГГ). Серед загальної структури закладів ресторанного господарства у Дубен-
ському рекреаційному районі переважають кафе (37,5 %) та бари (28,4 %). Для подальшого розвитку, зважаючи на сучасні економічні реалії, ГРГ району
необхідно: враховувати потреби споживачів, а саме визначити цільову аудиторію
за методикою 5W; систематично аналізувати діяльність конкурентів на основі
аналізу основних чинників, таких як якість продукції та послуг, кваліфікований
персонал, ціна та інше; проводити активну рекламно-інформаційну діяльність,
висвітлюючи імідж даних закладів; розробити єдиний підхід у зборі інформації
щодо територіальної дислокації закладів готельного та ресторанного господар-
ства та ін. Діяльність закладів ГРГ безпосередньо залежить від розвитку туристичної
галузі. Проте на сучасному етапі вона гальмується загостренням політичної й
економічної ситуації в державі, що відображається у зниженні статистичних по-
казників кількості туристів та закладів гостинності. Покращення економічної
та політичної ситуації, активна державна підтримка туристичної галузі, рекон-
струкція пам’яток історії, культури, мистецтва, архітектури, а також поліпшення
стану інфраструктури дадуть змогу активізувати розвиток туризму в регіоні. Таким чином, у Дубенському рекреаційному районі наявні об’єктивні переду-
мови для подальшого розвитку ГРГ. Наступним етапом у дослідженні ГРГ є аналіз рекреаційних мезорегіонів
Поліського рекреаційного регіону, з’ясування їх просторових диференціацій та
розвиток шляхів оптимізації. СПИСОК ПОСИЛАНЬ Алєшугіна, Н. О., & Зеленська, О. О. (2012). Тенденції та перспективи розвитку ринку рес-
торанних послуг м. Чернігів. Науковий вісник Чернігівського державного інституту
економіки і управління. Серія 1. Економіка, 1, 92–98. Демидівська районна державна адміністрація. (2019, 22 лютого). http://www.old.rv.gov.ua/
sitenew/demydivsk/ua/27769.htm. y
Дубенська районна державна адміністрація. (2019, 3 червня). http://www.old.r
sitenew/dubensk/ua/16509.htm. Єрко, І. В. (2016). Аналіз діяльності закладів ресторанного господарства Північно-Західно-
го економічного району України. Науковий вісник Східноєвропейського національного
університету імені Лесі Українки. Серія "Географічні науки", 15(340), 95–100. Кравченко, Н. О. (2007). Рекреаційне господарство Полісся: сучасний стан та перспективи
розвитку. МІЛАНІК. Курмаєв, П. Ю. (2015). Сучасні тенденції функціонування готельно-ресторанного господар-
ства України. Глобальні та національні проблеми економіки, 7, 160–163. http://global-
national.in.ua/archive/7-2015/35.pdf. Ляшук, М. М. (2014). Ресторанне господарство Поліського рекреаційного регіону: сучасний
стан. Географія та туризм, 31, 75–84. Мазур, С. А., & Прилуцький, А. М. (2019). Стан та перспективи розвитку готельно-ресто-
ранної справи в Україні. Ефективна економіка, 2. https://doi.org/10.32702/2307-2105-
2019.2.31. 116 Ресторанний і готельний консалтинг. Інновації. 2020 Том 3 № 1 Restaurant and hotel consulting. Innovations. 2020 Vol. 3 No 1 Мельник, І. М. (2016). Сучасні тенденції розвитку готельного господарства в Україні. Моло-
дий вчений, 7, 84–88. Млинівська районна державна адміністрація. (2019, 1 січня). http://www.old.rv.gov.ua/
sitenew/mlynivsk/ua/27858.htm. Нємець, Л. М., Корнус, А. О., & Корнус, О. Г. (2008). Територіальна концентрація закладів
обслуговування населення Сумської області. Вісник Харківського національного уні-
верситету імені В. Н. Каразіна. Серія "Геологія. Географія. Екологія", 824. http://dspace. univer.kharkov.ua/bitstream/123456789/3014/2/Nemets%2cKornus%2cKornus%201.pdf Радивилівська районна державна адміністрація. (2015, 5 листопада). http://www.old.rv.gov. ua/sitenew/radyvylivsk/ua/4589.htm. Романів, О. Я., Трусова, Т. С., & Бричка, Р. П. (2015). Розвиток готельного господарства
у ринкових умовах. Вісник Національного університету "Львіська політехніка". Серія
"Менеджмент та підприємництво в Україні: етапи становлення і проблеми розвит-
ку", 819, 427–433. Стратегія сталого розвитку міста Дубно до 2022 року. (2017). https://dubno-adm.gov.ua/
fileadmin/user_upload/Ekonomika/Strategija_Dubno_2020.pdf. Стратегія сталого розвитку міста Дубно до 2022 року. (2017). https://dubno-adm.gov.ua/
fileadmin/user_upload/Ekonomika/Strategija_Dubno_2020.pdf. Nemets, L. M., Kornus, A. O., & Kornus, O. H. (2008). Terytorialna kontsentratsiia zakladiv
obsluhovuvannia naselennia Sumskoi oblasti [The establishments of population service’s
territorial agglomerations of the Sumy region]. Visnyk of V.N. Karazin Kharkiv National
University. Series "Geology. Geography. Ecology", 824. http://dspace.univer.kharkov.ua/
bitstream/123456789/3014/2/ Nemets%2cKornus%2cKornus%201.pdf [in Ukrainian]. УДК 640.4(477.81) Николай Ляшук,
старший преподаватель,
Восточноевропейский национальный
университет имени Леси Украинки,
Луцк, Украина,
nicklyashuk@i.ua
https://orcid.org/0000-0002-4993-0191 Ирина Ерко,
кандидат географических наук,
Восточноевропейский национальный
университет имени Леси Украинки,
Луцк, Украина,
ierko@i.ua
https://orcid.org/0000-0002-5395-7557 Actual problems of hotel and restaurant business development
Актуальні проблеми розвитку готельно-ресторанного бізнесу Актуальні проблеми розвитку готельно-ресторанного бізнесу November 5). http://www.old.rv.gov.ua/sitenew/ radyvylivsk/ua/4589.htm [in Ukrainian]. Romaniv, O. Ya., Trusova, T. S., & Brychka, R. P. (2015). Rozvytok hotelnoho hospodarstva u
rynkovykh umovakh [Development of hospitality industry in market environment]. Bulletin of the Lviv Polytechnic National University. Series "Management and Entrepreneurship
in Ukraine: Stages of Formation and Problems of Development", 819, 427–433 [in Ukrainian]. Romaniv, O. Ya., Trusova, T. S., & Brychka, R. P. (2015). Rozvytok hotelnoho hospodarstva u
rynkovykh umovakh [Development of hospitality industry in market environment]. Bulletin of the Lviv Polytechnic National University. Series "Management and Entrepreneurship
in Ukraine: Stages of Formation and Problems of Development", 819, 427–433 [in Ukrainian]. Romaniv, O. Ya., Trusova, T. S., & Brychka, R. P. (2015). Rozvytok hotelnoho hospodarstva u
rynkovykh umovakh [Development of hospitality industry in market environment]. Bulletin of the Lviv Polytechnic National University. Series "Management and Entrepreneurship
in Ukraine: Stages of Formation and Problems of Development", 819, 427–433 [in Ukrainian]. Stratehiia staloho rozvytku mista Dubnо do 2022 roku [Dubnо City Sustainable Development Strategy
until
2022]. (2017). https://dubno-adm.gov.ua/fileadmin/user_upload/Ekonomika/
Strategija_Dubno_2020.pdf [in Ukrainian]. Stratehiia staloho rozvytku mista Dubnо do 2022 roku [Dubnо City Sustainable Development Strategy
until
2022]. (2017). https://dubno-adm.gov.ua/fileadmin/user_upload/Ekonomika/
Strategija_Dubno_2020.pdf [in Ukrainian]. Стаття надійшла до редакції 23.04.2020 Стаття надійшла до редакції 23.04.2020 Romaniv, O. Ya., Trusova, T. S., & Brychka, R. P. (2015). Rozvytok hotelnoho hospodarstva u
rynkovykh umovakh [Development of hospitality industry in market environment].
Bulletin of the Lviv Polytechnic National University. Series "Management and Entrepreneurship
in Ukraine: Stages of Formation and Problems of Development", 819, 427–433 [in Ukrainian]. REFERENCES Alieshuhina, N. O., & Zelenska, O. O. (2012). Tendentsii ta perspektyvy rozvytku rynku
restorannykh posluh m. Chernihiv [Trends and Prospects for the Development of the
Restaurant Services Market in Chernihiv] Scientific Journal of Chernihiv State Institute
of Economics and Management. Series 1. Economics, 1, 92–98 [in Ukrainian]. Demydivska raionna derzhavna administratsiia [Demydiv district state administration]. (2019,
February 22). http://www.old.rv.gov.ua/sitenew/ demydivsk/ua/27769.htm [in Ukrainian]. Demydivska raionna derzhavna administratsiia [Demydiv district state administration]. (2019,
February 22). http://www.old.rv.gov.ua/sitenew/ demydivsk/ua/27769.htm [in Ukrainian]. Dubenska raionna derzhavna administratsiia [Dubna District State Administration]. (2019, Juni 3). http://www.old.rv.gov.ua/sitenew/ dubensk/ua/16509.htm [in Ukrainian]. Dubenska raionna derzhavna administratsiia [Dubna District State Administration]. (2019, Juni 3). http://www.old.rv.gov.ua/sitenew/ dubensk/ua/16509.htm [in Ukrainian]. Jerko, I. V. (2016). Analiz diialnosti zakladiv restorannoho hospodarstva Pivnichno-Zakhidnoho
ekonomichnoho raionu Ukrainy [Analysis of the Institutions of Restaurant Business
in North-Western Economic Region of Ukraine]. Scientific Bulletin of the Lesia Ukrainka East
European National University. Geographical Sciences Series, 15(340), 95–100 [in Ukrainian]. Kravchenko, N. O. (2007). Rekreatsiine hospodarstvo Polissia: suchasnyi stan ta perspektyvy rozvytku
[Recreational Economy of Polesie: Current State and Prospects of Development]. MILANIK [in
Ukrainian]. Kurmaiev, P. Yu. (2015). Suchasni tendentsii funktsionuvannia hotelno-restorannoho
hospodarstva Ukrainy [Modern tendencies of functioning of hotel and restaurant economy
of Ukraine]. Global and National Problems of Economy, 7, 160–163. http://global-national. in.ua/archive/7-2015/35.pdf [in Ukrainian]. Liashuk, M. M. (2014). Restoranne hospodarstvo Poliskoho rekreatsiinoho rehionu: suchasnyi
stan [Restaurant of Polissya recreational region: current state]. Geography and Tourism,
31, 75–84 [in Ukrainian]. Mazur, S. A., & Prylutskyi, A. M. (2019). Stan ta perspektyvy rozvytku hotelno-restorannoi spravy
v Ukraini [The issues of the development of hotel and restaurant business in Ukraine]. Efektyvna ekonomika, 2. https://doi.org/10.32702/2307-2105-2019.2.31 [in Ukrainian]. Melnyk I. M. (2016). Suchasni tendentsii rozvytku hotelnoho hospodarstva v Ukraini [Current
trends in the development of the hotel industry in Ukraine]. Molodyi vchenyi, 7, 84–88 [in
Ukrainian]. Mlynivska raionna derzhavna administratsiia [Mlyniv District State Administration]. (2019, January
1). http://www.old.rv.gov.ua/ sitenew/mlynivsk/ua/27858.htm [in Ukrainian]. 117 Radyvylivska raionna derzhavna administratsiia [Radyvyliv District State Administration]. (2015,
November 5). http://www.old.rv.gov.ua/sitenew/ radyvylivsk/ua/4589.htm [in Ukrainian]. UDC 640.4(477.81) UDC 640.4(477.81) Mykola Lyashuk, Senior Lecturer,
Lesya Ukrainka Eastern European
National University,
Lutsk, Ukraine
nicklyashuk@i.ua
https://orcid.org/ https://orcid.org/0000-0002-4993-
0191 Iryna Ierko,
PhD in Geographic Sciences,
Lesya Ukrainka Eastern European
National University,
Lutsk, Ukraine,
ierko@i.ua
https://orcid.org/0000-0002-5395-7557 Keywords: hotel and restaurant industry, recreation district, hotel, restaurant, territorial
concentration index, localization index. Ресторанний і готельний консалтинг. Інновації. 2020 Том 3 № 1
Restaurant and hotel consulting. Innovations. 2020 Vol. 3 No 1 Ресторанний і готельний консалтинг. Інновації. 2020 Том 3 № 1
Restaurant and hotel consulting. Innovations. 2020 Vol. 3 No 1 СОВРЕМЕННЫЕ ТЕНДЕНЦИИ РАЗВИТИЯ ГОСТИНИЧНО-РЕСТОРАННОГО
БИЗНЕСА РЕКРЕАЦИОННОГО РАЙОНА Актуальность. Исследование гостинично-ресторанного хозяйства (ГРХ), составной
части сферы рекреации, является актуальным, поскольку наличие существующей сети за-
ведений ГРХ на определенной территории является важным фактором, необходимым для
определения путей последующего развития, усовершенствования организации и функ-
ционирования. Цель статьи – анализ функционирования заведений гостинично-ресто-
ранного хозяйства Дубенского рекреационного района Украины. Методы исследования. При проведении исследования использовались общенаучные методы: анализа, синтеза,
сравнения и статистический. Результаты исследования. Проанализировано современ-
ное состояние гостинично-ресторанного хозяйства Дубенского рекреационного района. Обнаружены места наибольшей и наименьшей дислокации предприятий гостинично-рес-
торанного хозяйства в районе. Исследованы территориальная концентрация и уровень
развития предприятий гостинично-ресторанного хозяйства Дубенского рекреационного
района путем расчета индекса территориальной концентрации и индекса локализации. 118 Restaurant and hotel consulting. Innovations. 2020 Vol. 3 No 1 Охарактеризованы перспективы развития заведений ГРХ исследуемого района. Выводы
и обсуждения. Исследование ГРХ Дубенского рекреационного района имело целью пока-
зать территориальные отличия в размещении и уровне развития ГРХ района. Наилучшими
показателями развития ГРХ характеризуются Демидовский административный район и г. Дубно, наихудшим – Млиновский административный район. Ключевые слова: гостинично-ресторанное хозяйство, рекреационный район, гостини-
ца, ресторан, индекс территориальной концентрации, индекс локализации. Ресторанний і готельний консалтинг. Інновації. 2020 Том 3 №
Restaurant and hotel consulting. Innovations. 2020 Vol. 3 No 1 р
Restaurant and hotel consulting. Innovations. 2020 Vol. 3 No 1 MODERN TRENDS of DEVELOPMENT of HOTEL AND RESTAURANT BUSINESS
of THE RECREATION DISTRICT Topicality. a study of the hotel and restaurant industry (HRI), an integral part of the
sphere of recreation, is relevant, since the existence of an existing network of HRI institutions
in a certain territory is an important factor necessary to determine ways for further development,
and to improve organization and functioning. The purpose of the article is the analysis and
functioning of hotel and restaurant industry of the Dubensky Recreation District of Ukraine. Research methods. During the study, general scientific methods were used: analysis, synthesis,
comparison, and statistical. The research results. The present state of the hotel and restaurant
industry of the Dubensky Recreation District has been analyzed. The locations of the largest
and smallest dislocation of hotel and restaurant enterprises in the district have been identified. The territorial concentration and the development level of the hotel and restaurant industry
enterprises of the Dubensky Recreation District were studied by calculating the territorial
concentration index and the localization index. The prospects for the development of HRI
institutions in the study district have been described. Conclusions and discussion. The study
of the HRI of the Dubensky Recreation District was aimed at showing territorial differences in the
location and development level of the HRI of the district. The best indicators of HRI development
are characterized by the Demydivsky administrative district and Dubno, the worst is Mlynivsky
administrative district. Keywords: hotel and restaurant industry, recreation district, hotel, restaurant, territorial
concentration index, localization index. 119
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†
‡
§
k
Department of Anatomy, Histology, Forensic Medicine and Orthopedic, Section of Histology,
Sapienza University of Rome, 00185 Rome, Italy; innocenti@generalifeitalia.it (F.I.);
sara.dipersio@ukmuenster.de (S.D.P.); marilenataggi@gmail.com (M.T.); rita.canipari@uniroma1.it (R.C.)
Endocrinology, Department of Clinical and Molecular Medicine, Sant’Andrea University Hospital,
Sapienza University of Rome, 00185 Rome, Italy; r.maggio@uniroma1.it (R.M.); pina85la@gmail.com (P.L.);
vincenzo.toscano@uniroma1.it (V.T.)
Correspondence: elena.vicini@uniroma1.it (E.V.); antonio.stigliano@uniroma1.it (A.S.);
Tel.: +39-6-4976-6757 (E.V.); +39-6-3377-5147 (A.S.)
Current address: Clinica Valle Giulia, GeneraLife IVF, 00197 Rome, Italy.
Current address: Centre of Reproductive Medicine and Andrology, University Hospital Münster,
48149 Münster, Germany.
In memoriam.
These authors contributed equally to this work.
Simple Summary: Mitotane (MTT) is the treatment of choice for adrenocortical carcinoma. Male
hypogonadism is often diagnosed in male patients treated with this drug. This research aims to
consider possible side effects induced by MTT in the testis and a hypothetical detrimental effect
deriving from androgenic deficiency. Furthermore, considering the increased potentiality of treatment
for adrenocortical carcinoma, we want to provide male patients of childbearing age, as in other
oncological diseases, the opportunity to consider a sperm cryopreservation program.
Citation: Innocenti, F.; Di Persio, S.;
Taggi, M.; Maggio, R.; Lardo, P.;
Toscano, V.; Canipari, R.; Vicini, E.;
Stigliano, A. Effect of Mitotane on
Male Gonadal Function. Cancers 2023,
15, 3234. https://doi.org/10.3390/
cancers15123234
Academic Editor: Naris Nilubol
Received: 3 May 2023
Revised: 2 June 2023
Abstract: Background: Clinical evidence has shown frequent hypogonadism following mitotane
(MTT) treatment in male patients with adrenocortical carcinoma. This study aimed to evaluate the
impact of MTT on male gonadal function. Methods: Morphological analysis of testes and testosterone
assays were performed on adult CD1 MTT-treated and untreated mice. The expression of key
genes involved in interstitial and tubular compartments was studied by real-time PCR. Moreover,
quantitative and qualitative analysis of spermatozoa was performed. Results: Several degrees of
damage to the testes and a significant testosterone reduction in MTT-treated mice were observed. A
significant decline in 3βHsd1 and Insl3 mRNA expression in the interstitial compartment confirmed
an impairment of androgen production. Fsh-R mRNA expression was unaffected by MTT, proving
that Sertoli cells are not the drug’s primary target. Sperm concentrations were significantly lower
in MTT-treated animals. Moreover, the drug caused a significant increase in the percentage of
spermatozoa with abnormal chromatin structures. Conclusion: MTT negatively affects the male
reproductive system, including changes in the morphology of testicular tissue and reductions in
sperm concentration and quality.
Accepted: 13 June 2023
Published: 18 June 2023
Keywords: mitotane; adrenocortical carcinoma; testis; testosterone; male gonadal function
Copyright: © 2023 by the authors.
1. Introduction
Licensee MDPI, Basel, Switzerland.
Adrenocortical carcinoma (ACC) is a rare and aggressive endocrine tumor with poor
oncological and endocrinological prognoses [1]. ACC affects about two million people per
year with a bimodal incidence and a peak incidence in pediatric and middle ages [1]. The
clinical picture is frequently characterized by high tumor cortisol secretion and sometimes
androgens with the typical signs of overt Cushing’s syndrome and a more complicated
endocrinological status [1]. In fact, the excess of cortisol interferes with the physiological
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Cancers 2023, 15, 3234. https://doi.org/10.3390/cancers15123234
https://www.mdpi.com/journal/cancers
Cancers 2023, 15, 3234
2 of 12
effects of other hormones and endocrine pathways [2]. This condition adversely impacts
many metabolic processes involving the hypothalamic-pituitary-gonadal axis, resulting in
hypogonadotropic hypogonadism [2].
Mitotane (1,1-dichloro)-2-(o_-chlorophenyl)-2-(p_-chlorophenyl)-ethane or o_0 ,p_0 -DDD)
(MTT) is an adrenolytic drug widely used in the advanced and adjuvant treatment of ACC
and sometimes in Cushing’s disease and neuroendocrine ACTH-secreting tumors [3,4].
Currently, it is the only drug approved by the US and European regulatory authorities (FDA,
EMA) to treat metastatic ACC. However, it is also used in adjuvant settings [1,3]. This drug
represents the medical gold standard in the treatment of ACC, and today, its clinical use has
become much more controlled and appropriate. Its mechanism of action, not yet completely
understood, inhibits the adrenal cortex’s steroidogenic enzymes, inducing a detrimental
effect on the fasciculata and reticularis zonae with a relative effect on the glomerular
zone [3]. Although MTT has been used for many years to treat ACC, few data exist on the
effects of this drug on other glandular epithelia with steroidogenic activity. In the past, the
greatest attention in the care of ACC was directed, above all, to the patient’s overall survival.
Currently, the progress made in knowledge about the disease and the improvement of MTT
management has considerably prolonged the survival chances of patients. Advances in
treatment programs unquestionably require better consideration of those aspects of ACC
care considered less relevant in the past. We previously demonstrated that MTT treatment
at therapeutic concentrations interferes with follicular development and endocrine ovarian
activity without inducing irreversible changes in the gland. Furthermore, a higher drug
concentration might lead to increased apoptosis of granulosa cells [5]. Interestingly, data
from mating experiments demonstrated that MTT increases breeding time but does not
preclude the possibility of procreative function [5]. However, increasing clinical evidence
has shown the occurrence of hypogonadism following treatment with this drug, more
frequently in male patients [6]. Androgenic deficiency negatively affects bone and muscle
metabolism, resulting in many other metabolic complications in men. These complications
add to the critical illness resulting from hypercortisolism and oncological disease. On
the other hand, the increased survival achieved by young patients should cause us to
consider the desire to procreate. Hence, the clinical endocrinologist is called to answer
these unanswered questions. To fill this gap, the present study evaluated the impact of
MTT treatment on male gonadal function in adult CD1 mice.
2. Materials and Methods
2.1. Animals
CD1 mice (Charles River, Como, Italy) were housed under controlled temperature
(25 ◦ C) and light conditions (12 h light/day) with at libitum access to food and water. At
30 days of age, adult male mice were treated for up to 45 days with 0.152 mg/kg MTT
(Sigma-Aldrich Co., St Louis, MO, USA). The length of MTT treatment was chosen to ensure
at least one cycle to the seminiferous epithelium (34.5 days in mice), while the drug dose
was chosen according to previously published papers [5,7]. The drug was resuspended in
dimethyl sulfoxide (DMSO) and administered in a final volume of 20 µL per day/animal
via intraperitoneal injection. Healthy mice were treated with the same volume of the vehicle
alone (DMSO) (Ctrl). Testosterone replacement therapy, 0.25 mg/mouse/week (Testoviron,
Bayer S.p.A., Leverkusen, Germany, 250 mg/mL), was administered by intramuscular
injection to a group of MTT-treated adult male animals (MTT + Test) or alone to a control
group (Test). The testosterone dose, based on data from Griffin et al. [8], reproduced the
physiological levels of testosterone found in male mice [9,10]. The hormone was resuspended in sunflower seed oil (Sigma-Aldrich) in a final volume of 50 µL per week/animal
to administer 0.25 mg/mouse/week. The animals were weighed daily and sacrificed by
cervical dislocation at the end of treatment. All animal procedures were approved by local
ethics committee for animal research.
Cancers 2023, 15, 3234
3 of 12
2.2. Quantitative Morphometric Evaluation of Seminiferous Tubules
For morphological analysis, the gonads were quickly removed, fixed in Bouin’s liquid,
paraffin-embedded, serially sectioned at 6 µm, and stained with carmalum. Testis sections
were examined under a bright-field microscope for quantification of morphological changes.
Pictures were taken from every fifth section of the whole testicle. All quantifications were
performed on stored images using Image J software. The seminiferous tubule diameter
and the area from about 70 sections of seminiferous tubules that were round or nearly
round were chosen and measured for each group. The software was calibrated by using
the ‘straight line’ icon and by extending a straight line over the length indicating a known
distance of 100 µm.
2.3. Immunofluorescence
For immunofluorescence analysis, the gonads were quickly removed, fixed in 4%
PFA paraffin-embedded, serially sectioned at 6 µm, and mounted on Polysine-TM slides
(Menzel-Glaser, Braunschweig, Germany). The slides were incubated with 1 M glycine at a
pH of 7.5 for 30 min at room temperature and then in a blocking solution of 1× phosphatebuffered saline (PBS) containing 1% w/v bovine serum albumin (BSA; Sigma-Aldrich) and
5% v/v normal donkey serum (Sigma-Aldrich) to minimize non-specific binding. The slides
were then incubated for 20 h at 4 ◦ C with 1:300 anti-3β-HSD goat polyclonal IgG (Santa
Cruz Biotechnology, Milano, Italy; sc-30820). After extensive washing in PBS, the slides
were incubated for 2 h at room temperature with 1:500 donkey anti-goat IgG (H+L) crossadsorbed secondary antibody from Alexa Fluor™ 488 (Thermo Fisher Scientific, Rome,
Italy). Nuclei were then counterstained with Hoechst 33342, and the slides were closed
with Vectashield mounting medium (Vector Laboratories, Inc., Newark, CA, USA). Pictures
were acquired and examined using a Zeiss Axioscope Imager 2 fluorescence microscope. In
the control samples, the primary antibody was substituted with rabbit pre-immune serum.
2.4. Isolation of Interstitial Cells from Seminiferous Tubules
The testes were removed from the scrotum, decapsulated, transferred to small Petri
dishes in M2 medium containing 0.18% trypsin, and then transferred to a shaking water
bath at 32 ◦ C under agitation (90 cycles/min) for 15 min to detach the interstitium. After
dissociation, the enzyme was diluted with medium, and the seminiferous tubules were
removed by gravity sedimentation. The tubules were washed again to detach the remaining interstitial cells, and the two supernatants were pooled together. The two fractions
corresponding to isolated seminiferous tubules and a supernatant enriched in Leydig cells
(LCs) were collected for further analysis.
2.5. Serum Testosterone Assay
Blood samples were collected under anesthesia via cardiac puncture from all the
mice after 45 days of treatment. Clots were removed by centrifugation at 2000× g for
10 min, and the resulting supernatant in serum was collected and stored at −20 ◦ C until
required for testosterone assay. Total plasma testosterone concentrations were measured
by a radioimmunoassay (RIA) kit (Diagnostic Products, Los Angeles, CA, USA) using
’Coat-a-count’ kits from Siemens Healthcare Diagnostics (Los Angeles, CA, USA) with a
sensitivity of 4 ng/dL.
2.6. RNA Extraction, Reverse Transcription, and Real-Time PCR
Total RNA was isolated using a silica gel-based membrane spin column (Rneasy Kit,
Qiagen S.p.A., Milan, Italy) from the whole gonads. Total RNA was reverse transcribed
using the M-MLV reverse transcriptase kit (Qiagen). Ribonucleic acid integrity and purity
were confirmed spectroscopically and by gel electrophoresis before use. Total RNA (1 µg)
was reverse transcribed in a final volume of 30 µL using the M-MLV Reverse Transcriptase
kit (Invitrogen, Milan, Italy); cDNA was diluted 1:2 in nuclease-free water, aliquoted, and
stored at −20 ◦ C. The presence of transcripts for insulin-like 3 (Insl3), 3β-hydroxysteroid
Cancers 2023, 15, 3234
4 of 12
dehydrogenase/isomerase type 1 (3β-Hsd1), and follicle stimulating hormone receptor (FshR) was evaluated by SYBR Green real-time PCR on an Applied Biosystems 7500 real-time
PCR system (Thermo Fisher Scientific, Rome, Italy), equipped with 96-well optical reaction
plates using SYBR Green Universal PCR Master Mix (Euroclone, Milan, Italy), by adding
0.3 µmol/L of each specific primer to a total volume of 20 µL reaction mixture according
to the manufacturer’s recommendations; negative controls contained water instead of
first-strand cDNA. Each sample was normalized to its β-actin content. The results were
expressed as arbitrary units (a.u.) calculated using the ∆∆Ct method. The primers used are
shown in Supplementary Table S1.
2.7. Sperm Extraction and Flow Cytometry Sperm Chromatin Structure Assay
For the MTT-induced DNA damage to sperm, a sperm chromatin structure assay
was performed, which evaluates low pH-induced denaturation of sperm nuclear DNA [8].
The right and left epididymis cauda were dissected, transferred individually into small
Petri dishes, minced in M2 medium, and incubated for 15 min at 37 ◦ C (95% air and 5%
CO2 ) to allow for the release of sperm for the determination of epidydimal sperm reserve
(ESR). Samples were diluted in a final volume of 1 mL, and the sperm concentration was
evaluated using a hemocytometer. The samples were then frozen at −80 ◦ C and thawed
at the analysis time. They were diluted to a concentration of 5 × 105 sperm/mL with
TNE buffer solution (0.15 M NaCl, 0.01 M Tris-HCL, 1 mM disodium EDTA, pH 7.4). One
hundred microliters of diluted samples were loaded onto the sorter in 12 × 7.5 mL BD
FACS tubes and mixed with 200 µL of low-pH detergent solution (0.1% Triton X-100, 0.15 M
NaCl, 0.08 N HCL, pH 1.2). After 30 s, the cells were stained with 600 µL of acridine orange
(AO) diluted in a buffer composed of 0.1 M citric acid, 0.2 M Na2 HPO4 , 1 mM EDTA, and
0.15 M NaCl with a pH of 6 until a final concentration of 6 µg/mL was obtained. Acridine
orange is a cell-permeant nucleic acid binding dye that emits green fluorescence when
bound to dsDNA and red fluorescence when bound to ssDNA or RNA. Therefore, live cells
have a typical green nucleus, and late apoptotic cells display condensed and fragmented
orange chromatin [11]. Samples were analyzed using flow cytometry (Dako Cyan ADP,
Beckman Coulter Inc., Milan, Italy). Green fluorescence was detected at 515–530 nm, and
red fluorescence was measured at 630–650 nm. The result allowed us to obtain a DNA
fragmentation index (DFI), and this quantification was performed with FlowJo software
(BD Bioscence, Milan, Italy)
2.8. Statistical Analyses
All experiments were repeated at least three times, and each experiment was performed at least in duplicate. Statistical analyses were performed using ANOVA followed
by the Tukey–Kramer test for comparisons of multiple groups or the two-tailed t-test
when comparing data derived from two groups. Values with p < 0.05 were considered
statistically significant.
3. Results
3.1. Mitotane Interferes with Testosterone Production
We did not observe differences in food intake with the different treatments; however,
MTT-treated animals displayed a smaller weight increase per day compared to control mice,
although the difference was not statistically significant. The replacement of testosterone
reversed this decrease (Figure 1a).
Serum testosterone levels in control mice, 5.8 + 1.4 ng/mL, were in line with normal
levels found in mice [9,10]. Serum testosterone was significantly lower in MTT-treated
animals compared to their control siblings (p < 0.05; Figure 1b), confirming the effect of the
pharmacological treatment on steroid production. The testosterone replacement therapy
restored the serum hormonal concentrations to levels comparable to those of untreated
animals. The group of animals treated with the MTT-testosterone in combination did not
differ from that treated with testosterone alone used as an internal control.
Cancers 2023, 15, 3234
therapy restored the serum hormonal concentrations to levels comparable to those of
untreated animals. The group of animals treated with the MTT-testosterone in
5 of 12
combination did not differ from that treated with testosterone alone used as an internal
control.
Figure 1.
1. Effect
Effect of
of mitotane
mitotane on:
on: (a)
(a) daily
daily body
body weight
weight increase;
increase; and
and (b)
(b) serum
serum testosterone
testosterone dosage
dosage
Figure
in control
control (Ctrl),
(Ctrl), mitotane
mitotane (MTT),
(MTT), MTT-testosterone
MTT-testosterone cotreated
cotreated (MTT
(MTT + Test), and testosterone (Test)
in
treatedanimals.
animals.The
Thevalues
valuesare
areexpressed
expressedas
asthe
themean
mean±± s.e.m.
s.e.m. with
with aa total
total animal
animal number
number of
of C
C == 10;
10;
treated
MTT
=
11;
M
+
T
=
6;
Test
=
6.
Statistical
analysis
was
performed
by
one-way
ANOVA,
followed
by
MTT = 11; M + T = 6; Test = 6. Statistical analysis was performed by one-way ANOVA, followed by
the Tukey–Kramer test. * p < 0.05; ** p < 0.01.
the Tukey–Kramer test. * p < 0.05; ** p < 0.01.
3.2. MTT
MTT Induces
Induces Detrimental
Detrimental Effects
Effects on
on Seminiferous
Seminiferous Tubules
Tubules
3.2.
Wenext
nextanalyzed
analyzed
the impact
of treatment
MTT treatment
on testis morphology.
The
We
the impact
of MTT
on testis morphology.
The histological
histological
analysis
Ctrl testes
showed tubules
seminiferous
tubules by
characterized
by alumen
wellanalysis
of Ctrl
testes of
showed
seminiferous
characterized
a well-defined
defined
lumenspermatogenesis
and complete spermatogenesis
(Figure
2a,a’ Ctrl).
Infrom
contrast,
testesgroup
from
and
complete
(Figure 2a,a’ Ctrl).
In contrast,
testes
the MTT
the MTTseveral
group degrees
showedofseveral
degrees
of damage:ofdisorganization
of the germinal
showed
damage:
disorganization
the germinal epithelium
and a
pronounced
alteration
of the spermatogenic
with a reduction
in aspermatozoa.
epithelium and
a pronounced
alteration of theprocess
spermatogenic
process with
reduction in
In
some
instances,
we
observed
an
absence
(Figure
2c,c’;
arrowhead)
or
an
enlargement
spermatozoa. In some instances, we observed an absence (Figure 2c,c’; arrowhead) or an
(Figure
2b,b’; (Figure
asterisk)2b,b’;
of theasterisk)
lumen, with
less compact
space
(Figure 2b–c’).
enlargement
of thea lumen,
with ainterstitial
less compact
interstitial
space
Morphometric
revealed that
the diameter
area
the seminiferous
tubules
of
(Figure 2b–c’).analysis
Morphometric
analysis
revealedand
that
theof diameter
and area
of the
treated
animalstubules
were slightly
smaller
but notwere
significantly
fromnot
those
of control
seminiferous
of treated
animals
slightly different
smaller but
significantly
animals.
the MTT-Test
group,
we did notinobserve
gross alterations
indid
tubule
differentInterestingly,
from those ofincontrol
animals.
Interestingly,
the MTT-Test
group, we
not
morphology
(Figure
2d,d’),
indicating
that
the
testosterone
supplementation
counteracted
observe gross alterations in tubule morphology (Figure 2d,d’), indicating that the
the
MTT-induced
damage to seminiferous
tubules.
testosterone
supplementation
counteracted
the MTT-induced damage to seminiferous
tubules.
3.3. Different Effect of MTT on Sertoli and Leydig Cell Gene Expression
To investigate MTT-dependent changes in germ and somatic cells, we evaluated
specific gene expression after isolating interstitium and seminiferous tubules as described
in the Materials and Methods section. To validate the efficacy of the seminiferous tubule
and interstitium separation method, we evaluated by real-time PCR the presence of mRNA
for Fsh-R and Insl3, specific markers of Sertoli (SCs) and LCs, respectively (Figure S1). As
shown in Figure S1, Insl3 mRNA was found mainly in the interstitium. In contrast, Fsh-R
mRNA was found in the tubules, demonstrating that we efficiently separated the two
cell compartments.
Then, we examined the effects of MTT on the expression levels of 3β-Hsd1, Insl3,
and Fsh-R. FSH is required for SC proliferation, maturation, and synthesis of specific SC
proteins. It is also important at the beginning of the first wave of spermatogenesis [12]. The
expression of mRNA from Fsh-R was unaffected by treatment, proving that SCs are not
the main target of the drug and that the alteration of spermatogenesis is unrelated to SC
damage (Figure 3c).
The molecular analysis of the interstitial compartment revealed the expected significant decline in mRNA expression of Insl3 (p < 0.01) and 3β-Hsd1 (p < 0.05) in MTTtreated mice, confirming the alteration of the cells responsible for androgen production
(Figure 3a,b). The expression of 3β-Hsd1 and Insl3 in the interstitial cells of testosteronecotreated mice was partially restored compared to those receiving MTT alone (Figure 3a,b).
Cancers 2023, 15, 3234
6 of 12
Immunofluorescence analysis performed on testicular sections stained with 3β-HSD
revealed a less intense fluorescence signal and fewer stained LCs in the MTT6 of 14
treated animals and recovery after testosterone cotreatment (Figure 3d).
antibody
Cancers 2023, 15, x FOR PEER REVIEW
Figure
Figure2.2.Photomicrographs
Photomicrographsofofsections
sectionsofoftestes
testesfrom
from(a,a’,
(a,a’,Ctrl)
Ctrl)control,
control,(b–c’,
(b–c’,MTT)
MTT)MTT-treated,
MTT-treated,
and
and(d,d’,
(d,d’,MTT
MTT++Test)
Test)MTT
MTTand
andtestosterone-treated
testosterone-treatedmice.
mice.Arrows:
Arrows:spermatozoa;
spermatozoa;asterisks:
asterisks:tubules
tubules
with
withenlarged
enlargedlumen;
lumen;arrowheads:
arrowheads:tubules
tubuleswith
withabsence
absenceofoflumen.
lumen.Scale
Scalebar
bar= =100
100µm.
μm.
3.4.
Negatively
Affects
Concentration
3.3.MTT
Different
Effect of
MTTSperm
on Sertoli
and Leydigand
CellQuality
Gene Expression
The
concentration
of
spermatozoa
collected
from
epididymis
cauda
was
signifTo investigate MTT-dependent changes in germthe
and
somatic cells,
we
evaluated
icantly
lower
in
MTT-treated
animals
than
in
the
control
group
(p
<
0.001;
Figure
4a).
specific gene expression after isolating interstitium and seminiferous tubules as described
Moreover, the administration of MTT caused a significant increase in the percentage of
in the Materials and Methods section. To validate the efficacy of the seminiferous tubule
spermatozoa with abnormal chromatin structures defined by the DNA fragmentation index
and interstitium separation method, we evaluated by real-time PCR the presence of
(%DFI) compared to the untreated animals (p < 0.001; Figure 4b). Significant protection was
mRNA for Fsh-R and Insl3, specific markers of Sertoli (SCs) and LCs, respectively (Figure
conferred by testosterone replacement therapy against the MTT-induced sperm damage; in
S1). As shown in Figure S1, Insl3 mRNA was found mainly in the interstitium. In contrast,
fact, both sperm concentration and viability were restored after testosterone administration
Fsh-R mRNA was found in the tubules, demonstrating that we efficiently separated the
(Figure 4a,b).
two cell compartments.
Then, we examined the effects of MTT on the expression levels of 3β-Hsd1, Insl3, and
Fsh-R. FSH is required for SC proliferation, maturation, and synthesis of specific SC
proteins. It is also important at the beginning of the first wave of spermatogenesis [12].
The expression of mRNA from Fsh-R was unaffected by treatment, proving that SCs are
Cancers 2023, 15, 3234
(Figure 3a,b). The expression of 3β-Hsd1 and Insl3 in the interstitial cells of testoste
cotreated mice was partially restored compared to those receiving MTT alone (F
3a,b).
Immunofluorescence analysis performed on testicular sections stained with 3β
antibody revealed a less intense fluorescence signal and fewer stained LCs in the
7 of 12
treated animals and recovery after testosterone cotreatment (Figure 3d).
3. Effects
of mitotane
on gene
expression
in isolated
seminiferous
tubules
and Leydi
Figure 3. EffectsFigure
of mitotane
on gene
expression
in isolated
seminiferous
tubules
and Leydig
cells
removed
from
untreated
mice
(Ctrl)
and
mice
treated
with
mitotane
(MTT),
MTT
and testost
removed from untreated mice (Ctrl) and mice treated with mitotane (MTT), MTT and testosterone
(MTT+Test)
together,
and
testosterone
(Test).
Expression
of
3β-Hsd1
(a)
and
Insl3
(b)
(MTT+Test) together, and testosterone (Test). Expression of 3β-Hsd1 (a) and Insl3 (b) in LCs, detectedin LCs, de
by real-time PCR in LCs within interstitial cells. Expression of Fsh-R (c) mRNA in mouse SCs w
by real-time PCR in LCs within interstitial cells. Expression of Fsh-R (c) mRNA in mouse SCs within
the seminiferous tubules detected by real-time PCR. Each sample was normalized to its β
the seminiferous tubules detected by real-time PCR. Each sample was normalized to its β-actin
content. Results are expressed as arbitrary units (a.u.) and are represented as the mean ± s.e
content. Results
are independent
expressed as experiments
arbitrary units
(a.u.)
are numbers
represented
as the
mean
three
with
totaland
animal
of Ctrl
= 15,
MTT±= s.e.m.
18 and MTT +
of three independent
experiments
with
total
animal
numbers
of
Ctrl
=
15,
MTT
=
18
andfollowed
MTT + by the T
12, and testosterone = 7. Statistical analysis was performed using ANOVA
Test = 12, and testosterone
analysis
performed
using ANOVA
followed by the analys
Kramer test;= *7.pStatistical
< 0.05 and
** p <was
0.01,
ns: no Significance.
Immunofluorescence
Representative
images
of
immunofluorescent
3β-HSD
staining
in
sections
testes
from
Tukey–Kramer
test;
*
p
<
0.05
and
**
p
<
0.01,
ns:
no
Significance.
Immunofluorescence
analysis
(d).
Cancers 2023, 15, x FOR PEER REVIEW
8ofof
14
treated
as
described
above.
Scale
bar
=
100
μm.
Representative images of immunofluorescent 3β-HSD staining in sections of testes from mice treated
as described above. Scale bar = 100 µm.
3.4. MTT Negatively Affects Sperm Concentration and Quality
The concentration of spermatozoa collected from the epididymis cauda
significantly lower in MTT-treated animals than in the control group (p < 0.001; Figur
Moreover, the administration of MTT caused a significant increase in the percenta
spermatozoa with abnormal chromatin structures defined by the DNA fragment
index (%DFI) compared to the untreated animals (p < 0.001; Figure 4b). Signi
protection was conferred by testosterone replacement therapy against the MTT-ind
sperm damage; in fact, both sperm concentration and viability were restored
testosterone administration (Figure 4a,b).
Figure4.4.Mean
Meanofofthe
thenumber
numberofofspermatozoa
spermatozoaininmice
miceinjected
injectedwith
withDMSO
DMSO(Ctrl),
(Ctrl),MTT,
MTT,MTT
MTTand
and
Figure
testosterone
together
(MTT
+
Test)
and
testosterone
(Test).
Results
are
expressed
as
number
testosterone together (MTT + Test) and testosterone (Test). Results are expressed as number of sper-of
spermatozoa per epididymis (a). Quantification of sperm with abnormal chromatin structures
matozoa per epididymis (a). Quantification of sperm with abnormal chromatin structures calculated
calculated by the evaluation of the parameter DNA fragmentation index (DFI). A high %DFI
by the evaluation of the parameter DNA fragmentation index (DFI). A high %DFI correspond to a
correspond to a large percentage of damaged germ cells. Results are expressed as arbitrary units
large
percentage
of are
damaged
germ cells.
expressed
as arbitrary
unitsexperiments
(a.u.) (b). Values
(a.u.)
(b). Values
represented
as theResults
mean are
± s.e.m.
of three
independent
with are
total
represented
as
the
mean
±
s.e.m.
of
three
independent
experiments
with
total
animal
numbers
of
animal numbers of Ctrl = 12, MTT = 12, MTT + Test = 7 and Test = 4. Statistical analysis was
Ctrl
=
12,
MTT
=
12,
MTT
+
Test
=
7
and
Test
=
4.
Statistical
analysis
was
performed
using
ANOVA
performed using ANOVA followed by the Tukey–Kramer test. ** p < 0.01, *** p < 0.001 vs. MTT.
followed by the Tukey–Kramer test. ** p < 0.01, *** p < 0.001 vs. MTT.
3.5. MTT Withdrawal Restores Testosterone Level but Not the Spermatogenesis
Next, we asked whether the MTT-induced effects were permanent or could be
reversed by MTT withdrawal. To this end, after 45 days of treatment, the Ctrl and MTT
groups were left for a further 30 days without any treatment and defined as the Ctrl-R
Cancers 2023, 15, 3234
8 of 12
3.5. MTT Withdrawal Restores Testosterone Level but Not the Spermatogenesis
Next, we asked whether the MTT-induced effects were permanent or could be reversed
by MTT withdrawal. To this end, after 45 days of treatment, the Ctrl and MTT groups
were left for a further 30 days without any treatment and defined as the Ctrl-R and MTT-R
groups, respectively. Animal weight was recorded every other day, as well as food intake.
After 30 days, one testis/animal was collected for morphological examination and the
contralateral testis for RNA extraction. A blood sample from each animal was retrieved to
evaluate serum testosterone levels.
We observed that specific genes for the LCs Insl3 and 3b-Hsd1 were comparable in
Ctrl-R and MTT-R mice (Figure 5a,b), as well as testosterone levels (Figure 5c). However,
the number of spermatozoa remained statistically lower in MTT-R mice compared to
Ctrl-R mice (p < 0.01; Figure 5d). In addition, cells with abnormal chromatin structures,
calculated by the evaluation of the parameter DNA fragmentation index (%DFI), were
significantly more common in MTT-R mice (p < 0.01; Figure 5e). Morphological evaluation
of seminiferous tubules in the MTT-R mice confirmed these latter data. Although the
morphology of the seminiferous tubules was partially
Cancers 2023, 15, x FOR PEER REVIEW
9 of 14 recovered, we observed a smaller
number of spermatozoa in the tubule sections (Figure 5f–h).
Figure 5. Effect
of MTT withdrawal
the expression of Insl3
and 3β-Hsd1
(b) in LCsof
removed
Figure
5. Effect
of MTTonwithdrawal
on(a)the
expression
Insl3 (a) and 3β-Hsd1 (b) in LCs removed
from untreated (Ctrl) and mitotane (MTT)-treated mice at 30 days after MTT removal, detected by
real-timeuntreated
PCR in LCs within
interstitial
Each sample
was normalized to itsmice
β-actin content.
from
(Ctrl)
andcells.
mitotane
(MTT)-treated
at 30 days after MTT removal, detected by
Results are expressed as arbitrary units (a.u.) and are represented as the mean ± s.e.m. of three
real-time
PCR inwith
LCs
within
interstitial
cells.
independent experiments
a total
animal number
of Ctrl = 6 and
MTT =Each
6. Serumsample
testosterone was normalized to its β-actin content.
concentration (c). Mean of the number of spermatozoa (d). Quantification of sperm with abnormal
Results
are
expressed
as
arbitrary
units
(a.u.)
and
are
represented
as the mean ± s.e.m. of three
chromatin structures calculated by the evaluation of the parameter DNA fragmentation index
(DFI)
(e). A high %DFI corresponds to a large percentage of damaged germ cells. Statistical analysis was
independent
experiments with a total animal number of Ctrl = 6 and MTT = 6. Serum testosterone
performed using Student’s t test. ** p < 0.01 vs. respective Ctrl-R. Testis morphology after MTT
withdrawal (f–h). Scale bar = 100 mm.
concentration
(c). Mean of the number of spermatozoa (d). Quantification of sperm with abnormal
chromatin structures calculated by the evaluation of the parameter DNA fragmentation index (DFI)
(e). A high %DFI corresponds to a large percentage of damaged germ cells. Statistical analysis was
performed using Student’s t test. ** p < 0.01 vs. respective Ctrl-R. Testis morphology after MTT
withdrawal (f–h). Scale bar = 100 mm.
Cancers 2023, 15, 3234
9 of 12
4. Discussion
Despite the high aggressiveness of ACC, there have been considerable advances in
knowledge about this tumor over the years, and overall survival from it is increasing.
This evidence leads us to consider many other parameters during disease management.
Unfortunately, information regarding the effects on male testes and fertility is still poor.
Many studies have supported evidence of toxic side effects of MTT involving both metabolic
and endocrine systems that may require treatment [13–16]. In a retrospective study in
50 Danish patients, Vikner et al. showed that total cholesterol increased significantly after
six months of treatment with MTT, raising LDL, HDL, and triglycerides [17]. Plasma
thyroxine decreased while TSH remained unchanged. Drug discontinuation restored the
average hormonal balance [17].
We previously demonstrated that MTT at therapeutic concentrations in female mice
interferes with follicular development and endocrine ovarian activity. We observed a
decrease in early antral follicle numbers with a subsequent increase in secondary follicles.
This effect was accompanied by altered steroidogenesis with a significant decrease in
mRNA expression of P450scc (Cyp11a1), which catalyzes the conversion of cholesterol to
pregnenolone, and in 17-alpha-hydroxylase (Cyp17a1), a key enzyme in the biosynthesis of
sex hormones localized in the endoplasmic reticulum of thecal cells. Ovulation was also
significantly impaired, with a reduction in the numbers of oocytes, corpora lutea, and pups
per litter in the treated animals [5].
In contrast, in males, the available data supporting the harmful effects of MTT have
been conflicting. Gentilin et al. demonstrated an inhibitory effect of MTT in mouse
gonadotroph cell lines [15]. According to the authors, MTT reduced cell viability, induced
apoptosis, and modified cell cycle phases and secretion in gonadotroph cells. These effects
could explain the lack of LH increase during MTT treatment despite the reduction in
testosterone levels [15]. Moreover, an increase in sex hormone-binding globulin (SHBG)
and cortisol-binding globulin (CBG) has been shown in males treated with MTT, ranging
from 4 to 6.5 daily for recurrent ACC [18]. It has also been reported that SHBG significantly
increases in the first six months after MTT treatment, followed by a progressive decline in
free testosterone concentrations and unchanged gonadotropin levels [13,16].
This study aimed to evaluate the effect of MTT on the male gonad, either on androgen
hormone synthesis or on testis morphological structure. In addition, possible detrimental
involvement in sperm parameters was evaluated.
Animals well tolerated the treatment with MTT, and no changes in weight were observed (Figure 1a). However, MTT treatment significantly reduced the plasma levels of total
testosterone (p < 0.05; Figure 1b). On the contrary, androgen replacement therapy reverted
the testosterone drop in MTT-treated mice by equilibrating the hormone level comparable
to mice treated with testosterone alone, used as an internal control. Morphometric analysis
revealed that the area and the diameter of seminiferous tubules of the treated animals were
slightly smaller than the control, even if not statistically significant. However, the testicular
tissue of MTT-treated mice displays different degrees of morpho-structural damage consistent with disorganization of germinal epithelium, either absence or enlargement of the
lumen, and a less compact interstitial space (Figure 2). Many data, in the past, recognized in
a variety of species, various degrees of testicular damage, followed by a decline in androgen
synthesis and an impairment of spermatogenesis by DDT (1,1 bis (p-chlorophenyl) 2,2,2
trichloroethane), an organochlorine insecticide, from which the MTT derives [19,20].
Data relating to the biochemical trend of testosterone during treatment of the patients
have been inconsistent, but all agree in attributing clinical overt hypogonadism to many of
them. A prospective study observed a biphasic trend of total testosterone levels in seven
male patients enrolled to evaluate MTT toxicity after adrenalectomy for ACC [18]. It was
characterized by a sharp increase in the first three months of treatment, followed by a
decrease in testosterone after six months. Instead, free testosterone levels were significantly
reduced [13]. Basile et al. [16], in a report describing unwanted hormonal and metabolic
effects of MTT in an adjuvant setting, reported hypogonadism in 12 of 35 male patients
Cancers 2023, 15, 3234
10 of 12
(34.3%) who required testosterone replacement therapy. A more recent paper supported
these data, showing an increase in total testosterone in the first six months, followed by
a decrease over time in 24 patients during a follow-up over eight years in a retrospective,
longitudinal study [6].
In this regard, we evaluated the effects of MTT on the different cellular compartments—the
interstitial tissue with LCs and the seminiferous epithelium with SCs and germ cells—to
better understand the cellular compartments on which MTT acts. As expected, we observed
a significant decline in mRNA expression of specific markers of LCs, including 3β-Hsd1, a
key enzyme involved in the biosynthesis of steroid hormones [21], and Insl3 [22] in MTTtreated mice, confirming a detrimental impact by MTT on LCs, as shown in Figure 3a,b.
These results are in accordance with immunofluorescence analysis performed on the testicular sections stained with 3β-HSD antibody, revealing a less intense fluorescence signal
and fewer LCs stained in MTT-treated animals (Figure 3d). The expression levels of 3βHsd1 and Insl3 were partially restored by administering testosterone compared to those
in mice receiving MTT alone (Figure 3a,b), highlighting the protective role of testosterone.
Conversely, we demonstrated that Fsh-R in SCs was unaffected after MTT administration,
suggesting that these cells were not the main target of the drug and that the alteration of
spermatogenesis was unrelated to SC damage (Figure 3c).
The present findings support, for the first time in an animal model, a primary effect of
MTT on the testis and a steroidogenic process regarding what occurs in adrenal [23] and
ovarian steroidogenesis [5].
Androgens are essential for male fertility and the maintenance of spermatogenesis.
In the testis, LCs produce testosterone, and SCs are the primary target for this androgen,
which is required to support germ cell development. Decreased levels of testosterone cause
impairment of spermatogenesis and prevent the progression of germ cells to meiosis. Cell
counts from MTT-treated animals demonstrated few spermatozoa in the tubular lumen. In
addition, the sperm concentrations collected from the tail epididymis were significantly
lower (p < 0.001; Figure 4a). Furthermore, MTT administration caused a significant increase
in the percentage of sperm with abnormal chromatin structures, defined by the DNA
fragmentation index (%DFI), compared to untreated animals (p < 0.001; Figure 4b). Indeed,
testosterone replacement therapy allowed for recovery of cell number concentrations,
demonstrating significant protection against MTT-induced sperm damage. These data
support the well-recognized role of testosterone as an essential factor for spermatogenesis
and male fertility [24,25].
Data from the literature and those presented here support the following case report describing a 41-year-old black man treated with MTT for ectopic ACTH-syndrome
who developed erectile dysfunction with a biochemical picture of hypergonadotropic hypogonadism (low testosterone and elevated plasmatic gonadotropins at baseline and an
exaggerated response to gonadotropin-releasing hormone at dynamic testing) and testes
reduced in size. Testicular biopsy showed damage to the seminiferous epithelium with
maturation arrest and azoospermia. Administration of testosterone enanthate improved
libido and the ability to produce semen [26]. All the data collected so far allow us to
hypothesize that MTT acts by inducing a direct toxic effect on the male gonad and through
the reduction in androgen hormone synthesis.
Finally, after one-month discontinuation of MTT treatment, we showed recovery of
the MTT-induced detrimental effects on the testes. The expression of LC-specific genes,
Insl3 and 3β-Hsd1, in MTT-R mice was comparable to the levels in Ctrl-R mice. Moreover,
complete recovery of testosterone levels and initial recovery in epididymal sperm numbers
were observed. This outcome suggests that, as for females, the MTT-induced damage to
male gonads could be reversed by MTT withdrawal [5].
The present study and the previous ones that described the indirect effects strongly
support the evidence that MTT can induce hypogonadism in males. We can hypothesize
that the mechanism through which the drug exerts its action is twofold, with a first acute
phase aimed directly at the testis with a harmful effect, either on the germinal and tubular
Cancers 2023, 15, 3234
11 of 12
epithelium or on the LCs, with a negative impact on androgen production. Furthermore, the
evidence from our study demonstrates that MTT induces an impairment of spermatogenesis
with increased DNA fragmentation. These data agree with those observed during clinical
practice [26]. Furthermore, they provide strong evidence of the risk to sperm quality in
patients treated with MTT, opening the consideration of semen cryopreservation before
starting therapy for those patients planning to procreate. A second indirect mechanism,
which would explain the hypogonadism induced by MTT, occurs at a later time after
prolonged administration of MTT. It predicts a dual effect at the level of the pituitary
gonadotropic cells and at the liver, resulting in an inhibitory gonadotropin release [15] and
in an increase in the circulating level of SHBG [6,13,16,18]. These conditions may lead to a
progressive decline in free testosterone and a paradoxical reduction in LH levels. These
data account for the effects of MTT clinically observed during therapy in patients with
ACC and hypercortisolism and for measures to be taken before and after MTT therapy.
However, to support this recommend, future clinical studies in male patients are needed to
confirm the findings.
5. Conclusions
For the first time, our study analyzed the effects of MTT treatment on the male
reproductive system using an in vivo model. It demonstrated that MTT induces adverse
effects on male mouse gonads, including changes in testis morphology and reduced sperm
concentration and quality.
Supplementary Materials: The following supporting information can be downloaded at:
https://www.mdpi.com/article/10.3390/cancers15123234/s1, Figure S1: Fsh-R and Insl3 expression
in seminiferous tubules and interstitium; Table S1: Sequence of oligonucleotides used as Real-time
PCR primers.
Author Contributions: Conceptualization, V.T., R.C., E.V. and A.S.; methodology, F.I., S.D.P., M.T. and
R.C.; software, R.C.; validation, E.V., R.C. and A.S.; formal analysis, M.T., E.V. and R.C.; investigation,
F.I., S.D.P., M.T., R.M., P.L., R.C. and A.S.; resources, E.V., R.C. and A.S.; data curation, R.C., E.V.
and A.S.; writing—original draft preparation, F.I.; writing—review and editing, R.C., E.V. and A.S.;
visualization, R.C. and A.S.; supervision, R.C., E.V. and A.S.; project administration, R.C. and A.S.;
funding acquisition, R.C., E.V. and A.S. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by Sapienza Progetti di Ateneo 2020, grant number RM120172B957E7DB
to A.S., and by the Ministry of Research under MIUR Grant PRIN 2017 2017TK7Z8L to E.V.
Institutional Review Board Statement: The animal study protocol was approved by the Local Ethics
Committee for Animal Research (672/2018-PR) of Sapienza University of Rome.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Acknowledgments: The authors wish to thank aipacus (Associazione Italiana Pazienti Cushing) for
sharing the aims of this study and encouraging it.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
Fassnacht, M.; Assie, G.; Baudin, E.; Eisenhofer, G.; de la Fouchardiere, C.; Haak, H.R.; de Krijger, R.; Porpiglia, F.; Terzolo, M.;
Berruti, A. Adrenocortical carcinomas and malignant phaeochromocytomas: ESMO-EURACAN Clinical Practice Guidelines for
diagnosis, treatment and follow-up. Ann. Oncol. 2020, 31, 1476–1490. [CrossRef]
Barbot, M.; Zilio, M.; Scaroni, C. Cushing’s syndrome: Overview of clinical presentation, diagnostic tools and complications. Best
Pract. Res. Clin. Endocrinol. Metab. 2020, 34, 101380. [CrossRef]
Stigliano, A.; Chiodini, I.; Giordano, R.; Faggiano, A.; Canu, L.; Della Casa, S.; Loli, P.; Luconi, M.; Mantero, F.; Terzolo, M.
Management of adrenocortical carcinoma: A consensus statement of the Italian Society of Endocrinology (SIE). J. Endocrinol.
Investig. 2016, 39, 103–121. [CrossRef]
Cancers 2023, 15, 3234
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
12 of 12
Baudry, C.; Coste, J.; Bou Khalil, R.; Silvera, S.; Guignat, L.; Guibourdenche, J.; Abbas, H.; Legmann, P.; Bertagna, X.; Bertherat, J.
Efficiency and tolerance of mitotane in Cushing’s disease in 76 patients from a single center. Eur. J. Endocrinol. 2012, 167, 473–481.
[CrossRef] [PubMed]
Innocenti, F.; Cerquetti, L.; Pezzilli, S.; Bucci, B.; Toscano, V.; Canipari, R.; Stigliano, A. Effect of mitotane on mouse ovarian follicle
development and fertility. J. Endocrinol. 2017, 234, 29–39. [CrossRef]
Delbarba, A.; Cosentini, D.; Facondo, P.; Lagana, M.; Pezzaioli, L.C.; Cremaschi, V.; Alberti, A.; Grisanti, S.; Cappelli, C.; Ferlin, A.;
et al. Androgen serum levels in male patients with adrenocortical carcinoma given mitotane therapy: A single center retrospective
longitudinal study. Front. Endocrinol. 2023, 14, 1128061. [CrossRef] [PubMed]
Cerquetti, L.; Bucci, B.; Carpinelli, G.; Lardo, P.; Proietti, A.; Saporito, R.; Rindi, G.; Petrangeli, E.; Toscano, V.; Stigliano, A.
Antineoplastic Effect of a Combined Mitotane Treatment/Ionizing Radiation in Adrenocortical Carcinoma: A Preclinical Study.
Cancers 2019, 11, 1768. [CrossRef]
Griffin, D.K.; Ellis, P.J.; Dunmore, B.; Bauer, J.; Abel, M.H.; Affara, N.A. Transcriptional profiling of luteinizing hormone
receptor-deficient mice before and after testosterone treatment provides insight into the hormonal control of postnatal testicular
development and Leydig cell differentiation. Biol. Reprod. 2010, 82, 1139–1150. [CrossRef]
Davidyan, A.; Pathak, S.; Baar, K.; Bodine, S.C. Maintenance of muscle mass in adult male mice is independent of testosterone.
PLoS ONE 2021, 16, e0240278. [CrossRef]
Machida, T.; Yonezawa, Y.; Noumura, T. Age-Associated Changes in Plasma Testosterone Levels in Male Mice and Their Relation
to Social Dominance or Subordinance. Horm. Behav. 1981, 15, 238–245. [CrossRef] [PubMed]
Renvoize, C.; Biola, A.; Pallardy, M.; Breard, J. Apoptosis: Identification of dying cells. Cell Biol. Toxicol. 1998, 14, 111–120.
[CrossRef]
Zirkin, B.R.; Awoniyi, C.; Griswold, M.D.; Russell, L.D.; Sharpe, R. Is FSH required for adult spermatogenesis? J. Androl. 1994, 15,
273–276.
Daffara, F.; De Francia, S.; Reimondo, G.; Zaggia, B.; Aroasio, E.; Porpiglia, F.; Volante, M.; Termine, A.; Di Carlo, F.; Dogliotti, L.;
et al. Prospective evaluation of mitotane toxicity in adrenocortical cancer patients treated adjuvantly. Endocr. Relat. Cancer 2008,
15, 1043–1053. [CrossRef] [PubMed]
Chortis, V.; Taylor, A.E.; Schneider, P.; Tomlinson, J.W.; Hughes, B.A.; O’Neil, D.M.; Libe, R.; Allolio, B.; Bertagna, X.; Bertherat,
J.; et al. Mitotane therapy in adrenocortical cancer induces CYP3A4 and inhibits 5alpha-reductase, explaining the need for
personalized glucocorticoid and androgen replacement. J. Clin. Endocrinol. Metab. 2013, 98, 161–171. [CrossRef]
Gentilin, E.; Mole, D.; Gagliano, T.; Minoia, M.; Ambrosio, M.R.; Degli Uberti, E.C.; Zatelli, M.C. Inhibitory effects of mitotane on
viability and secretory activity in mouse gonadotroph cell lines. Reprod. Toxicol. 2014, 45, 71–76. [CrossRef]
Basile, V.; Puglisi, S.; Calabrese, A.; Pia, A.; Perotti, P.; Berruti, A.; Reimondo, G.; Terzolo, M. Unwanted Hormonal and Metabolic
Effects of Postoperative Adjuvant Mitotane Treatment for Adrenocortical Cancer. Cancers 2020, 12, 2615. [CrossRef]
Vikner, M.E.; Krogh, J.; Daugaard, G.; Andreassen, M. Metabolic and hormonal side effects of mitotane treatment for adrenocortical
carcinoma: A retrospective study in 50 Danish patients. Clin. Endocrinol. 2021, 94, 141–149. [CrossRef] [PubMed]
Nader, N.; Raverot, G.; Emptoz-Bonneton, A.; Dechaud, H.; Bonnay, M.; Baudin, E.; Pugeat, M. Mitotane has an estrogenic effect
on sex hormone-binding globulin and corticosteroid-binding globulin in humans. J. Clin. Endocrinol. Metab. 2006, 91, 2165–2170.
[CrossRef]
Ben Rhouma, K.; Tebourbi, O.; Krichah, R.; Sakly, M. Reproductive toxicity of DDT in adult male rats. Hum. Exp. Toxicol. 2001, 20,
393–397. [CrossRef] [PubMed]
Veeramachaneni, D.N. Impact of environmental pollutants on the male: Effects on germ cell differentiation. Anim. Reprod. Sci.
2008, 105, 144–157. [CrossRef]
Rasmussen, M.K.; Ekstrand, B.; Zamaratskaia, G. Regulation of 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4) isomerase:
A review. Int. J. Mol. Sci. 2013, 14, 17926–17942. [CrossRef] [PubMed]
Roth, M.Y.; Lin, K.; Bay, K.; Amory, J.K.; Anawalt, B.D.; Matsumoto, A.M.; Marck, B.T.; Bremner, W.J.; Page, S.T. Serum insulin-like
factor 3 is highly correlated with intratesticular testosterone in normal men with acute, experimental gonadotropin deficiency
stimulated with low-dose human chorionic gonadotropin: A randomized, controlled trial. Fertil. Steril. 2013, 99, 132–139.
[CrossRef]
Tritos, N.A. Adrenally Directed Medical Therapies for Cushing Syndrome. J. Clin. Endocrinol. Metab. 2021, 106, 16–25. [CrossRef]
Smith, L.B.; Walker, W.H. The regulation of spermatogenesis by androgens. Semin. Cell Dev. Biol. 2014, 30, 2–13. [CrossRef]
[PubMed]
Ruwanpura, S.M.; McLachlan, R.I.; Meachem, S.J. Hormonal regulation of male germ cell development. J. Endocrinol. 2010, 205,
117–131. [CrossRef] [PubMed]
Sparagana, M. Primary hypogonadism associated with o,p’ DDD (mitotane) therapy. J. Toxicol. Clin. Toxicol. 1987, 25, 463–472.
[CrossRef]
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English
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HOUSING TYPES, POPULATION ATTRIBUTES AND THE DEVELOPMENT PATH OF HOUSING IN LARGE CITIES – A CASE STUDY OF ZHENGZHOU, CHINA
|
International journal of strategic property management
| 2,023
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International Journal of Strategic Property Management
ISSN: 1648-715X / eISSN: 1648-9179
2023 Volume 27 Issue 5: 317–331
https://doi.org/10.3846/ijspm.2023.20425 International Journal of Strategic Property Management
ISSN: 1648-715X / eISSN: 1648-9179
2023 Volume 27 Issue 5: 317–331
https://doi.org/10.3846/ijspm.2023.20425 International Journal of Strategic Property Management
ISSN: 1648-715X / eISSN: 1648-9179
2023 Volume 27 Issue 5: 317–331
https://doi.org/10.3846/ijspm.2023.20425 Received 16 March 2023; accepted 19 November 2023 Received 16 March 2023; accepted 19 November 2023 Abstract. Large-city housing costs are commonly attributed to excessive investment or financialization of the property
market. The primary cause of housing issues is the imbalance between housing supply and demand. In the context of the
urban housing market, the dynamic matching of housing supply and population type plays a crucial role in promoting
equilibrium, maximizing the effectiveness of the market’s long-term mechanism, and preserving the coordination and sta-
bility of the market. To illustrate this point, the paper analyzes the impact of different attributes of population on housing
demand from the perspective of dynamic matching between population attributes and housing types. Taking Zhengzhou
City in Henan Province as an example, we consider the relationship between population attributes and housing types in
the past, construct a theoretical model of population and housing type and anticipate the future supply scale of various
housing kinds. Ultimately, our findings provide a theoretical justification for curbing excessive financialization and housing
bubbles in large cities. Furthermore, we propose a practical method for urban housing to return to the essence of life from
the perspective of matching housing supply and demand. ords: urban housing, housing types, population attributes, population increment, development path, housing mat paralleled by the movement of people. This migration has
engendered a surge in the populace, thereby intensify-
ing the need for habitation in urban locales. As a result,
this has perpetually stimulated the escalation of housing
costs in Chinese cities. However, the housing supply in
big cities is inelastic (Howard & Liebersohn, 2021) and
does not match the growth of housing demand, which
tends to cause housing market failure. The inadequacies
of the housing market not only pose a threat to the eco-
nomic development at the local level, but also result in
prolonged commuting periods, diminished standards of
living, and impede social mobility for specific segments
of the population. In numerous countries, metropolitan
areas have witnessed a surge in the escalation of these ap-
prehensions in recent times (Rodriguez-Pose & Storper,
2020). Contemporary research has successfully identified
a plethora of factors responsible for the escalation in resi-
dential property prices. The factors in question are those
that induce a hike in housing demand and those that are
responsible for the amplification in housing supply. These
factors entail but are not limited to, construction costs, *Corresponding author. E-mail: lizhihui@zzu.edu.cn This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unre-
stricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright © 2023 The Author(s). Published by Vilnius Gediminas Technical University HOUSING TYPES, POPULATION ATTRIBUTES AND
THE DEVELOPMENT PATH OF HOUSING IN LARGE CITIES –
A CASE STUDY OF ZHENGZHOU, CHINA Zhifeng SHEN1, Jing LI1, Ahsan SIRAJ1, Shilpa TANEJA2, Zhihui LI1,* 1 School of Management, Zhengzhou University, Zhengzhou, China
2 School of Management, IILM University, Gurugram, India 1 School of Management, Zhengzhou University, Zhengzhou, China
2 School of Management, IILM University, Gurugram, India Introduction Our aim is to miti-
gate the escalation of housing prices by aligning different
housing types with specific population attributes and to
address the housing challenges faced by low- and mid-
dle-income individuals. To achieve this, we have gath-
ered and meticulously organized population and hous-
ing data from Zhengzhou, China. Using the SPSS data
analysis software, we have established a linear regression
equation to model the relationship between population
and housing. Based on this model, we have made projec-
tions for various population types from 2021 to 2035. Subsequently, we employed the same linear regression
equation to predict the data for different housing types. Our findings present a match between different popula-
tion attributes and housing types. We can able to find the
minimum housing needs as well as the future preferences
of different population attributes from a demand per-
spective, which can enable us to make supply-side efforts
to deliver housing options that cater to the specific needs
and attributes of the population. We believe that an effec-
tive match between population types and housing types
will provide a basis for decision-making for government
and relevant enterprises. geographic limitations, land policies, income levels, pop-
ulation expansion, housing preferences, tax implications,
and speculative demand, among others (Glaeser et al.,
2017; Hanink et al., 2012; Huang, 2004; Liang et al., 2016;
Yu, 2011; Yu & Huang, 2016). Some scholars have discovered a correlation between
demographic shifts and housing costs. Previous studies in
this area mainly point out population size (Ding, 2019),
population density (Miles, 2012), population structure
(Gevorgyan, 2019; Green & Lee, 2016; Levin et al., 2009;
Myers, 1990), labor mobility (Akbari & Aydede, 2012;
Wang et al., 2018), household demographic characteristics
(Eichholtz & Lindenthal, 2014; Lauster, 2008) and other
factors related to housing prices. Mulder (2006) posits the
existence of a symbiotic relationship between the popu-
lation and housing sectors. A burgeoning populace has
a significant impact on the demand for housing, which
in turn influences the spatial distribution of people, ei-
ther encouraging or impeding mobility. Concurrently, the
escalation of housing costs has resulted in a geographic
mismatch between residential areas and places of work,
culminating in the gradual suburbanization of the labor
force. Most urban inhabitants tend to reside in suburban
areas while working in the central business district, result-
ing in an increased commuting time between their abodes
and workplaces. Introduction Urban housing has long been a prominent concern for
those residing in metropolitan areas. Nonetheless, follow-
ing the implementation of housing market reform, hous-
ing prices in China have experienced a significant surge,
ultimately leading to the emergence of the wealth effect
pertaining to housing. Consequently, housing has gradu-
ally transformed from a mere consumer good to an invest-
ment product, with financialization further exacerbating
the issue of housing inequality. This, in turn, has come
to represent a crucial symbol of social differentiation be-
tween those of affluent and impoverished backgrounds,
ultimately serving as a pivotal mechanism that contrib-
utes to social inequality and differentiation (Li & Fan,
2020). In light of the issue of exorbitant residential costs,
the Chinese government has put forward the notion that
abodes should serve as dwellings rather than as commodi-
ties subject to speculation. Additionally, authorities have
advocated for the formation of a rental-purchase housing
framework. The expansion of metropolitan areas has been Copyright © 2023 The Author(s). Published by Vilnius Gediminas Technical University This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unre-
stricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Z. Shen et al. Housing types, population attributes and the development path of housing in large... Z. Shen et al. Housing types, population attributes and the development path of housing in large... 318 study is that we fully consider the matching relationship
between different attributes of population and housing
types, and propose corresponding suggestions based on
the existing mismatch between population attributes and
housing. For example, we take into account the emerging
youth groups in the city – college students and graduates. Because they are the target of competition for talent in
each city, cities will provide housing subsidy policies to
attract and retain these talents. The provision of reason-
ably priced housing, economical dwellings, metropolitan
villages, extended-stay rental units, and additional forms
of subsidized housing can decrease both procurement
demand and investment impetus. This not only resolves
the housing predicament of low- and moderate-income
individuals but concurrently curtails their anticipations
of future escalation in housing costs. In this study, we
investigate the correlation between population and hous-
ing dynamics in major urban areas. Introduction Additionally, there exists a causal connec-
tion between population migration and the volatility of
house prices. In the short term, the availability of urban
housing is constrained. The inflow of migrants will exacer-
bate the already high urban housing prices, which will, in
turn, lead to higher living expenses, ultimately impeding
population mobility. There is an inverted U relationship
between the proportion of the population in large cities
and urbanization (Liu et al., 2022). Among demographic
factors, both qualitatively and quantitatively, housing de-
mand varies with age of the population. This change deter-
mines future demand for housing. Over the past few de-
cades, many countries have experienced severe population
aging. In large cities facing the challenge of aging, there is
a contradictory relationship between population dynamics
and housing stock, with a surplus of housing stock that
is unable to meet the housing needs of the population. The mismatch between the existing housing stock and
the needs of the population stems from changes in de-
mographic and household structures in an aging society,
as well as changes in the urban housing stock and rental
system (Garha & Azevedo, 2021). The aforementioned oc-
currences are also prevalent within urban areas of China. W
hi k h
h
li
f
l i
d
i 1.2. Land market and housing supply Housing and land are institutionally inseparable. In the
early days, the government viewed land as an asset. The
commoditization of land became a means of generating
revenue and paved the way for the massive development
of commercial housing in China. However, the assetiza-
tion of land stimulated an increase in housing assetiza-
tion, making housing affordability an increasingly acute
problem in China’s cities, especially for those new entrants
to the housing market (Chen & Wu, 2022). Since 2008,
the importance of urban land has been increasing and the
supply of urban construction land has attracted great at-
tention and regulation by the government. Government
intervention in the land market generally takes two forms:
land use regulation and direct government control of land
supply. When government obtain direct control over resi-
dential land supply, it usually determines the quantity of
land supply based on the projected demand for housing
(Chiu, 2007). At a certain building density, the quantity of
land supply should be positively related to the quantity of
new housing supply. When there is an adequate supply of
land, developers are able to respond quickly to an increase
in housing price by building more housing units. By con-
trast, the responsiveness of new housing supply to change
in housing price will be constrained when land supply is
inadequate (Yan et al., 2014).i l
Moreover, the housing category plays a significant role
in producing the social-spatial segregation of urban in-
habitants. There exists a social segregation between house-
hold residents and mobile populations within the housing
area, where different housing categories typically occupy
distinct regions of the city, catering to diverse social and
economic groups. With regards to urban population mo-
bility, few scholars have discovered that the rise in the
floating population impacts the demand in the real estate
market. The immigration process comprises of stages, and
the process of settling immigrant populations in cities is
a dynamic process that involves three stages: arrival, set-
tlement, and stabilization (Llopis Alvarez & Muller-Eie,
2022). Newly arrived immigrants may be uncertain as to
whether they will establish permanent residence in the
city, and thus tend to prefer rental accommodations of
smaller size, as compared to long-term residents. Young
people, who have lived and worked in the city for an ex-
tended period, face the prospect of marriage and often
seek to purchase a house suitable for family life. 1.1. The impact of demographics on housing
demand In the late 1960s, scholars analyzed the relationship be-
tween demographic fluctuations and the housing con-
struction cycle and demonstrated the impact of demo-
graphic changes on total housing demand (Campbell,
1963). Among demographic factors, age structure is con-
sidered to be the most important factor in determining
the interaction between population and housing demand
(Myers & Pitkin, 2009). The effect of population growth
on housing demand is contingent upon age. The younger
demographic exhibits a greater degree of mobility among
new households, which subsequently leads to an ampli-
fication in the demand for fresh housing units. On the We think that the alignment of population and resi-
dential accommodations can effectively diminish the
demand for housing investments. While previous stud-
ies have considered the relationship between population
and housing, they have not revealed a match between
population attributes and housing type. In particular,
there is a lack of focus on those who rely on housing
security, who are generally economically weaker and find
it difficult to survive living in the city. The novelty of our International Journal of Strategic Property Management, 2023, 27(5): 317–331 319 other hand, an aging populace within society has a mark-
edly deleterious effect on housing demand (Malmberg,
2010). The advent of population groups into adulthood
engenders a surge in the exigency for housing. As these
individuals mature, they eventually succumb to mortality
or relocate to nursing homes, consequently bequeathing
a substantial number of unoccupied dwellings. This, in
turn, engenders a commensurate rise in the provision
of housing within the housing market. The aging pro-
cess might bring down house prices in the future. On
the contrary, considering the accelerated urban popula-
tion movement, the process of urbanization will generate
more housing demand, thereby promoting housing pric-
es (Chen et al., 2011). Several academics have conducted
research into the effects of urbanization on the real es-
tate industry, as well as the surge in inflexible demand
for housing that has resulted from the growth of urban
populations. Urban areas possess a greater potential for
attracting skilled individuals due to their developmental
benefits. Due to the widening income gap between rural
and urban residents, many farmers left their hometowns
and ventured to the major cities to seek employment op-
portunities. Moreover, the enhancement of educational
attainment has the capacity to augment the populace of
young homeowners (Myers et al., 2019). 1.1. The impact of demographics on housing
demand Studies show
that human capital agglomeration has a long-term posi-
tive effect on housing prices and economic development
(Yang & Pan, 2020). China’s housing reform is charac-
terized by a distinctive housing supply system that en-
genders significant differentiation in urban social space. The underlying factors responsible for this social-spatial
differentiation include the household registration system
(Zhao & Howden-Chapman, 2010), work unit character-
istics (Li & Wu, 2006), and the dual land system (Zhao,
2013). Hence, residents possessing local urban household
registration exhibit a greater degree of contentment with
their living conditions relative to the floating population.i constraints emanating from economic and social status,
as well as exclusionary systems, which make it difficult
for them to acquire housing in inflow areas. Therefore,
immigrants frequently opt for public rental housing as a
viable alternative to meet their housing needs. The inflow
of migrant populations contributes to urban development,
resulting in rapid escalation of urban housing prices, but
only a small percentage of migrants have been able to
purchase homes in the receiving city. Even if the migrant
population has a high income, it remains challenging to
obtain urban housing due to the high construction costs
and the long-life cycle of urban housing, resulting in a
less flexible housing stock that cannot accommodate the
needs of highly active populations and households. As
a result, the housing market responds less effectively to
demographic changes, leading to a mismatch between
population and housing. Addressing the mutual match-
ing of housing and population in cities is essential for the
promotion of social integration, as well as the achievement
of new urbanization strategies. 1.2. Land market and housing supply Skov-
gaard Nielsen (2017) has discovered that immigrants face When the government intensified its intervention in
the land market, the supply of residential land was severe-
ly restricted. When the supply of residential land decreas-
es, developers race to buy government land, making land
prices high, which in turn promotes higher costs for the
housing built. Despite rapidly rising home prices, more
than half of all households are still interested in buying
a new home. The central policy has introduced purchase
restriction policies in the hope of controlling housing de-
mand, yet it has not cooled the city’s real estate market, Z. Shen et al. Housing types, population attributes and the development path of housing in large... Z. Shen et al. Housing types, population attributes and the development path of housing in large... 320 this price is even higher than 30,000 yuan, which outstrips
the rise in the material index, such as Beijing and Shang-
hai. The burgeoning real estate investment sector is evi-
dent, as evidenced by the data released by the Zhengzhou
Bureau of Statistics indicating that the total sales area of
commercial housing was 209.99 million m2 before 2010,
which had risen to approximately 439.28 million m2 by
the end of 2020 (Henan Province Bureau of Statistics
[HAPBS], 2021). The proportion of investment has gradu-
ally increased from 22% in 2007 to 43% in 2017, with the
proportion of real estate investment remaining above 30%
since 2011. Housing prices have continued to increase
annually. The real estate market began to rise steadily in
2010, and by 2016 Zhengzhou house prices began to soar,
and since then the rise has eased. After nearly ten years
of development, the average price of commercial hous-
ing in Zhengzhou has reached 10,215 yuan/m2 in 2020
from 3,994 yuan/m2 in 2007. The high price of housing
has brought great pressure on the life of ordinary urban
residents. which stems from the fact that the localized policies
might be, to a certain extent, different from the central
government’s original guidelines. When local municipal
government formally issued its localized measures for im-
plementing the central government’s policies, the market
uncertainty induced by the State Council’s notice disap-
peared, confidence about the future growth of housing
prices was rebuilt up. 1.3. Analysis of urban housing types and problems China’s urban housing reform has resulted in a housing
system that is increasingly market-oriented. This shift has
given rise to the coexistence of numerous distinct hous-
ing types. China has implemented the dual housing supply
system consisting of commercial housing provided by the
private sector and social housing provided by the public
sector. The current housing supply mainly consists of pri-
vate sector housing (regular commercial housing), subsi-
dized housing (e.g., affordable housing), and public rental
housing (e.g., talent apartments). Commercial housing is
built by private developers and sold at market-determined
prices. Therefore, the supply of commercial housing is ex-
cessive and the prices are rising too fast. Affordable hous-
ing is planned and directed by the state. It has the charac-
teristics of economy, security, and practicability, and can
be suitable for the affordability of low- and middle-income
families. The talent apartment serves as a rental living fa-
cility that supports talent employment and provides tem-
porary living arrangements for entrepreneurs in a specific
location. The apartment follows the principle of renting
rather than selling, with a household size ranging from
10 to 200 m2 and a limited rental period. Its accommoda-
tion is primarily provided to professionals, doctors, and
both international and local university students who have
not purchased their own houses and are not entitled to
other subsidized housing. At present, China’s urban hous-
ing system faces some unresolved internal contradictions,
such as rapidly increasing prices for regular commercial
housing, insufficient coverage of subsidized housing and
public rental housing, and a mismatch of housing supply
and demand levels, calling into question the effectiveness
of existing land supply policies. Although land allocations
for residential development have increased, they have not
matched the growth in housing demand. Policies to regu-
late access to housing for urban migrants further add to
the complexity and can be socially destabilizing. 1.3.2. Insufficient coverage of affordable housing The proliferation of commercial housing is advancing
expeditiously, while the subsidized housing construction
that is spearheaded by the government is consistently un-
derprovided. Consequently, this has not only resulted in
a swift escalation of housing costs, but also in a dispar-
ity within the housing system. The supply of affordable
housing is relatively limited, and local governments tend
to restrict the resources for affordable housing to the local
household population. In other words, the threshold for
obtaining affordable housing is so high that it fails to cover
the large numbers of rural migrants and urban workers in
the process of urbanization (He et al., 2022). In addition,
cities with more dependence on land finance and higher
fiscal autonomy were less likely to devote land to afford-
able housing construction unless local governments’ reli-
ance on urban land-based interests is weakened. Although
the central government has issued a series of guaranteed
housing support policies to help ease the housing difficul-
ties of new citizens, young people, etc., local government
revenues are mainly dependent on land grant revenues,
and high land prices allow local governments to achieve
rapid short-term economic growth (Zou, 2014). In cities
with a high level of economic development, housing and
land prices are high, leading to high costs for sheltered
housing. It can be seen that resources for affordable hous-
ing are not well concentrated in areas with high rates of
population urbanization, and there is a certain degree of
spatial mismatch (He et al., 2022). 1.2. Land market and housing supply Some potential homebuyers who are
not excluded from the market by the limits seized the op-
portunity to purchase homes, therefore driving up hous-
ing prices. This was far from the expectations of policy
makers (Jia et al., 2018). 1.3.3. A mismatch between supply and demand levels The annual increase in the
count of individuals with university degrees results in a
new cohort of youthful migrants emerging from educa-
tional institutions. As they leave the educational system
for the first time, they find themselves in a transitional
phase characterized by economic vulnerability and the
need to further refine their skills across diverse domains. Consequently, this has prompted municipal authorities
across different cities to direct their attention towards
the housing predicament faced by these graduates, in an
endeavor to exert their utmost efforts in aiding the as-
piring citizens and young individuals, among others, in
mitigating the challenges encountered in securing hous-
ing. Urban housing prices affect the job location choice
of college graduates (Lin et al., 2022). Although most
cities have provided housing subsidy policies for gradu-
ates for talent introduction, the current housing prices
are still beyond the graduates’ financial ability (Li et al.,
2017), and most of them have to choose the form of tal-
ent apartments with rented rooms, becoming a new gen-
eration of housing difficulty body. The number of college
students in Zhengzhou has even exceeded one million. Although many schools provide cheap accommodation
for college students, growth in the number of college stu-
dents has increased demand for accommodation beyond
that which universities themselves can provide, provok-
ing private investor supply-side responses. The large
number of students seeking off-campus rental housing
has created pressure on the city’s rental housing market
(Sood & Vicino, 2023; Wilkinson & Greenhalgh, 2022). Although college students become graduates or perma-
nent residents of the city after a few years new groups of 2. Model construction and analysis Based on the aforementioned housing development issues
prevalent in major urban centers, the present study aims
to establish a model for the progression of housing advan-
tages in said areas, which aligns housing categories with
corresponding population demographics and fosters the
healthy growth of urban housing. To this end, regression
analysis was conducted on the factors influencing the sup-
ply and population distribution of housing in Zhengzhou,
utilizing data procured from “Henan Statistical Yearbook”
(HAPBS, 2021), “Zhengzhou Housing Security and Real
Estate Bureau 2021 Annual Report on Government In-
formation Disclosure” (Zhengzhou Housing Security and
Real Estate Bureau, 2021). 1.3.3. A mismatch between supply and demand levels Moreover, cities are now reforming
their household registration systems to attract talent,
and have enacted a number of subsidized settlement
policies. Therefore, the demand for housing by the reg-
istered population can well reflect the development of
the city. Migrant workers have an even more profound
impact on urban housing. They are constantly flowing
into cities and choosing urban life for the sake of their
children’s education and improved quality of life, which
has led to a growing urban population and rapidly rising
housing prices (Wang et al., 2017). However, the major-
ity of them are primarily occupied in occupations that
offer lower wages, and their absence of official residency
status poses a challenge in accessing affordable urban
housing, thereby exacerbating the severity and com-
plexity of the housing issue. The annual increase in the
count of individuals with university degrees results in a
new cohort of youthful migrants emerging from educa-
tional institutions. As they leave the educational system
for the first time, they find themselves in a transitional
phase characterized by economic vulnerability and the
need to further refine their skills across diverse domains. The permanent residents who have already integrated
into the city, formed their families and lived in the city
for a long time. They have a certain economic base and
can afford the price of general commercial housing. The
registered population is divided according to the place
of registration of the household, and they have the hous-
ing benefits of that city. The restriction on housing reg-
istration makes it easier for the registered population to
qualify for housing. Moreover, cities are now reforming
their household registration systems to attract talent,
and have enacted a number of subsidized settlement
policies. Therefore, the demand for housing by the reg-
istered population can well reflect the development of
the city. Migrant workers have an even more profound
impact on urban housing. They are constantly flowing
into cities and choosing urban life for the sake of their
children’s education and improved quality of life, which
has led to a growing urban population and rapidly rising
housing prices (Wang et al., 2017). However, the major-
ity of them are primarily occupied in occupations that
offer lower wages, and their absence of official residency
status poses a challenge in accessing affordable urban
housing, thereby exacerbating the severity and com-
plexity of the housing issue. 1.3.3. A mismatch between supply and demand levels Another significant factor contributing to elevated hous-
ing prices is the incongruity that exists between supply
and demand. A spatial partition between the demand for
housing and the supply of land exacerbates the predica-
ment of the dislocation between the demand and supply In previous years, data from China’s statistical yearbook
reveals that the average cost of commercial housing has
increased from 1854 yuan/m2 to 9980 yuan/m2 (National
Bureau of Statistics of China, 2020). In some mega-cities, International Journal of Strategic Property Management, 2023, 27(5): 317–331 321 students, and there are differences in the housing demand
among these different population attributes. h of housing. This predicament is gradually escalating. The
equilibrium between the housing supply and demand var-
ies considerably across different regions in China. Some
regions experience a scarcity of housing supply, while oth-
ers have an oversupply of housing in the market (Weng
& Pan, 2019). Furthermore, the present occupancy of
the subsidized housing primarily comprises of resettled
households, which strays from the initial aim of safe-
guarding the housing rights and interests of the low- and
middle-income urban groups. Additionally, the design of
the housing does not align with their living necessities. This incongruity between supply and demand will inevi-
tably result in a significant accumulation of commercial
housing. On one hand, certain individuals are incapable
of affording housing, while on the other, some high-in-
come individuals possess multiple residences. In order
to curb excessively high housing prices, China’s Central
Government has announced a policy of restricting home
purchases. While the purchase restriction policy reduced
house prices and transaction amounts, it did not affect the
housing investment or construction market. Heterogene-
ity exists across cities, with first- and second-tier cities,
as well as highly urbanized cities, experiencing significant
reductions in house prices following the implementation
of the policy, but the housing policy is less effective in
curbing speculative demand (Wu & Li, 2018). f
The permanent residents who have already integrated
into the city, formed their families and lived in the city
for a long time. They have a certain economic base and
can afford the price of general commercial housing. The
registered population is divided according to the place
of registration of the household, and they have the hous-
ing benefits of that city. The restriction on housing reg-
istration makes it easier for the registered population to
qualify for housing. 2.1. Variables selection and regression model In recent years, Zhengzhou has been developing continu-
ously, becoming a national central city and an interna-
tional comprehensive transportation hub, with a strong
population absorption force in the Central Plains. As a
result, population density has increased significantly and
housing has become an important issue. In previous stud-
ies on the relationship between population and housing,
some scholars have divided the population by household
size (Day, 2018), population structure (Choi et al., 2019),
and mobility status (Li et al., 2022). In this paper, from
the perspective of population attributes, we mainly select
the resident population, registered population, migrant
workers, university graduates, and the group of college Although many schools provide cheap accommodation
for college students, growth in the number of college stu-
dents has increased demand for accommodation beyond
that which universities themselves can provide, provok-
ing private investor supply-side responses. The large
number of students seeking off-campus rental housing
has created pressure on the city’s rental housing market
(Sood & Vicino, 2023; Wilkinson & Greenhalgh, 2022). Although college students become graduates or perma-
nent residents of the city after a few years, new groups of Shen et al. Housing types, population attributes and the development path of housing in large... Z. Shen et al. Housing types, population attributes and the development path of housing in large... 322 college students are also increasing every year, and they
still have an important impact on urban housing. hi Y1 = A + aX1 + bX2 + cX3 + dX4 + eX5;
Y2 = B + fX1 + gX2 + hX3 + iX4 + jX5;
Y3 = C + kX1 + lX2 + mX3 + nX4 + oX5, Therefore, we choose these five groups of population
as the independent variable, and the amount of new area
of general commercial housing, talent apartments and
affordable housing as the dependent variable for the dif-
ferent housing needs of these groups. We establish the
regression equation model of population attributes and
housing. The symbols are described in Table 1. where A, B, C, a, b..., o are unknown parameters. where A, B, C, a, b..., o are unknown parameters. 2.2. Data collection and organization The data pertaining to population and real estate utilized
in this study has been sourced from the appropriate official
government websites and platforms, namely, “Henan Sta-
tistical Yearbook” (HAPBS, 2021), “Zhengzhou Housing
Security and Real Estate Bureau 2021 Annual Report on
Government Information Disclosure” (Zhengzhou Hous-
ing Security and Real Estate Bureau, 2021). According to
the statistical data, the data on the population types and
housing types in Zhengzhou in the past ten years are ob-
tained. The specific data are shown in Table 2, and Table 3. Table 1. Representation of regression model variables
Symbol
Implication
Unit
X1
Permanent resident population
10,000
X2
Registered population
10,000
X3
Migrant workers
10,000
X4
University graduates
10,000
X5
College students
10,000
Y1
New commercial housing
10,000 m2
Y2
New talent apartments
10,000 m2
Y3
New affordable housing
10,000 m2 Table 1. Representation of regression model variables hi
A regression equation was established based on the re-
lationship between the dates from 2010 to 2020 and each
population type, and then the independent variable date
was substituted into the equation to obtain the popula-
tion type prediction data for each year. The forecast data Table 2. 2010–2019 population data
Date
Permanent resident
population
Registered population
Migrant workers
University graduates
College students
2010
866
734
115
56
102
2011
886
744
120
60
103
2012
903
758
119
67
116
2013
919
762
125
72
125
2014
938
785
130
80
137
2015
957
810
134
85
146
2016
972
827
141
91
157
2017
988
842
144
104
180
2018
1014
863
149
106
184
2019
1035
878
146
116
200
Table 3. 2010–2019 housing data
Date
New commercial housing
New talent apartments
New affordable housing
2010
1200
0
120
2011
1306
0
150
2012
1226
0
136
2013
1313
0.4
132
2014
1293
0.6
140
2015
1695
1
167
2016
2571
1.3
230
2017
2735
2.5
284
2018
3330
2.8
360
2019
3242
3
340 Table 2. 2010–2019 population data nternational Journal of Strategic Property Management, 2023, 27(5): 317–331 323 Table 4. 2.2. Data collection and organization Predicted data of Zhengzhou population type from 2021 to 2035
Date
Permanent resident
population
Registered population
Migrant workers
University graduates
College students
2021
1296
1054
183
135
215
2022
1340
1093
191
141
223
2023
1389
1132
198
149
235
2024
1434
1161
205
156
243
2025
1469
1182
212
164
252
2026
1506
1210
219
169
259
2027
1548
1249
226
175
266
2028
1580
1288
233
181
271
2029
1610
1327
240
186
278
2030
1640
1366
247
192
288
2031
1675
1405
254
198
297
2032
1706
1444
261
203
307
2033
1752
1474
270
209
317
2034
1789
1503
278
216
326
2035
1826
1536
288
225
336 Table 4. Predicted data of Zhengzhou population type from 2021 to 2035 Table 5. Forecast data for each housing type based on natural development trends Table 5. Forecast data for each housing type based on natural development trends
Date
New commercial housing
New talent apartments
New affordable housing
2021
3835.23
3.636
367.681
2022
4110.86
3.952
393.142
2023
4386.49
4.268
418.603
2024
4662.12
4.584
444.064
2025
4937.75
4.9
469.525
2026
5213.38
5.216
494.986
2027
5489.01
5.532
520.447
2028
5764.64
5.848
545.908
2029
6040.27
6.164
571.369
2030
6315.9
6.48
596.83
2031
6591.53
6.796
622.291
2032
6867.16
7.112
647.752
2033
7142.79
7.428
673.213
2034
7418.42
7.744
698.674
2035
7694.05
8.06
724.135 Z. Shen et al. Housing types, population attributes and the development path of housing in large... 324 Table 6. Relationship between the independent variables and the area of new commercial housing
Model
Unstandardized coefficient
Standardized coefficient
t
Sig. B
Std. Error
Beta
(Constant)
–5856.357
6823.789
–0.858
0.439
College students
–5.603
15.765
0.219
–0.355
0.740
Registered population
17.144
7.629
1.013
2.247
0.088
Migrant workers
33.221
30.833
0.546
1.077
0.342
Permanent resident population
–11.265
5.949
0.730
–1.893
0.131
University graduates
14.888
26.385
0.367
0.564
0.603 able 6. Relationship between the independent variables and the area of new commercial housing Table 7. Relationship between the independent variables and the area of new talent apartments
Model
Unstandardized coefficient
Standardized coefficient
t
Sig. B
Std. Error
Beta
(Constant)
6.382
12.424
0.514
0.635
College students
0.019
0.029
0.666
0.672
0.538
Permanent resident population
–0.014
0.011
0.815
–1.316
0.259
Registered population
–0.009
0.014
0.477
–0.659
0.546
Migrant workers
0.082
0.056
1.197
1.470
0.216
University graduates
0.018
0.048
0.402
0.385
0.720 Table 7. Relationship between the independent variables and the area of new talent apartments Table 8. Relationship between the independent variables and the area of new affordable housing
Model
Unstandardized coefficient
Standardized coefficient
t
Sig. B
Std. Error
Beta
(Constant)
–1081.066
1356.987
–0.797
0.470
College students
–2.593
3.135
1.068
–0.827
0.455
Permanent resident population
–0.756
1.183
0.516
–0.639
0.558
Registered population
1.858
1.517
1.156
1.225
0.288
Migrant workers
5.707
6.131
0.988
0.931
0.405
University graduates
1.526
5.247
0.395
0.291
0.786 Table 8. Relationship between the independent variables and the area of new affordable housing middle- and low-income groups to live in peace, and the
college group is also a major part of affordable housing. College students are unlikely to buy commercial housing in
the short term, and may not necessarily stay in Zhengzhou
in the future, so the correlation with the total area of new
commercial housing is low. Then we continue to analyze
the relationship between the independent variables and the
area of new talent apartments, as shown in Table 7. From the above tables, it can be seen that the sig val-
ues between the independent variables and the depend-
ent variable are all much greater than 0.05. Since there is
an inclusion relationship between the individual samples
and the phenomenon of multicollinearity between the
variables, we adopt the ridge regression analysis method
to determine the coefficients of the regression equation. Z. Shen et al. Housing types, population attributes and the development path of housing in large... From Table 7, it can be seen that the population of
migrant workers and the resident population of Zheng-
zhou have the most obvious influence on the area of new
talent apartments. With many universities purchasing tal-
ent apartments for their students or teachers, it leads to
an increased correlation between the number of college
students and the area of new talent apartments. Finally,
we analyzed the relationship between the respective vari-
ables and the area of new affordable housing, as shown
in Table 8. 2.3. Regression analysis of population type and
housing type in Zhengzhou from 2010 to 2019 of population types for Zhengzhou from 2021–2035 are
shown in Table 4. Considering that there may be multicollinearity problems
between variables, multivariate analysis is used to analyze
the multivariate linear relationship between all independ-
ent variables and dependent variables, as shown in Table 6. We forecast housing types from 2021 to 2035 based
on real estate trends. With the new commercial housing,
new talent apartments, and new affordable housing as the
dependent variables and the date as the independent vari-
able, we obtained the regression equations between the
three housing types and the date through linear regression
analysis by SPSS, and then calculated the forecast data of
housing types from 2021–2035, as shown in Table 5. It can be seen that the registered population of Zheng-
zhou has the greatest impact on the total area of new com-
mercial housing, and the demand for commercial housing
from non-local residents in Zhengzhou is also increasing. Z. Shen et al. Housing types, population attributes and the development path of housing in large... 2.4. Determination of regression equation
coefficients by ridge regression analysis Ridge regression analysis is carried out on the relationship
between the respective variables and the new commercial
housing area, the new talent apartments area, and the new
affordable housing area, among which: It can be seen that the registered population and col-
lege students in Zhengzhou are strongly correlated with the
new affordable housing. Affordable housing is mainly for f
PRP – Permanent resident population; RP – Registered
population; MW – Migrant workers; UG – University f
PRP – Permanent resident population; RP – Registered
population; MW – Migrant workers; UG – University International Journal of Strategic Property Management, 2023, 27(5): 317–331 325 graduates; CS – College students; CH – New commercial
housing area; TA – New talent apartments area; AH –
New affordable housing area. The ridge regression analysis is carried out on the re-
lationship between the independent variables and the new
affordable housing area. The analysis results are shown in
Tables 13 and 14. Ridge regression analysis on the relationship between
each variable and the new commercial housing area. The
analysis results are shown in Tables 9 and 10.h It can be concluded that the regression equation be-
tween the independent variables and the new affordable
housing area is as follows: The regression equation between the independent var-
iables with the new commercial housing area is derived
from the SPSS ridge regression operation: Y3 = –1281 – 7.6 · PRP + 1.9 · RP + 5.7 · MW +
1.5 · UG – 2.6 · CS. (3) (3) Y1 = –7450 – 11 · PRP + 17 · RP + 33 · MW +
15 · UG – 5 · CS. (1) Y1 = –7450 – 11 · PRP + 17 · RP + 33 · MW + It is discernible from the expressions of the aforemen-
tioned three equations that the analysis of these three sce-
narios is more intricate. This is unequivocally linked to the
fact that certain independent variables exhibit a decline
or negative growth rate; nonetheless, in general, we can
observe the degree of correlation between the independ-
ent variables and the dependent variable. There exists an
inverse correlation between diverse housing types and the
permanent resident population in Zhengzhou, which may
be attributed to the exorbitant housing prices and the ex-
clusionary effect. 3. Advantage path prediction analysis Previous research indicates a strong correlation between
population attributes and housing type. Therefore, it is
imperative that future studies focus on the alignment of
these two factors to alleviate housing difficulties. Such an
approach would not only facilitate a more precise and ap-
propriate housing supply for the market but also provide
a foundation for decision-making by the government and
private companies, ultimately leading to the development
of housing types that cater to market demands and meet
the needs of the populace. The future housing supply type
can be predicted by analyzing the previous regression
analysis of population and housing as well as the popu-
lation forecast data from 2021 to 2035, after which the
future population type and housing type can be further
examined. 2.4. Determination of regression equation
coefficients by ridge regression analysis The degree
of positive correlation between the population of college
graduates and the area of commercial housing is great-
er than that of talent apartments. Talent apartments are
primarily associated with attracting college graduates to
promote urban innovation and development. Most college
talents still place their aspirations on commercial housing. This could be due to the fact that talent apartments are not
suitable for family populations, and new talents require
more extensive commercial housing to settle in the city. In
the forthcoming times, it is imperative to provide appro-
priate apartments in accordance with the talent structure. For instance, certain smaller-sized apartments are suitable
for students. It may be feasible to moderately augment the
investment in apartments, enhance the quality of apart-
ments, and provide housing security for high-level talents. The groups inhabiting affordable housing mainly comprise
of middle- and low-income groups who migrate to Zheng-
zhou for work and household registration. Hence, it can
be deduced that when the price of commercial housing 2.4. Determination of regression equation
coefficients by ridge regression analysis Housing strays from residential attributes
and gravitates towards attracting investment capital and a (1) The ridge regression analysis is carried out on the re-
lationship between the respective variables and the new
talent apartments area. The analysis results are shown in
Tables 11 and 12. It can be concluded that the regression equation
between the independent variables and the new talent
apartments area is as follows: Y2 = –180 – 0.014 · PRP – 0.009 · RP +
0.825 · MW + 0.185 · UG + 0.193 · CS. (2) (2) Table 9. Model summary
R
R2
Adj. R2
SE
F
Sig. F
0.995154
0.990332
0.978247
130.704443
81.948674
0.000405
Table 10. Model paraments
Variable
B
SE(B)
Beta
B/SE(B)
PRP
–11.265154
5.949482
–0.730059
–1.893468
RP
17.144285
7.628710
1.013218
2.247337
MW
33.221186
30.833001
0.546448
1.077455
UG
14.888051
26.384950
0.366617
0.564263
CS
–5.603453
15.765017
–0.219237
–0.355436
Constant
–7450.357209
6823.788737
0.000000
–0.858227
Table 11. Model summary
R
R2
Adj. R2
SE
F
Sig. F
0.987448
0.975054
0.943872
0.237977
31.269497
0.002655
Table 12. Model paraments
Variable
B
SE(B)
Beta
B/SE(B)
PRP
–0.014252
0.010832
–0.814873
–1.315696
RP
–0.009157
0.013890
–0.477463
–0.659281
MW
0.082498
0.056139
1.197196
1.469540
UG
0.0184881
0.048040
0.401663
0.384854
CS
0.019298
0.028704
0.666138
0.672322
Constant
–180.382275
12.424274
0.000000
0.513694 Table 10. Model paraments 326
Z. Shen et al. Housing types, population attributes and the development path of housing in large... Table 13. Model summary
R
R2
Adj. R2
SE
F
Sig. F
0.978592
0.957641
0.904693
25.992046
18.086376
0.007520
Table 14. Model paraments
Variable
B
SE(B)
Beta
B/SE(B)
PRP
–0.756008
1.183121
–0.515707
–0.638994
RP
1.857748
1.517055
1.155650
1.224575
MW
5.706606
6.131489
0.988024
0.930705
UG
1.525563
5.246944
0.395423
0.290753
CS
–2.593451
3.135051
–1.068051
–0.827244
Constant
–1281.065901
1356.987123
0.000000
–0.796666 hen et al. Housing types, population attributes and the development path of housing in large... 326 ascends excessively, it is essential to construct high-quality
affordable housing to cater to people’s living security. mobile population. The association between commercial
housing and the population is more pronounced. Among
these, the migrant population evinces a stronger correla-
tion with commercial housing. The process of urbaniza-
tion has led to an upsurge in the number of migrants in
major cities, which signifies people’s yearning for supe-
rior housing and resources, consequently contributing to
the escalation in commercial housing prices. 3.1. Forecast data of new housing supply types from
2021 to 2035 Using SPSS, the regression equation between the new
housing types and the population attributes from 2010 to
2019 was obtained, as shown in Table 15. Table 15. The population attributes of Zhengzhou City are related to the regression relationship between various housing supplies
Regression equation types
Regression equation
The regression equation of commercial housing and
population types
Y1 = –7450 – 11 · PRP + 17 · RP + 33 · MW + 15 · UG – 5 · CS
Regression equation of talent apartments and each
population type
Y2 = –180 – 0.014 · PRP – 0.009 · RP + 0.825 · MW + 0.185 · UG + 0.193 · CS
Regression equation of affordable housing and each
population type
Y3 = –1281 – 7.6 · PRP + 1.9 · RP + 5.7 · MW + 1.5 · UG – 2.6 · CS Table 15. The population attributes of Zhengzhou City are related to the regression relationship between v International Journal of Strategic Property Management, 2023, 27(5): 317–331 327 Table 16. Forecast data of housing types in Zhengzhou from 2021–2035
Date
Commercial housing
Talent apartments
Affordable housing
2021
3442
13
439
2022
4159
25
436
2023
4574
33
498
2024
4990
40
560
2025
5405
48
622
2026
5821
56
685
2027
6236
64
747
2028
6652
71
809
2029
7067
79
871
2030
7483
87
933
2031
7898
94
995
2032
8314
102
1057
2033
8794
111
1131
2034
9957
100
1486
2035
10690
112
1584 Table 16. Forecast data of housing types in Zhengzhou from 2021–2035 housing needs of diverse talent groups, and align with the
size of the new population in Zhengzhou. Then use the regression equation between the hous-
ing types and various population types to calculate the
forecast data of each housing type from 2021 to 2035, as
shown in Table 16. 3.3. The matching of population attributes and
affordable housing Comparing the forecast data of each house type under
the natural development trend in Table 5 with the forecast
data under the development trend of the reference popula-
tion attributes in Table 16, it can be seen that the predicted
data for housing type, under the influence of demographic
type factors, has a great difference. This also explains the
necessity of studying the matching of population attributes
and housing type to a certain extent. Under the circumstance of demographic change, the do-
main of affordable housing is anticipated to exhibit an up-
ward trajectory in the forthcoming years. Affordable hous-
ing, which is a form of commercial housing that guaran-
tees its nature, is of paramount importance in addressing
the housing requirements of the populace, particularly
those belonging to the low- and middle-income strata. With an increasing influx of immigrants to Zhengzhou, af-
fordable housing, which is characterized by relatively low
prices and guaranteed nature, has emerged as a critical
welfare project that supports the process of urban expan-
sion and demographic growth. The construction and pro-
vision of affordable housing necessitate not just the leader-
ship of the government but also its implementation at the
micro-operational level. The government should augment
its endeavors in planning, construction, and investment
to ensure an orderly and adequate supply of affordable
housing. Furthermore, it should also take into account the
equitable distribution of affordable housing and devise a
gradient supply model to meet the needs of a wide range
of groups. More importantly, it also needs to consider the
spatial suitability of affordable housing, so that affordable
housing does not only exist in the suburban areas of the
city and meets people’s basic transportation needs. 3.2. The matching of population attributes and
talent apartment To enhance urban construction and development, mu-
nicipalities furnish elite apartments to entice high-caliber
talents to settle down. In 2017, the Zhengzhou adminis-
tration promulgated a talent policy to attract talents at all
levels to propel development. Given the surging housing
prices and rents in numerous cities, particularly first and
second-tier cities, lodging has emerged as a crucial con-
sideration for young individuals when selecting a city,
especially college graduates (Wang et al., 2021). Conse-
quently, providing talent apartments for college graduates
and other cohorts plays a pivotal role in aiding Zhengzhou
City’s talent acquisition scheme. As a result of the talent
apartment’s guaranteed and low price, the administration
shoulders the primary responsibility for the relevant con-
struction and supply. Thus, the administration ought to
consider fully the dynamic demand for talent apartments
from college graduates and other groups in the context of
talent acquisition, augment investment in the construction
and supply of talent apartments, to effectively satisfy the Z. Shen et al. Housing types, population attributes and the development path of housing in large... Z. Shen et al. Housing types, population attributes and the development path of housing in large... 328 Population attributes
Commercial housing
Affordable housing
Talent apartments
Breaking urban
hollowing out
Increase city
activity
Enhancing urban
innovation
Housing type
Match
Figure 1. Advantages of housing matching population
mechanism alleviate the housing difficulties faced by large cities and
presents an effective solution to returning houses to resi-
dential properties. This mechanism is pivotal to resolving
the supply and demand contradictions in large cities, as
well as the contradictions between residents’ income and
housing prices. Additionally, it serves as a crucial element
in addressing the contradiction between the government’s
reliance on land finance and the assurance of essential
housing needs for residents. The specific research findings
are outlined below. h Match (1) The development of forthcoming housing varieties is
influenced by the evolution of population varieties. When
the diversity of housing provision is aligned with the hous-
ing requirements of individuals, it can diminish the need
for housing investments. Individuals depend on afford-
able housing, low-cost housing, urban villages, and long-
term rental apartments to fulfill their basic needs, which
can mitigate the housing burden of people with low- and
middle-income and resolve their housing predicaments. The government ought to augment the supply of subsidized
housing. This subsidized housing can resolve the housing
predicament, diminish people’s aspirations for future hous-
ing price increments, and restore the housing provision to
the aspect of housing needs of diverse population types. Figure 1. Advantages of housing matching population
mechanism accelerating the development of its metropolitan region. The establishment of high-quality integrated development
of the Central Plains urban agglomeration depends on
satisfying the housing needs of the population as a funda-
mental and obligatory requirement. The provision of com-
mercial housing in an orderly manner is of paramount im-
portance. However, the assurance of adequate supply does
not entail a laissez-faire approach to the haphazard devel-
opment of commercial housing. Instead, it necessitates an
increase in effective supply and the provision of commer-
cial housing that is relevant to the type of population and
the demands of consumers. This will not only mitigate or
inhibit the problem of escalating housing prices due to a
decrease in supply in the residential market but also safe-
guard the housing needs of the population. Z. Shen et al. Housing types, population attributes and the development path of housing in large... This will foster an environment that is conducive to in-
novation and entrepreneurship, and promote the stability
of employment for local and foreign university students in
Zhengzhou. Such measures will enhance urban activism
and advance urban innovation.hii After the above analysis, the appropriate matching
mechanism between population attributes and housing
types is shown in Figure 1. Z. Shen et al. Housing types, population attributes and the development path of housing in large... While ensuring
the supply of commercial housing, it is also pertinent to
consider that continuous investment in the capital of the
commercial residential market will create further inflation-
ary pressure. Hence, the government must adopt adminis-
trative and legal measures to increase macro-control of the
real estate market. It is also crucial to collect data and man-
age the types of commercial housing developed by compa-
nies to effectively regulate the market inflow of commercial
housing, especially the supply of different types of housing
to correspond to the type of population. (2) There is a strong correlation between different
types of housing and urban population inflows and out-
flows. Over time, urban population inflows will generate
different intensities of demand for different types of hous-
ing, so the type of housing supply should be a dynamic
adjustment process. Planning housing supply according to
the different attributes of the population will be effective
in reducing housing prices.i (3) Matching housing classifications with the populace
and ensuring the fundamental housing needs of individ-
uals are met represents the crux of resolving the afore-
mentioned issues. One should augment the availability of
subsidized housing, as well as refine the entrance and exit
mechanisms pertaining to such housing, in order to sat-
isfy the housing needs of the majority of individuals with
middle to low-income levels. Simultaneously, it is impera-
tive to safeguard the land intended for elementary urban
construction. The government should further alleviate
restrictions on migration, while simultaneously tailoring
housing subsidies to individuals with distinctive talents. This will foster an environment that is conducive to in-
novation and entrepreneurship, and promote the stability
of employment for local and foreign university students in
Zhengzhou. Such measures will enhance urban activism
and advance urban innovation.hii (3) Matching housing classifications with the populace
and ensuring the fundamental housing needs of individ-
uals are met represents the crux of resolving the afore-
mentioned issues. One should augment the availability of
subsidized housing, as well as refine the entrance and exit
mechanisms pertaining to such housing, in order to sat-
isfy the housing needs of the majority of individuals with
middle to low-income levels. Simultaneously, it is impera-
tive to safeguard the land intended for elementary urban
construction. The government should further alleviate
restrictions on migration, while simultaneously tailoring
housing subsidies to individuals with distinctive talents. 3.4. The matching of population attributes and
commercial housing The largest sector of residential areas is currently com-
prised of commercial housing. Zhengzhou is presently Conclusions and discussion The present study’s investigation has demonstrated a close
correlation between population attributes and housing
type. At present, a severe asymmetry exists between the
supply and demand of housing. The scarcity of reasonably
priced housing, such as talent apartments and affordable
housing, has contributed to housing’s investment proper-
ties, thereby exacerbating the discrepancy between supply
and demand. The mismatch between urban populations
and housing will lead to the financialization of hous-
ing investment and an increase in housing prices. Prop-
erly aligning population attributes and housing type can The aforementioned findings hold significant reference
value for the formulation of China’s housing policy. Firstly,
the local government ought to construct a multi-tiered
housing supply system based on the population’s attributes
and quantity to cater to the varied requirements of diverse
groups. They must not only rationally plan the number
of self-owned, commercial housing, subsidized housing,
public rental housing, and dilapidated communities, etc., International Journal of Strategic Property Management, 2023, 27(5): 317–331 329 but also provide differentiated housing supply for different
groups to mitigate housing prices and address the housing
demands of low- and middle-income individuals, includ-
ing urban migrants and new citizens. Secondly, it is vital
to establish sound entry and exit mechanisms for subsi-
dized housing. The government should prohibit middle
and high-income individuals from acquiring subsidized
housing and extend the coverage scope for low-income
individuals. It should oversee the subsequent income of
family members who have already obtained guaranteed
housing so that those who no longer satisfy the require-
ments shall withdraw from guaranteed housing. Lastly, it
is imperative to foster the high-welfare talent settlement
policy and the reform of the household registration sys-
tem, and to guarantee that highly skilled talents can live
and work in Zhengzhou. The government should provide
eligible personnel with Provide guaranteed rental housing,
and give a certain area subsidy to those who rent market-
oriented rental housing, so as to take advantage of low
housing prices to retain talents and attract talents.h Grant [21YJCZH130]; Henan office of philosophy and
social science under Grant [2024-ZDJH-031]; Henan
office of philosophy and social science under Grant
[2023BZH010]. Acknowledgements Eichholtz, P., & Lindenthal, T. (2014). Demographics, human
capital, and the demand for housing. Journal of Housing Eco-
nomics, 26, 19–32. https://doi.org/10.1016/j.jhe.2014.06.002 Authors would like to thank the editors and anonymous
reviewers for their constructive comments and sugges-
tions. Garha, N. S., & Azevedo, A. B. (2021). Population and housing
(mis)match in Lisbon, 1981–2018. A challenge for an aging
society. Social Sciences, 10(3), 102. https://doi.org/10.3390/socsci10030102f References Akbari, A. H., & Aydede, Y. (2012). Effects of immigration on
house prices in Canada. Applied Economics, 44(13), 1645–
1658. https://doi.org/10.1080/00036846.2010.548788 Campbell, B. O. (1963). Long swings in residential construc-
tion: the postwar experience. The American Economic Re-
view, 53(2), 508–518. https://www.jstor.org/stable/1823891i Chen, J., & Wu, F. (2022). Housing and land financialization un-
der the state ownership of land in China. Land Use Policy, 112,
104844. https://doi.org/10.1016/j.landusepol.2020.104844 Chen, J., Guo, F., & Wu, Y. (2011). One decade of urban housing
reform in China: urban housing price dynamics and the role
of migration and urbanization, 1995-2005. Habitat Interna-
tional, 35(1), 1–8. https://doi.org/10.1016/j.habitatint.2010.02.003f https://doi.org/10.1016/j.habitatint.2010.02.003f Chiu, R. L. (2007). Planning, land and affordable housing in
Hong Kong. Housing Studies, 22(1), 63–81. https://doi.org/10.1080/02673030601024614 Choi, C., Jung, H., & Su, L. (2019). Population structure and
housing prices: evidence from Chinese provincial panel
data. Emerging Markets Finance and Trade, 55(1), 29–38. https://doi.org/10.1080/1540496X.2018.1496417 Day, C. (2018). Population and house prices in the United King-
dom. Scottish Journal of Political Economy, 65(2), 127–141. https://doi.org/10.1111/sjpe.12166 Ding, Y. (2019). Housing prices and population dynamics in ur-
ban China. Pacific Economic Review, 24(1), 27–45. https://doi.org/10.1111/1468-0106.12271 Author contributions Zhifeng Shen and Jing Li conceived the study. Zhifeng
Shen, Jing Li, Ahsan Siraj, Shilpa Taneja, and Zhihui Li
were responsible for the design and development of the re-
search. Zhihui Li and Jing Li were responsible for data col-
lection and data interpretation. Jing Li, Ahsan Siraj, and
Shilpa Taneja wrote the first draft, edited, and revised the
article. All authors have read and agreed to the published
version of the manuscript. Disclosure statement Authors declare that they do not have any competing finan-
cial, professional, or personal interests from other parties. g p
The research in this paper, undoubtedly, presents cer-
tain limitations. The paper employs data concerning the
population and housing in Zhengzhou City, Henan Prov-
ince. Nevertheless, the selection of the city sample is rela-
tively narrow. The focus of the study lies primarily in ex-
amining the relationship between housing demand across
various demographic attributes and the prevailing housing
supply. However, the variable of population income level
remains absent. Although it is noteworthy that guaran-
teed housing is primarily a policy benefiting low-income
groups, and we have included demographic variables such
as graduates and migrant workers as representatives of this
group, we find ourselves still lacking specific data to sub-
stantiate this claim. This deficiency arises from the chal-
lenge of obtaining income data for different attributes of
demographic groups. Consequently, it becomes impera-
tive to further investigate the impact of income level on
housing in subsequent studies. Furthermore, taking into
account the projected sluggish population growth and the
impending aging problem, the rate at which new housing
units will be developed is expected to decelerate. In order
to offer a broader range of housing security, the govern-
ment and relevant authorities must contemplate the reju-
venation of old housing structures as well as the provision
of long-term rental housing in the future. Additionally, we
plan to conduct a comparative analysis by selecting a large
dataset from multiple cities. Z. Shen et al. Housing types, population attributes and the development path of housing in large... https://doi.org/10.1080/10511482.2018.1452045 Jia, S., Wang, Y., & Fan, G. Z. (2018). Home-purchase limits and
housing prices: evidence from China. The Journal of Real Es-
tate Finance and Economics, 56(3), 386–409. https://doi.org/10.1007/s11146-017-9615-2 National Bureau of Statistics of China. (2020). China statistical
yearbook. China Statistics Press. Lauster, N. T. (2008). Better homes and families: housing mar-
kets and young couple stability in Sweden. Journal of Mar-
riage and Family, 70(4), 891–903. https://doi.org/10.1111/j.1741-3737.2008.00534.x Rodriguez-Pose, A., & Storper, M. (2020). Housing, urban growth
and inequalities: the limits to deregulation and upzoning in
reducing economic and spatial inequality. Urban Studies,
57(2), 223–248. https://doi.org/10.1177/0042098019859458h Levin, E., Montagnoli, A., & Wright, R. E. (2009). Demographic
change and the housing market: evidence from a comparison
of Scotland and England. Urban Studies, 46(1), 27–43. https://doi.org/10.1177/0042098008098635 Skovgaard Nielsen, R. (2017). The potentials of a strong social
housing sector: the case of Turks and Somalis in the Copen-
hagen housing market. Housing, Theory and Society, 34(4),
458–476. https://doi.org/10.1080/14036096.2016.1274678hi Li, C., & Fan, Y. (2020). Housing wealth inequality in urban
China: the transition from welfare allocation to market dif-
ferentiation. Journal of Chinese Sociology, 7(1), 1–17. https://doi.org/10.1186/s40711-020-00129-4 Sood, A., & Vicino, T. J. (2023). The politics of studentification: an
analysis of the student housing debate in Boston. Housing Policy
Debate, 1–24. https://doi.org/10.1080/10511482.2023.2236078 g
Wang, X. R., Hui, E. C. M., & Sun, J. X. (2017). Population mi-
gration, urbanization and housing prices: evidence from the
cities in China. Habitat International, 66, 49–56. https://doi.org/10.1016/j.habitatint.2017.05.010 Li, L. H., Wu, F., Dai, M., Gao, Y., & Pan, J. (2017). Housing
affordability of university graduates in Guangzhou. Habitat
International, 67, 137–147. https://doi.org/10.1016/j.habitatint.2017.07.007 Wang, X., Hui, E. C. M., & Sun, J. (2018). Population aging, mo-
bility, and real estate price: evidence from cities in China. Sus-
tainability, 10(9), 3140. https://doi.org/10.3390/su10093140 Li, T., Zhang, Y. T., Zhu, H. W., & Liu, P. J. (2022). Floating
population, housing security and family medical economic
risk. Sustainability, 14(18), 11220. https://doi.org/10.3390/su141811220 Wang, Y., Cui, C., Wang, Q., Ning, Y., & Yanag, Z. (2021). Mi-
gration of human capital in the context of vying for talent
competition: a case study of China’s “first class” university
graduates. Geographical Research, 40(3), 743–761. Li, Z., & Wu, F. (2006). Socio-spatial differentiation and residen-
tial inequalities in Shanghai: a case study of three neighbour-
hoods. Housing Studies, 21(5), 695–717. https://doi.org/10.1080/02673030600807365 Weng, G., & Pan, Y. (2019). Z. Shen et al. Housing types, population attributes and the development path of housing in large... Z. Shen et al. Housing types, population attributes and the development path of housing in large... 330 Glaeser, E., Huang, W., Ma, Y., & Shleifer, A. (2017). A real es-
tate boom with Chinese characteristics. Journal of Economic
Perspectives, 31(1), 93–116. Liu, Y., Gao, H., Cai, J., Lu, Y., & Fan, Z. (2022). Urbanization
path, housing price and land finance: international experience
and China’s facts. Land Use Policy, 113, 105866. https://doi.org/10.1016/j.landusepol.2021.105866 Llopis Alvarez, A., & Muller-Eie, D. (2022). Housing circum-
stances and quality of life among local and immigrant popu-
lation in Norwegian neighbourhoods. Journal of Housing and
the Built Environment, 37(1), 157–178. https://doi.org/10.1007/s10901-021-09853-w Green, R. K., & Lee, H. (2016). Age, demographics, and the
demand for housing, revisited. Regional Science and Urban
Economics, 61, 86–98. https://doi.org/10.1016/j.regsciurbeco.2016.09.005 https://doi.org/10.1016/j.regsciurbeco.2016.09.005 Hanink, D. M., Cromley, R. G., & Ebenstein, A. Y. (2012). Spatial
variation in the determinants of house prices and apartment
rents in China. Journal of Real Estate Finance and Economics,
45(2), 347–363. https://doi.org/10.1007/s11146-010-9262-3 Malmberg, B. (2010). Low fertility and the housing market: evi-
dence from Swedish regional data. European Journal of Popu-
lation / Revue Européenne de Démographie, 26(2), 229–244. https://doi.org/10.1007/s10680-009-9205-y He, C., Li, D., & Yu, J. (2022). Quantifying the spatial-temporal
variation of population urbanization and affordable housing
land in China. Land, 11(2), 259. https://doi.org/10.3390/land11020259 Miles, D. (2012). Population density, house prices and mortgage
design. Scottish Journal of Political Economy, 59(5), 444–466. https://doi.org/10.1111/j.1467-9485.2012.00589.x Mulder, C. H. (2006). Population and housing: a two-sided rela-
tionship. Demographic Research, 15, 401–412. https://doi.org/10.4054/DemRes.2006.15.13 Henan Province Bureau of Statistics. (2021). Henan statistical
yearbook. China Statistics Press. Howard, G., & Liebersohn, J. (2021). Why is the rent so darn
high? The role of growing demand to live in housing-sup-
ply-inelastic cities. Journal of Urban Economics, 124, 103369. https://doi.org/10.1016/j.jue.2021.103369 Myers, D. (1990). Housing demography: linking demographic
structure and housing markets. University of Wisconsin Press. Myers, D., & Pitkin, J. (2009). Demographic forces and turning
points in American city, 1950-2040. The Annals of the Ameri-
can Academy of Political and Social Science, 626(1), 91–111. https://doi.org/10.1177/0002716209344838h Huang, Y. (2004). Housing markets, government behaviors, and
housing choice: a case study of three cities in China. Environ-
ment and Planning A, 36(1), 45–68. https://doi.org/10.1068/a35158 Myers, D., Painter, G., Zissimopoulos, J., Lee, H., & Thunell, J. (2019). Simulating the change in young adult homeowner-
ship through 2035: effects of growing diversity and rising ed-
ucational attainment. Housing Policy Debate, 29(1), 126–142. Funding Gevorgyan, K. (2019). Do demographic changes affect house
prices? Journal of Demographic Economics, 85(4), 305–320. https://doi.org/10.1017/dem.2019.9 This work was supported by the Humanities and Social
Sciences research project of Ministry of Education under Z. Shen et al. Housing types, population attributes and the development path of housing in large... Spatial mismatch analysis of newly-
increased housing demand, housing and land supply in urban
areas. Geography and Geo-Information Science, 35(1), 75–81. Liang, W., Lu, M., & Zhang, H. (2016). Housing prices raise wag-
es: estimating the unexpected effects of land supply regulation
in China. Journal of Housing Economics, 33, 70–81. https://doi.org/10.1016/j.jhe.2016.07.002 Wilkinson, C., & Greenhalgh, P. (2022). Exploring student hous-
ing demand, supply side and planning policy responses in a
small university city: studentification in Durham, UK. Hous-
ing Policy Debate, 1–23. htt
//d i
/10 1080/10511482 2022 2137379 Lin, X., Zhong, J., Ren, T., & Zhu, G. (2022). Spatial-temporal ef-
fects of urban housing prices on job location choice of college
graduates: evidence from urban China. Cities, 126, 103690. https://doi.org/10.1016/j.cities.2022.103690 https://doi.org/10.1080/10511482.2022.2137379 Wu, Y., & Li, Y. (2018). Impact of government intervention in
the housing market: evidence from the housing purchase re- Wu, Y., & Li, Y. (2018). Impact of government intervention in
the housing market: evidence from the housing purchase re- International Journal of Strategic Property Management, 2023, 27(5): 317–331 331 striction policy in China. Applied Economics, 50(6), 691–705. https://doi.org/10.1080/00036846.2017.1340569 striction policy in China. Applied Economics, 50(6), 691–705. https://doi.org/10.1080/00036846.2017.1340569 Zhao, P. (2013). The impact of urban sprawl on social segrega-
tion in Beijing and a limited role for spatial planning. Tijd-
schrift voor Economische en Sociale Geografie, 104(5), 571–
587. https://doi.org/10.1111/tesg.12030 Yan, S., Ge, X. J., & Wu, Q. (2014). Government intervention in
land market and its impacts on land supply and new hous-
ing supply: evidence from major Chinese markets. Habitat
International, 44, 517–527. https://doi.org/10.1016/j.habitatint.2014.10.009 p
g
g
Zhao, P., & Howden-Chapman, P. (2010). Social inequalities in
mobility: the impact of the hukou system on migrants’ job
accessibility and commuting costs in Beijing. International
Development Planning Review, 32(3–4), 363–384. https://doi.org/10.3828/idpr.2010.13 Yang, Z., & Pan, Y. (2020). Human capital, housing prices,
and regional economic development: will “vying for talent”
through policy succeed? Cities, 98, 102577. https://doi.org/10.1016/j.cities.2019.102577 Zhengzhou Housing Security and Real Estate Bureau. (2021). Zhengzhou housing security and real estate bureau 2021 an-
nual report on government information disclosure. https://public.zhengzhou.gov.cn/D42Y/6238409.jhtmlf Yu, H. (2011). Size and characteristic of housing bubbles in Chi-
na’s major cities: 1999–2010. China & World Economy, 19(6),
56–75. https://doi.org/10.1111/j.1749-124X.2011.01266.x https://public.zhengzhou.gov.cn/D42Y/6238409.jhtml Zou, Y. (2014). Contradictions in China’s affordable housing
policy: goals vs. structure. Habitat International, 41, 8–16. https://doi.org/10.1016/j.habitatint.2013.06.001 Yu, H., & Huang, Y. (2016). Z. Shen et al. Housing types, population attributes and the development path of housing in large... Regional heterogeneity and the
trans-regional interaction of housing prices and inflation:
evidence from China’s 35 major cities. Urban Studies, 53(16),
3472–3492. https://doi.org/10.1177/0042098015617882
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Consumption of olive oil and adherence to the Mediterranean Food Pattern among academics of lusophone origin
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Tânia Graça1 , Juliana Almeida-de-Souza2 , Joana Margarida Bôto3 , Nuno Rodrigues2 , Vera Ferro-Lebres2 ,
Manuela Meireles2* 1Escola Superior de Saúde, Instituto Politécnico de Bragança, Bragança, Portugal
2Centro de Investigação de Montanha (CIMO), Instituto Politécnico de Bragança, Bragança, Portugal
3GreenUPorto – Sustainable Agrifood Production Research Center/Inov4Agro, Faculty of Nutrition and Food Sciences, University of Porto, Porto, Portugal. *Autor correspondente/Corresponding author: manuela.meireles@ipb.pt
Recebido/Received: 28-10-2021; Revisto/Revised: 24-01-2022; Aceite/Accepted: 02-02-2022 1Escola Superior de Saúde, Instituto Politécnico de Bragança, Bragança, Portugal
2Centro de Investigação de Montanha (CIMO), Instituto Politécnico de Bragança, Bragança, Portugal
3GreenUPorto – Sustainable Agrifood Production Research Center/Inov4Agro, Faculty of Nutrition and Food Sciences, University of Porto, Porto, Portugal. *Autor correspondente/Corresponding author: manuela.meireles@ipb.pt Recebido/Received: 28-10-2021; Revisto/Revised: 24-01-2022; Aceite/Accepted: 02-02-2022 Resumo Introdução: O azeite muito apreciado pelos consumidores pelas suas propriedades nutricionais e pelos seus aromas e
sabores, é um dos pilares da Dieta Mediterrânica (DM). Este padrão alimentar, característico dos países mediterrânicos,
é considerado um dos padrões alimentares mais saudáveis que existem. Muitos dos seus benefícios estão associados
ao consumo de alimentos com propriedades benéficas para a saúde, como é o caso do azeite. Objetivo: Avaliar
a adesão ao padrão alimentar mediterrânico e o consumo de azeite, numa amostra de estudantes e investigadores
portugueses e de origem lusófona. Material e Métodos: De forma a avaliar a adesão à Dieta Mediterrânica foi utilizado
o questionário MEDAS (Mediterranean Diet Adherence Screener) previamente traduzido e validado para a população
portuguesa, que inclui 14 questões relacionadas com a frequência do consumo de alimentos típicos e não típicos do
padrão alimentar mediterrânico, incluindo azeite. Este estudo teve como principal foco estudantes e investigadores
portugueses e estrangeiros. Resultados: Observou-se que 94,4% dos estudantes e investigadores portugueses e
86,5% dos estrangeiros utilizam azeite como principal gordura culinária, apresentando médias (DP) de ingestão diária
de 2,9 (2.3) e 3,9 (2,9) colheres de sopa respetivamente. A mediana (P25-P75) de adesão à DM foi de 8,0 (6,0-9,0) nos
participantes nacionais sendo significativamente superior dos internacionais 6,0 (4,5-7,5) (p=0,009). Conclusões: Este
estudo revelou que apesar do azeite ser a gordura de eleição mesmo entre estudantes e investigadores estrangeiros,
várias medidas podem ser tomadas para aumentar a adesão à Dieta Mediterrânica, em particular nestes últimos. Palavras-chave: dieta mediterrânica, azeite, comportamento alimentar, Portugal, estudantes. A r t i g o s C i e n t í f i c o s | S c i e n t i f i c A r t i c l e s A r t i g o s C i e n t í f i c o s | S c i e n t i f i c A r t i c l e s RevSALUS Vol. 4: Nº1
Doi: https://doi.org/10.51126/revsalus.v4i1.209 1. INTRODUÇÃO Poor eating habits and insufficient physical activity are
associated with the main risk factors for obesity, diabetes, and
other chronic diseases, especially if adopted in early adulthood
(Haire-Joshu et al., 2016; Spiegel et al., 2006; Salameh et al.,
2014). Adopting a balanced and healthy diet helps to prevent
some of the most prevalent chronic diseases in our society
(Menotti et al., 1999; Gómez-gracia et al., 2013; Sánchez-Villegas
et al., 2006; Aidoud et al., 2019). Consequently, government
organizations regularly publish dietary guidelines to promote
healthier eating patterns (Renzella et al., 2018). Many of these
recommendations are based on well-recognized healthy diets,
such as the Mediterranean Diet (MD) (Aidoud et al., 2019). The Mediterranean Diet pyramid was developed as a visual
adaptation of food-based dietary guidelines (Bach-Faig et al.,
2011). At the bottom of the pyramid are foods that should
be eaten most often, and the top includes those that should
be rarely eaten. It translates into a recommendation for a
higher intake of olive oil, fruits, nuts, vegetables and cereals;
moderate intake of fish and poultry; low consumption of dairy
products, red meats, processed meats and sweets, and even
the consumption of wine in moderation, only with the meals. In Portugal, the adaptation of the national food guide to the
recommendations of the Mediterranean Diet is reflected in the
New Mediterranean Food Wheel (Rodrigues et al. 2021). Hábitos alimentares inadequados e atividade física
insuficiente estão associados aos principais fatores de risco para
obesidade, diabetes e outras doenças crónicas, principalmente
se adotados no início da idade adulta (Haire-Joshu et al.,
2016; Spiegel et al., 2006; Salameh et al., 2014). Adotar uma
alimentação equilibrada e saudável ajuda a prevenir algumas
das doenças crónicas mais prevalentes na nossa sociedade
(Menotti et al., 1999; Gómez-gracia et al., 2013; Sánchez-Villegas
et al., 2006; Aidoud et al., 2019). Por conseguinte, as organizações
governamentais publicam regularmente diretrizes dietéticas
para promover padrões alimentares mais saudáveis (Renzella
et al., 2018). Inúmeras destas recomendações estão baseadas
em dietas saudáveis bem reconhecidas, como por exemplo a
Dieta Mediterrânica (DM) (Aidoud et al., 2019). A pirâmide da
Dieta Mediterrânica foi desenvolvida como adaptação visual
às diretrizes dietéticas baseadas em alimentos (Bach-Faig et
al., 2011). Na base da pirâmide encontram-se os alimentos que
devem ser consumidos com mais frequência e o topo inclui
aqueles que devem ser raramente consumidos. 1. INTRODUÇÃO Destaca-se a
recomendação relativa à maior ingestão de azeite, frutas, nozes,
vegetais e cereais; ingestão moderada de peixes e aves; baixo
consumo de laticínios, carnes vermelhas, carnes processadas
e doces, e ainda o consumo de vinho com moderação, apenas
com as refeições. Em Portugal, a adaptação do guia alimentar
nacional às recomendações da Dieta Mediterrânica reflete-se
na Nova Roda dos Alimentos Mediterrânica (Rodrigues et al. 2021). One of the distinguishing features of the Mediterranean
Diet is the use of extra virgin olive oil instead of solid fats, giving
primacy to a fat produced locally, in much of the Mediterranean
region. This dietary pattern has extra virgin olive oil as the
main source of fat, which, unlike other vegetable oils, contains
phenolic compounds that provide its unique aroma and flavor. This characteristic is due to olive oil being obtained through
a cold mechanical extraction of the olives, resulting in a dark
green product with an intense flavor (Visioli et al., 2001). Uma
das
características
diferenciadora
da
Dieta
Mediterrânica é o uso de azeite virgem extra em vez de gorduras
sólidas, dando primazia a uma gordura produzida localmente,
em grande parte da região Mediterrânica. Este padrão alimentar
tem o azeite virgem extra como a principal fonte de gordura,
que ao contrário de outros óleos vegetais, contém compostos
fenólicos que fornecem o seu aroma e sabor únicos. Esta
característica deve-se à obtenção de azeite ser feita através de
uma extração mecânica a frio das azeitonas, resultando num
produto de cor verde escura e de sabor intenso (Visioli et al.,
2001). Portugal é um país de características mediterrânicas, e
foi um dos países a apoiar a candidatura da DM a património
cultural imaterial da humanidade da UNESCO (Freitas et al.,
2015) . Portugal deveria assim apresentar uma elevada adesão
à Dieta Mediterrânica, mas a maioria da população portuguesa
tem um consumo abaixo do desejável de alguns alimentos
característicos, tais como hortícolas, frutas, leguminosas (Lopes
et al., 2016). Portugal is a country with Mediterranean characteristics
and was one of the countries to support the MD's candidacy
for UNESCO's Intangible Cultural Heritage of Humanity (Freitas
et al., 2015). Portugal should thus have high adherence to
the Mediterranean Diet, but the majority of the Portuguese
population has a below-desirable consumption of some
characteristic foods, such as vegetables, fruits and legumes
(Lopes et al., 2016). 1. INTRODUÇÃO The Polytechnic Institute of Bragança (IPB) is located within
the second-largest olive oil-producing region, a key element of
the Mediterranean Diet, and constituent of the dietary pattern
of local inhabitants. Considering also that the IPB receives
annually students and researchers from non-Mediterranean
countries, where high adherence to the MD is not expected,
there is scientific interest in comparing the level of adherence
to this dietary pattern in these foreign students. Therefore, this
study aimed to assess the adherence to the Mediterranean
dietary pattern and olive oil consumption among Portuguese
and foreign higher education students and researchers. O Instituto Politécnico de Bragança (IPB) situa-se dentro
da segunda maior região produtora de azeite, um elemento
fundamental da Dieta Mediterrânica, e constituinte do padrão
alimentar dos habitantes locais. Considerando também que o
IPB recebe anualmente estudantes e investigadores de países
não mediterrânicos, onde não é expectável elevada adesão à
DM, existe interesse científico em comparar o nível de adesão
a este padrão alimentar nestes alunos estrangeiros. Assim
sendo, este estudo teve como objetivo avaliar a adesão ao
padrão alimentar mediterrânico e o consumo de azeite, entre
estudantes e investigadores do ensino superior portugueses e
estrangeiros. Abstract Introduction: Olive oil is one of the pillars of the Mediterranean Diet (MD), highly appreciated by consumers for its
nutritional properties and its aromas and flavors. The MD, characteristic of Mediterranean countries, is considered
one of the healthiest existent eating patterns. Many of its benefits are associated with the consumption of foods with
beneficial properties for health, such as olive oil. Objective: To evaluate adherence to the Mediterranean dietary pattern
and consumption of olive oil, in a sample of Portuguese and Lusophone origin students and researchers. Material and
Methods: Adherence to the Mediterranean Diet was measured using the Mediterranean Diet Adherence Screener
(MEDAS) questionnaire, previously translated and validated for the Portuguese population, containing 14 questions
related to the frequency of consumption of typical and non-typical foods of the Mediterranean food pattern, including
olive oil. Results: It was observed that 94.4% of Portuguese participants and 86.5% of the foreign ones use olive oil
as their main culinary fat and reported mean (SD) daily ingestion of 2.9 (2.3) and 3.9 (2.9) tablespoons respectively. The median of adherence to MD was 8.0 (6.0-9.0) among national participants, being significantly higher than foreign
participants: 6.0 (4.5-7.5) (p=0.009). Conclusions: This study revealed that even though olive oil was the fat of choice
among both foreign students and researchers, several measures may be taken to raise the score of adherence to the
Mediterranean Diet, especially in the international community. Keywords: mediterranean diet, olive oil, feeding behavior, Portugal, students. RevSALUS Vol. 4: Nº1
Doi: https://doi.org/10.51126/revsalus.v4i1.209 RevSALUS Vol. 4: Nº1
Doi: https://doi.org/10.51126/revsalus.v4i1.209 R e v i s t a C i e n t í f i c a I n t e r n a c i o n a l R e v S A L U S 2.2 STATISTICAL ANALYSIS Data are presented as frequencies and percentages
for categorical variables and as means, standard deviation,
medians, and interquartile ranges for continuous variables. The
normality of distribution was measured using the Kolmogorov–
Smirnov test. The Mann-Whitney test was used for continuous
variables and Pearson's chi-square test for categorical variables. Differences were considered significant when p <0,05(IBM
2020). O consumo de azeite foi avaliado através do número de
colheres de sopa consumidas num dia reportadas no questionário
MEDAS, incluindo a quantidade usada para temperar saladas e
refeições fora de casa. 2.1 PARTICIPANTES E RECOLHA DE DADOS Este trabalho reflete um estudo transversal, realizado no
período de junho a julho de 2021. O estudo inclui estudantes
ou investigadores da comunidade académica do IPB de
nacionalidade portuguesa e de outras nacionalidades. Os
participantes foram recrutados de forma voluntária através
das associações de estudantes internacionais, centros de
investigação e dos professores para o preenchimento individual
do questionário, sendo toda a informação recolhida de forma
anónima. De forma a avaliar a adesão à DM foi utilizado um questionário
MEDAS (Mediterranean Diet Adherence Screener) previamente
traduzido e validado para a população portuguesa (Afonso,
Moreira, and Oliveira 2014; Gregório et al. 2020). Este continha
14 questões relacionadas com a frequência do consumo de
alimentos que determinam o nível de adesão ao padrão alimentar
mediterrânico, sendo 12 questões do consumo alimentar e
2 questões de hábitos alimentares considerados específicos
da Dieta Mediterrânica. A resposta a cada uma das questões é
pontuada com 1, no caso do cumprimento dos critérios definidos
como característicos deste tipo de alimentação, e com 0 pontos,
no caso de não cumprimento, sendo que a pontuação final pode
variar entre 0 e 14. A adesão ao padrão alimentar mediterrânico
foi definida em três níveis de acordo com os autores originais:
baixo (0-6), moderado (6-9) e alto (10-14) (Martínez-González et
al., 2012; Afonso et al., 2014). Olive oil consumption was assessed by the number
of tablespoons consumed in a day reported in the MEDAS
questionnaire, including the amount used to season salads and
meals outside the home. 2.2 ANÁLISE ESTATÍSTICA A análise estatística foi realizada através do programa
estatístico da IBM® Statistical Package for the Social Sciences
(SPSS®). Os dados são apresentados como frequências e
percentagens para variáveis categóricas e como médias, desvio
padrão, medianas e amplitudes interquartis para variáveis
contínuas. A normalidade da distribuição foi medida através
do teste de Kolmogorov–Smirnov. Foi utilizado o Teste Mann-
Whitney para variáveis contínuas e o Teste Qui-quadrado
de Pearson para variáveis categóricas. As diferenças foram
consideradas significativas quando p <0,05(IBM 2020). A r t i g o s C i e n t í f i c o s | S c i e n t i f i c A r t i c l e s the IPB's Portuguese and other Lusophone origin academic
community. Participants were recruited voluntarily through
the international student associations, research centers,
and professors. Information regarding sociodemographic
characteristics and the level of adherence to the Mediterranean
Diet was obtained through a self-completed questionnaire. The Mediterranean Diet Adherence Screener (MEDAS), a
tool previously translated and validated for the Portuguese
population was used to assess MD adherence (Afonso, Moreira,
and Oliveira 2014; Gregório et al. 2020). It contained 14 questions
related to the frequency of consumption of foods that determine
the level of adherence to the Mediterranean dietary pattern,
being 12 questions on food consumption and 2 questions on
eating habits considered specific to the Mediterranean diet. The
answer to each question is scored with 1, in case of compliance
with the criteria defined as characteristic of MD, and with 0
points, in case of non-compliance, and the final score may vary
between 0 and 14. Adherence to the Mediterranean dietary
pattern was defined in three levels according to the original
authors: low (0-6), moderate (6-9), and high (10-14) (Martínez-
González et al., 2012; Afonso et al., 2014). 3.1. SOCIODEMOGRAPHIC CHARACTERISTICS OF THE
PARTICIPANTS Este estudo respeitou os princípios da Declaração de
Helsínquia(WMA 2018) e foi aprovado pela comissão de Ética
do IPB (Parecer nº51/2021). Todos os participantes assinaram um
consentimento informado e toda a informação foi recolhida de
forma anónima, garantindo a privacidade dos participantes e
em acordo com o disposto no Regulamento Geral de Proteção
de Dados. The main sociodemographic characteristics of the
participants are shown in Table 1. A total of 76 Portuguese and
foreigner students and researchers from the IPB were surveyed,
being 18 (48.6%) female and 19 (51.4%) male of Portuguese
nationality, and 14 (35.9%) female and 25 (64.1%) male of
international nationality (Brazilian n=12, Cape Verdean n=23
and Guinean n=4). The mean age (SD) of participants was 24.08
(4.71) in Portuguese students, and 24.69 (5.04) in international
students. Most participants, both foreigner (94.9%) and
Portuguese (91.9%) were single. All participants had at least
secondary education and about half had higher education, 2.3 ETHICS This study respected the principles of the Declaration
of Helsinki (WMA 2018) and was approved by the Ethics
Committee of the IPB (Approval nº51/2021). All participants
signed an informed consent and all information was collected
anonymously, guaranteeing the privacy of the participants
under the provisions of the General Data Protection Regulation. 2.1 PARTICIPANTS AND DATA COLLECTION This work was a cross-sectional study, carried out from June
to July 2021. The study includes students or researchers from R e v i s t a C i e n t í f i c a I n t e r n a c i o n a l R e v S A L U S A r t i g o s C i e n t í f i c o s | S c i e n t i f i c A r t i c l e s with no significant differences between Portuguese and
international participants. Taking into account the Body Mass
Index, 34.3% of Portuguese participants and 26.3% of foreign
students were overweight/obese. participantes são apresentadas na Tabela 1. Um total de 76
estudantes e investigadores portugueses e estrangeiros do IPB
foram inquiridos, sendo 18 (48,6%) do sexo feminino e 19 (51,4%)
do sexo masculino de nacionalidade portuguesa, 14 (35,9%) do
sexo feminino e 25 (64,1%) do sexo masculino de nacionalidade
internacional (brasileira n=12, cabo-verdiana n=23 e guineense
n=4). A idade média (DP) dos participantes foi de 24,08 (4,71)
nos estudantes portugueses, e 24,69 (5,04) nos internacionais. A maioria dos participantes quer estrangeiros (94,9%) quer
portugueses (91,9%) eram solteiros. Todos os participantes
apresentavam pelo menos o ensino secundário e cerca da
metade o ensino superior, não havendo diferenças significativas
entre os participantes portugueses e internacionais. Tendo
em conta o Índice de Massa Corporal, 34,3% dos participantes
portugueses e 26,3% dos estudantes estrangeiros apresentavam
excesso de peso/ obesidade. Tabela/Table 1: Características sociodemográficas dos participantes/Sociodemographic characteristics of the participants. Total
Portugueses/Portuguese
Estrangeiros/Foreigners
Valor/Value -p
Sexo/Sex -n (%)
Feminino/Female
18 (48,6%)
14 (35,9%)
0,260
Masculino/Male
19 (51,4%)
25 (64,1%)
Idade/Age
Média/Mean ± SD
24,08 ± 4,71
24,69 ± 5,04
0,438
Estado civil/Marital status - n (%)
Solteiro(a)/Single
34 (91,9%)
37 (94,9%)
0,600
Casado ou a viver em união de facto/Married or in a registered
partnership
3 (8,1%)
2 (5,1%)
Nível de educação/Level of education - n (%)
Secundário/High school
16 (43,2%)
15 (39,4%)
0,740
Superior/University
21 (56,7%)
23 (60,5%)
Estado de Saúde/Health status - n (%)
Muito bom/Very good
21 (56,8%)
17 (43,6%)
0,251
Outros/Others
16 (43,2%)
22 (56,4%)
IMC/BMI
Média/Mean ± SD/
24,35 ± 4,33
23,27 ± 2,38
0,458
IMC/BMI categories - n (%)
Magreza/ Normal/Underweight/ Normal weight
23 (65,7%)
28 (73,6%)
0,458
Excesso de peso/Obesidade/Overweight/ Obesity
12 (34,3%)
10 (26,3%)
SD- Desvio Padrão; Outros= bom; nem bom, nem mau; fraco; muito fraco; não sabe (não responde)/SD- Standard deviation; IMC- Índice de Massa Corporal; Others=
good; neither good nor bad; weak; very weak; don't know (no answer); BMI- Body Mass Index. Tabela/Table 1: Características sociodemográficas dos participantes/Sociodemographic characteristics of the participants. 3.1. CARACTERÍSTICAS SOCIODEMOGRÁFICAS DOS
PARTICIPANTES As
principais
características
sociodemográficas
dos A r t i g o s C i e n t í f i c o s | S c i e n t i f i c A r t i c l e s only 10.5% of foreign participants did so, this difference being
statistically significant (p=0.032). There were no statistically
significant differences between groups regarding the amount
of olive oil consumed in a day, amount of fruit, legumes,
oleaginous fruits, red meat, hamburger or meat products,
butter, margarine, or cream consumed per day, amount of
pastries or glasses of wine consumed per week. (p=0,033). Relativamente ao consumo de peixe ou marisco,
30,6% dos participantes portugueses consumia ≥3 porções/
semana, enquanto que apenas 10,5% dos participantes
estrangeiros o fazia, sendo esta diferença estatisticamente
significativa (p=0,032). Não houve diferenças estaticamente
significativas relativamente à quantidade de azeite consumida
num dia, quantidade de fruta, leguminosas, frutos oleaginosos,
carne vermelha, hambúrguer ou produtos cárneos, manteiga,
margarina, ou natas consumidas por dia, quantidade de
produtos de pastelaria ou copos de vinho consumidos por
semana. (p=0,033). Relativamente ao consumo de peixe ou marisco,
30,6% dos participantes portugueses consumia ≥3 porções/
semana, enquanto que apenas 10,5% dos participantes
estrangeiros o fazia, sendo esta diferença estatisticamente
significativa (p=0,032). Não houve diferenças estaticamente
significativas relativamente à quantidade de azeite consumida
num dia, quantidade de fruta, leguminosas, frutos oleaginosos,
carne vermelha, hambúrguer ou produtos cárneos, manteiga,
margarina, ou natas consumidas por dia, quantidade de
produtos de pastelaria ou copos de vinho consumidos por
semana. Tabela/Table 2: Resultados do questionário (MEDAS)/Results of the questionnaire (MEDAS). Descrição das questões de adesão à Dieta Mediterrânica/
Description of the Mediterranean Diet adherence questions
Respostas com 1 ponto/
Answers with 1 point
Questões de adesão à dieta mediterrânica/MEDAS Questions
Critério para 1 ponto/
Criteria for punctuation
Portugueses/
Portuguese
Estrangeiros/
Foreigner
Valor/
value -p
1. Utiliza azeite como principal gordura culinária?/Do you use olive oil as main culinary
fat? Sim/Yes
34 (94,4%)
32 (86,5%)
0,248
2. Que quantidade de azeite consome num dia (incluindo uso para fritar, temperar
saladas,refeições fora de casa, etc.)?/How much olive oil do you consume in a given
day (including oil used for frying, salads, out-of-house meals, etc.)? ≥ 4 colheres de sopa/
tablespoons
10 (27,0%)
13 (35,1%)
0,451
3. Quantas porções de produtos hortícolas consome por dia? (1 porção: 200 g;
considere acompanhamentos como metade de uma porção)/How many vegetable
servings do you consume per day? (1 serving : 200 g , consider side dishes as hald of
serving? ≥ 2 (≥ 1 se for porção em cru
ou em salada/
if serving raw or in salad)
22 (61,1%)
11 (31,4%)
0,012
4. A r t i g o s C i e n t í f i c o s | S c i e n t i f i c A r t i c l e s Quantas peças de fruta (incluindo sumos de fruta natural) consome por dia?/How
many fruit units (including natural fruit juices) do you consume per day? ≥ 3
10 (27,8%)
10 (27,0%)
0,943
5. Quantas porções de carne vermelha, hambúrguer ou produtos cárneos (presunto,
salsicha, etc.) consome por dia? (1 porção: 100-150 g)/How many servings of red meat,
hamburger, or meat products (ham, sausage,etc.)do you consume per day? (1 portion:
100-150 g)
< 1
8 (21,6%)
8 (21,6%)
1,00
6. Quantas porções de manteiga, margarina, ou natas consome por dia? (1 porção:
12 g)/How many servings of butter, margarine, or cream do you consume per day? (1
serving: 12g)
< 1
24 (64,9%)
16 (43,2%)
0,062
7. Quantas bebidas açucaradas ou gaseificadas bebe por dia?/How many sweet or
carbonated beverages do you drink per day? < 1
28 (77,8%)
20 (54,1%)
0,033
8. Quantos copos de vinho bebe por semana?/How much wine do you drink per week? ≥ 7 copos/glasses
1 (2,7%)
3 (7,7%)
0,330
9. Quantas porções de leguminosas consome por semana? (1 porção: 150 g)/How
many servings of legumes do you consume per week? (1 portion: 150 g)
≥ 3
14 (37,8%)
16 (42,1%)
0,706
10. Quantas porções de peixe ou marisco consome por semana? (1 porção: 100-150 g
de peixe ou 4-5 unidades ou 200 g de marisco)/How many servings of fish/shellfish do
you consume per week? (1 serving: 100-150 g fish or 4-5 units or 200 g shellfish)
≥ 3
11 (30,6%)
4 (10,5%)
0,032
11. Quantas vezes por semana consome produtos de pastelaria ou doces comerciais
(não caseiros), como bolos, bolachas, biscoitos?/ How many times per week do you
consume commercial sweets or pastries (not homemade), such as cakes, cookies,
biscuits, or custard? < 3
31 (83,5%)
28 (75,7%)
0,386
12. Quantas porções de oleaginosas (nozes, amêndoas, incluindo amendoins)
consome por semana? (1 porção: 30 g)/How many servings of nuts (including peanuts)
do you consume per week? (1 serving 30 g)
≥ 3
8 (21,6%)
7 (18,9%)
0,772
13. Consome preferencialmente frango, peru ou coelho em vez de vaca, porco,
hambúrguer ou salsicha?/Do you preferentially consume chicken, turkey, or rabbit
meat instead of veal, pork, hamburguer, or sausage? Sim/Yes
32 (88,9%)
31 (81,6%)
0,377
14. A r t i g o s C i e n t í f i c o s | S c i e n t i f i c A r t i c l e s Quantas vezes por semana consome hortícolas, massa, arroz ou outros pratos
confecionados com um refogado (tomate, cebola, alho-francês ou alho e azeite)?/
How many times per week do you consume vegetables, pasta, rice, or other dishes
seasoned with sofrito (sauce made with tomato and onion, leek, or garlic and
simmered with olive oil)? ≥ 2
33 (94,3%)
33 (89,2%)
0,434
Tabela/Table 2: Resultados do questionário (MEDAS)/Results of the questionnaire (MEDAS). 3.2. QUESTIONÁRIO MEDAS As respostas a cada item do questionário MEDAS estão
apresentados na Tabela 2. Foi observado que 94,4% dos
participantes portugueses e 86,5% dos estrangeiros, utilizavam
azeite como a principal gordura culinária. Em relação à
quantidade de hortícolas consumidas diariamente, 61,1% dos
participantes portugueses consumia ≥2 porções/dia, enquanto
que apenas 31,4% dos estrangeiros consumia essa mesma
quantidade, tendo sido verificada uma diferença significativa
(p=0,012) entres eles. Relativamente ao consumo de bebidas
açucaradas 77,8% dos participantes portugueses e 54,1% dos
estrangeiros consumia <1 bebida açucarada/dia, tendo sido
verificada uma diferença significativa entre os dois grupos The results of each MEDAS questionnaire item are shown in
Table 2. It was observed that 94.4% of Portuguese participants
and 86.5% of foreigners used olive oil as the main cooking fat. Regarding the amount of vegetables consumed daily, 61.1%
of Portuguese participants consumed ≥2 portions/day, while
only 31.4% of foreigners consumed the same amount, with
a significant difference (p=0.012) between them. Regarding
the consumption of sugary drinks, 77.8% of Portuguese
participants and 54.1% of foreigners consumed <1 sugary
drink/day, with a significant difference between the two groups
(p=0.033). Regarding the consumption of fish or shellfish, 30.6%
of Portuguese participants consumed ≥3 portions/week, while R e v i s t a C i e n t í f i c a I n t e r n a c i o n a l R e v S A L U S A r t i g o s C i e n t í f i c o s | S c i e n t i f i c A r t i c l e s of adherence to the MD, a greater percentage of Portuguese
had moderate (78.4%) and high (8.1%) adherence than
foreigners (52.8% and 2.8% respectively); a higher percentage of
foreigners were classified as having a low adherence to the MD
(44.4%) compared to Portuguese (13.5%) (p=0.012). Regarding
the amount of olive oil consumed by the participants, there
was a median (P25-P75) of 2.0 (1-4) and an average (SD) of
3.1 (2.6) tablespoons, with no significant differences between
Portuguese and foreigners. of adherence to the MD, a greater percentage of Portuguese
had moderate (78.4%) and high (8.1%) adherence than
foreigners (52.8% and 2.8% respectively); a higher percentage of
foreigners were classified as having a low adherence to the MD
(44.4%) compared to Portuguese (13.5%) (p=0.012). Regarding
the amount of olive oil consumed by the participants, there
was a median (P25-P75) of 2.0 (1-4) and an average (SD) of
3.1 (2.6) tablespoons, with no significant differences between
Portuguese and foreigners. relativamente aos estrangeiros (6,0) (p=0,009). Em relação ao
nível de adesão à DM, uma maior percentagem de portugueses
apresentava uma adesão moderada (78,4%) e alta (8,1%) que
os estrangeiros (52,8% e 2,8% respetivamente); uma maior
percentagem de estrangeiros foram classificados com uma baixa
adesão à DM (44,4%) relativamente aos portugueses (13,5%)
(p=0,012). Em relação à quantidade de azeite consumida pelos
participantes, verificou-se uma mediana (P25-P75) de 2,0 (1-4)
e uma média (DP) de 3,1 (2,6) colheres de sopa, não havendo
diferenças significativas entre portugueses e estrangeiros. Tabela/Table 3: Adesão à Dieta Mediterrânica e consumo de azeite/Adherence to the Mediterranean Diet and olive oil consumption Nível de adesão à Dieta Mediterrânica (MEDAS)/Score of adherence to the Mediterranean Diet (MEDAS)
Total
Portugueses/Portuguese
Estrangeiros/Foreigner
Valor/value -p
Mediana/Median (IQR)
7.0 (5.5 -9.0)
8.0 (6-9)
6.0 (4.50-7.50)
0.009
Média/Mean ± SD
6.8 ± 2.0
7.4 ± 1.8
6.2 ± 2.0
Categorias de adesão à Dieta Mediterrânica/Categories of Mediterranean Diet adherence - n (%)
Total
Portugueses/Portuguese
Estrangeiros/Foreigner
Valor/value -p
Baixo/Low (0-5)
21 (27.6%)
5 (13.5%)
16 (44.4%)
0.012
Moderado/Moderate (6-9)
48 (63.2%)
29 (78.4%)
19 (52.8%)
Alto/High (10-14)
4 (5.3%)
3 (8.1%)
1 (2.8%)
Quantidade de azeite consumida (colheres sopa/dia)/Amount of olive oil consumed (tablespoons/day)
Total
Portugueses/Portuguese
Estrangeiros/Foreigner
Valor/value -p
Mediana/Median (IQR)
2.0 (1.0-4.0)
2.3 (0.5-3.5)
2.0 (1.0-4.0)
0.337
Média/Mean ± SD
3.1 ± 2.6
2.9 ± 2.3
3.9 ± 2.9
IQR - Intervalo interquartil/Interquartile range; SD- Desvio Padrão/Standard deviation. 4. DISCUSSÃO A DM tem grandes benefícios para a saúde, tais como a
diminuição de risco de doenças crónicas, como a diabetes e
hipertensão arterial, e da mortalidade geral, devido às suas
comprovadas características alimentares saudáveis (Kant et al.,
2004; Trichopoulou et al, 2003). Os estudantes do ensino superior
por serem mais jovens, normalmente são mais escolarizados e
mais saudáveis do que a população em geral, mas a passagem
para a vida independente pode causar muito stress e pode
influenciar as escolhas alimentares, especialmente se estiverem
a viver fora de casa ou fora do país de origem (Ansari et al., 2012;
Sharma et al., 2010). MD has great health benefits, such as the decreased risk of
chronic diseases like diabetes and hypertension, and decreased
overall mortality, due to its proven healthy eating characteristics (Kant et al., 2004; Trichopoulou et al, 2003). Higher
education students, being younger, are usually more educated
and healthier than the general population, but the transition to
independent living can cause a lot of stress and can influence
food choices, especially if they are living away from home or
away from their home country. (Ansari et al., 2012; Sharma et
al., 2010). In this study, the assessment of MD adherence and olive
oil consumption, among higher education students and
researchers, - Portuguese and foreigners- were evaluated using
the MEDAS questionnaire. Olive oil is considered as the fat of
choice in the MD and the results of the present study showed
that 94.4% of Portuguese and 86.5% of foreign participants use
olive oil as the main cooking fat. Similar results were found in
another study assessing MD adherence in Spanish university
students, where it was found that more than 90% of Spanish
students used olive oil as their main cooking fat (Cobo-Cuenca
et al., 2019). This similarity probably reflects the cultural heritage
of Mediterranean countries having olive oil as a central food
component. Because of its properties, olive oil is associated with
a reduced incidence of cardiovascular diseases and increased
HDL cholesterol, which proves to be a great advantage for the
health of university students (Covas et al., 2015; Gómez-gracia Neste estudo a avaliação da adesão à DM e o consumo de
azeite, entre estudantes e investigadores do ensino superior,
portugueses e de outras nacionalidades foram avaliados através
do questionário MEDAS. A r t i g o s C i e n t í f i c o s | S c i e n t i f i c A r t i c l e s MEDAS (Mediterranean Diet Adherence Score)- Questionário de adesão à Dieta Mediterrânica. Tabela/Table 3: Adesão à Dieta Mediterrânica e consumo de azeite/Adherence to the Mediterranean Diet and olive oil consumption 3.3. ADESÃO À DIETA MEDITERRÂNICA Os resultados da adesão à DM estão apresentados na
tabela 3. A adesão à DM foi observada através da ferramenta
MEDAS, onde os participantes apresentaram uma pontuação
mediana (P25-P75) de 7,0 (5,5-9,0) e uma média (DP) de 6,8 (2,0). Analisando a população por nacionalidades, os portugueses
apresentam uma maior mediana (8,0) de adesão à DM, The results of adherence to MD are shown in table 3. Adherence to MD was observed using the MEDAS tool, where
participants had a median score (P25-P75) of 7.0 (5.5-9.0) and a
mean (SD) of 6.8 (2.0). Analyzing the population by nationality,
the Portuguese have a higher median (8.0) of adherence to the
MD, compared to foreigners (6.0) (p=0.009). Regarding the level et al., 2013). et al., 2013). associado à redução da incidência de doenças cardiovasculares
e aumenta o colesterol HDL, o que demonstra ser uma grande
vantagem para a saúde dos estudantes universitários (Covas et
al., 2015; Gómez-gracia et al., 2013). Vegetable consumption is a basic component of MD, and
it was observed in the results of this study that the majority
(61.1%) of Portuguese participants consumed ≥ 2 servings/day. This prevalence was significantly (p=0.002) higher than that
found in the foreign participants, (31.4%), possibly explained
by foreign participants living away from their parents, having
a poorer family background, or coming from a lower average
income country (Vilela et al., 2014). A total different result was
found in another study assessing the MD adherence of the
Italian population using the PREDIMED questionnaire, which
observed that 55% of the participants consumed ≥ 2 servings/
day of vegetables. The difference between the prevalence
of foreign participants consuming ≥ 2 servings per day of
vegetables in this study and the results of the study with Italian
students may be due to them being from a country with a
high human development index (HDI), and also because the
study had older participants and the level of education was
higher (Tessari et al., 2021). In previous studies, another reason
given for the low consumption of vegetables in the university
population was the price and taste (Pinho et al., 2018). O consumo de hortícolas é um componente básico da
DM, e foi observado nos resultados deste estudo que a maioria
(61,1%) dos participantes portugueses consumia ≥ 2 porções/
dia. Esta prevalência foi significativamente (p=0,002) mais alta
do que a encontrada nos participantes estrangeiros, (31,4%),
esta diferença nos resultados pode dever-se ao facto de os
estudantes estrangeiros viverem longe dos pais, terem uma
origem familiar mais pobre ou virem de um país de rendimento
médio mais baixo (Vilela et al., 2014). Um resultado totalmente
diferente foi encontrado em outro estudo que avaliou a adesão à
DM da população italiana por meio do questionário PREDIMED,
que observou que 55% dos participantes consumiram ≥ 2
porções/dia de hortícolas. et al., 2013). A diferença entre a prevalência dos
participantes estrangeiros que consomem ≥ 2 porções por
dia de hortícolas neste estudo e os resultados do estudo com
estudantes italianos pode ser devida a estes serem de um país
com um elevado índice de desenvolvimento humano (IDH),
e também pelo estudo ter participantes com mais idade e o
nível de escolaridade ser maior (Tessari et al., 2021). Em estudos
anteriores, outro motivo apontado para o baixo consumo de
hortícolas na população universitária, foi o preço e sabor (Pinho
et al., 2018). É possível que os estudantes que vivem com os
pais se envolvam ainda mais na dieta tradicional, enquanto os
que ficam sozinhos tenham uma dieta com baixo consumo de
hortícolas. Esse resultado pode exigir uma maior promoção
de hortícolas em uma variedade de lugares frequentados por
estudantes (Peltzer et al., 2015). Students who live with their parents may engage even
more in the traditional diet, while those who stay alone have
a diet with low consumption of vegetables. This result may
call for greater promotion of vegetables in a variety of places
frequented by students (Peltzer et al., 2015). High consumption of sugary drinks is an important factor
in the development of obesity and diabetes. In this study, it
was observed that the majority of Portuguese participants
(77.8%) and foreign participants (54.1%) consumed <1 sugary
or carbonated beverage per day. A similar result was found in
another study that assessed MD adherence among Lebanese
university students, revealing that half of this population
(51.8%) consumed <1 sugary or carbonated beverage per day
(Karam et al., 2021). Participants in both studies had a high level
of education, which contributes to greater food literacy and
healthier food choices. However, many participants did not
meet this criterion reinforcing the importance to re-educate
college students about reducing the intake of sugar and sugary
foods, to prevent the development of chronic diseases, through
actions to promote healthier living (Meric et al., 2021). O consumo elevado de bebidas açucaradas é um fator
importante no desenvolvimento de obesidade e diabetes. Neste estudo observou-se que a maioria dos participantes
portugueses (77,8%) e dos participantes estrangeiros (54,1%)
consumia <1 de bebidas açucaradas ou gaseificadas por dia. Resultado semelhante foi encontrado em outro estudo que
avaliou a adesão à DM entre estudantes universitários libaneses,
revelando que metade desta população (51,8%) consumia
<1 de bebidas açucaradas ou gaseificadas por dia (Karam et
al., 2021). 4. DISCUSSÃO O azeite é considerado como a gordura
de eleição da dieta DM e os resultados do presente estudo
mostraram que 94,4% dos estudantes portugueses e 86,5%
dos estudantes estrangeiros utilizam o azeite como principal
gordura culinária. Este resultado foi semelhante ao encontrado
em outro estudo que avaliou a adesão à DM e sua associação
com a composição corporal e a aptidão física em estudantes
universitários espanhóis, onde se constatou que mais de
90% dos estudantes espanhóis usaram azeite como principal
gordura culinária (Cobo-Cuenca et al., 2019). Esta semelhança
nos resultados deve-se ao fato da utilização do azeite como
principal gordura culinária ser o património cultural dos
países mediterrânicos. Pelas suas propriedades, o azeite está R e v i s t a C i e n t í f i c a I n t e r n a c i o n a l R e v S A L U S 2013). Foram encontrados resultados semelhantes num estudo que
avaliou a adesão à DM e bem-estar subjetivo em uma amostra
de adultos portugueses, sendo a percentagem mais baixa
(17,1%) classificado como alta adesão à DM, a percentagem
mais alta (62,7%) classificada como moderada, e a restante
(20,2%) classificada como baixa adesão (Andrade et al., 2020). In a study assessing adherence to the Mediterranean
dietary pattern in students at the University of Malta, similar
results were observed, in which 20% had low MD adherence,
72% moderate adherence, and 8% high adherence (Mota
et al., 2005). Similar results were observed in studies that
evaluated MD adherence using the Kidmed questionnaire
(Mediterranean diet quality index in children and adolescents). It can be observed in a study that evaluated the adherence to
the Mediterranean Diet among secondary school students in
the province of Taranto, MD adherence was classified as high
adherence in 24.8%, as moderate in 56.4% and low in 18.8% of
the study population (Bonaccorsi et al., 2020). In another study
that assessed MD adherence in a sample of Tuscan adolescents
using the Kidmed questionnaire, a high MD adherence of 16.5%,
a moderate adherence of 60.5%, and a low adherence of 23%
were observed in their results, reinforcing the low prevalence
of adherence classified as high. Implementation of healthy
policies targeting children and adolescents, especially those
at highest risk is needed to promote healthier lifestyle choices. The nutritional patterns of the Mediterranean Diet should be
among these choices (Santomauro et al., 2014). Em um estudo que avaliou a adesão ao padrão alimentar
mediterrânico em estudantes da Universidade de Malta, foram
observados resultados semelhantes, em que a adesão à DM
foi de 20% para uma adesão baixa, 72% para uma adesão
moderada e 8% para uma adesão alta (Mota et al., 2005). Semelhantes resultados foram observados em estudos que
avaliaram a adesão à DM através do questionário Kidmed
(Índice de qualidade da dieta mediterrânea em crianças e
adolescentes). Pode-se observar em um estudo que avaliou a
adesão à dieta mediterrânea entre alunos do ensino secundário
na província de Taranto, a adesão à DM foi classificada como
uma adesão alta em 24,8%, como moderada em 56,4% e
baixa em 18,8% na população em estudo. Em relação ao score
Kidmed, a média foi de 5,9 ± 2,4 (Bonaccorsi et al., 2020). 2013). ligado às diferenças culturais, influências do ambiente social,
conveniência na preparação das refeições e no preço elevado
(Pieniak et al., 2010; Tuu et al., 2008; Maciel et al., 2015). De
forma a se poder aumentar a frequência de consumo de peixe
e marisco nos estudantes universitários, é relevante pensar em
campanhas que incentivem o seu consumo e que destaquem
os seus benefícios(Maciel et al., 2013). The median (P25-P75) MD adherence, assessed by the
questionnaire (MEDAS), was 7.0 (5.5-9.0) and the mean (SD) was
6.8 (2.0) (p= 0.009). Similar results regarding MD adherence in
university students were observed in two national studies: one
study that used a sample of Portuguese university students,
and observed a mean (SD) score of 6.8 (2.3) (Ferreira-Pêgo et
al., 2019), and another study that used a population from
central Lisbon and reported a mean (SD) score of 7.29 (2.15)
(Gregório et al., 2020). One of the highlights in the present
study was the low percentage of the population (5.3%) that was
classified as having high MD adherence, with most participants
being classified with moderate adherence (63.2%), and 27.6%
with low adherence. Similar results were found in a study that
assessed MD adherence and subjective well-being in a sample
of Portuguese adults, with the lowest percentage (17.1%)
classified as high MD adherence, the highest percentage
(62.7%) classified as moderate, and the remainder (20.2%)
classified as low adherence (Andrade et al., 2020). A mediana (P25-P75) de adesão à DM, avaliada pelo
questionário (MEDAS), foi de 7,0 (5,5-9,0) e a média (DP) de 6,8
(2,0) (p= 0,009). Resultados semelhantes em relação à adesão
à DM em estudantes universitários foram observados em dois
estudos nacionais: um estudo que utilizou uma amostra de
universitários Portugueses, e observou um score médio (DP)
de 6.8 (2.3) (Ferreira-Pêgo et al., 2019), e outro estudo que
utilizou uma população do centro de Lisboa e que relatou uma
pontuação média (DP) de 7,29 (2,15) (Gregório et al., 2020). De
destacar no presente estudo a baixa percentagem da população
(5,3%) que foi classificada como tendo uma alta adesão à DM,
sendo que a maioria dos participantes foi classificado com uma
adesão moderada (63,2%), e 27,6% com uma adesão baixa. 2013). Num
outro estudo que foi avaliou a adesão à DM numa amostra de
adolescentes toscanos através do questionário Kidmed, foi
observado em seus resultados uma adesão à DM alta de 16,5%,
uma adesão moderada de 60,5% e uma baixa adesão de 23%,
reforçando a baixa prevalência de adesão classificada como
elevada. É necessária a implementação de políticas saudáveis
voltadas para as crianças e os adolescentes, principalmente
para aqueles em maior risco de forma a promover escolhas de
estilos de vida mais saudáveis. Os padrões nutricionais da dieta
mediterrânea devem estar entre essas escolhas (Santomauro et
al., 2014). A baixa adesão à DM pode ter sido influenciada pelo
facto de os estudantes universitários viverem longe dos pais,
do país, e também pela falta de tempo e preço dos principais
elementos da DM (Vilela et al., 2014; Pinho et al., 2018). The low adherence to MD may have been influenced by the
fact that the university students live far away from their parents
and their country, and also by the lack of time and price of the
main elements of MD (Vilela et al., 2014; Pinho et al., 2018). Some limitations of this study must be acknowledged. First, the study sample is relatively small. Secondly, the absence
of literature evaluating MD adherence among international
students, namely of Brazilian and Cape Verdean nationality. The results of this study may serve as a basis for further studies
in this population. Even though it is a relatively small sample,
it allowed us to detect a low to moderate adherence to MD
among Portuguese and foreigner participants. et al., 2013). A semelhança nos resultados pode dever-se ao fato
dos participantes de ambos os estudos terem um alto nível de
escolaridade, o que contribui para uma maior literacia alimentar
e escolhas alimentares mais saudáveis. É importante reeducar
os estudantes universitários sobre a redução da ingestão de
açúcar e alimentos que contenham açúcar, a fim de prevenir
o desenvolvimento de doenças crónicas, através de ações de
promoção para uma vida mais saudável (Meric et al., 2021). The Mediterranean dietary pattern is characterized by
moderate consumption of fish and seafood ≥3 servings/week,
and it was possible to verify in the results of this study that less
than half (30.6%) of the Portuguese participants and only 10.5%
of the foreign participants consumed ≥3 servings/week of fish
or seafood. In previous studies, it was observed that 28.8% of
Lebanese university students consumed ≥3 servings/week
(Karam et al., 2021), and in another study with Italian university
students, the consumption of ≥3 servings/week was 15%
(Tessari et al., 2021). O padrão alimentar mediterrânico é caracterizado por um
consumo moderado de peixe e marisco ≥3 porções/ semana,
e foi possível verificar nos resultados deste estudo que menos
de metade (30,6%) dos participantes portugueses e que
apenas 10,5% dos participantes estrangeiros consumia ≥ 3
porções/semana de peixe ou mariscos. Em estudos realizados
anteriormente, foi observado que 28,8% dos estudantes
universitários libaneses consumiam ≥3 porções/ semana (Karam
et al., 2021), e noutro estudo com estudantes universitários
italianos o consumo de ≥3 porções/ semana foi de 15% (Tessari
et al., 2021). O baixo consumo de peixe e marisco pode estar The low consumption of fish and seafood may be linked
to cultural differences, influences of the social environment,
convenience in meal preparation, and high price (Pieniak et
al., 2010; Tuu et al., 2008; Maciel et al., 2015). To increase the
frequency of fish and seafood consumption among college
students, it is relevant to think about campaigns that encourage
their consumption and highlight their benefits (Maciel et al., CONTRIBUIÇÕES AUTORAIS Conceptualização: JAS, VFL e MM; Metodologia: JAS, VFL e
MM; Recursos: JAS, VFL e MM; Curadoria de dados: TG e MM;
Análise formal: TG, JB and MM; Redação- preparação do draft
original: TG e MM; Redação- revisão e edição: JAS, JB, NR, VFL
e MM. A r t i g o s C i e n t í f i c o s | S c i e n t i f i c A r t i c l e s olive oil use as the main cooking fat, as recommended by the
Mediterranean Diet, stands out. High adherence to MD has great
health benefits, such as reducing the risk of chronic diseases
like diabetes, hypertension, obesity and neurodegenerative
diseases. An improvement in the consumption of various
components of this diet is needed to increase adherence in the
university community, highlighting the need for intervention to
raise awareness of the importance and health benefits of MD. Health promotion programs and evidence-based educational
interventions are needed to promote healthier eating. Nutrition
education, provided widely and effectively, is one of the pillars
for shaping healthy eating behaviors and consequent health
promotion. deste estudo podem servir de base para novos estudos nesta
população. Mesmo sendo uma amostra relativamente pequena,
permitiu-nos detetar uma baixa a moderada adesão à DM em
académicos portugueses e internacionais. 5. CONCLUSÕES Em
conclusão,
este
estudo
mostra
uma
adesão
moderada a baixa à Dieta Mediterrânica nos estudantes e
investigadores portugueses e internacionais, sendo menor
nestes últimos. No entanto, na maioria dos participantes de
ambas as nacionalidades, destaca-se a elevada prevalência
do uso de azeite como principal gordura culinária, tal como
é recomendado pela Dieta Mediterrânica. Uma alta adesão à
DM tem grandes benefícios para a saúde, como a redução do
risco de doenças crónicas como a diabetes, hipertensão arterial,
obesidade, doenças neurodegenerativas. É necessária uma
melhoria no consumo de vários componentes desta dieta para
aumentar a adesão da comunidade universitária, salientando a
necessidade de intervenção para sensibilizar para a importância
e os benefícios para a saúde da DM. Programas de promoção da
saúde e intervenções educacionais baseadas em evidências são
necessários para promover uma alimentação mais saudável. A
educação nutricional, fornecida de forma ampla e eficiente, é
um dos pilares para a formação de comportamentos alimentares
saudáveis e consequente promoção da saúde. AGRADECIMENTOS Conceptualization: JAS, VFL and MM; Methodology: JAS,
VFL and MM; Resources: JAS, VFL and MM; Investigation: TG and
MM; Data curation and Formal analysis: TG, JB and MM; Writing
- original draft: TG and MM; Writing - review & editing: JAS, JB,
NR, VFL and MM. Os autores gostariam de agradecer a todos os
participantes que voluntariamente participaram neste estudo. Os autores agradecem também à Fundação para a Ciência e
Tecnologia (FCT, Portugal) pelo apoio financeiro através de
fundos nacionais FCT/MCTES ao CIMO (UIBD/00690/2020) e
pela bolsa 2021.051216.BD. Agradecem ainda ao Programa
Operacional Regional do Norte - NORTE 2020 pelo emprego
científico da doutorada Manuela Meireles através do Projecto
NORTE-06-3559-FSE-000188 “OleaChain: Competências para
a sustentabilidade e inovação da cadeia de valor do olival
tradicional no Norte Interior de Portugal”. ACKNOWLEDGMENTS The authors would like to thank to all the participant who
voluntarily took part in this study. Authors are also grateful to
the Foundation for Science and Technology (FCT, Portugal)
for financial support by national funds FCT/MCTES to CIMO
(UIBD/00690/2020) and PhD grant of JB (2021.051216.BD). Manuela Meireles has a scientific contract through the Project
“OleaChain: Skills for sustainability and innovation in the value
chain of traditional olive groves in the Northern Interior of
Portugal” (NORTE-06-3559-FSE-000188). 5. CONCLUSIONS Algumas limitações deste estudo devem ser reconhecidas. Em primeiro lugar, a amostra do estudo é relativamente
pequena. Em segundo lugar, a ausência de literatura que avalia
a adesão à DM entre estudantes internacionais, nomeadamente
de nacionalidade brasileira e cabo-verdiana. Os resultados In conclusion, this study shows moderate to low adherence
to the Mediterranean Diet in Portuguese and foreign students
and researchers, being lower in the foreign ones. However, in
most participants of both nationalities, the high prevalence of R e v i s t a C i e n t í f i c a I n t e r n a c i o n a l R e v S A L U S REFERÊNCIAS BIBLIOGRÁFICAS/REFERENCES Afonso L, Moreira T, Oliveira A. Índices de adesão ao padrão alimentar
mediterrânico – a base metodológica para estudar a sua relação
com a saúde. Factores de Risco 31:48-55, 2014. Afonso L, Moreira T, Oliveira A. Índices de adesão ao padrão alimentar
mediterrânico – a base metodológica para estudar a sua relação
com a saúde. Factores de Risco 31:48-55, 2014. Aidoud A, Ziane E, Vara L, et al. Changes in Mediterranean dietary
pattern of university students: A comparative study between
Spain and Algeria. Nutr Clin y Diet Hosp 39(2):26-33, 2019. Andrade V, Jorge R, García-Conesa MT, et al. Mediterranean diet
adherence and subjective well-being in a sample of portuguese
adults. Nutrients 12(12):1-15,2020. Ansari EW, Stock C, Mikolajczyk RT. Relationships between food
consumption and living arrangements among university students
in four European countries - A cross-sectional study. Nutr J 11(1):1-
7, 2012. A r t i g o s C i e n t í f i c o s | S c i e n t i f i c A r t i c l e s A r t i g o s C i e n t í f i c o s | S c i e n t i f i c A r t i c l e s Bach-Faig A, Berry EM, Lairon D, et al. Mediterranean diet pyramid
today. Science and cultural updates. Public Health Nutr
14(12A):2274-2284, 2011. Bonaccorsi G, Furlan F, Scocuzza M, Lorini C. Adherence to mediterranean
diet among students from primary and middle school in the
Province of Taranto, 2016–2018. Int J Environ Res Public Health
17(15):1-10, 2020. Cobo-Cuenca AI, Garrido-Miguel M, Soriano-Cano A, Ferri-Morales
A, Martínez-Vizcaíno V, Martín-Espinosa NM. Adherence to the
mediterranean diet and its association with body composition and
physical fitness in Spanish university students. Nutrients 11(11), 2019. Covas MI, De La Torre R, Fitó M. Virgin olive oil: A key food for
cardiovascular risk protection. Br J Nutr 113(S2): S19-S28, 2015. Ferreira-Pêgo C, Rodrigues J, Costa A, Sousa B. Adherence to the
Mediterranean diet in Portuguese university students. Biomed
Biopharm Res 16(1):41-49, 2019. Freitas A, Bernardes JP, Mateus MP, Braz N. Dimensions of the
Mediterranean Diet. Universidade do Algarve, 2015. Gómez-gracia E, Ruiz-gutiérrez V, Fiol M. Primary prevention of
cardiovascular disease with a mediterranean diet. Zeitschrift fur
Gefassmedizin 10(2):28, 2013. Gregório MJ, Rodrigues AM, Salvador C, et al. R e v i s t a C i e n t í f i c a I n t e r n a c i o n a l R e v S A L U S REFERÊNCIAS BIBLIOGRÁFICAS/REFERENCES Validation of the
telephone-administered version of the mediterranean diet adherence
screener (Medas) questionnaire. Nutrients 12(5):1-13, 2020. Haire-Joshu D, Tabak R. Preventing Obesity Across Generations:
Evidence for Early Life Intervention. Annu Rev Public Health
37:253, 2016. IBM. Propel research and analysis with a comprehensive statistical
software solution. IBM Corp, 2020. Kant AK. Dietary patterns and health outcomes. J Am Diet Assoc
104(4):615-635, 2004. Karam J, Bibiloni M, Serhan M. Adherence to Mediterranean Diet among
Lebanese University Students. Lancet 255(6614):1044, 2021. Lopes C, Torres D, Oliveira A, Severo M, Alarcão V, Guiomar S, Mota J,
Teixeira P, Rodrigues S, Lobato L, Magalhães V, Correia D, Carvalho
C, Pizarro A, Marques A, Vilela S, Oliveira L, Nicola P, Soares S, Ramos
E. Inquérito Alimentar Nacional e de Atividade Física, IAN-AF 2015-
2016: Relatório de resultados. Universidade do Porto, 2017. ISBN:
978-989-746-181-1. Disponível em: www.ian-af.up.pt. Maciel E da S, Savay-Da-Silva LK, Vasconcelos JS, Galvão JA, Sonati JG,
Silva D, Oetterer M. Application of exploratory factor analysis to
assess fish consumption in a university community. Food Sci
Technol 33(1):99-106, 2013. Maciel E da S, Savay-Da-Silva LK, Galvão JA, Oetterer M. Atributos de
qualidade do pescado relacionados ao consumo na cidade de
Corumbá, MS. Bol do Inst Pesca 41(1):199-206, 2015. Martínez-González MA, García-Arellano A, Toledo E, et al. A 14-item
mediterranean diet assessment tool and obesity indexes among
high-risk subjects: The PREDIMED trial. PLoS One 7(8), 2012. Menotti A, Kromhout D, Blackburn H, Fidanza F, Buzina R, Nissinen A. Food intake patterns and 25-year mortality from coronary heart
disease: Cross-cultural correlations in the Seven Countries Study. Eur J Epidemiol 15(6):507-515, 1999. Meric ÇS, Ayhan NY, Yilmaz HÖ. Evaluation of added sugar and sugar-
sweetened beverage consumption by university students. Kesmas
16(1):9-15, 2021. Mota L, Sorg I, Cornelis G, et al. Adherence to the Mediterranean dietary
pattern among University students. FEMS Microbiol Lett 222:103–
505, 2006. Peltzer K, Pengpid S. Correlates of healthy fruit and vegetable diet in
students in low, middle and high income countries. Int J Public
Health 60(1):79-90, 2015. Pieniak Z, Verbeke W, Scholderer J. Health-related beliefs and consumer
knowledge as determinants of fish consumption. J Hum Nutr Diet
23(5):480-488, 2010. A r t i g o s C i e n t í f i c o s | S c i e n t i f i c A r t i c l e s Pinho MGM, Mackenbach JD, Charreire H, Oppert JM, Bárdos H, Glonti
K, Rutter H, Compernolle S, De Bourdeaudhuij I, Beulens JWJ,
Brug J, Lakerveld J. Exploring the relationship between perceived
barriers to healthy eating and dietary behaviours in European
adults. Eur J Nutr 57(5):1761-1770, 2018. Renzella J, Townsend N, Jewell J, Breda J, Roberts N, Rayner M,
Wickramasinghe K. What national and subnational interventions
and policies based on are recommended or implemented in the
WHO European Region, and is there evidence of effectiveness in
reducing noncommunicable diseases? Health Evidence Network
Synthesis Reports 58 2018. Salameh P, Jomaa L, Issa C, et al. Assessment of dietary intake patterns
and their correlates among university students in Lebanon. Front
Public Heal 2:1-12, 2014. Sánchez-Villegas A, Bes-Rastrollo M, Martínez-González M, Serra-Majem
L. Adherence to a Mediterranean dietary pattern and weight gain in
a follow-up study: The SUN cohort. Int J Obes 30(2):350-358, 2006. Santomauro F, Lorini C, Tanini T, et al. Adherence to mediterranean diet
in a sample of tuscan adolescents. Nutrition 30(11-12):1379-1383, 2014. Sharma B, Harker M, Harker D, Reinhard K. Youth transition to university
in Germany and Australia: An empirical investigation of healthy
eating behaviour. J Youth Stud 13(3):353-367, 2010. Spiegel AM, Nabel EG. NIH research on obesity and type 2 diabetes:
providing the scientific evidence base for actions to improve
health. Nat Med 12(1):67-69, 2006. Tessari S, Casazza M, De Boni G, Bertoncello C, Fonzo M, Di Pieri
M, Russo F. Promoting health and preventing non-communicable
diseases: evaluation of the adherence of the Italian population to
the Mediterranean Diet by using the PREDIMED questionnaire. Ann Ig 33(4):337-346, 2021. Trichopoulou A, Costacou T, Bamia C, Trichopoulos D. Adherence to a
Mediterranean Diet and Survival in a Greek Population. N Engl J
Med 348(26):2599-608, 2003. Tuu HH, Olsen SO, Thao DT, Anh NTK. The role of norms in explaining
attitudes, intention and consumption of a common food (fish) in
Vietnam. Appetite 51(3):546-551, 2008. Vilela S, Santos S, Padrão P, Caraher M. Length of Migration and Eating
Habits of Portuguese University Students Living in London, United
Kingdom. Ecol Food Nutr 53(4):419-435, 2014. Visioli F, Galli C. The role of antioxidants in the Mediterranean diet. Lipids 36(SUPPL.):49-52, 2001. WMA. WMA Declaration of Helsinki – Ethical Principles for Medical
Research Involving Human Subjects – WMA – The World Medical
Association. WMA Declar Helsinki – Ethical Princ Med Res Involv
Hum Subj. Disponível em: https://www.wma.net/policies-post/
wma-declaration-of-helsinki-ethical-principles-for-medical-
research-involving-human-subjects/.
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https://www.nature.com/articles/s41375-024-02168-z.pdf
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English
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Che-1/miR-590-3p/TAZ axis sustains multiple myeloma disease
|
Leukemia
| 2,024
|
cc-by
| 4,109
|
TO THE EDITOR:
M l i l
l YAP genes by querying patient gene expression in the context of
disease evolution, using the International Staging System (ISS) as a
proxy. Notably, TAZ expression significantly decreased during the
progression of the MM (from stage 1 to stage 3) showing an
anticorrelation
with Che-1
levels
(Supplementary
Fig. S2A),
whereas we did not observe any significant trend associated with
the YAP gene (Supplementary Fig. S2B). Moreover, by using a
patient cohort from Oncomine database [8], we confirmed
CoMMpass results (Supplementary Fig. S2C). Accordingly, the
survival analysis of 639 MM patients (CoMMpass dataset) revealed
a high expression of TAZ strongly associated with a better
prognosis in MM (Fig.1C) confirmed by Oncomine database [8]
(Supplementary Figs. S2D, E). Multiple myeloma (MM) is a malignant disease of plasma cells that
produces high levels of monoclonal immunoglobulins. This
pathology involves among its clinical implications osteolytic bone
disease, which represents the major cause of mortality in patients
with MM [1]. YAP/TAZ co-factors belong to the HIPPO pathway,
which
plays
an
important
role
in
epidermal
and
dermal
development. TAZ, encoded by the WWTR1 gene, was found to
be downregulated in MM patients [2], and low TAZ expression is
correlated to a decrease in osteogenic potential of mesenchymal
stem cells from MM, supporting its putative involvement in bone
lesions present in MM patients [3]. Che-1/AATF (Che-1) is a protein
involved in various cellular pathways [4]. Recently, some studies
have shown an increase in its expression levels during MM
progression and its involvement in the survival of myeloma cells
[5–7]. By retrieving data from RNA-seq analysis of plasma cells
from a conditional Che-1 transgenic mouse model (Vk*Che-1) [5],
we observed TAZ among downregulated genes in Vk*Che-1 mice
(Fig. 1A). RT-qPCR and western blot analyses confirmed these
results, while no changes in YAP expression were observed (Fig. 1B
and Supplementary Fig. S1A). Further RT-qPCR analyses showed a
decrease of mRNA expression of two TAZ target genes, CTGF and
CYR61 (Supplementary Fig. S1B). Conversely, the RNA-seq analysis
performed in Che-1 depleted Kms27 MM cell [5] revealed an up-
regulation of TAZ and its targets, but not of YAP (Supplementary
Fig. S1C). These results were further confirmed at transcriptional
and post translational level in Kms27 and Kms18 MM cell lines
(Supplementary Fig. S1D), and similar results were also observed
by depleting Che-1 expression with another specific siRNA (siChe-
1B) (Supplementary Fig. Received: 14 June 2023 Revised: 1 February 2024 Accepted: 2 February 2024
1SAFU Laboratory, Department of Research, Advanced Diagnostics, and Technological Innovation, Translational Research Area, IRCCS Regina Elena National Cancer Institute,
Rome, Italy. 2Microbiology and Virology Unit, San Gallicano Dermatological Institute IRCCS, Rome, Italy. 3Laboratory of Cutaneous Physiopathology and Integrated Center of
Metabolomics Research, IRCCS San Gallicano Dermatological Institute, Rome, Italy. 4Hematology and Stem Cell Transplant Unit, IRCCS Regina Elena National Cancer Institute,
Rome, Italy. 5Unit Of Hematology, Stem Cell Transplantation, Fondazione Policlinico Universitario Campus Bio-Medico, Via Alvaro del Portillo, 200, 00128 Roma, Italy.
6Translational Oncology Research Unit, IRCCS Regina Elena National Cancer Institute, Via Elio Chianesi, 53, 00144 Rome, Italy. 7Department of Health Sciences, Magna Græcia
University of Catanzaro, 88100 Catanzaro, Italy. 8These authors contributed equally: Tiziana Bruno, Valeria Catena, Giacomo Corleone. ✉email: maurizio.fanciulli@ifo.it © The Author(s) 2024 Leukemia (2024) 38:877–882; https://doi.org/10.1038/s41375-024-02168-z Leukemia (2024) 38:877–882; https://doi.org/10.1038/s41375-024-02168-z Leukemia www.nature.com/leu Received: 14 June 2023 Revised: 1 February 2024 Accepted: 2 February 2024
Published online: 17 February 2024 LETTER
OPEN
Che-1/miR-590-3p/TAZ axis sustains multiple myeloma disease
Tiziana Bruno1,8, Valeria Catena1,8, Giacomo Corleone
1,8, Clelia Cortile
1, Maria Chiara Cappelletto2, Barbara Bellei3,
Francesca De Nicola1, Bruno Amadio1, Svitlana Gumenyuk4, Francesco Marchesi
4, Ombretta Annibali5, Giovanni Blandino
6,
Maurizio Fanciulli
1✉and Silvia Di Agostino
7 © The Author(s) 2024 © The Author(s) 2024 TO THE EDITOR:
M l i l
l Notably, Vk*Che-1 mice exhibited higher levels
of EIF4H mRNA and miR-590-3p when compared to their control
littermates (Fig. 1G), and a decrease of both EIF4H and miR-590-3p
expression was observed in Che-1 depleted MM cells (Supple-
mentary Fig. S4F). Interestingly, miR-590-3p (and EIF4H) and TAZ
expression were inversely correlated in MM cell lines (Supple-
mentary Figs. S4G, H). Accordingly, miR-590-3p overexpression
induced a reduction in TAZ levels in MM cell lines (Fig. 1H;
Supplementary Fig. S4I). To assess direct mRNA regulation on TAZ,
we cloned 3’-UTR of TAZ WT or mutant in a luciferase reporter
vector. As shown in (Supplementary Fig. S4J). g
Next, we evaluated the effects of the osteogenic differentiation
of two tissue-derived pluripotent mesenchymal stem cell lines,
ADSC1 and ADSC2. The staining of calcium deposits by using
Alizarine red S (ARS) confirmed that both these cells underwent an
increase in the mineralization process (Fig. 2F and Supplementary
Fig. S5C) accompanied by an upregulation of genes involved in
osteoblast differentiation (Supplementary Figs. S5B, E). Interest-
ingly, together with an increase of TAZ expression, we observed in
differentiated cells a significant decrease of both Che-1 and
miR590-3p levels (Fig. 2G and Supplementary Fig. 5D). Strikingly,
the presence of miR590-3p in the medium of these cells strongly
affected the differentiation process, decreasing calcium mineral
deposits (Figs. 2H, I and Supplementary Figs. S5F, G) and a
significant
reduction
of
TAZ
expression
and
differentiation
markers (Fig. 2J and Supplementary Fig. S5G). Importantly, similar
results were obtained overexpressing Che-1 in ADSC1 cells
(Supplementary Fig. S5I), observing an increase of EIF4H and
miR590-3p expression (Supplementary Fig. S5J), and an inhibition
of
TAZ
expression
and
osteogenic
differentiation
markers
(Supplementary Figs. S5K, L). pp
y
g
It has been described that TAZ expression in MM is inhibited
through its hypermethylation [2]. To compare the regulation of
TAZ by miR-590-3p with epigenetic regulation, we treated with
the demethylating agent 5-azacytidine MM cells transfected or not
with an anti-sense able to downregulate miR-590-3p levels. These
experiments showed that DNA demethylation has a greater
impact on TAZ expression than miR inhibition, but that the
combined treatments have an additive effect (Supplementary
Fig. S4K). Strikingly, ectopic overexpression of miR-590-3p in MM
cell lines was able to strongly counteract the increase of TAZ
expression at transcriptional and protein levels produced by Che-1
depletion, (Fig. 1I and Supplementary Figs. TO THE EDITOR:
M l i l
l Volcano plot displaying 2030 significantly upregulated (Red) and 759 downregulated (Blue) genes. x-axis: -log2(q value) which
approximates fold change (FC) obtained from the Wald test. Y-axis: -log10 (q-value) of significant genes. B Total cell extracts (TCEs) from
Vk*Che-1 bone marrow compared with control littermates were subjected to Western Blot analysis (WB) and probed for the indicated
antibodies (Abs). C Kaplan–Meier survival curve of overall survival (OS) in the CoMMpass cohort (N = 639) with TAZ expression as unique
variable. The patient cohort was divided into TAZ high (N = 312) (red) and TAZ low (N = 327) (black) by splitting the population at the TAZ
median expression threshold. The patients above the TAZ median expression in our cohort were assigned to TAZ High, while patients below
the median were assigned to TAZ low group. D Kms27 cell proliferation (105) of the time course at indicated points (Right), and WB analysis
(Left) with the indicated Abs after TAZ overexpression. E Volcano plot showing the up (Red) and down (Blue) regulated transcripts upon TAZ
overexpression (HA-TAZ). X-axis: Effect size represented by the B value; y-axis: -log10(FDR). Differential transcripts are defined as FDR < 10-3
and B value > |0.7 | . F ChIP-seq signal on EIF4H promoter of Che-1, RNA Pol II and H3K27ac in Kms27 MM cells from two independent
experiments. Signal enrichment scale is reported on the y-axis of the site. G RT-qPCR analysis of EIF4H and miR590-3p expression of bone
marrow from three Vk*Che-1 mice. Values were normalized to Actin expression. Error bars represent the standard error of three different
analyses. *P < 0. 05, ****P < 0.001. H Left: RT-qPCR analysis of TAZ expression in Kms27 MM cells transiently transfected with miR-590-3p or
Control mimic. Values were normalized to Actin expression. Error bars represent the standard error of three different experiments. ****P < 0.001. Right: TCEs extracted from Kms27 MM cells transfected as above were analyzed by WB for the indicated Abs. I Left: RT-qPCR
analysis of TAZ expression from Kms27 MM cells transiently transfected with siControl, siChe-1A, miR-590-3p or its mimic where indicated. Values were normalized to Actin expression. Error bars represent the standard error of three different experiments. **P < 0.01. Right: TCEs
extracted from Kms27 MM cells transfected as above were analyzed by WB for the indicated Abs. (LCL) (Fig. 2A). TO THE EDITOR:
M l i l
l Moreover, we found that both Vk*Che-1 and
Vk*Myc transgenic mouse model, which, through activating c-Myc
oncogene in maturing B cells, induces MM transformation [13],
also showed higher levels of the miR-590-3p in their bone marrow
than control littermates (Fig. 2B and Supplementary Fig. S5A). These findings were further strengthened by the evaluation of the
levels of miR-590-3p in the bone marrow
of MGUS and
symptomatic patients, finding a dramatic increase of this molecule
in patients with MM (Fig. 2C). Consistent with these findings,
Vk*Che-1 mesenchymal stem cells showed higher levels of miR
590-3p
compared
to
same
cells
from
control
mice
with
concomitant reduced levels of TAZ (Fig. 2D). Moreover, biochem-
ical markers analysis [14] in sera from Vk*Che-1 mice indicated a
strong increase in bone resorption associated with a decrease in
bone formation (Fig. 2E). observed that hsa-miR-590 is placed within the intron at 5’ of the
EIF4H gene, a translation initiation factor. Interestingly, Che-1 ChIP-
seq showed the presence of this protein on the EIF4H promoter
together with RNA Pol II and H3K27ac (Fig. 1F), confirmed by ChIP-
RT analyses in different MM cell lines (Supplementary Fig. S4D). Consistent with these results, overexpression of Che-1 was able to
activate a luciferase reporter containing EIF4H promoter (Supple-
mentary Fig. S4E). Notably, Vk*Che-1 mice exhibited higher levels
of EIF4H mRNA and miR-590-3p when compared to their control
littermates (Fig. 1G), and a decrease of both EIF4H and miR-590-3p
expression was observed in Che-1 depleted MM cells (Supple-
mentary Fig. S4F). Interestingly, miR-590-3p (and EIF4H) and TAZ
expression were inversely correlated in MM cell lines (Supple-
mentary Figs. S4G, H). Accordingly, miR-590-3p overexpression
induced a reduction in TAZ levels in MM cell lines (Fig. 1H;
Supplementary Fig. S4I). To assess direct mRNA regulation on TAZ,
we cloned 3’-UTR of TAZ WT or mutant in a luciferase reporter
vector. As shown in (Supplementary Fig. S4J). observed that hsa-miR-590 is placed within the intron at 5’ of the
EIF4H gene, a translation initiation factor. Interestingly, Che-1 ChIP-
seq showed the presence of this protein on the EIF4H promoter
together with RNA Pol II and H3K27ac (Fig. 1F), confirmed by ChIP-
RT analyses in different MM cell lines (Supplementary Fig. S4D). Consistent with these results, overexpression of Che-1 was able to
activate a luciferase reporter containing EIF4H promoter (Supple-
mentary Fig. S4E). TO THE EDITOR:
M l i l
l S1E), but not in other cell lines of different
origin (Supplementary Fig. S1F). pp
y
g
To directly assess the relevance of TAZ expression in MM, we
overexpressed this gene in two different MM cell lines observing
in both experiments a strong reduction of cell proliferation
(Fig. 1D and Supplementary Fig. S3A). Next, we performed RNA-
seq analysis and unbiased GSEA in Kms27 cells overexpressing
TAZ (Fig. 1E). Notably, the Hallmarks collection from the Molecular
Signatures Database (MSigDB) revealed a downregulation of
several pathways involved in cell proliferation (Supplementary
Figs. S3B, C). Indeed, E2F1, Myc and PI3K/Akt/MTOR target genes
were inhibited in TAZ overexpressing cells, conversely targets of
the
interferon
response
resulted
activated
(Supplementary
Figs. S3D). In agreement with these results, the inhibition of
Deptor or MKK6 expression strongly reduced Kms27, Kms18 or
RPMI8226 cell proliferation (Supplementary Fig. S3E). Next, we moved to identify the mechanism/s by which Che-1
regulates
TAZ
expression. Che-1
positively
regulates
gene
transcription, binding promoter genes and increasing histone
acetylation in MM cells [5]. Previous ChIP-seq of Che-1 experi-
ments [5] did not show Che-1 on the WWTR1 gene promoter,
although both RNA Polymerase II and histone acetylation were
present (Supplementary Fig. S4A). This evidence led us to consider
an indirect way by which Che-1 could inhibit TAZ expression. Since TAZ is a target of several miRNAs [9], we hypothesized this
activity as a possible mechanism. To test this hypothesis, we
identified by an in-silico analysis the miR-590-3p targeting a highly
conserved sequence contained in the 3’ UTR of TAZ (Supplemen-
tary Figs. S4B, C). Throughout a Genome Browser analysis, we Previous studies have demonstrated that Che-1 is required for
cell proliferation in MM [5, 6]. To evaluate whether TAZ regulation
is required in this function, we performed rescue experiments,
demonstrating how TAZ depletion at least partially recovers the
effects of Che-1 inhibition (Supplementary Fig. S1G). Interestingly,
we found a decrease of Che-1 mRNA and protein expression in
MM cells overexpressing TAZ (Supplementary Fig. S1H), p
g
pp
y
g
Afterwards, we evaluated ~1000 MM complete transcriptomes
from the CoMMpass study to evaluate the RNA levels of TAZ and T. Bruno et al. T. Bruno et al. 878 Leukemia (2024) 38:877 – 882 T. Bruno et al. 879 Fig. 1
Che-1 regulates TAZ expression in MM through miR590-3p. A Differential analysis of Wild type c57/BL6 mice vs Vk*Che-1 mice
transcriptome. REFERENCES 1. Liu H, Liu Z, Du J, He J, Lin P, Amini B, et al. Thymidine phosphorylase exerts
complex effects on bone resorption and formation in myeloma. Sci Transl Med. 2016;8:353ra113. 1. Liu H, Liu Z, Du J, He J, Lin P, Amini B, et al. Thymidine phosphorylase exerts
complex effects on bone resorption and formation in myeloma. Sci Transl Med. 2016;8:353ra113. 2. Grieve S, Wajnberg G, Lees M, Chacko S, Weir J, Crapoulet N, et al. TAZ functions
as a tumor suppressor in multiple myeloma by downregulating MYC. Blood Adv. 2019;3:3613–25. ACKNOWLEDGEMENTS High Throughput Sequencing data of Vk*Che mice and Kms27 MM cells were
retrieved from High Throughput Sequencing data (RNA-seq, ChIP-seq, ATAC-seq-
identifier numbers GSE149031) previously reported in Bruno et al. [5]. High
Throughput Sequencing data (RNA-seq,) were publicly deposited and are available
at GEO (GSE234642). All other row data supporting the findings of this study are
available at this link: https://gbox.garr.it/garrbox/f/647389073. These data were generated as part of the Multiple Myeloma Research Foundation
Personalized Medicine Initiatives (https://research.themmrf.org and www.themmrf.org). We would like to thank Dr. Andrea Sacconi for some bioinformatics analysis
suggestions and the nursing staff Gianluca Falzone, Marco Prete, Anna Attico and
Antonio Cicotto of the Hematology and Stem Cell Transplant Unit. This work was
supported: Italian Association for Cancer Research (AIRC) grant ID 22759 to G.B.; Italian
Association for Cancer Research (A.I.R.C.) grant ID 15255, and Italian Ministry of Health
(RF-2019-12368737) to M.F. G. Corleone has received funding from AIRC and from the
European Union’s Horizon 2020 research and innovation program under the Marie
Skłodowska‐Curie grant agreement No. 800924. TO THE EDITOR:
M l i l
l Terpos E, Dimopoulos MA, Sezer O, Roodman D, Abildgaard N, Vescio R, et al. The
use of biochemical markers of bone remodeling in multiple myeloma: a report of
the International Myeloma Working Group. Leukemia. 2010;24:1700–12. have shown that MM cells secrete this miRNA, and high levels of
this molecule are found in vivo both in the bone marrow of MM
mouse models and in patients. Finally, overexpression of either
miR-590-3p or Che-1 significantly inhibited TAZ expression in
mesenchymal stem cells along with osteoblastic differentiation. 14. Terpos E, Dimopoulos MA, Sezer O, Roodman D, Abildgaard N, Vescio R, et al. The
use of biochemical markers of bone remodeling in multiple myeloma: a report of
the International Myeloma Working Group. Leukemia. 2010;24:1700–12. TO THE EDITOR:
M l i l
l S4L–M), thus confirm-
ing that Che-1 inhibited TAZ expression in MM by inducing EIF4H/
miR-590-3p transcription. Bone diseases are a major feature of MM, and an upregulation
of osteoclastic activity with a concomitant decrease in osteoblasts
is considered the hallmark of pathology [10]. In this context, TAZ
plays an important role in osteoblastic differentiation [11] and MM
cells inhibit its expression through the release of several factors in
the bone marrow, such as TNF-α and FGF2 [12]. Therefore, based
on the obtained results, we hypothesized that miR-590-3p could
contribute to inhibit osteoblastic differentiation by downregulat-
ing the expression of TAZ. In this context, miR-590-3p was found
to be released at high levels in the culture medium of several MM
lines compared to the levels found in a lymphoblastoid cell line In conclusion, in this study, we identify a novel role of Che-1 in
MM pathogenesis. Indeed, by performing experiments in cell lines,
patient samples, and mice models, we found that Che-1 can
negatively control TAZ expression in MM by upregulating miR-
590-3p. Our
results
clearly
demonstrated
that
during
the
differentiation of mesenchymal cells into osteoblasts, alongside
the induction of TAZ and its target genes, there is also a
downregulation of both Che-1 and miR-590-3p. Furthermore, we Leukemia (2024) 38:877 – 882 T. Bruno et al. T. Bruno et al. 880 Leukemia (2024) 38:877 – 882 T. Bruno et al. 881 Fig. 2
Che-1/miR590-3p inhibits osteogenic differentiation of mesenchymal stem cells. A RT-qPCR for estimation of the expression level of
miR590-3p release in the medium of different MM cell lines compared to lymphoblastoid cells. The expression was normalized versus miRNA
RNU48. Error bars represent the standard error of three different experiments. ***P < 0.005. B miR590-3p expression in Vk*Che-1( = 4)
transgenic mice compared to their control littermates. The normalization was carried out using the mouse miRNA sno202 levels as an internal
control. *P < 0.05, **P < 0.01. C miR590-3p expression in a cohort of patients at different stages of the disease (MGUS = 5, Symptomatic=5). The human miRNA RNU48 levels were used as an internal control. ***P < 0.005. D WB analysis with the indicated Abs (left) and RT-qPCR of TAZ
expression (center) or miR-590-3p (right) in mesenchymal stem cells from Vk*Che-1 or control littermates bone marrow. Values were
normalized to Actin expression. Error bars represent the standard error of three different experiments. *P < 0. 05. AUTHOR CONTRIBUTIONS T.B. and S.D.A. conceived the rationale of the study. T.B., S.D.A., V.C. and M.C.C. designed and performed all the experiments. F.D.N. performed RNA-seq experiments. G.C. and C.C. carried out the bioinformatics analyses. T.B., S.D.A. and M.F. drafting the
initial manuscript and structuring the figures. S.G. and F.M. provided sample from
patients of Multiple Myeloma. B.B. isolated and manipulated mesenchymal stem cells
from specimens of adipose tissue from healthy donors. B.A. isolated mesenchymal
stem cells from mice bone marrows. O.A. provided MGUS samples from patients. G.B. discussed the experiments on TAZ and miR-590-3p mechanism. T.B., S.D.A., G.B. and
M.F. reviewed, modified, and edited the manuscript. All authors have read and
agreed to the published version of the manuscript. 3. Li B, Shi M, Li J, Zhang H, Chen B, Chen L, et al. Elevated tumor necrosis factor-alpha
suppresses TAZ expression and impairs osteogenic potential of Flk-1+ mesenchy-
mal stem cells in patients with multiple myeloma. Stem Cells Dev. 2007;16:921–30. 4. Iezzi S, Fanciulli M. Discovering Che-1/AATF: a new attractive target for cancer
therapy. Front Genet. 2015;6:141. 4. Iezzi S, Fanciulli M. Discovering Che-1/AATF: a new attractive target for cancer
therapy. Front Genet. 2015;6:141. 5. Bruno T, De Nicola F, Corleone G, Catena V, Goeman F, Pallocca M, et al. Che-1/
AATF-induced transcriptionally active chromatin promotes cell proliferation in
multiple myeloma. Blood Adv. 2020;4:5616–30. 5. Bruno T, De Nicola F, Corleone G, Catena V, Goeman F, Pallocca M, et al. Che-1/
AATF-induced transcriptionally active chromatin promotes cell proliferation in
multiple myeloma. Blood Adv. 2020;4:5616–30. 6. Desantis A, Bruno T, Catena V, De Nicola F, Goeman F, Iezzi S, et al. Che-1-induced
inhibition of mTOR pathway enables stress-induced autophagy. EMBO J. 2015;34:1214–30. 7. Bruno T, Corleone G, Catena V, Cortile C, De Nicola F, Fabretti F, et al. AATF/Che-1
localizes to paraspeckles and suppresses R-loops accumulation and interferon
activation in Multiple Myeloma. EMBO J. 2022;41:e109711. 7. Bruno T, Corleone G, Catena V, Cortile C, De Nicola F, Fabretti F, et al. AATF/Che-1
localizes to paraspeckles and suppresses R-loops accumulation and interferon
activation in Multiple Myeloma. EMBO J. 2022;41:e109711. COMPETING INTERESTS The authors declare no competing interests. 8. Agnelli L, Mosca L, Fabris S, Lionetti M, Andronache A, Kwee I, et al. A SNP
microarray and FISH-based procedure to detect allelic imbalances in multiple
myeloma: an integrated genomics approach reveals a wide gene dosage effect. Genes Chromosomes Cancer. 2009;48:603–14. TO THE EDITOR:
M l i l
l E ELISA assays of sera from
three VK*Che-1 or three wildtype (wt) mice. C-terminal cross-linking telopeptide of type I collagen (aCTx) (competitive-ELISA principle), bone-
specific alkaline phosphatase (BALP), osteocalcin (OC), and C-terminal cross-linking telopeptide of type I collagen generated by MMPs (ICTP)
(sandwich-ELISA principle). *P < 0.05, **P < 0.01. F Left: WB analysis of TCEs from ADSC1 mesenchymal cells subjected to osteogenic
differentiation and probed with the indicated Abs. Right: Two representatives Alizarin Red S staining images from ADSC1 mesenchymal cell
treated as in (F). Bottom Bar Plot represented the measure of absorbance of stain extraction at 405 nM. Error bars represent the standard error
of three different experiments **P < 0.01. G RT-qPCR analysis of the indicated genes (left) and miR590-3p (right) expression from ADSC1
mesenchymal cells induced as in F. Values were normalized to Actin expression. *P < 0.05, **P < 0.01. H Left: WB analysis of TCEs from ADSC1
mesenchymal cells induced for osteogenic differentiation and transiently transfected where indicated with miR-590-3p or its mimic, and
probed with specific Abs. Right Two representatives Alizarin Red S staining images. Bottom The measurement of the stain from Alizarine Red
experiments was performed as in (F). Error bars represent the standard error of three different experiments. *P < 0.05. RT-qPCR analysis for
miR590-3p (I) and TAZ (J) expression from ADSC1 mesenchymal cell treated as in (H). Error bars represent the standard error of three different
experiments. Values were normalized to Actin expression. *P < 0.05, ***P < 0.005. 13. Chesi M, Robbiani DF, Sebag M, Chng WJ, Affer M, Tiedemann R, et al. AID-
dependent activation of a MYC transgene induces multiple myeloma in a conditional
mouse model of post-germinal center malignancies. Cancer Cell. 2008;13:167–80. 14. Terpos E, Dimopoulos MA, Sezer O, Roodman D, Abildgaard N, Vescio R, et al. The
use of biochemical markers of bone remodeling in multiple myeloma: a report of
the International Myeloma Working Group. Leukemia. 2010;24:1700–12. 13. Chesi M, Robbiani DF, Sebag M, Chng WJ, Affer M, Tiedemann R, et al. AID-
dependent activation of a MYC transgene induces multiple myeloma in a conditional
mouse model of post-germinal center malignancies. Cancer Cell. 2008;13:167–80. 13. Chesi M, Robbiani DF, Sebag M, Chng WJ, Affer M, Tiedemann R, et al. AID-
dependent activation of a MYC transgene induces multiple myeloma in a conditional
mouse model of post-germinal center malignancies. Cancer Cell. 2008;13:167–80. 14. © The Author(s) 2024 ADDITIONAL INFORMATION Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s41375-024-02168-z. 9. Samji P, Rajendran MK, Warrier VP, Ganesh A, Devarajan K. Regulation of Hippo
signaling pathway in cancer: A MicroRNA perspective. Cell Signal. 2021;78:109858. Correspondence and requests for materials should be addressed to Maurizio
Fanciulli. 10. Terpos E, Ntanasis-Stathopoulos I, Gavriatopoulou M, Dimopoulos MA. Patho-
genesis of bone disease in multiple myeloma: from bench to bedside. Blood
Cancer J. 2018;8:7. Reprints and permission information is available at http://www.nature.com/
reprints 11. Varelas X. The Hippo pathway effectors TAZ and YAP in development, home-
ostasis and disease. Development. 2014;141:1614–26. 12. Kyriazoglou A, Ntanasis-Stathopoulos I, Terpos E, Fotiou D, Kastritis E, Dimopoulos
MA, et al. Emerging insights into the role of the hippo pathway in multiple myeloma
and associated bone disease. Clin Lymphoma Myeloma Leuk. 2020;20:57–62. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims
in published maps and institutional affiliations. Leukemia (2024) 38:877 – 882 T. Bruno et al. 882 Open Access This article is licensed under a Creative Commons
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http://cds.cern.ch/record/2718646/files/scoap3-fulltext.pdf
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English
| null |
First measurement of quarkonium polarization in nuclear collisions at the LHC
|
Physics letters. B
| 2,021
|
cc-by
| 17,255
|
1. Introduction Quarkonia, bound states of charm (c) and anticharm (c) or bot-
tom (b) and antibottom (b) quarks, represent an important tool
to test our understanding of quantum chromodynamics (QCD),
since their production process involves both perturbative and non-
perturbative aspects. At high energy, the creation of the heavy
quark-antiquark pair is a process that can be described using a
perturbative QCD approach, due to the large value of the charm
and bottom quark masses (mc ∼1.3 GeV/c2, mb ∼4.2 GeV/c2 [1]). However, the subsequent formation of the bound state is a non-
perturbative process that can be described only by empirical mod-
els or effective field theory approaches. Among those, models
based on Non-Relativistic QCD (NRQCD) [2] give the most suc-
cessful description of the production cross section, as measured
at high-energy hadron colliders (Tevatron, RHIC, LHC) [3–14]. In
the NRQCD approach, the non-perturbative aspects are parame-
terized via long-distance matrix elements (LDME), corresponding
to the possible intermediate color, spin and angular momentum
states of the evolving quark-antiquark pair. The values of LDMEs
need to be fitted on a subset of the available measurements and
can be then considered as universal quantities, in the sense that
they can be used in the calculation of production cross sections
and other observables corresponding, for example, to different col-
lision systems and energies. Other theory approaches, as the Color Among the various charmonium states, the J/ψ meson, with
quantum numbers J PC = 1−−, was the first to be discovered. It is
surely the most studied, also due to the sizeable decay branching
ratio to dilepton pairs ((5.961 ±0.033)% for the μ+μ−channel [1])
that represents an excellent experimental signature. While the J/ψ
production cross sections are well reproduced by NRQCD-based
models, it was soon realized that describing the measured polar-
ization of this state represents a much more difficult problem [18]. The polarization, corresponding to the orientation of the particle
spin with respect to a chosen axis, can be accessed via a study
of the polar (θ) and azimuthal (φ) production angles, relative to
that axis, of the two-body decay products in the quarkonium rest
frame. a r t i c l e
i n f o Article history:
Received 9 June 2020
Received in revised form 10 February 2021
Accepted 12 February 2021
Available online 22 February 2021
Editor: M. Doser The polarization of inclusive J/ψ and ϒ(1S) produced in Pb–Pb collisions at √sNN = 5.02 TeV at the LHC
is measured with the ALICE detector. The study is carried out by reconstructing the quarkonium through
its decay to muon pairs in the rapidity region 2.5 < y < 4 and measuring the polar and azimuthal angular
distributions of the muons. The polarization parameters λθ , λφ and λθφ are measured in the helicity
and Collins-Soper reference frames, in the transverse momentum interval 2 < pT < 10 GeV/c and pT <
15 GeV/c for the J/ψ and ϒ(1S), respectively. The polarization parameters for the J/ψ are found to be
compatible with zero, within a maximum of about two standard deviations at low pT, for both reference
frames and over the whole pT range. The values are compared with the corresponding results obtained
for pp collisions at √
s = 7 and 8 TeV in a similar kinematic region by the ALICE and LHCb experiments. Although with much larger uncertainties, the polarization parameters for ϒ(1S) production in Pb–Pb
collisions are also consistent with zero. © 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/). Funded by SCOAP3. Singlet Model [15], the Color Evaporation Model [16] and the kT-
factorization [17] are also used to describe the quarkonium pro-
duction process. First measurement of quarkonium polarization in nuclear collisions at
the LHC .ALICE Collaboration ⋆ Physics Letters B 815 (2021) 136146 Physics Letters B 815 (2021) 136146 ⋆E-mail address: alice -publications @cern .ch.
λφ = 0, λθφ = 0) and (λθ =
1, λφ = 0, λθφ = 0) correspond to the
so-called transverse and longitudinal polarizations, respectively. At
https://doi.org/10.1016/j.physletb.2021.136146
0370-2693/© 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Funded by
SCOAP3. by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Funded by 1. Introduction Further important progress includes a
quantitative evaluation of the contribution of feed-down processes
(J/ψ coming from the decay of χc and ψ(2S) states) on the polar-
ization observables [30]. It was shown that at NLO there are rather
large cancellations between contributions corresponding to the dif-
ferent possible combinations of the spin and angular momentum
of the intermediate cc states, reaching a more satisfactory descrip-
tion of the absence of polarization observed in the data [31]. How-
ever, those descriptions usually require the inclusion of both cross
section and polarization results in the fit of the LDME, leading to a
more limited predictive power on the polarization observables and
to large variations in the values of the extracted LDME values, de-
pending on the set of data used for their determination. Finally, the
description of the J/ψ production in the NRQCD framework was
recently extended to the low-pT region, and the polarization pa-
rameters were studied in a color glass condensate (CGC) + NRQCD
formalism, obtaining a fair agreement with LHC data at forward ra-
pidity [32]. Measurements of the polarization parameters are also
available for several bottomonium states, and in particular for the
ϒ(1S), ϒ(2S) and ϒ(3S) resonances, which were shown to exhibit
little or no polarization at LHC energies [33–35]. Approaches simi-
lar to that adopted for charmonium, which also need to take into
account the rather complex feed-down decay structure for these
states, lead to a fair agreement with the experimental results [36]. charmonium states (ψ(2S), χc). In nuclear collisions, since sup-
pression effects are expected to affect more strongly the less bound
states, the relative contribution of direct and feed-down production
would change with respect to that in pp collisions, and the overall
measured polarization may be different according to the poten-
tially different polarization of the various states [44,45]. On the
other hand, the contribution of the regeneration mechanism in the
J/ψ formation process by recombination of uncorrelated cc pairs is
likely to give rise to unpolarized production at low pT. Finally, the
possible presence of polarization is known to strongly affect the
acceptance for J/ψ detection in the dilepton decay (up to 20–30%
in ALICE [22]), and its measurement is an important requisite for
an unbiased evaluation of the absolute yields in nuclear collisions. 2. Experimental setup and data sample The measurement described in this Letter is performed with the
ALICE detector [46,47], whose main components are a central bar-
rel and a forward muon spectrometer. The latter covers the pseu-
dorapidity region −4 < η < −2.5 and is used to detect muon pairs
from quarkonium decays [48]. The muon spectrometer includes a
hadron absorber made of concrete, carbon and steel with a thick-
ness of 10 interaction lengths, followed by five tracking stations
(cathode-pad chambers), with the central one embedded inside
a dipole magnet with a 3 T·m field integral. Downstream of the
tracking system, an iron wall filters out the remaining hadrons as
well as low-momentum muons originating from pion and kaon de-
cays, and is followed by two trigger stations (resistive plate cham-
bers). Another forward detector, the V0 [49], composed of two
scintillator arrays located at opposite sides of the interaction point
(IP) and covering the pseudorapidity intervals −3.7 < η < −1.7
and 2.8 < η < 5.1, provides the minimum bias (MB) trigger which
is given by a coincidence of signals from the two sides. Among the
central barrel detectors, the two layers of the Silicon Pixel Detec-
tor (SPD), with |η| < 2 and |η| < 1.4 coverage, and corresponding
to the inner part of the ALICE Inner Tracking System (ITS) [50], are
used to determine the position of the interaction vertex. Finally,
the Zero Degree Calorimeters (ZDC) [51], located on either side of
the IP at ± 112.5 m along the beam axis, detect spectator nucle-
ons emitted at zero degrees with respect to the LHC beam axis and
are used to reject electromagnetic Pb–Pb interactions. g
p
[
]
In this Letter, we move a step forward by presenting the first
measurement of J/ψ and ϒ(1S) polarization in ultrarelativistic
heavy-ion interactions performed by the ALICE Collaboration by
studying Pb–Pb collisions at √sNN = 5.02 TeV. Such collisions rep-
resent an important source of information for the investigation of
the phase diagram of QCD [37], and in particular for the study of
the properties of the quark–gluon plasma (QGP), a state of mat-
ter where quarks and gluons are not confined inside hadrons [38]. 1. Introduction A first measurement of ϒ(1S) polarization in Pb–Pb collisions is
also presented in this Letter, even if the corresponding candidate
sample is smaller by a factor ∼30, leading to larger uncertainties. For such a state, considerations similar to those discussed for the
J/ψ should hold, except that the contribution of the regeneration
mechanism should be negligible due to the much lower multiplic-
ity of bottom quarks with respect to charm. The next sections of the Letter are organized as follows. Sec-
tion 2 contains a short description of the experimental apparatus
and some details on the data sample used in this analysis. The
analysis procedure and the evaluation of systematic uncertainties
are presented in Sec. 3, while the results on the J/ψ and ϒ(1S)
polarization parameters λθ , λφ and λθφ are shown in Sec. 4. The
conclusions are presented in Sec. 5. 1. Introduction How-
ever, those descriptions usually require the inclusion of both cross
section and polarization results in the fit of the LDME, leading to a
more limited predictive power on the polarization observables and
to large variations in the values of the extracted LDME values, de-
pending on the set of data used for their determination. Finally, the
description of the J/ψ production in the NRQCD framework was
recently extended to the low-pT region, and the polarization pa-
rameters were studied in a color glass condensate (CGC) + NRQCD
formalism, obtaining a fair agreement with LHC data at forward ra-
pidity [32]. Measurements of the polarization parameters are also
available for several bottomonium states, and in particular for the
ϒ(1S), ϒ(2S) and ϒ(3S) resonances, which were shown to exhibit
little or no polarization at LHC energies [33–35]. Approaches simi-
lar to that adopted for charmonium, which also need to take into
account the rather complex feed-down decay structure for these
states, lead to a fair agreement with the experimental results [36]. I
thi
L tt
t
f
d b
ti
th
fi
t leading order, the high-pT production is dominated by gluon frag-
mentation and therefore the J/ψ would be expected to be trans-
versely polarized [18]. However, the results from the CDF experi-
ment at Tevatron showed that the J/ψ exhibits a very small polar-
ization [20,21], an observation which was impossible to reconcile
with the NRQCD prediction. As of today, on the experimental side,
accurate results on inclusive and prompt (i.e., removing contribu-
tions from b-quark decays) J/ψ polarization have become available
at LHC energies [22–25]. They confirm that this state shows little
or no polarization in a wide rapidity (up to y = 4.5) and trans-
verse momentum region (from 2 to 70 GeV/c), with the exception
of the LHCb measurements at √
s = 7 TeV [24], where the value
λθ = −0.145 ± 0.027, corresponding to a weak longitudinal po-
larization, was obtained in the interval 2 < pT < 15 GeV/c and
2 < y < 4.5, in the helicity frame (its definition will be given later
in Sec. 3). On the theory side, a huge effort was pursued in order to
move to a complete next-to-leading order (NLO) description of the
J/ψ production process [26,27], and to the calculation of the po-
larization variables [28,29]. 1. Introduction Their angular distribution W (θ, φ) is parameterized as W (θ,φ) ∝
1
3 + λθ
×
1 + λθ cos2 θ + λφ sin2 θ cos 2φ + λθφ sin 2θ cosφ
,
(1) (1) with the polarization parameters λθ , λφ and λθφ corresponding
to various combinations of the elements of the spin density ma-
trix of J/ψ production [19]. In particular, the two cases (λθ = 1,
λφ = 0, λθφ = 0) and (λθ = −1, λφ = 0, λθφ = 0) correspond to the
so-called transverse and longitudinal polarizations, respectively. At Physics Letters B 815 (2021) 136146 ALICE Collaboration leading order, the high-pT production is dominated by gluon frag-
mentation and therefore the J/ψ would be expected to be trans-
versely polarized [18]. However, the results from the CDF experi-
ment at Tevatron showed that the J/ψ exhibits a very small polar-
ization [20,21], an observation which was impossible to reconcile
with the NRQCD prediction. As of today, on the experimental side,
accurate results on inclusive and prompt (i.e., removing contribu-
tions from b-quark decays) J/ψ polarization have become available
at LHC energies [22–25]. They confirm that this state shows little
or no polarization in a wide rapidity (up to y = 4.5) and trans-
verse momentum region (from 2 to 70 GeV/c), with the exception
of the LHCb measurements at √
s = 7 TeV [24], where the value
λθ = −0.145 ± 0.027, corresponding to a weak longitudinal po-
larization, was obtained in the interval 2 < pT < 15 GeV/c and
2 < y < 4.5, in the helicity frame (its definition will be given later
in Sec. 3). On the theory side, a huge effort was pursued in order to
move to a complete next-to-leading order (NLO) description of the
J/ψ production process [26,27], and to the calculation of the po-
larization variables [28,29]. Further important progress includes a
quantitative evaluation of the contribution of feed-down processes
(J/ψ coming from the decay of χc and ψ(2S) states) on the polar-
ization observables [30]. It was shown that at NLO there are rather
large cancellations between contributions corresponding to the dif-
ferent possible combinations of the spin and angular momentum
of the intermediate cc states, reaching a more satisfactory descrip-
tion of the absence of polarization observed in the data [31]. 3. Data analysis The J/ψ mass is kept free in the fits, while for each interval
(i, j) in (cosθ, φ) the width is fixed to σ i, j
J/ψ = σ i, j,MC
J/ψ
· (σJ/ψ/σ MC
J/ψ),
i.e., scaling the resonance width extracted from Monte Carlo (MC)
simulations (σ i, j,MC
J/ψ
) by the ratio between the width obtained
by fitting the angle-integrated spectrum in data (σJ/ψ) and MC
(σ MC
J/ψ ) for the pT interval under consideration. The parameters of
the non-Gaussian tails of the resonance are kept fixed to the MC
values. The ψ(2S) contribution, although comparatively negligible,
is also taken into account in the fits, with its width and mass
fixed in each fit to those of the J/ψ according to the relations
σψ(2S) = σJ/ψ ·σ MC
ψ(2S)/σ MC
J/ψ and mψ(2S) = mJ/ψ +mPDG
ψ(2S) −mPDG
J/ψ , with
the Particle Data Group (PDG) masses taken from Ref. [1]. In Fig. 1
(left) an example of a fit to the invariant-mass spectrum in the J/ψ
mass region is shown. Due to the stability of the extracted J/ψ pa-
rameters (mass, width), the fits were carried out directly on the
sum of the 2015 and 2018 invariant mass spectra. The J/ψ
and ϒ(1S)
candidates are formed by combining
opposite-sign muons reconstructed using the tracking algorithm
described in Ref. [48]. In order to reject tracks at the edge of
the spectrometer acceptance, the condition −4 < ημ < −2.5 is re-
quired. In addition, tracks must have a radial transverse position
at the end of the absorber in the range 17.6 < Rabs < 88.9 cm. This selection is applied to remove tracks passing through the in-
ner and denser part of the absorber, which are strongly affected
by multiple scattering. For each muon candidate, a match between
tracks reconstructed in the tracking system and track segments in
the muon trigger system is required. The J/ψ polarization parameters λθ , λφ and λθφ are studied as
a function of transverse momentum in the intervals 2 < pT < 4,
4 < pT < 6 and 6 < pT < 10 GeV/c. 2. Experimental setup and data sample Among the experimental observables studied in heavy-ion colli-
sions the suppression of heavy quarkonium production is a fun-
damental signal, since QGP formation prevents the binding of the
heavy-quark pair due to the screening of the color charge [39] and,
more generally, has strong effects on the spectral functions [40]. At
LHC energies, another mechanism, corresponding to the (re)gen-
eration of charmonium states in the QGP and/or when the sys-
tem hadronizes, becomes relevant [41,42], in particular at low pT,
due to the large charm-quark multiplicity (> 100 pairs in a cen-
tral Pb–Pb collision). The presence of a deconfined system may
in principle affect also the polarization of quarkonium states. In
Ref. [43] the observation of a partial transverse polarization for
the J/ψ was predicted in case of QGP formation, due to a modi-
fication of the non-perturbative effects in the high energy-density
phase. More generally, the observed prompt J/ψ are known to be a
mixture of direct production and decay products from higher-mass The analysis is based on events where, in addition to the
MB condition, two opposite-sign tracks are detected in the trig-
gering system of the muon spectrometer (dimuon trigger). The
dimuon trigger selects tracks each having a transverse momentum
above a threshold nominally set at pμ
T = 1 GeV/c, corresponding
to the value for which the single-muon trigger efficiency reaches
50% [52]. The single-muon trigger efficiency reaches a plateau
value of 98% at ∼2.5 GeV/c. The events are further characterized according to their central-
ity, i.e., the degree of geometric overlap of the colliding nuclei. It
is estimated by means of a Glauber model fit to the V0 signal am- 2 Physics Letters B 815 (2021) 136146 ALICE Collaboration ALICE Collaboration
Physics Letters B 815 (2021) 136146
Fig. 1. Examples of fits to the invariant-mass distributions in the helicity reference frame. The left plot corresponds to the J/ψ mass region, while on the right a fit to the
ϒ(1S) mass region is shown. The fits are performed using an extended Crystal Ball function for the resonance signals, while the background is parameterized with a variable
width Gaussian. Fi
1 E
l
f fi
h
i
i
di
ib
i
i
h
h li i
f
f Fig. 1. Examples of fits to the invariant-mass distributions in the helicity reference frame. 2. Experimental setup and data sample The left plot corresponds to the J/ψ mass region, while on the right a fit to the
ϒ(1S) mass region is shown. The fits are performed using an extended Crystal Ball function for the resonance signals, while the background is parameterized with a variable
width Gaussian. collision, and therefore the polarization can be evaluated with re-
spect to the momentum direction of the J/ψ itself. The φ = 0 plane
is the one containing the two beams in the J/ψ rest frame. plitude distribution [53,54], with more central events leading to a
larger signal in the V0. In this analysis, events corresponding to the
most central 90% of the inelastic Pb–Pb cross section are selected,
as for these events the MB trigger is fully efficient and the residual
contamination from electromagnetic processes is negligible. For each dimuon invariant-mass spectrum, the J/ψ raw yield
is obtained by means of a binned maximum likelihood fit in the
interval 2.1 < mμμ < 4.9 GeV/c2. The background continuum is
parameterized with a Gaussian distribution whose width varies
linearly with the mass or, alternatively, with a fourth degree poly-
nomial function times an exponential. The J/ψ signal is modeled
with a pseudo-Gaussian function or with a Crystal Ball function
with asymmetric tails on both sides of the peak [55]. The results of the analysis are obtained using the √sNN =
5.02 TeV Pb–Pb data samples collected by the ALICE experiment
during the years 2015 and 2018, corresponding to an integrated
luminosity Lint ∼750 μb−1. 3. Data analysis Indeed, although the differ-
ence between efficiencies calculated via MC or from data [59] is of
the order of 2%, a detailed investigation has shown no dependence
on the angular variables and therefore no effect on the polarization
parameters. rected for acceptance and efficiency, according to Eq. (1). For each
combination of signal and background shape used in the fit to the
dimuon invariant-mass spectra, a separate evaluation of the polar-
ization parameters is carried out and their average is taken as the
best estimate. The statistical uncertainty is given by the average
of the statistical uncertainties of the 2D fits, while the root mean
square of the results provides the systematic uncertainty on the
signal extraction, with the absolute values ranging between 0.002
and 0.039. The overall procedure described above was checked be-
forehand with a MC closure test. The 2D fits on the (cosθ, φ)
distributions only allow a determination of the absolute value of
λθφ, due to the presence of sin 2θ in the corresponding term that
induces an ambiguity in its sign. It is checked that the values of λθ
and λφ are stable against the choice of the sign of the λθφ term. In the following the λθφ values corresponding to the choice of a
positive sign are quoted. Fig. 2 illustrates an example of the fit to
the angular distributions. For better visibility, both the distribution
and the fitted function are projected along one dimension. p
Finally, the systematic uncertainty induced by the choice of
the pT and y distributions used as an input for the calculation
of A × ε is evaluated testing alternative pT and y parameteriza-
tions, which are obtained by varying within their uncertainties the
default distributions directly tuned on Pb–Pb data. The polariza-
tion parameters extracted with the modified values of A × ε are
compared with those obtained with the default input shapes and
the corresponding systematic uncertainty extracted in this way is
found to range between 0.001 and 0.030, with the largest value as-
signed to λHE
θ
for 2 < pT < 4 GeV/c. The influence of the choice of
the angular distributions of the J/ψ decay products for the A × ε
calculation is also investigated by means of an iterative procedure
on these input distributions. 3. Data analysis The effect is found to be negligible,
also due to the fact that the 2D correction procedure on the an-
gular variables is by definition relatively insensitive to the specific
choice of the corresponding distributions. A summary of the values
of all the absolute systematic uncertainties, which are considered
as uncorrelated as a function of pT, is reported in Table 1. The to-
tal systematic uncertainties are obtained, for each parameter and
pT interval, as the quadratic sum of the values. In addition to the systematic uncertainty related to the choice
of the mass shapes for signal and background, several other
sources are taken into account. First, an alternative procedure for
extracting the J/ψ signal is carried out, by keeping its width as a
free parameter in the invariant-mass fits. The corresponding results
for the polarization parameters are then obtained and the averages
of the values corresponding to fixing the width or not are taken as
the central values for λθ , λφ and λθφ. Half the difference between
the results obtained with free or MC-anchored widths is then con-
sidered as a further systematic uncertainty related to the signal
extraction. This uncertainty is found to be the leading contribution
to the total absolute systematic uncertainty on the polarization pa-
rameters, and ranges between 0.001 and 0.063, the latter value
corresponding to the uncertainty on λHE
θ
for 2 < pT < 4 GeV/c. A similar procedure is followed for the extraction of the ϒ(1S)
polarization parameters. Due to the smaller candidate sample, in-
tegrated values over the kinematic interval 2.5 < y < 4, pT <
15 GeV/c are obtained. The main difference with respect to the 2D
approach followed for the J/ψ is the use of a simultaneous fit to
the 1D angular distributions [23], after integration over the other
variables. The requirement pμ
T > 2 GeV/c, which helps reducing the
combinatorial background, is included [60]. The ϒ(1S) signal ex-
traction in the various cosθ and φ intervals is performed by means
of invariant-mass fits (see the right panel of Fig. 1 for an example). 3. Data analysis For each pT interval, a two–
dimensional (2D) grid of dimuon invariant-mass spectra is cre-
ated, corresponding to intervals in cosθ and φ, where θ and φ
are the polar and azimuthal emission angles, respectively, of the
decay products in the J/ψ rest frame, with respect to the refer-
ence axis. More in detail, the 2D grid covers the fiducial region
−0.8 < cosθ < 0.8 (17 intervals), 0.5 < φ < π −0.5 rad (8 inter-
vals, assuming a symmetric distribution around φ = π), with the
choice of the boundaries as well as the width of the intervals dic-
tated by acceptance considerations. The J/ψ raw yields as a function of the angular variables are
then corrected by the product of the acceptance and detector ef-
ficiency (A × ε), which is evaluated as a function of cosθ and φ
on a 2D grid via MC simulations. The J/ψ are generated accord-
ing to pT and y distributions directly tuned on data [56] via an
iterative procedure [57], and their decay muons are propagated in-
side a realistic description of the ALICE setup, based on GEANT
3.21 [58]. The misalignment of the detection elements and the
time-dependent status of each electronic channel during the data
taking period are taken into account as well. In the J/ψ generation
an isotropic distribution of decay products, corresponding to the
assumption of no polarization, is adopted. Due to the choice of rel-
atively small (cosθ, φ) intervals, the A × ε values for each interval
are quite insensitive to the specific angular distribution assumed
in the generation. The analysis is performed choosing two different reference sys-
tems for the determination of the angular variables. In the Collins-
Soper (CS) frame the z-axis is defined as the bisector of the angle
between the direction of one beam and the opposite of the direc-
tion of the other one in the rest frame of the decaying particle,
allowing therefore an evaluation of the polarization parameters
with respect to the direction of motion of the colliding hadrons. In the helicity (HE) reference frame the z-axis is given by the di-
rection of the decaying particle in the center-of-mass frame of the The three polarization parameters λθ , λφ and λθφ are obtained
through χ 2-minimization fits of the 2D J/ψ distributions, cor- 3 Physics Letters B 815 (2021) 136146 ALICE Collaboration Fig. 2. 3. Data analysis Fit to the J/ψ 2D angular distributions in the helicity reference frame projected along cosθ (left) and φ (right) for 2.5 < y < 4 and 4 < pT < 6 GeV/c. The displayed
uncertainties are statistical. Fig 2 Fit to the J/ψ 2D angular distributions in the helicity reference frame projec Fig. 2. Fit to the J/ψ 2D angular distributions in the helicity reference frame projected along cosθ (left) and φ (right) for 2.5 < y < 4 and 4 < pT < 6 GeV/c. The displayed
uncertainties are statistical. Fig. 2. Fit to the J/ψ 2D angular distributions in the helicity reference frame projected along cosθ (left) and φ (right) for 2.5 <
uncertainties are statistical. ns in the helicity reference frame projected along cosθ (left) and φ (right) for 2.5 < y < 4 and 4 < pT < 6 GeV/c. The displayed rected for acceptance and efficiency, according to Eq. (1). For each
combination of signal and background shape used in the fit to the
dimuon invariant-mass spectra, a separate evaluation of the polar-
ization parameters is carried out and their average is taken as the
best estimate. The statistical uncertainty is given by the average
of the statistical uncertainties of the 2D fits, while the root mean
square of the results provides the systematic uncertainty on the
signal extraction, with the absolute values ranging between 0.002
and 0.039. The overall procedure described above was checked be-
forehand with a MC closure test. The 2D fits on the (cosθ, φ)
distributions only allow a determination of the absolute value of
λθφ, due to the presence of sin 2θ in the corresponding term that
induces an ambiguity in its sign. It is checked that the values of λθ
and λφ are stable against the choice of the sign of the λθφ term. In the following the λθφ values corresponding to the choice of a
positive sign are quoted. Fig. 2 illustrates an example of the fit to
the angular distributions. For better visibility, both the distribution
and the fitted function are projected along one dimension. ing a significant reduction of the total systematic uncertainty with
respect to previous pp analyses [23]. Table 3 Table 3
ϒ(1S) polarization parameters in the helicity and Collins-Soper reference frames
measured in Pb–Pb collisions at √sNN = 5.02 TeV in the rapidity interval 2.5 < y <
4 and for transverse momentum pT < 15 GeV/c. The first uncertainty is statistical
and the second systematic. Helicity
Collins-Soper
λθ
−0.090 ± 0.395 ± 0.101
0.418 ± 0.526 ± 0.178
λφ
−0.094 ± 0.072 ± 0.020
−0.141 ± 0.087 ± 0.033
λθφ
−0.074 ± 0.099 ± 0.020
0.017 ± 0.113 ± 0.024 3.3σ in the interval 2 < pT < 4 GeV/c in the helicity reference
frame, where pp data [24] indicate a small but significant de-
gree of longitudinal polarization, while the Pb–Pb results favor a
slightly transverse polarization. In Pb–Pb collisions at LHC ener-
gies, a significant fraction of the detected J/ψ originates from the
recombination of cc pairs in the QGP phase or when the system
hadronizes. Moreover, the contribution from higher-mass charmo-
nium states decaying to J/ψ could vary between pp and Pb-Pb due
to different suppression effects for each state in nuclear collisions. Therefore, the observed hint for a different polarization in pp and
Pb–Pb might be a reflection of the different production and sup-
pression mechanisms in the two systems, but more precise data,
along with quantitative theory estimates, are needed for a def-
inite conclusion. It should also be noted that the ALICE results
refer to inclusive production, while LHCb has measured prompt
J/ψ. However, as discussed in Ref. [22], the size of the non-prompt
component is small in the covered pT region (of the order of 15%
at high pT) and its polarization was also measured to be small by
CDF (λHE
θ
∼−0.1 [21]), implying that the net effect of this source
on inclusive J/ψ polarization should be negligible. 3. Data analysis MC
λθ
2<pT<4
0.030
0.063
0.043
0.030
0.026
0.049
0.015
0.009
4<pT<6
0.017
0.046
0.040
0.024
0.002
0.052
0.018
0.007
6<pT<10
0.039
0.005
0.018
0.017
0.022
0.001
0.011
0.006
λφ
2<pT<4
0.007
0.030
0.004
0.002
0.024
0.010
0.020
0.003
4<pT<6
0.003
0.035
0.003
0.003
0.002
0.010
0.020
0.003
6<pT<10
0.002
0.009
0.001
0.002
0.005
0.013
0.011
0.002
λθφ
2<pT<4
0.021
0.029
0.024
0.001
0.013
0.010
0.017
0.015
4<pT<6
0.007
0.011
0.017
0.006
0.002
0.042
0.010
0.015
6<pT<10
0.020
0.019
0.007
0.008
0.007
0.042
0.003
0.013
Table 2
J/ψ polarization parameters, measured for Pb–Pb collisions at √sNN = 5.02 TeV, in the helicity and Collins-Soper
reference frames in the rapidity interval 2.5 < y < 4. The first uncertainty is statistical and the second systematic. pT (GeV/c)
Helicity
Collins-Soper
λθ
2<pT<4
0.218 ± 0.060 ± 0.087
−0.157 ± 0.049 ± 0.058
4<pT<6
0.151 ± 0.071 ± 0.068
−0.057 ± 0.059 ± 0.055
6<pT<10
−0.070 ± 0.068 ± 0.047
−0.008 ± 0.063 ± 0.026
λφ
2<pT<4
−0.029 ± 0.017 ± 0.031
0.061 ± 0.015 ± 0.033
4<pT<6
−0.013 ± 0.019 ± 0.036
0.047 ± 0.024 ± 0.023
6<pT<10
0.047 ± 0.021 ± 0.010
0.024 ± 0.032 ± 0.018
λθφ
2<pT<4
−0.124 ± 0.028 ± 0.043
−0.090 ± 0.027 ± 0.029
4<pT<6
−0.059 ± 0.030 ± 0.021
−0.040 ± 0.034 ± 0.046
6<pT<10
−0.025 ± 0.031 ± 0.030
0.018 ± 0.035 ± 0.044 Table 1
Summary of the absolute systematic uncertainties on the evaluation of the J/ψ polarization parameters. All the uncertainties are considered as uncorrelated as a function
of pT. Table 1
Summary of the absolute systematic uncertainties on the evaluation of the J/ψ polarization parameters. All the uncertainties are considered as uncorrelated as a function
of pT Table 2
J/ψ polarization parameters, measured for Pb–Pb collisions at √sNN = 5.02 TeV, in the helicity and Collins-Soper
reference frames in the rapidity interval 2.5 < y < 4. The first uncertainty is statistical and the second systematic. Table 2
J/ψ polarization parameters, measured for Pb–Pb collisions at √sNN = 5.02 TeV, in the helicity and Collins-Soper
reference frames in the rapidity interval 2.5 < y < 4. The first uncertainty is statistical and the second systematic. 3. Data analysis pT (GeV/c)
Helicity
Collins-Soper
λθ
2<pT<4
0.218 ± 0.060 ± 0.087
−0.157 ± 0.049 ± 0.058
4<pT<6
0.151 ± 0.071 ± 0.068
−0.057 ± 0.059 ± 0.055
6<pT<10
−0.070 ± 0.068 ± 0.047
−0.008 ± 0.063 ± 0.026
λφ
2<pT<4
−0.029 ± 0.017 ± 0.031
0.061 ± 0.015 ± 0.033
4<pT<6
−0.013 ± 0.019 ± 0.036
0.047 ± 0.024 ± 0.023
6<pT<10
0.047 ± 0.021 ± 0.010
0.024 ± 0.032 ± 0.018
λθφ
2<pT<4
−0.124 ± 0.028 ± 0.043
−0.090 ± 0.027 ± 0.029
4<pT<6
−0.059 ± 0.030 ± 0.021
−0.040 ± 0.034 ± 0.046
6<pT<10
−0.025 ± 0.031 ± 0.030
0.018 ± 0.035 ± 0.044 Table 2
J/ψ polarization parameters, measured for Pb–Pb collisions at √sNN = 5.02 TeV, in the helicity and Collins-Soper
reference frames in the rapidity interval 2.5 < y < 4. The first uncertainty is statistical and the second systematic. or, alternatively, with a second degree polynomial function times
an exponential. The systematic uncertainty on the signal extrac-
tion is calculated with the same procedure adopted for the J/ψ. An uncertainty related to the choice of the signal width has also
been considered, taken as the half-difference between the results
obtained with the prescription described above and using as an
alternative prescription the pure MC values. The uncertainty on
the trigger efficiency is negligible, due to the additional require-
ment on the single-muon transverse momentum which selects a
pT-region where the trigger efficiency is very high and its evalu-
ation via data and MC is consistent. Finally, the procedure for the
determination of the uncertainty related to the ϒ(1S) kinematic
distributions used in the MC is the same as for the J/ψ. The to-
tal systematic uncertainties for the ϒ(1S) analysis are reported in
Table 3, together with the results. 3. Data analysis The functions chosen for the resonances are the same as in the J/ψ
analysis (pseudo-Gaussian or Crystal Ball), the mass value is fixed
to that obtained from a fit to the integrated invariant-mass distri-
bution, while the width for each angular interval is fixed to the
MC value scaled by the ratio of the widths between data and MC
for the angle-integrated distributions. The tail parameters are fixed
to MC values. The small contribution from ϒ(2S) is also included
in the fits [60]. The background continuum is parameterized with
a Gaussian distribution whose width varies linearly with the mass Another source of systematic uncertainty is related to the eval-
uation of the trigger efficiency. The muon trigger response function
as a function of the single muon transverse momentum pμ
T can be
obtained via MC or with a procedure based on data [59]. Small
deviations are found for pμ
t < 2 GeV/c which induce an effect on
A × ε for the J/ψ. Therefore, the polarization parameters are re-
calculated with A × ε values weighted in such a way to account
for the deviations. The variation of the polarization parameters
between the different trigger efficiency estimates is taken as the
related systematic uncertainty, with values ranging from 0.001 to
0.043, the highest values being found for λHE
θ
in 2 < pT < 4 GeV/c. The systematic uncertainty related to the evaluation of the muon
tracking efficiency is found to be negligible for this analysis, allow- 4 Physics Letters B 815 (2021) 136146 ALICE Collaboration Table 1
Summary of the absolute systematic uncertainties on the evaluation of the J/ψ polarization parameters. All the uncertainties are considered as uncorrelated as a function
of pT. Helicity
Collins-Soper
Signal
J/ψ
Trigger
Input
Signal
J/ψ
Trigger
Input
pT (GeV/c)
extr. width
eff. MC
extr. width
eff. 4. Results The polarization parameters for J/ψ inclusive production in Pb–
Pb collisions at √sNN = 5.02 TeV in the helicity and Collins-Soper
reference frames are shown in Fig. 3 and the corresponding nu-
merical values are reported in Table 2. In Fig. 3, λθ , λφ and λθφ
are also compared with the LHCb [24] and ALICE [23] measure-
ments in pp collisions at √
s = 7 and 8 TeV, respectively. For all the pT intervals and in both reference frames the val-
ues of the polarization parameters exhibit at most slight deviations
from zero. In particular, λHE
θ
indicates a slight transverse polar-
ization at low pT (∼2.1σ effect, calculated using the Gaussian
approximation), while λCS
θ
shows a weak longitudinal polarization
(∼2.1σ ). When increasing pT, the central values of λθ become
close to zero. All values of λφ and λθφ are, in absolute value,
smaller than 0.1, except for λHE
θφ, which is −0.124 at low pT and
deviates from zero by ∼2.4σ . In Table 3 the values of the ϒ(1S) polarization parameters are
shown. The λθ values are consistent with zero, with large uncer-
tainties that prevent a firm conclusion on the absence of polariza-
tion in nuclear collisions. The λφ and λθφ values are also consistent
with zero. The relatively smaller uncertainties for these parameters
are related to a more uniform acceptance distribution as a function
of the azimuthal angular variable. When comparing with the pp results, no significant difference
is found with respect to ALICE results at √
s = 8 TeV, which are
compatible with zero. A significant difference is found with re-
spect to the higher-precision LHCb results at √
s = 7 TeV, reaching 5 Physics Letters B 815 (2021) 136146 ALICE Collaboration Fig. 3. Inclusive J/ψ polarization parameters as a function of transverse momentum for Pb–Pb collisions at √sNN = 5.02 TeV, compared with results obtained in pp collisions
by ALICE at √
s = 8 TeV [23] and by LHCb for prompt J/ψ at √
s = 7 TeV [24] (the LHCb markers were shifted horizontally by +0.3 GeV/c for better visibility) in the rapidity
interval 3 < y < 3.5. The error bars represent the total uncertainties for the pp results, while for Pb–Pb statistical and systematic uncertainties are plotted separately as a
vertical bar and a shaded box, respectively. 4. Results In the left part of the plot the polarization parameters in the helicity reference frame are reported, in the right those for the
Collins-Soper frame. Fig. 3. Inclusive J/ψ polarization parameters as a function of transverse momentum for Pb–Pb collisions at √sNN = 5.02 TeV, compared with results obtained in pp collisi Fig. 3. Inclusive J/ψ polarization parameters as a function of transverse momentum for Pb–Pb collisions at √sNN = 5.02 TeV, compared with results obtained in pp collisions
by ALICE at √
s = 8 TeV [23] and by LHCb for prompt J/ψ at √
s = 7 TeV [24] (the LHCb markers were shifted horizontally by +0.3 GeV/c for better visibility) in the rapidity
interval 3 < y < 3.5. The error bars represent the total uncertainties for the pp results, while for Pb–Pb statistical and systematic uncertainties are plotted separately as a
vertical bar and a shaded box, respectively. In the left part of the plot the polarization parameters in the helicity reference frame are reported, in the right those for the
Collins-Soper frame. 5. Conclusions The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared to
influence the work reported in this paper. The first measurement of the polarization parameters for J/ψ
production in nuclear collisions at LHC energies was carried out by
the ALICE Collaboration in Pb–Pb interactions at √sNN = 5.02 TeV. The λθ , λφ and λθφ parameters were evaluated in the helicity and
Collins-Soper reference frames in the rapidity interval 2.5 < y < 4
and in the transverse momentum interval 2 < pT < 10 GeV/c. All
the parameter values are close to zero, with a ∼2.1σ indication
for a small transverse polarization in the helicity frame at low pT,
and a corresponding indication for a small longitudinal polariza-
tion in the Collins-Soper frame (∼2.1σ effect). When comparing
these results with pp data taken at higher energy at the LHC, an
interesting feature is a significant difference in λHE
θ
with respect
to the LHCb results which showed instead a small longitudinal po-
larization in a similar kinematic domain. This first result obtained
for J/ψ in nuclear collisions and described in this Letter represents
therefore a starting point for future studies connecting such fea-
tures with the known differences in the production mechanisms
between pp and nucleus–nucleus collisions. Results were also ob-
tained for the first time for the ϒ(1S) polarization, integrated over
pT and y, showing, within the large uncertainties of the measure-
ment, values compatible with the absence of polarization. Physics Letters B 815 (2021) 136146 J. C 76 (4) (2016) 184,
arXiv:1509 .08258 [hep -ex]. [14] ATLAS Collaboration, G. Aad, et al., Measurement of Upsilon production in 7
TeV pp collisions at ATLAS, Phys. Rev. D 87 (5) (2013) 052004, arXiv:1211.7255
[hep -ex]. [15] M. Einhorn, S. Ellis, Hadronic production of the new resonances: probing gluon
distributions, Phys. Rev. D 12 (1975) 2007. [16] M. Gluck, J.F. Owens, E. Reya, Gluon contribution to hadronic J/ψ production,
Phys. Rev. D 17 (1978) 2324. [17] S. Baranov, A. Lipatov, Prompt charmonia production and polarization at LHC
in the NRQCD with kT -factorization. Part III: J/ψ meson, Phys. Rev. D 96 (3)
(2017) 034019, arXiv:1611.10141 [hep -ph]. [18] E. Braaten, B.A. Kniehl, J. Lee, Polarization of prompt J/ψ at the Tevatron, Phys. Rev. D 62 (2000) 094005, arXiv:hep -ph /9911436 [hep -ph]. [19] P. Faccioli, C. Lourenco, J. Seixas, H.K. Wohri, Towards the experimental clari-
fication of quarkonium polarization, Eur. Phys. J. C 69 (2010) 657–673, arXiv:
1006 .2738 [hep -ph]. [20] CDF Collaboration, T. Affolder, et al., Measurement of J/ψ and ψ(2S) polar-
ization in p ¯p collisions at √
s = 1.8 TeV, Phys. Rev. Lett. 85 (2000) 2886–2891,
arXiv:hep -ex /0004027 [hep -ex]. [21] CDF Collaboration, A. Abulencia, et al., Polarization of J/ψ and ψ(2S) mesons
produced in p ¯p collisions at √
s = 1.96-TeV, Phys. Rev. Lett. 99 (2007) 132001,
arXiv:0704 .0638 [hep -ex]. [22] ALICE Collaboration, B. Abelev, et al., J/ψ polarization in pp collisions at √
s =
7 TeV, Phys. Rev. Lett. 108 (2012) 082001, arXiv:1111.1630 [hep -ex]. [23] ALICE Collaboration, S. Acharya, et al., Measurement of the inclusive J/ ψ polar-
ization at forward rapidity in pp collisions at √
s = 8 TeV, Eur. Phys. J. C 78 (7)
(2018) 562, arXiv:1805 .04374 [hep -ex]. [24] LHCb Collaboration, R. Aaij, et al., Measurement of J/ψ polarization in pp col-
lisions at √
s = 7 TeV, Eur. Phys. J. C 73 (11) (2013) 2631, arXiv:1307.6379
[hep -ex]. [25] CMS Collaboration, S. Chatrchyan, et al., Measurement of the prompt J/ψ and
ψ(2S) polarizations in pp collisions at √
s = 7 TeV, Phys. Lett. B 727 (2013)
381–402, arXiv:1307.6070 [hep -ex]. [26] M. Butenschoen, B.A. Kniehl, Reconciling J/ψ production at HERA, RHIC, Teva-
tron, and LHC with NRQCD factorization at next-to-leading order, Phys. Rev. Lett. 106 (2011) 022003, arXiv:1009 .5662 [hep -ph]. United States of America. [27] Y.-Q. Physics Letters B 815 (2021) 136146 [3] ALICE Collaboration, S. Acharya, et al., Energy dependence of forward-rapidity
J/ψ and ψ(2S) production in pp collisions at the LHC, Eur. Phys. J. C 77 (6)
(2017) 392, arXiv:1702 .00557 [hep -ex]. [4] ALICE Collaboration, S. Acharya, et al., Inclusive J/ψ production at mid-rapidity
in pp collisions at √
s = 5.02 TeV, J. High Energy Phys. 10 (2019) 084, arXiv:
1905 .07211 [nucl -ex]. [5] LHCb Collaboration, R. Aaij, et al., Measurement of forward J/ψ production
cross-sections in pp collisions at √
s = 13 TeV, J. High Energy Phys. 10 (2015)
172, arXiv:1509 .00771 [hep -ex ]. [Erratum : JHEP05 ,063(2017 )]. [6] CMS Collaboration, A.M. Sirunyan, et al., Measurement of quarkonium produc-
tion cross sections in pp collisions at √
s = 13 TeV, Phys. Lett. B 780 (2018)
251–272, arXiv:1710 .11002 [hep -ex]. [7] ATLAS Collaboration, G. Aad, et al., Measurement of the differential cross-
sections of prompt and non-prompt production of J/ψ and ψ(2S) in pp colli-
sions at √
s = 7 and 8 TeV with the ATLAS detector, Eur. Phys. J. C 76 (5) (2016)
283, arXiv:1512 .03657 [hep -ex]. [8] PHENIX Collaboration, U. Acharya, et al., J/ψ and ψ(2S) production at forward
rapidity in p+p collisions at √
s = 510 GeV, Phys. Rev. D 101 (5) (2020) 052006,
arXiv:1912 .13424 [hep -ex]. [9] STAR Collaboration, J. Adam, et al., Measurements of the transverse-
momentum-dependent cross sections of J/ψ production at mid-rapidity in
proton+proton collisions at √
s = 510 and 500 GeV with the STAR detector,
Phys. Rev. D 100 (5) (2019) 052009, arXiv:1905 .06075 [hep -ex]. [10] CDF Collaboration, F. Abe, et al., J/ψ and ψ(2S) production in p ¯p collisions at
√
s = 1.8 TeV, Phys. Rev. Lett. 79 (1997) 572–577. [11] D0 Collaboration, S. Abachi, et al., J/ψ production in p ¯p collisions at √
s = 1.8-
TeV, Phys. Lett. B 370 (1996) 239–248. [12] LHCb Collaboration, R. Aaij, et al., Measurement of ϒ production in pp colli-
sions at √
s = 13 TeV, J. High Energy Phys. 07 (2018) 134, arXiv:1804 .09214
[hep -ex];
R. Aaij, et al., J. High Energy Phys. 05 (2019) 076, Erratum. R. Aaij, et al., J. High Energy Phys. 05 (2019) 076, Erratum. [13] ALICE Collaboration, J. Adam, et al., Inclusive quarkonium production at for-
ward rapidity in pp collisions at √
s = 8 TeV, Eur. Phys. Physics Letters B 815 (2021) 136146 dação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
and Universidade Federal do Rio Grande do Sul (UFRGS), Brazil;
Ministry of Education of China (MOEC), Ministry of Science &
Technology of China (MSTC) and National Natural Science Foun-
dation of China (NSFC), China; Ministry of Science and Educa-
tion and Croatian Science Foundation, Croatia; Centro de Aplica-
ciones Tecnológicas y Desarrollo Nuclear (CEADEN), Cubaenergía,
Cuba; Ministry of Education, Youth and Sports of the Czech Repub-
lic, Czech Republic; The Danish Council for Independent Research
| Natural Sciences, the Villum Fonden and Danish National Re-
search Foundation (DNRF), Denmark; Helsinki Institute of Physics
(HIP), Finland; Commissariat à l’Énergie Atomique (CEA) and Insti-
tut National de Physique Nucléaire et de Physique des Particules
(IN2P3) and Centre National de la Recherche Scientifique (CNRS),
France; Bundesministerium für Bildung und Forschung (BMBF)
and GSI Helmholtzzentrum für Schwerionenforschung GmbH, Ger-
many; General Secretariat for Research and Technology, Ministry of
Education, Research and Religions, Greece; National Research, De-
velopment and Innovation Office, Hungary; Department of Atomic
Energy, Government of India (DAE), Department of Science and
Technology, Government of India (DST), University Grants Com-
mission, Government of India (UGC) and Council of Scientific and
Industrial Research (CSIR), India; Indonesian Institute of Science,
Indonesia; Centro Fermi - Museo Storico della Fisica e Centro Studi
e Ricerche Enrico Fermi and Istituto Nazionale di Fisica Nucle-
are (INFN), Italy; Institute for Innovative Science and Technology,
Nagasaki Institute of Applied Science (IIST), Japanese Ministry of
Education, Culture, Sports, Science and Technology (MEXT) and
Japan Society for the Promotion of Science (JSPS) KAKENHI, Japan;
Consejo Nacional de Ciencia (CONACYT) y Tecnología, through
Fondo de Cooperación Internacional en Ciencia y Tecnología (FON-
CICYT) and Dirección General de Asuntos del Personal Academico
(DGAPA), Mexico; Nederlandse Organisatie voor Wetenschappelijk
Onderzoek (NWO), Netherlands; The Research Council of Norway,
Norway; Commission on Science and Technology for Sustainable
Development in the South (COMSATS), Pakistan; Pontificia Uni-
versidad Católica del Perú, Peru; Ministry of Science and Higher
Education, National Science Centre and WUT ID-UB, Poland; Ko-
rea Institute of Science and Technology Information and National
Research Foundation of Korea (NRF), Republic of Korea; Ministry
of Education and Scientific Research, Institute of Atomic Physics
and Ministry of Research and Innovation and Institute of Atomic
Physics, Romania; Joint Institute for Nuclear Research (JINR), Min-
istry of Education and Science of the Russian Federation, National
Research Centre Kurchatov Institute, Russian Science Foundation
and Russian Foundation for Basic Research, Russia; Ministry of Ed-
ucation, Science, Research and Sport of the Slovak Republic, Slo-
vakia; National Research Foundation of South Africa, South Africa;
Swedish Research Council (VR) and Knut & Alice Wallenberg Foun-
dation (KAW), Sweden; European Organization for Nuclear Re-
search, Switzerland; Suranaree University of Technology (SUT), Na-
tional Science and Technology Development Agency (NSDTA) and
Office of the Higher Education Commission under NRU project of
Thailand, Thailand; Turkish Atomic Energy Agency (TAEK), Turkey;
National Academy of Sciences of Ukraine, Ukraine; Science and
Technology Facilities Council (STFC), United Kingdom; National Sci-
ence Foundation of the United States of America (NSF) and United
States Department of Energy, Office of Nuclear Physics (DOE NP),
United States of America. Physics Letters B 815 (2021) 136146 Ma, K. Wang, K.-T. Chao, A complete NLO calculation of the J/ψ and ψ′
production at hadron colliders, Phys. Rev. D 84 (2011) 114001, arXiv:1012 .1030
[hep -ph]. [2] G.T. Bodwin, E. Braaten, G.P. Lepage, Rigorous QCD analysis of inclusive annihi-
lation and production of heavy quarkonium, Phys. Rev. D 51 (1995) 1125–1171,
arXiv:hep -ph /9407339; [1] Particle Data Group Collaboration, M. Tanabashi, et al., Review of particle
physics, Phys. Rev. D 98 (3) (2018) 030001. Physics Letters B 815 (2021) 136146 Bodwin, E. Braaten, G.P. Lepage, Phys. Rev. D 55 (1997) 5853, Erratum. Acknowledgements The ALICE Collaboration would like to thank all its engineers
and technicians for their invaluable contributions to the construc-
tion of the experiment and the CERN accelerator teams for the
outstanding performance of the LHC complex. The ALICE Collab-
oration gratefully acknowledges the resources and support pro-
vided by all Grid centres and the Worldwide LHC Computing Grid
(WLCG) collaboration. The ALICE Collaboration acknowledges the
following funding agencies for their support in building and run-
ning the ALICE detector: A. I. Alikhanyan National Science Labo-
ratory (Yerevan Physics Institute) Foundation (ANSL), State Com-
mittee of Science and World Federation of Scientists (WFS), Arme-
nia; Austrian Academy of Sciences, Austrian Science Fund (FWF):
[M 2467-N36] and Österreichische Nationalstiftung für Forschung,
Technologie und Entwicklung, Austria; Ministry of Communications
and High Technologies, National Nuclear Research Center, Azer-
baijan; Conselho Nacional de Desenvolvimento Científico e Tec-
nológico (CNPq), Financiadora de Estudos e Projetos (Finep), Fun- 6 ALICE Collaboration Physics Letters B 815 (2021) 136146 Physics Letters B 815 (2021) 136146 dação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
and Universidade Federal do Rio Grande do Sul (UFRGS), Brazil;
Ministry of Education of China (MOEC), Ministry of Science &
Technology of China (MSTC) and National Natural Science Foun-
dation of China (NSFC), China; Ministry of Science and Educa-
tion and Croatian Science Foundation, Croatia; Centro de Aplica-
ciones Tecnológicas y Desarrollo Nuclear (CEADEN), Cubaenergía,
Cuba; Ministry of Education, Youth and Sports of the Czech Repub-
lic, Czech Republic; The Danish Council for Independent Research
| Natural Sciences, the Villum Fonden and Danish National Re-
search Foundation (DNRF), Denmark; Helsinki Institute of Physics
(HIP), Finland; Commissariat à l’Énergie Atomique (CEA) and Insti-
tut National de Physique Nucléaire et de Physique des Particules
(IN2P3) and Centre National de la Recherche Scientifique (CNRS),
France; Bundesministerium für Bildung und Forschung (BMBF)
and GSI Helmholtzzentrum für Schwerionenforschung GmbH, Ger-
many; General Secretariat for Research and Technology, Ministry of
Education, Research and Religions, Greece; National Research, De-
velopment and Innovation Office, Hungary; Department of Atomic
Energy, Government of India (DAE), Department of Science and
Technology, Government of India (DST), University Grants Com-
mission, Government of India (UGC) and Council of Scientific and
Industrial Research (CSIR), India; Indonesian Institute of Science,
Indonesia; Centro Fermi - Museo Storico della Fisica e Centro Studi
e Ricerche Enrico Fermi and Istituto Nazionale di Fisica Nucle-
are (INFN), Italy; Institute for Innovative Science and Technology,
Nagasaki Institute of Applied Science (IIST), Japanese Ministry of
Education, Culture, Sports, Science and Technology (MEXT) and
Japan Society for the Promotion of Science (JSPS) KAKENHI, Japan;
Consejo Nacional de Ciencia (CONACYT) y Tecnología, through
Fondo de Cooperación Internacional en Ciencia y Tecnología (FON-
CICYT) and Dirección General de Asuntos del Personal Academico
(DGAPA), Mexico; Nederlandse Organisatie voor Wetenschappelijk
Onderzoek (NWO), Netherlands; The Research Council of Norway,
Norway; Commission on Science and Technology for Sustainable
Development in the South (COMSATS), Pakistan; Pontificia Uni-
versidad Católica del Perú, Peru; Ministry of Science and Higher
Education, National Science Centre and WUT ID-UB, Poland; Ko-
rea Institute of Science and Technology Information and National
Research Foundation of Korea (NRF), Republic of Korea; Ministry
of Education and Scientific Research, Institute of Atomic Physics
and Ministry of Research and Innovation and Institute of Atomic
Physics, Romania; Joint Institute for Nuclear Research (JINR), Min-
istry of Education and Science of the Russian Federation, National
Research Centre Kurchatov Institute, Russian Science Foundation
and Russian Foundation for Basic Research, Russia; Ministry of Ed-
ucation, Science, Research and Sport of the Slovak Republic, Slo-
vakia; National Research Foundation of South Africa, South Africa;
Swedish Research Council (VR) and Knut & Alice Wallenberg Foun-
dation (KAW), Sweden; European Organization for Nuclear Re-
search, Switzerland; Suranaree University of Technology (SUT), Na-
tional Science and Technology Development Agency (NSDTA) and
Office of the Higher Education Commission under NRU project of
Thailand, Thailand; Turkish Atomic Energy Agency (TAEK), Turkey;
National Academy of Sciences of Ukraine, Ukraine; Science and
Technology Facilities Council (STFC), United Kingdom; National Sci-
ence Foundation of the United States of America (NSF) and United
States Department of Energy, Office of Nuclear Physics (DOE NP), G.T. [1] Particle Data Group Collaboration, M. Tanabashi, et al., Review of particle
physics, Phys. Rev. D 98 (3) (2018) 030001.
[2] G.T. Bodwin, E. Braaten, G.P. Lepage, Rigorous QCD analysis of inclusive annihi-
lation and production of heavy quarkonium, Phys. Rev. D 51 (1995) 1125–1171,
arXiv:hep -ph /9407339; References Aaij, et al., Measurement of the ϒ polarizations in pp
collisions at √
s = 7 and 8 TeV, J. High Energy Phys. 12 (2017) 110, arXiv:
1709 .01301 [hep -ex]. [52] ALICE Collaboration, F. Bossu, M. Gagliardi, M. Marchisone, Performance of the
RPC-based ALICE muon trigger system at the LHC, J. Instrum. 7 (2012) T12002,
arXiv:1211.1948 [physics .ins -det ]. [PoSRPC2012 , 059(2012 )]. [36] H. Han, Y.-Q. Ma, C. Meng, H.-S. Shao, Y.-J. Zhang, K.-T. Chao, ϒ(nS) and χb(nP)
production at hadron colliders in nonrelativistic QCD, Phys. Rev. D 94 (1)
(2016) 014028, arXiv:1410 .8537 [hep -ph]. [53] ALICE Collaboration, B. Abelev, et al., Centrality determination of Pb-Pb col-
lisions at √sNN = 2.76 TeV with ALICE, Phys. Rev. C 88 (4) (2013) 044909,
arXiv:1301.4361 [nucl -ex]. [37] P. Braun-Munzinger, J. Wambach, The phase diagram of strongly-interacting
matter, Rev. Mod. Phys. 81 (2009) 1031–1050, arXiv:0801.4256 [hep -ph]. [54] ALICE Collaboration, Centrality determination in heavy ion collisions, ALICE-
PUBLIC-2018-011, http://cds .cern .ch /record /2636623. [38] P. Braun-Munzinger, V. Koch, T. Schäfer, J. Stachel, Properties of hot and dense
matter from relativistic heavy ion collisions, Phys. Rep. 621 (2016) 76–126,
arXiv:1510 .00442 [nucl -th]. [55] ALICE Collaboration, Quarkonium signal extraction in ALICE, ALICE-PUBLIC-
2015-006, https://cds .cern .ch /record /2060096. [39] T. Matsui, H. Satz, J/ψ suppression by quark-gluon plasma formation, Phys. Lett. B 178 (1986) 416–422. [56] ALICE Collaboration, S. Acharya, et al., Studies of J/ψ production at forward
rapidity in Pb-Pb collisions at √sNN = 5.02 TeV, J. High Energy Phys. 02 (2020)
041, arXiv:1909 .03158 [nucl -ex]. [40] M. Laine, A resummed perturbative estimate for the quarkonium spectral func-
tion in hot QCD, J. High Energy Phys. 05 (2007) 028, arXiv:0704 .1720 [hep -ph]. [41] P. Braun-Munzinger, J. Stachel, (Non)thermal aspects of charmonium produc-
tion and a new look at J/ψ suppression, Phys. Lett. B 490 (2000) 196–202,
arXiv:nucl -th /0007059 [nucl -th]. [57] ALICE Collaboration, S. Acharya, et al., Inclusive J/ψ production at forward and
backward rapidity in p-Pb collisions at √sNN = 8.16 TeV, J. High Energy Phys. 07 (2018) 160, arXiv:1805 .04381 [nucl -ex]. [42] R.L. Thews, M. Schroedter, J. Rafelski, Enhanced J/ψ production in deconfined
quark matter, Phys. Rev. C 63 (2001) 054905, arXiv:hep -ph /0007323 [hep -ph]. [58] R. Brun, F. Bruyant, F. Carminati, S. Giani, M. Maire, A. McPherson, G. Patrick,
L. Urban, GEANT: Detector Description and Simulation Tool; Oct 1994. References [28] M. Butenschoen, B.A. Kniehl, J/ψ
polarization at tevatron and LHC:
nonrelativistic-QCD factorization at the crossroads, Phys. Rev. Lett. 108 (2012)
172002, arXiv:1201.1872 [hep -ph]. [29] K.-T. Chao, Y.-Q. Ma, H.-S. Shao, K. Wang, Y.-J. Zhang, J/ψ polarization at
hadron colliders in nonrelativistic QCD, Phys. Rev. Lett. 108 (2012) 242004,
arXiv:1201.2675 [hep -ph]. 7 Physics Letters B 815 (2021) 136146 ALICE Collaboration [46] ALICE Collaboration, K. Aamodt, et al., The ALICE experiment at the CERN LHC,
J. Instrum. 3 (2008) S08002. [30] B. Gong, L.-P. Wan, J.-X. Wang, H.-F. Zhang, Polarization for prompt j/ψ and
ψ(2s) production at the tevatron and LHC, Phys. Rev. Lett. 110 (4) (2013)
042002, arXiv:1205 .6682 [hep -ph]. [47] ALICE Collaboration, B. Abelev, et al., Performance of the ALICE Experiment at
the CERN LHC, Int. J. Mod. Phys. A 29 (2014) 1430044, arXiv:1402 .4476 [nucl -
ex]. [31] Y. Feng, B. Gong, C.-H. Chang, J.-X. Wang, Remaining parts of the long-standing
J/ψ polarization puzzle, Phys. Rev. D 99 (1) (2019) 014044, arXiv:1810 .08989
[hep -ph]. [48] ALICE Collaboration, K. Aamodt, et al., Rapidity and transverse momentum de-
pendence of inclusive J/ψ production in pp collisions at √
s = 7 TeV, Phys. Lett. B 704 (2011) 442, arXiv:1105 .0380 [hep -ex]. [32] Y.-Q. Ma, T. Stebel, R. Venugopalan, J/ψ polarization in the CGC+NRQCD ap-
proach, J. High Energy Phys. 12 (2018) 057, arXiv:1809 .03573 [hep -ph]. [49] ALICE Collaboration, E. Abbas, et al., Performance of the ALICE VZERO system,
J. Instrum. 8 (2013) P10016, arXiv:1306 .3130 [nucl -ex]. [33] CMS Collaboration, S. Chatrchyan, et al., Measurement of the ϒ(1S), ϒ(2S) and
ϒ(3S) polarizations in pp collisions at √
s = 7 TeV, Phys. Rev. Lett. 110 (8)
(2013) 081802, arXiv:1209 .2922 [hep -ex]. [50] ALICE Collaboration, K. Aamodt, et al., Alignment of the ALICE Inner Tracking
System with cosmic-ray tracks, J. Instrum. 5 (2010) P03003, arXiv:1001.0502
[physics .ins -det]. [34] CMS Collaboration, V. Khachatryan, et al., ϒ(nS) polarizations versus particle
multiplicity in pp collisions at √
s = 7 TeV, Phys. Lett. B 761 (2016) 31–52,
arXiv:1603 .02913 [hep -ex]. [51] ALICE Collaboration, B. Abelev, et al., Measurement of the cross section for elec-
tromagnetic dissociation with neutron emission in Pb-Pb collisions at √sNN =
2.76 TeV, Phys. Rev. Lett. 109 (2012) 252302, arXiv:1203 .2436 [nucl -ex]. [35] LHCb Collaboration, R. References CERN
Program Library. CERN, Geneva, 1993, http://cds .cern .ch /record /1082634, Long
Writeup W5013. [43] B.L. Ioffe, D.E. Kharzeev, Quarkonium polarization in heavy ion collisions as a
possible signature of the quark gluon plasma, Phys. Rev. C 68 (2003) 061902,
arXiv:hep -ph /0306176 [hep -ph]. [59] ALICE Collaboration, B. Abelev, et al., Measurement of quarkonium production
at forward rapidity in pp collisions at √
s = 7 TeV, Eur. Phys. J. C 74 (8) (2014)
2974, arXiv:1403 .3648 [nucl -ex]. [44] H.-S. Shao, Y.-Q. Ma, K. Wang, K.-T. Chao, Polarizations of χc1 and χc2 in
prompt production at the LHC, Phys. Rev. Lett. 112 (18) (2014) 182003, arXiv:
1402 .2913 [hep -ph]. [60] ALICE Collaboration, S. Acharya, et al., ϒ suppression at forward rapidity in
Pb-Pb collisions at √sNN = 5.02 TeV, Phys. Lett. B 790 (2019) 89–101, arXiv:
1805 .04387 [nucl -ex]. [45] CMS Collaboration, A.M. Sirunyan, et al., Measurement of the χc1 and χc2
polarizations in proton-proton collisions at √
s = 8 TeV, arXiv:1912 .07706
[hep -ex]. ALICE Collaboration S. Acharya 141, D. Adamová 95, A. Adler 74, J. Adolfsson 81, M.M. Aggarwal 100, G. Aglieri Rinella 34,
M. Agnello 30, N. Agrawal 10,54, Z. Ahammed 141, S. Ahmad 16, S.U. Ahn 76, Z. Akbar 51, A. Akindinov 92,
M. Al-Turany 107, S.N. Alam 40,141, D.S.D. Albuquerque 122, D. Aleksandrov 88, B. Alessandro 59,
H.M. Alfanda 6, R. Alfaro Molina 71, B. Ali 16, Y. Ali 14, A. Alici 10,26,54, N. Alizadehvandchali 125,
A. Alkin 2,34, J. Alme 21, T. Alt 68, L. Altenkamper 21, I. Altsybeev 113, M.N. Anaam 6, C. Andrei 48,
D. Andreou 34, A. Andronic 144, M. Angeletti 34, V. Anguelov 104, C. Anson 15, T. Antiˇci´c 108, F. Antinori 57,
P. Antonioli 54, N. Apadula 80, L. Aphecetche 115, H. Appelshäuser 68, S. Arcelli 26, R. Arnaldi 59,
M. Arratia 80, I.C. Arsene 20, M. Arslandok 104, A. Augustinus 34, R. Averbeck 107, S. Aziz 78, M.D. Azmi 16,
A. Badalà 56, Y.W. Baek 41, S. Bagnasco 59, X. Bai 107, R. Bailhache 68, R. Bala 101, A. Balbino 30,
A. Baldisseri 137, M. Ball 43, S. Balouza 105, D. Banerjee 3, R. Barbera 27, L. Barioglio 25, G.G. Barnaföldi 145,
L.S. Barnby 94, V. Barret 134, P. Bartalini 6, C. Bartels 127, K. Barth 34, E. Bartsch 68, F. Baruffaldi 28,
N. Bastid 134, S. Basu 143, G. Batigne 115, B. Batyunya 75, D. Bauri 49, J.L. Bazo Alba 112, I.G. Bearden 89,
C. Beattie 146, C. Bedda 63, N.K. Behera 61, I. Belikov 136, A.D.C. Bell Hechavarria 144, F. Bellini 34,
R. Bellwied 125, V. Belyaev 93, G. Bencedi 145, S. Beole 25, A. Bercuci 48, Y. Berdnikov 98, D. Berenyi 145,
R.A. Bertens 130, D. Berzano 59, M.G. Besoiu 67, L. Betev 34, A. Bhasin 101, I.R. Bhat 101, M.A. Bhat 3,
H. Bhatt 49, B. Bhattacharjee 42, A. Bianchi 25, L. Bianchi 25, N. Bianchi 52, J. Bielˇcík 37, J. Bielˇcíková 95,
A. Bilandzic 105, G. Biro 145, R. Biswas 3, S. Biswas 3, J.T. Blair 119, D. Blau 88, C. Blume 68, G. Boca 139,
F. Bock 96, A. Bogdanov 93, S. Boi 23, J. Bok 61, L. Boldizsár 145, A. Bolozdynya 93, M. Bombara 38,
G. Bonomi 140, H. Borel 137, A. Borissov 93, H. Bossi 146, E. Botta 25, L. Bratrud 68, P. Braun-Munzinger 107,
M. Bregant 121, M. Broz 37, E. Bruna 59, G.E. ALICE Collaboration Bruno 33,106, M.D. Buckland 127, D. Budnikov 109,
H. Buesching 68, S. Bufalino 30, O. Bugnon 115, P. Buhler 114, P. Buncic 34, Z. Buthelezi 72,131, J.B. Butt 14, 8 Physics Letters B 815 (2021) 136146 ALICE Collaboration ALICE Collaboration
Physics Letters B 815 (2021) 13614
S.A. Bysiak 118, D. Caffarri 90, A. Caliva 107, E. Calvo Villar 112, J.M.M. Camacho 120, R.S. Camacho 45,
P. Camerini 24, F.D.M. Canedo 121, A.A. Capon 114, F. Carnesecchi 26, R. Caron 137, J. Castillo Castellanos 137,
A.J. Castro 130, E.A.R. Casula 55, F. Catalano 30, C. Ceballos Sanchez 75, P. Chakraborty 49, S. Chandra 141,
W. Chang 6, S. Chapeland 34, M. Chartier 127, S. Chattopadhyay 141, S. Chattopadhyay 110, A. Chauvin 23,
C. Cheshkov 135, B. Cheynis 135, V. Chibante Barroso 34, D.D. Chinellato 122, S. Cho 61, P. Chochula 34,
T. Chowdhury 134, P. Christakoglou 90, C.H. Christensen 89, P. Christiansen 81, T. Chujo 133, C. Cicalo 55,
L. Cifarelli 10,26, L.D. Cilladi 25, F. Cindolo 54, M.R. Ciupek 107, G. Clai 54,ii, J. Cleymans 124, F. Colamaria 53,
D. Colella 53, A. Collu 80, M. Colocci 26, M. Concas 59,iii, G. Conesa Balbastre 79, Z. Conesa del Valle 78,
G. Contin 24,60, J.G. Contreras 37, T.M. Cormier 96, Y. Corrales Morales 25, P. Cortese 31, M.R. Cosentino 123,
F. Costa 34, S. Costanza 139, P. Crochet 134, E. Cuautle 69, P. Cui 6, L. Cunqueiro 96, D. Dabrowski 142,
T. Dahms 105, A. Dainese 57, F.P.A. Damas 115,137, M.C. Danisch 104, A. Danu 67, D. Das 110, I. Das 110,
P. Das 86, P. Das 3, S. Das 3, A. Dash 86, S. Dash 49, S. De 86, A. De Caro 29, G. de Cataldo 53,
J. de Cuveland 39, A. De Falco 23, D. De Gruttola 10, N. De Marco 59, S. De Pasquale 29, S. Deb 50,
H.F. Degenhardt 121, K.R. Deja 142, A. Deloff 85, S. Delsanto 25,131, W. Deng 6, P. Dhankher 49, D. Di Bari 33,
A. Di Mauro 34, R.A. Diaz 8, T. Dietel 124, P. Dillenseger 68, Y. Ding 6, R. Divià 34, D.U. Dixit 19,
Ø. Djuvsland 21, U. Dmitrieva 62, A. Dobrin 67, B. Dönigus 68, O. Dordic 20, A.K. Dubey 141, A. Dubla 90,107,
S. Dudi 100, M. Dukhishyam 86, P. Dupieux 134, R.J. Ehlers 96, V.N. Eikeland 21, D. Elia 53, B. Erazmus 115,
F. Erhardt 99, A. ALICE Collaboration Erokhin 113, M.R. Ersdal 21, B. Espagnon 78, G. Eulisse 34, D. Evans 111, S. Evdokimov 91,
L. Fabbietti 105, M. Faggin 28, J. Faivre 79, F. Fan 6, A. Fantoni 52, M. Fasel 96, P. Fecchio 30, A. Feliciello 59,
G. Feofilov 113, A. Fernández Téllez 45, A. Ferrero 137, A. Ferretti 25, A. Festanti 34, V.J.G. Feuillard 104,
J. Figiel 118, S. Filchagin 109, D. Finogeev 62, F.M. Fionda 21, G. Fiorenza 53, F. Flor 125, A.N. Flores 119,
S. Foertsch 72, P. Foka 107, S. Fokin 88, E. Fragiacomo 60, U. Frankenfeld 107, U. Fuchs 34, C. Furget 79,
A. Furs 62, M. Fusco Girard 29, J.J. Gaardhøje 89, M. Gagliardi 25, A.M. Gago 112, A. Gal 136, C.D. Galvan 120,
P. Ganoti 84, C. Garabatos 107, J.R.A. Garcia 45, E. Garcia-Solis 11, K. Garg 115, C. Gargiulo 34, A. Garibli 87,
K. Garner 144, P. Gasik 105,107, E.F. Gauger 119, M.B. Gay Ducati 70, M. Germain 115, J. Ghosh 110,
P. Ghosh 141, S.K. Ghosh 3, M. Giacalone 26, P. Gianotti 52, P. Giubellino 59,107, P. Giubilato 28,
A.M.C. Glaenzer 137, P. Glässel 104, A. Gomez Ramirez 74, V. Gonzalez 107,143, L.H. González-Trueba 71,
S. Gorbunov 39, L. Görlich 118, A. Goswami 49, S. Gotovac 35, V. Grabski 71, L.K. Graczykowski 142,
K.L. Graham 111, L. Greiner 80, A. Grelli 63, C. Grigoras 34, V. Grigoriev 93, A. Grigoryan 1, S. Grigoryan 75,
O.S. Groettvik 21, F. Grosa 30,59, J.F. Grosse-Oetringhaus 34, R. Grosso 107, R. Guernane 79, M. Guittiere 115,
K. Gulbrandsen 89, T. Gunji 132, A. Gupta 101, R. Gupta 101, I.B. Guzman 45, R. Haake 146, M.K. Habib 107,
C. Hadjidakis 78, H. Hamagaki 82, G. Hamar 145, M. Hamid 6, R. Hannigan 119, M.R. Haque 63,86,
A. Harlenderova 107, J.W. Harris 146, A. Harton 11, J.A. Hasenbichler 34, H. Hassan 96, Q.U. Hassan 14,
D. Hatzifotiadou 10,54, P. Hauer 43, L.B. Havener 146, S. Hayashi 132, S.T. Heckel 105, E. Hellbär 68,
H. Helstrup 36, A. Herghelegiu 48, T. Herman 37, E.G. Hernandez 45, G. Herrera Corral 9, F. Herrmann 144,
K.F. Hetland 36, H. Hillemanns 34, C. Hills 127, B. Hippolyte 136, B. Hohlweger 105, J. Honermann 144,
D. Horak 37, A. Hornung 68, S. Hornung 107, R. Hosokawa 15,133, P. Hristov 34, C. Huang 78, C. Hughes 130,
P. Huhn 68, T.J. Humanic 97, H. Hushnud 110, L.A. ova 37, M. Krüger 68, E. Kryshen 98, M. Krzewicki 39, A.M. Kubera 97, V. Kuˇcera 34,61 ALICE Collaboration Husova 144, N. Hussain 42, S.A. Hussain 14, D. Hutter 39,
J.P. Iddon 34,127, R. Ilkaev 109, H. Ilyas 14, M. Inaba 133, G.M. Innocenti 34, M. Ippolitov 88, A. Isakov 95,
M.S. Islam 110, M. Ivanov 107, V. Ivanov 98, V. Izucheev 91, B. Jacak 80, N. Jacazio 34,54, P.M. Jacobs 80,
S. Jadlovska 117, J. Jadlovsky 117, S. Jaelani 63, C. Jahnke 121, M.J. Jakubowska 142, M.A. Janik 142,
T. Janson 74, M. Jercic 99, O. Jevons 111, M. Jin 125, F. Jonas 96,144, P.G. Jones 111, J. Jung 68, M. Jung 68,
A. Jusko 111, P. Kalinak 64, A. Kalweit 34, V. Kaplin 93, S. Kar 6, A. Karasu Uysal 77, D. Karatovic 99,
O. Karavichev 62, T. Karavicheva 62, P. Karczmarczyk 142, E. Karpechev 62, A. Kazantsev 88, U. Kebschull 74,
R. Keidel 47, M. Keil 34, B. Ketzer 43, Z. Khabanova 90, A.M. Khan 6, S. Khan 16, A. Khanzadeev 98,
Y. Kharlov 91, A. Khatun 16, A. Khuntia 118, B. Kileng 36, B. Kim 61, B. Kim 133, D. Kim 147, D.J. Kim 126,
E.J. Kim 73, H. Kim 17, J. Kim 147, J.S. Kim 41, J. Kim 104, J. Kim 147, J. Kim 73, M. Kim 104, S. Kim 18,
T. Kim 147, T. Kim 147, S. Kirsch 68, I. Kisel 39, S. Kiselev 92, A. Kisiel 142, J.L. Klay 5, C. Klein 68, J. Klein 34,59,
S. Klein 80, C. Klein-Bösing 144, M. Kleiner 68, A. Kluge 34, M.L. Knichel 34, A.G. Knospe 125, C. Kobdaj 116,
M.K. Köhler 104, T. Kollegger 107, A. Kondratyev 75, N. Kondratyeva 93, E. Kondratyuk 91, J. Konig 68,
S A Konigstorfer 105 PJ Konopka 34 G Kornakov 142 L Koska 117 O Kovalenko 85 V Kovalenko 113 S.A. Bysiak 118, D. Caffarri 90, A. Caliva 107, E. Calvo Villar 112, J.M.M. Camacho 120, R.S. Camacho 45,
P. Camerini 24, F.D.M. Canedo 121, A.A. Capon 114, F. Carnesecchi 26, R. Caron 137, J. Castillo Castellanos 137,
A.J. Castro 130, E.A.R. Casula 55, F. Catalano 30, C. Ceballos Sanchez 75, P. Chakraborty 49, S. Chandra 141,
W. Chang 6, S. Chapeland 34, M. Chartier 127, S. Chattopadhyay 141, S. Chattopadhyay 110, A. Chauvin 23,
C. Cheshkov 135, B. Cheynis 135, V. Chibante Barroso 34, D.D. Chinellato 122, S. Cho 61, P. Chochula 34,
T. Chowdhury 134, P. Christakoglou 90, C.H. Christensen 89, P. Christiansen 81, T. ALICE Collaboration Chujo 133, C. Cicalo 55,
L. Cifarelli 10,26, L.D. Cilladi 25, F. Cindolo 54, M.R. Ciupek 107, G. Clai 54,ii, J. Cleymans 124, F. Colamaria 53,
D. Colella 53, A. Collu 80, M. Colocci 26, M. Concas 59,iii, G. Conesa Balbastre 79, Z. Conesa del Valle 78,
G. Contin 24,60, J.G. Contreras 37, T.M. Cormier 96, Y. Corrales Morales 25, P. Cortese 31, M.R. Cosentino 123,
F. Costa 34, S. Costanza 139, P. Crochet 134, E. Cuautle 69, P. Cui 6, L. Cunqueiro 96, D. Dabrowski 142,
T. Dahms 105, A. Dainese 57, F.P.A. Damas 115,137, M.C. Danisch 104, A. Danu 67, D. Das 110, I. Das 110,
P. Das 86, P. Das 3, S. Das 3, A. Dash 86, S. Dash 49, S. De 86, A. De Caro 29, G. de Cataldo 53,
J. de Cuveland 39, A. De Falco 23, D. De Gruttola 10, N. De Marco 59, S. De Pasquale 29, S. Deb 50,
H.F. Degenhardt 121, K.R. Deja 142, A. Deloff 85, S. Delsanto 25,131, W. Deng 6, P. Dhankher 49, D. Di Bari 33,
A. Di Mauro 34, R.A. Diaz 8, T. Dietel 124, P. Dillenseger 68, Y. Ding 6, R. Divià 34, D.U. Dixit 19,
Ø. Djuvsland 21, U. Dmitrieva 62, A. Dobrin 67, B. Dönigus 68, O. Dordic 20, A.K. Dubey 141, A. Dubla 90,107,
S. Dudi 100, M. Dukhishyam 86, P. Dupieux 134, R.J. Ehlers 96, V.N. Eikeland 21, D. Elia 53, B. Erazmus 115,
F. Erhardt 99, A. Erokhin 113, M.R. Ersdal 21, B. Espagnon 78, G. Eulisse 34, D. Evans 111, S. Evdokimov 91,
L. Fabbietti 105, M. Faggin 28, J. Faivre 79, F. Fan 6, A. Fantoni 52, M. Fasel 96, P. Fecchio 30, A. Feliciello 59,
G. Feofilov 113, A. Fernández Téllez 45, A. Ferrero 137, A. Ferretti 25, A. Festanti 34, V.J.G. Feuillard 104,
J. Figiel 118, S. Filchagin 109, D. Finogeev 62, F.M. Fionda 21, G. Fiorenza 53, F. Flor 125, A.N. Flores 119,
S. Foertsch 72, P. Foka 107, S. Fokin 88, E. Fragiacomo 60, U. Frankenfeld 107, U. Fuchs 34, C. Furget 79,
A. Furs 62, M. Fusco Girard 29, J.J. Gaardhøje 89, M. Gagliardi 25, A.M. Gago 112, A. Gal 136, C.D. Galvan 120,
P. Ganoti 84, C. Garabatos 107, J.R.A. Garcia 45, E. Garcia-Solis 11, K. Garg 115, C. Gargiulo 34, A. Garibli 87,
K. Garner 144, P. Gasik 105,107, E.F. ALICE Collaboration Gauger 119, M.B. Gay Ducati 70, M. Germain 115, J. Ghosh 110,
P. Ghosh 141, S.K. Ghosh 3, M. Giacalone 26, P. Gianotti 52, P. Giubellino 59,107, P. Giubilato 28,
A.M.C. Glaenzer 137, P. Glässel 104, A. Gomez Ramirez 74, V. Gonzalez 107,143, L.H. González-Trueba 71,
S. Gorbunov 39, L. Görlich 118, A. Goswami 49, S. Gotovac 35, V. Grabski 71, L.K. Graczykowski 142,
K.L. Graham 111, L. Greiner 80, A. Grelli 63, C. Grigoras 34, V. Grigoriev 93, A. Grigoryan 1, S. Grigoryan 75,
O.S. Groettvik 21, F. Grosa 30,59, J.F. Grosse-Oetringhaus 34, R. Grosso 107, R. Guernane 79, M. Guittiere 115,
K. Gulbrandsen 89, T. Gunji 132, A. Gupta 101, R. Gupta 101, I.B. Guzman 45, R. Haake 146, M.K. Habib 107,
C. Hadjidakis 78, H. Hamagaki 82, G. Hamar 145, M. Hamid 6, R. Hannigan 119, M.R. Haque 63,86,
A. Harlenderova 107, J.W. Harris 146, A. Harton 11, J.A. Hasenbichler 34, H. Hassan 96, Q.U. Hassan 14,
D. Hatzifotiadou 10,54, P. Hauer 43, L.B. Havener 146, S. Hayashi 132, S.T. Heckel 105, E. Hellbär 68,
H. Helstrup 36, A. Herghelegiu 48, T. Herman 37, E.G. Hernandez 45, G. Herrera Corral 9, F. Herrmann 144,
K.F. Hetland 36, H. Hillemanns 34, C. Hills 127, B. Hippolyte 136, B. Hohlweger 105, J. Honermann 144,
D. Horak 37, A. Hornung 68, S. Hornung 107, R. Hosokawa 15,133, P. Hristov 34, C. Huang 78, C. Hughes 130,
P. Huhn 68, T.J. Humanic 97, H. Hushnud 110, L.A. Husova 144, N. Hussain 42, S.A. Hussain 14, D. Hutter 39,
J.P. Iddon 34,127, R. Ilkaev 109, H. Ilyas 14, M. Inaba 133, G.M. Innocenti 34, M. Ippolitov 88, A. Isakov 95,
M.S. Islam 110, M. Ivanov 107, V. Ivanov 98, V. Izucheev 91, B. Jacak 80, N. Jacazio 34,54, P.M. Jacobs 80,
S. Jadlovska 117, J. Jadlovsky 117, S. Jaelani 63, C. Jahnke 121, M.J. Jakubowska 142, M.A. Janik 142,
T. Janson 74, M. Jercic 99, O. Jevons 111, M. Jin 125, F. Jonas 96,144, P.G. Jones 111, J. Jung 68, M. Jung 68,
A. Jusko 111, P. Kalinak 64, A. Kalweit 34, V. Kaplin 93, S. Kar 6, A. Karasu Uysal 77, D. Karatovic 99,
O. Karavichev 62, T. Karavicheva 62, P. Karczmarczyk 142, E. Karpechev 62, A. Kazantsev 88, U. Kebschull 74,
R. Keidel 47, M. Keil 34, B. Ketzer 43, Z. Khabanova 90, A.M. Khan 6, S. Khan 16, A. ALICE Collaboration Khanzadeev 98,
Y. Kharlov 91, A. Khatun 16, A. Khuntia 118, B. Kileng 36, B. Kim 61, B. Kim 133, D. Kim 147, D.J. Kim 126,
E.J. Kim 73, H. Kim 17, J. Kim 147, J.S. Kim 41, J. Kim 104, J. Kim 147, J. Kim 73, M. Kim 104, S. Kim 18, 9 Physics Letters B 815 (2021) 136146 ALICE Collaboration C. Kuhn 136, P.G. Kuijer 90, L. Kumar 100, S. Kundu 86, P. Kurashvili 85, A. Kurepin 62, A.B. Kurepin 62,
A. Kuryakin 109, S. Kushpil 95, J. Kvapil 111, M.J. Kweon 61, J.Y. Kwon 61, Y. Kwon 147, S.L. La Pointe 39,
P. La Rocca 27, Y.S. Lai 80, M. Lamanna 34, R. Langoy 129, K. Lapidus 34, A. Lardeux 20, P. Larionov 52,
E. Laudi 34, R. Lavicka 37, T. Lazareva 113, R. Lea 24, L. Leardini 104, J. Lee 133, S. Lee 147, S. Lehner 114,
J. Lehrbach 39, R.C. Lemmon 94, I. León Monzón 120, E.D. Lesser 19, M. Lettrich 34, P. Lévai 145, X. Li 12,
X.L. Li 6, J. Lien 129, R. Lietava 111, B. Lim 17, V. Lindenstruth 39, A. Lindner 48, C. Lippmann 107, M.A. Lisa 97,
A. Liu 19, J. Liu 127, S. Liu 97, W.J. Llope 143, I.M. Lofnes 21, V. Loginov 93, C. Loizides 96, P. Loncar 35,
J.A. Lopez 104, X. Lopez 134, E. López Torres 8, J.R. Luhder 144, M. Lunardon 28, G. Luparello 60, Y.G. Ma 40,
A. Maevskaya 62, M. Mager 34, S.M. Mahmood 20, T. Mahmoud 43, A. Maire 136, R.D. Majka 146,i,
M. Malaev 98, Q.W. Malik 20, L. Malinina 75,iv, D. Mal’Kevich 92, P. Malzacher 107, G. Mandaglio 32,56,
V. Manko 88, F. Manso 134, V. Manzari 53, Y. Mao 6, M. Marchisone 135, J. Mareš 66, G.V. Margagliotti 24,
A. Margotti 54, A. Marín 107, C. Markert 119, M. Marquard 68, C.D. Martin 24, N.A. Martin 104,
P. Martinengo 34, J.L. Martinez 125, M.I. Martínez 45, G. Martínez García 115, S. Masciocchi 107,
M. Masera 25, A. Masoni 55, L. Massacrier 78, E. Masson 115, A. Mastroserio 53,138, A.M. Mathis 105,
O. Matonoha 81, P.F.T. Matuoka 121, A. Matyja 118, C. Mayer 118, F. Mazzaschi 25, M. Mazzilli 53,
M.A. Mazzoni 58, A.F. Mechler 68, F. Meddi 22, Y. Melikyan 62,93, A. Menchaca-Rocha 71, C. Mengke 6,
E. Meninno 29,114, A.S. Menon 125, M. Meres 13, S. ALICE Collaboration Mhlanga 124, Y. Miake 133, L. Micheletti 25,
L.C. Migliorin 135, D.L. Mihaylov 105, K. Mikhaylov 75,92, A.N. Mishra 69, D. Mi´skowiec 107, A. Modak 3,
N. Mohammadi 34, A.P. Mohanty 63, B. Mohanty 86, M. Mohisin Khan 16,v, Z. Moravcova 89,
C. Mordasini 105, D.A. Moreira De Godoy 144, L.A.P. Moreno 45, I. Morozov 62, A. Morsch 34, T. Mrnjavac 34,
V. Muccifora 52, E. Mudnic 35, D. Mühlheim 144, S. Muhuri 141, J.D. Mulligan 80, A. Mulliri 23,55,
M.G. Munhoz 121, R.H. Munzer 68, H. Murakami 132, S. Murray 124, L. Musa 34, J. Musinsky 64,
C.J. Myers 125, J.W. Myrcha 142, B. Naik 49, R. Nair 85, B.K. Nandi 49, R. Nania 10,54, E. Nappi 53, M.U. Naru 14,
A.F. Nassirpour 81, C. Nattrass 130, R. Nayak 49, T.K. Nayak 86, S. Nazarenko 109, A. Neagu 20,
R.A. Negrao De Oliveira 68, L. Nellen 69, S.V. Nesbo 36, G. Neskovic 39, D. Nesterov 113, L.T. Neumann 142,
B.S. Nielsen 89, S. Nikolaev 88, S. Nikulin 88, V. Nikulin 98, F. Noferini 10,54, P. Nomokonov 75,
J. Norman 79,127, N. Novitzky 133, P. Nowakowski 142, A. Nyanin 88, J. Nystrand 21, M. Ogino 82,
A. Ohlson 81,104, J. Oleniacz 142, A.C. Oliveira Da Silva 130, M.H. Oliver 146, C. Oppedisano 59,
A. Ortiz Velasquez 69, A. Oskarsson 81, J. Otwinowski 118, K. Oyama 82, Y. Pachmayer 104, V. Pacik 89,
S. Padhan 49, D. Pagano 140, G. Pai´c 69, J. Pan 143, S. Panebianco 137, P. Pareek 50,141, J. Park 61,
J.E. Parkkila 126, S. Parmar 100, S.P. Pathak 125, B. Paul 23, J. Pazzini 140, H. Pei 6, T. Peitzmann 63, X. Peng 6,
L.G. Pereira 70, H. Pereira Da Costa 137, D. Peresunko 88, G.M. Perez 8, S. Perrin 137, Y. Pestov 4,
V. Petráˇcek 37, M. Petrovici 48, R.P. Pezzi 70, S. Piano 60, M. Pikna 13, P. Pillot 115, O. Pinazza 34,54,
L. Pinsky 125, C. Pinto 27, S. Pisano 10,52, D. Pistone 56, M. Płosko ´n 80, M. Planinic 99, F. Pliquett 68,
M.G. Poghosyan 96, B. Polichtchouk 91, N. Poljak 99, A. Pop 48, S. Porteboeuf-Houssais 134, V. Pozdniakov 75,
S.K. Prasad 3, R. Preghenella 54, F. Prino 59, C.A. Pruneau 143, I. Pshenichnov 62, M. Puccio 34,
J. Putschke 143, S. Qiu 90, L. Quaglia 25, R.E. Quishpe 125, S. Ragoni 111, S. Raha 3, S. Rajput 101, J. ALICE Collaboration Rak 126,
A. Rakotozafindrabe 137, L. Ramello 31, F. Rami 136, S.A.R. Ramirez 45, R. Raniwala 102, S. Raniwala 102,
S.S. Räsänen 44, R. Rath 50, V. Ratza 43, I. Ravasenga 90, K.F. Read 96,130, A.R. Redelbach 39, K. Redlich 85,vi,
A. Rehman 21, P. Reichelt 68, F. Reidt 34, X. Ren 6, R. Renfordt 68, Z. Rescakova 38, K. Reygers 104,
A. Riabov 98, V. Riabov 98, T. Richert 81,89, M. Richter 20, P. Riedler 34, W. Riegler 34, F. Riggi 27, C. Ristea 67,
S.P. Rode 50, M. Rodríguez Cahuantzi 45, K. Røed 20, R. Rogalev 91, E. Rogochaya 75, D. Rohr 34,
D. Röhrich 21, P.F. Rojas 45, P.S. Rokita 142, F. Ronchetti 52, A. Rosano 56, E.D. Rosas 69, K. Roslon 142,
P. Rosnet 134, A. Rossi 28,57, A. Rotondi 139, A. Roy 50, P. Roy 110, O.V. Rueda 81, R. Rui 24, B. Rumyantsev 75,
A. Rustamov 87, E. Ryabinkin 88, Y. Ryabov 98, A. Rybicki 118, H. Rytkonen 126, O.A.M. Saarimaki 44,
R. Sadek 115, S. Sadhu 141, S. Sadovsky 91, K. Šafaˇrík 37, S.K. Saha 141, B. Sahoo 49, P. Sahoo 49, R. Sahoo 50,
S Sahoo 65 PK Sahu 65 J Saini 141 S Sakai 133 S Sambyal 101 V Samsonov 93,98 D Sarkar 143 10 Physics Letters B 815 (2021) 136146 ALICE Collaboration M. Sharma 101, N. Sharma 100, S. Sharma 101, O. Sheibani 125, K. Shigaki 46, M. Shimomura 83,
S. Shirinkin 92, Q. Shou 40, Y. Sibiriak 88, S. Siddhanta 55, T. Siemiarczuk 85, D. Silvermyr 81, G. Simatovic 90
G. Simonetti 34, B. Singh 105, R. Singh 86, R. Singh 101, R. Singh 50, V.K. Singh 141, V. Singhal 141,
T. Sinha 110, B. Sitar 13, M. Sitta 31, T.B. Skaali 20, M. Slupecki 44, N. Smirnov 146, R.J.M. Snellings 63,
C. Soncco 112, J. Song 125, A. Songmoolnak 116, F. Soramel 28, S. Sorensen 130, I. Sputowska 118,
J. Stachel 104, I. Stan 67, P.J. Steffanic 130, E. Stenlund 81, S.F. Stiefelmaier 104, D. Stocco 115,
M.M. Storetvedt 36, L.D. Stritto 29, A.A.P. Suaide 121, T. Sugitate 46, C. Suire 78, M. Suleymanov 14,
M. Suljic 34, R. Sultanov 92, M. Šumbera 95, V. Sumberia 101, S. Sumowidagdo 51, S. Swain 65, A. Szabo 13,
I. Szarka 13, U. Tabassam 14, S.F. Taghavi 105, G. Taillepied 134, J. Takahashi 122, G.J. Tambave 21,
S. Tang 6,134, M. ALICE Collaboration Tarhini 115, M.G. Tarzila 48, A. Tauro 34, G. Tejeda Muñoz 45, A. Telesca 34, L. Terlizzi 25,
C. Terrevoli 125, D. Thakur 50, S. Thakur 141, D. Thomas 119, F. Thoresen 89, R. Tieulent 135, A. Tikhonov 62,
A.R. Timmins 125, A. Toia 68, N. Topilskaya 62, M. Toppi 52, F. Torales-Acosta 19, S.R. Torres 37,
A. Trifiró 32,56, S. Tripathy 50,69, T. Tripathy 49, S. Trogolo 28, G. Trombetta 33, L. Tropp 38, V. Trubnikov 2,
W.H. Trzaska 126, T.P. Trzcinski 142, B.A. Trzeciak 37,63, A. Tumkin 109, R. Turrisi 57, T.S. Tveter 20,
K. Ullaland 21, E.N. Umaka 125, A. Uras 135, G.L. Usai 23, M. Vala 38, N. Valle 139, S. Vallero 59,
N. van der Kolk 63, L.V.R. van Doremalen 63, M. van Leeuwen 63, P. Vande Vyvre 34, D. Varga 145,
Z. Varga 145, M. Varga-Kofarago 145, A. Vargas 45, M. Vasileiou 84, A. Vasiliev 88, O. Vázquez Doce 105,
V. Vechernin 113, E. Vercellin 25, S. Vergara Limón 45, L. Vermunt 63, R. Vernet 7, R. Vértesi 145,
L. Vickovic 35, Z. Vilakazi 131, O. Villalobos Baillie 111, G. Vino 53, A. Vinogradov 88, T. Virgili 29,
V. Vislavicius 89, A. Vodopyanov 75, B. Volkel 34, M.A. Völkl 103, K. Voloshin 92, S.A. Voloshin 143,
G. Volpe 33, B. von Haller 34, I. Vorobyev 105, D. Voscek 117, J. Vrláková 38, B. Wagner 21, M. Weber 114,
S.G. Weber 144, A. Wegrzynek 34, S.C. Wenzel 34, J.P. Wessels 144, J. Wiechula 68, J. Wikne 20, G. Wilk 85,
J. Wilkinson 10,54, G.A. Willems 144, E. Willsher 111, B. Windelband 104, M. Winn 137, W.E. Witt 130,
J.R. Wright 119, Y. Wu 128, R. Xu 6, S. Yalcin 77, Y. Yamaguchi 46, K. Yamakawa 46, S. Yang 21, S. Yano 137,
Z. Yin 6, H. Yokoyama 63, I.-K. Yoo 17, J.H. Yoon 61, S. Yuan 21, A. Yuncu 104, V. Yurchenko 2, V. Zaccolo 24,
A. Zaman 14, C. Zampolli 34, H.J.C. Zanoli 63, N. Zardoshti 34, A. Zarochentsev 113, P. Závada 66,
N. Zaviyalov 109, H. Zbroszczyk 142, M. Zhalov 98, S. Zhang 40, X. Zhang 6, Z. Zhang 6, V. Zherebchevskii 113,
Y. Zhi 12, D. Zhou 6, Y. Zhou 89, Z. Zhou 21, J. Zhu 6,107, Y. Zhu 6, A. Zichichi 10,26, G. Zinovjev 2, N. Zurlo 140 M. Sharma 101, N. Sharma 100, S. Sharma 101, O. Sheibani 125, K. ALICE Collaboration Shigaki 46, M. Shimomura 83,
S. Shirinkin 92, Q. Shou 40, Y. Sibiriak 88, S. Siddhanta 55, T. Siemiarczuk 85, D. Silvermyr 81, G. Simatovic 90,
G. Simonetti 34, B. Singh 105, R. Singh 86, R. Singh 101, R. Singh 50, V.K. Singh 141, V. Singhal 141,
T. Sinha 110, B. Sitar 13, M. Sitta 31, T.B. Skaali 20, M. Slupecki 44, N. Smirnov 146, R.J.M. Snellings 63,
C. Soncco 112, J. Song 125, A. Songmoolnak 116, F. Soramel 28, S. Sorensen 130, I. Sputowska 118,
J. Stachel 104, I. Stan 67, P.J. Steffanic 130, E. Stenlund 81, S.F. Stiefelmaier 104, D. Stocco 115,
M.M. Storetvedt 36, L.D. Stritto 29, A.A.P. Suaide 121, T. Sugitate 46, C. Suire 78, M. Suleymanov 14,
M. Suljic 34, R. Sultanov 92, M. Šumbera 95, V. Sumberia 101, S. Sumowidagdo 51, S. Swain 65, A. Szabo 13,
I. Szarka 13, U. Tabassam 14, S.F. Taghavi 105, G. Taillepied 134, J. Takahashi 122, G.J. Tambave 21,
S. Tang 6,134, M. Tarhini 115, M.G. Tarzila 48, A. Tauro 34, G. Tejeda Muñoz 45, A. Telesca 34, L. Terlizzi 25,
C. Terrevoli 125, D. Thakur 50, S. Thakur 141, D. Thomas 119, F. Thoresen 89, R. Tieulent 135, A. Tikhonov 62,
A.R. Timmins 125, A. Toia 68, N. Topilskaya 62, M. Toppi 52, F. Torales-Acosta 19, S.R. Torres 37,
A. Trifiró 32,56, S. Tripathy 50,69, T. Tripathy 49, S. Trogolo 28, G. Trombetta 33, L. Tropp 38, V. Trubnikov 2,
W.H. Trzaska 126, T.P. Trzcinski 142, B.A. Trzeciak 37,63, A. Tumkin 109, R. Turrisi 57, T.S. Tveter 20,
K. Ullaland 21, E.N. Umaka 125, A. Uras 135, G.L. Usai 23, M. Vala 38, N. Valle 139, S. Vallero 59,
N. van der Kolk 63, L.V.R. van Doremalen 63, M. van Leeuwen 63, P. Vande Vyvre 34, D. Varga 145,
Z. Varga 145, M. Varga-Kofarago 145, A. Vargas 45, M. Vasileiou 84, A. Vasiliev 88, O. Vázquez Doce 105,
V. Vechernin 113, E. Vercellin 25, S. Vergara Limón 45, L. Vermunt 63, R. Vernet 7, R. Vértesi 145,
L. Vickovic 35, Z. Vilakazi 131, O. Villalobos Baillie 111, G. Vino 53, A. Vinogradov 88, T. Virgili 29,
V. Vislavicius 89, A. Vodopyanov 75, B. Volkel 34, M.A. Völkl 103, K. Voloshin 92, S.A. Voloshin 143,
G. Volpe 33, B. von Haller 34, I. Vorobyev 105, D. Voscek 117, J. Vrláková 38, B. Wagner 21, M. Weber 114,
S.G. ALICE Collaboration Weber 144, A. Wegrzynek 34, S.C. Wenzel 34, J.P. Wessels 144, J. Wiechula 68, J. Wikne 20, G. Wilk 85,
J. Wilkinson 10,54, G.A. Willems 144, E. Willsher 111, B. Windelband 104, M. Winn 137, W.E. Witt 130,
J.R. Wright 119, Y. Wu 128, R. Xu 6, S. Yalcin 77, Y. Yamaguchi 46, K. Yamakawa 46, S. Yang 21, S. Yano 137,
Z. Yin 6, H. Yokoyama 63, I.-K. Yoo 17, J.H. Yoon 61, S. Yuan 21, A. Yuncu 104, V. Yurchenko 2, V. Zaccolo 24,
A. Zaman 14, C. Zampolli 34, H.J.C. Zanoli 63, N. Zardoshti 34, A. Zarochentsev 113, P. Závada 66,
N. Zaviyalov 109, H. Zbroszczyk 142, M. Zhalov 98, S. Zhang 40, X. Zhang 6, Z. Zhang 6, V. Zherebchevskii 113,
Y. Zhi 12, D. Zhou 6, Y. Zhou 89, Z. Zhou 21, J. Zhu 6,107, Y. Zhu 6, A. Zichichi 10,26, G. Zinovjev 2, N. Zurlo 140 p
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Y. Zhi 12, D. Zhou 6, Y. Zhou 89, Z. Zhou 21, J. Zhu 6,107, Y. Zhu 6, A. Zichichi 10,26, G. Zinovjev 2, N. Zurlo 140 1 A.I. Alikhanyan National Science Laboratory (Yerevan Physics Institute) Foundation, Yerevan, Armenia
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13 Comenius University Bratislava, Faculty of Mathematics, Physics and Informatics, Bratislava, Slovakia
14 Comenius University Bratislava, Faculty of Mathematics, Physics and Informatics, Bratislava, Slovakia
14 COMSATS University Islamabad, Islamabad, Pakistan
15 Creighton University, Omaha, NE, United States
16 Department of Physics, Aligarh Muslim University, Aligarh, India
17 Department of Physics, Pusan National University, Pusan, Republic of Korea
18 Department of Physics, Sejong University, Seoul, Republic of Korea
19 Department of Physics, University of California, Berkeley, CA, United States
20 Department of Physics, University of Oslo, Oslo, Norway
21 Department of Physics and Technology, University of Bergen, Bergen, Norway
22 Dipartimento di Fisica dell’Università ‘La Sapienza’ and Sezione INFN, Rome, Italy
23 Dipartimento di Fisica dell’Università and Sezione INFN, Cagliari, Italy
24 Dipartimento di Fisica dell’Università and Sezione INFN, Trieste, Italy
25 Dipartimento di Fisica dell’Università and Sezione INFN, Turin, Italy
26 Dipartimento di Fisica e Astronomia dell’Università and Sezione INFN, Bologna, Italy
27 Dipartimento di Fisica e Astronomia dell’Università and Sezione INFN, Catania, Italy
28 Dipartimento di Fisica e Astronomia dell’Università and Sezione INFN, Padova, Italy
29 Dipartimento di Fisica ‘E.R. ALICE Collaboration Šafárik University, Košice, Slovakia y f
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43 Helmholtz-Institut für Strahlen- und Kernphysik, Rheinische Friedrich-Wilhelms-Universität Bonn, Bonn, Germany Energy Physics Group, Universidad Autónoma de Puebla, Puebla, M 47 Hochschule Worms, Zentrum für Technologietransfer und Telekommunikation (ZTT), Worms, Germany
48 52 INFN, Laboratori Nazionali di Frascati, Frascati, Italy 61 Inha University, Incheon, Republic of Korea 62 Institute for Nuclear Research, Academy of Sciences, Moscow, Russ 63 Institute for Subatomic Physics, Utrecht University/Nikhef, Utrecht, Netherlands 64 Institute of Experimental Physics, Slovak Academy of Sciences, Košice, Slovakia nstitute of Physics, Homi Bhabha National Institute, Bhubaneswar, India 66 Institute of Physics of the Czech Academy of Sciences, Prague, Czech Republic 67 Institute of Space Science (ISS), Bucharest, Romania 69 Instituto de Ciencias Nucleares, Universidad Nacional Autónoma de México, Mexico City, Mexico y
70 Instituto de Física, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Brazil 72 iThemba LABS, National Research Foundation, Somerset West, South Africa 73 Jeonbuk National University, Jeonju, Republic of Korea J
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78 Laboratoire de Physique des 2 Infinis, Irène Joliot-Curie, Orsay, France 78 Laboratoire de Physique des 2 Infinis, Irène Joliot-Curie, Orsay, Fran 79 Laboratoire de Physique Subatomique et de Cosmologie, Université Grenoble-Alpes, CNRS-IN2P3, Grenoble, France
80 79 Laboratoire de Physique Subatomique et de Cosmologie, Université 80 Lawrence Berkeley National Laboratory, Berkeley, CA, United States 81 Lund University Department of Physics, Division of Particle Physic 82 Nagasaki Institute of Applied Science, Nagasaki, Japan 82 Nagasaki Institute of Applied Science, Nagasaki, Japan 83 Nara Women’s University (NWU), Nara, Japan 83 Nara Women’s University (NWU), Nara, Japan y (
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84 National and Kapodistrian University of Athens, School of Science, Department of Physics, Athens, Greece 84 National and Kapodistrian University of Athens, School of Science, Department of Physics, Athens, Greece 85 National Centre for Nuclear Research, Warsaw, Poland 85 National Centre for Nuclear Research, Warsaw, Poland ational Cent e fo
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86 National Institute of Science Education and Research, Homi Bhabha National Institute, Jatni, India
87 86 National Institute of Science Education and Research, Homi Bhabha National Institute, Jatni, India 86 National Institute of Science Education and Research, 87 National Nuclear Research Center, Baku, Azerbaijan 88 National Research Centre Kurchatov Institute, Moscow, Russia 88 National Research Centre Kurchatov Institute, Moscow, Russia 89 Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark 89 Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark 90 Nikhef, National institute for subatomic physics, Amsterdam, Netherlands 90 Nikhef, National institute for subatomic physics, Amsterdam, Netherlands 91 NRC Kurchatov Institute IHEP, Protvino, Russia 92 NRC “Kurchatov” Institute – ITEP, Moscow, Russia 93 NRNU Moscow Engineering Physics Institute, Moscow, Russia g
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94 Nuclear Physics Group, STFC Daresbury Laboratory, Daresbury, United Kingdom 94 Nuclear Physics Group, STFC Daresbury Laboratory, Daresbury, United Kingdom 95 Nuclear Physics Institute of the Czech Academy of Sciences, ˇRež u Prahy, Czech Republic 95 Nuclear Physics Institute of the Czech Academy of Sciences, ˇRež u Prahy, Czech Republic 96 Oak Ridge National Laboratory, Oak Ridge, TN, United States 96 Oak Ridge National Laboratory, Oak Ridge, TN, United S 97 Ohio State University, Columbus, OH, United States 97 Ohio State University, Columbus, OH, United States 98 Petersburg Nuclear Physics Institute, Gatchina, Russia 98 Petersburg Nuclear Physics Institute, Gatchina, Russi 9 Physics department, Faculty of science, University of Zagreb, Zagreb, Cr 100 Physics Department, Panjab University, Chandigarh, India 100 Physics Department, Panjab University, Chandigarh, India 101 Physics Department, University of Jammu, Jammu, India 101 Physics Department, University of Jammu, Jammu, India 102 Physics Department, University of Rajasthan, Jaipur, India 103 Physikalisches Institut, Eberhard-Karls-Universität Tübingen, Tüb 103 Physikalisches Institut, Eberhard-Karls-Universität Tübingen, Tübingen, Germany
104 103 Physikalisches Institut, Eberhard-Karls-Universität Tübingen, Tübingen, Germany 104 Physikalisches Institut, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany 05 Physik Department, Technische Universität München, Munich, German 106 Politecnico di Bari, Bari, Italy ,
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107 Research Division and ExtreMe Matter Institute EMMI, GSI Helmholtzzentrum für Schwerionenforschung GmbH, Darmstadt, Germany
108 Rudjer Boškovi´c Institute Zagreb Croatia y
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111 School of Physics and Astronomy, University of Birmingham, Birmingham, United Kingdo 111 School of Physics and Astronomy, University of Birmingham, Birmingham, United Kingdom
112 112 Sección Física, Departamento de Ciencias, Pontificia Universidad Católica del Perú, Lima, Peru 112 Sección Física, Departamento de Ciencias, Pontificia Universidad Católica del Perú, Lima, Peru
113 113 St. ALICE Collaboration Caianiello’ dell’Università and Gruppo Collegato INFN, Salerno, Italy
30 Dipartimento DISAT del Politecnico and Sezione INFN, Turin, Italy
31 Dipartimento di Scienze e Innovazione Tecnologica dell’Università del Piemonte Orientale and INFN Sezione di Torino, Alessandria, Italy
32 Dipartimento di Scienze MIFT, Università di Messina, Messina, Italy
33 Dipartimento Interateneo di Fisica ‘M. Merlin’ and Sezione INFN, Bari, Italy
34 European Organization for Nuclear Research (CERN), Geneva, Switzerland
35 Faculty of Electrical Engineering, Mechanical Engineering and Naval Architecture, University of Split, Split, Croatia
36 Faculty of Engineering and Science, Western Norway University of Applied Sciences, Bergen, Norway
37 Faculty of Nuclear Sciences and Physical Engineering, Czech Technical University in Prague, Prague, Czech Republic 14 COMSATS University Islamabad, Islamabad, Pakistan 15 Creighton University, Omaha, NE, United States 16 Department of Physics, Aligarh Muslim University, Aligarh, India 17 Department of Physics, Pusan National University, Pusan, Republic o 18 Department of Physics, Sejong University, Seoul, Republic of Korea 21 Department of Physics and Technology, University of Bergen, Bergen, Norway 11 Physics Letters B 815 (2021) 136146 ALICE Collaboration 38 Faculty of Science, P.J. ALICE Collaboration Petersburg, Russia 114 Stefan Meyer Institut für Subatomare Physik (SMI), Vienna, Austria 114 Stefan Meyer Institut für Subatomare Physik (SMI), Vienna, Austria 115 SUBATECH, IMT Atlantique, Université de Nantes, CNRS-IN2P3, Nantes, France 115 SUBATECH, IMT Atlantique, Université de Nantes, CNRS-IN2P3, Nantes, France 116 Suranaree University of Technology, Nakhon Rat 116 Suranaree University of Technology, Nakhon Ratchasima, Thailan 117 Technical University of Košice, Košice, Slovakia 117 Technical University of Košice, Košice, Slovakia 12 Physics Letters B 815 (2021) 136146 ALICE Collaboration 118 The Henryk Niewodniczanski Institute of Nuclear Physics, Polish Academy of Sciences, Cracow, Poland
119 The University of Texas at Austin Austin TX United States 118 The Henryk Niewodniczanski Institute of Nuclear Physics, Polish Academy of Sciences, Cracow, Poland
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119 The University of Texas at Austin, Austin, TX, United States
120 Universidad Autónoma de Sinaloa, Culiacán, Mexico
121 Universidade de São Paulo (USP), São Paulo, Brazil
122 Universidade Estadual de Campinas (UNICAMP), Campinas, Brazil
123 Universidade Federal do ABC, Santo Andre, Brazil
124 University of Cape Town, Cape Town, South Africa
125 University of Houston, Houston, TX, United States
126 University of Jyväskylä, Jyväskylä, Finland
127 University of Liverpool, Liverpool, United Kingdom
128 University of Science and Technology of China, Hefei, China
129 University of South-Eastern Norway, Tonsberg, Norway
130 University of Tennessee, Knoxville, TN, United States
131 University of the Witwatersrand, Johannesburg, South Africa
132 University of Tokyo, Tokyo, Japan
133 University of Tsukuba, Tsukuba, Japan
134 Université Clermont Auvergne, CNRS/IN2P3, LPC, Clermont-Ferrand, France
135 Université de Lyon, Université Lyon 1, CNRS/IN2P3, IPN-Lyon, Villeurbanne, Lyon, France
136 Université de Strasbourg, CNRS, IPHC UMR 7178, F-67000 Strasbourg, France
137 Université Paris-Saclay Centre d’Etudes de Saclay (CEA), IRFU, Départment de Physique Nucléaire (DPhN), Saclay, France
138 Università degli Studi di Foggia, Foggia, Italy
139 Università degli Studi di Pavia, Pavia, Italy
140 Università di Brescia, Brescia, Italy
141 Variable Energy Cyclotron Centre, Homi Bhabha National Institute, Kolkata, India
142 Warsaw University of Technology, Warsaw, Poland
143 Wayne State University, Detroit, MI, United States
144 Westfälische Wilhelms-Universität Münster, Institut für Kernphysik, Münster, Germany
145 Wigner Research Centre for Physics, Budapest, Hungary
146 Yale University, New Haven, CT, United States
147 Yonsei University, Seoul, Republic of Korea 118 The Henryk Niewodniczanski Institute of Nuclear Physics, Polish Academy of Sc
119 The University of Texas at Austin, Austin, TX, United States
120 Universidad Autónoma de Sinaloa, Culiacán, Mexico
121 Universidade de São Paulo (USP), São Paulo, Brazil
122 Universidade Estadual de Campinas (UNICAMP), Campinas, Brazil
123 Universidade Federal do ABC, Santo Andre, Brazil
124 University of Cape Town, Cape Town, South Africa
125 University of Houston, Houston, TX, United States
126 University of Jyväskylä, Jyväskylä, Finland
127 University of Liverpool, Liverpool, United Kingdom
128 University of Science and Technology of China, Hefei, China
129 University of South-Eastern Norway, Tonsberg, Norway
130 University of Tennessee, Knoxville, TN, United States
131 University of the Witwatersrand, Johannesburg, South Africa
132 University of Tokyo, Tokyo, Japan
133 University of Tsukuba, Tsukuba, Japan
134 Université Clermont Auvergne, CNRS/IN2P3, LPC, Clermont-Ferrand, France
135 Université de Lyon, Université Lyon 1, CNRS/IN2P3, IPN-Lyon, Villeurbanne, Lyo
136 Université de Strasbourg, CNRS, IPHC UMR 7178, F-67000 Strasbourg, France
137 Université Paris-Saclay Centre d’Etudes de Saclay (CEA), IRFU, Départment de P
138 Università degli Studi di Foggia, Foggia, Italy
139 Università degli Studi di Pavia, Pavia, Italy
140 Università di Brescia, Brescia, Italy
141 Variable Energy Cyclotron Centre, Homi Bhabha National Institute, Kolkata, In
142 Warsaw University of Technology, Warsaw, Poland
143 Wayne State University, Detroit, MI, United States
144 Westfälische Wilhelms-Universität Münster, Institut für Kernphysik, Münster, G
145 Wigner Research Centre for Physics, Budapest, Hungary
146 Yale University, New Haven, CT, United States
147 Yonsei University, Seoul, Republic of Korea 118 The Henryk Niewodniczanski Institute of Nuclear Physics, Polish Academy of Sciences, Cracow, Poland
119 The University of Texas at Austin, Austin, TX, United States 119 The University of Texas at Austin, Austin, TX, United States 119 The University of Texas at Austin, Austin, TX, United State 120 Universidad Autónoma de Sinaloa, Culiacán, Mexico 120 Universidad Autónoma de Sinaloa, Culiacán, Mexico 121 Universidade de São Paulo (USP), São Paulo, Brazil 121 Universidade de São Paulo (USP), São Paulo, Brazil 122 Universidade Estadual de Campinas (UNICAMP), Campinas, Brazil rsité Clermont Auvergne, CNRS/IN2P3, LPC, Clermont-Ferrand, France 134 Université Clermont Auvergne, CNRS/IN2P3, LPC, Clermont-Ferrand, France 135 Université de Lyon, Université Lyon 1, CNRS/IN2P3, IPN-Lyon, Villeurbanne, Lyon, France
136 137 Université Paris-Saclay Centre d’Etudes de Saclay (CEA), IRFU, Départment de Physique Nucléaire (DPhN), Saclay, France
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y
141 Variable Energy Cyclotron Centre, Homi Bhabha National Institute, Kolkata, India 142 Warsaw University of Technology, Warsaw, Poland 143 Wayne State University, Detroit, MI, United States 144 Westfälische Wilhelms-Universität Münster, Institut für Kernphysik, Münster, Germany
145 145 Wigner Research Centre for Physics, Budapest, Hungary 146 Yale University, New Haven, CT, United States 147 Yonsei University, Seoul, Republic of Korea ALICE Collaboration Petersburg State University, St. i Deceased. ii Italian National Agency for New Technologies, Energy and Sustainable Economic Development (ENEA), Bologna, Italy. ii Italian National Agency for New Technologies, Energy and Sustainable Economic Development (ENEA), Bologna, Italy. iii Dipartimento DET del Politecnico di Torino, Turin, Italy. iii Dipartimento DET del Politecnico di Torino, Turin, Italy. iii Dipartimento DET del Politecnico di Torino, Turin, Italy. iv M.V. Lomonosov Moscow State University, D.V. Skobeltsyn Institute of Nuclear, Physics, Moscow, Russia. iv M.V. Lomonosov Moscow State University, D.V. Skobeltsy v Department of Applied Physics, Aligarh Muslim University, Aliga vi Institute of Theoretical Physics, University of Wroclaw, Poland. vi Institute of Theoretical Physics, University of Wroclaw, Poland. 13
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Human rights’ interdependence and indivisibility: a glance over the human rights to water and sanitation
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BMC international health and human rights
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(2019) 19:14 (2019) 19:14 Neves-Silva et al. BMC International Health and Human Rights
https://doi.org/10.1186/s12914-019-0197-3 Neves-Silva et al. BMC International Health and Human Rights
https://doi.org/10.1186/s12914-019-0197-3 Abstract Background: In 1948 the Universal Declaration of Human Rights was drawn up. The content of this document was
further reflected two treaties, the Covenant on Civil and Political Rights, and the Covenant on Economic, Social and
Cultural Rights. To try to maintain the interrelationship between the rights contained in each document, the idea
that all rights are interdependent and indivisible was stressed. Based on this vision, this study aimed to explore the
extent to which the violation of the human rights to water and sanitation interferes with the guarantee of other
rights, addressing the principles of interdependence and indivisibility. Methods: For that, 24 homeless, in the city of Belo Horizonte, Brazil, were interviewed. Individual and group
interviews were carried out in addition to participant observation. The content analysis was used in order to analyze
the data collected. Results: The research found that violation of the rights to water and sanitation promotes violation of other rights,
such as health and education rights, strengthening the view of rights’ interdependence and indivisibility. Conclusion: It is important to affirm that the protection of human rights must be consolidated at an operational
and normative level, aligned to concepts of indivisibility and interdependence as it has been proposed for
approximately seven decades. Keywords: Human rights, Water, Sanitation, Homeless Human rights’ interdependence and
indivisibility: a glance over the human
rights to water and sanitation Priscila Neves-Silva*
, Giselle Isabele Martins and Léo Heller © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Background * Correspondence: priscila.neves31@gmail.com
René Rachou Institute - Fiocruz Minas, Augusto de Lima Avenue, 1715 CEP:
30, Belo Horizonte, Minas Gerais 190-002, Brazil * Correspondence: priscila.neves31@gmail.com
René Rachou Institute - Fiocruz Minas, Augusto de Lima Avenue, 1715 CEP:
30, Belo Horizonte, Minas Gerais 190-002, Brazil Background individual freedoms and procedural guarantees of access
to justice and political participation. On the other hand,
the second group brings together the rights related to the
state of social welfare, for which the State must intervene
to ensure a more fair and egalitarian society [1, 2]. Last year, seven decades of the adoption of the Universal
Declaration of Human Rights, the main document that
underlies the elaboration of several international treaties
and covenants as well as serving as a model for many
national constitutions, were completed. Adopted in 1948
by 48 countries and reiterated in 1993 through the
Vienna Declaration of Human Rights signed by 171
countries, it remains the basis for many international
agreements. Despite of having two covenants, human rights must
be seeing as a system where all rights are interdepend-
ent, indivisible and interrelated. According to Minkler
and Sweeney [3] the vision of a comprehensive human
rights system was built on the understanding that, in
order to guarantee the dignity of the human person, all
human rights should be reached. In this way, the integral
human rights system is established where the violation
of one of them, either civil or political right; or eco-
nomic, social and cultural, damages the achievement of
the others. The drafting of the Universal Declaration of Human
Rights gave rise to two main Covenants, the International
Covenant on Civil and Political Rights; and the Inter-
national Covenant on Economic, Social and Cultural
Rights. The first one consists of a set of rights that aim at In the following decades, the main United Nations
General Assembly resolutions reaffirmed the vision of In the following decades, the main United Nati
General Assembly resolutions reaffirmed the vision
* Correspondence: priscila.neves31@gmail.com
René Rachou Institute - Fiocruz Minas, Augusto de Lima Avenue, 1715 CEP:
30, Belo Horizonte, Minas Gerais 190-002, Brazil
© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. The goal 6 meant to achieve universal access to
water and sanitation for all. By 2030 no one should be
left behind [19]. According to Payne [12] the basic rights, defined by
Shue [13], would be those that ensure self-respect and
survival, and are therefore necessary for the guarantee of
all others. For this author there were two types of basic
rights: the rights related to security and the ones related
to subsistence. Security rights refer to civil rights as
those that protect the human being from torture, rape,
assault and death. Subsistence would correspond to eco-
nomic, social and cultural rights such as access to water,
food, clothing, shelter and health care. Thus, when taken
together, these rights would be mutually indispensable
and equally necessary for the guarantee of other rights. Therefore, all other non-basic rights would rely on these
rights. The homeless live in a vulnerable situation and do not
have access to a minimally adequate housing [20]. The
United Nations estimates that, in the world, 100 million
people are homeless [20]. In Brazil, it is believed that
this population is around 101 thousand [21]. Walter’s [22] study on access to water and sanitation
for homeless in India verified that these people have the
HRWS violated, affecting, among other things, their
health and dignity. For this author the reach of the
HRWS for this population goes beyond technical ques-
tions demanding an understanding of how the vulner-
ability situation of these individuals was created and is
maintained, in this way, these people would be exposed
to structural and social iniquities that include the viola-
tion of rights. Some authors argue that the interdependence and in-
divisibility of human rights will be ensured when the
most vulnerable, socially excluded and marginalized
populations are included [2, 14]. For them, the eco-
nomic, social and cultural rights are more addressed to
this population, favoring the social inclusion and the
construction of a more egalitarian society. Thus, to neg-
lect these rights would be a reflection of societies
marked by injustices and social disparities. These findings are aligned to Udin et al. [23] who car-
ried out a survey in Dhaka, Bangladesh, and concluded
that the lack of access to water is related to a marginal
position in society, preventing the access to basic ser-
vices such as housing, education and health, besides
water and sanitation. Neves-Silva et al. BMC International Health and Human Rights (2019 Page 2 of 8 (2019) 19:14 Page 2 of 8 the interdependence of human rights – “All human
rights are universal, interdependent and interrelated. The international community must treat human rights
globally in a fair and equitable manner, on equal terms,
and with the same emphasis.” [4] - and the Vienna Dec-
laration, renewed and expanded this understanding by
stating that there is interdependence between human
rights, democracy and development. sanitation has distinct characteristics that specify it, and
that when separating it from the water it would be pos-
sible to give greater visibility to its peculiarities, it be-
came an independent right. Thus, resolution A/RES/70/
169 defined sanitation as a separate but integrated right
to the human right to water. General comment number 15 points out the normative
content of access to water and defines that the access to
this resource should be within the human rights frame-
work, therefore, it is necessary to ensure availability, qual-
ity and safety, accessibility, affordability and acceptability. For sanitation, hygiene, privacy and dignity must be en-
sured as well, as attested by the Special Rapporteur on the
Human Rights to Water and Sanitation [17] However, the indivisibility of human rights is a con-
tested point on which scholars disagree. Neier [5], Roth
[6], Nickel [7] challenge the effectiveness of economic,
social and cultural rights, and point out that a very large
number of human rights make it difficult to implement,
making it almost impractical. However, authors such as
Feinberg [8], Copp [9] and Sen [10, 11] point out that
the understanding of human rights as essential to pro-
vide the basic needs of individuals and guarantee free-
dom, justifies the role of economic, social and cultural
rights. In this scenario, Minkler and Sweeney [3] point
out that until this issue is solved, researchers should
focus on the human rights considered to be basic. The JMP report, published in 2017, which has been
monitoring global progress on access to water and sani-
tation, showed that 844 million people lack access to
basic water and 2.3 billion lack access to basic sanitation. Vulnerable populations are the ones that suffer more
from the lack of those services [18]. It is necessary to re-
member that 193 Member States from United Nations,
including Brazil, signed the Sustainable Development
Goals. For these authors, this lack of ac-
cess reflects an asymmetric distribution of power and
opportunity, and vulnerability is a result of the inequities
in power relations that would lead to rights’ violation. Hence, these individuals would face difficulties accessing
subsistence rights, but also claiming them. In this sce-
nario, the authors draw attention to the importance of
the concept of indivisibility of human rights. For them,
the guarantee of HRWS should be rooted on the Within this scenario, one finds the Human Rights to
Water and Sanitation (HRWS), recognized by the United
Nations Assembly in 2010, through resolution A/RES/
64/262 [15], which are closely related to the right to a
dignified life. The right to water is described in General
Comment N° 15 [16], adopted by the Committee on
Economic, Social and Cultural Rights, and affirms that it
is the right of all, without discrimination, to access safe
water for drinking, cooking and hygiene of the home
and
belongings. In
2015,
after
understanding
that Neves-Silva et al. BMC International Health and Human Rights (2019) 19:14 Page 3 of 8 Page 3 of 8 Page 3 of 8 concept of a right-based approach, which broadly acts in
all the rights that are mostly denied to vulnerable and
marginalized people. approached people were suspicious and did not want to
participate or were drunk and/or drugged, and it was
not possible to interview them. The saturation sampling
technique, in which the absence of new themes and the
repetition of content in the interviews are indicative that
the main ideas have already been raised, was used to de-
fine the number of interviewees [29]. Neves-Silva and Heller [24], evaluated homeless’ access
to water and sanitation in Belo Horizonte, Brazil, and
verified that the lack of these accesses implies, among
other things, in health problems, physical and psycho-
logical violence by the society and public administration,
mainly municipal officers, and the risk of violence
against women. In addition, it has been found that the
normative content of both the right to water and sanita-
tion is generally not respected, as are human right prin-
ciples such as non-discrimination, access to information
and participation. These findings reinforce the import-
ance of discussing HRWS using the concepts of inter-
dependence and indivisibility. Methods This is a qualitative research with homeless living in the
city of Belo Horizonte. The qualitative methodology was
chosen in order to comprehend better the reality in
which the homeless live, and to get their perspective
about it [25]. Data collection Individual interviews and one group interview were used
to gather data, those two techniques were used as com-
plementary. This helps to better understand the reality
[25]. These interviews, carried out from May through
July 2016, followed a semi-structured script, elaborated
from the researchers’ experience and literature review,
and contained general questions about street life, ques-
tions about the normative content of HRWS (availability,
quality and safety, accessibility, affordability and accept-
ability), and issues that addressed some of the principles
of human rights (non-discrimination, information/trans-
parency and social participation) (Additional file 1). Description of study area Initially, a pilot interview was carried out to assess the
script and any question that were not well understood
by the participant were modify. After the necessary
changes were made, researchers went to the field. On
the first day in the field, a participant observation [19]
was made in a public space downtown where many
homeless live. The choice of this square is due to the
high number of homeless that pass through or live there
and the presence of a water source. During the partici-
pant observation, made in two days, the two researchers
assessed how homeless used the water source located in
the square and how was the routine of the people who
lived there. The participant observation was essential to
have a better understanding about the ways this group
uses water and sanitation services. Belo Horizonte is the 6th most populous city in Brazil. The 2017 Population and Household Census indicated
that its population is 2. 523. 794 [26]. It is located at the
southeast and it is the fourth wealthiest city in the coun-
try. The main economic activities are services like finan-
cial
services
and
trade. Nevertheless,
the
homeless
population in the city is growing and data from the Third
Census on the Homeless Population and Migrants, carried
out in 2013, showed that there were 1827 homeless people
in the city; an increase of 57% compared with the 2005
census [27]. It is estimated that at the end of 2017, 4.500
people were living in the streets [28]. In order to carry out the group interview, a contact was
initially made with the Pastoral de Rua de Belo Horizonte,
an organization linked to the Catholic Church, acting to
promote dignity to homeless, and that has been operating
in Belo Horizonte since 1987., who promptly received us
and pointed out that they were not aware of any other re-
search that had approached homeless and the access to
water and sanitation. Every week, at the Pastoral’s prem-
ises a meeting takes place along with the homeless. Re-
searchers attended those meetings for 2 months and
presented the research to the group, which was very
interested. Therefore, based on the idea of a comprehensive hu-
man rights system and the importance of access to eco-
nomic, social and cultural rights by the marginalized
population with a view to establishing a more egalitarian
society, this article aims to explore the extent to which
violation of HRWS of homeless population at Belo Hori-
zonte, Brazil, interferes with the guarantee of other
rights, addressing the principles of interdependence and
indivisibility among them. Data processing and analysis The interviews were recorded, transcribed and analyzed
by the two researchers that collected them. The tech-
nique of content analysis was used [30], through which
the information collected was systematized into thematic
categories. This involved reading through participants
responses and assigning codes to specific aspects of the
responses. “Stinky homeless entering on an establishment asking
for a glass of water? Come on! There’s no purpose on
that.” GF/HL 3. “When you are homeless and arrive all dirty, you are
hardly looked at. People do not pay attention at you.”
HL 7. In order to reduce the influence of the researchers’
own backgrounds, conceptions and perceptions of the
topic during the interview and the analytical process, the
interviews and participant observations were done by
the two researchers. Both of them had a field book, and
all data analyses were done by the two researchers inde-
pendently and then compared. “The society sees the homeless as a burden, someone
who litters the city.” HL 8. “We have to remove the garbage! Get out garbage.”
HL 5. A code book was then developed, discussed and ac-
cepted by the researchers. Then, the codes were grouped
into themes: the discrimination due to lack of access to
water and sanitation; the lack of access to education and
health; the lack of access to leisure activities; the lack of
social participation; the lack of access to information. Respondent statements, which are especially expressive,
informative and representative, were selected and are
presented as quotes. Selection of participants Men and women living in the streets, aged > 18 years
old, participated in the study. Twelve people were inter-
viewed individually, out of which, five were women, and
the interviews were carried out on the streets or in the
premises of the Pastoral de Rua. The group interview
was carried out at the Pastoral’s premises and included Participants were selected according to a criterion of ac-
cessibility, after participant observation. Approaches in
the street took place in the central region of the munici-
pality, which concentrates the largest number of home-
less [27]. The approaches were made introducing the
researchers and the purposes of the research. Some Neves-Silva et al. BMC International Health and Human Rights (2019) 19:14 Page 4 of 8 (2019) 19:14 Page 4 of 8 Neves-Silva et al. BMC International Health and Human Rights twelve participants, out of which, four were women. The
24 participants in the study were aged between 20 and
55 years old and the time elapsed since they became
homeless ranged from 15 days to 30 years. access to water and sanitation services, and the associ-
ation society makes between them and rubbish, promote
the construction of the stigma and strengthen their
marginalization
and
exclusion. The
exclusion
and
marginalization suffered by them result in lack of access
to many places, and in many circumstances they are like
invisible to other people. Ethical considerations Gathered data is confidential and the anonymity of the par-
ticipants has been guaranteed, so that their names will not
be unveiled in any situation. Participants that were indi-
vidually interviewed were identified with the abbreviation
HL (Homeless) followed by the serial number in which they
were interviewed. Those who participated in the group
interview were identified as EG/HL followed by the partici-
pation order number. Participants were informed about the
project and invited to participate voluntarily, signing the In-
formed Consent Form. They were certified that they could
feel free to leave the study at any time. The survey herein
was approved by the Research Ethics Committee from the
René-Rachou Research Center – under protocol CAAE
49209515.0.0000.50.91. “This is the second time I try to go back to school,
but I was ashamed because I couldn’t get the money
to take a shower so not to arrive stinking at school.”
HL12. They also face some difficulties when looking for
health services. Health professionals, in some cases, ask
them to take a shower or change clothes before the ap-
pointment. As they have difficulties in finding places
that allow them to take a bath or clean their belongs,
they avoid going to the health care center. “When he/she gets sick and looks for a hospital, it’s
not everyone who likes to take care of him/her, either
… he/she has to be clean to be taken care of, if he/she
gets dirty, is looked at with an ugly face.” GF/HL7. The lack of access to education and health Through the reports of the homeless, it is possible to see
that the absence of HRWS has repercussions on rights’
guarantee, such as education and health. As they are
seen as dirty, stinking and using dirty cloths, they feel
bad going to school and staying in the classroom. There-
fore, as access to education is, in many cases, limited,
they will face many barriers to create opportunities to
improve their lives. The lack of civil and political rights As homeless are seen as dirty and their cloths and be-
longings with a bad smell, they are discriminated and
this affects the way they are treated by society. Some
state officials, in special the police, are not prepared to
deal with them and their particularities, necessities and
way of living. This results in approaches done with vio-
lence and inhumanity. Theirs belongings are taking off
and the police usually take their documents which pre-
vent them to vote. This affect the access to the right to
vote and the enjoyment of other political and civil rights,
as they have problems to get their documents. “I’m nothing; I have right to nothing.” GF/HL 3. The discrimination due to lack of access to water and
sanitation Lack of access to water and sanitation results in discrim-
ination and more difficulty to access water. The dirty
person is not allowed to enter in an establishment to ask
for water and also are treated as garbage by society. Therefore, the participants pointed out that lack of “If homeless gets sick, he/she goes to the hospital but
he/she is not admitted because he/she got there dirty;
you have to hunt down a place to take a shower, and
where are you going to take a shower? At the bus (2019) 19:14 Neves-Silva et al. BMC International Health and Human Rights Page 5 of 8 Page 5 of 8 Page 5 of 8 The lack of access to information The lack of access to information station if you have no money you do not take it. In
the spout, for you to leave here and go there to take a
shower and then come back to the hospital, you have
already missed the appointment. At the Station
Square, if you take a bath in the fountain, the water is
returnable and you will get skin diseases. If you go to
take a shower in another corner, by the time you
manage to come back to the hospital, the doctor will
say, “your record has already been canceled” and you
stay ill. That’s the way it is.” GF/HL3. Also, they cannot participate actively in decision make
process as they do not have access to information and
they feel that they do not have rights. As they are seen
as dirty, and wearing dirty cloths, they are compared to
trash, they are invisible and they are not part of the soci-
ety. For these reasons, they are not informed, and, in
many cases, not allowed to participate in meetings to
discuss public policies. This reinforces discrimination
and put them away from decision make processes, which
helps to increase the feeling of having no rights. “The government never calls, and will never call,
homeless to discuss the water issue. We are garbage,
the cancer of the world. When publicized, it is only
done among higher classes, we the street class, we are
invisible and have no access. We are the
untouchables. Society sees us and runs away: "no, it is
full of animals, garbage out there”. HL 5. Discussion These reports are aligned to what many authors point
out: the full rights’ accomplishment must be approached
from the indivisibility and interdependence point of
view. Failure to implement some human rights can cause
harm to others, in which case it is important to check
the damages and risks that can be created when certain
rights are not guaranteed. The relationship between ac-
cess to water and sanitation and the accomplishment of
other rights is evident when homeless report that the
lack of these services often prevents them from attend-
ing public establishments. “They try, in all ways, to oust homeless, taking our
belongings, our documents.” HL 5. “I’ve been trying to get my documents for four
months. The police carry my belongings; no one
wants to help homeless, they rather undermine us.”
GF/HL 2. “At the elections I do not vote.” HL 4. Nickel(7) argues that indivisibility and interdependence
are not the same thing since indivisibility supposes a
much stronger form of interdependence, in what it
claims a bidirectional support, where the guarantee of a
right would only exist if the other is implemented, and
vice versa, ensuring a reciprocity in the relationship. The
author understands that interdependence occurs when
one right depends on another to be guaranteed but there
would be no reciprocity, that is, one right would be
dependent on another right, but this other right would
not need the first to be guaranteed, with that there was
no bidirectional support, it would be unidirectional. Thus, for this author, several rights are interdependent,
but they are not indivisible. (2019) 19:14 Page 6 of 8 Page 6 of 8 Neves-Silva et al. BMC International Health and Human Rights (2019) 19:14 hand, the lack of access to education, health and housing
can result in HRWS violation. Failure to accomplish
education and health rights, especially education, pre-
vents homeless from re-entering society, what would im-
prove access to water and sanitation, housing and other
economic, social and cultural rights, as well as of politic
and civil ones. Therefore, one can say that there is a
strong and bidirectional relationship between these
rights, so they are interdependent and indivisible. For
this reason, one can say that the quality of implementa-
tion of HRWS interferes in the quality of implementa-
tion of the human rights framework. hand, the lack of access to education, health and housing
can result in HRWS violation. Failure to accomplish
education and health rights, especially education, pre-
vents homeless from re-entering society, what would im-
prove access to water and sanitation, housing and other
economic, social and cultural rights, as well as of politic
and civil ones. Therefore, one can say that there is a
strong and bidirectional relationship between these
rights, so they are interdependent and indivisible. For
this reason, one can say that the quality of implementa-
tion of HRWS interferes in the quality of implementa-
tion of the human rights framework. lack of bathing or clean clothes and belongings? How to
assert the right to participation if they cannot access
information? The full exercise of political rights presupposes an active
participation of the subjects and promotes the empower-
ment of the most vulnerable populations, improving their
capacity for pressure, articulation and mobilization, aim-
ing at a better implementation of economic, social and
cultural rights. On the other hand, as Udin et al. [23],
Walter [22] and Neves-Silva and Heller [24] pointed out,
marginalized and excluded populations, who have their
rights, among them HRWS, violated, have greater diffi-
culty to participate in decision-making processes. Thus,
one can see the importance of the concepts of indivisibil-
ity and interdependence of human rights. In addition, when it comes to HRWS, it can be said
that it is a subsistence right, since its recognition is an-
chored in the right to a quality life, and Shue [13] under-
stands
livelihood
rights
are
indispensable
for
the
accomplishment of many other rights. (2019) 19:14 It is important to draw attention to the study carried
out by Leblang et al. [32] that demonstrated the positive
intersection between democracy and economic freedom. It can be said, then, that economic-social vulnerability
leads to the vulnerability of civil and political rights and
reduced participation in democratic spaces. "Denial of
economic freedom, in the form of extreme poverty, ren-
ders the person vulnerable to violations of other forms of
freedom. [...]. The denial of economic freedom implies the
denial of social and political freedom "Amartya Sen [33]. The study also shows how violation of HRWS pro-
motes violation of civil and political rights, showing that
HRWS interdependence with other rights is a reality that
should not be neglected. Besides, violation of the HRWS
also inhibits the active participation of homeless people
in society. They can’t participate in leisure activities and
have difficulty accessing public places. This finding coincides with Kaufman’s [31] study, in
which he assesses the interdependence and indivisibility
between the so-called first generation (civil and political)
and the second generation (economic and social) rights,
and finds that they are interrelated. However, it chal-
lenges the findings of Leblang et al. [32] that found a
negative correlation between livelihood and security
right. Therefore, as stated by Paul Farmer [34], human rights
were created to protect the most vulnerable people. Hence, all documents drawn up on the basis of this
framework will only be valid if they are used to protect
the persons most at risk of having their rights violated. One of the limitations of this study was the difficulty
to interview some groups of homeless due to their alco-
hol and drug abuse. It is also important to consider that
the findings in this study came from qualitative research,
which was based, mainly, on what was reported by the
participants. Therefore, they are more indicative than
representative of the situation. Notwithstanding, import-
ant lessons could be drawn from the findings in this
paper. In particular, the results of the study show how
access to water and sanitation, in this context, is import-
ant in order to guarantee other rights. Future research
could be done in other contexts to see if similar findings
come out. The HRWS violation, also, helps to strengthen the
stigma and prejudice that put these people at the mar-
gins of society. (2019) 19:14 As reported, they are seen as junk, as
dirt, as something that can be disposed, that promotes
the loss of self-esteem and favors social exclusion. Quane [14] states that besides being considered indivis-
ible and interdependent in what refers to implementation,
it is important to understand that rights are also inter-
dependent and indivisible in their content. For this, the
author draws attention to the right to participation. Every-
one can invoke direct participation in what refers to “the
right to effectively participate in public life” [14], but the
right to participation is embedded in several other rights,
such as in HRWS, and is more generally represented in
the covenants. Thus, for the author, the boundaries that
existed between right contents were undone and instead,
the degree of interdependence and indivisibility was
broadened and no longer confined to a specific covenant,
but reflected a trend of the international protection sys-
tem of the process of interaction between human rights. The lack of access to leisure activities The lack of access to water and sanitation also prevents
homeless from participating in social life and leisure ac-
tivities. They are not allowed to get into public places as
municipal theater or central market, and, sometimes,
they are removed from public spaces when there is a
special event in that area. For them, they are excluded
because they are dirty and not smelly. “A homeless cannot get into the Palácio das Artes
(theater in the city). If he/she is dirty, he/she is not
allowed to enter the Palácio das Artes, if he/she is
dirty, he/she cannot enter the Central Market.” HL 5. “A homeless cannot get into the Palácio das Artes
(theater in the city). If he/she is dirty, he/she is not
allowed to enter the Palácio das Artes, if he/she is
dirty, he/she cannot enter the Central Market.” HL 5. Nevertheless, one can say that HRWS, in the context
herein evaluated, fall within the two concepts, inter-
dependence and indivisibility. This is because the lack of
access to water and sanitation echoes in the violation of
other rights, such as health and education. On the other “If a party is thrown at the Station Square, homeless
are removed from the area. They are not allowed to
stay near the party. To enter you must be well
dressed, be clean and smelly.” HL 8. Conclusions The Economic, Social and Cultural covenant’s recogni-
tion impacts on a commitment to social integration,
solidarity and equality, favoring the protection of vulner-
able populations, so that basic needs are not conditional
on charity and welfare programs, but are defined as a
right. Within this set of rights are the HRWS. These
rights, as discussed, are essential in securing other rights. Therefore, in order for us to talk about democracy and
social participation, it is necessary that public policies However, how to you claim for these rights when indi-
viduals are barred from entering public spaces due to Neves-Silva et al. BMC International Health and Human Rights Page 7 of 8 Page 7 of 8 (2019) 19:14 turn towards vulnerable populations, respecting their
particularities and specificities, and giving them oppor-
tunities and guaranteeing their rights. Received: 4 June 2018 Accepted: 27 February 2019 Competing interests 28. PBH. Prefeitura de Belo Horizonte. Prefeitura apresenta plano para
moradores em situação de rua. https://prefeitura.pbh.gov.br/noticias/
prefeitura-apresenta-plano-para-moradores-em-situacao-de-rua. Accessed 19
july 2018. 28. PBH. Prefeitura de Belo Horizonte. Prefeitura apresenta plano para
moradores em situação de rua. https://prefeitura.pbh.gov.br/noticias/
prefeitura-apresenta-plano-para-moradores-em-situacao-de-rua. Accessed 19
july 2018. Competing interests
The authors declare that they have no competing interests. Abbreviations Abbreviations
HRWS: Human Rights to Water and Sanitation 12. Payne M. Henry Shue on Basic Rights. Essays in Philosophy: 2008; 9(2):1–3. 13. Shue H. Basic rights: subsistence, affluence and US foreign policy. United
Kindow: Princeton University Press; 1996. 13. Shue H. Basic rights: subsistence, affluence
Kindow: Princeton University Press; 1996. References Thus, for vulnerable populations to be truly contem-
plated, advances are needed in the conceptual expansion
of human rights, incorporating the basic needs of social
justice, and affirming the integral, indivisible and inter-
dependent perspective that exists among them. 1. Baets A. History of human rights. International encyclopedia of the Social &
Behavioral. Sciences. 2015;2(11):367–74. 1. Baets A. History of human rights. International encyclopedia of the Social &
Behavioral. Sciences. 2015;2(11):367–74. 2. Trindade AAC. The interdependence of all human rights – obstacles and
challenges to their implementation: UNESCO; 1998. 2. Trindade AAC. The interdependence of all human rights – obstacles and
challenges to their implementation: UNESCO; 1998. 3. Minkler L, Sweeney S. On the invisibility and interdependence of
rights in developing countries. Hum Rights Q. 2011;33:351–96. 3. Minkler L, Sweeney S. On the invisibility and interdependence of b
rights in developing countries. Hum Rights Q. 2011;33:351–96. Therefore, it is important to affirm that the protection
of human rights must be consolidated at an operational
and normative level, aligned to concepts of indivisibility
and interdependence as it has been proposed for ap-
proximately seven decades. 4. UN. United Nations. Vienna Declaration and Programme of Action. Geneva:
United Nations; 1993. 4. UN. United Nations. Vienna Declaration and Programme of Action. Geneva:
United Nations; 1993. 5. Neier A. Social and economic rights: a critique. Human Rights Brief. 2006;
13(2):1–3. 5. Neier A. Social and economic rights: a critique. Human Rights Brief. 2006;
13(2):1–3. 6. Roth K. Defending economic, social and cultural rights: practical issues
faced by international human rights organization. Hum Rights Q. 2004;26(1):
63–73. 6. Roth K. Defending economic, social and cultural rights: practical issues
faced by international human rights organization. Hum Rights Q. 2004;26(1):
63–73. 7. Nickel JW. Rethinking indivisibility: towards a theory of supporting relations
between human rights. Hum Rights Q. 2008;30(4):984–1001. 7. Nickel JW. Rethinking indivisibility: towards a theory of supporting relations
between human rights. Hum Rights Q. 2008;30(4):984–1001. Funding 15. UNGA. United Nations General Assembly. Human Right to Water and
Sanitation. UN Document A/RES/64/292. Geneva:UNGA; 2010. Centro de Aperfeiçoamento de Pessoal de Nível Superior (CAPS). The
founding sponsors had no role in the design of the study; in the collection,
analyses, or interpretation of data; in the writing of the manuscript, and in
the decision to publish the results. 16. OHCHR. Office of the High Commissioner for human rights. General
comment no. 15: the right to water (arts. 11 and 12 of the covenant). Geneva: OHCHR; 2010. 17. UN. United Nation. Human right to safe drinking water and sanitation. Geneva: United Nations; 2015. Availability of data and materials 18. WHO/UNICEF Joint monitoring program for water supply and sanitation. Progress on drinking water and sanitation. Update 2017. Geneva:
WHO,2017. The data that supports the findings of this study will not be made publicly
available, due to participant confidentiality restrictions. Interested
investigators may contact the researchers to explore data sharing options. The data that supports the findings of this study will not be made publicly
available, due to participant confidentiality restrictions. Interested investigators may contact the researchers to explore data sharing options. 19. UN. United Nations. Resolution adopted by the General Assembly on 25
September 2015. Transforming our world: the 2030 Agenda for Sustainable
Development . Geneva: United Nations.2015. Acknowledgements Kindow: Princeton University Press; 1996. We would like to thank the participants of the interviews for sharing their
valuable insights. 14. Quane HA. Further dimension to the interdependence and indivisibility of
human rights? Recent developments concerning the rights of indigenous
peoples. Harvard Hum Rights J. 2012;(25):49–83. Authors’ contributions
S
d
b
d PNS and LH contributed to the study design. PNS and GIM conducted the
interviews and analyzed the data. PNS reviewed the relevant literature and
drafted the manuscript which was edited and critically revised by LH. All
authors read and approved the final manuscript. 20. UN. United Nations. Report of the Special Rapporteur on adequate housing
as a component of the right to an adequate standard of living, and on the
right to non-discrimination in this context. In: United Nations. Geneve; 2015. 21. Instituto de Pesquisa Econômica Aplicada IPEA. Texto para discussão
estimativa da população em situação de rua no Brasil. In: Brasília: Rio de
Janeiro: Ipea; 2016. Consent for publication Not applicable. 27. PBH. Prefeitura de Belo Horizonte. Terceiro censo da População em Situação
de Rua e Migrantes; 2014. 27. PBH. Prefeitura de Belo Horizonte. Terceiro censo da População em Situação
de Rua e Migrantes; 2014. Ethics approval and consent to participate Ethical approval was obtained from the Rene Rachou Institute ethics
committee under protocol CAAE 49209515.0.0000.50.91. the homeless
population gave signed consent for participation in the study as a whole,
including the interviews. Information about the study, potential harms and
benefits, the purpose of the study and the right to withdraw were
emphasized, both in the written material and directly to the homeless
population before the interviews started. Anonymity and confidentiality on
the audio-recordings and the transcripts were secured by using subject iden-
tifier codes instead of names. 22. Walters V. Urban homelessness and the right to water and sanitation:
experiences from India’s cities. Water Policy. 2014;16:755–72. 22. Walters V. Urban homelessness and the right to water and sanitation:
experiences from India’s cities. Water Policy. 2014;16:755–72. 23. Uddin SMN, et al. Water sanitation and hygiene for homeless people. J
Water Health. 2016;14(1):47–51. 23. Uddin SMN, et al. Water sanitation and hygiene for homeless people. J
Water Health. 2016;14(1):47–51. 24. Neves-Silva P, Heller L. “We only have access as a favor, don’t we?” The
perception of homeless population on the human rights to water and
sanitation. Cad. Saúde Pública. 2018;34(3):e00024017. 24. Neves-Silva P, Heller L. “We only have access as a favor, don’t we?” The
perception of homeless population on the human rights to water and
sanitation. Cad. Saúde Pública. 2018;34(3):e00024017. 25. Minayo MCS. O desafio do conhecimento. Pesquisa qualitativa em saúde. Editora:Hucitec; 2014. 25. Minayo MCS. O desafio do conhecimento. Pesquisa qualitativa em saúde. Editora:Hucitec; 2014. 26. IBGE. Instituto Brasileiro de Geografia e Estatística. Brasil em síntese: https://
cidades.ibge.gov.br/brasil/mg/belo-horizonte/panorama. Accessed 19 july 2018. 26. IBGE. Instituto Brasileiro de Geografia e Estatística. Brasil em síntese: https://
cidades.ibge.gov.br/brasil/mg/belo-horizonte/panorama. Accessed 19 july 2018. Additional file 8. Feinberg J. The nature and value of rights. J Value lnq. 1970;4(4):243–60. 8. Feinberg J. The nature and value of rights. J Value lnq. 1970;4(4):243–60. 9. Coop D. The right to an adequate standard of living: justice, autonomy and
the basic needs. Social Philosophy and Policy Foundation. 1992;9(1):231–26. 9. Coop D. The right to an adequate standard of living: justice, autonomy and
the basic needs. Social Philosophy and Policy Foundation. 1992;9(1):231–26. Additional file 1: Interview Guide. Questions made during the interviews. (DOCX 19 kb) 10. Sen A. Development as Freedom. New York: First Anchor Books Edition;
2000. 10. Sen A. Development as Freedom. New York: First Anchor Books Edition;
2000. 11. Sen A. Elements of a theory of human right. Philos Public Aff. 2004;32(4):
316–56. 11. Sen A. Elements of a theory of human right. Philos Public Aff. 2004;32(4):
316–56. Abbreviations
HRWS: Human Rights to Water and Sanitation Abbreviations
HRWS: Human Rights to Water and Sanitation Neves-Silva et al. BMC International Health and Human Rights (2019) 19:14 Publisher’s Note 29. Fontanella BJB, Ricas J, Turato ER. Amostragem por saturação em pesquisas
qualitativas em saúde: contribuições teóricas. Cad Saúde Pública. 2008;24(1):
17–27. 29. Fontanella BJB, Ricas J, Turato ER. Amostragem por saturação em pesquisas
qualitativas em saúde: contribuições teóricas. Cad Saúde Pública. 2008;24(1):
17–27. Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. Neves-Silva et al. BMC International Health and Human Rights (2019) 19:14 Neves-Silva et al. BMC International Health and Human Rights (2019) 19:14 Page 8 of 8 Page 8 of 8 30. Bardin L. Análise de Conteúdo. São Paulo: Edições. 2011:70. 34. Health FP. Human rights, and pathologies of power: the new war on the
poor. Berkeley, CA. London: University of California Press; 2007.
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Seed quantity affects the fungal community composition detected using metabarcoding
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Scientific reports
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Seed quantity affects the fungal
community composition detected
using metabarcoding
OPEN Funda Oskay
1,7, Anna Maria Vettraino
2,7*, H. Tuğba Doğmuş
3, Asko Lehtijärvi4,
Stephen Woodward
5 & Michelle Cleary
6 Pest introductions via trade in tree seed may result from a lack of adequate survey and validation
protocols. Developing better diagnostic protocols to identify potentially harmful pests and pathogens
in forest tree seed is of critical importance. High-throughput sequencing-based barcoding and
metabarcoding provide effective tools for screening potentially harmful organisms in various plant
materials, including seeds. However, the sample size needed to detect the total microorganism
diversity of a community is a major challenge in microbiome studies. In this work, we examined
how increasing sample size (ranging between 100 and 1000 seeds) influences diversity of fungal
communities detected by high throughput sequencing in Pinus sylvestris seeds. Our results showed
that as sample size increased, fungal alpha diversity also increased. Beta-diversity estimators
detected significant differences between the mycobiota from different samples. However, taxonomic
and functional diversity were not correlated with sample size. In addition, we found that increasing
the number of PCR replicates resulted in a higher abundance of plant pathogens. We concluded that
for the purpose of screening for potentially harmful pathogens using HTS, greater efforts should be
made to increase the sample size and replicates when testing tree seed. Increased connectivity and globalization have greatly accelerated the frequency and magnitude of biological inva-
sions around the globe1. Huge numbers of plants and plant products, many imported from developing countries,
circulate within the market, potentially enhancing the spread of alien pests and pathogens2,3. For example, plant
trade accounts for 57% of pest introductions into Europe alone3. Long-term impacts of invasive alien forest pests and pathogens, including costs for management and derived
economic loss, grow inexorably as the number of introductions increases. Phytosanitary requirements for inter-
national trade are established by the International Plant Protection Convention (IPPC) through International
Standards for Phytosanitary Measures (ISPMs) in order to allow safe plant trade, help prevent the introduction
and spread of invasive alien species and advise appropriate measures for control when pests become established. National Plant Protection Organizations (NPPOs) follow the IPPC recommendations and implement ISPMs
in administrative and legislative procedures4. Based on IPPC recommendations (Agreement on the Applica-
tion of Sanitary and Phytosanitary Measures), a new European Plant Health Law (Regulation 2016/2031) came
into force in December 2019 to ensure safe trade and help mitigate climate change impacts on plant health. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Seed quantity affects the fungal
community composition detected
using metabarcoding
OPEN The new rules were implemented with several acts, including lists of regulated pests, high-risk plants and plant
products and priority pests. Although this new regulation introduces more effective measures than previously
in place for the protection of the European territory, discrepancies among member states in the application of
the regulations could result in a weakening of the EU biosecurity status5. Another issue not considered in the
new legislation is the possibility that alien pests and pathogens enter states on either non- or unknown hosts
and asymptomatic hosts6,7. y
p
As with the previous EU Plant Health Directive (2000/29/EC), EU Regulation 2016/2031 does not provide
phytosanitary rules for trade in all forest seeds. Based on the pest risk analysis issued by the European Food
and Environment Safety Agency (EFSA), contaminated seeds can be pathways for the potential introduction of 1Faculty of Forestry, Çankırı Karatekin University, 18200 Çankırı, Turkey. 2Department for Innovation in Biological,
Agro‑Food and Forest Systems, University of Tuscia, Via S. Camillo de Lellis, Snc, 01100 Viterbo, Italy. 3Faculty
of Forestry, Isparta University of Applied Sciences, 32260 Isparta, Turkey. 4Isparta University of Applied Sciences,
Sütçüler Prof. Dr. Hasan Gürbüz Vocational School, 32950 Isparta, Turkey. 5School of Biological Sciences, University
of Aberdeen, Cruickshank Building, Aberdeen AB24 3UU, Scotland, UK. 6Southern Swedish Forest Research
Centre, Swedish University of Agricultural Sciences, Sundsvägen 3, 230 53 Alnarp, Sweden. 7These authors
contributed equally: Funda Oskay and Anna Maria Vettraino. *email: vettrain@unitus.it | https://doi.org/10.1038/s41598-022-06997-9 Scientific Reports | (2022) 12:3060 www.nature.com/scientificreports/ Figure 1. Rarefaction curves of the OTU numbers at 97% similarity for each sample. In the legend, numbers
indicate replicates (1, 2, 3) and letters the number of seeds analyzed (A = 100, B = 200; D = 600, E = 800; F = 1000
seeds). Figure 1. Rarefaction curves of the OTU numbers at 97% similarity for each sample. In the legend, numbers
indicate replicates (1, 2, 3) and letters the number of seeds analyzed (A = 100, B = 200; D = 600, E = 800; F = 1000
seeds). quarantine pathogens to disease-free areas. Recently, Cleary, et al.8 and Franic, et al.9 highlighted that the threat
posed by this pathway may be underestimated. Especially for gymnosperms, the production of planting stock is
largely through seed, sourced locally or elsewhere, including through e-commerce. Thus, without seed inspec-
tion, seed trade could lead to new pest introductions. quarantine pathogens to disease-free areas. Seed quantity affects the fungal
community composition detected
using metabarcoding
OPEN Recently, Cleary, et al.8 and Franic, et al.9 highlighted that the threat
posed by this pathway may be underestimated. Especially for gymnosperms, the production of planting stock is
largely through seed, sourced locally or elsewhere, including through e-commerce. Thus, without seed inspec-
tion, seed trade could lead to new pest introductions. Recent DNA-based approaches to identify pests and pathogens in plants and plant products, in particular
high-throughput sequencing methods (HTS), have the potential to improve surveys and enable screening for
potentially harmful microorganisms in large sample sets7,10–12. These methods involve several steps from the field
to species detection, with high risks of pitfalls12,13. The sample size needed to adequately cover the diversity and
community structure of microorganisms is acknowledged as a major challenge in plant microbiome studies13–15. Standard protocols for detection of specific pathogens have been developed by international bodies such as the
European Plant Protection Organization (EPPO), International Seed Testing Association (ISTA) and the Food
and Agriculture Organization (FAO)-IPPC. These protocols can define the sample size needed to detect the
organism in question. For example, with Fusarium circinatum Nirenberg and O’Donnell, the causal agent of
pine pitch canker, EPPO diagnostic protocol PM 7/91 takes into consideration the risk of seed contamination
(EPPO Standard PM 7/91)16. All steps in the diagnostic process are covered, including that the number of seeds
to be tested in a lot should be defined statistically and depends on the specific diagnostic method chosen for
pathogen identification. Since counting seeds is demanding, the EPPO protocol suggests that seeds need to be
weighed. The mean thousand seed weight suggested, however, ranges from 4 g for Pinus banksiana up to 895 g
for P. pinea. In contrast, international rules for seed testing by ISTA17 recommended a minimum of 400 seeds
for detecting F. circinatum in Pinus spp. and Pseudotsuga menziesii. For culture dependent detection of this
pathogen, sampling strategy using 400 seeds was deemed appropriate to detect an infection of at least 1% with
a 95% confidence level18. Seed quantity affects the fungal
community composition detected
using metabarcoding
OPEN However, it remains unknown what sample volume might be needed to adequately
capture the microbial diversity in forest seeds and to inform inspectors about the presence of potentially harmful
pathogens when high-throughput sequencing is used.h p
g
g
g p
q
g
The aim of this work was to determine if the total fungal community detected in Pinus sylvestris varies in
samples with different numbers of seeds analysed, hypothesizing that the full coverage is obtained when more
seeds are tested. Results
Ill
i The pairwise comparison of samples based on the
Chao-1 index supported the distinction of samples in two group: Group 1 (samples A and B; extracted from
equal to or less than 200 seeds) and Group 2 (samples D, E and F; samples obtained from greater than 600 seeds)
(Student t-test, P < 0.05), and were then confirmed by the ANOVA test (post-hoc Tukey test; P < 0.05). Conversely,
Shannon and Simpson indices did not differ significantly between samples (Student T-test; P > 0.05) (Fig. 3). Fungal diversity among replicates. The average of core OTUs (number of OTUs in all replicates) among
three replicates was 20 ± 3%, with no significant differences among samples (Chi-square test, P > 0.05). About
66% (A) to 57% (F) of unique OTUs were only captured in a single sub-sample replicate, and no correlation
with sample size was found (Pearson’s correlation coefficient, P > 0.05). OTUs richness (total number of OTUs)
in a sample increased with additional replicates, regardless of the number of seeds sampled. A single replicate
captured approximately 50% of the richness. Adding a second replicate and a third replicate led to the apparent
richness increasing by approximately 30% and 20%, respectively (Fig. 4). The OTUs present in a single replicate
were mainly low-abundant taxa (< 50 reads).h y
The threshold of proportional abundance required for an OTU to be detected in all 3 PCR replicates was 10%
of the total reads of that OTU. The coefficient of variation of α-diversity indices (% CV) among replicates was
not associated with sample size (Linear regression; P < 0.05). Fungal community structure at OTU level. Differences between communities were evaluated using as
explanatory variable the OTUs abundance by the two non-parametric tests of analysis of similarities (ANOSIM)
and PERMANOVA. Both analysis confirmed that communities were separated with high dissimilarity between
groups (P = 0.0001; 1000 permutations) and separation was large (ANOSIM R = 0.69 and PERMANOVA F-sta-
tistics = 3.8). The potential effects of technical variation on the differentiation of microbial communities at different sample
sizes were determined using Non-Metric Multidimensional Scaling analysis (NMDS). Replicates mainly grouped
according to the number of seeds (Fig. 5). Samples A and B partially overlapped, whereas samples D, E and F
were well separated. Fungal community structure at the taxonomic level. OTUs retrieved from seed samples were classi-
fied in four phyla: Ascomycota, Basidiomycota, Chytridiomycota and Mucoromycota. Results
Ill
i Illumina MiSeq sequencing data. After removal of low-quality reads, short-length, chimeras, plant-
derived contaminations and clustering at a 97% similarity level, a total of 1,070,176,680 high-quality reads was
obtained. The DNA of sample C was of poor quality and was left out from the analysis. The libraries contained
between 1,597,479 and 16,656,791 reads. Fungal α‑diversity between different amounts of seed samples. All rarefaction curves approached
the saturation plateau, which indicated that the sequencing effort detected the full diversity of the fungal com-
munities (Fig. 1). The correlation between sequencing depth and number of OTUs was not significant (Pear-
son’s correlation coefficient r = 0.003; P > 0.05). However, rarefaction curves demonstrated that 15 replicates were
characterized by a different abundance of OTUs, with the highest values for sample F3.h f
The number of OTUs per sample (A–F) ranged from 101 to 178, with a total of 625 unique OTUs across the
dataset. There was a linear correlation between sample size and number of OTUs (Linear regression, R2 = 0.90;
P < 0.05). Approximately 14% of the OTUs (87) were shared among all samples (Fig. 2). With increasing sam-
ple size, higher numbers of unique OTUs were observed (Pearson’s correlation coefficient r = 0.98, P = 0.002). https://doi.org/10.1038/s41598-022-06997-9 Scientific Reports | (2022) 12:3060 | www.nature.com/scientificreports/ Figure 2. Venn diagram showing the number of shared and unique OTUs among differently sized seed samples
(A = 100, B = 200; D = 600, E = 800; F = 1000 seeds) (left) and the unique OTUs abundance for each replicate of
different samples (right). Figure 2. Venn diagram showing the number of shared and unique OTUs among differently sized seed samples
(A = 100, B = 200; D = 600, E = 800; F = 1000 seeds) (left) and the unique OTUs abundance for each replicate of
different samples (right). However, no correlation between the abundance of reads of unique OTUs and sample size was recorded (Pearson’s
correlation coefficient r = 0.7; P > 0.05). The number of unique OTUs increased by approximately 70% from sam-
ple A and B (Group 1) (approximately 50 unique OTUs/sample) to samples D, E and F (Group 2), (approximately
85 unique OTUs/sample) (Fig. 2). The α-diversity species richness, based on Chao-1, Shannon and Simpson
indices, tended to increase as sample size increased (Fig. 3). Results
Ill
i Approximately 9% of the
OTUs remained unclassified (range: 1–28% among samples). All samples appeared dominated by Ascomycota,
which accounted for an average of 90% (range: 88–97% among samples) of the total fungal diversity in the sam-
ples, followed by Basidiomycota (range: 0–1% among samples). The relative abundances (read abundance) of
Chytridiomycota and Mucoromycota were lower than 0.00001% in all samples. The remaining sequences (9%),
consistently detected in all samples, were mostly affiliated to uncultured fungi, matching members of “Fungi
spp.”i pp
At the order level, approximately 34% of OTUs were unclassified. Diaporthales and Pleosporales were the
most abundant orders with about 14% and 20% of the total reads and they were equally present in all samples
(Chi-square test; P > 0.05). https://doi.org/10.1038/s41598-022-06997-9 Scientific Reports | (2022) 12:3060 | www.nature.com/scientificreports/ ntificreports/
At the genus level a total of 148 genera were found The genera Sydo
Figure 3. The α-diversity of fungi in Scots pine seed, based on Chao-1 (A
indices. (A = 100, B = 200; D = 600, E = 800; F = 1000 seeds). Differences am
same letters indicate no significant differences (T-test; P < 0.05). A
h
l
l
l f 1 8
f
d Th
S d
i d
i
d h f
l
Figure 3. The α-diversity of fungi in Scots pine seed, based on Chao-1 (A), Shannon (B) and Simpson (C)
indices. (A = 100, B = 200; D = 600, E = 800; F = 1000 seeds). Differences among five samples were analyzed. The
same letters indicate no significant differences (T-test; P < 0.05). Figure 3. The α-diversity of fungi in Scots pine seed, based on Chao-1 (A), Shannon (B) and Simpson (C)
indices. (A = 100, B = 200; D = 600, E = 800; F = 1000 seeds). Differences among five samples were analyzed. The
same letters indicate no significant differences (T-test; P < 0.05). Figure 3. The α-diversity of fungi in Scots pine seed, based on Chao-1 (A), Shannon (B) and Simpson (C)
indices. (A = 100, B = 200; D = 600, E = 800; F = 1000 seeds). Differences among five samples were analyzed. The
same letters indicate no significant differences (T-test; P < 0.05). At the genus level, a total of 148 genera were found. The genera Sydowia dominated the fungal community
(8% of the total OTUs), followed by Alternaria (2% of the total OTUs). Results
Ill
i [A = 100 seeds (
), B = 200 seeds (
), D = 600 seeds (
), E = 800 seeds (
)
and F = 1000 seeds (
)]. Figure 5. Non-metric multidimensional scaling (NMDS) ordinations based on Bray–Curtis similarities of
OTU-based fungal community structures found in P. sylvestris seed samples (A = 100, B = 200; D = 600, E = 800;
F = 1000 seeds) (Stress value = 0.13). Figure 5. Non-metric multidimensional scaling (NMDS) ordinations based on Bray–Curtis similarities of
OTU-based fungal community structures found in P. sylvestris seed samples (A = 100, B = 200; D = 600, E = 800;
F = 1000 seeds) (Stress value = 0.13). only in one sample. These reads represent a total of 48% (n = 155) of the taxa identified to species level, and
equally distributed among samples (Chi-square test, P > 0.05). Most of the OTUs identified at the species level
were considered cosmopolitan species. However, one OTU (matching Pestalotiopsis citrina) has been never
detected in Europe and was equally distributed in different samples. only in one sample. These reads represent a total of 48% (n = 155) of the taxa identified to species level, and
equally distributed among samples (Chi-square test, P > 0.05). Most of the OTUs identified at the species level
were considered cosmopolitan species. However, one OTU (matching Pestalotiopsis citrina) has been never
detected in Europe and was equally distributed in different samples. Fungal community structure at the functional level. The fungal community colonizing P. sylvestris
seeds was assessed in terms of fungal guilds and trophic modes. The community was included in six trophic
categories with confidence levels including “highly probable” and “probable”, with the category “other” including
“possible” and “unclassified” guilds: pathotroph,athotroph-saprotroph, pathotroph-symbiotroph, saprotroph,
saprotroph-symbiotroph, symbiotroph. The relative read abundance of saprotroph fungi was higher than that of
other groups (mean of the 15 replicates was 56%; range 36–87%). Pathotroph fungi, pathotroph-symbiotroph
and pathotroph-saprotroph were less represented (12.4%, 5.2%, 2.6% of the total reads, respectively). There
were no significant correlations between fungal functional groups (trophic modes and guilds) and seed sam-
ples. Table 1 shows the relative richness of the most abundant functional guilds (> 1% in at least one sample)
among samples. The functional guild “Plant Pathogen” included 35 genera out of the 148 recorded genera (24%). Results
Ill
i However, approximately 27 genera, known to include plant pathogenic species, were classified in other guilds. The genus Alternaria, for example, was included in the Animal Pathogen-Endophyte-Plant Pathogen-Wood
Saprotroph guild. The abundance (number of OTUs) of plant pathogens in a sample increased with additional
replicates, for all samples (Fig. 6). However, the richness in terms of reads assigned to plant pathogens was not
significantly affected by sample size (Pearson’s correlation coefficient r = 0.78; P > 0.05). Results
Ill
i A total of 32 genera were common to all
samples, while 60 genera were detected only on one of the five samples analyzed.i p
g
yi
p
y
Of the 625 OTUs, 155 were identified at the species level with Sydowia polyspora being the most abundant
(55% of total reads) followed by Dothiorella sarmentorum (15% of total reads), Epiccoccum nigrum (6% of total
reads) Alternaria alternata (4% of total reads) and Alternaria brassicae (3% of total reads). Species composition
differed between samples, although 14 taxa (Alternaria alternata, Cryptococcus pinus, C. tephrensis, Dothiorella
sarmentorum, Epicoccum nigrum, Exobasidium maculosum, Fusarium avenaceum, Nectria balsamea, Pleuroceras
pseudoplatani, Rosellinia aquila, Sarocladium strictum, Stagonospora pseudopaludosa, Sydowia polyspora, Vishni-
acozyma carnescens) were common in all samples, with a minimum occupancy in the dataset of approximately
0.0005% of total reads. A minimum portion of the total reads (0.01%) was identified as rare species, i.e. present https://doi.org/10.1038/s41598-022-06997-9 Scientific Reports | (2022) 12:3060 | tificreports/
Figure 4. Relative increase in OTUs completeness with each added replication (a total of three) for different
amounts of seed samples. [A = 100 seeds (
), B = 200 seeds (
), D = 600 seeds (
), E = 800 seeds (
)
and F = 1000 seeds (
)]. Figure 5. Non-metric multidimensional scaling (NMDS) ordinations based on Bray–Curtis similarities of
OTU-based fungal community structures found in P. sylvestris seed samples (A = 100, B = 200; D = 600, E = 800;
F = 1000 seeds) (Stress value = 0.13). www.nature.com/scientificreports/ Figure 4. Relative increase in OTUs completeness with each added replication (a total of three) for different
amounts of seed samples. [A = 100 seeds (
), B = 200 seeds (
), D = 600 seeds (
), E = 800 seeds (
)
and F = 1000 seeds (
)]. Figure 4. Relative increase in OTUs completeness with each added replication (a total of three) for different
amounts of seed samples. [A = 100 seeds (
), B = 200 seeds (
), D = 600 seeds (
), E = 800 seeds (
and F = 1000 seeds (
)]. Figure 4. Relative increase in OTUs completeness with each added replication (a total of three) for different
amounts of seed samples. Discussionh The high-throughput sequencing methodology used in the current study combined with FUNGuild analyses
allowed detection of the DNA of a range of fungal taxa and demonstrated that fungal community diversity
depends on the sampling intensity of seeds. This means that current protocols used in phytosanitary screening
with a standard sample size (e.g. the EPPO requirement on number of seeds) may not account totally for all
possible fungal taxa. https://doi.org/10.1038/s41598-022-06997-9 Scientific Reports | (2022) 12:3060 | www.nature.com/scientificreports/ Table 1. Relative richness of the most abundant functional guilds (> 1% in at least one sample) among samples
reported as mean (SD). Different letters after the SD indicate significance at p < 0.05 (Tukey post hoc test). Functional guild
Samples
A
B
D
E
F
Animal Pathogen
0% (0)b
0% (0) b
0% (0)b
0% (0)b
3% (0.016)a
Plant Pathogen
15% (0.06)ac
5% (0.03)bc
8% (0.06)bc
24% (0.09)a
7% (0.04)bc
Undefined Saprotroph
68% (0.21)ab
70% (0.09)a
48% (0.07)ab
53% (0.139)ab
39% (0.03)ab
> 1 guild
3% (0.01)a
7% (0.05)a
12% (0.07)a
7% (0.08)a
7% (0.10)a
Other
14% (0.15)ab
17% 7 (0.11)ab
37% 7(0.03)a
16% (0.13)ab
38% (0.13)a Table 1. Relative richness of the most abundant functional guilds (> 1% in at least one sample) among samples
reported as mean (SD). Different letters after the SD indicate significance at p < 0.05 (Tukey post hoc test). Table 1. Relative richness of the most abundant functional guilds (> 1% in at least one sample) among samples
reported as mean (SD). Different letters after the SD indicate significance at p < 0.05 (Tukey post hoc test). Figure 6. Relative increase in plant pathogens completeness with each added replication (a total of three) for
different amounts of seed samples [A = 100 seeds (
), B = 200 seeds (
), D = 600 seeds (
), E = 800 seeds
(
) and F = 1000 seeds (
)]. Figure 6. Relative increase in plant pathogens completeness with each added replication (a total of three) for
different amounts of seed samples [A = 100 seeds (
), B = 200 seeds (
), D = 600 seeds (
), E = 800 seeds
(
) and F = 1000 seeds (
)]. To our knowledge, this is the first work showing that sample size can affect the measurement and inter-
pretation of biodiversity metrics in tree seeds. Discussionh Safe trade of tree seeds requires adequate surveys and validated
protocols in which each step should be well-defined, including the size of samples to be tested. Our results sup-
port the findings of previous eDNA studies that the choice of sample size should be considered in detection and
monitoring studies. Results showed that samples have significant variability in their fungal microbiotas (beta-
diversity) but samples A and B had less variability than samples D through F (NMDS, Chao index). Moreover,
larger samples (D-F) accumulated more rare OTUs. Penton, et al.15 argued that optimal diversity was detected in
large soil samples (10 g soil), capturing rare taxa and decreasing replicate variation, compared with 0.25 and 1 g
samples. Similarly, Nascimento, et al.19, considering the effects of sample volumes in HTS of aquatic sediments,
showed that large samples (> 1% of the sampled area) were required to obtain robust diversity measurement of
both metazoan and non-metazoan eukaryotes. y
It remains a critical question whether variation among replicates can be fully overcome by increasing sample
size in community studies. Several studies focused on the effect of PCR replicates on ecological measures, also
with inconsistent results. While many studies using different numbers of PCR replicates reported differences
between taxonomic profiles20–22, Smith and Peay23 concluded that increasing the number of PCR replicates
does not affect ecological measures, especially the alpha diversity. However, according to Kauserud et al.24, the
consistency between replicates might depend on the diversity and complexity of the fungal community consid-
ered. The impact of stochasticity during PCR might be higher where a high number of different fungi (and PCR
templates) are present. In our study, PCR replicates increased the possibility to detect new OTUs. In accordance with Shirazi et al.24,
our study also showed that rare taxa were unique to one or a few replicates of a sample. Moreover, a high value
of abundance was not the limit factor for an OTU to be present in all replicates. Thought collectively the unique
OTUs account for a small proportion of the total microbiome, we cannot exclude they could play an important
role in plant health. p
It is worth noticing, that increasing the number of replicates in sampling comes at a cost in terms of finance
and time. Nevertheless, replicate numbers should be defined based on sample characteristics. Methods
l Pinus sylvestris seeds, 0.5 kg, collected in 2015 were obtained from a seed orchard in Sweden (Lilla Istad, Öland
Island). The pre-determined thousand seed weight (TSW) of the seed lot was 6.2 g, with 100% purity, and a ger-
mination rate and viability of 98.50 and 99.75%, respectively. Seeds were not sterilized prior to DNA analyses in
order to retain the microbial community on the seed coat which may in itself, serve as an introduction pathway
for pathogens. DNA extraction, PCR and Illumina amplicon sequencing. Six samples, containing 100, 200, 400,
600, 800 and 1000 seeds (samples A-F), were randomly sorted from the seed lot by weighing the corresponding
amount calculated according to the TSW (0.62 to 6.2 g). To allow for small scale heterogeneity within the seed
lot, 0.62 g (100 seeds) was taken from the lot at each time and added to the relevant sample until the required
final weight was attained (i.e.; 1 × 0.62 g seeds for sample A, 6 × 0.62 g for sample D, 10 × 0.62 g for sample F). Prior to sampling, the 0.5 kg seed lot was transferred to a plastic box, which was surface sterilized using 2%
NaOCl (for 10 min) and 70% EtOH (for 5 min), and thoroughly mixed between sub-samplings. Seeds were lyo-
philized for 48 h in 50 ml Falcon tubes, before homogenizing samples to fine powder in a 25 ml metal grinding
container containing a 6 mm diameter stainless steel ball using a mixer mill (Retsch GmbH and Co., KG, Haan,
Germany). The metal container and balls were cleaned thoroughly between samples. A single DNA extraction
was carried out for each of the six samples, on 30 mg homogenized tissue, using the E.Z.N.A. SP Plant DNA Kit
(Omega Bio-tek, Doraville, Georgia) following the manufacturer’s protocol for dry samples. DNA was quantified
using a NanoDrop® (ND-2000 UV/vis Spectrophotometer, Thermo Fisher Scientific Inc., Wilmington, Delaware,
USA).hi The ITS2 region of the rDNA was amplified by PCR using the primers fITS7 (GTGARTCATCGAATCTTT
G)26 and ITS4 (TCCTCCGCTTATTGATATGC)27. For each sample, three replicate PCRs were conducted. Prim-
ers were modified by different 5’ identifier sequences of nine base pairs (Macrogen Inc, Seoul, Korea). Reactions
were carried out in 50 µl volumes each containing the following reaction components: 5 ng. Methods
l μl−1 template DNA,
200 μM dNTPs; 750 μM MgCl2; 10 units Phusion Hot Start polymerase (New England BioLabs, Ipswich, MA,
USA), and 200 nM each primer in 1 × buffer. All amplification reactions were performed in an Eppendorf Master
Cycler®. The PCR program began with denaturation at 98 °C for 3 min, followed by 31 cycles of 98 °C for 30 s,
annealing at 57 °C for 30 s and 72 °C for 30 s, with a final extension step at 72 °C for 7 min. PCR products were
purified using Agencourt AMPure XP (Agencourt Bioscience Corp, Massachusetts USA) and quantified using
a Qubit 3.0 Fluorometer with the Qubit dsDNA HS Assay Kit (Invitrogen, Carlsbad, CA, USA). After quanti-
fication, PCR products were pooled in an equimolar mix and sent for Illumina sequencing, one lane of Miseq
2 × 300 bp (Macrogen Inc, Seoul, Korea). Bioinformatics and analysis of sequence data. Sequences were processed using QIIME version
1.7.028. Poor quality reads (< Q20, minimum length = 150 bp, homopolymer = 6 bp) were removed. All sequence
files were combined into a single fasta file. Operational taxonomic units [OTUs, sensu29] were clustered with
97% similarity cut-off using UPARSE30 and chimeric sequences identified and removed in de-novo mode using
UCHIME with the default parameters (λ = 2, Minimum score (h) = 0.28; (β) = 8.0; n = 1.4, minimum number of
diffs in a segment = 3; minimum divergence = 0.8)31. To reduce the effects of sequence number variation, samples
were rarefied to the minimum sequencing depth (1 597 479 reads, sample B3) through a subset of randomly
selected reads prior to downstream analysis8. OTUs were assigned to taxonomic affinities using the Basic Local
Alignment Search Tool (BLAST) at the NCBI website (based on > 97% sequence similarity). OTUs were only
assigned to species level if the query sequence matched database sequences from fungal isolates (including at
least one vouchered specimen) where percentage sequence identity was > 97% and then performed a manual
check of the E-values (E−100), to ensure no contradictions among different species within the same genus. At
lower percentage sequence identity, the query read was identified to genus (identity 95–96%) family (identity
90–94%) order (identity 80–88%) and class level (identity < 80%)10. Non fungal-sequences were removed. Comparisons of microbial communities and statistical analysis. www.nature.com/scientificreports/ dominated fungal communities in seed (relative abundance between 38.5 and 69.5%), which is in agreement
with data from previous work on pine seeds8.i In this study, we confirm that HTS can be used to detect alien species and plant pathogens. In addition,
increasing the sample size will result in a higher number of rare OTUs, and one cannot exclude the possibility
that they may be potentially harmful plant pathogens. Thus, while there may be limitations to surveys in terms
of resource, in the case of phytosanitary inspections, greater efforts should be made to increase the sample size
and replicates when testing tree seed. We recognize that the practical application of the HTS method, considering the number of samples and
replicates that can be processed, will never capture the full diversity in samples, and screening only the DNA
(as opposed to RNA) cannot discern the viability of fungal taxa detected. In any case, this method is useful as
a rapid and first screening to detect potentially harmful pathogens from which more targeted diagnostics can
then be performed (e.g. selective media for culturing, qPCR assays or RNAseq) to confirm identity and viability
of pathogens. Albeit we should be aware that those taxa that are ‘unknown’ or yet to be described will inevitably
escape our attention. Discussionh Ficetola, et al.25
examining ancient DNA estimated that at least eight PCR replicates should be analysed for samples suspected
as having low detection probability. Shirazi et al.24 found that even 24 PCR replicates may be insufficient to
catalogue biodiversity fully in soil samples and then they recommended a minimum of two PCR replicates to
differentiate between sites. f
In terms of the taxonomy and functional composition, fungal communities of P. sylvestris seeds were similar
to those described previously8 with the most abundant fungal phylum being Ascomycota. Similarly, a high pres-
ence of Diaporthales and Pleosporales was already reported in pine seeds8. At the species level, Sydowia polyspora Scientific Reports | (2022) 12:3060 | https://doi.org/10.1038/s41598-022-06997-9 www.nature.com/scientificreports/ Weighted OTU overlap = (SOTUS1 + SOTUS2 + SOTUS3)/TOTUs Weighted OTU overlap = (SOTUS1 + SOTUS2 + SO where SOTUS1 = the number of sequences within the shared OTUs of replicate 1 shared with other OTUs;
SOTUS2 = the number of sequences within the shared OTUs of replicate 2 shared with other OTUs; SOTUS3 = the
number of sequences within the shared OTUs of replicate 3 shared with other OTUs; TOTUs = total number of
sequences from replicates 1, 2, and 3.fi where SOTUS1 = the number of sequences within the shared OTUs of replicate 1 shared with other OTUs;
SOTUS2 = the number of sequences within the shared OTUs of replicate 2 shared with other OTUs; SOTUS3 = the
number of sequences within the shared OTUs of replicate 3 shared with other OTUs; TOTUs = total number of
sequences from replicates 1, 2, and 3.fi Dispersion of the variation among replicates was estimated using the coefficient of variation, or ratio of
standard deviation to the mean, with the formula CV = s/x̄, where s = standard deviation and x̄ = mean. The
higher the value of CV the more dispersion is present. When comparing the OTUs abundance across samples,
a Chi-square test was usually used. The identity of OTUs was assessed considering ≥ 97% sequence identify,
and ≥ 90% coverage as positive hits.i Each OTU was classified into an ecological guild using FUNGuild34 to assess how much variation in com-
munity composition was represented by sample size. Those OTUs that were identified with a confidence level
designated as “possible”33 were included in the category “other” together with the OTUs which remained unclas-
sified. Trophic modes and guilds were described for the OTUs that were identified with a confidence level des-
ignated as “highly probable” or “probable”33. In addition, OTUs which were placed in more than one guild were
included in the “ > 1 guild” category35. Finally, relative abundances of microbial taxa and guilds in each sample
were calculated and compared using the Chi-square test. The significance level was set at 5%.hi hi
The geographical distribution of OTUs identified to a species level was checked using the USDA Fun
Database36. Data were analysed in R version 3.5.137 using vegan package version 2.438, ggplot239 and VennDiagram
package40. Received: 21 July 2021; Accepted: 10 February 2022 References 1. Prospero, S. & Cleary, M. Effects of host variability on the spread of invasive forest diseases. Forests 8, 80. https://doi.org/10.3390/
f8030080 (2017). 2. Liebhold, A. M., Brockerhoff, E. G., Garrett, L. J., Parke, J. L. & Britton, K. O. Live plant imports: The major pathway for forest
insect and pathogen invasions of the US. Front. Ecol. Environ. 10, 135–143 (2012). g
3. Santini, A. et al. Biogeographical patterns and determinants of invasion by forest pathogens in Europe. New Phytol. 197, 238–250. https://doi.org/10.1111/j.1469-8137.2012.04364.x (2013). p
g
j
4. Vettraino, A., Potting, R. & Raposo, R. EU legislation on forest plant health: An overview with a focus on Fusarium circinatum. Forests https://doi.org/10.3390/f9090568 (2018). p
g
(
)
5. Eschen, R. et al. Phytosanitary inspection of woody plants for planting at European Union entry points: a practical enquiry. Biol
Invasions 17, 2403–2413. https://doi.org/10.1007/s10530-015-0883-6 (2015). 6 Roques A et al Temporal and interspecific variation in rates of spread for insect species invading Europe during the last 200 years Invasions 17, 2403–2413. https://doi.org/10.1007/s10530-015-0883-6 (2015). 6. Roques, A. et al. Temporal and interspecific variation in rates of spread for insect species invading Europe during the last 200 years. Biol Invasions 18 907 920 https //doi org/10 1007/s10530 016 1080 y (2016) p
g
6. Roques, A. et al. Temporal and interspecific variation in rates of spread for insect species invading Europe during the last 200 years. Biol. Invasions 18, 907–920. https://doi.org/10.1007/s10530-016-1080-y (2016). 6. Roques, A. et al. Temporal and interspecific variation in rates of spread for insect species invading Europe during the last 200 years
Biol. Invasions 18, 907–920. https://doi.org/10.1007/s10530-016-1080-y (2016).h p
g
y (
)
7. Vettraino, A. M., Li, H. M., Eschen, R., Morales-Rodriguez, C. & Vannini, A. The sentinel tree nursery as an early warning system
for pathway risk assessment: Fungal pathogens associated with Chinese woody plants commonly shipped to Europe. PLoS ONE
12, e0188800. https://doi.org/10.1371/journal.pone.0188800 (2017). p
g
j
p
(
)
8. Cleary, M. et al. Cryptic risks to forest biosecurity associated with the global movement of commercial seed. Forests 10, 459. https://
doi.org/10.3390/f10050459 (2019). g
9. Franic, I. et al. Are traded forest tree seeds a potential source of nonnative pests?. Ecol. Appl. 29, e01971. https://doi.org/10.1002/
eap.1971 (2019). p
0. Vettraino, A. et al. Sentinel trees as a tool to forecast invasions of alien plant pathogens. PLoS ONE 10, e0120571. https://doi.org/
10.1371/journal.pone.0120571 (2015). j
p
11. Roques, A. Methods
l A rarefaction curve plot was gen-
erated showing the number of OTUs versus the number of sequences (reads). The α-diversity of fungal commu-
nities was determined using Chao-1, Simpson (D) and the Shannon diversity (H’) indices. Pairwise comparison
was done between individual samples using t-test which identified two distinct groups. The values of indices https://doi.org/10.1038/s41598-022-06997-9 Scientific Reports | (2022) 12:3060 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ were compared between groups using the ANOVA test with Tukey post hoc analysis (P > 0.05). Linearity of the
association of the α-diversity and sample size was determined by linear regression. Correlation between the
number of unique OTUs and sample size was assessed by the Pearson’s correlation analysis. Venn diagrams were
constructed to visualize the shared fungal communities among all samples. To explore relationships between
the fungal community structures and numbers of seeds, PERMANOVA32 and ANOSIM33 analyses were imple-
mented using a Bray–Curtis distance matrix and the OTUs abundance as explanatory variable. The ANOSIM
test statistic (R) ranges from 0 to 1, where the value 0 indicates random grouping between samples and the value
1 indicates 100% dissimilarity between samples. y
p
Fungal community composition among samples and replicates was visualized using Non-Metric Dimensional
Scaling (NMDS) on a Bray–Curtis distance matrix. g
y
Several approaches were used to assess the reproducibility of replicates. To estimate OTUs overlap among
hree replicates the following formula was applied: www.nature.com/scientificreports/ 34. Nguyen, N. H. et al. FUNGuild: An open annotation tool for parsing fungal community datasets by ecological guild. Fungal Ecol. 20, 241–248. https://doi.org/10.1016/j.funeco.2015.06.006 (2016).t 35. Veach, A. M., Stokes, C. E., Knoepp, J., Jumpponen, A. & Baird, R. Fungal communities and functional guilds shift along an eleva-
tional gradient in the Southern Appalachian Mountains. Microb. Ecol. 76, 156–168. https://doi.org/10.1007/s00248-017-1116-6
(2018). 36. Farr, D. & Rossman, A. (United States Department of Agriculture–Agricultural Research Service, 2021) https://nt.ars-grin
fungaldatabases. g
37. A Language and Environment for Statistical Computing. v. R version 3.5. 1 (R Foundation for Statistical Computing, 2018). 38. Oksanen, J. et al. vegan: Community Ecology Package. R package version 2.4–2. (2017). kham, H. Elegant Graphics for Data Analysis (ggplot2) (Springer-V g
p
f
y
ggp
p
g
g
40. Chen, H. VennDiagram: Generate High-Resolution Venn and Euler plots. R package version 1.6. 17. (2016). www.nature.com/scientificreports/ www.nature.com/scientificreports/ O. PM 7/91 (1): Gibberella circinata. EPPO Bull. 39, 298–309 (200 ( )
(
)
7. ISTA. International rules for seed testing. Seed Health Methods 2020. 7 -009: Detection of Fusarium circinatum in Pinus spp. (pine)
d P
d t
i ii (D
l
fi ) S
d htt
//
dt t
/
l
d/
/
/ISTASH
th d 20207 009 df (2020) 17. ISTA. International rules for seed testing. Seed Health Methods 2020. 7 -009: Detection of Fusarium circinatum in Pinus spp. (pine)
and Pseudotsuga menziesii (Douglas fir) Seed. https://www.seedtest.org/upload/cms/user/ISTASHmethods20207-009.pdf (2020).i 7. ISTA. International rules for seed testing. Seed Health Methods 2020. 7 -009: Detection of Fusarium circinatum in Pinus spp. (pine)
and Pseudotsuga menziesii (Douglas fir) Seed. https://www.seedtest.org/upload/cms/user/ISTASHmethods20207-009.pdf (2020)
8 Ioos, R et al Test performance study of diagnostic procedures for identification and detection of Gibberella circinata in pine seeds 18. Ioos, R. et al. Test performance study of diagnostic procedures for identification and detection of Gibberella circinata in pine seeds
in the framework of a EUPHRESCO project. EPPO Bull. 43, 267–275. https://doi.org/10.1111/epp.12037 (2013).f 8. Ioos, R. et al. Test performance study of diagnostic procedures for identification and detection of Gibberella circinata in pine seeds
in the framework of a EUPHRESCO project. EPPO Bull. 43, 267–275. https://doi.org/10.1111/epp.12037 (2013).f in the framework of a EUPHRESCO project. EPPO Bull. 43, 267–275. https://doi.org/10.1111/epp.12037 (2013).f p
j
p
g
pp
9. Nascimento, F. J. A., Lallias, D., Bik, H. M. & Creer, S. Sample size effects on the assessment of eukaryotic diversity and community
structure in aquatic sediments using high-throughput sequencing. Sci. Rep. 8, 11737. https://doi.org/10.1038/s41598-018-30179-1
(2018).f (
)
0. Shirazi, S., Meyer, R. S. & Shapiro, B. Revisiting the effect of PCR replication and sequencing depth on biodiversity metrics in
environmental DNA metabarcoding. Ecol. Evol. 11, 15766–15779 (2021). g
1. Alberdi, A., Aizpurua, O., Gilbert, M. T. P. & Bohmann, K. Scrutinizing key steps for reliable metabarcoding of environmenta
samples. Methods Ecol. Evol. 9, 134–147 (2018). p
2. Murray, D. C., Coghlan, M. L. & Bunce, M. From benchtop to desktop: Important considerations when designing amplicon
sequencing workflows. PLoS ONE 10, e0124671 (2015). q
gl
23. Smith, D. P. & Peay, K. G. Sequence depth, not PCR replication, improves ecological inference from next generation DNA sequenc-
ing. PLoS ONE 9, e90234 (2014). g
4. Kauserud, H., Kumar, S., Brysting, A. K., Nordén, J. & Carlsen, T. Acknowledgements g
We gratefully acknowledge COST Action FP 1406 “Pine pitch canker—strategies for management of Gibberella
circinata in greenhouses and forests” (PINESTRENGTH), for supporting F.O. in this work. M.C. is supported by
The Swedish Research Council FORMAS (grant number 2018-00966). S.W. was part-funded by the UK Forestry
Commission. Permission has been obtained for collection of seeds for the study. www.nature.com/scientificreports/ High consistency between replicate 454 pyrosequencing analyses
of ectomycorrhizal plant root samples. Mycorrhiza 22, 309–315 (2012). y
p
p
y
5. Ficetola, G. F. et al. Replication levels, false presences and the estimation of the presence/absence from eDNA metabarcoding data
Mol. Ecol. Resour. 15, 543–556. https://doi.org/10.1111/1755-0998.12338 (2015).i p
g
26. Ihrmark, K. et al. New primers to amplify the fungal ITS2 region–evaluation by 454-sequencing of artificial and natural communi-
ties. FEMS Microbiol. Ecol. 82, 666–677. https://doi.org/10.1111/j.1574-6941.2012.01437.x (2012).i 26. Ihrmark, K. et al. New primers to amplify the fungal ITS2 regio p
g
j
(
)
27. White, T., Bruns, T., Lee, S. & Taylor, J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In
PCR Protocols: A Guide to Methods and Applications (eds Innis, M. A. et al.) (Academic Press, 1990). 27. White, T., Bruns, T., Lee, S. & Taylor, J. Amplification and direct sequencing of fungal ribosomal RNA genes for phy
PCR Protocols: A Guide to Methods and Applications (eds Innis M A et al ) (Academic Press 1990) 8. Caporaso, J. G. et al. QIIME allows analysis of high-throughput community sequencing data. Nat. Methods 7, 335–336. https://
doi.org/10.1038/nmeth.f.303 (2010).i 29. Blaxter, M. et al. Defining operational taxonomic units using 29. Blaxter, M. et al. Defining operational taxonomic units using DNA barcode data. Philos. Trans. R. Soc. B 360, 1935–1943 (2005). 30 Ed
R C UPARSE Hi hl
OTU
f
i
bi l
li
d N
M h d 10 996 998 h
//d i
/ 29. Blaxter, M. et al. Defining operational taxonomic units using DNA barcode data. Philos. Trans. R. Soc. B 360, 1935–1943 (2005). 30. Edgar, R. C. UPARSE: Highly accurate OTU sequences from microbial amplicon reads. Nat. Methods 10, 996–998. https://doi.org/
h
(
) 9. Blaxter, M. et al. Defining operational taxonomic units using DNA barcode data. Philos. Trans. R. Soc. B 360, 1935–1943 (2005)
0. Edgar, R. C. UPARSE: Highly accurate OTU sequences from microbial amplicon reads. Nat. Methods 10, 996–998. https://doi.org/
10.1038/nmeth.2604 (2013). 1. Edgar, R. C., Haas, B. J., Clemente, J. C., Quince, C. & Knight, R. UCHIME improves sensitivity and speed of chimera detection
Bioinformatics 27, 2194–2200. https://doi.org/10.1093/bioinformatics/btr381 (2011). 32. Anderson, M. J. A new method for non-parametric multivariate analysis of variance. Austral. Ecol. 26, 32–46. https://doi.or
1046/j.1442-9993.2001.01070.x (2001). j
33. Clarke, K. R. Non-parametric multivariate analyses of changes in community structure. Aust. J. Ecol. 18, 117–143 (1993). References et al. Planting sentinel European trees in eastern Asia as a novel method to identify potential insect pest invaders. PLoS
ONE 10, e0120864. https://doi.org/10.1371/journal.pone.0120864 (2015).i p
g
j
p
2. Tedersoo, L., Drenkhan, R., Anslan, S., Morales-Rodriguez, C. & Cleary, M. High-throughput identification and diagnostics of
pathogens and pests: Overview and practical recommendations. Mol. Ecol. Resour. 19, 47–76. https://doi.org/10.1111/1755-0998
12959 (2019). 13. Lindahl, B. D. et al. Fungal community analysis by high-throughput sequencing of amplified markers: A user’s guide. New Ph
199, 288–299. https://doi.org/10.1111/nph.12243 (2013). p
g
p
(
)
14. Ambardar, S., Singh, H. R., Gowda, M. & Vakhlu, J. Comparative metagenomics reveal phylum level temporal and spatial changes
in mycobiome of belowground parts of Crocus sativus. PLoS ONE 11, e0163300. https://doi.org/10.1371/journal.pone.0163300
(2016). (
)
15. Penton, C. R., Gupta, V. V., Yu, J. & Tiedje, J. M. Size matters: Assessing optimum soil sample size for fungal and bacterial com-
munity structure analyses using high throughput sequencing of rRNA gene amplicons. Front. Microbiol. 7, 824. https://doi.org/
10.3389/fmicb.2016.00824 (2016). https://doi.org/10.1038/s41598-022-06997-9 Scientific Reports | (2022) 12:3060 | Author contributions A.M.V., S.W., F.O. and M.C. conceived the ideas and designed the experimental plan; M.C. carried out project
administration and funding acquisition; F.O. performed the laboratory work; A.M.V. performed the bioinfor-
matics, statistical analyses and data interpretation; A.M.V. prepared the original draft and M.C., S.W., F.O., A.L. and H.T.D. contributed the writing of the manuscript with critical input and gave final approval for publication. M.C., F.O. and A.M.V. worked with the revisions. A.M.V. and F.O. as first authors contributed equally to this work. Open Access This article is licensed under a Creative Commons Attribution 4.0 International
License, which permits use, sharing, adaptation, distribution and reproduction in any medium or
format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the
Creative Commons licence, and indicate if changes were made. The images or other third party material in this
article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the
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permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from
the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Additional information Correspondence and requests for materials should be addressed to A.M.V. Correspondence and requests for materials should be addressed to A.M.V. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations. https://doi.org/10.1038/s41598-022-06997-9 Scientific Reports | (2022) 12:3060 | www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International
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Photo-response of Two-Dimensional Ruddlesden-Popper Perovskite Films for Photovoltaics
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Photo-response of Two-Dimensional Ruddlesden-Popper
Perovskite Films for Photovoltaics Perovskite Films for Photovoltaics
Gao Wu1, Molang Cai1,2, and Songyuan Dai1,2,
1State Key Laboratory of Alternate Electrical Power System with Renewable Energy Sources, North China Electric Power University,
Beijing 102206, P. R. China Gao Wu1, Molang Cai1,2, and Songyuan Dai1,2, ,
g
gy
1State Key Laboratory of Alternate Electrical Power System with Renewable Energy Sources, North China Electric Power University,
Beijing 102206, P. R. China 1State Key Laboratory of Alternate Electrical Power System with Renewable Energy Sources, North China Electric P
Beijing 102206, P. R. China y Laboratory of Novel Thin-Film Solar Cells, North China Electric Power University, Beijing, 102206, P. R. China 2Beijing Key Laboratory of Novel Thin-Film Solar Cells, North China Electric Power University, Beijing, 102206, P. R. China Abstract. Two-dimensional (2D) Ruddlesden-Popper (RP) perovskites have emerged as a prospective
candidate to address the instability issues of traditional perovskite solar cells. However, the mechanisms of
charge carrier transport of 2D perovskite films obtained by the solution process still remain elusive. In this
work, we proposed a novel characterization technique based on the Kelvin probe force microscopy (KPFM)
to investigate the micro-scale morphology and surface potential (SP) of the BA2MA3Pb4I13 films. In
additionally, a Xenon laser source was adopted to realize the in-situ scanning of the light response of the
perovskite film. The obvious increase in surface potential values in the same scanning area before and after
white light illumination indicated the emergence of photo-generated charge carriers. Based on the unique
photophysical properties and form formation features of the hot-cast BA2MA3Pb4I13 films, we fabricated the
2D perovskite solar cells (PSCs) with an efficiency of 10.95%. As a result, the in-situ KPFM is capable to
serve as an effective approach to investigating the charge carrier behaviors in the 2D perovskites for
photovoltaic applications. Firstly, due to the dielectric contrast of inorganic slabs
and organic spacers, the exciton binding energy of 2D
RP perovskites is relatively high, resulting in excitonic
features and hinder the separation of electron-hole
pairs[8]. Secondly, the insulating spacer cations and the
fabrication crafts determine the self-assembly of 2D RP
perovskite and always result in the multiple phase
structure, leading to the multiple quantum-wells (MQWs)
that greatly influence the charge carrier transport
behaviors in the 2D perovskite solar cells (2D PSCs)[9-10]. Photo-response of Two-Dimensional Ruddlesden-Popper
Perovskite Films for Photovoltaics Therefore, effective and intuitive characterization
methods on the photo-response behaviors in the 2D RP
perovskite films are urgently needed for further
elucidating the mechanisms of the photo-induced charge
carriers in the 2D PSCs. © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0
(http://creativecommons.org/licenses/by/4.0/). Corresponding author: molangcai@ncepu.edu.cn, sydai@ncepu.edu.cn E3S Web of Conferences 257, 03020 (2021)
AESEE 2021 E3S Web of Conferences 257, 03020 (2021)
AESEE 2021 https://doi.org/10.1051/e3sconf/202125703020 Photo-response of Two-Dimensional Ruddlesden-Popper
Perovskite Films for Photovoltaics
Gao Wu1, Molang Cai1,2, and Songyuan Dai1,2,
1State Key Laboratory of Alternate Electrical Power System with Renewable Energy Sources, North China Electric Power University
Beijing 102206, P. R. China Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0
ses/by/4.0/). 2 Experimental For the photovoltaic performance of 2D PSCs,
current density-voltage (J-V) characteristics of the
devices were measured by a solar simulator (Newport
Class AAA) based on a Xenon lamp (150 W) and
Keithley 2420 source under the radiation of 100
mW/cm2 in the ambient environment. The active area of
each cell was 0.043 cm2. The external quantum
efficiency (EQE) curves of the device were tested using
a spectral response test system (SOFN Instruments Co.,
Ltd.). 1 Introduction To date, organic-inorganic hybrid perovskites (OIHPs)
have emerged as a series of promising semiconductor
materials[1-2]. Therefore, OIHPs have been extensively
investigated for various optoelectronic applications,
among which the rapidly evolved perovskite solar cells
(PSCs) have drawn tremendous attention in photovoltaic
(PV) research field, with the certificated power
conversion efficiency (PCE) exceeding 25%[3]. However,
the commercialization of PSCs has been impeded by the
instability issues when the devices are operating at
realistic conditions[4]. Hence, researchers have proposed
the two-dimensional (2D) perovskites as a promising
candidate for obtaining highly stable PSCs[5]. g
g y
2D perovskites are derived from 3D perovskites that
are generally described with the structural formula of
ABX3, with insulating organic spacers tailoring the 3D
perovskite octahedral framework into several (denoted as
an integer n, hereinafter) blocks of separated slabs. Generally, when the bulky organic spacer (R) is
monovalent, the type of 2D perovskites with the
chemical formula of R2An-1BnX3n+1 is recognized as 2D
Ruddlesden-Popper (RP) perovskites[6]. Owing to the
intercalation of the hydrophobic bulky spacers, 2D
perovskites exhibit distinct properties such as quantum
confinement effects, high tunability and outstanding
stability[7]. However, there are two major issues limiting
the photovoltaic applications of 2D RP perovskites. In this work, we proposed a novel characterization
technique based on the Kelvin probe force microscopy
(KPFM) to realize the in-situ scanning of the
morphology
and
surface
potential
(SP)
profiles
simultaneously. The in-situ KPFM techniques are able to
detect the nanoscale morphology and electronic signals
of the 2D RP perovskite film at high resolution without
causing damages to samples. The emergence of the
photo-generated
charge
carriers
was
successfully
revealed from the remarkable increase in the SP values
before and after the white light illumination. In addition,
the typical film formation features of the BA2MA3Pb4I13
films were investigated. Based on the characterization https://doi.org/10.1051/e3sconf/202125703020 E3S Web of Conferences 257, 03020 (2021) E3S Web of Conferences 257, 03020 (2021)
AESEE 2021 graphite copper Kα radiation (k=1.5418 Å) with the 2θ
range
from
5°
to
55°. The
steady-state
photoluminescence (PL) spectroscopy was conducted
with an integrated spectral scanning test system
(MStarter 100, Metatest Corporation). results, we fabricated 2D PSCs with an efficiency of
10.95%. results, we fabricated 2D PSCs with an efficiency of
10.95%. 2.4 The in-situ KPFM Measurements The in-situ KPFM platform was established on basis of
the scanning probe microscopy system (Agilent
Technologies 5500) coupled with a Xenon laser light
source (LSH-X150, Zolix Instruments Co., Ltd). The
topography and surface potential profiles of the 2D RP
perovskite film were characterized simultaneously at the
amplitude-modulation KPFM (AM-KPFM) mode and
Pt/Ir-coated conductive tips (Nanosensors PPPEFM-20,
~5 N/m) were used. The in-situ KPFM measurements
were carried out on the same area without changing the
sample under different illumination conditions, as
depicted in Fig. 1. The profiling of topography and SP of
the BA2MA3Pb4I13 film was conducted within a range of
3×3 μm2, which presented the “dark-state” surface
charge distribution of the BA2MA3Pb4I13 film. Then, the
Xenon laser light (8.48 A, light intensity: ~200 cd/mm2,
outlet size: 0.5×1.6 mm2) was continuously applied to
the sample for 30 s and afterwards the scanning started
again in the same area, which revealed the “light-
induced-state” surface charge distribution of the film[11]. 2.2 Device Fabrication The 2D perovskite solar cells were fabricated based on
an inverted planar configuration (glass/ITO/PTAA/
BA2MA3Pb4I13/C60/BCP/Ag). The ITO substrates were
sequentially rinsed as described above. For the
deposition of PTAA hole transport layer (HTL), 75 μL
of the PTAA/ chlorobenzene (2 mg/mL) solution were
dripped to the substrates, followed by a spin coating
process at 6000 rpm for 30 s. A thermal annealing of
100 °C for 10 min was processed to assist the
crystallization
of
PTAA. The
fabrication
of
BA2MA3Pb4I13 active layer was hot-cast as mentioned
above. The electron transport layer (ETL) of C60 and the
BCP layer with the thicknesses of 30 nm and 9 nm
respectively were successively deposited on top of the
perovskite layer by thermal evaporation. Finally, 80 nm
of Ag cathode was evaporated through masks and the
fabrication of 2D PSCs was completed. Figure 1. Schematic illustration of the in-situ KPFM
measurements on the 2D RP perovskite film in the dark (Xenon
light off) and under white light illumination (Xenon light on). Figure 1. Schematic illustration of the in-situ KPFM Figure 1. Schematic illustration of the in-situ KPFM
meas rements on the 2D RP pero skite film in the dark (Xen Figure 1. Schematic illustration of the in-situ KPFM
measurements on the 2D RP perovskite film in the dark (Xenon
light off) and under white light illumination (Xenon light on). measurements on the 2D RP perovskite film in the dark (Xenon
light off) and under white light illumination (Xenon light on). measurements on the 2D RP perovskite film in the dark (Xenon
light off) and under white light illumination (Xenon light on). 3 Results and Discussion A field-emission scanning electron microscopy (SEM,
SU8010, Hitachi) was used to obtain the top-view
morphology images of the BA2MA3Pb4I13 film. The UV–
vis absorption curve of the film was measured with a
spectrophotometer (UV-3600 Plus, Shimadzu). X-ray
diffraction (XRD) pattern of the film was recorded by X-
ray diffractometer (Rigaku D max-2500) equipped with 2.1 Film Preparation A hot-casting method was adopted to fabricate the 2D
RP perovskite film and the photovoltaic device. The
precursor solution of BA2MA3Pb4I13 was prepared by
dissolving BAI, MAI and PbI2 in the mixed solvents of
DMF: DMSO (volume radio is 97:3) with the molar
concentrations of 0.4, 0.6 and 0.8 mol/L and the solution
was stirred overnight in a nitrogen-purged glove box. g
g
p
g
g
The patterned ITO-coated substrates (15×15 mm2)
were rinsed by sequential sonication for 15 min in each
of the lotions including detergent, acetone, isopropanol,
ethanol and deionized water. Afterwards, the clean
substrates were dried under nitrogen flow and exposed to
ozone plasma for 15 min. For the hot-casting of the 2D
RP perovskite film, the substrates were preheated on a
hotplate at 60 °C for 10 min. Then, the preheated
substrate was quickly transferred to the spin coater and
80 μL of the perovskite precursor solution was dropped
to the substrates, followed by the spinning procedure at
4800 rpm for 20 s. After the spinning process, a thermal
annealing of 100 °C for 10 min was processed to remove
the solvent from the film. Notably, the spin coating was
operated in the fume hood at the environmental
temperature of ~20 °C and relative humidity of ~25%. 3.1 Basic Film and Device Characterizations A summary of photovoltaic parameters of 2D PSCs
based on the hot -cast BA2MA3Pb4I13. Table 1. A summary of photovoltaic parameters of 2D PSCs
based on the hot -cast BA2MA3Pb4I13. Cell
Structure
PCE
(%)
VOC
(V)
JSC
(mA/cm2)
FF
Jint
(mA/cm2)
p-i-n
inverted
structure
10.95
1.02
15.35
0.70
14.93 3.1 Basic Film and Device Characterizations Fig. 2a showed the top-view SEM images of the
BA2MA3Pb4I13 film. The hot-cast film exhibited a
uniform and compact surface morphology. Distinctive 2 2 E3S Web of Conferences 257, 03020 (2021)
AESEE 2021 https://doi.org/10.1051/e3sconf/202125703020 from the morphology of 3D perovskites consisting of
grains with regular shapes[12], it was difficult to observe
grains and boundaries in the 2D RP perovskite film. In
addition, there existed cracks in the surface of the film,
which indicated the specific film formation features. from the morphology of 3D perovskites consisting of
grains with regular shapes[12], it was difficult to observe
grains and boundaries in the 2D RP perovskite film. In
addition, there existed cracks in the surface of the film,
which indicated the specific film formation features. The 2D PSCs based on the BA2MA3Pb4I13 active
layer was fabricated with the inverted planar structure of
glass/ITO/PTAA/perovskite/C60/BCP/Ag, as shown in
the inset of Fig. 2e. A reasonable PCE of 10.95% was
recorded from current density-voltage (J-V) scanning, as
shown in Fig. 2e, with an open-circuit voltage (VOC) of
1.02 V, short-circuit current density (JSC) of 15.35
mA/cm2 and fill factor (FF) of 0.70 (Table 1). The
external quantum efficiency (EQE) and integrated
current density (Jint) of 14.93 mA/cm2 were shown in Fig. 2f, well agreed with the measured JSC from J-V scanning. Compared with other reports[16,17], the VOC of 2D PSCs is
generally high which exceeds 1.0 V, while the JSC will
be the key to further improving the PCE. The UV-Vis absorption curve was depicted in Fig. 2b. Exciton-like absorption peaks at ~642 nm and ~607 nm
were observed, indicating the exsistence of the low n-
value phases (n=4 and 3, respectively) in the hot-cast
BA2MA3Pb4I13 film. Hence, the UV-Vis absorption
curve clarified three dominant species in the 2D RP
perovskite film from low n-value to high n-value[13-15]. The X-ray diffraction (XRD) pattern of the
BA2MA3Pb4I13 film was shown in Fig. 2c. Three
diffraction peaks at 14.08°, 28.31° and 44.74° were well
resolved, which matched the diffraction peak positions
of (111), (202) and (313) crystallographic planes from
the 2D RP perovskites[12]. However, according to the
XRD results on the 3D perovskite MAPbI3, the
characteristic diffraction peaks were located at 14.05°,
28.45° and 43.13° [13], which was close to the results in
Fig. 2c. We assumed that this might result from the
dominant 3D-like phases (n→∞) on top of the perovskite
film. y
g
Table 1. 3.2 in-situ KPFM Measurements on the Photo-
Response of the 2D RP perovskite film The steady-state PL spectroscopy on the 2D RP
perovskite film deposited on glass also confirmed the
unique phase distribution phenomenon. As Fig. 2d
showed, the PL spectrum was dominated by the
emissions at 3D-like phases (n→∞, around 740 nm),
while no PL emmision peaks in the short wavelength
region. This indicated that the 3D-like species tended to
accumulate on the top of the perovskite film. Through
the hot-casting method, the vertical phase distribution
with the low n-value (2D) and high n-value (3D-like)
phases accumulated at the bottom and the top of
perovskite film could facilitate the charge transfer across
the device[15]. Fig. 3 showed the topography images and surface
potential (SP) profiles of the BA2MA3Pb4I13 film in the
dark state and under illumination condition. As Fig. 3a
and d showed, the calculated root-mean-square (RMS)
values of roughness of the BA2MA3Pb4I13 film before
and after Xenon laser illumination were 7.55 nm and
7.26 nm, which was in accord with the SEM image (Fig. 2a) that exhibited a less undulating surface of the 2D RP
film. More importantly, the calculated RMS values were
quite similar and the topography images were essentially
identical, which proved that the scanning was conducted
in the same position of the sample with high credibility. Figure 2. (a) Top-view SEM image, (b) XRD pattern, (c) UV-Vis absorption curve and (d) steady-state PL spectra of the
BA2MA3Pb4I13 film. The insets in Fig. 2d showed the incident 488 nm laser excited the top surface of the film. (e) J-V curve and (f)
EQE spectra of 2D PSCs based on the hot-cast BA2MA3Pb4I13. The inset in Fig. 2e showed the inverted structure of the 2D PSCs. Figure 2. (a) Top-view SEM image, (b) XRD pattern, (c) UV-Vis absorption curve and (d) steady-state PL spectra of the
BA2MA3Pb4I13 film. The insets in Fig. 2d showed the incident 488 nm laser excited the top surface of the film. (e) J-V curve and (f)
EQE spectra of 2D PSCs based on the hot-cast BA2MA3Pb4I13. The inset in Fig. 2e showed the inverted structure of the 2D PSCs. 3 3 E3S Web of Conferences 257, 03020 (2021)
AESEE 2021 E3S Web of Conferences 257, 03020 (2021)
AESEE 2021 https://doi.org/10.1051/e3sconf/202125703020 Notably, the histograms of SP distribution in the
dark and light-induced states were depicted in Fig. 3c
and f. 3.2 in-situ KPFM Measurements on the Photo-
Response of the 2D RP perovskite film Peng, et al., Advanced Materials,
30, 1703487 (2018). 5. Y. Chen, Y. Sun, J. Peng, et al., Advanced Materials,
30, 1703487 (2018). 4 Conclusion 6. D. Luo, R. Su, W. Zhang, et al., Nat. Rev. Mater. 5,
44–60 (2020). 6. D. Luo, R. Su, W. Zhang, et al., Nat. Rev. Mater. 5,
44–60 (2020). In this work, we proposed a novel characterization
technique based on the Kelvin probe force microscopy
(KPFM) coupled with a Xenon laser source to realize the
in-situ scanning. The emergence of the photo-generated
charge carriers was successfully revealed from the
remarkable increase in the SP values in the same
scanning area before and after the white light
illumination. In addition, the typical film morphology
and multiple phase distribution of the BA2MA3Pb4I13
films were investigated. The fabricated 2D PSCs with an
efficiency of 10.95%. As a result, the establishment of
in-situ KPFM platform paves the way to the research on
the mechanisms of carrier behaviors in 2D perovskites. 7. B. Wang, J. Iocozzia, M. Zhang, et al., Chem. Soc. Rev. 48, 4854–4891 (2019). 8. L. He, Z. Xiao, X. Yang, et al., J. Mater. Sci. 55, 7691–7701 (2020). 9. S. Deng, E. Shi, L. Yuan, et al., Nat. Commun. 11,
664 (2020). 10. G. Wu, X. Li, J. Zhou, et al., Adv. Mater. 31,
1903889 (2019). 11. C. Lu, Z. Hu, Y. Wang, et al., Adv. Mater. Interfaces 6, 1801253 (2019). 12. H. Peng, M. Cai, J. Zhou, et al., Sol. RRL 4,
2000216 (2020). 3.2 in-situ KPFM Measurements on the Photo-
Response of the 2D RP perovskite film From the two histograms, it was confirmed that
the overall SP values were improved after the Xenon
laser excitation. The SP interval in the dark state was
~0.05-0.27 V, while that in the light-induced state was
~0.21-0.48 V. Also, the calculated mean surface
potential (SPmean) of the BA2MA3Pb4I13 film increased
dramatically from 0.18 V to 0.34 V after the Xenon laser
illumination, in accord with the analyses above. By unifying the scale bars of the two SP profiles
(Fig. 3b and e), we found that the whole SP values of the
BA2MA3Pb4I13 film in the scanning regions improved
dramatically after the illumination. This was ascribed to
the generation of charge carriers when the perovskite
film absorbed light. The photo-induced charge carriers
would significantly influence the built-in electric field in
the perovskite film[11]. Since the 2D RP perovskite film
was deposited on glass, enormous electron-hole pairs
generated might well be confined in the perovskite and
then recombine due to the high exciton binding energy. Figure 3. (a, d) Topography images and (b, e) surface potential profiles of the BA2MA3Pb4I13 film in the dark state and light-induced
state within the range of 3×3 μm2. (c, f) Histograms from the SP values of the BA2MA3Pb4I13 film in the dark and light-induced states. 5
Y Ch
Y S
J P
t l Ad
d M t i l Figure 3. (a, d) Topography images and (b, e) surface potential profiles of the BA2MA3Pb4I13 film in the dark state and light-induced
state within the range of 3×3 μm2. (c, f) Histograms from the SP values of the BA2MA3Pb4I13 film in the dark and light-induced states. 5
Y Chen Y Sun J Peng et al Advanced Materials Figure 3. (a, d) Topography images and (b, e) surface potential profiles of the BA2MA3Pb4I13 film in the dark state and light-induced
state within the range of 3×3 μm2. (c, f) Histograms from the SP values of the BA2MA3Pb4I13 film in the dark and light-induced states. Figure 3. (a, d) Topography images and (b, e) surface potential profiles of the BA2MA3Pb4I13 film in the dark state and light-induced
state within the range of 3×3 μm2. (c, f) Histograms from the SP values of the BA2MA3Pb4I13 film in the dark and light-induced states. 4 Conclusion
5. Y. Chen, Y. Sun, J. Peng, et al., Advanced Materials,
30, 1703487 (2018). 5. Y. Chen, Y. Sun, J. 4. S. P. Dunfield, L. Bliss, F. Zhang, et al., Advanced
Energy Materials, 10, 1904054 (2020). References 13. Y. Lin, Y. Fang, J. Zhao, et al., Nat. Commun. 10,
1008 (2019). 1. F. Liu, J. Zhu, J. Wei, et al., Appl. Phys. Lett. 104,
253508 (2014). 1. F. Liu, J. Zhu, J. Wei, et al., Appl. Phys. Lett. 104,
253508 (2014). 14. G. Wu, T. Yang, X. Li, et al., Matter 4, 582-599
(2021). 2. G. Grancini, M. K. Nazeeruddin, Nat. Rev. Mater. 4,
4–22 (2019). 2. G. Grancini, M. K. Nazeeruddin, Nat. Rev. Mater. 4,
4–22 (2019). 15. T. He, S. Li, Y. Jiang, et al., Nat Commun 11, 1672
(2020). 3. NREL, Best Research-Cell Efficiencies, (accessed:
April 2021). 3. NREL, Best Research-Cell Efficiencies, (accessed:
April 2021). 16. M. S. de la Fuente, S. Kaur, Q. Hu, et al., ACS
Energy Lett. 5, 109–116 (2020). 4. S. P. Dunfield, L. Bliss, F. Zhang, et al., Advanced
Energy Materials, 10, 1904054 (2020). 17. H. Tsai, W. Nie, J. C. Blancon, et al., Nature 536,
312–316 (2016). 4 4
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English
| null |
Supplementary Figures from Aspirin Use Reduces the Risk of Aggressive Prostate Cancer and Disease Recurrence in African-American Men
| null | 2,023
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cc-by
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A
C -re a tiv e p ro te in (m g /L )
A fric a n -A m e ric a n
(n = 1 0 0 )
a v g . a g e : 6 2 .7
E u r o p e a n -A m e ric a n
(n = 1 0 0 )
a v g . a g e : 6 3 .5
0
5
1 0
1 5
2 0
P = 0 .0 3
µ = 3 .5 m g /L
µ = 2 .4 m g /L
B
C -re a tiv e p ro te in (m g /L )
A fric a n -A m e ric a n
(n = 1 0 0 )
a v g . a g e : 6 5 .8
E u r o p e a n -A m e ric a n
(n = 1 0 0 )
a v g . a g e : 6 6 .8
0
5
1 0
1 5
2 0
P = 0 .0 6 7
µ = 2 .9 m g /L
µ = 2 .1 m g /L
Supplementary Figure 1. Increased C-reac
American (AA) men. Differences in CRP pla
(EA) men. CRP was measured in plasma sa
(100 AA, 100 EA). (A) CRP levels are signif
patients. (B) CRP plasma levels are higher i
i i
ll
i
ifi
T
id d M A
C -re a tiv e p ro te in (m g /L )
A fric a n -A m e ric a n
(n = 1 0 0 )
a v g . a g e : 6 2 .7
E u r o p e a n -A m e ric a n
(n = 1 0 0 )
a v g . a g e : 6 3 .5
0
5
1 0
1 5
2 0
P = 0 .0 3
µ = 3 .5 m g /L
µ = 2 .4 m g /L
B
C -re a tiv e p ro te in (m g /L )
A fric a n -A m e ric a n
(n = 1 0 0 )
a v g . a g e : 6 5 .8
E u r o p e a n -A m e ric a n
(n = 1 0 0 )
a v g . a g e : 6 6 .8
0
5
1 0
1 5
2 0
P = 0 .0 6 7
µ = 2 .9 m g /L
µ = 2 .1 m g /L
Supplementary Figure 1. Increased C-reac
American (AA) men. Differences in CRP pla
(EA) men. CRP was measured in plasma sa
(100 AA, 100 EA). (A) CRP levels are signif
patients. (B) CRP plasma levels are higher i
i i
ll
i
ifi
T
id d M A C -re a tiv e p ro te in (m g /L )
A fric a n -A m e ric a n
(n = 1 0 0 )
a v g . a g e : 6 2 .7
E u r o p e a n -A m e ric a n
(n = 1 0 0 )
a v g . a g e : 6 3 .5
0
5
1 0
1 5
2 0
P = 0 .0 3
µ = 3 .5 m g /L
µ = 2 .4 m g /L C -re a t
A fric a n -A m e ric a n
(n = 1 0 0 )
a v g . a g e : 6 2 .7
E u r o p e a n -A m e ric a n
(n = 1 0 0 )
a v g . A
C -re a tiv e p ro te in (m g /L )
A fric a n -A m e ric a n
(n = 1 0 0 )
a v g . a g e : 6 2 .7
E u r o p e a n -A m e ric a n
(n = 1 0 0 )
a v g . a g e : 6 3 .5
0
5
1 0
1 5
2 0
P = 0 .0 3
µ = 3 .5 m g /L
µ = 2 .4 m g /L
B
C -re a tiv e p ro te in (m g /L )
A fric a n -A m e ric a n
(n = 1 0 0 )
a v g . a g e : 6 5 .8
E u r o p e a n -A m e ric a n
(n = 1 0 0 )
a v g . a g e : 6 6 .8
0
5
1 0
1 5
2 0
P = 0 .0 6 7
µ = 2 .9 m g /L
µ = 2 .1 m g /L
Supplementary Figure 1. Increased C-reac
American (AA) men. Differences in CRP pla
(EA) men. CRP was measured in plasma sa
(100 AA, 100 EA). (A) CRP levels are signif
patients. (B) CRP plasma levels are higher i
i i
ll
i
ifi
T
id d M a g e : 6 3 .5
0
B
C -re a tiv e p ro te in (m g /L )
A fric a n -A m e ric a n
(n = 1 0 0 )
a v g . a g e : 6 5 .8
E u r o p e a n -A m e ric a n
(n = 1 0 0 )
a v g . a g e : 6 6 .8
0
5
1 0
1 5
2 0
P = 0 .0 6 7
µ = 2 .9 m g /L
µ = 2 .1 m g /L
Supplementary Figure 1. Increased C-reactive protein (CRP) plasma level in African-
American (AA) men. Differences in CRP plasma level between AA and European-American
(EA) men. CRP was measured in plasma samples of cases (100 AA, 100 EA) and controls
(100 AA, 100 EA). (A) CRP levels are significantly higher in AA patients compared with EA
patients. (B) CRP plasma levels are higher in AA controls compared with EA controls, albeit
not statistically significant. Two-sided Mann-Whitney U test. µ = mean. B
C -re a tiv e p ro te in (m g /L )
A fric a n -A m e ric a n
(n = 1 0 0 )
a v g . a g e : 6 5 .8
E u r o p e a n -A m e ric a n
(n = 1 0 0 )
a v g . a g e : 6 6 .8
0
5
1 0
1 5
2 0
P = 0 .0 6 7
µ = 2 .9 m g /L
µ = 2 .1 m g /L B Supplementary Figure 1. Increased C-reactive protein (CRP) plasma level in African-
American (AA) men. Differences in CRP plasma level between AA and European-American
(EA) men. CRP was measured in plasma samples of cases (100 AA, 100 EA) and controls
(100 AA, 100 EA). (A) CRP levels are significantly higher in AA patients compared with EA
patients. (B) CRP plasma levels are higher in AA controls compared with EA controls, albeit
not statistically significant. Two-sided Mann-Whitney U test. µ = mean.
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https://openalex.org/W3035371187
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http://uu.diva-portal.org/smash/get/diva2:1508639/FULLTEXT01
|
English
| null |
Variability in epilimnion depth estimations in lakes
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Hydrology and earth system sciences
| 2,020
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cc-by
| 15,138
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Correspondence: Harriet L. Wilson (wilsonh@dkit.ie) Correspondence: Harriet L. Wilson (wilsonh@dkit.ie) Received: 13 May 2020 – Discussion started: 10 June 2020
Revised: 22 September 2020 – Accepted: 5 October 2020 – Published: 24 November 2020 Abstract. The epilimnion is the surface layer of a lake typi-
cally characterised as well mixed and is decoupled from the
metalimnion due to a steep change in density. The concept of
the epilimnion (and, more widely, the three-layered structure
of a stratified lake) is fundamental in limnology, and calcu-
lating the depth of the epilimnion is essential to understand-
ing many physical and ecological lake processes. Despite the
ubiquity of the term, however, there is no objective or generic
approach for defining the epilimnion, and a diverse number
of approaches prevail in the literature. Given the increas-
ing availability of water temperature and density profile data
from lakes with a high spatio-temporal resolution, automated
calculations, using such data, are particularly common, and
they have vast potential for use with evolving long-term glob-
ally measured and modelled datasets. However, multi-site
and multi-year studies, including those related to future cli-
mate impacts, require robust and automated algorithms for
epilimnion depth estimation. In this study, we undertook a
comprehensive comparison of commonly used epilimnion
depth estimation methods, using a combined 17-year dataset,
with over 4700 daily temperature profiles from two Euro-
pean lakes. Overall, we found a very large degree of variabil-
ity in the estimated epilimnion depth across all methods and
thresholds investigated and for both lakes. These differences,
manifesting over high-frequency data, led to fundamentally
different understandings of the epilimnion depth. In addition,
estimations of the epilimnion depth were highly sensitive to
small changes in the threshold value, complex thermal wa- ter column structures, and vertical data resolution. These re-
sults call into question the custom of arbitrary method se-
lection and the potential problems this may cause for studies
interested in estimating the ecological processes occurring
within the epilimnion, multi-lake comparisons, or long-term
time series analysis. We also identified important systematic
differences between methods, which demonstrated how and
why methods diverged. These results may provide rationale
for future studies to select an appropriate epilimnion defi-
nition in light of their particular purpose and with aware-
ness of the limitations of individual methods. Correspondence: Harriet L. Wilson (wilsonh@dkit.ie) While there
is no prescribed rationale for selecting a particular method,
the method which defined the epilimnion depth as the shal-
lowest depth, where the density was 0.1 kg m−3 more than
the surface density, may be particularly useful as a generic
method. 1
Introduction The “epilimnion depth”, “mixed layer”, or “top of the metal-
imnion” are common terms in limnology, typically referring
to the deepest point of the surface layer of a stratified lake,
which is characterised as quasi-uniform in terms of physical
and biogeochemical properties and overlying a layer of steep
vertical gradients. Incoming heat to a lake, received at the
lake surface, expands water above 3.98 ◦C, resulting in den-
sity stratification. Convective cooling at the surface and me- Harriet L. Wilson1, Ana I. Ayala2, Ian D. Jones3, Alec Rolston4, Don Pierson2, Elvira de Eyto5, Hans-Peter Grossart6,
Marie-Elodie Perga7, R. Iestyn Woolway1, and Eleanor Jennings1 Harriet L. Wilson1, Ana I. Ayala2, Ian D. Jones3, Alec Rolston4, Don Pierson2, Elvira de Eyto5, Hans-Peter Grossart6,
Marie-Elodie Perga7, R. Iestyn Woolway1, and Eleanor Jennings1 1Centre for Freshwater and Environmental Studies, Dundalk Institute of Technology, Dundalk, Co. Louth, Ireland
2Department of Ecology and Genetics, Limnology, Uppsala University, Uppsala, Sweden
3Biological and Environmental Sciences, Faculty of Natural Sciences, University of Stirling, Stirling, UK
4An Fóram Uisce – The Water Forum, Nenagh, Co. Tipperary, Ireland
5Marine Institute Catchment Research Facility, Furnace, Newport, Co. Mayo, Ireland
6Institute for Biochemistry and Biology, Potsdam University, Potsdam, Germany
7University of Lausanne, Faculty of Geoscience and Environment, 1015 Lausanne, Switzerland H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes 5560 chanical energy injected by the wind drive vertical mixing
(Wüest and Lorke, 2003). These competing surface fluxes
result in a warm well-mixed layer of water that interacts dy-
namically with the atmosphere (Monismith and MacIntyre,
2009). The vertical propagation of energy manifested at the
lake surface is constrained by the steep density gradients in
the metalimnion, which act to decouple the epilimnion from
the deep hypolimnion. As such, it has become foundational
in limnology to consider a stratified lake as consisting of
three well-defined layers: a turbulent epilimnion (diffusiv-
ity typically 10−5 to 10−2 m2 s−1), the stable metalimnion
(5 × 10−8 to 10−6 m2 s−1), and the quiescent hypolimnion
(3 × 10−6 to 10−4 m2 s−1) (Wüest and Lorke, 2009). The
discretisation of these layers, however, is understood to be
essentially theoretical, since micro-profile studies show that
the conditions within layers are not uniform and exact cut-
offs between layers do not necessarily exist (Imberger, 1985;
Jonas et al., 2003; Tedford et al., 2014; Kraemer, 2020). The
definition of the epilimnion depth is thus inherently subjec-
tive but has profound importance in limnology. also introduce new challenges for estimating metrics such as
the epilimnion depth. Such large quantities of data can limit
a user’s capacity to examine individual profiles and therefore
require robust automated algorithms with low computational
expense (Read et al., 2011; Pujoni et al., 2019). p
(
,
;
j
,
)
Despite the ubiquity of the epilimnion depth, there is no
consistent determining method used in limnology. The epil-
imnion depth can be defined in terms of many variables
(e.g. water temperature, water density, turbulence estima-
tions, surface fluxes, biogeochemical properties), represents
different temporal scales of variability (e.g. inter-annual to
sub-daily), and can be calculated using a range of numerical
approaches (e.g. sigmoidal functions, threshold algorithms)
(Brainard and Gregg, 1996; Thomson and Fine, 2003; Kara
et al., 2003; De Boyer et al., 2004; Lorbacher et al., 2006;
Gray et al., 2020). A particularly common approach in lim-
nology, due to the availability of the required data, is to define
the epilimnion using water temperature profile data. How-
ever, inconsistencies exist between studies which use water
temperature (e.g. Zorzal-Almeida et al., 2017; Strock et al.,
2017) or water density (e.g. Read et al., 2011; Obrador et al.,
2014). H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes The depth of the epilimnion is
also used for estimating the transfer of oxygen, received at
the lake surface, to deeper layers, sustaining aerobic life and
preventing anoxia (Foley et al., 2012; Schwefel et al., 2016). The increasing availability of high-frequency measured
and simulated data, coupled with collaborative networks of
lake scientists, offers a great potential for broadening our un-
derstanding of the epilimnion depth. Water temperature pro-
file data collected at frequent intervals on automatic monitor-
ing buoys in lakes are becoming increasingly available (Jen-
nings et al., 2012; de Eyto et al., 2016; Marcé et al., 2016). In
addition, the collation of these datasets globally through col-
laborative initiatives such as GLEON (http://gleon.org/, last
access: 19 November 2020) and NETLAKE (https://www. dkit.ie/netlake, last access: 19 November 2020) (Weathers
et al., 2013; Jennings et al., 2017) and modelling initiatives
such as ISIMIP2b (Ayala et al., 2020) broaden the poten-
tial for long-term multi-lake studies. However, these datasets Quantifying the vertical extent of the epilimnion is cru-
cial for understanding many of the physical, chemical, and
biological processes in lakes. Although the epilimnion is dif-
ferentiated from the typically shallower layer that is actively
mixing (Gray et al., 2020), the depth of the epilimnion in-
dicates the volume and properties of the water that is influ-
enced by air–water interactions. It is therefore essential for
interpreting the physical response of lakes to long-term atmo-
spheric changes (Lorbacher et al., 2006; Persson and Jones,
2008; Flaim et al., 2016) and extreme climatic events (Jen-
nings et al., 2012; Calderó-Pascual et al., 2020), and it is even
required for predicting the local climate for very large lakes
(Thiery et al., 2015). The epilimnion depth is also critical
for the estimation of algal light availability, nutrient fluxes,
and epilimnetic water temperatures, which determine pho-
tosynthesis rates and establish the basis of the food web in a
lake (MacIntyre, 1993; Diehl et al., 2002; Berger et al., 2006;
Bouffard and Wüest, 2018). The depth of the epilimnion is
also used for estimating the transfer of oxygen, received at
the lake surface, to deeper layers, sustaining aerobic life and
preventing anoxia (Foley et al., 2012; Schwefel et al., 2016). The increasing availability of high-frequency measured
and simulated data, coupled with collaborative networks of
lake scientists, offers a great potential for broadening our un-
derstanding of the epilimnion depth. H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes Often the epilimnion depth is defined as the location
where the change in water temperature or density exceeds a
defined threshold. However, studies vary in the value selected
which may be defined in absolute units (e.g. Andersen et al.,
2017) or gradients between consecutive sensors (e.g. Lam-
ont et al., 2004). A particularly prevalent method in recent
studies is the “meta.top” function proposed in the R package
“rLakeAnalyzer” (Read et al., 2011). In contrast, epilimnion
depth definitions based on actual turbulence measurements
are uncommon. Compared with long-term water temperature
datasets, there are relatively few turbulent eddy diffusivity
measurements in lakes, typically using micro-profiling meth-
ods conducted over a small time period (e.g. Imberger, 1985;
Tedford et al., 2014). Other methods of estimating vertical
eddy diffusivity, from water temperature data, e.g. the Jassby
and Powell (1975) heat-flux method, are restricted to use be-
low the epilimnion and photic zone. Vertical turbulence pro-
files, however, as well as water temperature profiles, are esti-
mated by some hydrodynamic lake models (Goudsmit et al.,
2002, Dong et al., 2019). Such modelled data, therefore, of-
fer a tool for assessing commonly used water temperature-
or density-based methods in comparison to turbulence-based
methods. Quantifying the vertical extent of the epilimnion is cru-
cial for understanding many of the physical, chemical, and
biological processes in lakes. Although the epilimnion is dif-
ferentiated from the typically shallower layer that is actively
mixing (Gray et al., 2020), the depth of the epilimnion in-
dicates the volume and properties of the water that is influ-
enced by air–water interactions. It is therefore essential for
interpreting the physical response of lakes to long-term atmo-
spheric changes (Lorbacher et al., 2006; Persson and Jones,
2008; Flaim et al., 2016) and extreme climatic events (Jen-
nings et al., 2012; Calderó-Pascual et al., 2020), and it is even
required for predicting the local climate for very large lakes
(Thiery et al., 2015). The epilimnion depth is also critical
for the estimation of algal light availability, nutrient fluxes,
and epilimnetic water temperatures, which determine pho-
tosynthesis rates and establish the basis of the food web in a
lake (MacIntyre, 1993; Diehl et al., 2002; Berger et al., 2006;
Bouffard and Wüest, 2018). 2.2
Measured data In this study, we used a total of 4783 daily water tempera-
ture profiles from Lough Feeagh (n = 2778) and Erken lake
(n = 2005). Profiles were collected at frequent intervals on
moored automatic monitoring buoys, and from these the
mean daily profiles were calculated. On Lough Feeagh, ver-
tical water temperature measurements were collected every
2 min for the period 2004–2017 at depths of 0.9, 2.5, 5, 8,
11, 14, 16, 18, 20, 22, 27, 32, and 42 m using submerged
platinum resistance thermometers (PRTs) (PT100 1/10DIN,
Lab Facility, Bognor Regis, United Kingdom) (de Eyto et al.,
2016, 2020). On Erken, temperature profile data were col-
lected at 1 min intervals at depths of 0.5 to 15 m in 0.5 m in-
tervals, using Type T thermocouple sensors using a Campbell
Scientific AM416 multiplexer and CR10 data logger (Pier-
son et al., 2011). The topmost sensor data were excluded to
match the topmost sensor in Lough Feeagh. In Erken lake,
the monitoring buoy was manually deployed each year prior
to or just after the onset of stratification to avoid damage from
the seasonal ice cover; therefore, the number of observa-
tions varied annually. To ensure data were consistent for both
lakes, data were divided into a subset from 1 April to 31 Oc-
tober. To address the issue of large data gaps, years when less
than 70 % of the data between April and October were avail-
able (> 150 d) were excluded from the analysis. The remain-
ing years were 2004, 2005, 2006, 2011, 2012, 2013, 2014,
2015, 2016, and 2017 for Lough Feeagh and 2002, 2005,
2008, 2009, 2014, 2015, and 2017 for Erken lake. Water den-
sity (kg m−3) was calculated from water temperature (◦C) us-
ing rLakeAnalyzer (Read et al., 2011), with the Martin and
McCutcheon (1999) equation, assuming negligible effects of
soluble material. In this study, we undertook an in-depth comparison of
methods commonly used for the estimation of epilimnion
depth using high-frequency multi-year data for water tem-
perature profiles, collected with automated monitoring buoys
from two European lakes: Lough Feeagh (Ireland) and Erken
lake (Sweden). In addition to estimates based on these mea-
sured data, we used simulated data output from a lake model
to compare water-temperature- and turbulence-based meth-
ods and to assess the influence of vertical sensor resolu-
tion. H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes sity gradient (0.056 kg m−3 m−1) compared to Lough Feeagh
(0.016 kg m−3 m−1). on the level of day-to-day variability in epilimnion depth and
demonstrate how methods perform over a continuum of wa-
ter column conditions. In addition, through the vast number
of measured profiles, high-frequency data offer a more robust
comparison of methods than that previously demonstrated
with manually collected datasets; even when aggregated to
the daily time step, high-frequency data are more represen-
tative of the sub-daily variability (Marcé et al., 2016). Given
the potential of multi-lake comparison and longitudinal stud-
ies, methods are required to perform consistently across tem-
poral and spatial ranges rather than being tailored specifically
to one lake or period of time. Therefore, the sensitivity of dif-
ferent methods to temporal and spatial characteristics, such
as water column structure and vertical resolution of data mea-
surements, is essential for assessing which methods are most
suitable for future analysis (Fee et al., 1996; Thomson and
Fine, 2003; Lorbacher et al., 2006; Pujoni et al., 2019). 2.2
Measured data The objectives of this study were to (1) compare water-
temperature- and water-density-based estimates of the epil-
imnion depth; (2) compare a range of common methods and
threshold values; (3) assess the sensitivity of individual meth-
ods to the threshold value, the water column structure, and
the vertical sensor resolution; and (4) to compare profile-
based methods to turbulence-derived estimates using lake
modelled data. Meteorological data were required to drive a physical
hydrodynamic model (GOTM, Global Ocean Turbulence
Mode; Burchard et al., 1999), including wind speed (m s−1),
atmospheric pressure (hPa), air temperature (◦C), relative hu-
midity (%), cloud cover (dimensionless, 0–1), short-wave
radiation (W m−2), and precipitation (mm d−1). For Erken
lake, air temperature, wind speed, and short-wave radiation
were collected from the Malma Island meteorological station
on the lake at 1 min intervals and averaged to 60 min inter-
vals. Mean sea level pressure, relative humidity, and precip-
itation were measured at the Svanberga meteorological sta-
tion located 400 m from the lake shore at 60 min intervals. Cloud cover was recorded from Svenska Högarna station,
located 69 km south-east of Erken lake. At Lough Feeagh,
wind speed, air temperature, short-wave radiation, mean sea
level pressure, relative humidity, and precipitation were mea-
sured in the meteorological station next to the lake (de Eyto
et al., 2020). Cloud cover was recorded at Knock Airport,
located 50 km east from Lough Feeagh. H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes Water temperature pro-
file data collected at frequent intervals on automatic monitor-
ing buoys in lakes are becoming increasingly available (Jen-
nings et al., 2012; de Eyto et al., 2016; Marcé et al., 2016). In
addition, the collation of these datasets globally through col-
laborative initiatives such as GLEON (http://gleon.org/, last
access: 19 November 2020) and NETLAKE (https://www. dkit.ie/netlake, last access: 19 November 2020) (Weathers
et al., 2013; Jennings et al., 2017) and modelling initiatives
such as ISIMIP2b (Ayala et al., 2020) broaden the poten-
tial for long-term multi-lake studies. However, these datasets The diversity of epilimnion depth definitions and arbitrary
selection process suggest that methods may be used inter-
changeably and are relatively insensitive to the threshold
value used. However, recent studies have begun to recog-
nise large inconsistencies between different definitions and
the potential problems this may cause, although, so far in
limnology, analysis has been restricted to a small number
of manual profiles (Gray et al., 2020) and a limited number
of methods (Pujoni et al., 2019). Although lower temporal
resolution data are sufficient for investigating seasonal pat-
terns, high-frequency data can be used to gain information https://doi.org/10.5194/hess-24-5559-2020 Hydrol. Earth Syst. Sci., 24, 5559–5577, 2020 5561 H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes 5562 Figure 1. Bathymetric map of Lough Feeagh in Ireland (a) and Erken lake in Sweden (b), where the grey stars show the locations of the
automatic monitoring buoys used for measuring high-frequency water temperature profiles in both lakes. Long-term mean water temperatures
for each Julian day for all measured depths are shown for Lough Feeagh (profiles = 2778, years = 10) (c) and Erken lake (profiles = 2005,
years = 7) (d). Figure 1. Bathymetric map of Lough Feeagh in Ireland (a) and Erken lake in Sweden (b), where the grey stars show the locations of the
automatic monitoring buoys used for measuring high-frequency water temperature profiles in both lakes. Long-term mean water temperatures
for each Julian day for all measured depths are shown for Lough Feeagh (profiles = 2778, years = 10) (c) and Erken lake (profiles = 2005,
years = 7) (d). 2.3
Simulated data Table 1. Lake model performance evaluation, showing the percent-
age relative error (%), root mean squared error (◦C), and Nash–
Sutcliffe efficiency for Lough Feeagh (profiles = 1016, years = 5)
and Erken lake (profiles = 1449, years = 7). The Global Ocean Turbulence Model (GOTM), adapted for
use in lakes, simulates small-scale turbulence and vertical
mixing (Burchard et al., 1999; Sachse et al., 2014; Moras
et al., 2019; Ayala et al., 2020) and was used to sim-
ulate daily profiles of water temperature (◦C) and verti-
cal eddy diffusivity (m−2 s−1) for Erken lake and Lough
Feeagh. GOTM was calibrated using 4 years of data (2006–
2009 for Erken lake and 2008–2011 for Lough Feeagh),
including 1-year spin-up followed by 3 years of calibra-
tion. The calibrated model parameters were surface heat-flux
factor (shf_factor), short-wave radiation factor (swr_factor),
wind factor (wind_factor), minimum turbulent kinetic en-
ergy (k_min), and e-folding depth for visible fraction of
light (g2) (see Table S1 in the Supplement for the calibrated
values). During calibration, the model was run approximately
5000 times to obtain a stable solution. The validation pe-
riod was 7 years for Erken lake (2010–2016) and 4 years
for Lough Feeagh (2012–2015). For both the calibration and
validation, daily mean water temperatures were simulated
when GOTM was forced using measured mean hourly data. Model simulated profiles of mean daily water temperature
were then compared to measured mean daily water temper-
ature profiles. Model performance was evaluated by com- Statistic
Lough Feeagh
Erken lake
Calibration
Validation
Calibration
Validation
PRE (%)
−0.48
0.47
−1.85
1.36
RMSE (◦C)
0.67
1.18
0.53
0.55
NSE
0.97
0.92
0.98
0.97 paring mean daily modelled and measured temperature pro-
files, and the model efficiency coefficients used were percent
relative error (PRE), root mean squared error (RMSE), and
Nash–Sutcliffe efficiency (NSE) (Nash and Sutcliffe, 1970). Overall, there was a good model fit for both lakes (Table 1). 2.1
Study sites We used data from two European temperate lakes, Lough
Feeagh (53◦56′ N, 9◦34′ W) in Ireland and Erken lake
(59◦51′ N, 18◦36′ E) in Sweden (Fig. 1). The lakes differ
in many characteristics, including depth, surface area, and
sensor deployment resolution, providing an opportunity to
assess method performance in different lake-specific con-
ditions. Lough Feeagh is located on the west coast of Ire-
land and is a cold monomictic, oligotrophic, and humic
lake with a surface area of 3.9 km2, maximum depth of
45 m, and mean depth of 14.5 m (de Eyto et al., 2016). Erken lake is located in east central Sweden near the Baltic
coast and is a dimictic, mesotrophic, clear lake with a sur-
face area of 24 km2, maximum depth of 21 m, and mean
depth of 9 m (Yang et al., 2016). In addition, Erken lake
has a substantially greater mean summer top–bottom den- https://doi.org/10.5194/hess-24-5559-2020 Hydrol. Earth Syst. Sci., 24, 5559–5577, 2020 H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes 2.4.2
Gradient from the surface method (M2) In M2, the epilimnion depth was defined as the shallowest
depth where the density gradient between consecutively mea-
sured depths exceeded the threshold value. M2 can be de-
scribed as 2.4
Definitions for the epilimnion depth We selected four epilimnion depth definitions that are com-
monly used in limnology and were computationally efficient
for multi-year automated high-frequency data. These meth-
ods we describe as profile-based methods (M1–M4) (Fig. 2). In addition, we calculated epilimnion depth using a method
for modelled data only (M5). In our analysis, epilimnion https://doi.org/10.5194/hess-24-5559-2020 Hydrol. Earth Syst. Sci., 24, 5559–5577, 2020 H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes
5563
Figure 2. Schematic of epilimnion depth methods used in this study, including the range of threshold values for each method and the input
data type (i.e. water temperature, density profile, or lake modelled data). H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes 5563 Figure 2. Schematic of epilimnion depth methods used in this study, including the range of threshold values for each method and the input
data type (i.e. water temperature, density profile, or lake modelled data). exceeded, the epilimnion depth was defaulted to the deep-
est value (Lorbacher et al., 2006). Epilimnion depth esti-
mates calculated with water temperature used the same type
of equation (Eq. 1) but with temperature rather than density
and noting that temperature decreases with depth. The only
threshold value used for temperature was 1 ◦C. depth was expressed relative to the water surface and is there-
fore always a negative value. The range of thresholds used for
each method was selected based on the values found within
the literature (see Table 1 in Gray et al., 2020). We made
no assumption of the conditions below the deepest measured
depth; therefore, the deepest estimated epilimnion depth was
limited to the maximum measured depth for each lake (42 m
in Lough Feeagh and 15 m in Erken lake). 2.4.1
Absolute difference from the surface
method (M1) In M1, the epilimnion depth was defined as the shallowest
depth where the density was a given “threshold” value more
than the surface density (Fig. 2), with the surface density (ρ1)
approximated as the density at the topmost sensor deploy-
ment: 0.9 m in Lough Feeagh and at 1 m in Erken lake. We
used a linear interpolation method to estimate the epilimnion
depth on a continuous depth scale for all methods (Read et
al., 2011), which assumed a linear relationship of densities
between the first measured depth which exceeded the thresh-
old (zi+1) and the preceding measured depth (zi). The nu-
merical scheme can be described (using notation from Read
et al., 2011) as follows: ze = zi1 +
1ρ/1z −∂ρ
∂zi1
zi1+1 −zi1
∂ρ
∂zi1+1 −
∂ρ
∂zi1
! ,
(2) (2) where zi1 is the midpoint between zi and zi+1,
∂ρ
∂zi1 is
the density gradient between zi and zi+1, and 1ρ/1z is
the threshold value (kg m−3 m−1). The threshold values for
all gradient methods (i.e. M2–M4) ranged from 0.025 to
0.2 kg m−3 m−1 at intervals of 0.025 kg m−3 m−1. https://doi.org/10.5194/hess-24-5559-2020 2.4.4
rLakeAnalyzer (M4) In M4, the epilimnion depth was defined using the rLakeAna-
lyzer function “meta.depths” (relating to output “meta.top”),
which used the same numerical scheme as M3, Eq. (3), but
differed in certain assumptions (Read et al., 2011). Firstly,
in M4, the epilimnion depth was prohibited from extend-
ing below the depth of the pycnocline. Therefore, for pro-
files where the predefined threshold value was less than the
maximum density gradient, the epilimnion depth defaulted to
the maximum density gradient. This differed from the other
methods where, for such profiles, the epilimnion depth was
defaulted to the deepest measured depth. Secondly, a user-
defined filter (“mixed.cutoff” object) was used to remove
profiles which were not sufficiently stratified to identify an
epilimnion depth. We used the default filter value, which re-
moved profiles where the overall water temperature range
was less than 1 ◦C. For the days which did not meet the fil-
ter value (and no epilimnion depth was identified), we set the
epilimnion depth to the deepest measured depth (i.e. no epil-
imnion depth) to ensure each method had the same number
of data points for comparison with other methods. 2.4.5
Modelled turbulence method (M5) The modelled turbulence method (M5) used the GOTM lake
model simulated profile estimates of vertical eddy diffusiv-
ity (m2 s−1). In M5, the epilimnion depth was defined as the
first depth, from the lake surface, where the vertical eddy dif-
fusivity fell below the predefined threshold value, and it was
described as ze = zi +
1Kz −Kzi
zi+1 −zi
Kzi+1 −Kzi
,
(4) (4) where Kz is vertical eddy diffusivity (m2 s−1) and 1Kz is
the threshold value (m2 s−1). The thresholds ranged from
10−5 to 10−4 m2 s−1 at intervals of 10−5 m2 s−1, based on
the values described in Wüest and Lorke (2009) and MacIn-
tyre and Melack (2009). 2.5.2
Comparison between water-density-based
methods (M1–M4) Following this, we compared water-density-based epilimnion
depth estimates, using all four methods (M1–M4) and the
range of thresholds described earlier. Using data from both
sites, we considered overall variability (i.e. how much do
estimates vary between all methods and all thresholds),
variability within each individual method using different
threshold definitions (i.e. how sensitive are estimates to the
threshold value selected), and variability between methods
(i.e. what systematic differences exist between pairs of meth-
ods). Given that we had a total of 32 time series to compare,
4 methods each with 8 threshold values, it was necessary to
compute summary statistics for each of them. Therefore, the
following statistics were calculated for all 32 time series, for
the period from 1 April to 31 October each year, and then
averaged across all years. Firstly, we calculated the mean
epilimnion depth and presented the values for all methods
and all threshold values. We also summarised these statis-
tics for each method, showing the mean, standard error of
mean, minimum (shallowest), maximum (deepest) and in-
terquartile range for each method, to demonstrate differences
between methods. A large interquartile range in epilimnion
depth estimates indicated high sensitivity to the threshold
value. Secondly, we calculated the percentage of days with
available data, when the epilimnion depth was detected above
the deepest measured depth. This demonstrated differences
between methods in regards to the stratified period. Thirdly,
we calculated the percentage of days with available data
when the epilimnion depth was detected above the maximum
density gradient or pycnocline. By definition the epilimnion
should have relatively small density gradients and should not
be equal or deeper than the pycnocline; however, automated
methods have been found to regularly encroach on the met-
alimnion (Lorbacher et al., 2006). We therefore used this
metric to investigate how frequently epilimnion depth esti- H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes calculated the change in density that would result from a
1 ◦C increase in the water temperature and then subtracted
0.1 kg m−3. Positive values indicated that a 1 ◦C increase in
the water temperature resulted in a greater than 0.1 kg m−3
change in water density, while negative values indicated a
less than 0.1 kg m−3 change in water density. Secondly, we
compared water-temperature- and water-density-based esti-
mates of the epilimnion depth. To do this, we calculated the
difference between the mean water-density-derived estimate
and the water-temperature-derived estimate for each Julian
day. Positive differences indicated that the water-density-
derived estimate was shallower than the water-temperature-
derived estimate, while negative values were deeper. For all
analyses of measured data, the total numbers of observa-
tions were used for Lough Feeagh (n = 2778, years = 10)
and Erken lake (n = 2005, years = 7). calculated the change in density that would result from a
1 ◦C increase in the water temperature and then subtracted
0.1 kg m−3. Positive values indicated that a 1 ◦C increase in
the water temperature resulted in a greater than 0.1 kg m−3
change in water density, while negative values indicated a
less than 0.1 kg m−3 change in water density. Secondly, we
compared water-temperature- and water-density-based esti-
mates of the epilimnion depth. To do this, we calculated the
difference between the mean water-density-derived estimate
and the water-temperature-derived estimate for each Julian
day. Positive differences indicated that the water-density-
derived estimate was shallower than the water-temperature-
derived estimate, while negative values were deeper. For all
analyses of measured data, the total numbers of observa-
tions were used for Lough Feeagh (n = 2778, years = 10)
and Erken lake (n = 2005, years = 7). ze = zi1 +
1ρ/1z −∂ρ
∂zi1
zi1 −zi1+1
∂ρ
∂zi1 −
∂ρ
∂zi1+1
! . (3) (3) 2.4.3
Gradient from the pycnocline method (M3) ze = zi + ((ρ1 + 1ρ) −ρi)
zi+1 −zi
ρi+1 −ρi
,
(1) (1) In M3, the epilimnion depth was defined as the deepest depth
where the density between consecutively measured depths
exceeded the threshold value, starting from the depth of the
maximum density gradient (hereafter the “pycnocline”) as
the reference depth and moving to successively shallower
measured depths. M3 can be described by where z is depth (m), ρ is water density (kg m−3), and 1ρ is
the threshold value (kg m−3). The threshold values for the
absolute method, M1 only, ranged from 0.025 to 0.2 kg m−3
at intervals of 0.025 kg m−3. For all methods excluding the
rLakeAnalyzer method (M4), if the threshold value was not Hydrol. Earth Syst. Sci., 24, 5559–5577, 2020 https://doi.org/10.5194/hess-24-5559-2020 5564 2.5.3
Sensitivity of epilimnion depth to water column
structure and vertical sensor resolution There were large systematic differences between the epil-
imnion depth calculated using a water-temperature-based
method compared to values calculated using a water-density-
based method (Fig. 3). Due to the non-linear relationship be-
tween water density and water temperature, the difference
in density induced by a water temperature increase of 1 ◦C
(water column mean) varied seasonally, with the pattern dif-
fering between sites (Fig. 3a). We found that on average,
during the spring (April–May), when water column temper-
atures in both lakes were relatively low, a change of 1 ◦C
resulted in a water density change of less than 0.1 kg m−3,
as shaded in blue. As a result of this anomaly, estimates of
the epilimnion depth that were based on water temperature
data were shallower compared to those calculated using the
water density method (Fig. 3b). In contrast, in general from
June to October for both sites, a change of 1 ◦C in water tem-
perature induced a change in water density of greater than
0.1 kg m−3, as shaded in red, which resulted in estimates of
the epilimnion depth which were deeper when using water
temperature method compared to those estimated using wa-
ter density. Based on the long-term daily means, the differ-
ences in the estimates of epilimnion depth between the two
methods ranged from 3 to 5 m for Lough Feeagh and from
2 to 4 m for Erken lake. We also assessed the sensitivity of the profile-based meth-
ods to changes in the water column structure and the ver-
tical sensor resolution of measured data. For the water col-
umn structure sensitivity analysis, we calculated the long-
term mean epilimnion depth estimate for each Julian day
for all 32 method/threshold time series. For each method,
using all thresholds, we calculated the range for each Ju-
lian day. The range in estimates was presented alongside the
top–bottom density gradient for each Julian day to investi-
gate whether threshold sensitivity varied temporally and with
water column structure. For the vertical sensor deployment
resolution sensitivity analysis, we compared simulated wa-
ter density profiles for both lakes at two different resolutions. High-resolution data were resolved to 0.5 m for both lakes. Low-resolution data were subset to a mean of one sensor per
3 m, using the measured depths for Lough Feeagh and data
from 1, 2.5, 5, 8, and 13 m for Erken lake. 2.5.3
Sensitivity of epilimnion depth to water column
structure and vertical sensor resolution We then calcu-
lated the difference between the April–October mean epil-
imnion depth for the high- and low-resolution data. Meth-
ods where the high- and low-resolution data produced very
different estimates were regarded as having high sensitiv-
ity to the vertical resolution of the data, while methods with
small differences indicated low sensitivity. For all analyses
using simulated data, the total numbers of observations were
used for Lough Feeagh (n = 1016, years = 5) and Erken lake
(n = 1449, years = 7). 3.2
Comparison between water-density-based
methods (M1–M4) Inspection of water column profiles highlighted key differ-
ences in the performance of methods M1–M4 (Fig. 4). In a
stratified profile, with a well-defined three-layered water col-
umn profile, there was often strong agreement on the epil-
imnion depth between all methods and thresholds (Fig. 4a). In contrast, when the measured temperature profile was more
complex, i.e. at times when there was some stratification
close to the surface or when a secondary pycnocline had de-
veloped close to the surface, there was less agreement on the
estimates of the epilimnion depth between methods (Fig. 4b). 2.5.1
Comparison between a water-temperature- and
water-density-derived methods To compare water-temperature- and water-density-based es-
timates of the epilimnion depth, we used M1 only and used a
water temperature threshold of 1 ◦C with a density threshold
of 0.1 kg m−3 for both sites. Firstly, we investigated the rela-
tionship between 1 ◦C and 0.1 kg m−3 throughout the year. To do this, we calculated the long-term mean water col-
umn temperature for each Julian day. For each day, we then https://doi.org/10.5194/hess-24-5559-2020 Hydrol. Earth Syst. Sci., 24, 5559–5577, 2020 H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes 5565 culated the mean April–October epilimnion depth for each
method. We then calculated the difference between the turbu-
lence method (M5) and each of the four profile-based meth-
ods (M1–M4). We also presented the mean Pearson’s cor-
relation coefficients between each method and M5 (e.g. for
all threshold combinations between M5 and M1). These re-
sults indicated the extent to which profile-based methods
were able to characterise active mixing penetration, within
a hydrodynamic model setting, rather than confirming which
method was more reliable for predicting the “true” mixing
depth. mates calculated by each method erroneously extended into
the metalimnion. Pearson’s correlation coefficients were also calculated for
all possible combinations between the 32 time series to quan-
tify the degree of association between them, without using
any estimates of significance (Thomson and Fine, 2003; Riv-
etti et al., 2017). The full correlation matrices were calcu-
lated and then for, clarity, we presented only the mean Pear-
son’s correlation coefficient for each method, representing
the mean correlation for all possible combinations between
threshold values. This indicated the extent to which chang-
ing the threshold value influenced the temporal patterns. We
also presented the mean Pearson’s correlation coefficients
between each pair of methods (e.g. for all threshold combi-
nations between M1 and M2) to demonstrate method agree-
ment. 2.5.4
Comparison with modelled turbulence
method (M5) Finally, we assessed how each profile-based method com-
pared against the turbulence-based estimates. For this anal-
ysis, both water density and vertical eddy diffusivity profile
data were derived by using the GOTM lake model. Then,
using the same procedures as the measured data, we cal- https://doi.org/10.5194/hess-24-5559-2020 Hydrol. Earth Syst. Sci., 24, 5559–5577, 2020 H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes 5566 y
p
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Figure 3. Long-term mean for each Julian day of the difference in water density (kg m−3) induced by an increase of 1 ◦C in water tem-
perature, relative to 0.1 kg m−3 (black line with the red shaded area demonstrating when the change induced by an increase of 1 ◦C change
was greater than 0.1 kg m−3 and the blue shaded area for when it was less than 0.1 kg m−3) (a). The long-term mean for each Julian day of
epilimnion depth calculated using a water temperature threshold of 1 ◦C (the black line) compared to a water density threshold of 0.1 kg m−3
(shaded area, with the red shaded areas demonstrating when water density estimates were shallower and the blue shaded area for when they
were deeper) (b) for Lough Feeagh and Erken lake. Figure 3. Long-term mean for each Julian day of the difference in water density (kg m−3) induced by an increase of 1 ◦C in water tem-
perature, relative to 0.1 kg m−3 (black line with the red shaded area demonstrating when the change induced by an increase of 1 ◦C change
was greater than 0.1 kg m−3 and the blue shaded area for when it was less than 0.1 kg m−3) (a). The long-term mean for each Julian day of
epilimnion depth calculated using a water temperature threshold of 1 ◦C (the black line) compared to a water density threshold of 0.1 kg m−3
(shaded area, with the red shaded areas demonstrating when water density estimates were shallower and the blue shaded area for when they
were deeper) (b) for Lough Feeagh and Erken lake. For such profiles, estimates of the epilimnion depth calcu-
lated with the absolute difference from the surface method,
M1, were typically staggered at linear intervals along the
profile, depending on the exact threshold value. In contrast,
the estimated epilimnion depth calculated using the gradi-
ent methods (M2–M4) had a tendency to cluster at discrete
locations on the profile. 2.5.4
Comparison with modelled turbulence
method (M5) Therefore, a small change in the
threshold value induced either no difference at all in the epil-
imnion depth or at other times a very large difference. For
profiles with low water column stability, there was partic-
ularly large differences in the estimated epilimnion depth
calculated using different methods, reflecting differing un-
derlying assumptions (Fig. 4c). For M3, for example, the
epilimnion depth was defaulted to the deepest depth when
the threshold value was not exceeded, as was also the case
for methods M1 and M2. In contrast, however, in M4, near-
isothermal profiles often met the “mixed.cutoff” filter condi-
tion (i.e. water column range > 1 ◦C), whilst still not having
sufficient density gradients to meet the user threshold value. As a result, in M4, the epilimnion depth was defaulted to the
pycnocline, which, given the small density gradients, was of-
ten found at a very shallow depth. thresholds (Fig. 5). Considering that all the time series es-
timates for Lough Feeagh (left side) and Erken lake (right
side) were presumed to estimate the same theoretical loca-
tion, they would ideally all produce exactly the same tem-
poral patterns. Instead, the differences were large enough to
obscure the annual patterns and hinder the ability to com-
pare between the two lakes. The overall mean epilimnion
depth estimate using methods M1–M4 and all thresholds
was −28.1 m (standard error SE = 0.6 m, interquartile range
IQR = 19.0 m) for Lough Feeagh and −11.0 m (SE = 0.1 m,
IQR = 2.3 m) for Erken lake. The overall variability between
all estimates was particularly high for Lough Feeagh, where
the April–October mean epilimnion depth ranged by 36.9 m
(−4.6 to −41.5 m), while in Erken lake estimates ranged by
5.2 m (−7.8 to −13.0 m) (Fig. 6a). There were evident systematic differences between meth-
ods. In both lakes, the mean April–October epilimnion depth
for each method was shallowest for M1 and was on av-
erage shallower by 17.0, 16.6, and 2.2 m compared with
methods M2–M4 in Lough Feeagh and 1.2, 1.7, and 0.8 m
in Erken lake (Table 2). Hydrol. Earth Syst. Sci., 24, 5559–5577, 2020 2.5.4
Comparison with modelled turbulence
method (M5) The minimum (shallowest) esti-
mates of the April–October mean for gradient methods (M2–
M4) were comparable in magnitude to the maximum (deep-
est) estimate for the absolute difference from the surface Time series results demonstrated the extent of the variabil-
ity in epilimnion depth estimates between all methods and https://doi.org/10.5194/hess-24-5559-2020 Hydrol. Earth Syst. Sci., 24, 5559–5577, 2020 H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes https://doi.org/10.5194/hess-24-5559-2020 https://doi.org/10.5194/hess-24-5559-2020 H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes 5567 gure 4. An example of water column profiles with the epilimnion depth estimates superimposed (horizontal lines) for all profile-based
ilimnion depth methods calculated using the full range of thresholds for each. The water columns can be categorised as a three-layered
ater column structure (a), an intensely stratified profile (b), and a near-isothermal profile (c), which are all from Erken lake only. Figure 4. An example of water column profiles with the epilimnion depth estimates superimposed (horizontal lines) for all profile-based
epilimnion depth methods calculated using the full range of thresholds for each. The water columns can be categorised as a three-layered
water column structure (a), an intensely stratified profile (b), and a near-isothermal profile (c), which are all from Erken lake only. Table 2. Summary of statistics for each method, showing the mean (m) and standard error of the mean in brackets, minimum (i.e. shal-
lowest estimate) (m), maximum (i.e. deepest estimate) (m), interquartile range (IQR, m) of the April–October epilimnion depth estimates
(summarised from the results shown in Fig. 6a), and the mean Pearson’s correlation coefficient (r) for each method, representing the mean
correlation for all possible combinations between threshold values for Lough Feeagh and Erken lake. Lough Feeagh
Erken lake
Method
Mean
Min
Max
IQR
r
Mean
Min
Max
IQR
r
(m)
(m)
(m)
(m)
(m)
(m)
(m)
(m)
M1
−19.0 (0.8)
−4.6
−25.4
7.3
0.77
−10.0 (0.2)
−7.8
−11.2
1.3
0.92
M2
−35.9 (0.9)
−19.7
−41.4
6.5
0.48
−11.3 (0.2)
−8.4
−12.9
1.7
0.78
M3
−36.5 (0.8)
−22.4
−41.5
6.1
0.49
−11.8 (0.2)
−10.0
−13.0
1.2
0.82
M4
−21.1 (0.5)
−19.7
−21.5
0.3
1.00
−11.9 (0.1)
−10.1
−11.3
0.5
0.99 https://doi.org/10.5194/hess-24-5559-2020
Hydrol. Earth Syst. Sci., 24, 5559–5577, 2020 https://doi.org/10.5194/hess-24-5559-2020 Hydrol. Earth Syst. Sci., 24, 5559–5577, 2020 H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes 5569 H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes
5569
Figure 6. Mean April–October epilimnion depth (m) (a); percentage of stratified days, defined as days when the epilimnion depth was
identified as shallower than the lake maximum measured depth (%) (where a larger percentage value indicated a higher occurrence of days
identified as stratified) (b); and percentage of days when the epilimnion depth was above the pycnocline, defined as the number of days when
the epilimnion was identified at a depth shallower than the maximum density gradient (where a larger percentage value indicated a lower
occurrence of days erroneously extending into the metalimnion) (c) for Lough Feeagh and Erken lake. Figure 6. Mean April–October epilimnion depth (m) (a); percentage of stratified days, defined as days when the epilimnion depth was
identified as shallower than the lake maximum measured depth (%) (where a larger percentage value indicated a higher occurrence of days
identified as stratified) (b); and percentage of days when the epilimnion depth was above the pycnocline, defined as the number of days when
the epilimnion was identified at a depth shallower than the maximum density gradient (where a larger percentage value indicated a lower
occurrence of days erroneously extending into the metalimnion) (c) for Lough Feeagh and Erken lake. than the difference between the last two thresholds (0.175
and 0.2 kg m−3 m−1) in both lakes. depth estimates for each method was very high for M2, M1,
and M3, indicating high threshold sensitivity in these meth-
ods. Method M4 had a substantially lower interquartile range
than all other methods and a very high mean Pearson’s corre-
lation coefficient, indicating that both the mean value and the
temporal pattern of the epilimnion depth were only weakly
influenced by the threshold value. In both lakes, methods M2
and M1, where the epilimnion depth was defined from the
surface downwards, had a higher interquartile range in es-
timates calculated with different threshold values compared
to methods M3 and M4, where the epilimnion was defined
from the pycnocline upwards. M1, however, had higher mean
Pearson’s correlation coefficient than M2 and M3, indicating
that the temporal pattern of the epilimnion depth was less
influenced by the threshold value. In general, the threshold
sensitivity of each method reduced with increasing threshold
size. That is, the changes in the epilimnion depth occurring
between threshold values decreased with increasing thresh-
old value (Fig. H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes H. L. Wilson et al.: Variability in epilimnion depth estimations in lak 5568 y
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Figure 5. Daily epilimnion depth estimates using measured data for 2017 from Lough Feeagh and Erken lake, showing estimates from
all profile-based epilimnion depth methods, including M1, the absolute difference from the surface method (a); M2, the gradient from the
surface method (b); M3, the gradient from the pycnocline method (c); and M4, the rLakeAnalyzer method (d), which are calculated using
the full range of thresholds and for each lake. Figure 5. Daily epilimnion depth estimates using measured data for 2017 from Lough Feeagh and Erken lake, showing estimates from
all profile-based epilimnion depth methods, including M1, the absolute difference from the surface method (a); M2, the gradient from the
surface method (b); M3, the gradient from the pycnocline method (c); and M4, the rLakeAnalyzer method (d), which are calculated using
the full range of thresholds and for each lake. Table 3. Mean of all Pearson’s correlation coefficients calculated
for each pair of methods (e.g. for all threshold combinations be-
tween M1 and M2) for Lough Feeagh and Erken lake. method, M1. The mean Pearson’s correlation coefficient be-
tween each pair of methods also demonstrated that certain
method pairs had greater temporal agreement than other pairs
(Table 3). The full correlation matrices are available in Ta-
ble S2. Method pairs M3–M2 and M4–M1 had particularly
high Pearson correlation coefficients for both lakes, sug-
gesting these methods produced similar temporal trends. In
Erken lake all method pairs had higher Pearson’s correlation
coefficients than Lough Feeagh. Lough Feeagh
Erken lake
Method
r
r
M2–M1
0.35
0.77
M3–M1
0.33
0.74
M3–M2
0.55
0.8
M4–M1
0.68
0.81
M4–M2
0.29
0.71
M4–M3
0.3
0.75 The selection of a threshold value proved to be very im-
portant in the estimation of the epilimnion depth. For all
methods, smaller threshold values produced shallower esti-
mates of the mean April–October epilimnion depth, while
larger threshold values produced deeper estimates (Fig. 6a). Methods with a wide range between the shallowest (mini-
mum) and deepest (maximum) estimate demonstrated high
sensitivity to the threshold value (Table 2). For both lakes,
the interquartile range in the mean April–October epilimnion Hydrol. Earth Syst. Sci., 24, 5559–5577, 2020 H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes 5570 5570
H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes
Figure 7. Interquartile range between the shallowest and deepest estimate for each method calculated from long-term daily mean epilimnion
depth estimates for each Julian day, when a wide range suggests high threshold sensitivity and a narrow range suggests low sensitivity (a);
long-term daily mean water density gradient, calculated based on the surface and maximum measured depths (b) for Lough Feeagh and
Erken lake. Figure 7. Interquartile range between the shallowest and deepest estimate for each method calculated from long-term daily mean epilimnion
depth estimates for each Julian day, when a wide range suggests high threshold sensitivity and a narrow range suggests low sensitivity (a);
long-term daily mean water density gradient, calculated based on the surface and maximum measured depths (b) for Lough Feeagh and
Erken lake Figure 7. Interquartile range between the shallowest and deepest estimate for each method calculated from long-term daily mean epilimnion
depth estimates for each Julian day, when a wide range suggests high threshold sensitivity and a narrow range suggests low sensitivity (a);
long-term daily mean water density gradient, calculated based on the surface and maximum measured depths (b) for Lough Feeagh and
Erken lake. water density induced large difference between estimates cal-
culated using small thresholds compared with large threshold
values. In contrast, methods M2 and M3 had the highest in-
terquartile range in estimates occurring during the peak sum-
mer months. Even during peak summer in Lough Feeagh,
gradients in the water column were relatively small (Fig. 7b),
which resulted in a very large differences between the small-
est threshold values which found a near-surface epilimnion
depth and the largest thresholds that often found no epil-
imnion depth at all and therefore defaulting to the deepest
depth. In Erken lake, the water density gradients were typ-
ically much larger, and methods M1–M3 all peaked during
May and June, when gradients in the water column were typ-
ically increasing but prone to fluctuations. Compared with all
other methods, M4 produced a substantially lower interquar-
tile range in the epilimnion depth throughout the year, since
as long as the “mixed.cutoff” filter was met, the epilimnion
depth was defaulted to the pycnocline if the threshold was
not exceeded and thus largely reducing the ability for large
differences to occur. H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes The interquartile range in epilimnion
depth estimates for M4 was highest during the peak sum-
mer months, which was when the epilimnion depth was typi-
cally shallowest and more frequently defined by the threshold
value rather than defaulting to the pycnocline. dient, indicated that some methods may be less prone to erro-
neously estimating the epilimnion depth in the metalimnion
compared with others (Fig. 6b). For both lakes, M1 had the
highest number of days when the epilimnion depth was lo-
cated above the pycnocline, suggesting that on average the
method extended into the metalimnion less frequently than
other methods. In Lough Feeagh, all gradient methods, M2–
M4, had very high range occurring between the different
threshold values. In Lough Feeagh, gradient methods calcu-
lated with a threshold value greater than 0.1 kg m−3 m−1 re-
sulted in an average of 0 d when the epilimnion depth was
located above the pycnocline. https://doi.org/10.5194/hess-24-5559-2020 H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes 6a). For example, for M2, the difference in the
April–October mean epilimnion depth between the first two
thresholds (0.025 and 0.05 kg m−3 m−1) was much greater The percentage of stratified days, defined as days when the
epilimnion depth was identified as shallower than the deepest
measured depth, demonstrated the extent to which different
methods/thresholds influenced the stratified period (Fig. 6b). For M4, the percentage of stratified days remained static re-
gardless of the threshold value, because the epilimnion depth
was detected for all profiles where the water column temper-
ature range was more than 1 ◦C, regardless of the threshold
used. For all other methods, the number of stratified days
decreased with increasing threshold value. For M1 the dif-
ference in stratified days between threshold values was small
compared to both gradient methods M2 and M3, particularly
in Lough Feeagh. For example, in Lough Feeagh, for M3, the
number of stratified days calculated using a threshold value
of 0.025 kg m−3 m−1 was 125, while for threshold values
greater than 0.075 kg m−3 m−1, the mean number of strati-
fied days per annum decreased to less than 38. The percentage of days when the epilimnion depth was
located above the pycnocline, defined as days when the epil-
imnion depth was identified above the maximum density gra- https://doi.org/10.5194/hess-24-5559-2020 Hydrol. Earth Syst. Sci., 24, 5559–5577, 2020 H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes 3.4
Sensitivity of epilimnion depth to vertical sensor
resolution Table 5. Difference in mean April–October epilimnion depth esti-
mates (m) between each profile-based method (M1–M4) calculated
using lake modelled data and the modelled turbulence method (M5)
and mean of all Pearson’s correlation coefficients (r) calculated for
each profile-based method and M5 (e.g. for all threshold combi-
nations between M5 and M1) for Lough Feeagh and Erken lake. Positive differences indicate that the modelled turbulence method is
shallower. Table 5. Difference in mean April–October epilimnion depth esti-
mates (m) between each profile-based method (M1–M4) calculated
using lake modelled data and the modelled turbulence method (M5)
and mean of all Pearson’s correlation coefficients (r) calculated for
each profile-based method and M5 (e.g. for all threshold combi-
nations between M5 and M1) for Lough Feeagh and Erken lake. Positive differences indicate that the modelled turbulence method is
shallower. The vertical resolution of water density data was found to
have a systematic influence on the estimation of the epil-
imnion depth for all methods (Table 4). Overall, the modelled
higher vertical resolution data resulted in shallower estimates
of the epilimnion depth relative to the estimates made with
the modelled low-resolution data. For Lough Feeagh, the re-
sults showed that the annual mean April–October epilimnion
depth estimates using high-resolution data were 0.1, 3.2, 3.2,
and 0.5 m shallower than those using low-resolution data for
methods M1–M4, while in Erken lake they were 0.0, 1.2,
1.0, and 0.2 m shallower. Methods M1 and M4 had substan-
tially smaller differences between high- and low-resolution
estimates compared with M2 and M3. In particular, M1 had
almost no difference between high- and low-resolution data,
indicating that this method had very low sensitivity to the
vertical sensor deployment. Method
Lough Feeagh
Erken lake
Difference
r
Difference
r
in mean
in mean
epilimnion
epilimnion
depth (m)
depth (m)
M5–M1
1.3
0.90
0.0
0.89
M5–M2
11.0
0.55
1.0
0.73
M5–M3
11.2
0.54
1.1
0.72
M5–M4
1.3
0.88
0.4
0.85 H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes Table 4. Mean April–October epilimnion depth estimates (m) derived using high-resolution and low-resolution modelled water temperature
data, with standard error of the mean in brackets, and the difference calculated between the high-resolution and low-resolution estimates (m)
for Lough Feeagh and Erken lake. Table 4. Mean April–October epilimnion depth estimates (m) derived using high-resolution and low-resolution modelled water temperature
data, with standard error of the mean in brackets, and the difference calculated between the high-resolution and low-resolution estimates (m)
for Lough Feeagh and Erken lake. Lough Feeagh
Erken lake
Method
High-resolution
Low-resolution
Difference
High-resolution
Low-resolution
Difference
mean (m)
mean (m)
in mean
mean (m)
mean (m)
in mean
epilimnion
epilimnion
depth (m)
depth (m)
M1
−22.1 (0.7)
−22.2 (0.7)
0.1
−10.9 (0.1)
−10.9 (0.1)
0.0
M2
−31.7 (1.1)
−34.9 (1.2)
3.2
−11.9 (0.2)
−13.1 (0.2)
1.2
M3
−32.0 (1.1)
−35.2 (1.1)
3.2
−12.1 (0.2)
−13.1 (0.2)
1.0
M4
−22.1 (0.6)
−22.6 (0.6)
0.5
−11.3 (0.1)
−11.5 (0.1)
0.2 3.5
Comparison with modelled turbulence
method (M5) strong agreement with M5. In contrast, M2 and M3 had
much weaker agreement with M5, in terms of both the April–
October epilimnion depth estimate and the Pearson’s corre-
lation coefficients. strong agreement with M5. In contrast, M2 and M3 had
much weaker agreement with M5, in terms of both the April–
October epilimnion depth estimate and the Pearson’s corre-
lation coefficients. In general, the modelled turbulence method had very low
sensitivity to the threshold value compared with the profile-
based methods also calculated using modelled data. A time
series comparison of all modelled results is available in
Fig. S1 (in the Supplement). For both lakes, we found that
the modelled turbulence method produced shallower esti-
mates than modelled profile-based methods (Table 5). In
Lough Feeagh, the mean April–October epilimnion depth
estimate using the modelled turbulence method M5 was
−20.8 m, which was 1.3, 11.0, 11.2, and 1.3 m shallower
than methods M1–M4, while in Erken lake the M5 estimate
was −11.0 m, which was 0.0, 1.0, 1.1, and 0.4 m shallower. In both lakes, M1 had the strongest agreement with M5,
demonstrated by both the mean difference (1.3 m in Lough
Feeagh and 0.0 m in Erken lake) and the highest Pearson’s
correlation coefficient in Lough Feeagh (r = 0.90) and Erken
lake (r = 0.89). This was followed by M4, which also had 3.3
Sensitivity of epilimnion depth to water column
structure For all methods, threshold sensitivity fluctuated seasonally,
although it varied in a pattern (Fig. 7). Threshold sensitiv-
ity was shown by the interquartile range between the epil-
imnion depth estimates calculated for all threshold values. In
Lough Feeagh, M1 had a smaller interquartile range in epil-
imnion depth estimates during the peak summer months of
June, July, and August compared with months when the onset
and overturn of stratification commonly occurred. During pe-
riods of transient stratification, the stability of the water col-
umn was often low, but frequent changes in the near-surface https://doi.org/10.5194/hess-24-5559-2020 Hydrol. Earth Syst. Sci., 24, 5559–5577, 2020 5571 H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes oxygen or nutrients) between the epilimnion and the
metalimnion, since both terms are influenced by the epil-
imnion depth (Giling et al., 2017; Gray et al., 2020). In an idealised stratified profile, the epilimnion is por-
trayed as near uniform in water temperature or density and
clearly delineated from a well-defined metalimnion. How-
ever, many measured profiles, at least within this study, did
not conform to this idealised three-layered structure. Instead
the thermal water column structure was often more com-
plex, including multiple pycnoclines, near-surface micro-
stratification layers, and blurred boundaries between the epil-
imnion and metalimnion. One approach to this issue is to fil-
ter out appropriate water column profiles or apply functions
that coerce the profile into the expected structure (Read et al.,
2011; Pujoni et al., 2019; Gray et al., 2020). However, our
analysis of temporally high-resolution time series data em-
phasised that rather than jumping from states, such as strat-
ified or isothermal, changes in the water column occurred
over an evolving continuum and often fluctuated between
states. Similarly, the distinction between additional layers,
such as the primary or secondary pycnocline, is fraught with
the same issues of arbitrariness as discussed (Read et al.,
2011). This study demonstrates that when epilimnion depth
estimation methods, which are theorised for a three-layered
water column, are applied to nonconforming water columns,
they diverge widely on the location of the epilimnion depth,
and at times may not even be underpinned by the same the-
oretical assumptions. Since none of these methods can be
considered the true definition of the epilimnion depth, it is
necessary to understand the degree to which methods differ. Improved understanding of their systematic differences will
facilitate the use of methods that appropriately capture dif-
ferent processes, such as air–water exchanges, thermocline
entrainment, or the suspension of materials. Due to the re-
alised complexities of observed and aggregated profile data,
we may benefit from new approaches to water column dis-
cretisation that consider the vast proportion of profiles which
do not conform neatly to the three-layered paradigm. An important difference was also found between methods
detecting the layer that is isothermal relative to the surface
and methods detecting the point that is isothermal relative to
the steep gradients of the metalimnion, which has not been
well considered in the literature. H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes 5572 ing findings between studies which use different methods or
even just different thresholds. The magnitude of variability
also casts ambiguity on the calculation of key biogeochemi-
cal and ecological processes in a lake that rest on the assump-
tion that the layers of a lake are well defined, including cal-
culations of metabolic rates and oxygen fluxes (e.g. Coloso
et al., 2011; Foley et al., 2012; Obrador et al., 2014; Winslow
et al., 2016). northern temperate lakes due to the large annual water tem-
perature ranges (Maberly et al., 2020). Pronounced differ-
ences in the estimation of the epilimnion depth were also
found within estimates derived using the same water den-
sity input data. Typically, for the range of common thresh-
olds used in this study, the absolute difference from the sur-
face method, M1, produced shallower estimates relative to
gradient-based methods. In addition, the difference between
these methods was particularly large when the vertical res-
olution of the data was low. This suggests that studies us-
ing gradient-based methods, particularly those using coarse
vertical data, may have a deep bias relative to those using
an absolute method and, consequently, were more prone to
erroneously extending into the metalimnion. In addition, as
may be expected, the use of larger threshold values also
produced systematically deeper estimates of the epilimnion
depth. Surprisingly, however, the magnitudes of these differ-
ences were on par with those occurring between methods. The implications of a shallow or deep bias may be far reach-
ing, particularly given that various biological and ecologi-
cal metrics have already been found to be highly sensitive to
changes in the epilimnion depth (Coloso et al., 2011; Gray et
al., 2020). For example, a deeper estimate of the epilimnion
depth would systematically lead to a larger ratio between the
epilimnion and euphotic depth compared with a shallower es-
timate, which if used to understand the development of a phy-
toplankton bloom, could lead to contradictory results (Huis-
man et al., 1999). Alternatively, water-temperature-based es-
timates typically resulted in earlier stratification, which could
indicate a longer duration of phytoplankton in a shallower
epilimnion. The implications of a seasonal or deep/shallow
biases may be even more important for computing fluxes
(e.g. 4
Discussion The concept of the epilimnion (and, more widely, the three-
layered structure of a stratified lake) is fundamental in lim-
nology. Yet, despite the ubiquity of the term, there is no ob-
jective or generic approach for defining the epilimnion, and
a diverse number of approaches prevail in the literature. In
a comprehensive analysis of high-frequency multi-year data
from two lakes, this study has highlighted the extent to which
common water-temperature-profile-based epilimnion depth
estimates differ. The level of variability in epilimnion depth
estimates calculated using common methods and threshold
values was exceedingly high. This result calls into ques-
tion the practice of arbitrary method selection and compar- https://doi.org/10.5194/hess-24-5559-2020 Hydrol. Earth Syst. Sci., 24, 5559–5577, 2020 H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes In gen-
eral, a suitable threshold is any value that can be reason-
ably considered homogenous while also within the limit of
sensor detection (De Boyer et al., 2004). However, all the
threshold values used in this study met these criteria yet pro-
duced fundamentally different epilimnion depth estimations
and temporal patterns. Although it may be unreasonable to
suggest a “universal” threshold value, a given study may find
a threshold that is less problematic than other values. In gen-
eral, we found that the sensitivity of the epilimnion depth to
the threshold value decreased with the increasing size of the
threshold. That is, for small thresholds the impact of chang-
ing the threshold value was greater than larger thresholds
for the same incremental change. This may suggest that for
studies using smaller threshold values, the results are more
threshold dependent than those using large threshold values. However, larger threshold values had a greater frequency of
the epilimnion depth estimates being below the maximum
density gradient, suggesting that larger threshold values tend
to extend into the stable depths of the metalimnion more reg-
ularly and hence somewhat explaining the lower threshold
sensitivity. The trade-off between threshold sensitivity and
encroachment into the metalimnion points towards a mid-
range threshold, such as 0.1 kg m−3 or 0.1 kg m−3 m−1, as
potentially being more reliable than large or small thresholds. Regardless of the method selected, however, all water
temperature- or density-based methods are limited in their
ability to indicate actual mixing processes. Our results using
the lake modelled turbulence data demonstrated that even in
a modelled environment, epilimnion depth estimates were in-
consistent between the different methods and threshold val-
ues studied and that turbulence-based methods generally re-
sulted in a shallower epilimnion depth estimate. These find-
ings highlight the important but subtle difference between the
layer detected by water density profiles (i.e. has been recently
well mixed and therefore has little resistance to further mix-
ing due to the lack of density gradients) and the layer that is
actively mixing, determined only through directly measured
turbulence (Gray et al., 2020). Similarly, micro-profiling
studies have shown that the actively mixing layer can be
substantially shallower than the layer determined through
water temperature profile data (McIntyre, 1993; Tedford et
al., 2014). H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes very little on the threshold value selected. Instead, the se-
lection of the filter (defaulted to a water column range of
> 1 ◦C), which was unique to this method, determined the
number of stratified days and largely influenced the other
bulk statistics. This also resulted in the epilimnion being
identified even when the threshold was not exceeded, which
in some instances could have the effect of muting relative
temporal changes in the epilimnion depth. In contrast, for the
other methods, the threshold was used to determine whether
the water column was considered to be stratified; therefore,
the stratification period was highly sensitive to the thresh-
old value, similarly to the other bulk statistics. Ultimately,
these results suggest that the stratification period calculated
in different studies or for different regions cannot be com-
pared unless identical definitions are used. The method most
appropriate for identifying the stratified period has been con-
sidered in other studies (Woolway et al., 2014; Engelhardt
and Kirillin, 2014; however, our results offer some additional
insights. The results suggest that the use of water density
metrics, such as epilimnion depth estimates, in combination
with traditional water-temperature-based definitions of strat-
ification, is incompatible, given the non-linear relationship
between temperature and density. In addition, estimations of
the epilimnion depth (and the variability among definitions)
may be particularly relevant for understanding the stratified
period, since it is often assumed that the onset of stratifica-
tion marks the decoupling of the epilimnion from the deeper
layers, determining the duration of nutrient limitations in the
epilimnion and oxygen limitations in the hypolimnion (Mac-
Intyre, 1993; Foley et al., 2012; Schwefel et al., 2016). even turbulence-based methods are inherently arbitrary, as
there is no objective threshold value (Monismith and Macin-
tyre, 2009). Many of the ecological applications of the epil-
imnion depth have the underlying assumption that enough
mixing is occurring in the epilimnion to keep the relevant
organisms or particles suspended within the layer. However,
whether mixing is actually occurring and to what extent are
not directly described by epilimnion depth estimations de-
rived using water temperature or density profile data; in fact,
previous studies have found water density estimates of the
epilimnion depth to be relatively poor indicators for the ho-
mogeneity of other ecological variables (Gray et al., 2020). The selection of a suitable threshold value is far more
important than previously attributed in limnology. H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes M1 and M2, defined from
the surface downwards, were more prone to the detection of
a shallow secondary pycnocline compared with M3 and M4. Instead, M3 and M4, defined from the pycnocline upwards,
prioritised the relative difference between the metalimnion
and the surface. From a theoretical point of view, processes
related to the air–water interface could be better suited to
methods identifying the isothermal layer, while processes re-
lated to the entrainment of deep water into the epilimnion are
more suited to top of the metalimnion methods. A large systematic difference was found between water
temperature and water density. Due to the non-linear rela-
tionship between water density and temperature, the use of
water temperature was equivalent to using different density
threshold values throughout the year, resulting in a distinct
shift in the stratification period. Although water density gra-
dients are driven by temperature changes in lakes and are
also calculated from water temperature estimates, water den-
sity directly influences mixing processes and is therefore rec-
ommended for estimating the epilimnion depth (Read et al.,
2011; Gray et al., 2020). The implications of using a water-
temperature-based method may be particularly enhanced in The selection of an epilimnion method also had surpris-
ingly large consequences for understanding the stratification
period, which is widely used for quantifying the impact of
climate change on lakes (Livingstone, 2003; Butcher et al.,
2015; Ayala et al., 2020). Notably, the mean epilimnion depth
and number of stratified days calculated using M4 depended https://doi.org/10.5194/hess-24-5559-2020 Hydrol. Earth Syst. Sci., 24, 5559–5577, 2020 H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes 5573 H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes value. Therefore, the M1 method may be particularly useful
as a generic method. ful as a “generic” method, due in particular to the very low
sensitivity to the vertical sensor resolution compared with all
other gradient-based methods. This finding is in agreement
with previous oceanography studies that have similarly found
gradient methods to be highly sensitive to vertical resolution
(e.g. Lorbacher et al., 2006; Thomson and Fine, 2003). In
addition, however, the performance and threshold sensitiv-
ity of all methods also fluctuated temporally as influenced
by changes in the water column structure. Assessment of the
uncertainty associated with epilimnion depth estimates may
be useful, particularly for studies comparing the epilimnion
depth between periods of time that vary in stratification in-
tensity. Code and data availability. The analysis codes and output data
are stored in HydroShare http://www.hydroshare.org/resource/
26dbc260405b4bb9b3ac16ec55432684 (last access: 19 Novem-
ber 2020) (Wilson et al., 2020). Source code of the model GOTM is
freely available online at https://gotm.net/ (last access: 25 June 2020
(GOTM, 2020). The data used in this study from Lough Feeagh are
available online at https://doi.org/10.20393/6C4760C2-7392-4347-
8555-28BA0DAD0297 (de Eyto et al., 2020). Although long-term epilimnion depth trends are only
rarely reported directly (e.g. Hondzo and Stefan, 1993; Fee
et al., 1996; Sahoo et al., 2013), they are embedded in our
understanding of many climate-related variables. For exam-
ple, the epilimnion depth plays a key role in modulating the
effects of eutrophication, browning, and climate change on
lake water surface and epilimnetic temperatures (Persson and
Jones, 2008; Flaim et al., 2016; Strock et al., 2017; Bar-
tosiewicz et al., 2019). As such, changes in the epilimnion
depth may enhance or mute the effect of increasing incom-
ing heat on water surface temperatures and therefore may
be particularly important in explaining temporal and spatial
anomalies in surface temperature trends. Given the results
of this study, it may be that long-term trends calculated using
different metrics relate to fundamentally different parts of the
water column that may be undergoing different changes due
to climate change. Therefore, the strength and even the di-
rection of long-term trends in the epilimnion may be highly
dependent on the definition used (Yang and Wang, 2009; So-
mavilla et al., 2017). Supplement. The supplement related to this article is available on-
line at: https://doi.org/10.5194/hess-24-5559-2020-supplement. Author contributions. The original study concept was devised
by IDJ, MEP, EJ, and DP. H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes Funding was acquired by EJ, DP, IDJ,
HPG, EdE and RIW. EJ and AR were the project administrators. Data were curated by EdE and DP. The investigation was carried
out by HLW, AIA, IJ, EJ, DP, HPG and RIW. The formal analy-
sis was done by HLW and AIA. HLW was supervised by EJ, IDJ,
HPG, DP and AR. The draft was written by HLW, and all authors
contributed to the review and editing process. Competing interests. The authors declare that they have no conflict
of interest. Acknowledgements. This research was carried out as part of the
MANTEL Innovative Training Network, which has received fund-
ing from the European Union’s Horizon 2020 research and in-
novation programme under the Marie Skłodowska-Curie grant
agreement no. 722518 (H2020-MSCA-ITN-2016). In addition,
R. Iestyn Woolway received funding from the European Union’s
Horizon 2020 research and innovation programme under the
Marie Skłodowska-Curie grant agreement no. 791812. The long-
term monitoring program of Lough Feeagh is enabled by the field
staff of the Marine Institute (Ireland), and we gratefully acknowl-
edge their support. The monitoring program at Erken lake received
financial support from the Swedish Infrastructure for Ecosystem
Science (SITES). H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes Micro-profile studies also demonstrate that within
seemingly uniform layers there are micro-stratification lay-
ers, delineated by temperature differences as small as 0.02 ◦C
(Imberger, 1985; Shay and Gregg, 1986; MacIntyre, 1993;
Jonas et al., 2003), which can be sufficient to isolate in-
termediate layers from atmospheric wind shear and cool-
ing (Pernica et al., 2014). Although our results are not di-
rectly indicative of measured data, they demonstrate how One of the main goals behind the global collection of
high-frequency data in lakes is to understand how physical
processes vary between lakes, which indicates how different
lakes may respond to changing climatic conditions (Weath-
ers et al., 2013; Kraemer et al., 2015; Woolway and Mer-
chant, 2019). In order to understand this, we require methods
that perform consistently between lakes and over longitudi-
nal scales. The differences between the two lakes studied, in
particular variability in water column structure, the strength
of density gradients, and the vertical resolution of sensor de-
ployment influenced the level of agreement between epil-
imnion depth methods. Overall, Erken lake had much greater
agreement among methods than Lough Feeagh. In particular,
we found this to be related to the difference in vertical reso-
lution of the measured data between sites. Of all the methods
considered in this study, our results suggest that the absolute
difference from the surface method, M1, might be more use- https://doi.org/10.5194/hess-24-5559-2020 Hydrol. Earth Syst. Sci., 24, 5559–5577, 2020 5574 5
Conclusion This study has demonstrated that different definitions of
the epilimnion depth lead to different locations of the epil-
imnion depth in the water column and produce very dif-
ferent and contradictory temporal patterns. These results
have wide-reaching relevance in limnology, including stud-
ies interested in metabolism, eutrophication, and hypolim-
netic anoxia. The sensitivity of epilimnion depth methods
to temporal and spatial characteristics, such as morphol-
ogy, water column structure, and vertical resolution of data
measurements may also pose challenges for studies inter-
ested in long-term trends or global lake comparison studies. While there is no prescribed rationale for selecting a particu-
lar method, the M1 method, defined as the shallowest depth
where the density was 0.1 kg m−3 more than the surface den-
sity, was shown to be particularly insensitive to the vertical
sensor resolution of water temperature data, while the tempo-
ral pattern was relatively robust to changes in the threshold Financial support. This research has been supported by the Eu-
ropean Union’s Horizon 2020 Marie Skłodowska-Curie grant
no. 722518 (H2020-MSCA-ITN-2016) and grant no. 791812
(R. Iestyn Woolway). References Andersen, M. R., Sand-Jensen, K., Woolway, R. I., and Jones, I. D.:
Profound daily vertical stratification and mixing in a small, shal-
low, wind-exposed lake with submerged macrophytes, Aquat. Sci., 79, 395–406, https://doi.org/10.1007/s00027-016-0505-0,
2017. Diehl, S., Berger, S., Ptacnik, R., and Wild, A.: Phytoplankton,
light, and nutrients in a gradient of mixing depths: field ex-
periments, Ecology, 83, 399–411, https://doi.org/10.1890/0012-
9658(2002)083[0399:PLANIA]2.0.CO;2, 2002. Dong, F., Mi, C., Hupfer, M., Lindenschmidt, K. E., Peng, W., Liu,
X., and Rinke, K.: Assessing vertical diffusion in a stratified lake
using a three dimensional hydrodynamic model, Hydrol. Pro-
cess., 34, 1131–1143, https://doi.org/10.1002/hyp.13653, 2019. Ayala, A. I., Moras, S., and Pierson, D. C.: Simulations of future
changes in thermal structure of Lake Erken: proof of concept
for ISIMIP2b lake sector local simulation strategy, Hydrol. Earth
Syst. Sci., 24, 3311–3330, https://doi.org/10.5194/hess-24-3311-
2020, 2020. Engelhardt, C. and Kirillin, G.: Criteria for the onset and breakup of
summer lake stratification based on routine temperature measure-
ments, Fundament. Appl. Limnol./Arch. Hydrobiol., 184, 183–
194, https://doi.org/10.1127/1863-9135/2014/0582, 2014. Bartosiewicz, M., Przytulska, A., Lapierre, J. F., Laurion, I.,
Lehmann, M. F., and Maranger, R.: Hot tops, cold bottoms:
Synergistic climate warming and shielding effects increase car-
bon burial in lakes, Limnol. Oceanogr. Lett., 4, 132–144,
https://doi.org/10.1002/lol2.10117, 2019. Fee, E. J., Hecky, R. E., Kasian, S. E. M., and Cruikshank, D. R.: Ef-
fects of lake size, water clarity, and climatic variability on mixing
depths in Canadian Shield lakes, Limnol. Oceanogr., 41, 912–
920, https://doi.org/10.4319/lo.1996.41.5.0912, 1996. Berger,
S. A.,
Diehl,
S.,
Kunz,
T. J.,
Albrecht,
D.,
Oucible,
A. M.,
and
Ritzer,
S.:
Light
supply,
plank-
ton biomass, and seston stoichiometry in a gradient of
lake
mixing
depths,
Limnol. Oceanogr.,
51,
1898–1905,
https://doi.org/10.4319/lo.2006.51.4.1898, 2006. Flaim, G., Eccel, E., Zeileis, A., Toller, G., Cerasino, L., and
Obertegger, U.: Effects of re-oligotrophication and climate
change on lake thermal structure, Freshwater Biol., 61, 1802–
1814, https://doi.org/10.1111/fwb.12819, 2016. Bouffard, D. and Wüest, A.: Mixing in stratified lakes and
reservoirs,
in:
Mixing
and
dispersion
in
flows
Domi-
nated by Rotation and Buoyancy, Springer, Cham, 61–88,
https://doi.org/10.1007/978-3-319-66887-1_3, 2018. Foley, B., Jones, I. D., Maberly, S. C., and Rippey, B.: Long-term
changes in oxygen depletion in a small temperate lake: effects
of climate change and eutrophication, Freshwater Biol., 57, 278–
289, https://doi.org/10.1111/j.1365-2427.2011.02662.x, 2012. Brainerd, K. E. and Gregg, M. C.: Surface mixed and mix-
ing layer depths, Oceanogr. Literat. Rev., 4, 1521–1544,
https://doi.org/10.1016/0967-0637(95)00068-H, 1996. Giling, D. P., Staehr, P. A., Grossart, H. References P., Andersen, M. R.,
Boehrer, B., Escot, C., Evrendilek, F., Gómez-Gener, L., Honti,
M., Jones, I. D., and Karakaya, N.: Delving deeper: Metabolic
processes in the metalimnion of stratified lakes, Limnol. Oceanogr., 62, 1288–1306, https://doi.org/10.1002/lno.10504,
2017. Burchard, H., Bolding, K., and Villarreal, M. R.: GOTM. a Gen-
eral Ocean Turbulence Model, Theory, implementation and test
cases, Technical Report EUR 18745 EN, European Commission,
1999. GOTM: Software, available at: https://gotm.net/portfolio/software/,
last access: 25 June 2020. Butcher, J. B., Nover, D., Johnson, T. E., and Clark, C. M.:
Sensitivity
of
lake
thermal
and
mixing
dynam-
ics to climate change,
Climatic Change, 129, 295–305,
https://doi.org/10.1029/2010WR009104, 2015. Goudsmit, G.H., Burchard, H., Peeters, F., and Wüest, A.: Ap-
plication of k-ϵ turbulence models to enclosed basins: The
role of internal seiches, J. Geophys. Res.-Oceans, 107, 3230,
https://doi.org/10.1029/2001JC000954, 2002. Calderó-Pascual, M., de Eyto, E., Jennings, E, Dillane, M., An-
dersen, M., Kelly, S., Wilson, H., and McCarthy, V.: Effects of
consecutive extreme weather events on a temperate dystrophic
lake: A detailed insight into physical, chemical and biological
responses, Water, 12, 1411, https://doi.org/10.3390/w12051411,
2020. Gray, E., Mackay, E. B., Elliott, J. A., Folkard, A. M., and Jones, I. D.: Wide-spread inconsistency in estimation of lake mixed depth
impacts interpretation of limnological processes, Water Res.,
168,
115136,
https://doi.org/10.1016/j.watres.2019.115136,
2020. Coloso, J. J., Cole, J. J., and Pace, M. L.: Short-term variation in
thermal stratification complicates estimation of lake metabolism,
Aquat. Sci., 73, 305–315, https://doi.org/10.1007/s00027-010-
0177-0, 2011. Hondzo, M. and Stefan, H. G.: Regional water temperature charac-
teristics of lakes subjected to climate change, Climatic Change,
24, 187–211, https://doi.org/10.1007/BF01091829, 1993. Huisman, J. E. F., van Oostveen, P., and Weissing, F. J.: Critical
depth and critical turbulence: two different mechanisms for the
development of phytoplankton blooms, Limnol. Oceanogr, 44,
1781–1787, https://doi.org/10.4319/lo.1999.44.7.1781, 1999. de Boyer Montégut, C., Madec, G., Fischer, A. S., Lazar,
A., and Iudicone, D.: Mixed layer depth over the global
ocean:
An
examination
of
profile
data
and
a
profile-
based climatology, J. Geophys. Res.-Oceans, 109, C12003,
https://doi.org/10.1029/2004JC002378, 2004. Imberger, J.: The diurnal mixed layer, Limnol. Oceanogr., 30, 737–
770, https://doi.org/10.4319/lo.1985.30.4.0737, 1985. de Eyto, E., Jennings, E., Ryder, E., Sparber, K., Dillane,
M., Dalton, C., and Poole, R.: Response of a humic lake
ecosystem to an extreme precipitation event: physical, chem-
ical, and biological implications, Inland Waters, 6, 483–498,
https://doi.org/10.1080/IW-6.4.875, 2016. Jassby, A. and Powell, T.: Vertical patterns of eddy diffusion during
stratification in Castle Lake, California, Limnol. Financial support. This research has been supported by the Eu-
ropean Union’s Horizon 2020 Marie Skłodowska-Curie grant
no. 722518 (H2020-MSCA-ITN-2016) and grant no. 791812
(R. Iestyn Woolway). Financial support. This research has been supported by the Eu-
ropean Union’s Horizon 2020 Marie Skłodowska-Curie grant
no. 722518 (H2020-MSCA-ITN-2016) and grant no. 791812
(R. Iestyn Woolway). Financial support. This research has been supported by the Eu-
ropean Union’s Horizon 2020 Marie Skłodowska-Curie grant
no. 722518 (H2020-MSCA-ITN-2016) and grant no. 791812
(R. Iestyn Woolway). Review statement. This paper was edited by Anas Ghadouani and
reviewed by two anonymous referees. https://doi.org/10.5194/hess-24-5559-2020 Hydrol. Earth Syst. Sci., 24, 5559–5577, 2020 H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes 5575 stitute, Ireland, https://doi.org/10.20393/6C4760C2-7392-4347-
8555-28BA0DAD0297, 2020. H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes 5576 57, 589–601, https://doi.org/10.1111/j.1365-2427.2011.02729.x,
2012. 57, 589–601, https://doi.org/10.1111/j.1365-2427.2011.02729.x,
2012. Monismith, S. G. and MacIntyre, S.: The surface mixed layer
in lakes and reservoirs, in: Encyclopedia of inland waters,
edited by: Linkens, G. E., Academic Press, Oxford, 636–650,
https://doi.org/10.1016/B978-012370626-3.00078-8, 2009. Jennings, E., de Eyto, E., Laas, A., Pierson, D., Mircheva, G., Nau-
moski, A., Clarke, A., Healy, M., Šumberová, K., and Langen-
haun, D.: The NETLAKE Metadatabase – A Tool to Support
Automatic Monitoring on Lakes in Europe and Beyond, Limnol. Oceanogr. Bull., 26, 95–100, https://doi.org/10.1002/lob.10210,
2017. Moras, S., Ayala, A. I., and Pierson, D. C.: Historical modelling of
changes in Lake Erken thermal conditions, Hydrol. Earth Syst. Sci., 23, 5001–5016, https://doi.org/10.5194/hess-23-5001-2019,
2019. Nash, J. E. and Sutcliffe, J. V.: River flow forecasting through con-
ceptual models part I – A discussion of principles, J. Hydrol., 10,
282–290, https://doi.org/10.1016/0022-1694(70)90255-6, 1970. Jonas, T., Stips, A., Eugster, W., and Wüest, A.: Observations of a
quasi shear-free lacustrine convective boundary layer: Stratifica-
tion and its implications on turbulence, J. Geophys. Res.-Oceans,
108, 3328, https://doi.org/10.1029/2002JC001440, 2003. Obrador,
B.,
Staehr,
P. A.,
and
Christensen,
J. P.:
Ver-
tical
patterns
of
metabolism
in
three
contrasting
stratified
lakes,
Limnol. Oceanogr.,
59,
1228–1240,
https://doi.org/10.4319/lo.2014.59.4.1228, 2014. Kara, A. B., Rochford, P. A., and Hurlburt, H. E.: Mixed layer depth
variability over the global ocean, J. Geophys. Res.-Oceans, 108,
3079, https://doi.org/10.1029/2000JC000736, 2003. Pernica, P., Wells, M. G., and MacIntyre, S.: Persistent weak ther-
mal stratification inhibits mixing in the epilimnion of north-
temperate Lake Opeongo, Canada, Aquat. Sci., 76, 187–201,
https://doi.org/10.1007/s00027-013-0328-1, 2014. Kraemer, B. M.: Rethinking discretization to advance limnol-
ogy amid the ongoing information explosion, Water Res., 178,
115801, https://doi.org/10.1016/j.watres.2020.115801, 2020. Kraemer, B. M., Anneville, O., Chandra, S., Dix, M., Kuu-
sisto, E., Livingstone, D. M., Rimmer, A., Schladow, S. G.,
Silow, E., Sitoki, L. M., and Tamatamah, R.: Morphom-
etry and average temperature affect lake stratification re-
sponses to climate change, Geophys. Res. Lett., 42, 4981–4988,
https://doi.org/10.1002/2015GL064097, 2015. Persson, I. and Jones, I. D.: The effect of water colour on lake hy-
drodynamics: A modelling study, Freshwater Biol., 53, 2345–
2355, https://doi.org/10.1111/j.1365-2427.2008.02049.x, 2008. Pierson, D. C., Weyhenmeyer, G. A., Arvola, L., Benson, B.,
Blenckner, T., Kratz, T., Livingstone, D. M., Markensten, H.,
Marzec, G., Pettersson, K., and Weathers, K.: An automated
method to monitor lake ice phenology, Limnol. Oceanogr.:
Meth., 9, 74–83, https://doi.org/10.4319/lom.2010.9.0074, 2011. Lamont, G., Laval, B., Pawlowicz, R., Pieters, R., and Lawrence, G. H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes A.: Physical mechanisms leading to upwelling of anoxic bottom
water in Nitinat Lake, in: 17th ASCE Engineering Mechanisms
Conference, 13–16 June 2004, University of Delaware, Newark,
USA, 2004. Pujoni, D. G., Brighenti, L. S., Bezerra-Neto, J. F., Barbosa,
F. A., Assunção, R. M., and Maia-Barbosa, P. M.: Model-
ing vertical gradients in water columns: A parametric autore-
gressive approach, Limnol. Oceanogr.: Meth., 17, 320–329,
https://doi.org/10.1002/lom3.10316, 2019. Livingstone,
D. M.:
Impact
of
secular
climate
change
on
the
thermal
structure
of
a
large
temperate
cen-
tral
European
lake,
Climatic
Change,
57,
205–225,
https://doi.org/10.1023/A:1022119503144, 2003. Read, J. S., Hamilton, D. P., Jones, I. D., Muraoka, K., Winslow,
L. A., Kroiss, R., Wu, C. H., and Gaiser, E.: Derivation
of lake mixing and stratification indices from high-resolution
lake buoy data, Environ. Model. Softw., 26, 1325–1336,
https://doi.org/10.1016/j.envsoft.2011.05.006, 2011. Lorbacher, K., Dommenget, D., Niiler, P. P., and Köhl, A.:
Ocean mixed layer depth: A subsurface proxy of ocean at-
mosphere variability, J. Geophys. Res.-Oceans, 111, C07010,
https://doi.org/10.1029/2003JC002157, 2006. Maberly, S. C., O’Donnell, R. A., Woolway, R. I., Cutler, M. E.,
Gong, M., Jones, I. D., Merchant, C. J., Miller, C. A., Politi,
E., Scott, E. M., and Thackeray, S. J.: Global lake thermal
regions shift under climate change, Nat. Commun., 11, 1–9,
https://doi.org/10.1038/s41467-020-15108-z, 2020. Rivetti, I., Boero, F., Fraschetti, S., Zambianchi, E., and Li-
onello, P.: Anomalies of the upper water column in the
Mediterranean
Sea,
Global
Planet. Change,
151,
68–79,
https://doi.org/10.1016/j.gloplacha.2016.03.001, 2017. Sachse, R., Petzoldt, T., Blumstock, M., Moreira Martinez,
S., Pätzig, M., Rücker, J., Janse, J., Mooij, W. M., and
Hilt, S.: 485 Extending one-dimensional models for deep
lakes to simulate the impact of submerged macrophytes
on water quality, Environ. Model. Softw., 61, 410–423,
https://doi.org/10.1016/j.envsoft.2014.05.023, 2014. MacIntyre,
S.:
Vertical
mixing
in
a
shallow,
eutrophic
lake:
Possible
consequences
for
the
light
climate
of
phytoplankton,
Limnol. Oceanogr.,
38,
798–817,
https://doi.org/10.4319/lo.1993.38.4.0798, 1993. MacIntyre, S. and Melack, J. M.: Mixing Dynamics in Lakes
Across Climatic Zones, in: Encyclopedia of inland waters,
edited by: Linkens, G. E., Academic Press, Oxford, 603–612,
https://doi.org/10.1016/B978-012370626-3.00040-5, 2009. Sahoo, G. B., Schladow, S. G., Reuter, J. E., Coats, R., Dettinger,
M., Riverson, J., Wolfe, B., and Costa-Cabral, M.: The response
of Lake Tahoe to climate change, Climatic Change, 116, 71–95,
https://doi.org/10.1007/s10584-012-0600-8, 2013. Marcé, R., George, G., Buscarinu, P., Deidda, M., Dunalska, J.,
de Eyto, E., Flaim, G., Grossart, H. References Oceanogr., 20,
530–543, https://doi.org/10.4319/lo.1975.20.4.0530, 1975. Jennings, E., Jones, S., Arvola, L., Staehr, P. A., Gaiser, E., Jones,
I. D., Weathers, K. C., Weyhenmeyer, G. A., Chiu, C. Y., and
De Eyto, E.: Effects of weather-related episodic events in lakes:
an analysis based on high-frequency data, Freshwater Biol., de Eyto, E., Dillane, M., Moore, T., Wilson, H., Cooney, J.,
Hughes, P., Murphy, M., Nixon, P., Sweeney, D., and Poole,
R.: Lough Feeagh water temperature profiles, Marine In- Hydrol. Earth Syst. Sci., 24, 5559–5577, 2020 https://doi.org/10.5194/hess-24-5559-2020 H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes 5577 Oceanogr.,
16,
1777–1798,
https://doi.org/10.1175/1520-
0485(1986)016<1777:CDTMIT>2.0.CO;2, 1986. Oceanogr.,
16,
1777–1798,
https://doi.org/10.1175/1520-
0485(1986)016<1777:CDTMIT>2.0.CO;2, 1986. Winslow, L. A., Zwart, J. A., Batt, R. D., Dugan, H. A., Woolway,
R. I., Corman, J. R., Hanson, P. C., and Read, J. S.: LakeMetab-
olizer: an R package for estimating lake metabolism from free-
water oxygen using diverse statistical models, Inland Waters, 6,
622-636, https://doi.org/10.1080/IW-6.4.883, 2016. Somavilla, R., González-Pola, C., and Fernández-Diaz, J.: The
warmer the ocean surface, the shallower the mixed layer. How
much of this is true?, J. Geophys. Res.-Oceans, 122, 7698–7716,
https://doi.org/10.1002/2017JC013125, 2017. Woolway, R. I. and Merchant, C. J.: Worldwide alteration of lake
mixing regimes in response to climate change, Nat. Geosci., 12,
271–276, https://doi.org/10.1038/s41561-019-0322-x, 2019. Strock, K. E., Theodore, N., Gawley, W. G., Ellsworth, A. C., and
Saros, J. E.: Increasing dissolved organic carbon concentrations
in northern boreal lakes: Implications for lake water transparency
and thermal structure, J. Geophys. Res.-Biogeo., 122, 1022–
1035, https://doi.org/10.1002/2017JG003767, 2017. Woolway, R. I., Maberly, S. C., Jones, I. D., and Feuchtmayr, H.: A
novel method for estimating the onset of thermal stratification in
lakes from surface water measurements, Water Resour. Res., 50,
5131–5140, https://doi.org/10.1002/2013WR014975, 2014. Tedford, E. W., MacIntyre, S., Miller, S. D., and Czikowsky, M. J.: Similarity scaling of turbulence in a temperate lake dur-
ing fall cooling, J. Geophys. Res.-Oceans, 119, 4689–4713,
https://doi.org/10.1002/2014JC010135, 2014. Wüest,
A. and
Lorke,
A.. Small-scale
hydrodynam-
ics
in
lakes,
Annu. Rev. Fluid
Mech.,
35,
373–412,
https://doi.org/10.1146/annurev.fluid.35.101101.161220, 2003. Thiery, W., Davin, E. L., Panitz, H. J., Demuzere, M., Lher-
mitte, S., and Van Lipzig, N.: The impact of the African Great
Lakes on the regional climate, J. Climate, 28, 4061–4085,
https://doi.org/10.1175/JCLI-D-14-00565.1, 2015. Wüest, A. and Lorke, A.: Small-Scale Turbulence and Mixing:
Energy Fluxes in Stratified Lakes, in: Encyclopedia of Inland
Waters, Elsevier B. V., Amsterdam, the Netherlands, 628–635,
https://doi.org/10.1016/B978-012370626-3.00084-3, 2009. Yang, H. and Wang, F.: Revisiting the thermocline depth
in
the
equatorial
Pacific,
J. Climate,
22,
3856–3863,
https://doi.org/10.1175/2009JCLI2836.1, 2009. Thomson, R. E. and Fine, I. V.: Estimating mixed layer
depth
from
oceanic
profile
data,
J. Atmos. Ocean. Tech.,
20,
319–329,
https://doi.org/10.1175/1520-
0426(2003)020<0319:EMLDFO>2.0.CO;2, 2003. Yang, Y., Colom, W., Pierson, D., and Pettersson, K.: Water col-
umn stability and summer phytoplankton dynamics in a tem-
perate lake (Lake Erken, Sweden), Inland Waters, 6, 499–508,
https://doi.org/10.1080/IW-6.4.874, 2016. Weathers, K. C., Hanson, P. C., Arzberger, P., Brentrup, J.,
Brookes, J., Carey, C. C., Gaiser, E., Gaiser, E., Hamilton, D. P., Hong, G. H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes P., Istvanovics, V., Lenhardt,
M., and Moreno-Ostos, E.: Automatic high frequency monitoring
for improved lake and reservoir management, Environ. Sci. Tech-
nol., 50, 10780–10794, https://doi.org/10.1021/acs.est.6b01604,
2016. Schwefel, R., Gaudard, A., Wüest, A., and Bouffard, D.: Ef-
fects of climate change on deepwater oxygen and winter mix-
ing in a deep lake (Lake Geneva): Comparing observational
findings and modeling, Water Resour. Res., 52, 8811–8826,
https://doi.org/10.1002/2016WR019194, 2016. Martin, J. and McCutcheon, M.: Hydrodynamics and Transport for
Water Quality Modeling, Lewis Publishers, New York, USA,
1999. Shay,
T. J. and
Gregg,
M. C.:
Convectively
driven
turbulent
mixing
in
the
upper
ocean,
J. Phys. Hydrol. Earth Syst. Sci., 24, 5559–5577, 2020 https://doi.org/10.5194/hess-24-5559-2020 Hydrol. Earth Syst. Sci., 24, 5559–5577, 2020 https://doi.org/10.5194/hess-24-5559-2020 H. L. Wilson et al.: Variability in epilimnion depth estimations in lakes S., and Ibelings, B.: The Global Lake Ecolog-
ical Observatory Network (GLEON): the evolution of grass-
roots network science, Limnol. Oceanogr. Bull., 22, 71–73,
https://doi.org/10.1002/lob.201322371, 2013. Zorzal-Almeida, S., Soininen, J., Bini, L. M., and Bicudo,
D. C.: Local environment and connectivity are the main
drivers of diatom species composition and trait variation in
a set of tropical reservoirs, Freshwater Biol., 62, 1551–1563,
https://doi.org/10.1111/fwb.12966, 2017. Wilson, H., Ayala, A. I., Rolston, A., Pierson, D., de Eyto,
E., Grossart, H., Perga, M., Woolway, I., and Jennings, E.:
Variability in epilimnion depth estimations in lakes, Hy-
droShare, available at: https://www.hydroshare.org/resource/
26dbc260405b4bb9b3ac16ec55432684/, last access: 19 Novem-
ber 2020. Hydrol. Earth Syst. Sci., 24, 5559–5577, 2020 https://doi.org/10.5194/hess-24-5559-2020
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�ber einige neue Verbindungen von Stickstoff und Wasserstoff mit Metallen
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Monatshefte für Chemie
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Uber einige neue Verbindungen yon Stickstoff
und Wasserstoff mit Netallen
(VorI/iufige Mitteilung)
von
F. W . Dafert und R. Miklauz.
( V o r g e t e g t in der S i t z u n g a m 24. J u n i 1909.)
Nach den Beobachtungen von H. M o i s s a n 1 beginnt
Calciumnitrid, das im Wasserstoffstrom erhitzt wird, oberhalb
Rotglut in Hydrid fiberzugehen. ,,Seine Farbe wird immer
heller und wenn man die Temperatur steigert, nimmt die
gebildete Menge yon Hydrid zu.<, Gelegentlich yon Versuchen
technischer Natur erschien es uns angezeigt, diesen ProzeI3
n/iher zu studieren und vor allem quantitativ zu verfolgen,
5.hnlich wie dies, freilich in anderer Richtung und zu anderen
Zwecken, F. H a b e r und G. v a n O o r d t 2 getan haben. Die
letztgenannten Autoren fassen ihre Beobachtungen in den Satz
zusammen: ,,Es wurde gezeigt, dab die umkehrbare Reaktion
C % N , + 3 H ~ ~ 3 C a H 2 + N . sich bei Rotglut leicht verwirklicht
und mit der Reaktion N , + 3 H , ~ 2 N H 3 verkniipft. In l )bereinstimmung mit der Theorie tritt bei der Einwirkung yon
Wasserstoff auf Calciumnitrid Ammoniak in nachweisbaren
Mengen auf, bei der Einwirkung yon Stickstoff auf Calciumhydrid abet nicht. Das Temperaturgebiet, in welchem die
Calciumverbindungen als ZwischenkSrper bei der Ammoniaksynthese aus den Elementen bentitzt werden kSnnen, liegt z u
hoch, als dab erhebliche Ausbeuten an Ammoniak mit ihrer
1 H. M o i s s a n ,
Compt. rend., 127,31 u n d 4 9 8 (1898).
2 F. H a b e r und O. v a n O o r d t , Zeitschrift ffir anorgan. Chemie, 4d:
357 (1905).
Chemie-Heft Nr. 8.
46
t350
F.W. Dafert
und R. M i k l a u z ,
Hilfe erreichbar w~iren.<< Bei der Behandlung von Nitrid mit
W a s s e r s t o f f erhielten sie nicht mehr als 15 Teile Hydrid auf
100 Teile unver/inderten Nitrids, und w e n n sie Stickstoff auf das
H y d r i d einwirken liel3en, ein Produkt, das eine ziemlich ~ihnliche
Z u s a m m e n s e t z u n g hatte. Es gelang ihnen somit nicht, das
Nitrid vollstg,ndig in Hydrid und umgekehrt, das Hydrid in
Nitrid zu verwandeln.
Wir sind bei unseren Arbeiten, die - - wie gleich bier
betont sei - - ungew/3hnliche experimentelle Schwierigkeiten
bieten und daher nur langsam fortschreiten, gleichfalls v o n d e r
A n n a h m e ausgegangen, dal3 es sich bei der U m w a n d l u n g des
Calciumnitrids in Calciumhydrid tats/ichlich um einen einfachen
A u s t a u s c h des Stickstoffs gegen Wasserstoff handelt, erkannten
a b e t alsbald, daft dies nur bedingt richtig ist. Erhitzt man reines
Calciumnitrid in vollkommen trockenem und yon allen sch~idlichen Nebenbestandteilen befreitem Wasserstoffgas innerhalb
bestimmter, ziemlich weiter T e m p e r a t u r g r e n z e n (urn 700 ~ C.),
so tritt nicht die erwartete Gewichtsabnahme ein, sondern unter
lebhafter Wasserstoffabsorption eine Gewichts z u n a h m e, die bei
u n s e r e n Versuchen yon 2" 16 bis 2" 8 4 % schwankte. Daraus geht
hervor, dab zunS.chst nicht das Calciumnitrid zersetzt wird,
sondern, daft tiberraschenderweise eine Addition yon Wasserstoff
z u m Calciumnitrid stattfindet. Hierbei entsteht, wie sich gezeigt
hat, eine ganz neue chemische Verbindung, deren ~iul3ere Merkmale sehr charakteristisch sind. Ihre Farbe ist im Unterschiede
vom Hydrid und dem d u n k e l r o t b r a u n e n Nitrid z i t r o n e n - b i s
o r a n g e g e l b , ein Umstand, der die von frtiheren Beobachtern 1
bemerkte Gelbf/irbung des Nitrids im Wasserstoffstrom bei
Rotglut erkl~irt. Die Substanz verg.ndert sich in der N/ihe der
Bildungstemperatur, soweit unsere Hilfsmittel dies bis jetzt
festzustellen erlaubten, weder durch Erhitzen im Wasserstoffstrom, noch dutch die E i n w i r k u n g yon Stickstoff in nennenswertem Ausmal3, zeichnet sich aber durch eine merkwtirdige
Eigenschaft, durch starke Lichtempfindlichkeit, aus. Das zerstreute Tageslicht verwandelt sie nach und nach, das Sonnenlicht sehr rasch ohne merkliche GewichtsverS, nderung in einen
1 F. H a b e r
und G. v a n O o r d t ,
a. a. 0.~ p. 363.
Wasserstoff- und Stickstoffverbindungen mit Metallen.
651
g r a u s c h w a r z e n K6rper, aus dem beim E r w g r m e n im Stickstoffstrom a n s c h e i n e n d die ursprtingliche gelbe S u b s t a n z regeneriert
wird. Mit W a s s e r bildet sich A m m o n i a k und W a s s e r s t o f f g a s .
Im Sauerstoffstrom verbrennt diese V e r b i n d u n g n u r schwierig
und u n v o l l k o m m e n , so daft die E l e m e n t a r a n a l y s e auf bisher noch
nicht t i b e r w u n d e n e Hindernisse stSi3t. W i r mui3ten uns damit
begntigen, den Calciumgehalt direkt zu ermitteln und zur vor1/iufigen Beurteilung der Z u s a m m e n s e t z u n g die W e r t e heranzuziehen, welche sich ftir den Stickstoff aus der Menge d e s
bei der B e h a n d l u n g der S u b s t a n z mit W a s s e r abgespalteten
A m m o n i a k s und ftir den W a s s e r s t o f f aus der G e w i c h t s z u n a h m e
berechnen. Mit den g a s v o l u m e t r i s c h g e f u n d e n e n Zahlen fiir
W a s s e r s t o f f ltif3t sich nicht gut operieren, weil sie je nach der
u n b e k a n n t e n Konstitution der betreffenden S u b s t a n z entweder
zur H/ilfte oder voll in R e c h n u n g zu setzen sind. 1 Auf diesem
W e g e gelangen wir zu dem Schlusse, daft die g e l b e S u b s t a n z
durchschnittlich enth~tlt:
Calcium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Stickstoff . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Wasserstoff ...........................
Eisen, Silicium, Kohle, S a u e r s t o f f u s w . . . . . .
78" 05 ~
16" 66
2" 50
2" 79
Daraus b e r e c h n e t sich ftir die reine S u b s t a n z :
Calcium . . . . . . . . . . . . . . . . . . . . . .
Stickstoff . . . . . . . . . . . . . . . . . . . . .
Wasserstoff ...................
80" 3 ~
17" 1
2" 6
G a n z /ihnliche W e r t e e r g e b e n sich ftir das s c h w a r z e
Umwandlungsprodukt.
Es w~ire verfrtiht, aus diesen provisorischen Zahlen endgtiltige Schltisse auf die Konstitution ziehen z u wollen; immerhin
gestatten sie, uns eine Vorstellung yon den Vorg/ingen zu bilden,
welche sich m~Sglicherweise hier abspieten. Die Z u s a m m e n x Wie sieh F. H a b e r und v a n O o r d t mit diesem Umstande abgefunden
haben, ist nicht reeht ersichtlich. Sie berechnen ihre Wasserstoffzahlen nach der
Gleichung CaH2-+-H20 = CaO-t-H4, wiihrend doch aueh Ca3(NH2)2-b 3H20
= 3CaO-+-2NH3--t-HI, ja sogar CaNH--t-H~O = CaO-+-NH a in Betracht
kommt.
46*
652
F~ W. I ) ~ f e r t urtd R., M i k l a u ~ , ,
s e t z u n g entspficb_t am: bestea der Formel Caa;N2 H , mit felgenden
theoretischen C-ehaiten:
Calcium . . . . . . . . . . . . . . . . . . . . . .
Stickstoff . . . . . . . . . . . . . . . . . . . . .
Wasserstoff . . . . . . . . . . . . . . . . . . .
78" 96 ~
I8-40
2-64
Es wfirde sich demnach, woffir die giatte Addition spricht,
zun~ichst aus dem Calciumnitrid ein auch theoretisch interessantes, in der Hitze best~indiges T r i c a l c i u m a m i d
bilden:
Ca~ N~ + H ~ --. Ca 8 (NH2) ~.
Letzteres zerfgltt im Sonnenlicht in C a l c i u m i m f d
Calciumhydrid:
und
Ca 3 (NH~)2 - - 2 C a N H + CaH~.
Zu Gunste.n der Annahme, dab ein solches Tricalciumamid
existiert und eine - - unter den Bedingungen unserer Versuche - - sehr feste Verbindung darstetlt, spricht das Verhalten
des Hydrids gegen Stickstoff. Leitet man Stickstoff fiber
Calciumhydrid, so nimmt dessen Gewicht unter Wasserstoffabspaltung betr~ichtIich zu u n d e s bildet sich eine in ihren
~.ul3eren Merkmalen nach mit dem aus Nitrid hergestellten
Produkt sehr &hnliche oder identische Substanz, ebenso w e n n
man Gemenge yon Wasserstoff und Sfickstoff auf metallisches
Calcium einwirken lg.13t. Die genaue Ermittlung der Gewichtsz u n a h m e ist uns aber in diesen F~illen noch nicht gelungen,
sicher konnten wit nur beobachten, daft die Zunahme beim
Calciumhydrid nicht unter 1 7 % betrS.gt, wiihrend die T h e o r i e
20" 6 ~ e r f o r d e r t
Um in einem gewissen Sinne die Richtigkeit der gemachtet)
Beobachtungen z u fiberprtifen, studierten wit das Verhalten
anderer Metallnitride gegen Wasserstoff. Magnesiumnitrid zeigt
keine Neigung, W a s s e r s t o f f zu addieren, daffir fanden wir eine
urn, so gr6fiere bei Lithiumnitrid. Nach L. O u v r a r d I e n t w i c k e k
Lithiumnitrid beim Erhitzen im Wasserstoffstrom Ammoniak,
Lithitxmhydrid liefert dagegen nach G u n t z ~ beim B e h a n d e l n
1 L. O u v r a r d , Compt. rend., 11d~ 121 (1892).
2 O u n t z , ebenda, 122, 146 (1896), und 123, 997 (1896).
WassetstOff- und Stickstvffverbindungen mtt Metallen.
653
mit Stizksto~f unter Wasserstoffentwickl~ang Nitrid. G u n t z
schliel3t aus seinen Beobachtungen fiber die Bildungsw/irmen
des Li{hiumnitrids u n d -hydrids: ,,que l'hydrog~ne doit d4composer l'azoture de lithium et c'est ce q u e l'on constate assez
facilement en chauffant Li3Az dans u n courant d'hydrog~ne, car
on obtient de l ' h y d m r e e t de lithium faisant effervescence avec
l'eau en ddgageant de l'hydrog~ne. On peut c e p e n d a n t obtenir
la %action inverse et d6composer L i H dans un courant d'azote,
mais cette %action tient g la dissociation g haute temperature
de l'hydrure de lithium chauffd dans un courant d'azote qui
se combine en m6tal devenu libre.<, Alle diese Angaben
bedtirfen der Erg~inzung und Berichtigung.
Das s c h w a r z b r a u n e Lithiumnitrid nimmt beim Erwgtrmen
im Wasserstoffstrom unter bestimmten Verh~iltnissen sehr tebhaft W a s s e r s t o f f auf, und zwar durchschnittlich 5"40% , umgekehrt addiert Lithiumhydrid mindestens 51"090/0 Stickstoff.
Die entsprechenden weil3en Additionsprodukte zeigen in ihrem
Verhalten unleugbare ,~hnlichkeit mit jenen des Calciums. Der
F a r b e n u m s c h l a g erfolgt hier yon weil3 fiber r o s a nach rot, und
zwar noch schneller als beim Calcium. Die Regenerierung der
weil3en Form aus der roten ist uns bisher nicht gelungen, wohl
aber haben wir festgestellt, dal~ ein erheblicher Anteit des in
der letzteren enthaltenen Wasserstoffs bei der Behandlung mit
W a s s e r n i c h t als solch er in Freiheit gesetzt wird, also vermutlich
Imidwasserstoff ist. Die aus dem Lithiumnitrid entstehende
Verbindung muf~ etwa 5" 2 % W a s s e r s t o f f enthalten, was wiederum am besten mit dem T r i l i t h i u m a m i d
L i s N H . (Theorie
5 " 4 % ; Z u n a h m e bei der Bildung aus dem Hydrid 54" 1 ~ ) tibereinstimmt. Die Elementaranalyse bietet in diesem Falle weniger
Schwierigkeiten, um so grSflere die Darstellung einigermal3en
reiner Pr~iparate, weil Lithium und Lithiumhydrid alle Gefiif~e
heftig angreifen und man nur unter A n w e n d u n g gewisser Kunstgriffe dem Ziele niiher kommt. Das Verhalten des hypothetischen
Trilithiumamids gegen Licht l~iflt sich am einfachsten ebenfalls
als Imidbildung auffassen: Li 3 NH~ ~__ Li e NH + Li H. Die intensiv
rote Verbindung w~ire das bisher u n b e k a n n t e L i t h i u m i m i d
Lie NH in Mischung mit Lithiumhydrid, Fragen, auf die wit
selbstverstiindlich seinerzeit ausftihrlich zurtickkommen werden.
654
F.W. Dafertu.R. Miklauz, Wasserstoff- undStickstoffverb, etc.
Wir haben Grund z u r Annahme, da(3 auch andere Metallnitride sich analog verhalten und daft sich das yon uns einstweilen als Trilithiumamid a n g e s p r o c h e n e Additionsprodukt des
Wasserstoffs zum Lithiumnitrid, seiner grogen Reaktionsf/ihigkeit halber, als Mittel zur Herstellung direkt nicht erh/iltlicher
Hydronitride eignet. Z w e c k dieser vorl~iufigen Mitteilung ist, uns
das einscial/igige Arbeitsgebiet z u sichem.
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https://openalex.org/W2376087921
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https://pure.rug.nl/ws/files/56014082/7351985.pdf
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Activation Mechanism of LRRK2 and Its Cellular Functions in Parkinson’s Disease
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Parkinson's disease
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cc-by
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University of Groningen
Activation Mechanism of LRRK2 and Its Cellular Functions in Parkinson's Disease
Rosenbusch, Katharina E.; Kortholt, Arjan
Published in: University of Groningen
Activation Mechanism of LRRK2 and Its Cellular Functions in Parkinson's Disease
Rosenbusch, Katharina E.; Kortholt, Arjan
Published in: University of Groningen Activation Mechanism of LRRK2 and Its Cellular Functions in Parkinson's Disease
Rosenbusch, Katharina E.; Kortholt, Arjan Published in:
Parkinsons disease IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from
it. Please check the document version below. Document Version
Publisher's PDF, also known as Version of record IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from
it. Please check the document version below. Document Version Link to publication in University of Groningen/UMCG research database Citation for published version (APA):
Rosenbusch, K. E., & Kortholt, A. (2016). Activation Mechanism of LRRK2 and Its Cellular Functions in
Parkinson's Disease. Parkinsons disease, 2016, Article ID 7351985. https://doi.org/10.1155/2016/7351985 Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the
author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Department of Cell Biochemistry, University of Groningen, Nijenborgh 7, 9747 AG Groningen, Netherlands Department of Cell Biochemistry, University of Groningen, Nijenborgh 7, 9747 AG Groningen, Netherlands Correspondence should be addressed to Arjan Kortholt; a.kortholt@rug.nl Correspondence should be addressed to Arjan Kortholt; a.kortholt@rug.nl
Received 24 December 2015; Accepted 19 April 2016
Academic Editor: Jos´e Manuel Bravo-San Pedro Received 24 December 2015; Accepted 19 April 2016 Academic Editor: Jos´e Manuel Bravo-San Pedro Copyright © 2016 K. E. Rosenbusch and A. Kortholt. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited. Human LRRK2 (Leucine-Rich Repeat Kinase 2) has been associated with both familial and idiopathic Parkinson’s disease (PD). Although several LRRK2 mediated pathways and interaction partners have been identified, the cellular functions of LRRK2 and
LRRK2 mediated progression of PD are still only partially understood. LRRK2 belongs to the group of Roco proteins which
are characterized by the presence of a Ras-like G-domain (Roc), a C-terminal of Roc domain (COR), a kinase, and several
protein-protein interaction domains. Roco proteins exhibit a complex activation mechanism involving intramolecular signaling,
dimerization, and substrate/effector binding. Importantly, PD mutations in LRRK2 have been linked to a decreased GTPase and
impaired kinase activity, thus providing putative therapeutic targets. To fully explore these potential targets it will be crucial to
understand the function and identify the pathways responsible for LRRK2-linked PD. Here, we review the recent progress in
elucidating the complex LRRK2 activation mechanism, describe the accumulating evidence that link LRRK2-mediated PD to
mitochondrial dysfunction and aberrant autophagy, and discuss possible ways for therapeutically targeting LRRK2. Copyright The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license. More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-
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Parkinson’s Disease
Volume 2016, Article ID 7351985, 8 pages
http://dx.doi.org/10.1155/2016/7351985 Hindawi Publishing Corporation
Parkinson’s Disease
Volume 2016, Article ID 7351985, 8 pages
http://dx.doi.org/10.1155/2016/7351985 Hindawi Publishing Corporation
Parkinson’s Disease
Volume 2016, Article ID 7351985, 8 pages
http://dx.doi.org/10.1155/2016/7351985 2. LRRK2 Kinase and GTPase Activity Cytosol LRRK2 has two bona fide enzymatic activities via its Roc
(GTPase) and kinase domain. Several studies have shown that
the Serine/Threonine specific kinase activity is responsible for
LRRK2-mediated PD symptoms, including the degeneration
of nigrostriatal dopaminergic neurons and the formation
of Lewy bodies [2, 4, 12–14]. While PD-mutated LRRK2
triggers increased inclusion body formation in SH-SY5Y and
cell death in primary rat cortical neurons, both of these
phenotypes were diminished upon introduction of a LRRK2
kinase dead mutation [15]. For a long time, the function of
the kinase domain has been considered as the main output
of LRRK2. However, only for the G2019S PD mutation,
representing the most common pathogenic point mutation,
an increased phosphorylation activity has been reported [16–
18]. For other pathogenic mutations, inconsistent, modest,
or no effect on kinase activity has been shown [16–18]. Fur-
thermore, PD mutations in LRRK2 probably have different
defects in its activation mechanism and it is unclear if all
pathogenic effects are mediated via the kinase domain [17–
19]. Also the enzymatic activity of the Roc domain is affected
in LRRK2-mediated PD-mutants and recent data strongly
suggest that PD mutations in both Roc and COR domains
result in decreased GTP hydrolysis [18, 20–24]. The Roc
domain of LRRK2 belongs to the family of small G-proteins
which are GTP binding proteins switching between an active
GTP- and inactive GDP-bound state (Figure 2) [25]. Studies
with both LRRK2 and an amoebic homologue revealed that
a functional Roc domain is essential for kinase activity and
disruption of Roc or the kinase domain by a single point
mutation leads to the complete inactivation of the protein
[15, 22, 23, 26]. In vivo studies with LRRK2 G2019S showed
that primary neurons possess a lower level of toxicity after
the GTPase function was abolished [27]. Further studies
confirmed that GTPase activity is central for neuronal toxicity
and LRRK2 pathobiology in human cell lines and model
organisms [20–22]. However, the data prove the involvement
of both enzymatic activities in the onset of PD and imply a
present cross-talk between the two domains. Input/dimerization Pi
GDP
GDP
GTP
GTP
∼P
Substrate
+ATP
+ADP
GDP
Roc
COR
LRR
Kinase
Kinase active
Membrane
GTP Membrane Substrate Kinase active Kinase active Figure 2: Proposed model of the activation mechanism of LRRK2. 1. Introduction Figure 1: Schematic diagram of the domain architecture of LRRK2. Above, the segregating mutations of LRRK2
hown (arrows). ARM: Armadillo repeats, ANK: Ankyrin repeats, LRR: leucine-rich repeats, and WD: WD40 rep Figure 1: Schematic diagram of the domain architecture of LRRK2. Above, the segregating mutations of LRRK2 in Parkinson’s disease are
shown (arrows). ARM: Armadillo repeats, ANK: Ankyrin repeats, LRR: leucine-rich repeats, and WD: WD40 repeats. Pi
Input/dimerization
GDP
GDP
GDP
GTP
GTP
∼P
Substrate
+ATP
+ADP
GDP
Roc
COR
LRR
Kinase
Kinase active
Cytosol
Membrane
GTP
GDP
Figure 2: Proposed model of the activation mechanism of LRRK2. LRRK2 activation is at least regulated by three different mechanisms:
cycling between (1) an almost inactive monomer and active dimer
at the membrane, (2) intramolecular activation, and (3) binding of
input/substrate to the N- and C-terminal domains. Input/dimerization
GDP
Cytosol
GDP and autophagy pathways, and discuss possible ways to thera-
peutically target LRRK2-mediated PD. 2. LRRK2 Kinase and GTPase Activity LRRK2 activation is at least regulated by three different mechanisms:
cycling between (1) an almost inactive monomer and active dimer
at the membrane, (2) intramolecular activation, and (3) binding of
input/substrate to the N- and C-terminal domains. accumulating evidence suggests the involvement of at least
three different mechanisms: dimerization in close association
with localization, intramolecular activation, and binding of
input/substrate to the N- and C-terminal domains (Figures 2
and 3). LRRK2 is monomeric and almost inactive in the cytosol,
while it is predominantly dimeric and active when localized
at the membrane [28–32]. Membrane enriched LRRK2 dis-
played an enhanced molecular mass as well as a 8.4 times 1. Introduction LRRK2 is a large (286 kDa) and complex protein with a
unique multiple-domain architecture (Figure 1), consisting of
Armadillo repeats (ARM), Ankyrin repeats (ANK), leucine-
rich repeats (LRR), a Ras of complex proteins (Roc), a C-
terminal of Roc (COR), a kinase domain, and WD40 repeats
[2, 6, 7]. Parkinson’s disease (PD) is a progressive motor disorder that
is caused by the degeneration of dopaminergic neurons in
the midbrain. The prevalence of PD increases with age, with
2% of individuals over the age of 80 being affected thereby
representing the second most common neurodegenerative
disorder worldwide [1–3]. Causations are various and mostly
divided into a sporadic form without a clear trigger and a
familial form in which a genetic factor is involved. The mono-
genic form of PD is caused by a single mutation in a reces-
sively or dominantly inherited gene. It has been found in
sporadic as well as familial PD and accounts for approx-
imately 3–5% and 30% of the cases, respectively [4, 5]. Mutations in SCNA and LRRK2 (Leucine-Rich Repeat Kinase
2) are a specific subset of familial PD as they are autosomal-
dominant with LRRK2 representing the most common cause
of inherited PD [5]. It belongs to the Roco family of proteins,
which constitutes a novel family of Ras-like G-proteins being
conserved in almost all kingdoms of life [6–8]. Over 40 LRRK2 mutations have been identified repre-
senting risk factors for PD [9–11]. Most of the verified patho-
genic PD-linked LRRK2 mutations are accumulated around
the central core of the protein; one is found in the LRR,
one in the Roc domain (with multiple substitutions), one in
the COR domain, and two in the kinase domain (Figure 1). The multiple disease-linked mutations in LRRK2 represent
a unique opportunity to explore the activation mechanism
of the protein, its misregulation in PD, and the underlying
molecular mechanisms of genetic and sporadic PD. In this review, we will focus on the recent progress in
elucidating the complex LRRK2 activation mechanism, high-
light the evidence for a role of LRRK2 in the mitochondrial Parkinson’s Disease 2 2
Parkinsons Disease
ARM
LRR
Roc
LRRK2
R1441C/G/H
Y1699C
G2019S
I2020T
ANK
COR
Kinase
WD
286 kDA
Figure 1: Schematic diagram of the domain architecture of LRRK2. Above, the segregating mutations of LRRK2 in Parkinson’s disease are
shown (arrows). ARM: Armadillo repeats, ANK: Ankyrin repeats, LRR: leucine-rich repeats, and WD: WD40 repeats. 3. LRRK2 Activation Mechanism The exact molecular mechanism by which the catalytic
activity of LRRK2 is regulated remains unknown; however, Parkinson’s Disease 3 Early
endosome
Late
endosome
Vesicle like
structures
14-3-3
Rab5
Rab7
NAADP
NAADP
Dimer
Monomer
(a)
Mitophagy
Rab7
Lysosome
AMPK
NAADP
NAADP
Bcl-2
Bcl-2
DLP1
(b)
Lysosome
Autophagy
Autophagosome
MEK1/2
ERK1/2
Rab7
Cell fate/
apoptosis
(c)
Figure 3: Proposed pathways regulating LRRK2-mediated mitochondrial homeostasis and autophagy. Early
endosome
Late
endosome
Vesicle like
structures
14-3-3
Rab5
Rab7
NAADP
NAADP
Dimer
Monomer
(a) NAADP Monomer Rab5 Rab7 (a) NAADP AMPK Mitophagy
Rab7
Lysosome
AMPK
NAADP
Bcl-2
(b) MEK1/2 Bcl-2 ERK1/2 Lysosome Rab7
NAADP Rab7 Autophagy (c) (b) Figure 3: Proposed pathways regulating LRRK2-mediated mitochondrial homeostasis and autophagy. higher kinase activity in comparison to the cytosolic LRRK2
suggesting that localization is dependent on and affects
phosphorylation [31, 33, 34]. Structural studies with bacterial
Roco proteins have revealed that the COR domain functions
as an essential dimerization device [35]. During dimerization,
the catalytic machinery for the GTPase reaction is being
formed by complementation of the active site of one protomer
with the other protomer [33, 35]. COR truncated proteins
that are not able to dimerize have a drastically lower (700
times) GTPase activity. Interestingly, abolishing dimerization
also alters autophosphorylation levels, indicating that both
enzymatic activities are critically dependent on dimerization
[35–38]. In this way, the intramolecular GTPase reaction
functions as a timing device for the activation and biological
functions of Roco proteins. Interestingly, a recent study has
shown that mutations of known phosphorylation sides in
the G-domain affect both kinase and GTPase activity [30]. Together the data suggest that the Roc-COR tandem is reg-
ulating kinase activity, the kinase is regulating the GTPase
activity of Roc, and both events are critically involved in
LRRK2 cellular distribution. higher kinase activity in comparison to the cytosolic LRRK2
suggesting that localization is dependent on and affects
phosphorylation [31, 33, 34]. Structural studies with bacterial
Roco proteins have revealed that the COR domain functions
as an essential dimerization device [35]. During dimerization,
the catalytic machinery for the GTPase reaction is being
formed by complementation of the active site of one protomer
with the other protomer [33, 35]. COR truncated proteins
that are not able to dimerize have a drastically lower (700
times) GTPase activity. Interestingly, abolishing dimerization
also alters autophosphorylation levels, indicating that both
enzymatic activities are critically dependent on dimerization
[35–38]. 4. LRRK2-Mediated Mitochondrial
Dysfunction, Autophagy, and Cell Death Numerous potential LRRK2 mediated pathways have been
identified; however, much about its cellular functions and
LRRK2 mediated progression of PD remains unknown. Accumulating evidence links LRRK2-mediated PD to mito-
chondrial dysfunction and aberrant autophagy (Figure 3)
[48–51]. LRRK2 transfected HEK-293T cells showed a 10%
enhanced localization of LRRK2 to the outer but not inner
mitochondrial membrane [51]. The morphology and inter-
connectivity of mitochondria in skin samples of G2019S car-
rier patients were detected to be abnormal, most likely due
to dysregulated fission and fusion events [50]. Analysis of
the substantia nigra of patients with idiopathic PD revealed
a glutathione depletion and mitochondrial complex-I defi-
ciency, both representing known indicators of oxidative stress
[52]. Furthermore, polymorphism in mtDNA (mitochondrial
DNA) and aberrant levels of the neurotoxin MPP+ (1-methyl-
4-phenylpyridinium) and its precursor MPTP (1-methyl-
4-phenyl-1,2,3,6-tetrahydropyridine) were found in patient
samples, various model organisms, and human culture cell
lines [53]. In neurons it was shown that LRRK2 colocalizes
with the Dynamin like protein 1 (DLP1), a known mitochon-
drial fission factor (Figure 3). Expression of LRRK2 G2019S
and R1441C in neurons induced mitochondrial fragmenta-
tion and increased their interaction rate with DLP1 which also
displayed higher phosphorylation levels, resulting, among
others, in an enhanced level of reactive oxygen species (ROS). All these defects could be rescued by silencing of DLP1,
suggesting a LRRK2/DLP1 pathway regulating mitochondrial
fission events and their clearance [51, 54]. Localization of
LRRK2 is not limited to mitochondrial structures but was [
]
Several other PD associated proteins, including 𝛼-
synuclein, Parkin, DJ-1, PINK1, and HtrA2, have been linked
to similar defects in mitochondria regulation and autophagy
[37, 61, 62]. Parkin, a known regulator of mitochondrial
clearance, and AMPK seem to be directly involved in an
alternative or parallel pathway as overexpression acted pro-
tectively against cellular toxicity in fly dopaminergic neu-
rons expressing mutated LRRK2 [61]. Mutations in Parkin
and PINK1 (PTEN-induced kinase 1), both mitochondria
regulating proteins, have been found in sporadic as well as
autosomal recessive PD and result in severe mitochondrial
abnormalities and cell death [63]. The parallel expression
of PD-LRRK2 in PINK1 and DJ-1 deficient fly cells or
mice neurons with abnormal 𝛼-synuclein activity leads to
an increase of respective pathogenic phenotypes [64, 65]. Deletion of LRRK2 acts in a neuroprotective way towards
𝛼-synuclein mediated effects in mouse models [65]. 3. LRRK2 Activation Mechanism In this way, the intramolecular GTPase reaction
functions as a timing device for the activation and biological
functions of Roco proteins. Interestingly, a recent study has
shown that mutations of known phosphorylation sides in
the G-domain affect both kinase and GTPase activity [30]. LRRK2 dimerization and activation is regulated by the N-
and C-terminal LRRK2 protein-protein interaction domains. Cellular studies with LRRK2 and related Roco proteins lack-
ing the N- or C-terminus suggested their essential role for sig-
naling in vivo [7, 39]. Deletion of the WD40 repeats led to impaired dimer
formation accompanied with diminished kinase activity and
aberrant protein localization [40]. Recent data suggest that
the N-terminus inhibits LRRK2 kinase activity, since deletion
of the terminus resulted in increased LRRK2 autophosphory-
lation levels when expressed in human cell lines [32]. On the
contrary, LRRK2 G2019S PD mutation displayed increased
kinase activity with a lower level of autophosphorylation of 4 Parkinson’s Disease the N-terminus (S910/935∼P) [32, 41]. Although the N- and
C-terminus of LRRK2 have an essential role in vivo, they
are not required for kinase activity in vitro [7, 39]. This
might suggest that the N- and C-terminal protein-protein
interaction domains regulate LRRK2 activity by binding to
upstream and/or downstream effectors. In this perspective,
it has been shown that the N-terminal segment of LRRK2
interacts in a phosphorylation dependent manner with the
ubiquitous regulatory protein 14-3-3. Disruption of the phos-
phorylation sides S910 and S935 blocks 14-3-3 binding and
leads to the delocalization of LRRK2 from the membrane and
its accumulation in the cytosol (Figure 3) [34, 41]. Recently,
members of the Rab family of small GTPases have been
identified as valid LRRK2 interactors and substrates [42–44]. In vivo studies confirmed direct binding, most likely medi-
ated via the N-terminus, and colocalization of LRRK2 with
Rab5 and Rab7, suggesting an involvement in degradative
and endocytic membrane trafficking (Figure 3). Strikingly,
the PD mutation G2019S disrupted molecular trafficking
and colocalization with Rab7, resulting in the formation of
aberrant endosomal structures and endosomal/lysosomal
localization thus interfering with the cellular degradative traf-
ficking pathway of organelles [45, 46]. Furthermore, LRRK2
binding to Rab32 is regulating its localization to lysosomes as
well as mitochondria [47]. found at a variety of additional membranes, including mul-
tivesicular bodies (MVBs) representing autophagic vacuoles
(AVs) [55]. 3. LRRK2 Activation Mechanism Consistently, being involved in the regulation
of the endosomal-autophagic pathway, expression of PD-
mutated LRRK2 triggered the accumulation of (abnormal)
MVBs and AVs via misbalancing the induction of macroau-
tophagy and maturation of AVs to lysosomes (Figure 3) [55]. Furthermore, expression of LRRK2 G2019S in human cell
lines led to the shortening of neurite length and an increase
in autophagic vacuole levels [15, 56].h The pathways regulating and linking LRRK2 PD-medi-
ated mitochondrial dysregulation and abnormal autophagy
are only partly identified but most likely include the activa-
tion of the autophagy regulating protein 5AMP-activated
protein kinase (AMPK) [45, 57]. The abnormal kinase activity
of LRRK2 G2019S in human cell lines leads to an increased
level of phosphorylated AMPK, which subsequently results
in enhanced levels of autophagosomes [58]. The mitogen-
activated protein kinases (MAPK) cascade may represent
another important pathway regulating LRRK2-mediated
autophagy. In addition to enhanced autophagic activity and
cell death, cells expressing LRRK2 G2019S also showed a
threefold increase in protein turnover and a higher level
of phosphorylated MAPK/ERK. Incubation with a specific
inhibitor of MEK1/2 (U0126) was sufficient to rescue the aber-
rant phenotypes of the LRRK2 G2019S cells [56, 59]. It was
suggested that LRRK2 induces autophagy via the activation
of NAADP (nicotinic acid adenine dinucleotide phosphate)
receptors, which are involved in the calcium efflux from
endosomes [58]. The mitochondrial antiapoptotic protein,
Bcl-2, might represent the connection between LRRK2-
induced dysregulated mitochondrial homeostasis and auto-
phagy. Expression of phosphorylated Bcl-2 rescues both the
mitochondrial and autophagy defects of LRRK2 G2019S cells
[60]. 6. Summary Recent studies have shed light on the complex activation
mechanism of LRRK2 and revealed highly precise and exact
timed interactions on both intra- and intermolecular levels. These multiple layers of regulation and enzyme activities
within one protein make LRRK2 an interesting therapeutic
target. To further explore these therapeutic targets, it will be
essential to completely characterize the molecular activation
mechanism. Biochemical and structural characterization of
LRRK2 and/or related Roco proteins can give important
information about the dimerization mechanism, how the
kinase domain regulates GTPase activity, how LRRK2 activity
is regulated by binding of input or substrate to the LRR
and WD40 domains, and how the PD mutations influence
the complex regulatory mechanism. Recent data strongly
suggest that LRRK2 dysfunction in PD results in mitochon-
drial defects and autophagy. However, the precise underlying
mechanisms are still not well understood and many questions
about the cellular function of LRRK2 remain to be addressed,
including at which (inter)cellular membrane LRRK2 is acti-
vated and if common underlying pathways of familial PD
are existing. To answer these questions, it will be crucial to
identify physiological kinase substrate(s) and upstream and
downstream regulators. Understanding how other domains of LRRK2 modulate
its activity is an important but rather neglected field in LRRK2
research and not a focus of industries. However, the PD
causing mutations are found in nearly all domains of LRRK2
leading to the same well described symptoms. Furthermore,
as recent data suggest that different PD mutations have
diverse defects with regard to the activation mechanism, they
might require specified ways of inhibition for the purpose of
drug development [72, 74, 77]. The authors declare that they have no competing interests. The authors declare that they have no competing interests. Alternative approaches targeting further LRRK2 domains
and sites of its complex activation mechanism, including the
N- and C-terminus, the catalytic GTPase activity of Roc,
LRRK2 localization, dimerization, or allosteric modulation of
the kinase domain, might significantly improve therapeutic
benefits (Figure 2).h 4. LRRK2-Mediated Mitochondrial
Dysfunction, Autophagy, and Cell Death DJ-1 is
only partially able to rescue the phenotypes of PINK1 mutated
neurons but, vice versa, overexpression of both Parkin and
PINK1 restores the abnormal mitochondrial morphologies of
DJ-1 deficient cells, suggesting a present connection between
the involved pathways [66, 67]. Altogether it might suggest
the presence of common PD-pathogenic pathways that result
in mitochondrial dysfunction and autophagy. 5 Parkinson’s Disease Acknowledgments The authors thank Franz Ho for his careful proofreading
of the paper. This work is supported by the Michael J. Fox
foundation for Parkinson’s research and a NWO-VIDI grant
to Dr. Arjan Kortholt. i
g
The LRRK2 mutations in the Roc (R1441C/G/H) and
COR (Y1699C) domain have a decreased GTPase activity
and a functional LRRK2 G-domain is essential for LRRK2
activation, suggesting GTPase activity forms an interesting
therapeutic target [20, 78–80]. Targeting the G-domain could
be done by using small compounds that bind and interfere
with nucleotide binding, resemble the GDP-bound off-state,
or increase the GTPase cycle. Recently, the first LRRK2 GTP
binding inhibitors, compounds 68 and 70, were identified and
proved to inhibit both GTPase and kinase activity in vitro as
well as in vivo and thereby attenuated neuronal degeneration
in human cell lines/rodent tissues [14]. Importantly, FX2149, a
novel analog of 68, even displayed an around two times higher
brain inhibition efficiency in a rodent model organism [81].h 5. Therapeutic Targeting of LRRK2 active monomeric cytosolic state and a high active dimeric
membrane bound state. Importantly, since LRRK2 activation
is dependent on membrane localization and dimerization,
inhibiting either of these properties may be a good therapeu-
tic approach. The major focus of academia and industry is the development
of kinase inhibitors as potential therapeutics for LRRK2-
mediated PD. Almost all clinical kinase inhibitors are used for
short-time treatment in the cancer field and for immunologi-
cal, neurological, and infectious diseases, where side effects
caused by high dosage are tolerated [68]. In contrast, for
the long-term treatment of chronic diseases such as LRRK2-
associated PD no potential toxic side effects can be present. Several highly specific and brain penetrant LRRK2 kinase
inhibitors were identified but have yet to be optimized in
order to qualify as drug candidates for therapeutic treatment
[69–72]. Our structures of a humanized D. discoideum Roco4
kinase domain bound to the common inhibitors LRRK2-
IN-1 or Compound 19 revealed a highly similar binding
mechanism and gave important information for potential
optimization [73]. However, accumulation in peripheral tis-
sues, especially kidneys and lungs, and related drug induced
toxicity are still a major and common problem for all LRRK2
kinase inhibitors [72, 74, 75]. In rodent models, enhanced
dosages of the recent highly specific and brain penetrant
LRRK2 kinase inhibitors GNE-7915 and GNE-0877 are well
tolerated over a longer time period; however, they induced
the cytoplasmic accumulation of lysosome-related organelles
in the lungs of nonhuman primates [76]. The major focus of academia and industry is the development
of kinase inhibitors as potential therapeutics for LRRK2-
mediated PD. Almost all clinical kinase inhibitors are used for
short-time treatment in the cancer field and for immunologi-
cal, neurological, and infectious diseases, where side effects
caused by high dosage are tolerated [68]. In contrast, for
the long-term treatment of chronic diseases such as LRRK2-
associated PD no potential toxic side effects can be present.i References Cookson, “The role of leucine-rich repeat kinase 2
(LRRK2) in Parkinson’s disease,” Nature Reviews Neuroscience,
vol. 11, no. 12, pp. 791–797, 2010. [33] S. Sen, P. J. Webber, and A. B. West, “Dependence of leucine-
rich repeat kinase 2 (LRRK2) kinase activity on dimerization,”
The Journal of Biological Chemistry, vol. 284, no. 52, pp. 36346–
36356, 2009. [18] A. R. Esteves, R. H. Swerdlow, and S. M. Cardoso, “LRRK2, a
puzzling protein: insights into Parkinson’s disease pathogene-
sis,” Experimental Neurology, vol. 261, pp. 206–216, 2014. [19] R. D. Mills, T. D. Mulhern, F. Liu, J. G. Culvenor, and H.-C. Cheng, “Prediction of the repeat domain structures and impact
of parkinsonism-associated variations on structure and func-
tion of all functional domains of leucine-rich repeat kinase 2
(LRRK2),” Human Mutation, vol. 35, no. 4, pp. 395–412, 2014. [34] R. J. Nichols, N. Dzamko, N. A. Morrice et al., “14-3-3 Binding to
LRRK2 is disrupted by multiple Parkinson’s disease-associated
mutations and regulates cytoplasmic localization,” Biochemical
Journal, vol. 430, no. 3, pp. 393–404, 2010. [35] K. Gotthardt, M. Weyand, A. Kortholt, P. J. M. Van Haastert, and
A. Wittinghofer, “Structure of the Roc-COR domain tandem
of C. tepidum, a prokaryotic homologue of the human LRRK2
Parkinson kinase,” The EMBO Journal, vol. 27, no. 16, pp. 2239–
2249, 2008. [20] A. Biosa, A. Trancikova, L. Civiero et al., “GTPase activity
regulates kinase activity and cellular phenotypes of parkinson’s
disease-associated LRRK2,” Human Molecular Genetics, vol. 22,
no. 6, pp. 1140–1156, 2013. [21] K. Stafa, A. Trancikova, P. J. Webber, L. Glauser, A. B. West, and
D. J. Moore, “GTPase activity and neuronal toxicity of Parkin-
son’s disease-associated LRRK2 is regulated by ArfGAP1,” PLoS
Genetics, vol. 8, no. 2, Article ID e1002526, 2012. [36] E. Greggio, J.-M. Taymans, E. Y. Zhen et al., “The Parkinson’s
disease kinase LRRK2 autophosphorylates its GTPase domain
at multiple sites,” Biochemical and Biophysical Research Commu-
nications, vol. 389, no. 3, pp. 449–454, 2009. [22] Y. Xiong, C. E. Coombes, A. Kilaru et al., “GTPase activity plays
a key role in the pathobiology of LRRK2,” PLoS Genetics, vol. 6,
no. 4, 2010. [37] J. Deng, P. A. Lewis, E. Greggio, E. Sluch, A. Beilina, and M. R. Cookson, “Structure of the ROC domain from the Parkinson’s
disease-associated leucine-rich repeat kinase 2 reveals a dimeric
GTPase,” Proceedings of the National Academy of Sciences of the
United States of America, vol. 105, no. 5, pp. References [1] C. G. Goetz, “The history of Parkinson’s disease: early clinical
descriptions and neurological therapies,” Cold Spring Harbor
Perspectives in Medicine, vol. 1, no. 1, Article ID a008862, 2011. [2] J. C. D¨achsel and M. J. Farrer, “LRRK2 and Parkinson disease,”
Archives of Neurology, vol. 67, no. 5, pp. 542–547, 2010.h [3] A. J. Lees, J. Hardy, and T. Revesz, “Parkinson’s disease,” The
Lancet, vol. 373, no. 9680, pp. 2055–2066, 2009. [4] W. P. Gilks, P. M. Abou-Sleiman, S. Gandhi et al., “A common
LRRK2 mutation in idiopathic Parkinson’s disease,” The Lancet,
vol. 365, no. 9457, pp. 415–416, 2005. [5] C. Klein and A. Westenberger, “Genetics of Parkinson’s disease,”
Cold Spring Harbor Perspectives in Medicine, vol. 2, Article ID
a008888, 2012. fi
The N- and C-terminal segments of LRRK2 contain sev-
eral protein-protein interaction domains which are involved
in regulating kinase activity, oligomerization, and/or local-
ization. As described above, LRRK2 cycles between a low fi
The N- and C-terminal segments of LRRK2 contain sev-
eral protein-protein interaction domains which are involved
in regulating kinase activity, oligomerization, and/or local-
ization. As described above, LRRK2 cycles between a low [6] I. Mar´ın, W. N. van Egmond, and P. J. M. van Haastert, “The
Roco protein family: a functional perspective,” The FASEB
Journal, vol. 22, no. 9, pp. 3103–3110, 2008. 6 Parkinson’s Disease [25] I. R. Vetter and A. Wittinghofer, “The guanine nucleotide-bind-
ing switch in three dimensions,” Science, vol. 294, no. 5545, pp. 1299–1304, 2001. [7] W. N. van Egmond and P. J. M. van Haastert, “Characteriza-
tion of the Roco protein family in Dictyostelium discoideum,”
Eukaryotic Cell, vol. 9, no. 5, pp. 751–761, 2010. [8] L. Bosgraaf and P. J. M. Van Haastert, “Roc, a Ras/GTPase
domain in complex proteins,” Biochimica et Biophysica Acta
(BBA)—Molecular Cell Research, vol. 1643, no. 1–3, pp. 5–10,
2003. [26] J.-M. Taymans, R. Vancraenenbroeck, P. Ollikainen et al.,
“LRRK2 kinase activity is dependent on LRRK2 gtp binding
capacity but independent of LRRK2 GTP binding,” PLoS ONE,
vol. 6, no. 8, Article ID e23207, 2011. [9] H. Deng, W. Le, Y. Guo et al., “Genetic analysis of LRRK2 muta-
tions in patients with Parkinson disease,” Journal of the Neuro-
logical Sciences, vol. 251, no. 1-2, pp. 102–106, 2006. [27] W. W. Smith, Z. Pei, H. Jiang, V. L. Dawson, T. M. Dawson, and
C. A. References Ross, “Kinase activity of mutant LRRK2 mediates neu-
ronal toxicity,” Nature Neuroscience, vol. 9, no. 10, pp. 1231–1233,
2006. [10] D. Trabzuni, M. Ryten, W. Emmett et al., “Fine-mapping,
gene expression and splicing analysis of the disease associated
LRRK2 locus,” PLoS ONE, vol. 8, no. 8, Article ID e70724, 2013. [28] N. G. James, M. A. Digman, E. Gratton et al., “Number and
brightness analysis of LRRK2 oligomerization in live cells,”
Biophysical Journal, vol. 102, no. 11, pp. L41–L43, 2012. [11] V. Bonifati, “Parkinsonism and related disorders genetics of
Parkinson’s disease—state of the art, 2013,” Parkinsonism &
Related Disorders, vol. 20, pp. S23–S28, 2014. [29] J. Schapansky, J. D. Nardozzi, F. Felizia, and M. J. LaVoie,
“Membrane recruitment of endogenous LRRK2 precedes its
potent regulation of autophagy,” Human Molecular Genetics,
vol. 23, no. 16, Article ID ddu138, pp. 4201–4214, 2014. [12] B. Thomas and M. F. Beal, “Parkinson’s disease,” Human Molec-
ular Genetics, vol. 16, no. 2, pp. R183–R194, 2007. [13] A. Zimprich, S. Biskup, P. Leitner et al., “Mutations in LRRK2
cause autosomal-dominant parkinsonism with pleomorphic
pathology,” Neuron, vol. 44, no. 4, pp. 601–607, 2004. [30] P. J. Webber, A. D. Smith, S. Sen, M. B. Renfrow, J. A. Mobley,
and A. B. West, “Autophosphorylation in the leucine-rich repeat
kinase 2 (LRRK2) GTPase domain modifies kinase and GTP-
binding activities,” Journal of Molecular Biology, vol. 412, no. 1,
pp. 94–110, 2011. [14] T. Li, D. Yang, S. Zhong et al., “Novel LRRK2 GTP-binding
inhibitors reduced degeneration in Parkinson’s disease cell and
mouse models,” Human Molecular Genetics, vol. 23, no. 23, pp. 6212–6222, 2014. [31] Z. Berger, K. A. Smith, and M. J. Lavoie, “Membrane localiza-
tion of LRRK2 is associated with increased formation of the
highly active lrrk2 dimer and changes in its phosphorylation,”
Biochemistry, vol. 49, no. 26, pp. 5511–5523, 2010. [15] E. Greggio, S. Jain, A. Kingsbury et al., “Kinase activity is
required for the toxic effects of mutant LRRK2/dardarin,”
Neurobiology of Disease, vol. 23, no. 2, pp. 329–341, 2006. [16] V. S. Anand and S. P. Braithwaite, “LRRK2 in Parkinson’s
disease: biochemical functions,” FEBS Journal, vol. 276, no. 22,
pp. 6428–6435, 2009. [32] E. Greggio, I. Zambrano, A. Kaganovich et al., “The Parkinson
disease-associated leucine-rich repeat kinase 2 (LRRK2) is a
dimer that undergoes intramolecular autophosphorylation,”
The Journal of Biological Chemistry, vol. 283, no. 24, pp. 16906–
16914, 2008. [17] M. R. References 1499–1504, 2008. [23] G. Ito, T. Okai, G. Fujino et al., “GTP binding is essential to
the protein kinase activity of LRRK2, a causative gene product
for familial Parkinson’s disease,” Biochemistry, vol. 46, no. 5, pp. 1380–1388, 2007. [38] J. Liao, C.-X. Wu, C. Burlak et al., “Parkinson disease-associated
mutation R1441H in LRRK2 prolongs the ‘active state’ of
its GTPase domain,” Proceedings of the National Academy of
Sciences of the United States of America, vol. 111, no. 11, pp. 4055–
4060, 2014. [24] A. B. West, D. J. Moore, C. Choi et al., “Parkinson’s disease-
associated mutations in LRRK2 link enhanced GTP-binding
and kinase activities to neuronal toxicity,” Human Molecular
Genetics, vol. 16, no. 2, pp. 223–232, 2007. 7 Parkinson’s Disease [39] C. Iaccarino, C. Crosio, C. Vitale, G. Sanna, M. T. Carr`ı, and
P. Barone, “Apoptotic mechanisms in mutant LRRK2-mediated
cell death,” Human Molecular Genetics, vol. 16, no. 11, pp. 1319–
1326, 2007. [55] J. Alegre-Abarrategui, H. Christian, M. M. P. Lufino et al.,
“LRRK2 regulates autophagic activity and localizes to specific
membrane microdomains in a novel human genomic reporter
cellular model,” Human Molecular Genetics, vol. 18, no. 21, pp. 4022–4034, 2009. [40] N. D. Jorgensen, Y. Peng, C. C.-Y. Ho et al., “The WD40 domain
is required for LRRK2 neurotoxicity,” PLoS ONE, vol. 4, no. 12,
Article ID e8463, 2009. [56] S. V. Sambasivarao, “Role of autophagy in G2019S-LRRK2-
associated neurite shortening in differentiated SH-SY5Y cells,”
Journal of Neurochemistry, vol. 18, pp. 1199–1216, 2013. [41] A. Reynolds, E. A. Doggett, S. M. Riddle, C. S. Lebakken, and
R. J. Nichols, “LRRK2 kinase activity and biology are not uni-
formly predicted by its autophosphorylation and cellular phos-
phorylation site status,” Frontiers in Molecular Neuroscience, vol. 7, article 54, 2014. [57] T. D. Papkovskaia, K.-Y. Chau, F. Inesta-Vaquera et al., “G2019s
leucine-rich repeat kinase 2 causes uncoupling protein-medi-
ated mitochondrial depolarization,” Human Molecular Genetics,
vol. 21, no. 19, pp. 4201–4213, 2012. [42] P. Novick and M. Zerial, “The diversity of Rab proteins in vesicle
transport,” Current Opinion in Cell Biology, vol. 9, no. 4, pp. 496–
504, 1997. [58] P. G´omez-Suaga, B. Luz´on-Toro, D. Churamani et al., “Leucine-
rich repeat kinase 2 regulates autophagy through a calcium-
dependent pathway involving NAADP,” Human Molecular
Genetics, vol. 21, no. 3, Article ID ddr481, pp. 511–525, 2012. [43] A. Beilina, I. N. Rudenko, A. References Kaganovich et al., “Unbiased
screen for interactors of leucine-rich repeat kinase 2 supports a
common pathway for sporadic and familial Parkinson disease,”
Proceedings of the National Academy of Sciences of the United
States of America, vol. 111, no. 7, pp. 2626–2631, 2014. [59] J. M. Bravo-San Pedro, M. Niso-Santano, R. G´omez-S´anchez
et al., “The LRRK2 G2019S mutant exacerbates basal autophagy
through activation of the MEK/ERK pathway,” Cellular and
Molecular Life Sciences, vol. 70, no. 1, pp. 121–136, 2013. [44] M. Steger, F. Tonelli, G. Ito et al., “Phosphoproteomics reveals
that Parkinson’s disease kinase LRRK2 regulates a subset of Rab
GTPases,” eLife, vol. 5, Article ID e12813, 28 pages, 2016. [60] Y.-C. Su, X. Guo, and X. Qi, “Threonine 56 phosphorylation
of Bcl-2 is required for LRRK2 G2019S-induced mitochondrial
depolarization and autophagy,” Biochimica et Biophysica Acta
(BBA)—Molecular Basis of Disease, vol. 1852, no. 1, pp. 12–21,
2015. [45] P. G´omez-Suaga, P. Rivero-R´ıos, E. Fdez et al., “LRRK2 delays
degradative receptor trafficking by impeding late endosomal
budding through decreasing Rab7 activity,” Human Molecular
Genetics, vol. 23, no. 25, pp. 6779–6796, 2014. [61] L. Hang, J. Thundyil, and K.-L. Lim, “Mitochondrial dysfunc-
tion and Parkinson disease: a Parkin-AMPK alliance in neuro-
protection,” Annals of the New York Academy of Sciences, vol. 1350, pp. 37–47, 2015. [46] M. W. Dodson, T. Zhang, C. Jiang, S. Chen, and M. Guo, “Roles
of the Drosophila LRRK2 homolog in Rab7-dependent lysoso-
mal positioning,” Human Molecular Genetics, vol. 21, no. 6, pp. 1350–1363, 2012. [62] L. Vande Walle, M. Lamkanfi, and P. Vandenabeele, “The mito-
chondrial serine protease HtrA2/Omi: an overview,” Cell Death
and Differentiation, vol. 15, no. 3, pp. 453–460, 2008. [47] D. Waschb¨usch, H. Michels, S. Strassheim et al., “LRRK2 trans-
port is regulated by its novel interacting partner Rab32,” PLoS
ONE, vol. 9, no. 10, Article ID e111632, 2014. [63] H. Deng, M. W. Dodson, H. Huang, and M. Guo, “The Parkin-
son’s disease genes pink1 and parkin promote mitochondrial
fission and/or inhibit fusion in Drosophila,” Proceedings of the
National Academy of Sciences of the United States of America,
vol. 105, no. 38, pp. 14503–14508, 2008. [48] S. Biskup, D. J. Moore, F. Celsi et al., “Localization of LRRK2
to membranous and vesicular structures in mammalian brain,”
Annals of Neurology, vol. 60, no. 5, pp. 557–569, 2006. [64] K. Venderova, G. Kabbach, E. References Abdel-Messih et al., “Leucine-
rich repeat kinase 2 interacts with Parkin, DJ-1 and PINK-1 in a
Drosophila melanogaster model of Parkinson’s disease,” Human
Molecular Genetics, vol. 18, no. 22, pp. 4390–4404, 2009. [49] A. H. V. Schapira, J. M. Cooper, D. Dexter, J. B. Clark, P. Jenner,
and C. D. Marsden, “Mitochondrial complex I deficiency in
Parkinson’s disease,” Journal of Neurochemistry, vol. 54, no. 3,
pp. 823–827, 1990. [65] X. Lin, L. Parisiadou, X.-L. Gu et al., “Leucine-rich repeat kinase
2 regulates the progression of neuropathology induced by Par-
kinson’s-disease-related mutant 𝛼-synuclein,” Neuron, vol. 64,
no. 6, pp. 807–827, 2009. [50] H. Mortiboys, K. K. Johansen, J. O. Aasly, and O. Bandmann,
“Mitochondrial impairment in patients with Parkinson disease
with the G2019S mutation in LRRK2,” Neurology, vol. 75, no. 22,
pp. 2017–2020, 2010. [66] I. Irrcher, H. Aleyasin, E. L. Seifert et al., “Loss of the Parkinson’s
disease-linked gene DJ-1 perturbs mitochondrial dynamics,”
Human Molecular Genetics, vol. 19, no. 19, pp. 3734–3746, 2010. [51] A. B. West, D. J. Moore, S. Biskup et al., “Parkinson’s disease-
associated mutations in leucine-rich repeat kinase 2 augment
kinase activity,” Proceedings of the National Academy of Sciences
of the United States of America, vol. 102, no. 46, pp. 16842–16847,
2005. [67] K. J. Thomas, M. K. McCoy, J. Blackinton et al., “DJ-1 acts in
parallel to the PINK1/parkin pathway to control mitochondrial
function and autophagy,” Human Molecular Genetics, vol. 20,
no. 1, pp. 40–50, 2011. [52] P. B´enit, S. Lebon, M. Chol, I. Giurgea, A. R¨otig, and P. Rustin,
“Mitochondrial complex I deficiency in humans,” Current
Genomics, vol. 5, no. 2, pp. 137–146, 2004. [68] P. Cohen, “Protein kinases—the major drug targets of the
twenty-first century?” Nature Reviews Drug Discovery, vol. 1, no. 4, pp. 309–315, 2002. [53] M. T. Lin and M. F. Beal, “Mitochondrial dysfunction and
oxidative stress in neurodegenerative diseases,” Nature, vol. 443,
no. 7113, pp. 787–795, 2006. [69] A. D. Reith, P. Bamborough, K. Jandu et al., “GSK2578215A; a
potent and highly selective 2-arylmethyloxy-5-substitutent-N-
arylbenzamide LRRK2 kinase inhibitor,” Bioorganic and Medic-
inal Chemistry Letters, vol. 22, no. 17, pp. 5625–5629, 2012. [54] J. Niu, M. Yu, C. Wang, and Z. Xu, “Leucine-rich repeat kinase
2 disturbs mitochondrial dynamics via dynamin-like protein,”
Journal of Neurochemistry, vol. 122, no. 3, pp. 650–658, 2012. Parkinson’s Disease 8 [70] N. Ramsden, J. Perrin, Z. References Ren et al., “Chemoproteomics-based
design of potent LRRK2-selective lead compounds that attenu-
ate Parkinson’s disease-related toxicity in human neurons,” ACS
Chemical Biology, vol. 6, no. 10, pp. 1021–1028, 2011. [71] H. G. Choi, J. Zhang, X. Deng et al., “Brain penetrant LRRK2
inhibitor,” ACS Medicinal Chemistry Letters, vol. 3, no. 8, pp. 658–662, 2012. [72] X. Deng, N. Dzamko, A. Prescott et al., “Characterization of
a selective inhibitor of the Parkinson’s disease kinase LRRK2,”
Nature Chemical Biology, vol. 7, no. 4, pp. 203–205, 2011. [73] B. K. Gilsbach, A. C. Messias, G. Ito et al., “Structural character-
ization of LRRK2 inhibitors,” Journal of Medicinal Chemistry,
vol. 58, no. 9, pp. 3751–3756, 2015. [74] X. Deng, H. G. Choi, S. J. Buhrlage, and N. S. Gray, “Leucine-
rich repeat kinase 2 inhibitors: a patent review (2006–2011),”
Expert Opinion on Therapeutic Patents, vol. 22, no. 12, pp. 1415–
1426, 2012. [75] A. A. Estrada and Z. K. Sweeney, “Chemical biology of leucine-
rich repeat kinase 2 (LRRK2) inhibitors,” Journal of Medicinal
Chemistry, vol. 58, no. 17, pp. 6733–6746, 2015. [76] R. N. Fuji, M. Flagella, M. Baca et al., “Effect of selective
LRRK2 kinase inhibition on nonhuman primate lung,” Science
Translational Medicine, vol. 7, no. 273, Article ID 273ra15, 2015. [77] M. Jaleel, R. J. Nichols, M. Deak et al., “LRRK2 phosphorylates
moesin at threonine-558: characterization of how Parkinson’s
disease mutants affect kinase activity,” Biochemical Journal, vol. 405, no. 2, pp. 307–317, 2007. [78] J.-M. Taymans, “The GTPase function of LRRK2,” Biochemical
Society Transactions, vol. 40, no. 5, pp. 1063–1069, 2012. [79] P. A. Lewis, E. Greggio, A. Beilina, S. Jain, A. Baker, and M. R. Cookson, “The R1441C mutation of LRRK2 disrupts GTP
hydrolysis,” Biochemical and Biophysical Research Communica-
tions, vol. 357, no. 3, pp. 668–671, 2007. [80] X. Li, Y.-C. Tan, S. Poulose, C. W. Olanow, X.-Y. Huang, and Z. Yue, “Leucine-rich repeat kinase 2 (LRRK2)/PARK8 possesses
GTPase activity that is altered in familial Parkinson’s disease
R1441C/G mutants,” Journal of Neurochemistry, vol. 103, no. 1,
pp. 238–247, 2007. [81] T. Li, X. He, J. M. Thomas et al., “A novel GTP-binding inhibitor,
FX2149, attenuates LRRK2 toxicity in Parkinson’s disease mod-
els,” PLoS ONE, vol. 10, no. 3, Article ID e0122461, pp. 1–15, 2015. References Submit your manuscripts at
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https://openalex.org/W2589888688
|
https://zenodo.org/records/1744980/files/IJICR-3-112.pdf
|
English
| null |
Immunosuppressive and Cytotoxic Potential of Flavonoids from Medicinal Plants: Preliminary Investigation for Anticancer Activity
|
International journal of immunotherapy and cancer research
| 2,017
|
cc-by
| 4,306
|
Abstract Keywords: Calotropis gigantea; Adhatoda vasica; A
integrifolia; Hepatitis B vaccine; Flavonoid Introduction: Attention related to scientifi c and research interest towards naturally derived
compounds from medicinal plant products as they are considered to have less toxic side effects as
compared to current treatments such as chemotherapy. Medicinal plant produces naturally occurring
secondary metabolites especially fl avonoids which are being investigated for their anticancer activities
leading to the development of new clinical drugs. https://www.peertechz.com Objective- In this regard, immunological studies were conducted to investigate its immunosuppressive
and cytotoxic activity of fl avonoids from the leaves of Calotropis gigantea, Adhatoda vasica and Artocarpus
integrifolia on human whole blood and peripheral blood mononuclear cells (PBMC) against hepatitis B
vaccine containing surface antigen (HBsAg). Methods: for these studies, our group evaluated variable doses of fl avonoids extracted from the
leaves of Calotropis gigantea, Adhatoda vasica and Artocarpus integrifolia and estimated blood counts
(lymphocytes, monocytes and granulocytes count using fl ow cytometry); nitric oxide production (using
Griess reagent assay) and proliferation assay against HBsAg. Results: The results of these studies claimed that these fl avonoids showed sudden decline in
monocytes and granulocytes count; nitric oxide production and also showed its cytotoxicity in the form of
proliferation at higher doses as compared to control. Conclusion: Overall, these fl avonoids showed immunosuppressive as well as cytotoxic effect against
HBsAg and also probable candidate for anticancer agent. Immunosuppressive and Cytotoxic
Potential of Flavonoids from Medicinal
Plants: Preliminary Investigation for
Anticancer Activity Dates: Received: 05 December, 2016; Accepted: 30
January, 2017; Published: 01 February, 2017 *Corresponding author: Amit Gupta, Doctor,
Assistant Professor, Department of Immunology
and Virology, Vidya Pratishthan’s School
of Biotechnology (VSBT), Research Centre,
Affi liated to Savitribai Phule Pune University,
Baramati, Maharashtra, India, E-mail: Research Article Department of Immunology and Virology, Vidya
Pratishthan’s School of Biotechnology (VSBT)
Research Centre, Affi liated to Savitribai Phule Pune
University, Maharashtra, India apy
ISSN: 2455-8591
DOI
CC By https://www.peertechz.com Sample collection Fresh leaves of these medicinal plants (Calotropis gigantea,
Adhatoda vasica and Artocarpus integrifolia) were collected from
nakshatra garden, Vidya Pratishthan, Baramati, Maharashtra
India. Materials and Methods In cytotoxicity assay, similar volume of fresh medium were
added and again incubated the plate for another 24 h. After
incubation, add MTT (5 mg/ml; 10 μl) solution and again
incubate it for another 3-4 h. Thereafter, centrifuging the
plate, discard the supernatant and appearance of formazon
crystals settled at the bottom. These crystals were dissolved in
dimethyl sulphoxide and the optical density was measured at
570 nm [11-14]. Introduction The liver can
extend the conjugation of the fl avonoid by adding sulfate
group, methyl group, or both. The addition of these groups
which increases the circulatory elimination time and probably
also decreases its toxicity. According to the literature, lot of
fl avonoids are reported from these medicinal plant products
e.g. only one fl avonoid was isolated from the leaves of Juniperus
procera and could be identifi ed as; 3′,4′,3,7-tetrahydroxyfl avone
[9], new fl avonoid were also isolated from the stem bark of
Choerospondias axillaries, the fruit of which was used for the
treatment of cardiovascular diseases [10] etc. solution and then add ethyl acetate (20 ml) and distilled water
(40 ml) in the ratio of 1:2. Incubate for overnight and then
observed two separating layers i.e. upper layer containing
ethyl acetate (evaporate it) and lower layer containing dried
extracts (fl avonoids) that are reported or settled at the bottom. For determining the fl avonoid content qualitatively in these
medicinal plants, small quantity of lead acetate solution is
added and yellow precipitation if appeared it indicates the
presence of fl avonoid [4]. All three medicinal plants showed
positive results related to fl avonoid content. Introduction generally considered as non-nutrients [4]. Generally, these
fl avonoid intakes can range between 50 and 800 mg a day,
depending on the diet – consumption of vegetables, fruit, red
wine, tea, unfi ltered beer, etc. The absorption of the dietary
fl avonoids liberated from the food by chewing will depend on its
physicochemical properties (i.e. molecular size, confi guration,
lipophilicity, solubility etc.) [4-6]. Cancer is one of the major public health concern in all
over the world. Several medicinal plants were screened and
determined some anti-cancer agents i.e. taxol, vinblastine,
vincristine, campothecin derivatives etc. Other promising anti-
cancer agents that are reported i.e. fl avopiridol, roscovitine,
betulinic acid, silvestrol etc., [1]. In addition, there is another
group that derived from medicinal plant products in the form
of secondary metabolites i.e. fl avonoids and their synthetic
analogs are generally used in the treatment of ovarian, breast,
cervical, pancreatic and prostate cancer [1-3]. Generally, these fl avonoid should be absorbed in small
intestine or has to go to the colon before absorption and also
depends upon the structure of fl avonoid. Most fl avonoids,
except for the subclass of catechins, are present in medicinal
plants but they are actually bound to sugars as b-glycosides. As per the literature, fl avonoid glycosides converted into
aglycan form and they are easily absorbed in small intestine
[6-8]. The hydrophilic fl avonoid glucosides such as quercetin Flavonoids (formed from the aromatic amino acids
i.e. phenylalanine, tyrosine, and malonate) are one of the
important constituent in the human diet, although they are 007 Citation: Gupta A, Chavan RN (2017) Immunosuppressive and Cytotoxic Potential of Flavonoids from Medicinal Plants: Preliminary Investigation for Anticancer
Activity. Int J Immunother Cancer Res 3(1): 007-0011. DOI: http://doi.org/10.17352/2455-8591.000012 Citation: Gupta A, Chavan RN (2017) Immunosuppressive and Cytotoxic Potential of Flavonoids from Medicinal Plants: Preliminary Investigation for Anticancer
Activity. Int J Immunother Cancer Res 3(1): 007-0011. DOI: http://doi.org/10.17352/2455-8591.000012 Gupta and Chavan (2017) are transported across the small intestine by the intestinal
Na+-dependent glucose cotransporter (SGLT1). More than
4000 varieties of fl avonoids isolated from various medicinal
plants which is already identifi ed. The most important effect
of these fl avonoids is the scavenging of oxygen-derived free
radicals that is reported. In vitro experimental systems also
showed that fl avonoids possess anti-allergic, anti-viral and
anti-carcinogenic properties [7,8]. The fl avonoid is bound
to albumin and then transported to the liver. Cytotoxicity assay and estimation of nitric oxide pro-
duction Cytotoxicity assay and estimation of nitric oxide pro-
duction Briefl y, human PBMC (106 cells/ml; 100 μl; separated
through Ficoll–Hypaque gradient centrifugation) were plated
in 96 well plate and pre-incubated with HBsAg (20 μg/ml; 10
μl) and then treated with variable concentration of fl avonoids
(12.5 – 50 mg/ml; 50 μl). After addition of specifi c antigen and
test samples, incubate the plate for another 24h at 37ºC (carbon
dioxide incubator). The plates were centrifuged at 2500 rpm
for 10 minutes and then supernatant (100 μl) was collected for
estimation of nitric oxide [11-15]. Estimation of blood counts For these studies, exposure of variable doses of fl avonoids
(12.5 – 50 mg/ml; 50 μl) on human whole blood (n = 5; EDTA
human blood samples collected from Mangal Pathology lab,
Baramati) along with or without HBsAg (20 μg/ml; 10 μl) using
forward and side scatter representing different phenotypes i.e. lymphocytes (R1), monocytes (R2) and granulocytes (R3) count
that are analyzed using cell quest software (BD FACS Calibur). For these studies, addition of ACK lysing solution (1X, 2 ml)
after incubation of these fl avonoids extracted from the leaves
of Calotropis gigantea, Adhatoda vasica and Artocarpus integrifolia
with or without HBsAg and then followed by incubation for
another 10 min. Afterwards, the supernatant was aspirated
after centrifuging and washed two times with PBS. Finally,
pellet dissolved in PBS and observed the cells through fl ow
cytometer [11-15]. One of the medicinal plants, Calotropis gigantea (Rui;
family Asclepiadacea) have showed special attraction because
of major phytochemical i.e. Calotropin that are present and
showed various immunopharmacological activities [11,12]
i.e. anti-infl ammatory,
antimicrobial,
anti-diabetic
etc. Adhatoda vasica (Adulsa; family Acanthaceae), medicinal plant
products especially leaves showed various medicinal properties
and it will protect our immune system against number of
infectious diseases [13,14] e.g. cold, cough, asthma, malaria,
bleeding disorder, reduces blood sugar level etc. Artocarpus
integrifolia (Phanas; family Moraceae), medicinal plant which
is reported as an important source or requirement of edible
fruit and is widely used in traditional medicines. In Ayurveda,
Artocarpus integrifolia possessed antimicrobial, anti-peptic
ulcer,
cardiovascular
(anti-diabetic),
anti-oxidant
and
immunomodulatory properties [15]. This is the fi rst preliminary
study related to anticancer activity of fl avonoids isolated from
medicinal plants especially leaves in order to determined its
immunomodulatory (immunostimulator/immunosuppressor)
effect against specifi c protein antigen. Citation: Gupta A, Chavan RN (2017) Immunosuppressive and Cytotoxic Potential of Flavonoids from Medicinal Plants: Preliminary Investigation for Anticancer
Activity. Int J Immunother Cancer Res 3(1): 007-0011. DOI: http://doi.org/10.17352/2455-8591.000012 Cytotoxicity assay The effect of variable doses of fl avonoids on determining its
cytotoxicity in human PBMC’s against specifi c protein antigen
i.e. HBsAg as shown in Figure 2. The results of these studies
showed that these fl avonoids showed declined in proliferation
at higher doses after treating with PBMC as compared to
control. (C) (C)
Figure 1: Effect of variable doses of fl avonoids on human whole blood. Flow
cytometric analysis of fl avonoids extracted from A) Calotropis gigantean B)
Adhatoda vasica C) Artocarpus integrifolia in order to lymphocytes, monocytes and
granulocytes count in human whole blood which is exposed with HBsAg (20 μg/ml;
10 μl). After treatment with fl avonoids, lysed the cells with red cell lysis buffer and
wash the cells with phosphate buffered saline and analyzed the samples (10000
events, cell quest software) through fl ow cytometer. Extraction of fl avonoids For these studies, PBMC cell culture supernatant was
mixed with similar volume of Griess reagent (1% sulfanilamide
and 0.1% naphthylethylene diamine dihydrochloride in 2.5%
phosphoric acid) and incubated the plates at room temperature
for another 10 minutes and absorbance at 540 nm was measured
by spectrophotometer. The fresh culture medium (RPMI
containing 10 % fetal bovine serum) was used as a blank. The
nitrite quantity was determined from a sodium nitrite standard
curve. For fl avonoid extraction, using 10 g of fresh plant leaves
of Calotropis gigantea, Adhatoda vasica and Artocarpus integrifolia
were squashed with liquid nitrogen to prepare plant leaves
powder. Afterwards, leaves powder of these medicinal plants
were refl ux with methanol (80 %; 100 ml) for 2 h at 100 ºC. After
incubation, cool down the solution (another 1 h incubation)
and fi lter it using whatman fi lter paper. Filtrate solution
were collected containing constituents of plant powdered 008 Citation: Gupta A, Chavan RN (2017) Immunosuppressive and Cytotoxic Potential of Flavonoids from Medicinal Plants: Preliminary Investigation for Anticancer
Activity. Int J Immunother Cancer Res 3(1): 007-0011. DOI: http://doi.org/10.17352/2455-8591.000012 Citation: Gupta A, Chavan RN (2017) Immunosuppressive and Cytotoxic Potential of Flavonoids from Medicinal Plants: Preliminary Investigation for Anticancer
Activity. Int J Immunother Cancer Res 3(1): 007-0011. DOI: http://doi.org/10.17352/2455-8591.000012 Citation: Gupta A, Chavan RN (2017) Immunosuppressive and Cytotoxic Potential of Flavonoids from Medicinal Plants: Preli
Activity. Int J Immunother Cancer Res 3(1): 007-0011. DOI: http://doi.org/10.17352/2455-8591.000012 Gupta and Chavan (2017) Nitric oxide production The effect of variable doses of fl avonoids on nitric oxide
production in human PBMC’s as shown in Figure 3. The results
showed that these fl avonoids showed declined in nitric oxide at
higher doses after treating with PBMC as compared to control. Figure 1: Effect of variable doses of fl avonoids on human whole blood. Flow
cytometric analysis of fl avonoids extracted from A) Calotropis gigantean B)
Adhatoda vasica C) Artocarpus integrifolia in order to lymphocytes, monocytes and
granulocytes count in human whole blood which is exposed with HBsAg (20 μg/ml;
10 μl). After treatment with fl avonoids, lysed the cells with red cell lysis buffer and
wash the cells with phosphate buffered saline and analyzed the samples (10000
events, cell quest software) through fl ow cytometer. Estimation of lymphocytes, monocytes and granulo-
cytes count Estimation of lymphocytes, monocytes and granulo-
cytes count Estimation of lymphocytes, monocytes and granulo-
cytes count The result of these studies showed that these fl avonoids
extracted from the leaves of Calotropis gigantea, Adhatoda
vasica and Artocarpus integrifolia showed dramatically decreased
in monocytes as well as granulocytes count as compared to
control (Figure 1). In other words, these fl avonoids showed
immunosuppressive and cytotoxic effect at higher doses
against specifi c protein antigen. (B) (B) Statistical analysis Values are expressed as Mean ± S.E. The difference between
control and fl avonoids extracted from Calotropis gigantea,
Adhatoda vasica and A. integrifolia which is determined through
Bonferroni multiple comparison test (one way ANOVA test). Citation: Gupta A, Chavan RN (2017) Immunosuppressive and Cytotoxic Potential of Flavonoids from Medicinal Plants: Preliminary Investigation for Anticancer
Activity. Int J Immunother Cancer Res 3(1): 007-0011. DOI: http://doi.org/10.17352/2455-8591.000012 Results (A) Discussion DOI: http://doi.org/10.17352/2455-8591.000012 Gupta and Chavan (2017)
0
0.2
0.4
0.6
0.8
1
1.2
Calotropis gigantea
Adhatoda vasica
Artocarpus integrifolia
OD at 570 nm (Mean ± S.E.)
Control
HBsAg
12.5
25
50
***
**
***
**
*
Figure 2: Effect of variable doses of fl avonoids (12.5-50 mg/ml; 50 μl) extracted
from the leaves of medicinal plants (Calotropis gigantean, Adhatoda vasica and
Artocarpus integrifolia) on human PBMC proliferation assay. Values are expressed
as Mean ± S.E. The difference between the control, standard and treated
samples of fl avonoids is determined by one way ANOVA test (Bonferroni multiple
comparison test). 0
2
4
6
8
10
12
14
Calotropis gigantea
Adhatoda vasica
Artocarpus integrifolia
NO, μM (Mean ± S.E.)
Control
HBsAg
12.5
25
50
***
**
***
**
*
Figure 3: Effect of variable doses of fl avonoids (12.5-50 mg/ml; 50 μl) extracted
from the leaves of medicinal plants (Calotropis gigantean, Adhatoda vasica and
Artocarpus integrifolia) on nitric oxide production from human PBMC cell culture
supernatant. Values are expressed as Mean ± S.E. The difference between the
control, standard and treated samples of fl avonoids is determined by one way
ANOVA test (Bonferroni multiple comparison test). 0
0.2
0.4
0.6
0.8
1
1.2
Calotropis gigantea
Adhatoda vasica
Artocarpus integrifolia
OD at 570 nm (Mean ± S.E.)
Control
HBsAg
12.5
25
50
***
**
***
**
*
Figure 2: Effect of variable doses of fl avonoids (12.5-50 mg/ml; 50 μl) extracted
from the leaves of medicinal plants (Calotropis gigantean, Adhatoda vasica and
Artocarpus integrifolia) on human PBMC proliferation assay. Values are expressed
as Mean ± S.E. The difference between the control, standard and treated
samples of fl avonoids is determined by one way ANOVA test (Bonferroni multiple
comparison test). 0
0.2
0.4
0.6
0.8
1
1.2
Calotropis gigantea
Adhatoda vasica
Artocarpus integrifolia
OD at 570 nm (Mean ± S.E.)
Control
HBsAg
12.5
25
50
***
**
***
**
* uncontrolled nitric oxide production could also be a therapeutic
target [21]. From these studies, these medicinal plants i.e. Calotropis gigantea, Adhatoda vasica and Artocarpus integrifolia
showed inhibition of proliferation and nitric oxide production
at higher doses as compared to control. In other words, these
fl avonoids showed some drastic decreased in nitric oxide
production at higher doses and showed its therapeutic effect. In addition, these fl avonoids of these medicinal plants showed
inhibitory effects on HBsAg lymphocyte proliferation. Discussion Medicinal plants have been found as important contributors
to the pharmaceutical, agriculture and food industries. In
recent times, researchers focused on medicinal plants to
explore its various immunobiological activities all over
the world. These medicinal plants are generally composed
of primary (carbohydrates, proteins, fats etc.) as well as
secondary metabolites (alkaloids, fl avonoids, terpenoids
etc.). These secondary metabolites especially fl avonoids are
considered to be phenolic compounds which are normally
present in the pigments of fruit, vegetables, green tea and red
wine [16,17]. The fi rst fl avonoid isolated from citrus fruits and
was discovered by the Nobel Prize winner Albert Szent Gyorgyi
who called them vitamin P. These fl avonoids have shown series
of important reactions which can interact with drug transport,
interfere with cycline-dependent cell cycle regulation, inhibit
protein glycosylation or affect the function of platelets [16, 17]. rich in foods and vegetables containing fl avonoids reduces the
risk of chronic diseases especially cancer [1, 16, 17]. As per the literature survey, consumption of isofl avones
are directly associated with decreased risk of estrogen related
cancers and vascular diseases whereas total fl avonoids including
fl avanones, and fl avonols were inversely proportional to oral
and laryngeal cancers [18, 19]. In addition, reduced risk rate
of colorectal cancer was detected in high intake of fl avones,
isofl avones, anthocyanidins and fl avonols. In contrast, there
is no association between fl avonoids and prostate cancer
reported or emerged whereas inverse association was found
between proanthocyanidins and colorectal cancer [20]. In this
regard, we focused on various medicinal plants pertaining
to fl avonoids isolated from medicinal plants and determined
its immunopharmacological activity against specifi c protein
antigen. Cancer chemoprevention, by the use of natural and dietary
agents especially fl avonoids that can reverse, suppress or
prevent carcinogenic progression, has become an appealing
strategy to combat the dogma associated with increasing cases
of cancers worldwide. Number of epidemiological studies were
reported and conducted that long-term consumption of diet Modulation (stimulatory or suppressive) of the immune Modulation (stimulatory or suppressive) of the immune 009 Citation: Gupta A, Chavan RN (2017) Immunosuppressive and Cytotoxic Potential of Flavonoids from Medicinal Plants: Preliminary Investigation for Anticancer
Activity. Int J Immunother Cancer Res 3(1): 007-0011. Discussion From
these results, it is confi rmed that fl avonoids from Calotropis
gigantea, Adhatoda vasica and Artocarpus integrifolia showed
immunosuppressive and cytotoxic effect against HBsAg. Figure 2: Effect of variable doses of fl avonoids (12.5-50 mg/ml; 50 μl) extracted
from the leaves of medicinal plants (Calotropis gigantean, Adhatoda vasica and
Artocarpus integrifolia) on human PBMC proliferation assay. Values are expressed
as Mean ± S.E. The difference between the control, standard and treated
samples of fl avonoids is determined by one way ANOVA test (Bonferroni multiple
comparison test). Conclusion The present study has shown the immunosuppressive and
cytotoxic potential of fl avonoid extracted from the leaves of
Calotropis gigantea, Adhatoda vasica and Artocarpus integrifolia
by inhibiting cellular immunity in the form of nitric oxide
production, blood counts and proliferation assay. These
fi ndings also suggested that these fl avonoids from medicinal
plants suppress the immune system and also be one of the
probable candidate like other chemotherapeutic agents. Further
detailed studies of these fl avonoids are required which might
establish a possible mechanism for anticancer activity. 0
2
4
6
8
10
12
14
Calotropis gigantea
Adhatoda vasica
Artocarpus integrifolia
NO, μM (Mean ± S.E.)
Control
HBsAg
12.5
25
50
***
**
***
**
* 0
2
4
6
8
10
12
14
Calotropis gigantea
Adhatoda vasica
Artocarpus integrifolia
NO, μM (Mean ± S.E.)
Control
HBsAg
12.5
25
50
***
**
***
**
*
Figure 3: Effect of variable doses of fl avonoids (12.5-50 mg/ml; 50 μl) extracted
from the leaves of medicinal plants (Calotropis gigantean, Adhatoda vasica and
Artocarpus integrifolia) on nitric oxide production from human PBMC cell culture
supernatant. Values are expressed as Mean ± S.E. The difference between the
control, standard and treated samples of fl avonoids is determined by one way
ANOVA test (Bonferroni multiple comparison test). References 1. Ochwang’I DO, Kimwele CN, Oduma JA, Gathumbi PK, Mbaria JM, et al. (2014) Medicinal plants used in treatment and management of cancer in
Kakamega County Kenya. J Ethnopharmacology 151: 1040–1055. Link:
https://goo.gl/rWw3eb Artocarpus integrifolia Adhatoda vasica Figure 3: Effect of variable doses of fl avonoids (12.5-50 mg/ml; 50 μl) extracted
from the leaves of medicinal plants (Calotropis gigantean, Adhatoda vasica and
Artocarpus integrifolia) on nitric oxide production from human PBMC cell culture
supernatant. Values are expressed as Mean ± S.E. The difference between the
control, standard and treated samples of fl avonoids is determined by one way
ANOVA test (Bonferroni multiple comparison test). 2. Romagnolo DF, Selmin OI (2012) Flavonoids and cancer prevention:
a review of the evidence. J Nutr Gerontol Geriatr 31: 206–238. Link:
https://goo.gl/vr9CdO 2. Romagnolo DF, Selmin OI (2012) Flavonoids and cancer prevention:
a review of the evidence. J Nutr Gerontol Geriatr 31: 206–238. Link:
https://goo.gl/vr9CdO 3. Costa-Lotufo LV, Khan MTH, Ather A, Wilke DV, Jimenez PC, et al. (2005)
Studies of the anticancer potential of plants used in Bangladeshi folk
medicine. J Ethnopharmacology 99: 21–30. Link: https://goo.gl/xCesVs 3. Costa-Lotufo LV, Khan MTH, Ather A, Wilke DV, Jimenez PC, et al. (2005)
Studies of the anticancer potential of plants used in Bangladeshi folk
medicine. J Ethnopharmacology 99: 21–30. Link: https://goo.gl/xCesVs system by these cytostatic agents is emerging as a major
area in Immunopharmacology, especially in those cases
where undesired immunosuppression is the result of therapy. These studies were conducted in human whole blood and
peripheral blood mononuclear cells in order to estimate its
immunosuppressive and cytotoxic activity. In this study, there
is declined in the number of monocytes and granulocytes
count in human whole blood after treating with variable doses
of fl avonoids which is confi rmed through fl ow cytometric
analysis and the results showed that these fl avonoids from
these medicinal plants showed immunosuppressive and
cytotoxic activity against specifi c protein antigen i.e. HBsAg as
compared to control and standard. 4. Gupta A, Chaphalkar SR (2015) Immunosuppressive activity of fl avonoids
isolated from Terminalia arjuna, Prosopis spicigera and Mimusops elengi. Int J
Res Pharmacy and Science 5: 14–17. Link: https://goo.gl/YgJ10d 4. Gupta A, Chaphalkar SR (2015) Immunosuppressive activity of fl avonoids
isolated from Terminalia arjuna, Prosopis spicigera and Mimusops elengi. Int J
Res Pharmacy and Science 5: 14–17. Link: https://goo.gl/YgJ10d 5. Citation: Gupta A, Chavan RN (2017) Immunosuppressive and Cytotoxic Potential of Flavonoids from Medicinal Plants: Preliminary Investigation for Anticancer
Activity. Int J Immunother Cancer Res 3(1): 007-0011. DOI: http://doi.org/10.17352/2455-8591.000012 References Gupta A, Chaphalkar SR (2015) Anti-infl ammatory activity of fl avonoids from
medicinal plants against hepatitis B vaccine antigen on human peripheral
blood mononuclear cells. Asian J Pharmac Clinical Res 3: 728–732. Link:
https://goo.gl/Jmy2T0 5. Gupta A, Chaphalkar SR (2015) Anti-infl ammatory activity of fl avonoids from
medicinal plants against hepatitis B vaccine antigen on human peripheral
blood mononuclear cells. Asian J Pharmac Clinical Res 3: 728–732. Link:
https://goo.gl/Jmy2T0 6. Spencer JP (2008) Flavonoids: modulators of brain function. British J
Nutrition 99: 60–77. Link: https://goo.gl/ipkieq 6. Spencer JP (2008) Flavonoids: modulators of brain function. British J
Nutrition 99: 60–77. Link: https://goo.gl/ipkieq 7. Kumar S, Pathania AS, Saxena AK, Vishwakarma RA, Ali A, et al. (2013) The
anticancer potential of fl avonoids isolated from the stem bark of Erythrina
suberosa through induction of apoptosis and inhibition of STAT signaling
pathway in human leukaemia HL-60 cells. Chemico Biol Interactions 205:
128–137. Link: https://goo.gl/lmNJ8y As per literature, nitric oxide production is mainly released
by human PBMC and played a considerable role especially in the
pathophysiology of hormonal immune system [21]. Actually,
diseases related to animal or human can be characterized or
diagnosed through lack or excess of nitric oxide production
from human PBMC or animal peritoneal macrophages. In some
circumstances, protection against decline in constitutive nitric
oxide production or stimulation in the vasculature may intercept
the development of vascular disease, while inhibition of 8. Agati G, Azzarello E, Pollastri S, Tattini M (2012) Flavonoids as antioxidants
in plants: Location and functional signifi cance. Plant Science 196: 67–76. Link: https://goo.gl/3h2Uhk 9. Mujwah AA, Mohammed MA, Ahmed MH (2010) First isolation of a fl avonoid
from Juniperus procera using ethyl acetate extract. Arabian J Chem 3: 85-88. Link: https://goo.gl/UNUy7R 0010 Citation: Gupta A, Chavan RN (2017) Immunosuppressive and Cytotoxic Potential of Flavonoids from Medicinal Plants: Preliminary Investigation for Anticancer
Activity. Int J Immunother Cancer Res 3(1): 007-0011. DOI: http://doi.org/10.17352/2455-8591.000012 Gupta and Chavan (2017) Artocarpus integrifolia. Int J Pharma sciences research 6: 1–5. Link:
https://goo.gl/HC6r5i 10. Li CW, Cui CB (2014) One new and nine Known Flavonoids from
Choerospondias axillaries and their in vitro antitumor, anti-hypoxia
and
antibacterial
activities. Molecules
19:
21363–21377. Link:
https://goo.gl/Nrl3Qq 16. Braca A, Sortino C, Politi M, Morelli I, Mendez J (2002) Antioxidant activity of
fl avonoids from Licania licaniaeflora. J Ethnopharmacol 79: 379–381. Link:
https://goo.gl/FfbgWt 11. Gupta A, Chaphalkar SR (2016) Antidiabetic activity of Calotropis gigantea in
human whole blood. References J Disease Global Health 6: 107–112. 17. Heber D (2014) Vegetables, fruits and phytoestrogens in the prevention of
diseases. J Postgrad Med 50: 145–149. Link: https://goo.gl/ojPyiX 12. Gupta A, Chaphalkar SR (2016) Inhibition of antigen specifi c T cell population
using Calotropis gigantea and Terminalia arjuna. J Biology nature 5: 14–19. Link: https://goo.gl/V2WfOk 18. Arai Y, Watanabe S, Kimira M, Shimoi K, Mochizuki R, et al. (2000) Dietary
intakes of fl avonols, fl avones and isofl avones by Japanese women and the
inverse correlation between quercetin intake and plasma LDL cholesterol
concentration. J Nutr 130: 2243–2250. Link: https://goo.gl/8AnWPC 13. Gupta A, Chitte R, Chaphalkar SR (2016) Comparative evaluation of
aqueous extract and protease from Adhatoda vasica for determining its
immunosuppressive activity. Indo American J Pharmaceutical Sci 3: 821-
825. Link: https://goo.gl/nPZoMF 19. Birt DF, Hendrich S, Wang W (2001) Dietary agents in cancer prevention:
fl avonoids and isofl avonoids. Pharmacol Ther 90: 157–177. Link:
https://goo.gl/M2bGQe 14. Gupta A, Chaphalkar SR (2015) Immunosuppressive activity of saponin from
the leaves of Adhatoda vasica. Int J Institutional Pharmacy and Life Sciences
5: 137–145. 20. Batra P, Sharma AK (2013) Anti-cancer potential of fl avonoids: recent trends
and future perspectives. Biotech 3: 439–459. Link: https://goo.gl/e674sp 21. Makchuchit S, Itharat A, Tewtrakul S (2010) Antioxidant and nitric oxide
inhibition activities of Thai medicinal plants. J Med Assoc Thai 93: 227-235. Link: https://goo.gl/f0Y3wh 15. Gupta A, Chaphalkar SR (2015) Immunosuppressive activity of crude
saponins from the leaves of Calotropis gigantea, Calamus rotang and 15. Gupta A, Chaphalkar SR (2015) Immunosuppressive activity of crude
saponins from the leaves of Calotropis gigantea, Calamus rotang and 0011 Citation: Gupta A, Chavan RN (2017) Immunosuppressive and Cytotoxic Potential of Flavonoids from Medicinal Plants: Preliminary Investigation for Anticancer
Activity. Int J Immunother Cancer Res 3(1): 007-0011. DOI: http://doi.org/10.17352/2455-8591.000012 Citation: Gupta A, Chavan RN (2017) Immunosuppressive and Cytotoxic Potential of Flavonoids from Medicinal Plants: Preliminary Investigation for Anticancer
Activity. Int J Immunother Cancer Res 3(1): 007-0011. DOI: http://doi.org/10.17352/2455-8591.000012
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Hydrological soil properties control tree regrowth after forest disturbance in the forest steppe of central Mongolia
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Soil
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cc-by
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Hydrological soil properties control tree regrowth after forest
disturbance in the forest-steppe of central Mongolia We found a significant difference in soil texture between soils under DWIR and
20
DNOR. Sand generally dominated the soil texture, but soils under DWIR had more silt and clay compared to soils under
DNOR. Soil pF curves showed that soils under DWIR had higher plant-available field capacity than soils under DNOR. In
addition, hydraulic conductivity was higher in the uppermost horizons of soils under DWIR compared to soils under DNOR. Chemical properties of the soils under DWIR and DNOR showed no significant differences. chemical, physical, and hydrological analysis. We found a significant difference in soil texture between soils under DWIR and
20
DNOR. Sand generally dominated the soil texture, but soils under DWIR had more silt and clay compared to soils under
DNOR. Soil pF curves showed that soils under DWIR had higher plant-available field capacity than soils under DNOR. In
addition, hydraulic conductivity was higher in the uppermost horizons of soils under DWIR compared to soils under DNOR. Chemical properties of the soils under DWIR and DNOR showed no significant differences. We conclude that the differences in post-disturbance tree regrowth are mainly caused by soil hydrological properties. High
25
plant-available field capacity is the key factor for forest recovery under semi-arid conditions. High hydraulic conductivity in
the uppermost soil horizons can further support tree regrowth, because it reduces evaporation loss and competition of larch
saplings with grasses and herbs for water. Another important factor is human impact, particularly grazing on cleared forest
sites, which often keeps seedlings from growing and thus inhibits forest recovery. Permafrost was absent at all studied disturbed We conclude that the differences in post-disturbance tree regrowth are mainly caused by soil hydrological properties. High
25
plant-available field capacity is the key factor for forest recovery under semi-arid conditions. High hydraulic conductivity in
the uppermost soil horizons can further support tree regrowth, because it reduces evaporation loss and competition of larch
saplings with grasses and herbs for water. Another important factor is human impact, particularly grazing on cleared forest
sites, which often keeps seedlings from growing and thus inhibits forest recovery. Permafrost was absent at all studied disturbed sites (DWIR, DNOR). We thus conclude that it is not a major factor for the post-disturbance tree-regrowth pattern, although
30
it does contribute to water availability in summer. sites (DWIR, DNOR). Hydrological soil properties control tree regrowth after forest
disturbance in the forest-steppe of central Mongolia Florian Schneider1, Michael Klinge1, Jannik Brodthuhn1, Tino Peplau2, Daniel 1Department of Physical Geography, University of Göttingen, Göttingen, 37077, Germany
2Thünen Institute of Climate-Smart Agriculture, Braunschweig, 38116, Germany Abstract. The central Mongolian forest-steppe is a sensitive ecotone, commonly affected by disturbances such as logging and
forest fires. In addition, intensified drought events aggravate stress on the trees that are anyway at their drier limit in the forest-
10
steppe. Climate change increases evapotranspiration and reduces the distribution of discontinuous permafrost. The motivation
for this study came about through our previous observation that forest stands show great differences with respect to their
recovery after disturbance by fire or logging. Sometimes, no regrowth of trees takes place at all. As water availability is the
main limiting factor of forest growth in this region, we hypothesized that differences in hydrological soil properties control the forest fires. In addition, intensified drought events aggravate stress on the trees that are anyway at their drier limit in the forest-
10
steppe. Climate change increases evapotranspiration and reduces the distribution of discontinuous permafrost. The motivation
for this study came about through our previous observation that forest stands show great differences with respect to their
recovery after disturbance by fire or logging. Sometimes, no regrowth of trees takes place at all. As water availability is the
main limiting factor of forest growth in this region, we hypothesized that differences in hydrological soil properties control the forest-recovery pattern. 15
To test this hypothesis, we analysed soil properties under forests, predominantly consisting of Siberian larch (Larix sibirica
Ledeb.), in the forest-steppe of the northern Khangai Mountains in central Mongolia. We distinguished four vegetation
categories: 1. near-natural forest (FOR), 2. steppe close to the forest (STE), 3. disturbed forest with regrowth of trees (DWIR),
and 4. disturbed forest showing no regrowth of trees (DNOR). 54 soil profiles were described in the field and sampled for soil forest-recovery pattern. 15
To test this hypothesis, we analysed soil properties under forests, predominantly consisting of Siberian larch (Larix sibirica
Ledeb.), in the forest-steppe of the northern Khangai Mountains in central Mongolia. We distinguished four vegetation
categories: 1. near-natural forest (FOR), 2. steppe close to the forest (STE), 3. disturbed forest with regrowth of trees (DWIR),
and 4. disturbed forest showing no regrowth of trees (DNOR). 54 soil profiles were described in the field and sampled for soil chemical, physical, and hydrological analysis. https://doi.org/10.5194/soil-2020-52
Preprint. Discussion started: 5 October 2020
c⃝Author(s) 2020. CC BY 4.0 License. Hydrological soil properties control tree regrowth after forest
disturbance in the forest-steppe of central Mongolia We thus conclude that it is not a major factor for the post-disturbance tree-regrowth pattern, although
30
it does contribute to water availability in summer. sites (DWIR, DNOR). We thus conclude that it is not a major factor for the post-disturbance tree-regrowth pattern, although
30
it does contribute to water availability in summer. Keywords: Siberian larch, permafrost, forest fire, human impact, climate change Keywords: Siberian larch, permafrost, forest fire, human impact, climate change 1 1 https://doi.org/10.5194/soil-2020-52
Preprint. Discussion started: 5 October 2020
c⃝Author(s) 2020. CC BY 4.0 License. 1 Introduction The forest-steppe in central Mongolia is an ecotone at the transition between the Siberian Taiga in the north and the Gobi
35
Desert in the south, responding sensitively to climatic, ecological and anthropogenic disturbances. Water availability is the
key factor that determines, where forest patches can exist within this ecotone. Climate change is a major threat for the forest-
steppe due to aggravated drought stress (Allen et al., 2010). In Mongolia, intensified summer droughts became more frequent
over the last century (Batima et al., 2005; Dashkhuu et al., 2015). Siberian larch (Larix sibirica Ledeb.), which is the dominant over the last century (Batima et al., 2005; Dashkhuu et al., 2015). Siberian larch (Larix sibirica Ledeb.), which is the dominant
tree species in the Mongolian forest-steppe, suffers from these intensified droughts (Dulamsuren et al., 2009; 2010; 2011;
40
Chenlemuge et al., 2015b; 2015a). Furthermore, summer droughts increase the risk of fires that disturb the vegetation structure
in forests and reduce the size of the forest patches (Kharuk et al., 2008). This causes a negative effect on the forest microclimate,
which in turn intensifies the sensitivity of the forest to drought stress (Khansaritoreh et al., 2017a). In addition to fire, also
human impact affects the structure of the forest stands. Logging and forest pasture reduce the forest size, open the forest stand
and hamper post-disturbance forest regrowth (Hilbig, 1987; Khishigjargal et al., 2013; Tsogtbaatar, 2013; Dulamsuren et al.,
45 tree species in the Mongolian forest-steppe, suffers from these intensified droughts (Dulamsuren et al., 2009; 2010; 2011;
40
Chenlemuge et al., 2015b; 2015a). Furthermore, summer droughts increase the risk of fires that disturb the vegetation structure
in forests and reduce the size of the forest patches (Kharuk et al., 2008). This causes a negative effect on the forest microclimate,
which in turn intensifies the sensitivity of the forest to drought stress (Khansaritoreh et al., 2017a). In addition to fire, also
human impact affects the structure of the forest stands. Logging and forest pasture reduce the forest size, open the forest stand and hamper post-disturbance forest regrowth (Hilbig, 1987; Khishigjargal et al., 2013; Tsogtbaatar, 2013; Dulamsuren et al.,
45
2014). Meltwater from the active layer above permafrost patches increases the soil water availability and can thus support the survival
of trees during drought events in summer (Sugimoto et al., 2002; Ishikawa et al., 2005; Churakova et al., 2016). 1 Introduction Permafrost in
Mongolia is discontinuous and is mainly limited to forests. However, climate change is currently reducing the area of permafrost in Mongolia (Sharkhuu and Sharkhuu, 2012). After forest disturbance by fire or logging, permafrost degrades and
50
may only reconstitute under certain conditions (Klinge et al., submitted). The loss of this additional water source leads to
enhanced stress of trees during droughts. Decreased water supply and increased drought stress, raises the importance of soil properties in this ecotone even further. Soil-
water distribution in the landscape after a rainfall event is generally controlled by bedrock, sediment cover, slope morphology permafrost in Mongolia (Sharkhuu and Sharkhuu, 2012). After forest disturbance by fire or logging, permafrost degrades and
50
may only reconstitute under certain conditions (Klinge et al., submitted). The loss of this additional water source leads to
enhanced stress of trees during droughts. Decreased water supply and increased drought stress, raises the importance of soil properties in this ecotone even further. Soil-
water distribution in the landscape after a rainfall event is generally controlled by bedrock sediment cover slope morphology and vegetation. In addition, plant-available field capacity and hydraulic conductivity of the soils influence the amount of water
55
available at a site. Moreover, water repellency can reduce water infiltration, especially after fire events (DeBano, 2000; Doerr
et al., 2000). Several studies have been conducted on soil distribution and soil properties in the Mongolian forest-steppe (Opp
and Hilbig, 2003; Maximovich, 2004; Lehmkuhl et al., 2011). Nandintsetseg and Shinoda (2011) modelled soil moisture
conditions in Mongolia based on data from meteorological stations from 1986 until 2005. They obtained a gradient of and vegetation. In addition, plant-available field capacity and hydraulic conductivity of the soils influence the amount of water
55
available at a site. Moreover, water repellency can reduce water infiltration, especially after fire events (DeBano, 2000; Doerr
et al., 2000). Several studies have been conducted on soil distribution and soil properties in the Mongolian forest-steppe (Opp
and Hilbig, 2003; Maximovich, 2004; Lehmkuhl et al., 2011). Nandintsetseg and Shinoda (2011) modelled soil moisture
conditions in Mongolia based on data from meteorological stations from 1986 until 2005. They obtained a gradient of increasing soil moisture from southwest to northeast during summer. Haase (1963) studied soils in relation to altitudinal zones
60
in the Khangai Mountains, including particle size distribution, chemical soil properties, and water content. 1 Introduction Yet, he did not analyse soil-hydrological properties. During previous fieldwork in the Khangai Mountains, in the central Mongolian forest-steppe, we observed that forest regrowth
70
after disturbance does not proceed equally. The post-disturbance development of forest stands with apparently same site
conditions ranges from spontaneous dense regrowth to no regrowth at all. As the trees are at their drier limit in this sensitive
ecotone, we assumed that water availability is the main factor that controls forest regrowth after disturbance, whereas nutrient
availability plays a less important role. Since both factors are related to soil properties, we established the following
hypotheses:
75 During previous fieldwork in the Khangai Mountains, in the central Mongolian forest-steppe, we observed that forest regrowth
70
after disturbance does not proceed equally. The post-disturbance development of forest stands with apparently same site
conditions ranges from spontaneous dense regrowth to no regrowth at all. As the trees are at their drier limit in this sensitive
ecotone, we assumed that water availability is the main factor that controls forest regrowth after disturbance, whereas nutrient
availability plays a less important role. Since both factors are related to soil properties, we established the following
hypotheses:
75 1) Silty soil texture leads to high plant-available field capacity and is thus favourable for post-disturbance tree regrowth. 1) Silty soil texture leads to high plant-available field capacity and is thus favourable for post-dis 1) Silty soil texture leads to high plant-available field capacity and is thus favourable for post-disturbance tree regrowth. 2) High hydraulic conductivity in the uppermost parts of the soils supports post-disturbance forest regrowth, as rapid water
infiltration to some depth reduces evaporation and ground vegetation transpiration 2) High hydraulic conductivity in the uppermost parts of the soils supports post-disturbance forest regrowth, as rapid water
infiltration to some depth reduces evaporation and ground-vegetation transpiration. 3) High nutrient stocks, for example after fire events, may support post-disturbance forest regrowth, unless nutrient stocks are
anyway well above the requirements of Siberian larch. In this case, no effect is to be expected. 0 80 4) Permafrost may increase water availability for trees through the continuous water release at the melting front above the
permafrost table over the summer season. As water above the permafrost table cannot infiltrate downwards, interflow may
occur above the permafrost table. This process leads to increased water availability, especially in concave slope positions
where interflow converges. 1 Introduction He reported forest
vegetation on soils with higher water content and predominant steppe vegetation on soils with lower water content. However,
he focused mainly on general soil description and did not report any further details on soil-vegetation relationships. Krasnoshchekov (2008, 2010) described physical and chemical soil properties in the East Khubsugul region and the Khangai increasing soil moisture from southwest to northeast during summer. Haase (1963) studied soils in relation to altitudinal zones
60
in the Khangai Mountains, including particle size distribution, chemical soil properties, and water content. He reported forest
vegetation on soils with higher water content and predominant steppe vegetation on soils with lower water content. However,
he focused mainly on general soil description and did not report any further details on soil-vegetation relationships. Krasnoshchekov (2008, 2010) described physical and chemical soil properties in the East Khubsugul region and the Khangai 60 increasing soil moisture from southwest to northeast during summer. Haase (1963) studied soils in relation to altitudinal zones
60
in the Khangai Mountains, including particle size distribution, chemical soil properties, and water content. He reported forest
vegetation on soils with higher water content and predominant steppe vegetation on soils with lower water content. However,
he focused mainly on general soil description and did not report any further details on soil-vegetation relationships. Krasnoshchekov (2008, 2010) described physical and chemical soil properties in the East Khubsugul region and the Khangai Mountains. Sympilova and Gyninova (2012) conducted similar research in the Selenga Mountains. However, in both studies
65
only the soils were characterised, without linking them to the vegetation pattern. Sommer (2000) analysed soils of the forest- Mountains. Sympilova and Gyninova (2012) conducted similar research in the Selenga Mountains. However, in both studies
65
only the soils were characterised, without linking them to the vegetation pattern. Sommer (2000) analysed soils of the forest- Mountains. Sympilova and Gyninova (2012) conducted similar research in the Selenga Mountains. However, in both studies
65
only the soils were characterised, without linking them to the vegetation pattern. Sommer (2000) analysed soils of the forest- 2 2 https://doi.org/10.5194/soil-2020-52
Preprint. Discussion started: 5 October 2020
c⃝Author(s) 2020. CC BY 4.0 License. steppe of the Turgen-Charchiraa-Mountains in north-western Mongolia. He focused on soil-chemical aspects and did not find
a clear relationship between forest distribution and soil properties. He concluded that forest distribution was climate-controlled
and disturbed by pastoral pressure and logging. 1 Introduction Thus, permafrost may support post-disturbance forest regrowth also at sites with otherwise
unfavourable soil-hydrological conditions for forest regrowth. 85 85 2.1 Study area The study area was located in the central Mongolian forest-steppe, in the northern Khangai Mountains, near the town
Tosontsengel (Fig. 1). The geological basement predominantly consists of Permian acidic plutonic and metamorphic
sedimentary rock, with some occurrences of Carboniferous mafic rock (Academy of Sciences of Mongolia, Academy of
90
Sciences of USSR, 1990). Slope debris with inmixed aeolian sand and silt provides the parent material for the soils in the
region. The widespread aeolian deposits originate from lacustrine sediments of big lakes that dried out during the glacial
periods in the Basin of Great Lakes, located between the Mongolian Altai and the Khangai Mountains (Grunert et al., 2000;
Lehmkuhl and Haselein, 2000; Klinge and Lehmkuhl, 2013). 90 3 3 https://doi.org/10.5194/soil-2020-52
Preprint. Discussion started: 5 October 2020
c⃝Author(s) 2020. CC BY 4.0 License. 4 95 4 https://doi.org/10.5194/soil-2020-52
Preprint. Discussion started: 5 October 2020
c⃝Author(s) 2020. CC BY 4.0 License. Figure 1: Sentinel image (2019/07/31) of the study area with soil profile positions. The colours indicate the four vegetation categories
that were used for selecting the soil profile locations: near-natural forest (FOR, green), steppe close to the forest (STE, grey),
disturbed forest with regrowth of trees (DWIR, blue), and disturbed forest showing no regrowth of trees (DNOR, orange). The soil
profiles are listed in Table 1 of the appendix. Right top: Physical map of Mongolia, showing the location of the study area (white
rectangle). 100 The climate is cold, semi-arid, and highly continental. In the town Tosontsengel, in the northern part of the study area, mean
monthly temperatures range from -31.7 °C in January to 14.9 °C in July (Fig. 2). Mean annual precipitation is 200-500 mm. Rainfall concentrates in summer, because of low-pressure cells that are blown in with the westerlies (Batima et al., 2005). The
mean annual temperature of -5.9 °C supports discontinuous permafrost in the study area (Bonan and Shugart, 1989). Permafrost
105
patches occur on forested slopes, where the canopy protects them from solar radiation, and the organic layer insulates them
from high air temperatures in summer (Dashtseren et al., 2014). In summer, the forests benefit from an additional water supply
by meltwater from the active layer above the permafrost (Zhang et al., 2011). The climate is cold, semi-arid, and highly continental. The climate is cold, semi-arid, and highly continental. In the town Tosontsengel, in the northern part of the study area, mean
monthly temperatures range from -31.7 °C in January to 14.9 °C in July (Fig. 2). Mean annual precipitation is 200-500 mm.
Rainfall concentrates in summer, because of low-pressure cells that are blown in with the westerlies (Batima et al., 2005). The
mean annual temperature of -5.9 °C supports discontinuous permafrost in the study area (Bonan and Shugart, 1989). Permafrost
105
patches occur on forested slopes, where the canopy protects them from solar radiation, and the organic layer insulates them
from high air temperatures in summer (Dashtseren et al., 2014). In summer, the forests benefit from an additional water supply
by meltwater from the active layer above the permafrost (Zhang et al., 2011). 2.1 Study area In the town Tosontsengel, in the northern part of the study area, mean
monthly temperatures range from -31.7 °C in January to 14.9 °C in July (Fig. 2). Mean annual precipitation is 200-500 mm. Rainfall concentrates in summer, because of low-pressure cells that are blown in with the westerlies (Batima et al., 2005). The
mean annual temperature of -5.9 °C supports discontinuous permafrost in the study area (Bonan and Shugart, 1989). Permafrost
105
patches occur on forested slopes, where the canopy protects them from solar radiation, and the organic layer insulates them
from high air temperatures in summer (Dashtseren et al., 2014). In summer, the forests benefit from an additional water supply
by meltwater from the active layer above the permafrost (Zhang et al., 2011). 105 5 5 https://doi.org/10.5194/soil-2020-52
Preprint. Discussion started: 5 October 2020
c⃝Author(s) 2020. CC BY 4.0 License. https://doi.org/10.5194/soil-2020-52
Preprint. Discussion started: 5 October 2020
c⃝Author(s) 2020. CC BY 4.0 License. Figure 2: Climate diagram for Tosontsengel, Mongolia (data source: meteorological station of Tosontsengel, Mongolia; National
110
Statistical Office of Mongolia). Figure 2: Climate diagram for Tosontsengel, Mongolia (data source: meteorological station of Tosontsengel, Mongolia; National
0
Statistical Office of Mongolia). Figure 2: Climate diagram for Tosontsengel, Mongolia (data source: meteorological station of Tosontsengel, Mongolia; National
110
Statistical Office of Mongolia). The vegetation of the study area is dominated by steppe. Only north-facing slopes and some valley positions are covered by
forest, predominately consisting of Siberian larch (Larix sibirica Ledeb.) (Hilbig, 1987; Tsogtbaatar, 2013). The understory of The vegetation of the study area is dominated by steppe. Only north-facing slopes and some valley positions are covered by
forest, predominately consisting of Siberian larch (Larix sibirica Ledeb.) (Hilbig, 1987; Tsogtbaatar, 2013). The understory of these forests varies with respect to proportions of grasses, herbs, mosses and shrubs (commonly including e.g. Vaccinium vitis-
115
idea and Lonicera altaica). High evapotranspiration combined with low precipitation and relief-dependant differences in
insolation control the vegetation pattern (Schlütz et al., 2008; Hais et al., 2016). The semi-arid conditions promote frequent
forest fires in this region (Goldammer, 2002; Hessl et al., 2016). The last two severe fire events in the study area happened in
1996 and 2002. A timber factory was established in the 1960s in Tosontsengel. Thus, logging strongly affected the forest stands in the study
120
area. 2.1 Study area Since the 1990s, industrial logging is abandoned, but illegal logging is still common (Lkhagvadorj et al., 2013). Moreover, A timber factory was established in the 1960s in Tosontsengel. Thus, logging strongly affected the forest stands in the study
120
area. Since the 1990s, industrial logging is abandoned, but illegal logging is still common (Lkhagvadorj et al., 2013). Moreover, A timber factory was established in the 1960s in Tosontsengel. Thus, logging strongly affected the forest stands in the study
120
area. Since the 1990s, industrial logging is abandoned, but illegal logging is still common (Lkhagvadorj et al., 2013). Moreover, 6 https://doi.org/10.5194/soil-2020-52
Preprint. Discussion started: 5 October 2020
c⃝Author(s) 2020. CC BY 4.0 License. pastoral pressure increased over the last decades (Lkhagvadorj et al., 2013). Logging, followed by grazing, led to a reduction
of the forested area, especially at its lower boundary and at the forest edges. pastoral pressure increased over the last decades (Lkhagvadorj et al., 2013). Logging, followed by grazing, led to a reduction
of the forested area, especially at its lower boundary and at the forest edges. 2.2 Field work The site selection within the study area was based on four categories of vegetation: near-natural forest (FOR), steppe close to
125
the forest (STE), disturbed forest with regrowth of trees (DWIR) and disturbed forest showing no regrowth of trees (DNOR)
(Figs. 1 and 3). In addition, we distinguished three categories of disturbance intensity. “Low intensity” included e.g. logging
of single trees. “Moderate” included logging of patches within a forest stand and fires that did not affect the whole forest stand. “Severe” included clear cutting and fires that destroyed the whole forest stand (Tab. A1, appendix). To reduce the effects of The site selection within the study area was based on four categories of vegetation: near-natural forest (FOR), steppe close to
125
the forest (STE), disturbed forest with regrowth of trees (DWIR) and disturbed forest showing no regrowth of trees (DNOR)
(Figs. 1 and 3). In addition, we distinguished three categories of disturbance intensity. “Low intensity” included e.g. logging
of single trees. “Moderate” included logging of patches within a forest stand and fires that did not affect the whole forest stand. “Severe” included clear cutting and fires that destroyed the whole forest stand (Tab. A1, appendix). To reduce the effects of factors that were not in the focus of this study, we kept geology, exposition (only north-facing slopes), inclination, and slope
130
morphology as similar as possible. The elevation of the studied soil profiles ranged from 1850 to 2100 m a.s.l., which is well
below the upper tree line of approx. 2500 m a.s.l. (Klinge et al., 2018). 54 soil profiles were described according to the FAO
Guidelines for Soil Description (FAO, 2006) and classified according to WRB (IUSS Working Group WRB, 2015). The
presence of permafrost was documented as well. The soil profiles were sampled horizon-wise, whereby thick horizons were factors that were not in the focus of this study, we kept geology, exposition (only north-facing slopes), inclination, and slope
130
morphology as similar as possible. The elevation of the studied soil profiles ranged from 1850 to 2100 m a.s.l., which is well
below the upper tree line of approx. 2500 m a.s.l. (Klinge et al., 2018). 54 soil profiles were described according to the FAO
Guidelines for Soil Description (FAO, 2006) and classified according to WRB (IUSS Working Group WRB, 2015). The
presence of permafrost was documented as well. 2.3 Laboratory analyses Soil pF curves were determined in four to five replicates, using a pressure device (ecoTech) (Deutsches
160
Institut für Normung e.V., 2014). Water repellency was analysed on five replicates for each top-soil sample (Doerr, 1998). The sample was placed in a petri dish and the surface was manually smoothened. The time was measured until a drop of
distilled water, released from a pipette at 5 cm above the sample surface, infiltrated into the sample. 2.3 Laboratory analyses The soil samples were dried at 40 °C and passed through a 2 mm sieve. Approximately 10 g of each sample was dried at 105
°C to gravimetrically determine the remaining water contents for correcting all analytical data obtained from the 40 °C-dried
samples. An aliquot of each sample was milled. Total C and N contents were analysed on the milled samples using a CHN
145
analyser (LecoTruSpec). Carbonate contents were also analysed on the milled samples using the Scheibler method (Blume et
al., 2011). Soil organic carbon (SOC) contents were calculated by subtracting soil inorganic C (obtained from carbonate
analysis) from total C. Soil pH was measured in 1M KCl solution at a soil:solution ratio of 1:5. Exchangeable Al, Fe, Mn, Ca,
Mg, K, and Na were extracted with 1 M ammonium chloride solution and measured by use of an ICP-OES (Thermo Scientific) (Lüer and Böhmer, 2000). The effective cation exchange capacity (ECEC) was calculated as the sum of negative charges
150
occupied by these exchangeable cations. Pre-treatments for particle size distribution analysis were as follows: Soil organic
matter was removed by 30 % H2O2, carbonates were dissolved, if necessary, by 10 % hydrochloric acid, and micro-aggregates
were dispersed by 0.4 M sodium pyrophosphate (Deutsches Institut für Normung e.V., 2002; Durner and Iden, 2011). The
three sand fractions were separated by sieving, and the silt and clay fractions were determined by use of a sedigraph (micromeritics). Bulk density was estimated in the field (FAO, 2006) and measured on undisturbed samples in the laboratory. 155
All element concentrations in the fine earth were converted to element stocks per m² considering rock-fragment content and
bulk density. Hydraulic conductivity and pF curves were determined on 41 of the soil profiles under FOR, DWIR and DNOR. Hydraulic
conductivity was measured in five replicates, using a permeability device with falling water head (Deutsches Institut für Normung e.V., 2012). Soil pF curves were determined in four to five replicates, using a pressure device (ecoTech) (Deutsches
160
Institut für Normung e.V., 2014). Water repellency was analysed on five replicates for each top-soil sample (Doerr, 1998). The sample was placed in a petri dish and the surface was manually smoothened. The time was measured until a drop of
distilled water, released from a pipette at 5 cm above the sample surface, infiltrated into the sample. Normung e.V., 2012). 2.2 Field work The soil profiles were sampled horizon-wise, whereby thick horizons were subdivided for the sampling, such that the maximum thickness included in one sample did not exceed 30 centimetres. We also
135
carried out in situ measurements of saturated hydraulic conductivity, using a compact constant head permeameter (Amoozegar
and Warrick, 1986; USDA, 2014). The measurements were done in five replicates. In addition, soil horizons with low rock-
fragment contents were sampled with 100 cm³ and 250 cm³ steel cylinders for laboratory measurements of pF curves and
hydraulic conductivity, respectively. subdivided for the sampling, such that the maximum thickness included in one sample did not exceed 30 centimetres. We also
135
carried out in situ measurements of saturated hydraulic conductivity, using a compact constant head permeameter (Amoozegar
and Warrick, 1986; USDA, 2014). The measurements were done in five replicates. In addition, soil horizons with low rock-
fragment contents were sampled with 100 cm³ and 250 cm³ steel cylinders for laboratory measurements of pF curves and
hydraulic conductivity, respectively. 7 7 https://doi.org/10.5194/soil-2020-52
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c⃝Author(s) 2020. CC BY 4.0 License. 8 140 8 https://doi.org/10.5194/soil-2020-52
Preprint. Discussion started: 5 October 2020
c⃝Author(s) 2020. CC BY 4.0 License. 2.4 Data processing and statistical analysis 𝑥1 = 𝑖𝑓 (𝑢𝑝𝑝𝑒𝑟 𝑏𝑜𝑢𝑛𝑑𝑎𝑟𝑦> 10) {𝑁𝐴} 𝑒𝑙𝑠𝑒 {
𝑖𝑓 (𝑙𝑜𝑤𝑒𝑟 𝑏𝑜𝑢𝑛𝑑𝑎𝑟𝑦<= 10) {(𝑙𝑜𝑤𝑒𝑟 𝑏𝑜𝑢𝑛𝑑𝑎𝑟𝑦−𝑢𝑝𝑝𝑒𝑟 𝑏𝑜𝑢𝑛𝑑𝑎𝑟𝑦)/10}
𝑒𝑙𝑠𝑒 {(10 −𝑢𝑝𝑝𝑒𝑟 𝑏𝑜𝑢𝑛𝑑𝑎𝑟𝑦)/10}},
(1)
80 180 (1) 𝑥2 = 𝑖𝑓 (𝑢𝑝𝑝𝑒𝑟 𝑏𝑜𝑢𝑛𝑑𝑎𝑟𝑦> 10) {𝑁𝐴} 𝑒𝑙𝑠𝑒 {
𝑖𝑓 (𝑙𝑜𝑤𝑒𝑟 𝑏𝑜𝑢𝑛𝑑𝑎𝑟𝑦<= 10) {1}
𝑒𝑙𝑠𝑒 {((𝑙𝑜𝑤𝑒𝑟 𝑏𝑜𝑢𝑛𝑑𝑎𝑟𝑦−𝑢𝑝𝑝𝑒𝑟 𝑏𝑜𝑢𝑛𝑑𝑎𝑟𝑦) −(𝑙𝑜𝑤𝑒𝑟 𝑏𝑜𝑢𝑛𝑑𝑎𝑟𝑦−10))/(𝑙𝑜𝑤𝑒𝑟 𝑏𝑜𝑢𝑛𝑑𝑎𝑟𝑦− (2) 𝑢𝑝𝑝𝑒𝑟 𝑏𝑜𝑢𝑛𝑑𝑎𝑟𝑦)}}
(2)
185
We conducted all statistical analyses (i) on the entire data set of each profile, and (ii) on only the data of the uppermost 10 cm
of each profile. All analyses and graphical representations were performed with the R packages “aqp”, “ggplot2” and “ggpubr”
(Beaudette et al., 2013; Wickham, 2016; Kassambara, 2019). Principle component analyses (PCAs) were calculated and
graphically depicted with the R packages “FactoMineR” and “factoextra” (Lê et al., 2008; Kassambara and Mundt, 2019). The 𝑢𝑝𝑝𝑒𝑟 𝑏𝑜𝑢𝑛𝑑𝑎𝑟𝑦)}}
(2)
185
We conducted all statistical analyses (i) on the entire data set of each profile, and (ii) on only the data of the uppermost 10 cm
of each profile. All analyses and graphical representations were performed with the R packages “aqp”, “ggplot2” and “ggpubr”
(Beaudette et al., 2013; Wickham, 2016; Kassambara, 2019). Principle component analyses (PCAs) were calculated and
graphically depicted with the R packages “FactoMineR” and “factoextra” (Lê et al., 2008; Kassambara and Mundt, 2019). The PCAs were used to identify relations between the variables and the samples. The PCA of the uppermost 10 cm of the soils also
190
included inclination, exposition and elevation. Linear regression models were used to compare the data obtained for soils under
DWIR and DNOR. The results, including 95 % confidence intervals, described the differences between the soils under DWIR
and DNOR. PCAs were used to identify relations between the variables and the samples. The PCA of the uppermost 10 cm of the soils also
190
included inclination, exposition and elevation. Linear regression models were used to compare the data obtained for soils under
DWIR and DNOR. The results, including 95 % confidence intervals, described the differences between the soils under DWIR
and DNOR. 2.4 Data processing and statistical analysis We described and sampled the soil profiles down to either the bedrock or permafrost, resulting in a maximum depth of 180
165
cm. According to the literature, the roots of Siberian larch can reach this depth (Kapper, 1954; Albenskiy et al., 1956). Therefore, we included all soil horizons in the calculations of element stocks to evaluate the nutrient availability for larch trees
at a site. To assess in addition the site conditions also for tree seedlings without fully developed root system, we only used the
properties of the uppermost 10 cm of the soils. For this purpose, we calculated the weighted means of the following variables for the uppermost 10 cm of the soils: content of rock fragments, bulk density, H+ concentration (log (10-pH)), carbonate content,
170
clay content, silt content, sand content, and ECEC (first group of variables). Element contents in the fine earth of each horizon
were converted to element stocks per horizon (considering rock fragment content and bulk density), and were summed up over 9 9 https://doi.org/10.5194/soil-2020-52
Preprint. Discussion started: 5 October 2020
c⃝Author(s) 2020. CC BY 4.0 License. the uppermost 10 cm of the soils. This was done for exchangeable Al, Ca, K, Na, Mg, Mn, Fe, SOC, and total N (TN) (second
group of variables). the uppermost 10 cm of the soils. This was done for exchangeable Al, Ca, K, Na, Mg, Mn, Fe, SOC, and total N (TN) (second
group of variables). All statistical analyses were carried out with the R project for statistical computing (R Core Team, 2014). The share of each
175
horizon in the uppermost 10 cm of the soil was used for the weighting, based on formula 𝑥1 (1) for the first group of variables,
and formula 𝑥2 (2) for the second group of variables. All statistical analyses were carried out with the R project for statistical computing (R Core Team, 2014). The share of each
175
horizon in the uppermost 10 cm of the soil was used for the weighting, based on formula 𝑥1 (1) for the first group of variables,
and formula 𝑥2 (2) for the second group of variables. 3.2 Chemical and physical soil properties
225 3.2 Chemical and physical soil properties
225
Comparison of the entire soil profiles (down to either the bedrock or permafrost) showed that soils under STE had significantly
higher contents of carbonates and exchangeable Ca than soils under all other vegetation categories (Fig. 4), whereas soils under
FOR had significantly higher contents of exchangeable Fe compared to soils under all other vegetation categories. There were
no statistically significant differences between soils under DWIR and DNOR with respect to rock-fragment contents, SOC and p y
p
p
Comparison of the entire soil profiles (down to either the bedrock or permafrost) showed that soils under STE had significantly
higher contents of carbonates and exchangeable Ca than soils under all other vegetation categories (Fig. 4), whereas soils under
FOR had significantly higher contents of exchangeable Fe compared to soils under all other vegetation categories. There were
no statistically significant differences between soils under DWIR and DNOR with respect to rock-fragment contents, SOC and Comparison of the entire soil profiles (down to either the bedrock or permafrost) showed that soils under STE had significantly
higher contents of carbonates and exchangeable Ca than soils under all other vegetation categories (Fig. 4), whereas soils under
FOR had significantly higher contents of exchangeable Fe compared to soils under all other vegetation categories. There were
no statistically significant differences between soils under DWIR and DNOR with respect to rock-fragment contents, SOC and TN stocks, pH, contents of exchangeable Mg, K, Na, Al, Mn, and ECEC. However, soils under DWIR and DNOR differed
230
significantly in their particle size distribution: Soils under DWIR had higher silt and clay contents, whereas soils under DNOR
had higher sand contents. Soils under FOR had similar silt contents as soils under DWIR, whereas soils under STE had similar
sand contents as soils under DNOR. Bulk density of soils under FOR and DWIR was lower than that of soils under DNOR. Yet, there was only a significant difference in bulk density between soils under FOR and DNOR but not between soils under TN stocks, pH, contents of exchangeable Mg, K, Na, Al, Mn, and ECEC. However, soils under DWIR and DNOR differed
230
significantly in their particle size distribution: Soils under DWIR had higher silt and clay contents, whereas soils under DNOR
had higher sand contents. 3.1 Field observations
195 Disturbance in DWIR and DNOR varied between moderate and severe. Most of the
sites were impacted by both fire and logging; yet, some profiles were only affected by one type of disturbance (Tab. A1, (profiles 1, 2, 7, 8, 9), whereas cryoturbation was not found at any of the investigated sites, most likely because of insufficient
215
moisture. Charcoal was present in most of the topsoils. Human impact affected most of the sites, except for the near-natural forest sites (FOR) that showed either no or only minor
disturbance such as cutting of single trees. Disturbance in DWIR and DNOR varied between moderate and severe. Most of the
sites were impacted by both fire and logging; yet, some profiles were only affected by one type of disturbance (Tab. A1, (profiles 1, 2, 7, 8, 9), whereas cryoturbation was not found at any of the investigated sites, most likely because of insufficient
215
moisture. Charcoal was present in most of the topsoils. Human impact affected most of the sites, except for the near-natural forest sites (FOR) that showed either no or only minor
di
b
h
i
f i
l
Di
b
i DWIR
d DNOR
i d b
d
d
M
f h appendix). 220
Permafrost was only encountered in soil profiles under near-natural forest (FOR). The depth of the permafrost table varied
between 60 cm and 100 cm in soils on slope debris under closed forests, and between 140 cm and 180 cm in soils on sandy
deposits under open forests (Tab. A1, appendix). In soils under STE, DWIR, and DNOR no permafrost was present above the
bedrock that was typically encountered between 80 cm and 110 cm soil depth. appendix). 220
Permafrost was only encountered in soil profiles under near-natural forest (FOR). The depth of the permafrost table varied
between 60 cm and 100 cm in soils on slope debris under closed forests, and between 140 cm and 180 cm in soils on sandy
deposits under open forests (Tab. A1, appendix). In soils under STE, DWIR, and DNOR no permafrost was present above the
bedrock that was typically encountered between 80 cm and 110 cm soil depth. pp
)
Permafrost was only encountered in soil profiles under near-natural forest (FOR). 3.1 Field observations
195 The Holocene colluvial deposits had very low contents of rock fragments
that typically consisted of fine to medium gravel. At three sites, only a thin sediment layer covered the bedrock (profiles 3, 47,
52). As an exception, the parent material at one of the forest sites (profiles 1, 2, 7, 8, 9) consisted of a slope deposit with
extremely high proportions of aeolian sand. Phaeozems and Cambisols were the most common soils under the near-natural forests (FOR) and disturbed areas (DWIR,
210
DNOR). In addition, Cryosols occurred above permafrost patches. Soils under steppe (STE) included Phaeozems, Chernozems,
and Kastanozems. The soil structure of the A horizons was generally granular, the B horizons had subangular or angular blocky
structure, and the C horizons had single grain, massive or rock structure. Rock fragments in several C horizons showed
manganese or carbonate pendants (Tab. A1, appendix). Bioturbation by rodents was observed in the soils at one forest site Phaeozems and Cambisols were the most common soils under the near-natural forests (FOR) and disturbed areas (DWIR,
210
DNOR). In addition, Cryosols occurred above permafrost patches. Soils under steppe (STE) included Phaeozems, Chernozems,
and Kastanozems. The soil structure of the A horizons was generally granular, the B horizons had subangular or angular blocky
structure, and the C horizons had single grain, massive or rock structure. Rock fragments in several C horizons showed
manganese or carbonate pendants (Tab. A1, appendix). Bioturbation by rodents was observed in the soils at one forest site Phaeozems and Cambisols were the most common soils under the near-natural forests (FOR) and disturbed areas (DWIR,
210
DNOR). In addition, Cryosols occurred above permafrost patches. Soils under steppe (STE) included Phaeozems, Chernozems,
and Kastanozems. The soil structure of the A horizons was generally granular, the B horizons had subangular or angular blocky
structure, and the C horizons had single grain, massive or rock structure. Rock fragments in several C horizons showed
manganese or carbonate pendants (Tab. A1, appendix). Bioturbation by rodents was observed in the soils at one forest site (profiles 1, 2, 7, 8, 9), whereas cryoturbation was not found at any of the investigated sites, most likely because of insufficient
215
moisture. Charcoal was present in most of the topsoils. Human impact affected most of the sites, except for the near-natural forest sites (FOR) that showed either no or only minor
disturbance such as cutting of single trees. 3.1 Field observations
195 The bedrock in the study area predominantly consisted of granite, with some minor occurrences of gneiss. Thick aeolian sand
sheets locally covered the lower slopes and valley bottoms along west-east running valleys (Fig. 1). Thus, carbonates that were
observed in some of the soils were supposed to originate from aeolian deposits. The parent materials of the soils on the slopes generally consisted of a vertical succession of two to three sediment layers. The The bedrock in the study area predominantly consisted of granite, with some minor occurrences of gneiss. Thick aeolian sand
sheets locally covered the lower slopes and valley bottoms along west-east running valleys (Fig. 1). Thus, carbonates that were
observed in some of the soils were supposed to originate from aeolian deposits. The parent materials of the soils on the slopes generally consisted of a vertical succession of two to three sediment layers The lowermost one was typically a solifluction layer, characterised by a high content of rock fragments with sizes ranging from
200
fine gravel to angular stones. This layer was usually overlain by another slope deposit, consisting of a mixture of weathering
products of the bedrock, and aeolian sand and silt. This slope deposit showed lower rock-fragment contents than the underlying
one, but similar rock-fragment sizes. The proportions of sand and silt in the upper slope deposit varied both within a profile 10 https://doi.org/10.5194/soil-2020-52
Preprint. Discussion started: 5 October 2020
c⃝Author(s) 2020. CC BY 4.0 License. and between the profiles. In places, a Holocene colluvial deposit with varying thickness covered the slope deposits described
above. Our profiles 4, 5 and 38 predominantly consisted of Holocene colluvium, whereas most of the other profiles included
205
either no or only a thin Holocene colluvial deposit. The Holocene colluvial deposits had very low contents of rock fragments
that typically consisted of fine to medium gravel. At three sites, only a thin sediment layer covered the bedrock (profiles 3, 47,
52). As an exception, the parent material at one of the forest sites (profiles 1, 2, 7, 8, 9) consisted of a slope deposit with
extremely high proportions of aeolian sand. and between the profiles. In places, a Holocene colluvial deposit with varying thickness covered the slope deposits described
above. Our profiles 4, 5 and 38 predominantly consisted of Holocene colluvium, whereas most of the other profiles included
205
either no or only a thin Holocene colluvial deposit. 3.1 Field observations
195 The depth of the permafrost table varied
between 60 cm and 100 cm in soils on slope debris under closed forests, and between 140 cm and 180 cm in soils on sandy
deposits under open forests (Tab. A1, appendix). In soils under STE, DWIR, and DNOR no permafrost was present above the
bedrock that was typically encountered between 80 cm and 110 cm soil depth. 3.2 Chemical and physical soil properties
225 Soils under STE concentrated around sand and bulk density, thereby slightly
shifting to the side of exchangeable Ca, Mg, K, and SOC and TN stocks. Soils under DWIR plotted closer to silt than to sand,
275
and mostly fell into the cluster of ECEC, exchangeable Mg, K, and SOC and TN stocks. Soils under DNOR plotted between
those under STE and DWIR. The confidence interval of the soils under FOR was clearly separated from all other confidence
intervals. That of the soils under DWIR was also separated and showed only a minor overlap with the one of soils under
DNOR. The confidence intervals of the soils under STE and DNOR showed a considerable overlap. In the PCA of the data for the uppermost 10 cm of the soil profiles, the first two factors explained 54.5 % of the total variance
265
(Fig. 6 bottom). Sand content and bulk density on one hand, made up one axis together with silt content on the other hand. Rock-fragment content and exchangeable Fe and Al on one hand, made up a second axis together with pH on the other hand. In addition, a cluster of clay content, SOC and TN stocks, ECEC, and exchangeable Mg, K and Mn occurred. The factor
elevation also fell into this cluster, but with a minor contribution. The contributions of inclination, exposition, carbonate In the PCA of the data for the uppermost 10 cm of the soil profiles, the first two factors explained 54.5 % of the total variance
265
(Fig. 6 bottom). Sand content and bulk density on one hand, made up one axis together with silt content on the other hand. Rock-fragment content and exchangeable Fe and Al on one hand, made up a second axis together with pH on the other hand. In addition, a cluster of clay content, SOC and TN stocks, ECEC, and exchangeable Mg, K and Mn occurred. The factor
elevation also fell into this cluster, but with a minor contribution. The contributions of inclination, exposition, carbonate content, and exchangeable Na were negligible. In general, the data for the uppermost 10 cm of the soils under the four
270
vegetation categories (lower PCA) plotted more separately from each other than those for the entire soil profiles (upper PCA). 3.2 Chemical and physical soil properties
225 Soils under FOR had similar silt contents as soils under DWIR, whereas soils under STE had similar
sand contents as soils under DNOR. Bulk density of soils under FOR and DWIR was lower than that of soils under DNOR. Yet, there was only a significant difference in bulk density between soils under FOR and DNOR but not between soils under DWIR and DNOR. 235 11 https://doi.org/10.5194/soil-2020-52
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c⃝Author(s) 2020. CC BY 4.0 License. https://doi.org/10.5194/soil-2020-52
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c⃝Author(s) 2020. CC BY 4.0 License. 12 12 https://doi.org/10.5194/soil-2020-52
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c⃝Author(s) 2020. CC BY 4.0 License. Figure 4: Properties of the entire soil profiles (down to either the bedrock or permafrost) under the four vegetation categories (n =
305): near-natural forest (FOR, green), steppe close to the forest (STE, grey), disturbed forest with regrowth of trees (DWIR, blue)
and disturbed forest showing no regrowth of trees (DNOR, orange). P values indicated above the plots were calculated for differences
between the arithmetic means (ns: p > 0.05; *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001; ****: p ≤ 0.0001). Horizontal bars = medians,
boxes = first and third quartiles, points = outliers, violins = data distributions. 240 Comparison of only the uppermost 10 cm of each soil profile (for evaluating the soil conditions for tree seedlings) suggested
that soils under DWIR had larger stocks of exchangeable Mg and K, SOC and TN than soils under DNOR, however without
significant differences (Fig. 5). The other chemical soil properties showed no differences between soils under DWIR and
245
DNOR. In contrast, particle size distribution showed again a significant difference between soils under DWIR and DNOR. Soils under STE had the highest sand contents, followed by soils under DNOR. Soils under DWIR had the highest clay and
silt contents, followed by soils under FOR. Bulk densities and rock-fragment contents of the soils of the four vegetation
categories were similar. 13 https://doi.org/10.5194/soil-2020-52
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c⃝Author(s) 2020. CC BY 4.0 License. 14 14 250 250 14 https://doi.org/10.5194/soil-2020-52
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c⃝Author(s) 2020. CC BY 4.0 License. 3.2 Chemical and physical soil properties
225 Figure 5: Properties of the uppermost 10 cm of the soil profiles under the four vegetation categories (n = 54): near-natural forest
(FOR, green), steppe close to the forest (STE, grey), disturbed forest with regrowth of trees (DWIR, blue) and disturbed forest
showing no regrowth of trees (DNOR, orange). P values indicated above the plots were calculated for differences between the
arithmetic means (NS.: p = 1; ns: p > 0.05; *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001; ****: p ≤ 0.0001). Horizontal bars = medians,
boxes = first and third quartiles, points = outliers, violins = data distributions. 255 In the principle component analysis (PCA) of the data for the entire soil profiles, the first two factors explained 56.3 % of the
total variance (Fig. 6 top). The variables sand content, rock-fragment content and bulk density on one hand, made up one axis
together with clay and silt contents on the other hand. Most of the data scattered around this axis, regardless of vegetation
category. Exchangeable Al and Fe on one hand, made up a second axis together with pH, carbonate content, and exchangeable
260
Ca and Na on the other hand. Exchangeable Al and Fe did not contribute much to the PCA, yet, the soils under FOR clustered
around them. On the other hand, some soils under STE, DNOR and FOR clustered around pH, carbonate content, and In the principle component analysis (PCA) of the data for the entire soil profiles, the first two factors explained 56.3 % of the
total variance (Fig. 6 top). The variables sand content, rock-fragment content and bulk density on one hand, made up one axis
together with clay and silt contents on the other hand. Most of the data scattered around this axis, regardless of vegetation In the principle component analysis (PCA) of the data for the entire soil profiles, the first two factors explained 56.3 % of the
total variance (Fig. 6 top). The variables sand content, rock-fragment content and bulk density on one hand, made up one axis
together with clay and silt contents on the other hand. Most of the data scattered around this axis, regardless of vegetation category. Exchangeable Al and Fe on one hand, made up a second axis together with pH, carbonate content, and exchangeable
260
Ca and Na on the other hand. 3.2 Chemical and physical soil properties
225 Exchangeable Al and Fe did not contribute much to the PCA, yet, the soils under FOR clustered
around them. On the other hand, some soils under STE, DNOR and FOR clustered around pH, carbonate content, and
exchangeable Ca and Na. In addition, another cluster around SOC and TN stocks, and exchangeable Mg, K and Mn, occurred
close to the axis of clay and silt contents. Mainly soils under DWIR but also some soils under STE fell into this cluster. category. Exchangeable Al and Fe on one hand, made up a second axis together with pH, carbonate content, and exchangeable
260
Ca and Na on the other hand. Exchangeable Al and Fe did not contribute much to the PCA, yet, the soils under FOR clustered
around them. On the other hand, some soils under STE, DNOR and FOR clustered around pH, carbonate content, and
exchangeable Ca and Na. In addition, another cluster around SOC and TN stocks, and exchangeable Mg, K and Mn, occurred
close to the axis of clay and silt contents. Mainly soils under DWIR but also some soils under STE fell into this cluster. In the PCA of the data for the uppermost 10 cm of the soil profiles, the first two factors explained 54.5 % of the total variance
265
(Fig. 6 bottom). Sand content and bulk density on one hand, made up one axis together with silt content on the other hand. Rock-fragment content and exchangeable Fe and Al on one hand, made up a second axis together with pH on the other hand. In addition, a cluster of clay content, SOC and TN stocks, ECEC, and exchangeable Mg, K and Mn occurred. The factor
elevation also fell into this cluster, but with a minor contribution. The contributions of inclination, exposition, carbonate
content, and exchangeable Na were negligible. In general, the data for the uppermost 10 cm of the soils under the four
270
vegetation categories (lower PCA) plotted more separately from each other than those for the entire soil profiles (upper PCA). The arithmetic means and 95 % confidence intervals of the data for the uppermost 10 cm of the soils under the four vegetation
categories (smaller dots and shaded ellipses) confirmed the differences and clear separation. Soils under FOR plotted around
the axes of sand and exchangeable Fe and Al. 3.2 Chemical and physical soil properties
225 The arithmetic means and 95 % confidence intervals of the data for the uppermost 10 cm of the soils under the four vegetation
categories (smaller dots and shaded ellipses) confirmed the differences and clear separation. Soils under FOR plotted around
the axes of sand and exchangeable Fe and Al. Soils under STE concentrated around sand and bulk density, thereby slightly shifting to the side of exchangeable Ca, Mg, K, and SOC and TN stocks. Soils under DWIR plotted closer to silt than to sand,
275
and mostly fell into the cluster of ECEC, exchangeable Mg, K, and SOC and TN stocks. Soils under DNOR plotted between
those under STE and DWIR. The confidence interval of the soils under FOR was clearly separated from all other confidence
intervals. That of the soils under DWIR was also separated and showed only a minor overlap with the one of soils under
DNOR. The confidence intervals of the soils under STE and DNOR showed a considerable overlap. 15 https://doi.org/10.5194/soil-2020-52
Preprint. Discussion started: 5 October 2020
c⃝Author(s) 2020. CC BY 4.0 License. 16 16
0 280 16 https://doi.org/10.5194/soil-2020-52
Preprint. Discussion started: 5 October 2020
c⃝Author(s) 2020. CC BY 4.0 License. Figure 6: Top: PCA of the properties of the entire soil profiles under the four vegetation categories (n = 303): near-natural forest
(FOR, green), steppe close to the forest (STE, grey), disturbed forest with regrowth of trees (DWIR, blue) and disturbed forest
showing no regrowth of trees (DNOR, orange). Bottom: PCA of the properties of the uppermost 10 cm of the soil profiles under the
four vegetation categories (n = 52). In addition, inclination, exposition and elevation were included in the second PCA. Arithmetic
means and 95 % confidence intervals of the data for soils under each vegetation category are represented by a smaller circle and a
shaded ellipse, respectively. SOC = soil organic carbon, TN = total nitrogen, ECEC = effective cation exchange capacity, BD = bulk
density, and Ca, Mg, K, Na, Al, Fe, and Mn = exchangeable cations. 285 Based on the data presented in Figures 4-6, we chose the most relevant variables for direct comp under DWIR and DNOR, using multiple linear regression models (Fig. 7). We found a significant difference in particle size
290
distribution, both for the data of the entire soil profiles (Fig. 7 top) and for the uppermost 10 cm of the soil profiles (Fig 7. bottom). 3.2 Chemical and physical soil properties
225 Soils under DNOR had clearly more sand, and less silt and clay than soils under DWIR. The soils did not exhibit
significant differences with respect to any other soil properties. All confidence intervals overlapped with the zero line, and all
p values were >0.05. The only relevant observation was that the uppermost 10 cm of the soil profiles under DNOR tended to
have smaller stocks of SOC and exchangeable K, compared to those under DWIR, however without significant differences
295 under DWIR and DNOR, using multiple linear regression models (Fig. 7). We found a significant difference in particle size
290
distribution, both for the data of the entire soil profiles (Fig. 7 top) and for the uppermost 10 cm of the soil profiles (Fig 7. bottom). Soils under DNOR had clearly more sand, and less silt and clay than soils under DWIR. The soils did not exhibit
significant differences with respect to any other soil properties. All confidence intervals overlapped with the zero line, and all
p values were >0.05. The only relevant observation was that the uppermost 10 cm of the soil profiles under DNOR tended to have smaller stocks of SOC and exchangeable K, compared to those under DWIR, however without significant differences
295
either. 17 https://doi.org/10.5194/soil-2020-52
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c⃝Author(s) 2020. CC BY 4.0 License. Figure 7: Top: Estimates and 95 % confidence intervals of linear regression models, comparing the entire soil profiles under
disturbed forest showing no regrowth of trees (DNOR, orange) to those under disturbed forest with regrowth of trees (DWIR, blue)
(n = 305). P values indicated below the plots were calculated for the differences between the estimates for soils under DNOR and
DWIR. Data for exchangeable Mg were left out for better readability (estimate: +4.5; confidence intervals: +21.2/-12.2; p = 0.596). Bottom: Estimates and 95 % confidence intervals of linear regression models, comparing the uppermost 10 cm of the soil profiles
under DNOR (orange) to those under DWIR (blue) (n = 54). P values indicated below the plots were calculated for the differences
between the estimates for soils under DNOR and DWIR. Data for exchangeable Mg were left out for better readability (estimate: -
32.2; confidence intervals: +16.5/-80.9; p = 0.184). BD = bulk density, SOC = soil organic carbon, TN = total nitrogen, K =
exchangeable potassium. for a final statement on differences between the sites with respect to this factor.
320 4.1 Chemical soil properties In general, Siberian larch has low requirements with respect to chemical soil properties (Dylis, 1947). It can grow on a wide
range of soils and may colonise even rather fresh sediments (Timoshok et al., 2014). Siberian larch can also adapt to low
nutrient availability by enhanced soil nutrient exploitation (Lebedev, 2012; Chernyshenkо and Vasilyev, 2019). Most
importantly, it needs nitrogen (Lebedev and Lebedev, 2010; Chernyshenkо and Vasilyev, 2019), like many other larch species
335
including Larix gmelinii (Schulze et al., 1995; Liang et al., 2014), Larix kaempferi (Leyton, 1956; Kayama et al., 2009) and
hybrids such as Larix x eurolepis (Pâques, 1994). Also a lack in K and Mg may limit the growth of larch species on poor,
sandy soils (Leyton, 1956; Phu, 1975), whereas growth limitation by insufficient P supply has not yet been reported. Generally,
differences in the vitality and growth of larch are more commonly caused by climatic or hydrological differences than by nutrient limitations (Fiedler et al., 1980; Stüber, 1998; Viers et al., 2013). 340
Because of the low nutrient requirements of Siberian larch and since we did not detect any significant differences in the
chemical properties of soils under disturbed forest with regrowth of trees (DWIR) and under disturbed forest showing no
regrowth of trees (DNOR), we conclude that chemical soil properties are not responsible for the differences in post-disturbance
regrowth of Siberian larch in our study area. The uppermost 10 cm of the soils under FOR had even significantly smaller N nutrient limitations (Fiedler et al., 1980; Stüber, 1998; Viers et al., 2013). 340
Because of the low nutrient requirements of Siberian larch and since we did not detect any significant differences in the
chemical properties of soils under disturbed forest with regrowth of trees (DWIR) and under disturbed forest showing no
regrowth of trees (DNOR), we conclude that chemical soil properties are not responsible for the differences in post-disturbance
regrowth of Siberian larch in our study area. The uppermost 10 cm of the soils under FOR had even significantly smaller N stocks and lower contents of exchangeable Ca, Mg and K than the uppermost 10 cm of the soils under DNOR. Nevertheless,
345
saplings and young trees were growing in the FOR areas, which confirms that the nutrient supply of all analysed soils fulfilled
the needs of Siberian larch. 3.3 Hydrological soil properties In general, all soils had high hydraulic conductivities, due to their predominantly sandy texture. A site-wise comparison
between soils under DWIR and DNOR showed that soils under DWIR had higher hydraulic conductivities in their uppermost horizons (Fig. 8 bottom). For example, the hydraulic conductivities of soil profiles 31 and 32 (DNOR) were below 1.5 10-3 cm
310
s-1 whereas those of profiles 33 – 36 (DWIR) ranged between 1.5 and 3 10-3 cm s-1. Profiles 38 and 40 (DNOR) showed
hydraulic conductivities between 2.5 10-3 cm s-1 and 3.5 10-3 cm s-1 in their uppermost horizons, while the hydraulic
conductivities of profiles 37 and 41 (DWIR) exceeded 5 10-3 cm s-1. Soils under FOR showed similar hydraulic conductivities,
with a maximum of 3.5 10-3 cm s-1 (Fig. 8 top). Soils under FOR had lower plant-available field capacities than soils under DWIR and DNOR (Fig. 8). Except for one subsoil horizon, plant-available field capacity ranged from 12 vol. % to 22 vol. %
315
in soils under FOR, and from 12 vol. % to 33 vol. % in soils under DWIR and DNOR. The differences between soils under
DWIR and DNOR became clearer in a site-wise comparison. For example, plant-available field capacities of profiles 39 and
40 (DNOR) ranged between 12 vol. % and 17 vol. %, whereas profile 37 (DWIR) had plant-available field capacities between
17 vol. % and 26 vol. %. However, the limited number of samples analysed for plant-available field capacity does not allow 18 https://doi.org/10.5194/soil-2020-52
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c⃝Author(s) 2020. CC BY 4.0 License. 19 https://doi.org/10.5194/soil-2020-52
Preprint. Discussion started: 5 October 2020
c⃝Author(s) 2020. CC BY 4.0 License. Figure 8: Hydraulic conductivities (a) and plant-available field capacities (b) of soils under near-natural forest (FOR), and hydraulic
conductivities (c) and plant-available field capacities (d) of soils under disturbed forest with regrowth of trees (DWIR; left) and
under disturbed forest showing no regrowth of trees (DNOR; right). Soil horizons in light grey colour were not subjected to these
measurements. 325 Water repellency of topsoils under different vegetation categories did not show any differences (Fig. A2, appendix). Topsoil
samples from all vegetation categories included samples, on which the water drop only persisted for one or a few seconds, and
samples on which the drop persisted for more than 1000 seconds. 4.1 Chemical soil properties Furthermore, the nutrient stocks were similar or even higher compared to those reported from other
Siberian larch forests and from forests of various other larch species (Fiedler et al., 1980; Hwang and Son, 2006; Kayama et
al., 2009; Park et al., 2009; Lebedev and Lebedev, 2010; Watanabe et al., 2012; Wang et al., 2014). 4.2 Physical soil properties and soil hydrology
350 we did not observe a significant difference in water repellency between the four vegetation categories, possibly because of the
380
high frequency of fires in in our study area, and associated enhanced erosion (Goldammer, 2002; Hessl et al., 2012). As water
repellency occurred irregularly across the whole study area, including DWIR and DNOR, it seemed to be not relevant for the
post-disturbance tree recovery pattern in our study area. we did not observe a significant difference in water repellency between the four vegetation categories, possibly because of the
380
high frequency of fires in in our study area, and associated enhanced erosion (Goldammer, 2002; Hessl et al., 2012). As water
repellency occurred irregularly across the whole study area, including DWIR and DNOR, it seemed to be not relevant for the
post-disturbance tree recovery pattern in our study area. 4.2 Physical soil properties and soil hydrology
350 Soils under DWIR had significantly more silt and clay, and thus higher plant-available field capacity (Amelung et al., 2018)
than soils under DNOR, which were considerably sandier. As mean annual precipitation in Tosontsengel is only 200-250 mm,
lack of water represents a major limitation for tree growth (Dulamsuren et al., 2009; 2010; 2011; Chenlemuge et al., 2015b; Soils under DWIR had significantly more silt and clay, and thus higher plant-available field capacity (Amelung et al., 2018)
than soils under DNOR, which were considerably sandier. As mean annual precipitation in Tosontsengel is only 200-250 mm,
lack of water represents a major limitation for tree growth (Dulamsuren et al., 2009; 2010; 2011; Chenlemuge et al., 2015b; 20 https://doi.org/10.5194/soil-2020-52
Preprint. Discussion started: 5 October 2020
c⃝Author(s) 2020. CC BY 4.0 License. 2015a). Under these climatic conditions, it is plausible that soil texture and corresponding plant-available field capacity are
dominant factors controlling the post-disturbance tree regrowth pattern. The measured plant-available field capacities
355
confirmed the difference between the soils under DWIR and DNOR. Moreover, the data of the entire soil profiles showed that
the texture of soils under DWIR was similar to that of soils under near-natural forest (FOR), and that the texture of soils under
DNOR was similar to that of soils under steppe (STE). Also, the PCA confirmed the similarities between soils under DNOR
and STE, thus pointing to a potential risk of DNOR to shift from forest to steppe vegetation. Such potential shift has already 2015a). Under these climatic conditions, it is plausible that soil texture and corresponding plant-available field capacity are
dominant factors controlling the post-disturbance tree regrowth pattern. The measured plant-available field capacities
355
confirmed the difference between the soils under DWIR and DNOR. Moreover, the data of the entire soil profiles showed that
the texture of soils under DWIR was similar to that of soils under near-natural forest (FOR), and that the texture of soils under
DNOR was similar to that of soils under steppe (STE). Also, the PCA confirmed the similarities between soils under DNOR
and STE, thus pointing to a potential risk of DNOR to shift from forest to steppe vegetation. Such potential shift has already been predicted for the forest-steppe in the Khentey Mountains, Mongolia (Dulamsuren and Hauck, 2008; Dulamsuren et al.,
360
2009). Hydraulic conductivity was higher in the uppermost horizons of soils under DWIR compared to those under DNOR. 4.2 Physical soil properties and soil hydrology
350 (2014), who reported absence of
permafrost five years after severe forest fire. The same authors moreover measured increased soil moisture in soils of burned this experiment, these results confirm the competitiveness of grasses for water in the upper soil horizons and the relevance of
370
soil hydraulic conductivity for tree growth in water-limited environments. We did not detect a positive effect of permafrost on post-disturbance tree regrowth, as permafrost was neither encountered
under DWIR nor under DNOR. This is in agreement with observations by Kopp et al. (2014), who reported absence of
permafrost five years after severe forest fire. The same authors moreover measured increased soil moisture in soils of burned sites, compared to soils under forest, which they attributed to the absence of tree transpiration. In contrast, Park et al. (2009)
375
stated a decrease in soil moisture after fire and logging, arguing that evaporation loss from the bare soils and deterioration of
physical soil properties exceed the decrease in transpiration. In our study area, we also observed rather decreased soil moisture
at sites disturbed by fire and logging, compared to near-natural forest sites. An additional decline in soil moisture after fire can
be induced by water repellency that may increase surface run off (DeBano, 2000; Mataix-Solera and Doerr, 2004). However, sites, compared to soils under forest, which they attributed to the absence of tree transpiration. In contrast, Park et al. (2009)
375
stated a decrease in soil moisture after fire and logging, arguing that evaporation loss from the bare soils and deterioration of
physical soil properties exceed the decrease in transpiration. In our study area, we also observed rather decreased soil moisture
at sites disturbed by fire and logging, compared to near-natural forest sites. An additional decline in soil moisture after fire can
be induced by water repellency that may increase surface run off (DeBano, 2000; Mataix-Solera and Doerr, 2004). However, we did not observe a significant difference in water repellency between the four vegetation categories, possibly because of the
380
high frequency of fires in in our study area, and associated enhanced erosion (Goldammer, 2002; Hessl et al., 2012). As water
repellency occurred irregularly across the whole study area, including DWIR and DNOR, it seemed to be not relevant for the
post-disturbance tree recovery pattern in our study area. 4.2 Physical soil properties and soil hydrology
350 Trees
may benefit from this difference for two reasons. Firstly, rapid infiltration through the uppermost horizons reduces evaporation
loss. Secondly, grasses and herbs have a dense but shallow root system and compete with tree roots for water at shallow soil depth (Müller-Hohenstein, 1981; Breckle et al., 1994). As tree roots reach deeper down the soil, they benefit from rapid water
365
infiltration below the depth of the roots of grasses and herbs. Lange et al. (2015) carried out irrigation experiments on grass-
dominated south-facing slopes and forest-dominated north-facing slopes in the Mongolian forest-steppe. On the south-facing
slopes, most of the water was either taken up by grass or evaporated, and the remaining water reached only 5 cm soil depth. On the north-facing slopes, the water percolated down to the permafrost table. Although the contrasting exposition also affected depth (Müller-Hohenstein, 1981; Breckle et al., 1994). As tree roots reach deeper down the soil, they benefit from rapid water
365
infiltration below the depth of the roots of grasses and herbs. Lange et al. (2015) carried out irrigation experiments on grass-
dominated south-facing slopes and forest-dominated north-facing slopes in the Mongolian forest-steppe. On the south-facing
slopes, most of the water was either taken up by grass or evaporated, and the remaining water reached only 5 cm soil depth. On the north-facing slopes, the water percolated down to the permafrost table. Although the contrasting exposition also affected depth (Müller-Hohenstein, 1981; Breckle et al., 1994). As tree roots reach deeper down the soil, they benefit from rapid water
365
infiltration below the depth of the roots of grasses and herbs. Lange et al. (2015) carried out irrigation experiments on grass-
dominated south-facing slopes and forest-dominated north-facing slopes in the Mongolian forest-steppe. On the south-facing
slopes, most of the water was either taken up by grass or evaporated, and the remaining water reached only 5 cm soil depth. On the north-facing slopes, the water percolated down to the permafrost table. Although the contrasting exposition also affected this experiment, these results confirm the competitiveness of grasses for water in the upper soil horizons and the relevance of
370
soil hydraulic conductivity for tree growth in water-limited environments. We did not detect a positive effect of permafrost on post-disturbance tree regrowth, as permafrost was neither encountered
under DWIR nor under DNOR. This is in agreement with observations by Kopp et al. 4.3 Further relevant environmental factors Other factors besides soil hydrology that influence the post-disturbance tree regrowth pattern, include in particular relief and
385
human activity. Relief-induced water gains in concave positions may support tree growth even where soil properties seem to 21 https://doi.org/10.5194/soil-2020-52
Preprint. Discussion started: 5 October 2020
c⃝Author(s) 2020. CC BY 4.0 License. be unfavourable. Similarly, water losses through divergence in convex positions may hamper tree growth, even where soil
properties seem to be suitable. Also, small tree patches that survived a disturbance, can have a positive influence on post-
disturbance tree regrowth. In our study area, we observed enhanced fructification of trees that had survived forest fires,
initiating tree regrowth in belts around small tree patches that had persisted. We explain this observation by the shading of the
390
remaining adult trees that reduces evaporation and creates a more even microclimate on the ground, and by increased
availability of intact seeds in the direct surrounding of mature trees (Dugarjav, 2006). In contrast, human activity, especially logging and pastoral pressure, may inhibit post-disturbance forest recovery
(Khishigjargal et al., 2013; Dulamsuren et al., 2014; Khansaritoreh et al., 2017b). In particular, goats grazing in areas of burned be unfavourable. Similarly, water losses through divergence in convex positions may hamper tree growth, even where soil
properties seem to be suitable. Also, small tree patches that survived a disturbance, can have a positive influence on post-
disturbance tree regrowth. In our study area, we observed enhanced fructification of trees that had survived forest fires,
initiating tree regrowth in belts around small tree patches that had persisted. We explain this observation by the shading of the
390
remaining adult trees that reduces evaporation and creates a more even microclimate on the ground, and by increased In contrast, human activity, especially logging and pastoral pressure, may inhibit post-disturbance forest recovery
(Khishigjargal et al., 2013; Dulamsuren et al., 2014; Khansaritoreh et al., 2017b). In particular, goats grazing in areas of burned forest or in forest stands that have previously been opened by partial logging, hamper the growth of seedlings and damage
395
young trees (Sankey et al., 2006). Thus, human impact and soil hydrology are the two key factors controlling the post-
disturbance tree recovery pattern (Fig. 9). forest or in forest stands that have previously been opened by partial logging, hamper the growth of seedlings and damage
395
young trees (Sankey et al., 2006). 4.3 Further relevant environmental factors Figure 9: Possible development pathways of disturbed forest areas in the Mongolian forest-steppe, with respect to hydrological soil
properties and further environmental factors. 4.3 Further relevant environmental factors Thus, human impact and soil hydrology are the two key factors controlling the post-
disturbance tree recovery pattern (Fig. 9). Loss of the shading by the forest canopy, of the forest microclimate and the insulating organic layer in turn lead to permafrost
degradation. Also, the presently ongoing climate change accelerates permafrost decline in Mongolia. Thus, this essential soil disturbance tree recovery pattern (Fig. 9). Loss of the shading by the forest canopy, of the forest microclimate and the insulating organic layer in turn lead to permafrost
degradation. Also, the presently ongoing climate change accelerates permafrost decline in Mongolia. Thus, this essential soil water reservoir is likely to disappear (Sharkhuu and Sharkhuu, 2012). As the meltwater from the active layer above the
400
permafrost table can support tree growth even at sites where soil water storage is below the threshold for tree growth,
permafrost decline may also contribute to the decrease in forest area in the Mongolian forest-steppe. In some of our investigated
sites, this has already happened. Klinge et al. (2020) estimated that the potential forest distribution in the Khangai Mountains is three times larger than the water reservoir is likely to disappear (Sharkhuu and Sharkhuu, 2012). As the meltwater from the active layer above the
400
permafrost table can support tree growth even at sites where soil water storage is below the threshold for tree growth,
permafrost decline may also contribute to the decrease in forest area in the Mongolian forest-steppe. In some of our investigated
sites, this has already happened. Klinge et al. (2020) estimated that the potential forest distribution in the Khangai Mountains is three times larger than the Klinge et al. (2020) estimated that the potential forest distribution in the Khangai Mountains is three times larger than the
actual forest area. This discrepancy indicates that forest fire and human activity already caused a considerable decline of
405
forested areas. actual forest area. This discrepancy indicates that forest fire and human activity already caused a considerable decline of
405
forested areas. actual forest area. This discrepancy indicates that forest fire and human activity already caused a considerable decline of
405
forested areas. 22 22 https://doi.org/10.5194/soil-2020-52
Preprint. Discussion started: 5 October 2020
c⃝Author(s) 2020. CC BY 4.0 License. Figure 9: Possible development pathways of disturbed forest areas in the Mongolian forest-steppe, with respect to hydrological soil
properties and further environmental factors. 5 Conclusions
410 This study showed that the post-disturbance regrowth pattern of trees in the central Mongolian forest-steppe, a highly sensitive,
semi-arid ecotone, is largely controlled by soil hydrology. With respect to our hypotheses established in the beginning, we
conclude that: 1) Differences in post-disturbance tree regrowth depend mainly on soil texture. Loamy soils have high plant-available field
capacity and are thus able to store sufficient amounts of water for post-disturbance tree regrowth. In contrast, sandy soils
415
cannot store enough water for recovery of tree vegetation under the present environmental conditions. 2) Post-disturbance tree regrowth is favoured by higher hydraulic conductivity in the uppermost soil horizons, as rapid
percolation through the upper soil horizons reduces evaporation loss. In addition, trees benefit from the reduced competition
for water with grasses and herbs that results from rapid water percolation into the subsoil. 3) Nutrient limitation is not relevant for the uneven post-disturbance tree regrowth pattern in our study area, as the dominant
420
tree species, Siberian larch, has rather low nutrient requirements. 3) Nutrient limitation is not relevant for the uneven post-disturbance tree regrowth pattern in our study area, as the dominant
420
tree species, Siberian larch, has rather low nutrient requirements. 23 https://doi.org/10.5194/soil-2020-52
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c⃝Author(s) 2020. CC BY 4.0 License. 4) Meltwater from the active layer above the permafrost table provides additional water for near-natural forests. However, it
cannot be a relevant factor for the uneven post-disturbance regrowth pattern of Siberian larch in our study area, because
permafrost was encountered neither under disturbed forests with nor without tree regrowth. Under the given climatic conditions, water limitation is the predominant factor controlling tree growth in the semi-arid
425
Mongolian forest-steppe. Therefore, even small differences in soil properties that lead to an increase in the amount of plant-
available water, can be decisive for post-disturbance tree regrowth. Disturbances by severe fire or clear-cutting result in loss of permafrost and thus loss of an additional water reservoir during
summer. Such additional water source can be critical at sites, where soil water storage alone is slightly below the threshold for tree growth. Therefore, such sites are particularly prone to an irreversible shift from forest to steppe after strong disturbance,
430
involving loss of permafrost. 5 Conclusions
410 Moreover, we expect that many sites where soil hydrology allows for post-disturbance tree
regrowth under the present climatic conditions, will lose their ability for post-disturbance forest recovery in the course of the
ongoing climate change. Climate projections predict that the decrease of permafrost will continue, and drought events will become both more frequent Climate projections predict that the decrease of permafrost will continue, and drought events will become both more frequent
and more intensive. At the same time, it seems unlikely that the pressure on forests in the Mongolian forest-steppe by fire,
435
logging and grazing will decrease, unless explicit and effective measures are taken. Consequences of this unfavourable
interplay will most likely include a decrease in the growth rates of trees, and a further considerable and irreversible decline of
forested area. Therefore, regulation of human impact on the forests in the Mongolian forest-steppe is mandatory, if a further
irreversible loss of forest area shall be avoided. and more intensive. At the same time, it seems unlikely that the pressure on forests in the Mongolian forest-steppe by fire,
435
logging and grazing will decrease, unless explicit and effective measures are taken. Consequences of this unfavourable
interplay will most likely include a decrease in the growth rates of trees, and a further considerable and irreversible decline of
forested area. Therefore, regulation of human impact on the forests in the Mongolian forest-steppe is mandatory, if a further
irreversible loss of forest area shall be avoided. Table A1: General information on the soil profiles. Abbreviations are according to the Guidelines for Soil Description (FAO, 2006).
*: If permafrost is present, the depth of the permafrost table equals the profile depth. 6 Appendix A
440 Table A1: General information on the soil profiles. Abbreviations are according to the Guidelines for Soil Description (FAO, 2006). *: If permafrost is present, the depth of the permafrost table equals the profile depth. Table A1: General information on the soil profiles. Abbreviations are according to the Guidelines for Soil Description (FAO, 2006). *: If permafrost is present, the depth of the permafrost table equals the profile depth. 24 https://doi.org/10.5194/soil-2020-52
Preprint. Discussion started: 5 October 2020
c⃝Author(s) 2020. CC BY 4.0 License. 6 Appendix A
440 Discussion started: 5 October 2020
c⃝Author(s) 2020. CC BY 4.0 License. Figure A2: Violin plots and boxplots for water repellency (expressed as water drop infiltration time in seconds; n = 53), elevat
= 54), inclination (n = 54), and exposition (n = 54) of the soils under the four vegetation categories near-natural forest (FOR, g
steppe close to the forest (STE, grey), disturbed forest with regrowth of trees (DWIR, blue) and disturbed forest showing no reg
of trees (DNOR, orange). P values indicated above the plots were calculated for differences between the arithmetic means (NS
1; ns: p > 0.05; *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001; ****: p ≤ 0.0001). 0 445 Figure A2: Violin plots and boxplots for water repellency (expressed as water drop infiltration time in seconds; n = 53), elevation (n
= 54), inclination (n = 54), and exposition (n = 54) of the soils under the four vegetation categories near-natural forest (FOR, green),
steppe close to the forest (STE, grey), disturbed forest with regrowth of trees (DWIR, blue) and disturbed forest showing no regrowth
of trees (DNOR, orange). P values indicated above the plots were calculated for differences between the arithmetic means (NS.: p =
1; ns: p > 0.05; *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001; ****: p ≤ 0.0001). 450 6 Appendix A
440 Vegetation
category
Profile ID
Coordinates
N
Coordinates E Elevation
[m a.s.l.]
Inclination
[%]
Exposition
[°]
Bedrock
Main sediment
type
Pendants on rock
fragments
Profile
depth [cm]
Presence of
permafrost*
Disturbance
by fire
Disturban
by loggin
FOR
1
48° 38.681'
98° 22.141'
1915
24.5
320
granite
sand sheet
no
165
yes
no
no
FOR
2
48° 38.700'
98° 22.034'
1907
26.5
340
granite
sand sheet
no
140
yes
no
no
STE
3
48° 38.929'
98° 22.122'
1882
28.5
301
granite
bedrock
no
90
no
no
no
STE
4
48° 38.377'
98° 22.202'
1917
25.75
292
granite
colluvium
no
90
no
no
no
STE
5
48° 38.736'
98° 21.853'
1851
20
307
granite
colluvium
no
110
no
no
no
STE
6
48° 38.216'
98° 22.408'
1979
24.5
300
granite/gneiss
slope deposit
no
120
no
no
no
FOR
7
48° 38.181'
98° 22.487'
2007
31
304
granite/gneiss
sand sheet
no
105
no
no
low
FOR
8
48° 38.206'
98° 22.511'
2022
32
305
granite/gneiss
sand sheet
no
120
no
no
low
FOR
9
48° 38.770'
98° 22.074'
1868
16
301
granite
sand sheet
no
180
yes
no
moderate
FOR
10
48° 38.748'
98° 22.169'
1900
26.5
303
granite
slope deposit
no
100
no
no
moderate
FOR
11
48° 37.766'
98° 21.215'
1938
23.5
335
granite/gneiss
slope deposit
manganese
60
yes
no
no
FOR
12
48° 37.775'
98° 21.277'
1943
28.5
317
granite/gneiss
slope deposit
no
80
yes
no
no
FOR
13
48° 37.258'
98° 21.517'
1974
27.5
335
granite/gneiss
slope deposit
manganese
60
yes
no
no
FOR
14
48° 37.322'
98° 21.624'
1969
27
326
granite/gneiss
slope deposit
no
80
yes
no
no
FOR
15
48° 37.728'
98° 21.233'
1947
25
324
granite/gneiss
slope deposit
manganese
94
yes
no
no
FOR
16
48° 37.787'
98° 21.210'
1913
23.5
357
granite/gneiss
slope deposit
manganese
66
yes
no
low
FOR
17
48° 37.816'
98° 21.208'
1908
22
359
granite/gneiss
slope deposit
no
80
yes
no
low
FOR
18
48° 37.755'
98° 21.280'
1951
34.5
351
granite/gneiss
slope deposit
manganese
100
yes
no
no
FOR
19
48° 37.799'
98° 21.268'
1930
30
358
granite/gneiss
slope deposit
manganese
80
yes
no
no
FOR
20
48° 37.825'
98° 21.271'
1914
24
1
granite/gneiss
slope deposit
no
65
yes
no
no
FOR
21
48° 37.700'
98° 21.276'
1990
39
315
granite/gneiss
slope deposit
no
90
yes
no
no
FOR
22
48° 37.830'
98° 21.208'
1900
11
334
granite/gneiss
slope deposit
no
90
yes
no
low
FOR
23
48° 37.736'
98° 21.290'
1977
37
345
granite/gneiss
slope deposit
no
100
yes
no
no
FOR
24
48° 37.843'
98° 21.256'
1908
12
352
granite/gneiss
slope deposit
no
90
yes
no
low
FOR
25
48° 37.649'
98° 21.231'
1994
28.5
336
granite/gneiss
slope deposit
no
70
no
no
no
FOR
26
48° 37.669'
98° 21.199'
1982
29
346
granite/gneiss
slope deposit
no
70
yes
no
no
FOR
27
48° 37.697'
98° 21.184'
1977
29
356
granite/gneiss
slope deposit
no
58
yes
no
no
FOR
28
48° 37.725'
98° 21.161'
1957
24.5
352
granite/gneiss
slope deposit
no
80
yes
no
no
FOR
29
48° 37.768'
98° 21.135'
1943
25.5
353
granite/gneiss
slope deposit
no
80
yes
no
no
FOR
30
48° 37.787'
98° 21.127'
1915
15
351
granite/gneiss
slope deposit
no
70
yes
no
low
DNOR
31
48° 27.636'
98° 13.686'
2046
22
316
granite/gneiss
slope deposit
calcium carbonate
100
no
severe
moderate
DNOR
32
48° 27.640'
98° 13.730'
2055
25.5
344
granite/gneiss
slope deposit
calcium carbonate
80
no
severe
moderate
DWIR
33
48° 27.549'
98° 13.576'
2055
25
312
granite/gneiss
slope deposit
calcium carbonate
80
no
severe
moderate
DWIR
34
48° 27.595'
98° 13.723'
2079
32.5
332
granite/gneiss
slope deposit
manganese, calcium
carbonate
90
no
severe
moderate
DWIR
35
48° 27.597'
98° 10.886'
2077
26.5
322
granite/gneiss
slope deposit
no
90
no
severe
moderate
DWIR
36
48° 27.609'
98° 10.914'
2069
29.5
320
granite/gneiss
slope deposit
no
100
no
severe
moderate
DWIR
37
48° 28.784'
98° 19.246'
2088
35
324
granite/gneiss
slope deposit
no
110
no
severe
moderate
DNOR
38
48° 28.790'
98° 19.074'
2004
41
328
granite/gneiss
colluvium
no
100
no
severe
moderate
DNOR
39
48° 28.732'
98° 18.980'
2018
36
328
granite/gneiss
slope deposit
calcium carbonate
115
no
severe
severe
DNOR
40
48° 28.335'
98° 19.140'
2040
36.5
320
granite/gneiss
slope deposit
manganese, calcium
carbonate
100
no
severe
moderate
DWIR
41
48° 28.345'
98° 19.228'
2053
42.5
330
granite/gneiss
slope deposit
calcium carbonate
110
no
severe
moderate
DNOR
42
48° 33.181'
98° 13.362'
1893
23
330
granite
slope deposit
no
110
no
no
severe
DNOR
43
48° 24.506'
98° 18.295'
1912
35
17
granite
slope deposit
no
125
no
no
severe
DWIR
44
48° 48.525'
98° 19.850'
2093
25.5
320
granite/gneiss
slope deposit
no
100
no
severe
severe
DNOR
45
48° 48.371'
98° 19.351'
2059
23
324
granite/gneiss
slope deposit
no
90
no
severe
severe
DNOR
46
48° 48.400'
98° 19.136'
2002
28
330
granite/gneiss
slope deposit
manganese
105
no
severe
severe
DWIR
47
48° 52.582'
98° 19.482'
2006
32.5
315
granite/gneiss
bedrock
manganese
80
no
moderate
moderate
STE
48
48° 16.462'
98° 21.224'
2045
3
305
granite
slope deposit
calcium carbonate
130
no
no
no
DWIR
49
48° 16.438'
98° 20.856'
2043
26.5
325
granite
slope deposit
manganese
90
no
severe
moderate
DWIR
50
48° 22.427'
98° 38.329'
2039
23
357
granite
slope deposit
no
105
no
severe
moderate
DNOR
51
48° 22.293'
98° 38.313'
2098
34
4
granite
slope deposit
no
110
no
severe
moderate
DWIR
52
48° 22.113'
98° 38.247'
2213
30.5
355
granite
bedrock
no
52
no
severe
no
DWIR
53
48° 41.506'
98° 27.930'
1934
45
290
gneiss
slope deposit
manganese
95
no
moderate
moderate
DWIR
54
48° 41.471'
98° 28.073'
2033
44.5
291
gneiss
slope deposit
manganese
75
no
moderate
moderate Eutric Cambisol (Katoarenic, Bathygelic, Epiloamic, Nechic,
Ochric, Endoraptic)
Eutric Cambisol (Katoarenic, Bathygelic, Amphiloamic, Nechic,
Ochric)
Skeletic Cambic Endoleptic Phaeozem (Amphiarenic, Epiloamic,
Nechric)
Endoskeletic Phaeozem (Amphiarenic, Colluvic, Epiloamic,
Nechric, Epiraptic)
Endoskeletic Chernozem (Katoarenic, Cambic, Colluvic,
Amphiloamic, Amphiraptic)
Endoskeletic Endoleptic Phaeozem (Katoarenic, Colluvic,
Epiloamic, Nechric, Epiraptic)
Eutric Endoskeletic Cambisol (Katoarenic, Epiloamic, Nechic,
Ochric, Epiraptic)
Eutric Endoskeletic Cambisol (Katoarenic, Amphiloamic, Nechic,
Ochric, Endoraptic)
Cambic Phaeozem (Katoarenic, Colluvic, Katoloamic, Nechic,
Endoraptic, Bathyprotocalcic, Bathygleyic, Bathyturbic)
Haplic Phaeozem (Endoarenic, Colluvic, Amphiloamic, Nechric,
Amphiraptic)
Endoskeletic Cambic Folic Reductaquic Cryosol (Epiarenic,
Eutric, Amphiloamic, Nechic, Ochric, Epiraptic)
Endoskeletic Folic Reductaquic Cryosol (Amphiarenic, Eutric,
Epiloamic, Nechic, Ochric, Amphiraptic)
Endoskeletic Folic Reductaquic Cryosol ( Eutric, Loamic, Nechic,
Ochric, Epiraptic)
Endoskeletic Folic Cryosol (Epiarenic, Eutric, Amphiloamic,
Nechic, Ochric, Epiraptic)
Endoskeletic Cambic Folic Reductaquic Cryosol (Amphiarenic,
Eutric, Amphiloamic, Nechic, Ochric, Epiraptic)
Cambic Folic Reductaquic Cryosol (Amphiarenic, Eutric,
Amphiloamic, Nechic, Ochric, Epiraptic)
Endoskeletic Cambic Reductaquic Cryosol (Endoarenic, Eutric,
Epiloamic, Nechic, Ochric, Amphiraptic)
Endoskeletic Cambic Reductaquic Cryosol (Epiarenic, Eutric,
Amphiloamic, Nechic, Ochric, Epiraptic)
Endoskeletic Folic Reductaquic Cryosol (Eutric, Loamic, Nechic,
Ochric, Epiraptic)
Endoskeletic Cambic Folic Reductaquic Cryosol (Endoarenic,
Eutric, Anoloamic, Nechic, Ochric, Endoraptic)
Endoskeletic Reductaquic Cryosol (Amphiarenic, Eutric,
Amphiloamic, Nechic, Ochric, Epiraptic)
Endoskeletic Folic Reductaquic Cryosol (Endoarenic, Eutric,
Amphiloamic, Nechic, Ochric, Amphiraptic)
Endoskeletic Reductaquic Cryosol (Amphiarenic, Eutric,
Epiloamic, Nechic, Ochric, Epiraptic)
Endoskeletic Folic Reductaquic Cryosol (Endoarenic, Eutric,
Amphiloamic, Nechic, Ochric, Epiraptic)
Eutric Endoskeletic Cambisol (Gelic, Gelistagnic, Loamic,
Nechric, Ochric, Epiraptic)
Endoskeletic Folic Reductaquic Cryosol (Epiarenic, Eutric,
Amphiloamic, Nechic, Ochric, Epiraptic)
Cambic Folic Cryosol (Eutric, Loamic, Nechic, Ochric, Epiraptic)
Endoskeletic Folic Reductaquic Cryosol (Amphiarenic, Eutric,
Amphiloamic, Nechic, Ochric, Epiraptic)
Endoskeletic Folic Reductaquic Cryosol (Epiarenic, Eutric,
Amphiloamic, Nechic, Ochric, Epiraptic)
Endoskeletic Cambic Folic Reductaquic Cryosol (Eutric, Loamic,
Nechic, Ochric, Epiraptic)
Eutric Endoskeletic Cambisol (Endoarenic, Amphiloamic,
Nechric, Ochric, Amphiraptic)
Eutric Endoskeletic Cambisol (Endoarenic, Colluvic, Epiloamic,
Nechric, Ochric, Epiraptic)
Eutric Endoskeletic Cambisol (Colluvic, Loamic, Nechric, Ochric,
Epiraptic)
Eutric Endoskeletic Cambisol (Protocalcic, Colluvic, Loamic,
Nechric, Ochric, Amphiraptic)
Eutric Endoskeletic Cambisol (Loamic, Nechric, Ochric,
Epiraptic)
Eutric Endoskeletic Cambisol (Loamic, Nechric, Ochric,
Epiraptic)
Eutric Endoskeletic Cambisol (Amphiarenic, Colluvic, Epiloamic,
Nechric, Ochric, Epiraptic)
Endoskeletic Phaeozem (Amphiarenic, Colluvic, Epiloamic,
Nechric, Ochric, Amphiraptic)
Eutric Endoskeletic Cambisol (Amphiarenic, Colluvic, Epiloamic,
Nechric, Ochric, Amphiraptic)
Eutric Endoskeletic Cambisol (Amphiarenic, Colluvic, Epiloamic,
Nechric, Ochric, Amphiraptic)
Eutric Endoskeletic Cambisol (Amphiarenic, Colluvic, Epiloamic,
Nechric, Ochric, Amphiraptic)
Haplic Kastanozem (Amphiarenic, Cambic, Colluvic, Epiloamic,
Nechric, Epiraptic)
Skeletic Phaeozem (Katoarenic, Colluvic, Amphiloamic, Nechric,
Amphiraptic)
Eutric Endoskeletic Cambisol (Amphiarenic, Epiloamic, Nechric,
Ochric, Amphiraptic)
Eutric Endoskeletic Cambisol (Amphiarenic, Epiloamic, Nechric,
Ochric, Epiraptic)
Eutric Endoskeletic Cambisol (Katoarenic, Epiloamic, Nechric,
Ochric, Amphiraptic)
Eutric Endoskeletic Endoleptic Cambisol (Amphiarenic,
Amphiloamic, Nechric, Ochric, Epiraptic)
Haplic Kastanozem (Cambic, Loamic, Amphiraptic)
Eutric Endoskeletic Cambisol (Amphiarenic, Epiloamic, Nechric,
Ochric, Epiraptic)
Endoskeletic Phaeozem (Katoarenic, Colluvic, Amphiloamic,
Nechric, Amphiraptic)
Endoskeletic Phaeozem (Katoarenic, Colluvic, Amphiloamic,
Nechric, Amphiraptic)
Endoskeletic Endoleptic Phaeozem (Loamic, Nechric, Epiraptic)
Skeletic Endoleptic Phaeozem (Amphiarenic, Epiloamic, Nechric,
Epiraptic)
Endoskeletic Endoleptic Phaeozem (Amphiarenic, Epiloamic,
Nechric, Epiraptic) 25 https://doi.org/10.5194/soil-2020-52
Preprint. Acknowledgements We thank Dr. Choimaa Dulamsuren and Prof. Uudus Bayarsaikhan for their invaluable help in organising and conducting our
460
fieldwork, and Ms. Daramragchaa Tuya for her great support of our research. We are also grateful to our Mongolian colleagues
Mr. Amarbayasgalan, Mr. Enkhjargal, Mr. Enkh-Agar, and Ms. Munkhtuya; we appreciated their hospitality and help with the
fieldwork. Our thanks also go to the German students Janin Klaassen, Kim Lena Arndt and Tim Rollwage for their great
commitment during the fieldwork in Mongolia. Furthermore, we want to thank Dr. Jürgen Grotheer, Petra Voigt, Anja Södje We thank Dr. Choimaa Dulamsuren and Prof. Uudus Bayarsaikhan for their invaluable help in organising and conducting our
460
fieldwork, and Ms. Daramragchaa Tuya for her great support of our research. We are also grateful to our Mongolian colleagues
Mr. Amarbayasgalan, Mr. Enkhjargal, Mr. Enkh-Agar, and Ms. Munkhtuya; we appreciated their hospitality and help with the
fieldwork. Our thanks also go to the German students Janin Klaassen, Kim Lena Arndt and Tim Rollwage for their great
commitment during the fieldwork in Mongolia. Furthermore, we want to thank Dr. Jürgen Grotheer, Petra Voigt, Anja Södje We thank Dr. Choimaa Dulamsuren and Prof. Uudus Bayarsaikhan for their invaluable help in organising and conducting our
460
fieldwork, and Ms. Daramragchaa Tuya for her great support of our research. We are also grateful to our Mongolian colleagues
Mr. Amarbayasgalan, Mr. Enkhjargal, Mr. Enkh-Agar, and Ms. Munkhtuya; we appreciated their hospitality and help with the
fieldwork. Our thanks also go to the German students Janin Klaassen, Kim Lena Arndt and Tim Rollwage for their great
commitment during the fieldwork in Mongolia. Furthermore, we want to thank Dr. Jürgen Grotheer, Petra Voigt, Anja Södje and Eike Sebode for their excellent work in the laboratory. We also thank Lukáš Banyi for the essential support in using the R
465
project for statistical computing. The project was funded by the Deutsche Forschungsgemeinschaft (DFG), project number 385460422. and Eike Sebode for their excellent work in the laboratory. We also thank Lukáš Banyi for the essential support in using the R
465
project for statistical computing. The project was funded by the Deutsche Forschungsgemeinschaft (DFG), project number 385460422. Competing interests: The authors declare that they have no conflict of interest. The authors declare that they have no conflict of interest. Authors contribution: Michael Klinge and Daniela Sauer designed the research project. All authors carried out the field work together. Florian
Schneider, Tino Peplau and Jannik Brodthuhn carried out the laboratory work. Florian Schneider prepared the manuscript with
455
contributions from all co-authors. Allen, C. D., Macalady, A. K., Chenchouni, H., Bachelet, D., McDowell, N., Vennetier, M., Kitzberger, T., Rigling, A.,
Breshears, D. D., Hogg, E. H., Gonzalez, P., Fensham, R., Zhang, Z., Castro, J., Demidova, N., Lim, J.-H., Allard, G.,
475
Running, S. W., Semerci, A., and Cobb, N.: A global overview of drought and heat-induced tree mortality reveals
emerging climate change risks for forests, Forest Ecology and Management, 259, 660–684,
https://doi.org/10.1016/j.foreco.2009.09.001, 2010. Code/Data availability: See supplementary material (data.xlsx). See supplementary material (data.xlsx). 26 https://doi.org/10.5194/soil-2020-52
Preprint. Discussion started: 5 October 2020
c⃝Author(s) 2020. CC BY 4.0 License. Academy of Sciences of Mongolia, Academy of Sciences of USSR: National Atlas of the Peoples Republic of Mongolia,
470
Ulaanbaatar, Moscow, 1990.
Albenskiy, A. V., Krylov, G. V., Logginov, B. J., and Scherlin, J. D.: Usage of fast growing breeds in field protection forest
cultivation (in Russian), Moskow, 1956. Academy of Sciences of Mongolia, Academy of Sciences of USSR: National Atlas of the Peoples Republic of Mongolia,
470
Ulaanbaatar, Moscow, 1990.
Albenskiy A V Krylov G V Logginov B J and Scherlin J D : Usage of fast growing breeds in field protection forest References 495
Chenlemuge, T., Schuldt, B., Dulamsuren, C., Hertel, D., Leuschner, C., and Hauck, M.: Stem increment and hydraulic
architecture of a boreal conifer (Larix sibirica) under contrasting macroclimates, Trees, 29, 623–636,
https://doi.org/10.1007/s00468-014-1131-x, 2015a. Chenlemuge, T., Dulamsuren, C., Hertel, D., Schuldt, B., Leuschner, C., and Hauck, M.: Hydraulic properties and fine root Chenlemuge, T., Schuldt, B., Dulamsuren, C., Hertel, D., Leuschner, C., and Hauck, M.: Stem increment and hydraulic
architecture of a boreal conifer (Larix sibirica) under contrasting macroclimates, Trees, 29, 623–636,
https://doi.org/10.1007/s00468-014-1131-x, 2015a. architecture of a boreal conifer (Larix sibirica) under contrasting macroclimates, Trees, 29, 623–636,
https://doi.org/10.1007/s00468-014-1131-x, 2015a. Chenlemuge, T., Dulamsuren, C., Hertel, D., Schuldt, B., Leuschner, C., and Hauck, M.: Hydraulic properties and fine root
mass of Larix sibirica along forest edge-interior gradients, Acta Oecologica, 63, 28–35,
500
https://doi.org/10.1016/j.actao.2014.11.008, 2015b. Chernyshenkо, O. V. and Vasilyev, S. B.: Mineral nutrition specificity of coniferous trees on industrial waste discharge of
Egorievsk phosphorite deposit (in Russian), FB, 46–53, https://doi.org/10.18698/2542-1468-2019-5-46-53, 2019. Churakova, O. V., Shashkin, A. V., Siegwolf, R. T.W., Spahni, R., Launois, T., Saurer, M., Bryukhanova, M. V., Benkova, Chenlemuge, T., Dulamsuren, C., Hertel, D., Schuldt, B., Leuschner, C., and Hauck, M.: Hydr Chenlemuge, T., Dulamsuren, C., Hertel, D., Schuldt, B., Leuschner, C., and Hauck, M.: Hydraulic properties and fine root
mass of Larix sibirica along forest edge-interior gradients, Acta Oecologica, 63, 28–35,
500
https://doi.org/10.1016/j.actao.2014.11.008, 2015b. Chernyshenkо, O. V. and Vasilyev, S. B.: Mineral nutrition specificity of coniferous trees on industrial waste discharge of
Egorievsk phosphorite deposit (in Russian), FB, 46–53, https://doi.org/10.18698/2542-1468-2019-5-46-53, 2019. Chenlemuge, T., Dulamsuren, C., Hertel, D., Schuldt, B., Leuschner, C., and Hauck, M.: Hydraulic properties and fine root
mass of Larix sibirica along forest edge-interior gradients, Acta Oecologica, 63, 28–35,
500
https://doi.org/10.1016/j.actao.2014.11.008, 2015b. mass of Larix sibirica along forest edge-interior gradients, Acta Oecologica, 63, 28–35,
500
https://doi.org/10.1016/j.actao.2014.11.008, 2015b. Chernyshenkо, O. V. and Vasilyev, S. B.: Mineral nutrition specificity of coniferous trees on industrial waste discharge of
Egorievsk phosphorite deposit (in Russian), FB, 46–53, https://doi.org/10.18698/2542-1468-2019-5-46-53, 2019. Churakova, O. V., Shashkin, A. V., Siegwolf, R. T.W., Spahni, R., Launois, T., Saurer, M., Bryukhanova, M. V., Benkova, https://doi.org/10.1016/j.actao.2014.11.008, 2015b. Chernyshenkо, O. V. and Vasilyev, S. B.: Mineral nutrition specificity of coniferous trees on industrial waste discharge of
Egorievsk phosphorite deposit (in Russian), FB, 46–53, https://doi.org/10.18698/2542-1468-2019-5-46-53, 2019. Churakova, O. V., Shashkin, A. V., Siegwolf, R. T.W., Spahni, R., Launois, T., Saurer, M., Bryukhanova, M. V., Benkova, A. V., Kuptsova, A. References Academy of Sciences of Mongolia, Academy of Sciences of USSR: National Atlas of the Peoples Republic of Mongolia,
470
Ulaanbaatar, Moscow, 1990. Albenskiy, A. V., Krylov, G. V., Logginov, B. J., and Scherlin, J. D.: Usage of fast growing breeds in field protection forest
cultivation (in Russian), Moskow, 1956. Allen C D Macalady A K Chenchouni H Bachelet D McDowell N Vennetier M Kitzberger T Rigling A Academy of Sciences of Mongolia, Academy of Sciences of USSR: National Atlas of the Peoples Republic of Mongolia,
470
Ulaanbaatar, Moscow, 1990. Albenskiy, A. V., Krylov, G. V., Logginov, B. J., and Scherlin, J. D.: Usage of fast growing breeds in field protection forest
cultivation (in Russian), Moskow, 1956. Allen, C. D., Macalady, A. K., Chenchouni, H., Bachelet, D., McDowell, N., Vennetier, M., Kitzberger, T., Rigling, A.,
Breshears, D. D., Hogg, E. H., Gonzalez, P., Fensham, R., Zhang, Z., Castro, J., Demidova, N., Lim, J.-H., Allard, G.,
475
Running, S. W., Semerci, A., and Cobb, N.: A global overview of drought and heat-induced tree mortality reveals
emerging climate change risks for forests, Forest Ecology and Management, 259, 660–684,
https://doi.org/10.1016/j.foreco.2009.09.001, 2010. Breshears, D. D., Hogg, E. H., Gonzalez, P., Fensham, R., Zhang, Z., Castro, J., Demidova, N., Lim, J.-H., Allard, G.,
475
Running, S. W., Semerci, A., and Cobb, N.: A global overview of drought and heat-induced tree mortality reveals
emerging climate change risks for forests, Forest Ecology and Management, 259, 660–684,
https://doi.org/10.1016/j.foreco.2009.09.001, 2010. 27 https://doi.org/10.5194/soil-2020-52
Preprint. Discussion started: 5 October 2020
c⃝Author(s) 2020. CC BY 4.0 License. https://doi.org/10.5194/soil-2020-52
Preprint. Discussion started: 5 October 2020
c⃝Author(s) 2020. CC BY 4.0 License. Amelung, W., Blume, H.-P., Fleige, H., Horn, R., Kandeler, E., Kögel-Knabner, I., Kretzschmar, R., Stahr, K., and Wilke,
B.-M.: Scheffer/Schachtschabel Lehrbuch der Bodenkunde (in German), 17., überarbeitete und ergänzte Auflage,
Springer Spektrum, Berlin, 750 pp., 2018. Amelung, W., Blume, H.-P., Fleige, H., Horn, R., Kandeler, E., Kögel-Knabner, I., Kretzschmar, R., Stahr, K., and Wilke, Amelung, W., Blume, H.-P., Fleige, H., Horn, R., Kandeler, E., Kögel-Knabner, I., Kretzschmar, Amelung, W., Blume, H.-P., Fleige, H., Horn, R., Kandeler, E., Kögel-Knabner, I., Kretzschmar, R., Stahr, K., and Wilke,
B.-M.: Scheffer/Schachtschabel Lehrbuch der Bodenkunde (in German), 17., überarbeitete und ergänzte Auflage,
480
Springer Spektrum, Berlin, 750 pp., 2018. Amoozegar, A. and Warrick, A. W.: Hydraulic Conductivity of Saturated Soils: Field Methods, American Socienty of
Agronomy-Soil Science Society of America, 735–770, 1986. References Batima, P., Natsagdorj, L., Gombluudev, P., and Erdenetsetseg, B.: Observed climate change in Mongolia, Assessments of 480 Amoozegar, A. and Warrick, A. W.: Hydraulic Conductivity of Saturated Soils: Field Methods, American Socienty of
Agronomy-Soil Science Society of America, 735–770, 1986. Batima, P., Natsagdorj, L., Gombluudev, P., and Erdenetsetseg, B.: Observed climate change in Mongolia, Assessments of
Impacts and Adaptations of Climate Change, 1–26, 2005. Batima, P., Natsagdorj, L., Gombluudev, P., and Erdenetsetseg, B.: Observed climate change in Mongolia, Assessments of
I
d Ad
i
f Cli
Ch
1 26 2005
5 Impacts and Adaptations of Climate Change, 1–26, 2005. 485
Beaudette, D. E., Roudier, P., and O'Geen, A. T.: Algorithms for quantitative pedology: A toolkit for soil scientists,
Computers & Geosciences, 52, 258–268, https://doi.org/10.1016/j.cageo.2012.10.020, 2013. Blume, H.-P., Stahr, K., and Leinweber, P.: Bodenkundliches Praktikum (in German): Eine Einführung in pedologisches
Arbeiten für Ökologen, insbesondere Land- und Forstwirte, und für Geowissenschaftler, 3., neubearb. Aufl., Spektrum Computers & Geosciences, 52, 258–268, https://doi.org/10.1016/j.cageo.2012.10.020, 2013. Blume, H.-P., Stahr, K., and Leinweber, P.: Bodenkundliches Praktikum (in German): Eine Einführung in pedologisches
Arbeiten für Ökologen, insbesondere Land- und Forstwirte, und für Geowissenschaftler, 3., neubearb. Aufl., Spektrum Blume, H.-P., Stahr, K., and Leinweber, P.: Bodenkundliches Praktikum (in German): Eine Einführung in pedologisches
Arbeiten für Ökologen, insbesondere Land- und Forstwirte, und für Geowissenschaftler, 3., neubearb. Aufl., Spektrum
Akademischer Verlag, Heidelberg, 255 pp., 2011. 490 Akademischer Verlag, Heidelberg, 255 pp., 2011. 490
Bonan, G. B. and Shugart, H. H.: Environmental Factors and Ecological Processes in Boreal Forests, Annu. Rev. Syst., 20,
1–28, 1989. Breckle, S.-W., Agachanjanz, O., Rahmann, M., and Walter, H.: Spezielle Ökologie der Gemäßigten und Arktischen Zonen
Euro-Nordasiens (in German): Zonobiom VI-IX, 2., überarb. Aufl., UTB für Wissenschaft Große Reihe, Bd. 3, Fischer, Bonan, G. B. and Shugart, H. H.: Environmental Factors and Ecological Processes in Boreal Forests, Annu. Rev. Syst., 20,
1–28, 1989. 1–28, 1989. Breckle, S.-W., Agachanjanz, O., Rahmann, M., and Walter, H.: Spezielle Ökologie der Gemäßigten und Arktischen Zonen
Euro-Nordasiens (in German): Zonobiom VI-IX, 2., überarb. Aufl., UTB für Wissenschaft Große Reihe, Bd. 3, Fischer, Breckle, S.-W., Agachanjanz, O., Rahmann, M., and Walter, H.: Spezielle Ökologie der Gemäßigten und Arktischen Zonen
Euro-Nordasiens (in German): Zonobiom VI-IX, 2., überarb. Aufl., UTB für Wissenschaft Große Reihe, Bd. 3, Fischer,
Stuttgart, Jena, 726 pp., 1994. 495 Stuttgart, Jena, 726 pp., 1994. References V., Peylin, P., Vaganov, E. A., Masson-Delmotte, V., and Roden, J.: Application of eco-
505
physiological models to the climatic interpretation of δ13C and δ18O measured in Siberian larch tree-rings,
Dendrochronologia, 39, 51–59, https://doi.org/10.1016/j.dendro.2015.12.008, 2016. Dashkhuu, D., Kim, J. P., Chun, J. A., and Lee, W.-S.: Long-term trends in daily temperature extremes over Mongolia,
Weather and Climate Extremes, 8, 26–33, https://doi.org/10.1016/j.wace.2014.11.003, 2015. Dashkhuu, D., Kim, J. P., Chun, J. A., and Lee, W.-S.: Long-term trends in daily temperature extremes over Mongolia,
Weather and Climate Extremes, 8, 26–33, https://doi.org/10.1016/j.wace.2014.11.003, 2015. Dashtseren, A., Ishikawa, M., Iijima, Y., and Jambaljav, Y.: Temperature Regimes of the Active Layer and Seasonally
510
Frozen Ground under a Forest-Steppe Mosaic, Mongolia, Permafrost and Periglac. Process., 25, 295–306,
https://doi.org/10.1002/ppp.1824, 2014. 28 https://doi.org/10.5194/soil-2020-52
Preprint. Discussion started: 5 October 2020
c⃝Author(s) 2020. CC BY 4.0 License. https://doi.org/10.5194/soil-2020-52
Preprint. Discussion started: 5 October 2020
c⃝Author(s) 2020. CC BY 4.0 License. DeBano, L.F.: The role of fire and soil heating on water repellency in wildland environments: a review, Journal of
H d
l
231 232 195 206 htt
//d i
/10 1016/S0022 1694(00)00194 3 2000 DeBano, L.F.: The role of fire and soil heating on water repellency in wildland environments: a review, Journal of
Hydrology, 231-232, 195–206, https://doi.org/10.1016/S0022-1694(00)00194-3, 2000. Deutsches Institut für Normung e.V.: Bodenbeschaffenheit - Bestimmung des Wasserrückhaltevermögens - Laborverfahren
5
(in German), Beuth Verlag GmbH, Berlin, 2014. Deutsches Institut für Normung e.V.: Bodenbeschaffenheit - Physikalische Laboruntersuchungen - Teil 9: Bestimmung der Deutsches Institut für Normung e.V.: Bodenbeschaffenheit - Bestimmung des Wasserrückhaltevermögens - Laborverfahren
515
(in German), Beuth Verlag GmbH, Berlin, 2014. Deutsches Institut für Normung e.V.: Bodenbeschaffenheit - Physikalische Laboruntersuchungen - Teil 9: Bestimmung der
Wasserdurchlässigkeit in wassergesättigten Stechzylinderproben (in German), 13.080.20;65.060.35, Beuth Verlag
GmbH, Berlin, 2012. Deutsches Institut für Normung e.V.: Bodenbeschaffenheit - Bestimmung des Wasserrückhaltevermögens - Laborverfahren
5
(in German), Beuth Verlag GmbH, Berlin, 2014. Deutsches Institut für Normung e.V.: Bodenbeschaffenheit - Physikalische Laboruntersuchungen - Teil 9: Bestimmung der
Wasserdurchlässigkeit in wassergesättigten Stechzylinderproben (in German), 13.080.20;65.060.35, Beuth Verlag
GmbH, Berlin, 2012. Deutsches Institut für Normung e.V.: Bodenbeschaffenheit - Bestimmung der Partikelgrößenverteilung in Mineralböden -
520
Verfahren mittels Siebung und Sedimentation (in German), Beuth Verlag GmbH, Berlin, 2002. Deutsches Institut für Normung e.V.: Bodenbeschaffenheit - Bestimmung der Partikelgrößenverteilung in Mineralböden -
520
Verfahren mittels Siebung und Sedimentation (in German), Beuth Verlag GmbH, Berlin, 2002. Doerr, S. H., Shakesby, R. References A., and Walsh, R.P.D.: Soil water repellency: its causes, characteristics and hydro-
geomorphological significance, Earth-Science Reviews, 51, 33–65, https://doi.org/10.1016/S0012-8252(00)00011-8,
2000. Verfahren mittels Siebung und Sedimentation (in German), Beuth Verlag GmbH, Berlin, 2002. Doerr, S. H., Shakesby, R. A., and Walsh, R.P.D.: Soil water repellency: its causes, characteristics and hydro-
geomorphological significance, Earth-Science Reviews, 51, 33–65, https://doi.org/10.1016/S0012-8252(00)00011-8,
2000. Doerr, S. H.: On standardizing the ‘Water Drop Penetration Time’ and the ‘Molarity of an Ethanol Droplet’ techniques to
525
classify soil hydrophobicity: A case study using medium textured soils, Earth Surf. Process. Landforms, 23, 663–668,
https://doi.org/10.1002/(SICI)1096-9837(199807)23:7<663:AID-ESP909>3.0.CO;2-6, 1998. Dugarjav, C.: Larch forests of Mongolia (in Mongolian), Bembi San, Ulan Bator, 2006. p
g
(
)
(
)
;
,
Dugarjav, C.: Larch forests of Mongolia (in Mongolian), Bembi San, Ulan Bator, 2006. Dulamsuren, C. and Hauck, M.: Spatial and seasonal variation of climate on steppe slopes of the northern Mongolian
mountain taiga, Grassland Science, 54, 217–230, https://doi.org/10.1111/j.1744-697X.2008.00128.x, 2008. 530
Dulamsuren, C., Khishigjargal, M., Leuschner, C., and Hauck, M.: Response of tree-ring width to climate warming and
selective logging in larch forests of the Mongolian Altai, Journal of Plant Ecology, 7, 24–38,
https://doi.org/10.1093/jpe/rtt019, 2014. Dulamsuren, C., Hauck, M., Leuschner, H. H., and Leuschner, C.: Climate response of tree-ring width in Larix sibirica
growing in the drought-stressed forest-steppe ecotone of northern Mongolia, Annals of Forest Science, 68, 275–282,
535
https://doi.org/10.1007/s13595-011-0043-9, 2011. Dulamsuren, C., Hauck, M., and Leuschner, C.: Recent drought stress leads to growth reductions in Larix sibirica in the
western Khentey, Mongolia, Global Change Biology, 95, 3024-3035, https://doi.org/10.1111/j.1365-2486.2009.02147
2010. Dulamsuren, C. and Hauck, M.: Spatial and seasonal variation of climate on steppe slopes of the northern Mongolian
mountain taiga, Grassland Science, 54, 217–230, https://doi.org/10.1111/j.1744-697X.2008.00128.x, 2008. 0 mountain taiga, Grassland Science, 54, 217–230, https://doi.org/10.1111/j.1744-697X.2008.00128.x, 2008. 530
Dulamsuren, C., Khishigjargal, M., Leuschner, C., and Hauck, M.: Response of tree-ring width to climate warming and
selective logging in larch forests of the Mongolian Altai, Journal of Plant Ecology, 7, 24–38,
https://doi.org/10.1093/jpe/rtt019, 2014. Dulamsuren, C., Hauck, M., Leuschner, H. H., and Leuschner, C.: Climate response of tree-ring width in Larix sibirica
growing in the drought-stressed forest-steppe ecotone of northern Mongolia, Annals of Forest Science, 68, 275–282,
535
https://doi.org/10.1007/s13595-011-0043-9, 2011. Dulamsuren, C., Hauck, M., and Leuschner, C.: Recent drought stress leads to growth reductions in Larix sibirica in the
western Khentey, Mongolia, Global Change Biology, 95, 3024-3035, https://doi.org/10.1111/j.1365-2486.2009.02147.x,
2010. References Dulamsuren, C., Hauck, M., Bader, M., Osokhjargal, D., Oyungerel, S., Nyambayar, S., Runge, M., and Leuschner, C.:
540
Water relations and photosynthetic performance in Larix sibirica growing in the forest-steppe ecotone of northern
Mongolia, Tree physiology, 29, 99–110, https://doi.org/10.1093/treephys/tpn008, 2009. Durner, W. and Iden, S. C.: Skript Bodenphysikalische Versuche (in German), Skript, Institut für Geoökologie, Abteilung
Bodenkunde und Bodenphysik,, TU Braunschweig, Braunschweig, 2011. Durner, W. and Iden, S. C.: Skript Bodenphysikalische Versuche (in German), Skript, Institut für Geoökologie, Abteilung
Bodenkunde und Bodenphysik,, TU Braunschweig, Braunschweig, 2011. Dylis, N. V.: Siberian larch (in Russian), Moskow, 1947. 545
Guidelines for soil description 4 [rev ] ed FAO Rome 97 pp 2006 Dylis, N. V.: Siberian larch (in Russian), Moskow, 1947. 545
Guidelines for soil description, 4., [rev.] ed., FAO, Rome, 97 pp., 2006. 29 https://doi.org/10.5194/soil-2020-52
Preprint. Discussion started: 5 October 2020
c⃝Author(s) 2020. CC BY 4.0 License. https://doi.org/10.5194/soil-2020-52
Preprint. Discussion started: 5 October 2020
c⃝Author(s) 2020. CC BY 4.0 License. Fiedler, H. J., Hunger, W., Müller, K., and Skodawessely, K.: Zu den Beziehungen zwischen Boden, Ernährungszustand und
Wuchsleistung bei Lärche (Larix decidua Mill.) (in German), Flora, 170, 1–19, https://doi.org/10.1016/S0367-
2530(17)31187-8, 1980. Goldammer, J. G.: Fire Situation in Mongolia (IFFN No. 26) | GFMC, http://gfmc.online/iffn/country/mn/mn_11.html, last
550
access: 14 October 2019, 2002. Grunert, J., Lehmkuhl, F., and Walther, M.: Paleoclimatic evolution of the Uvs Nuur basin and adjacent areas (Western
Mongolia), Quaternary International, 65-66, 171–192, https://doi.org/10.1016/S1040-6182(99)00043-9, 2000. Haase, G.: Die Höhenstufen der Böden im Changai (MVR) (in German), Z. Pflanzenernaehr. Dueng. Bodenk., 102, 113– Goldammer, J. G.: Fire Situation in Mongolia (IFFN No. 26) | GFMC, http://gfmc.online/iffn/country/mn/mn_11.html, last
50
access: 14 October 2019, 2002. Goldammer, J. G.: Fire Situation in Mongolia (IFFN No. 26) | GFMC, http://gfmc.online/iffn/country/mn/mn_11.html, last
550
access: 14 October 2019, 2002. Grunert, J., Lehmkuhl, F., and Walther, M.: Paleoclimatic evolution of the Uvs Nuur basin and adjacent areas (Western Grunert, J., Lehmkuhl, F., and Walther, M.: Paleoclimatic evolution of the Uvs Nuur basin and adjacent areas (Western
Mongolia), Quaternary International, 65-66, 171–192, https://doi.org/10.1016/S1040-6182(99)00043-9, 2000. H
G Di H h
f
d
B d
i
Ch
i (MVR) (i G
) Z Pfl
h
D
B d
k 102 113 Haase, G.: Die Höhenstufen der Böden im Changai (MVR) (in German), Z. Pflanzenernaehr. Dueng. Bodenk., 102, 113–
127, https://doi.org/10.1002/jpln.19631020205, 1963. 555 127, https://doi.org/10.1002/jpln.19631020205, 1963. 555 127, https://doi.org/10.1002/jpln.19631020205, 1963. References 555
Hais, M., Chytrý, M., and Horsák, M.: Exposure-related forest-steppe: A diverse landscape type determined by topography
and climate, Journal of Arid Environments, 135, 75–84, https://doi.org/10.1016/j.jaridenv.2016.08.011, 2016. Hessl, A. E., Brown, P., Byambasuren, O., Cockrell, S., Leland, C., Cook, E., Nachin, B., Pederson, N., Saladyga, T., and
Suran, B.: Fire and climate in Mongolia (1532-2010 Common Era), Geophys. Res. Lett., 43, 6519–6527, Hais, M., Chytrý, M., and Horsák, M.: Exposure-related forest-steppe: A diverse landscape type determined by topography
and climate, Journal of Arid Environments, 135, 75–84, https://doi.org/10.1016/j.jaridenv.2016.08.011, 2016. Hessl, A. E., Brown, P., Byambasuren, O., Cockrell, S., Leland, C., Cook, E., Nachin, B., Pederson, N., Saladyga, T., and
Suran, B.: Fire and climate in Mongolia (1532-2010 Common Era), Geophys. Res. Lett., 43, 6519–6527, https://doi.org/10.1002/2016GL069059, 2016. 560
Hessl, A. E., Ariya, U., Brown, P., Byambasuren, O., Green, T., Jacoby, G., Sutherland, E. K., Nachin, B., Maxwell, R. S.,
Pederson, N., Grandpré, L. de, Saladyga, T., and Tardif, J. C.: Reconstructing fire history in central Mongolia from tree-
rings, Int. J. Wildland Fire, 21, 86, https://doi.org/10.1071/WF10108, 2012. Hilbig, W.: Zur Problematik der ursprünglichen Waldverbreitung in der Mongolischen Volksr Hilbig, W.: Zur Problematik der ursprünglichen Waldverbreitung in der Mongolischen Volksrepublik Ergebnisse der
Mongolisch - Deutschen Biologischen Expeditionen seit 1962, Nr. 181 (in German), Flora, 179, 1–15,
65
https://doi.org/10.1016/S0367-2530(17)30213-X, 1987. Hilbig, W.: Zur Problematik der ursprünglichen Waldverbreitung in der Mongolischen Volksrepublik Ergebnisse der Mongolisch - Deutschen Biologischen Expeditionen seit 1962, Nr. 181 (in German), Flora, 179, 1–15,
565
https://doi.org/10.1016/S0367-2530(17)30213-X, 1987. Hwang, J. and Son, Y.: Short-term effects of thinning and liming on forest soils of pitch pine and Japanese larch plantations
in central Korea, Ecological Research, 21, 671–680, https://doi.org/10.1007/s11284-006-0170-1, 2006. Ishikawa, M., Sharkhuu, N., Zhang, Y., Kadota, T., and Ohata, T.: Ground Thermal and Moisture Conditions at the Southern
Boundary of Discontinuous Permafrost, Mongolia, Permafrost Periglac. Process., 16, 209–216,
570
https://doi org/10 1002/ppp 483 2005 Mongolisch - Deutschen Biologischen Expeditionen seit 1962, Nr. 181 (in German), Flora, 179, 1–15,
565
https://doi.org/10.1016/S0367-2530(17)30213-X, 1987. Mongolisch - Deutschen Biologischen Expeditionen seit 1962, Nr. 181 (in German), Flora, 179, 1–15,
565
https://doi.org/10.1016/S0367-2530(17)30213-X, 1987. Hwang, J. and Son, Y.: Short-term effects of thinning and liming on forest soils of pitch pine and Japanese larch plantations
in central Korea, Ecological Research, 21, 671–680, https://doi.org/10.1007/s11284-006-0170-1, 2006. References Khishigjargal, M., Dulamsuren, C., Lkhagvadorj, D., Leuschner, C., and Hauck, M.: Contrasting responses of seedling and
sapling densities to livestock density in the Mongolian forest-steppe, Plant Ecol, 214, 1391–1403,
90
https://doi.org/10.1007/s11258-013-0259-x, 2013. Klinge, M., Schneider, F., Dulamsuren, C., Arndt, K., Bayarsaikhan, U., and Sauer, D.: Interrelations between vegetation,
natural and anthropogenic disturbances, and discontinuous permafrost in the forest-steppe of central Mongolia, Earth
Surface Processes and Landforms, submitted. Klinge, M. and Lehmkuhl, F.: Geomorphology of the Tsetseg Nuur basin, Mongolian Altai – lake development, fluvial
595
sedimentation and aeolian transport in a semi-arid environment, Journal of Maps, 9, 361–366,
https://doi.org/10.1080/17445647.2013.783513, 2013. Klinge, M., Dulamsuren, C., Schneider, F., Erasmi, S., Hauck, M., Bayarsaikhan, U., and Sauer, D.: Modelled potential
forest area in the forest-steppe of central Mongolia is about three times of actual forest area, 2020. Klinge, M. and Lehmkuhl, F.: Geomorphology of the Tsetseg Nuur basin, Mongolian Altai – lake development, fluvial
595
sedimentation and aeolian transport in a semi-arid environment, Journal of Maps, 9, 361–366,
https://doi.org/10.1080/17445647.2013.783513, 2013. https://doi.org/10.1080/17445647.2013.783513, 2013. Klinge, M., Dulamsuren, C., Schneider, F., Erasmi, S., Hauck, M., Bayarsaikhan, U., and Sauer, D.: Modelled potential
forest area in the forest-steppe of central Mongolia is about three times of actual forest area, 2020. Klinge, M., Dulamsuren, C., Erasmi, S., Karger, D. N., and Hauck, M.: Climate effects on vegetation vitality at the treeline
600
of boreal forests of Mongolia, Biogeosciences, 15, 1319–1333, https://doi.org/10.5194/bg-15-1319-2018, 2018. Kopp, B. J., Minderlein, S., and Menzel, L.: Soil Moisture Dynamics in a Mountainous Headwater Area in the
Discontinuous Permafrost Zone of northern Mongolia, Arctic, Antarctic, and Alpine Research, 46, 459–470,
https://doi.org/10.1657/1938-4246-46.2.459, 2014. Klinge, M., Dulamsuren, C., Erasmi, S., Karger, D. N., and Hauck, M.: Climate effects on vegetation vitality at the treeline
600
of boreal forests of Mongolia, Biogeosciences, 15, 1319–1333, https://doi.org/10.5194/bg-15-1319-2018, 2018. Kopp, B. J., Minderlein, S., and Menzel, L.: Soil Moisture Dynamics in a Mountainous Headwater Area in the
Discontinuous Permafrost Zone of northern Mongolia, Arctic, Antarctic, and Alpine Research, 46, 459–470,
https://doi.org/10.1657/1938-4246-46.2.459, 2014. Discontinuous Permafrost Zone of northern Mongolia, Arctic, Antarctic, and Alpine Research, 46, 459–470,
https://doi.org/10.1657/1938-4246-46.2.459, 2014. Krasnoshchekov, Y. N.: Soils and the Soil Cover of Mountainous Tundra and Forest Landscapes in the Central Khangai of
605
Mongolia, Eurasian Soil Sc., 43, 117–126, https://doi.org/10.1134/S1064229310020018, 2010. Krasnoshchekov, Y. References I hik
M Sh kh
N Zh
Y K d t
T
d Oh t
T G
d Th
l
d M i t
C
diti
t th S
th https://doi.org/10.1016/S0367-2530(17)30213-X, 1987. Hwang, J. and Son, Y.: Short-term effects of thinning and liming on forest soils of pitch pine and Japanese larch plantations
in central Korea, Ecological Research, 21, 671–680, https://doi.org/10.1007/s11284-006-0170-1, 2006. Hwang, J. and Son, Y.: Short-term effects of thinning and liming on forest soils of pitch pine and Japanese larch plantations
in central Korea, Ecological Research, 21, 671–680, https://doi.org/10.1007/s11284-006-0170-1, 2006. Ishikawa, M., Sharkhuu, N., Zhang, Y., Kadota, T., and Ohata, T.: Ground Thermal and Moisture Conditions at the Southern Ishikawa, M., Sharkhuu, N., Zhang, Y., Kadota, T., and Ohata, T.: Ground Thermal and Moisture Conditions at the Southern
Boundary of Discontinuous Permafrost, Mongolia, Permafrost Periglac. Process., 16, 209–216,
570
https://doi org/10 1002/ppp 483 2005 Boundary of Discontinuous Permafrost, Mongolia, Permafrost Periglac. Process., 16, 209–216,
570
https://doi.org/10.1002/ppp.483, 2005. IUSS Working Group WRB: World reference base for soil resources 2014: International soil classification system for
naming soils and creating legends for soil maps, World soil resources reports, 106, FAO, Rome, 181 pp., 2015. IUSS Working Group WRB: World reference base for soil resources 2014: International soil classification system for
naming soils and creating legends for soil maps, World soil resources reports, 106, FAO, Rome, 181 pp., 2015. Kapper, O. G.: Conifer Species (in Russian): Forestry Characteristics, Moskow, Leningrad, 1954. Kapper, O. G.: Conifer Species (in Russian): Forestry Characteristics, Moskow, Leningrad, 1954. Kassambara, A.: ggpubr: 'ggplot2' Based Publication Ready Plots, 2019. 575 Kassambara, A.: ggpubr: 'ggplot2' Based Publication Ready Plots, 2019. 575
Kassambara, A. and Mundt, F.: factoextra: Extract and Visualize the Results of, 2019. Kayama, M., Makoto, K., Nomura, M., Satoh, F., and Koike, T.: Nutrient dynamics and carbon partitioning in larch
seedlings (Larix kaempferi) regenerated on serpentine soil in northern Japan, Landscape Ecol Eng, 5, 125–135,
https://doi.org/10.1007/s11355-009-0069-4, 2009. Kassambara, A.: ggpubr: 'ggplot2' Based Publication Ready Plots, 2019. 575
Kassambara, A. and Mundt, F.: factoextra: Extract and Visualize the Results of, 2019. Kassambara, A. and Mundt, F.: factoextra: Extract and Visualize the Results of, 2019. Kayama, M., Makoto, K., Nomura, M., Satoh, F., and Koike, T.: Nutrient dynamics and carbon partitioning in larch
seedlings (Larix kaempferi) regenerated on serpentine soil in northern Japan, Landscape Ecol Eng, 5, 125–135,
https://doi.org/10.1007/s11355-009-0069-4, 2009. 30 https://doi.org/10.5194/soil-2020-52
Preprint. Discussion started: 5 October 2020
c⃝Author(s) 2020. References CC BY 4.0 License. https://doi.org/10.5194/soil-2020-52
Preprint. Discussion started: 5 October 2020
c⃝Author(s) 2020. CC BY 4.0 License. Khansaritoreh, E., Dulamsuren, C., Klinge, M., Ariunbaatar, T., Bat-Enerel, B., Batsaikhan, G., Ganbaatar, K., Saindovdon,
580
D., Yeruult, Y., Tsogtbaatar, J., Tuya, D., Leuschner, C., and Hauck, M.: Higher climate warming sensitivity of Siberian
larch in small than large forest islands in the fragmented Mongolian forest steppe, Global Change Biology, 23, 3675–
3689, https://doi.org/10.1111/gcb.13750, 2017a. Khansaritoreh, E., Dulamsuren, C., Klinge, M., Ariunbaatar, T., Bat-Enerel, B., Batsaikhan, G., Ganbaatar, K., Saindovdon,
580
D., Yeruult, Y., Tsogtbaatar, J., Tuya, D., Leuschner, C., and Hauck, M.: Higher climate warming sensitivity of Siberian
larch in small than large forest islands in the fragmented Mongolian forest steppe, Global Change Biology, 23, 3675–
3689, https://doi.org/10.1111/gcb.13750, 2017a. Khansaritoreh, E., Eldarov, M., Ganbaatar, K., Saindovdon, D., Leuschner, C., Hauck, M., and Dulamsuren, C.: Age
structure and trends in annual stem increment of Larix sibirica in two neighboring Mongolian forest–steppe regions
585
differing in land use history, Trees, 31, 1973–1986, https://doi.org/10.1007/s00468-017-1601-z, 2017b. 5 Khansaritoreh, E., Dulamsuren, C., Klinge, M., Ariunbaatar, T., Bat-Enerel, B., Batsaikhan, G., Ganbaatar, K., Saindovdon,
580
D., Yeruult, Y., Tsogtbaatar, J., Tuya, D., Leuschner, C., and Hauck, M.: Higher climate warming sensitivity of Siberian
larch in small than large forest islands in the fragmented Mongolian forest steppe, Global Change Biology, 23, 3675–
3689, https://doi.org/10.1111/gcb.13750, 2017a. Khansaritoreh, E., Eldarov, M., Ganbaatar, K., Saindovdon, D., Leuschner, C., Hauck, M., and Dulamsuren, C.: Age
structure and trends in annual stem increment of Larix sibirica in two neighboring Mongolian forest–steppe regions
585
differing in land use history, Trees, 31, 1973–1986, https://doi.org/10.1007/s00468-017-1601-z, 2017b. Kharuk, V. I., Ranson, K. J., and Dvinskaya, M. L.: Wildfires dynamic in the larch dominance zone, Geophys. Res. Lett., 35,
1–6, https://doi.org/10.1029/2007GL032291, 2008. Khishigjargal, M., Dulamsuren, C., Lkhagvadorj, D., Leuschner, C., and Hauck, M.: Contrasting responses of seedling and
sapling densities to livestock density in the Mongolian forest-steppe, Plant Ecol, 214, 1391–1403,
590
https://doi.org/10.1007/s11258-013-0259-x, 2013. Klinge, M., Schneider, F., Dulamsuren, C., Arndt, K., Bayarsaikhan, U., and Sauer, D.: Interrelations between vegetation,
natural and anthropogenic disturbances, and discontinuous permafrost in the forest-steppe of central Mongolia, Earth
Surface Processes and Landforms, submitted. differing in land use history, Trees, 31, 1973–1986, https://doi.org/10.1007/s00468-017-1601-z, 2017b. Kharuk, V. I., Ranson, K. J., and Dvinskaya, M. L.: Wildfires dynamic in the larch dominance zone, Geophys. Res. Lett., 35,
1–6, https://doi.org/10.1029/2007GL032291, 2008. References and Haselein, F.: Quaternary paleoenvironmental change on the Tibetan Plateau and adjacent areas (Western
China and Western Mongolia), Quaternary International, 65-66, 121–145, https://doi.org/10.1016/S1040-
6182(99)00040 3 2000 6182(99)00040-3, 2000. 620
Lehmkuhl, F., Hilgers, A., Fries, S., Hülle, D., Schlütz, F., Shumilovskikh, L., Felauer, T., and Protze, J.: Holocene
geomorphological processes and soil development as indicator for environmental change around Karakorum, Upper
Orkhon Valley (Central Mongolia), CATENA, 87, 31–44, https://doi.org/10.1016/j.catena.2011.05.005, 2011. Leyton, L.: The relationship between the growth and mineral composition of the foliage of Japanese larch (Larix leptolepis, Murr.), Plant Soil, 7, 167–177, https://doi.org/10.1007/BF01343725, 1956. 625
Liang, M., Sugimoto, A., Tei, S., Bragin, I. V., Takano, S., Morozumi, T., Shingubara, R., Maximov, T. C., Kiyashko, S. I.,
Velivetskaya, T. A., and Ignatiev, A. V.: Importance of soil moisture and N availability to larch growth and distribution
in the Arctic taiga-tundra boundary ecosystem, northeastern Siberia, Polar Science, 8, 327–341,
https://doi.org/10.1016/j.polar.2014.07.008, 2014. Lkhagvadorj, D., Hauck, M., Dulamsuren, C., and Tsogtbaatar, J.: Twenty Years After Decollectivization: Mobile Livestock
630
Husbandry and Its Ecological Impact in the Mongolian Forest-Steppe, Hum Ecol, 41, 725–735,
https://doi.org/10.1007/s10745-013-9599-3, 2013. Lüer, B. and Böhmer, A.: Comparison between percolation and extraction with 1 M NH4Cl solution to determine the
effective cation exchange capacity (CECeff) of soils, Journal of Plant Nutrition and Soil Science, 163, 555–557, 2000. Lkhagvadorj, D., Hauck, M., Dulamsuren, C., and Tsogtbaatar, J.: Twenty Years After Decollectivization: Mobile Livestock
630
Husbandry and Its Ecological Impact in the Mongolian Forest-Steppe, Hum Ecol, 41, 725–735,
https://doi.org/10.1007/s10745-013-9599-3, 2013. Lüer, B. and Böhmer, A.: Comparison between percolation and extraction with 1 M NH4Cl solution to determine the
effective cation exchange capacity (CECeff) of soils, Journal of Plant Nutrition and Soil Science, 163, 555–557, 2000. Mataix-Solera, J. and Doerr, S.H.: Hydrophobicity and aggregate stability in calcareous topsoils from fire-affected pine
635
forests in southeastern Spain, Geoderma, 118, 77–88, https://doi.org/10.1016/S0016-7061(03)00185-X, 2004. Maximovich S V : Geography and Ecology of Cryogenic Soils of Mongolia in: Cryosols: Permafrost-Affected Soils edited Mataix-Solera, J. and Doerr, S.H.: Hydrophobicity and aggregate stability in calcareous topsoils from fire-affected pine
635
forests in southeastern Spain, Geoderma, 118, 77–88, https://doi.org/10.1016/S0016-7061(03)00185-X, 2004. Maximovich, S. V.: Geography and Ecology of Cryogenic Soils of Mongolia, in: Cryosols: Permafrost-Affected Soils, edited
by: Kimble, J. M., Springer, Berlin, Heidelberg, 253–274, https://doi.org/10.1007/978-3-662-06429-0_13, 2004. Müller-Hohenstein, K.: Die Landschaftsgürtel der Erde (in German), 2., durchges. Aufl., Teubner-Studienbücher der Geographie, Teubner, Stuttgart, 204 pp., 1981. 640
Nandintsetseg, B. References N.: Soils and the Soil Cover of Mountainous Tundra and Forest Landscapes in the Central Khangai of
605
Mongolia, Eurasian Soil Sc., 43, 117–126, https://doi.org/10.1134/S1064229310020018, 2010. Krasnoshchekov, Y. N.: Soil cover of mountain forests in the East Khubsugul region of Mongolia, Eurasian Soil Sc., 41,
694 703 https://doi org/10 1134/S106422930807003X 2008 Krasnoshchekov, Y. N.: Soils and the Soil Cover of Mountainous Tundra and Forest Landscapes in the Central Khangai of
605
Mongolia, Eurasian Soil Sc., 43, 117–126, https://doi.org/10.1134/S1064229310020018, 2010. Krasnoshchekov, Y. N.: Soil cover of mountain forests in the East Khubsugul region of Mongolia, Eurasian Soil Sc., 41,
694–703, https://doi.org/10.1134/S106422930807003X, 2008. Lange, J., Kopp, B. J., Bents, M., and Menzel, L.: Tracing variability of run-off generation in mountainous permafrost of Krasnoshchekov, Y. N.: Soil cover of mountain forests in the East Khubsugul region of Mongolia, Eurasian Soil Sc., 41,
694–703, https://doi.org/10.1134/S106422930807003X, 2008. Lange, J., Kopp, B. J., Bents, M., and Menzel, L.: Tracing variability of run-off generation in mountainous permafrost of
semi-arid north-eastern Mongolia, Hydrol. Process., 29, 1046–1055, https://doi.org/10.1002/hyp.10218, 2015. 610 610 semi-arid north-eastern Mongolia, Hydrol. Process., 29, 1046–1055, https://doi.org/10.1002/hyp.10218, 2015. 610
Lê, S., Josse, J., and Husson, F.: FactoMineR: An R Package for Multivariate Analysis, Journal of Statistical Software, 25,
1–18, 2008. 31 https://doi.org/10.5194/soil-2020-52
Preprint. Discussion started: 5 October 2020
c⃝Author(s) 2020. CC BY 4.0 License. Lebedev, E. V.: Quantitative Measurements of Net Productivity of Photosynthesis, Mineral Nutrition and Biological
Productivity of Larix sibirica During Ontogenesis in the Areal of Southern Altai (in Russian), Herald of Altai state
agricultural university, 12, 61–66, 2012. 615 Lebedev, V. M. and Lebedev, E. V.: Impact of nitrogen forms on photosynthesis, mineral nutrition and biological
productivity of plants as Siberian larch and European spruce (in Russian), Forestry Bulletin, 6, 14–18, 2010. Lebedev, V. M. and Lebedev, E. V.: Impact of nitrogen forms on photosynthesis, mineral nutrition and biological
productivity of plants as Siberian larch and European spruce (in Russian), Forestry Bulletin, 6, 14–18, 2010. Lehmkuhl, F. and Haselein, F.: Quaternary paleoenvironmental change on the Tibetan Plateau and adjacent areas (Western productivity of plants as Siberian larch and European spruce (in Russian), Forestry Bulletin, 6, 14–18, 2010. Lehmkuhl, F. and Haselein, F.: Quaternary paleoenvironmental change on the Tibetan Plateau and adjacent areas (Western
China and Western Mongolia), Quaternary International, 65-66, 121–145, https://doi.org/10.1016/S1040- Lehmkuhl, F. References A., Woo, S. Y., and Zoyo, D.: Ecological Indicators of Forest Degradation after Forest
Fire and Clear-cutting in the Siberian Larch (Larix sibirica) Stand of Mongolia, Jour. Korean For. Soc., 98, 609–617,
2009. 650 Phu, T. D.: Nutrient requirements of planted European larch in Quebec, Plant Soil, 42, 109–117,
https://doi org/10 1007/BF02186977 1975 R Core Team: R: A language and environment for statistical computing, R Foundation for Statisti Sankey, T. T., Montagne, C., Graumlich, L., Lawrence, R., and Nielsen, J.: Lower forest–grassland ecotones and 20th
655
Century livestock herbivory effects in northern Mongolia, Forest Ecology and Management, 233, 36–44,
https://doi.org/10.1016/j.foreco.2006.05.070, 2006. Sankey, T. T., Montagne, C., Graumlich, L., Lawrence, R., and Nielsen, J.: Lower forest–grassland ecotones and 20th
655
Century livestock herbivory effects in northern Mongolia, Forest Ecology and Management, 233, 36–44,
https://doi.org/10.1016/j.foreco.2006.05.070, 2006. Schlütz, F., Dulamsuren, C., Wieckowska, M., Mühlenberg, M., and Hauck, M.: Late Holocene vegetation history suggests
natural origin of steppes in the northern Mongolian mountain taiga, Palaeogeography, Palaeoclimatology, Schlütz, F., Dulamsuren, C., Wieckowska, M., Mühlenberg, M., and Hauck, M.: Late Holocene vegetation history suggests
natural origin of steppes in the northern Mongolian mountain taiga, Palaeogeography, Palaeoclimatology, Palaeoecology, 261, 203–217, https://doi.org/10.1016/j.palaeo.2007.12.012, 2008. 660
Schulze, E.-D., Schulze, W., Koch, H., Arneth, A., Bauer, G., Kelliher, F. M., Hollinger, D. Y., Vygodskaya, N. N.,
Kusnetsova, W. A., Sogatchev, A., Ziegler, W., Kobak, K. I., and Issajev, A.: Aboveground biomass and nitrogen
nutrition in a chronosequence of pristine Dahurian Larix stands in eastern Siberia, Can. J. For. Res., 25, 943–960,
https://doi.org/10.1139/x95-103, 1995. Sharkhuu, N. and Sharkhuu, A.: Effects of Climate Warming and Vegetation Cover on Permafrost of Mongolia, in: Eurasian
665
Steppes. Ecological Problems and Livelihoods in a Changing World, edited by: Werger, M. J.A. and van Staalduinen,
M. A., Springer Netherlands, Dordrecht, 445–472, https://doi.org/10.1007/978-94-007-3886-7_17, 2012. Sommer, M.: Die Lärchenwälder der Gebirgs-Waldsteppe im Nordwesten der Mongolei (in German): Ökologische
Bestandsaufnahmen und Synthese, Dissertation, 2000. Sommer, M.: Die Lärchenwälder der Gebirgs-Waldsteppe im Nordwesten der Mongolei (in German): Ökologische
Bestandsaufnahmen und Synthese, Dissertation, 2000. Stüber, V.: Untersuchungen zum standortbezogenen Jugendwachstum der europäischen Lärche (Larix europaea, Dc; Larix
670
decidua, L.) (in German), Zugl.: Göttingen, Univ., Diss., 1998, Cuvillier, Göttingen, 147 pp., 1998. References and Shinoda, M.: Seasonal change of soil moisture in Mongolia: its climatology and modelling, Int. J. Climatol., 31, 1143–1152, https://doi.org/10.1002/joc.2134, 2011. National Statistical Office of Mongolia: Mongolian Statistical Information Service, http://1212.mn/, last access: 6 November
2019. National Statistical Office of Mongolia: Mongolian Statistical Information Service, http://1212.mn/, last access: 6 November
2019. National Statistical Office of Mongolia: Mongolian Statistical Information Service, http://1212.mn/, last access: 6 November
2019. Opp, C. and Hilbig, W.: Regular distribution patterns of soils and plant communities in northern Central Asia with special
645
consideration of the Uvs-Nuur-Basin (in German), Petermanns Geographische Mitteilungen, 147, 16–23, 2003. Opp, C. and Hilbig, W.: Regular distribution patterns of soils and plant communities in northern Central Asia with special
645
consideration of the Uvs-Nuur-Basin (in German), Petermanns Geographische Mitteilungen, 147, 16–23, 2003. 32 https://doi.org/10.5194/soil-2020-52
Preprint. Discussion started: 5 October 2020
c⃝Author(s) 2020. CC BY 4.0 License. Pâques, L. E.: Relationship between foliar nutrient concentrations and growth of hybrid larch (Larix × eurolepsis Henry),
Forest Ecology and Management, 63, 153–167, https://doi.org/10.1016/0378-1127(94)90108-2, 1994. Pâques, L. E.: Relationship between foliar nutrient concentrations and growth of hybrid larch (Larix × eurolepsis Henry),
Forest Ecology and Management, 63, 153–167, https://doi.org/10.1016/0378-1127(94)90108-2, 1994. Park, Y. D., Lee, D. K., Stanturf, J. A., Woo, S. Y., and Zoyo, D.: Ecological Indicators of Forest Degradation after Forest
Fire and Clear-cutting in the Siberian Larch (Larix sibirica) Stand of Mongolia, Jour. Korean For. Soc., 98, 609–617,
650
2009. Phu, T. D.: Nutrient requirements of planted European larch in Quebec, Plant Soil, 42, 109–117,
https://doi.org/10.1007/BF02186977, 1975. R Core Team: R: A language and environment for statistical computing, R Foundation for Statistical, Vienna, 2014. Pâques, L. E.: Relationship between foliar nutrient concentrations and growth of hybrid larch (Larix × eurolepsis Henry),
Forest Ecology and Management, 63, 153–167, https://doi.org/10.1016/0378-1127(94)90108-2, 1994. Park, Y. D., Lee, D. K., Stanturf, J. A., Woo, S. Y., and Zoyo, D.: Ecological Indicators of Forest Degradation after Forest
Fire and Clear-cutting in the Siberian Larch (Larix sibirica) Stand of Mongolia, Jour. Korean For. Soc., 98, 609–617,
650
2009. Phu, T. D.: Nutrient requirements of planted European larch in Quebec, Plant Soil, 42, 109–117,
https://doi.org/10.1007/BF02186977, 1975. R Core Team: R: A language and environment for statistical computing, R Foundation for Statistical, Vienna, 2014. Park, Y. D., Lee, D. K., Stanturf, J. A., Woo, S. Y., and Zoyo, D.: Ecological Indicators of Forest Park, Y. D., Lee, D. K., Stanturf, J. References Sugimoto A Yanagisawa N Naito D and Fujita Noboru Maximov Trofim C : Importance of permafrost as a source of Stüber, V.: Untersuchungen zum standortbezogenen Jugendwachstum der europäischen Lärche (Larix europaea, Dc; Larix
670
decidua, L.) (in German), Zugl.: Göttingen, Univ., Diss., 1998, Cuvillier, Göttingen, 147 pp., 1998. Sugimoto, A., Yanagisawa, N., Naito, D., and Fujita, Noboru, Maximov, Trofim C.: Importance of permafrost as a source of
water for plants in east Siberian taiga, Ecological Research, 17, 493–593, 2002. Sympilova, D. P. and Gyninova, A. B.: Soils of the subtaiga landscapes on the northern spurs of the Tsagan-Daban Ridge in Stüber, V.: Untersuchungen zum standortbezogenen Jugendwachstum der europäischen Lärche (Larix europaea, Dc; Larix
70
decidua, L.) (in German), Zugl.: Göttingen, Univ., Diss., 1998, Cuvillier, Göttingen, 147 pp., 1998. g
g
g
pp
Sugimoto, A., Yanagisawa, N., Naito, D., and Fujita, Noboru, Maximov, Trofim C.: Importance of permafrost as a source of
water for plants in east Siberian taiga, Ecological Research, 17, 493–593, 2002. Sympilova, D. P. and Gyninova, A. B.: Soils of the subtaiga landscapes on the northern spurs the Selenga Mountains, Eurasian Soil Sc., 45, 231–236, https://doi.org/10.1134/S1064229312030118, 2012. 675
Timoshok, E. E., Timoshok, E. N., and Skorokhodov, S. N.: Ecology of Siberian Stone Pine (Pinus sibirica Du Tour) and
Siberian Larch (Larix sibirica Ledeb.) in the Altai Mountain Glacial Basins, Russ J Ecol, 45, 194–200,
https://doi.org/10.1134/S1067413614030138, 2014. 33 https://doi.org/10.5194/soil-2020-52
Preprint. Discussion started: 5 October 2020
c⃝Author(s) 2020. CC BY 4.0 License. Tsogtbaatar, J.: Deforestation and Reforestation of Degraded Forestland in Mongolia, in: The Mongolian Ecosystem Tsogtbaatar, J.: Deforestation and Reforestation of Degraded Forestland in Mongolia, in: The Mongolian Ecosystem
Network: Environmental Issues Under Climate and Social Changes, edited by: Yamamura, N., Fujita, N., and Maekawa
680
A., Springer Japan; Imprint; Springer, Tokyo, 83–98, 2013. USDA: Soil Survey Field and Laboratory Methods Manual: oil Survey Investigations Report No. 51, Version 2.0. R., 2014. Viers, J., Prokushkin, A. S., Pokrovsky, O. S., Auda, Y., Kirdyanov, A. V., Beaulieu, E., Zouiten, C., Oliva, P., and Dupré,
B.: Seasonal and spatial variability of elemental concentrations in boreal forest larch foliage of Central Siberia on
continuous permafrost, Biogeochemistry, 113, 435–449, https://doi.org/10.1007/s10533-012-9770-8, 2013. 685
Wang, H.-M., Wang, W.-J., Chen, H., Zhang, Z., Mao, Z., and Zu, Y.-G.: Temporal changes of soil physic-chemical
properties at different soil depths during larch afforestation by multivariate analysis of covariance, Ecology and
evolution, 4, 1039–1048, https://doi.org/10.1002/ece3.947, 2014. References Watanabe, M., Ryu, K., Kita, K., Takagi, K., and Koike, T.: Effect of nitrogen load on growth and photosynthesis of
seedlings of the hybrid larch F1 (Larix gmelinii var. japonica×L. kaempferi) grown on serpentine soil, Environmental
690
and Experimental Botany, 83, 73–81, https://doi.org/10.1016/j.envexpbot.2012.04.011, 2012. Wickham, H.: ggplot2: Elegant graphics for data analysis, Use R!, Springer, Cham, 260 pp., 2016. Zhang, N., Yasunari, T., and Ohta, T.: Dynamics of the larch taiga–permafrost coupled system in Siberia under climate
change, Environ. Res. Lett., 6, 24003, https://doi.org/10.1088/1748-9326/6/2/024003, 2011. 680 Network: Environmental Issues Under Climate and Social Changes, edited by: Yamamura, N., Fujita, N., and Maekawa,
A., Springer Japan; Imprint; Springer, Tokyo, 83–98, 2013. Viers, J., Prokushkin, A. S., Pokrovsky, O. S., Auda, Y., Kirdyanov, A. V., Beaulieu, E., Zouiten, C., Oliva, P., and Dupré,
B.: Seasonal and spatial variability of elemental concentrations in boreal forest larch foliage of Central Siberia on
continuous permafrost, Biogeochemistry, 113, 435–449, https://doi.org/10.1007/s10533-012-9770-8, 2013. 685 Wang, H.-M., Wang, W.-J., Chen, H., Zhang, Z., Mao, Z., and Zu, Y.-G.: Temporal changes of soil physic-chemical
properties at different soil depths during larch afforestation by multivariate analysis of covariance, Ecology and
evolution, 4, 1039–1048, https://doi.org/10.1002/ece3.947, 2014. Watanabe, M., Ryu, K., Kita, K., Takagi, K., and Koike, T.: Effect of nitrogen load on growth and photosynthesis of
seedlings of the hybrid larch F1 (Larix gmelinii var. japonica×L. kaempferi) grown on serpentine soil, Environmental
and Experimental Botany, 83, 73–81, https://doi.org/10.1016/j.envexpbot.2012.04.011, 2012. 690 Wickham, H.: ggplot2: Elegant graphics for data analysis, Use R!, Springer, Cham, 260 pp., 2016 Zhang, N., Yasunari, T., and Ohta, T.: Dynamics of the larch taiga–permafrost coupled system in Siberia under climate
change, Environ. Res. Lett., 6, 24003, https://doi.org/10.1088/1748-9326/6/2/024003, 2011. 695 34 34
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English
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Tumor size, stage and grade alterations of urinary peptidome in RCC
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Journal of translational medicine
| 2,015
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cc-by
| 9,088
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© 2015 Chinello et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/
publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Among them, several play a possible role in tumorigenesis, progression and aggressiveness. These results could
be a useful starting point for future studies aimed at verifying their possible use in the managements of RCC patients. Keywords: Mass spectrometry, Proteomics, Urine, Renal cell carcinoma, Tumour size, Tumour progression, Stage,
Grade, Cancer, Peptidomics Conclusions: We identified several peptides whose urinary abundance varied according to tumour size, stage and
grade. Among them, several play a possible role in tumorigenesis, progression and aggressiveness. These results could
be a useful starting point for future studies aimed at verifying their possible use in the managements of RCC patients. Keywords: Mass spectrometry, Proteomics, Urine, Renal cell carcinoma, Tumour size, Tumour progression, Stage,
G d
C
P
tid
i Conclusions: We identified several peptides whose urinary abundance varied according to tumour size, stage and
grade. Among them, several play a possible role in tumorigenesis, progression and aggressiveness. These results could
be a useful starting point for future studies aimed at verifying their possible use in the managements of RCC patients. be a useful starting point for future studies aimed at verifying their possible use in the managements of RCC patients. Keywords: Mass spectrometry, Proteomics, Urine, Renal cell carcinoma, Tumour size, Tumour progression, Stage,
Grade, Cancer, Peptidomics Chinello et al. J Transl Med (2015) 13:332
DOI 10.1186/s12967-015-0693-8 Open Access Tumor size, stage and grade alterations
of urinary peptidome in RCC Clizia Chinello1*†, Marta Cazzaniga1†, Gabriele De Sio1, Andrew James Smith1, Angelica Grasso2,
Bernardo Rocco2, Stefano Signorini3, Marco Grasso4, Silvano Bosari5, Italo Zoppis6, Giancarlo Mauri6
and Fulvio Magni1 *Correspondence: clizia.chinello@unimib.it
†Clizia Chinello and Marta Cazzaniga contributed equally
1 Department of Health Science, School of Medicine, University of Milano-
Bicocca (UNIMIB), Via Cadore, 48, 20900 Monza, Italy
Full list of author information is available at the end of the article Abstract Background: Several promising biomarkers have been found for RCC, but none of them has been used in clinical
practice for predicting tumour progression. The most widely used features for predicting tumour aggressiveness still
remain the cancer stage, size and grade. Therefore, the aim of our study is to investigate the urinary peptidome to
search and identify peptides whose concentrations in urine are linked to tumour growth measure and clinical data. Methods: A proteomic approach applied to ccRCC urinary peptidome (n = 117) based on prefractionation with
activated magnetic beads followed by MALDI-TOF profiling was used. A systematic correlation study was performed
on urinary peptide profiles obtained from MS analysis. Peptide identity was obtained by LC–ESI–MS/MS. Background: Several promising biomarkers have been found for RCC, but none of them has been used in clinical
practice for predicting tumour progression. The most widely used features for predicting tumour aggressiveness still
remain the cancer stage, size and grade. Therefore, the aim of our study is to investigate the urinary peptidome to
search and identify peptides whose concentrations in urine are linked to tumour growth measure and clinical data. Methods: A proteomic approach applied to ccRCC urinary peptidome (n = 117) based on prefractionation with Methods: A proteomic approach applied to ccRCC urinary peptidome (n = 117) based on prefractionation with
activated magnetic beads followed by MALDI-TOF profiling was used. A systematic correlation study was performed
on urinary peptide profiles obtained from MS analysis. Peptide identity was obtained by LC–ESI–MS/MS. Results: Fifteen, twenty-six and five peptides showed a statistically significant alteration of their urinary concen-
tration according to tumour size, pT and grade, respectively. Furthermore, 15 and 9 signals were observed to have
urinary levels statistically modified in patients at different pT or grade values, even at very early stages. Among them,
C1RL, A1AGx, ZAG2G, PGBM, MMP23, GP162, ADA19, G3P, RSPH3, DREB, NOTC2 SAFB2 and CC168 were identified. Results: Fifteen, twenty-six and five peptides showed a statistically significant alteration of their urinary concen-
tration according to tumour size, pT and grade, respectively. Furthermore, 15 and 9 signals were observed to have
urinary levels statistically modified in patients at different pT or grade values, even at very early stages. Among them,
C1RL, A1AGx, ZAG2G, PGBM, MMP23, GP162, ADA19, G3P, RSPH3, DREB, NOTC2 SAFB2 and CC168 were identified. Conclusions: We identified several peptides whose urinary abundance varied according to tumour size, stage and
grade. Background cancer will have a recurrence [2]. Although partial or
total surgical resection are the gold standard treatments
for
patients
with
localized
RCC,
approximately
20–40 % of them will have progression during one-year
follow-up period. Neither chemotherapy nor radiation
therapy is effective for the patients with metastases [1]. Renal cell cancer (RCC) accounts for about 4 % of all
adult cancers and is the most dangerous of all urological
malignancies with a median survival time of about
13 months, with less than 10 % of patients surviving
more than 5 years. There are about 210,000 new cases
and 100,000 deaths diagnosed worldwide every year, and
the number of patients is increasing rapidly [1]. About
30 % of patients already have metastasis at the time of
diagnosis and 30–40 % of patients with localized kidney f
Over the last decade, most of kidney tumours are
revealed incidentally during unrelated clinical investigation,
owing to the widespread use of abdominal imaging,
including ultrasonography and computed tomography. Moreover, laboratory examinations accompanied by urine
analysis in search for haematuria are prompted for every
suspicion of RCC [3]; however, there are still no other
cancer type- or stage specific biomarkers appropriate for
clinical usage in urine analysis [4]. On the other hand, the *Correspondence: clizia.chinello@unimib.it
†Clizia Chinello and Marta Cazzaniga contributed equally
1 Department of Health Science, School of Medicine, University of Milano-
Bicocca (UNIMIB), Via Cadore, 48, 20900 Monza, Italy
Full list of author information is available at the end of the article Page 2 of 11 Chinello et al. J Transl Med (2015) 13:332 tissues from kidneys belonging to different stages
and grades as well as the non-neoplastic specimen
counterparty [10]. In particular, they have studied the
proteome of kidney tissue of 27 patients (pT1, pT2, pT3)
and 9 adjacent normal tissue by 2D-DIGE, identifying
several proteins correlating with pT. Lebdaj et al. found a significant association between expression of
transforming growth factor beta-induced (TGFBI) in
tissue with tumour category pT3-pT4, Fuhrman grades
III and IV and tumour mass size >4 cm [16]. Moreover,
Morrisey et al. have observed a correlation between
the neoplasm size and the pre-nephrectomy urinary
levels of AQP1 and PLIN2. A correlation was found
also for both markers with tumour stage but not with
grade [17]. The identification of biomarkers predicting
treatment response could avoid unnecessary costs,
prevent side effects, and help in choosing optimal patient
management. Background Considering that cancer progression is reflected by
tumour stages, Junker et al. have examined neoplastic Chemicals and standardsi Profiling Kit 1000 C8-MB, α-cyano-4-hydroxycinnamic
acid (CHCA), Protein Calibration Standard I (Prot-
Mix I) and Peptide Calibration Standard II (PepMix II)
were provided by Bruker Daltonics GmbH (Bremen,
Germany). Background In fact, peptidomics analysis has
detected the presence of clusters of urinary peptides able
not only to differentiate RCC patients from controls [11]
but even benign from malignant kidney solid masses [12]. Following surgery, TNM (Tumour-Node-Metastasis)
staging and Fuhrman grade as well as performance
status and serum markers (haemoglobin, calcium, lactate
dehydrogenase, platelets, neutrophils and C-reactive
protein) are the most used factors applied so far for
predicting tumour progression and recurrence [13]. Many authors have therefore investigated the role of
prognostic biomarkers attempting to improve patient’s
risk stratification, and better predict disease recurrence
and associated mortality [13, 14]. Quite recently, a
predictive model based on the following independent
prognostic factors has been published by Tosco et al. [15]: primary tumour T stage ≥3, primary tumour
Fuhrman grade ≥3, nonpulmonary metastases, disease-
free interval ≤12 mo and multiorgan metastases. Considering that cancer progression is reflected by
tumour stages, Junker et al. have examined neoplastic Rapid progress has been made to understand
tumorigenesis, but the key events leading to the
dysfunction of the control of cellular proliferation still
remain unclear. Since alterations of these regulatory
processes can be caused not only by altered gene
expression but also by proteins, proteomics investigations
are of special importance to detect patterns of disease-
associated
protein
alterations. Recently,
we
and
other researchers used these approaches to identify
differentially expressed proteins by comparing non-
neoplastic and neoplastic kidney tissue as possible
biomarkers [8–10]. Moreover, biomarker discovery for
RCC is not only feasible in the tissue proteome but also
in urinary peptidome. In fact, peptidomics analysis has
detected the presence of clusters of urinary peptides able
not only to differentiate RCC patients from controls [11]
but even benign from malignant kidney solid masses [12]. g
g
y
[
]
Following surgery, TNM (Tumour-Node-Metastasis)
staging and Fuhrman grade as well as performance
status and serum markers (haemoglobin, calcium, lactate
dehydrogenase, platelets, neutrophils and C-reactive
protein) are the most used factors applied so far for
predicting tumour progression and recurrence [13]. Many authors have therefore investigated the role of
prognostic biomarkers attempting to improve patient’s
risk stratification, and better predict disease recurrence
and associated mortality [13, 14]. Quite recently, a
predictive model based on the following independent
prognostic factors has been published by Tosco et al. [15]: primary tumour T stage ≥3, primary tumour
Fuhrman grade ≥3, nonpulmonary metastases, disease-
free interval ≤12 mo and multiorgan metastases. Background Recently, progress in the chemotherapeutic
treatment of renal cell carcinomas has been achieved
with the development of targeted molecular therapies
[16]. Currently, several promising molecular biomarkers
have been found, but none of them have been introduced
in clinical practice. Consequently, the most powerful tool
for predicting tumour aggressiveness still remains stage,
size and grade [16]. However, as it stands, no systematic
investigation has been carried out to highlight if part of
the urinary peptidome not only suggests the presence of
a tumour mass in kidney [11, 12] but may also reflect its
progression. Therefore, in this study we investigated the
urinary peptidome to search for and identify peptides
with a urinary expression alteration according to pT,
tumour dimension and grade to be employed as a
possible starting point for future more specific studies
aimed at ccRCC management. increase of RCC incidence is also associated with the rise
in incidental detection of localized small renal masses. About 20 % of small masses are benign and they are likely
to have pathological characteristics of low Fuhrman grade
and clear cell type. In addition, small renal masses are
increasingly detected in elderly patients who probably
have comorbidities and are a high-risk group for active
treatment like surgery [5]. Consequently, indications
for watchful-waiting in small renal kidney cancers are
consistently expanding [6]. As the management possibilities
are enhancing, the current role of renal mass biopsy is also
increasing. Thus, despite the associated risks (bleeding,
tumor seeding, etc.), renal mass biopsy is recommended
for active surveillance in most cases except in watchful-
waiting candidates, in patients with imaging or clinical
characteristics indicative of pathology as syndromes
and in cases whereby conservative management is not
contemplated [7]. p
[ ]
Rapid progress has been made to understand
tumorigenesis, but the key events leading to the
dysfunction of the control of cellular proliferation still
remain unclear. Since alterations of these regulatory
processes can be caused not only by altered gene
expression but also by proteins, proteomics investigations
are of special importance to detect patterns of disease-
associated
protein
alterations. Recently,
we
and
other researchers used these approaches to identify
differentially expressed proteins by comparing non-
neoplastic and neoplastic kidney tissue as possible
biomarkers [8–10]. Moreover, biomarker discovery for
RCC is not only feasible in the tissue proteome but also
in urinary peptidome. Urine collection and handling procedure Urine samples were collected from patients the day
before surgery at “Ospedale Maggiore Policlinico”
Foundation (Milan, Italy), San Gerardo Hospital
(Monza, Italy) and those from healthy volunteers at
Desio Hospital (Desio, Italy). All subjects had signed an
informed consent prior to sample donation and analyses
were carried out in agreement with the Declaration
of Helsinki. Study protocols and procedures were Chinello et al. J Transl Med (2015) 13:332 Page 3 of 11 approved by the local ethic committee (U.O. Comitato
di Etica e Sperimentazione Farmaci Direzione Scientifica
Fondazione IRCCS Ca’Granda Ospedale Maggiore
Policlinico, Milano and Comitato Etico Azienda
Ospedaliera San Gerardo, Monza, Italy). Second morning
midstream urine was collected in sterile urine tubes
(Anicrin s.r.l., Italy) [18]. approved by the local ethic committee (U.O. Comitato
di Etica e Sperimentazione Farmaci Direzione Scientifica
Fondazione IRCCS Ca’Granda Ospedale Maggiore
Policlinico, Milano and Comitato Etico Azienda
Ospedaliera San Gerardo, Monza, Italy). Second morning
midstream urine was collected in sterile urine tubes
(Anicrin s.r.l., Italy) [18]. Peptide sequencing by nLC–ESI–MS/MS Peptide sequencing by nLC–ESI–MS/MS p
q
g y
/
Identity of the endogenous peptides present in RPC8
enriched fractions were assessed by nLC–ESI–MS/
MS analysis of the ccRCC patients urine pool (n = 80)
prepared as previously described [19]. Briefly, before
injection
MB
fractionated
samples
underwent
a
purification step from salts and beads performed using
Ziptip™ μ-C18 Pipette Tips (Millipore Corp, Bedford,
MA, USA) as already reported [19]. Desalted fractions
were chromatographically separated into a Dionex
UltiMate 3000 rapid separation (RS) LC nano system
(Thermo Scientific, Germany) and analysed with an
online-coupled Impact HD™ mass spectrometer (Bruker
Daltonics, Germany). Prefractionation of urine samples was performed as
already described [12]. Briefly, 40 µL of the urine sam-
ple was incubated for 1 min in a magnetic separator with
80 µL of binding buffer and 5 µL of RPC8 magnetic beads. The beads were then washed twice with 45 µL and once
with 30 µL of washing solution. Finally, the peptides were
released with 10 µL of elution solution (50 % Acetonitrile
in water). A small amount of the eluates was used for the
automated spotting onto an AnchorChip™ target (Bruker
Daltonics, Germany). Peptides were loaded onto a µ-precolumn (Dionex,
Acclaim PepMap 100 C18, cartridge, 300 µm i.d. Urine collection and handling procedure × 5 mm,
5 µm), followed by a multistep 360 min gradient at
a flow rate of 300 nl/min on the analytical 50 cm nano
column (Dionex, 0.075 mm ID, Acclaim PepMap100,
C18, 2 µm). A ramp from 4 to 35 % in 245 min of mobile
phase B (0.1 % FA/80 % CHCN) was used. The column
was connected to a nanoBooster CaptiveSpray™ (Bruker
Daltonics). The mass spectrometer was operated in
the data-dependent-acquisition mode to automatically
switch between full scan MS and MS/MS acquisition in
CID mode (N2 was used as collision gas). The number
of precursor ions was automatically adjusted to fit into a
fixed cycle time of 5 s, and IDAS (Intensity Dependent
Acquisition Speed) and RT2 (RealTime Re-Think) func-
tionalities were applied. The tune parameters were set as
already described [12] in order to better assist the frag-
mentation of larger endogenous peptides. In addition, two pools were obtained by mixing 40 µL
of urine of each subject and used for peptide identifica-
tion. The two pools were manually purified with mag-
netic beads following the same steps and proportion
described for the automatic procedure. MALDI‑TOF peptide profilinghi The analysis of purified urine samples was performed in
linear mode (MALDI-LM) and reflector mode (MALDI-
RM) using an UltrafleXtreme™ MALDI-TOF/TOF
instrument (Bruker Daltonics, Germany) [19]. Multiple spectra comparison was performed with
ClinProTools™ Software v. 2.2 (Bruker Daltonics, Ger-
many) after normalization. Statistical elaboration was
conducted on the mean spectrum generated from each
subject dataset. A S/N threshold of 3 and zero level inte-
gration type were used as parameters in order to obtain a
list of peaks with their area, calculated on the total aver-
age spectrum. XML peaklists were processed using an in-house
Mascot search engine (v2.4.1). Database searching was
restricted to human Swiss-Prot (accessed Apr 2015,
548,208 sequences; 195,282,524 residues). Mass toler-
ances were set at 20 ppm for MS and 0.05 Da for MS/
MS. No enzyme and any fixed modification was set in the
search parameters. Acetyl (N-term), Acetyl (K), Deami-
dation (NQ), Oxidation (HW), Oxidation (M) were set as
variable modifications in the Mascot search parameters. Mascot score thresholds and decoy database were used as
peptide level filters of peptide significance (FDR <5 %). Expression profile analysis and statistical analysis Statistical analysis was conducted as already described
[12]. Initially normality and homogeneity of variance
(Shapiro–Wilk’s, Barlett and Leven’s test for normality
and homogeneity testing) were evaluated, followed by
suitable parametric or non-parametric tests for groups
comparisons (Equal Variance t test for Normal Data
with Equal Variances, Unequal Variance (Welch) t-test
for Normal Data with Unequal Variances, Mann–
Whitney U-test (Wilcoxon) for Non-normal Data with
Equal Variances and Kolmogorov–Smirnov test for
Non-normal Data with Unequal Variances). Peptide
urinary expression variation according to clinical data
was computed using Spearman rank-order correlation
(Spearman’s rho). All tests were applied using 0.05 as the
significance level. Urinary peptides differentially expressed according
to histological data To investigate the possible alterations of the urinary
levels of peptides according to pT and Grade, statisti-
cal analysis was applied to the peptidomic profiles of the
RCC patients divided into four groups. Histological analysis was performed on patients based
upon the Fuhrman grading system, sarcomatoid and
cystic differentiation, tumour necrosis, microvascularity
and urinary infiltration. Dimension of the tumour mass
was in the range from 1.5 to 18 cm. RCC patients were
divided in four groups according to pT (group 1 = pT1a;
2 = pT1b; 3 = pT2a and 4 = pT ≥ 2b) or Grade values
(1, 2, 3 and 4). Urine samples collected from 137 healthy
subjects (81 men, 56 women) were used for statistical
analysis [12]. Fifteen peptides were observed to have urinary lev-
els statistically different in patients at different pT val-
ues (Additional file 2: Table S2B, Figs. 1, 2), and nine of
them were significantly also varied in patients compared
with control subjects (Additional file 2: Table S2B, second
column). Furthermore, considering only the compari-
son between patients with tumour stage pT1a and pT1b,
fourteen peptides showed a statistically significant altera-
tion (Additional file 4: Table S4). i
On the contrary, nine peptides (Additional file 3:
Table S3B, Figs. 1, 2) showed a statistically significant
difference between patients having a tumour at different
grades, with seven of them also varied in patients ver-
sus healthy subjects (Additional file 3: Table S3B, second
column). Urinary peptides altered according to Gradei Only five peptides showed a statistically significant alter-
ation between their urinary concentration and grade
(Additional file 3: Table S3A). Four peptides were sig-
nificantly differentially expressed between patients and
controls (Additional file 3: Table S3A, second column). Only one of them showed a positive variation while for
the other four it was negative with a value of about −0.2. Peptide urinary expression altered according to clinical
data To investigate the possible association between urinary
levels of peptides and tumour size, pT and grade, statisti-
cal analysis was applied to the peptidomic profiles of the
ccRCC patients. Peptides with a urinary expression cor-
relating with age were not considered [12]. Clinical data and study design Urine collected from 117 clear cell RCC patients (ccRCC)
(72 men, 45 women) were used in the present study. Fisher test did not reveal any gender dependence in the
studied cohort. The mean age for patients was 64.05
with a range of 33–87 years. Patients were classified
according to the 2009 TNM system classification and
their clinical characteristics are described in Table 1 [12]. Page 4 of 11 Chinello et al. J Transl Med (2015) 13:332 Table 1 Patients clinical characteristics according to the
2009 TNM (tumour-node-metastasis) system classification
No. of patients
ALL
117
Mean ± SD age at diagnosis
64.05 ± 11.16
Median age at diagnosis (range)
33–87
Tumor mass in cm (range)
1.5–18
Gender
Males
72
Females
45
Staging
pT1a
45
pT1b
39
pT2a
23
pT2b
1
pT3a
9
pT3b
0
Grade
1
6
2
79
3
20
4
3 Peptide urinary expression altered according to pT Peptide urinary expression altered according to pT
Twenty-six peptides displayed a statistically significant
variation between their urinary concentration and pT
(Additional file 2: Table S2A). Fifteen of these peptides
were differentially expressed in RCC compared with
controls (Additional file 2: Table S2A, second column). Notably, only five of them showed a negative alteration
(range from −0.202 to −0.326) while for the other
twenty-one the variation was positive (range from 0.203
to 0.27). Peptide identification Identity of the urinary peptides in the C8-magnetic beads
pre-purified fractions was obtained by nanoLC-ESI–MS/
MS. Several peptides could be identified and among
them, fifteen signals having a variation linked to clinical
data were recognized (Additional file 5: Table S5). Urinary peptides varied depending on tumour sizei The ion at m/z
1934.2 could be generated from two different peptides,
whose masses could not be unequivocally assigned
observing the isotope cluster obtained in MALDI-RM:
the fragment 1152ERLEEAGGATSAQIEMNK1169 deriving
from the MYH1_HUMAN/MYH4_HUMAN or/and
the
fragment
40LGAPAVPAWSAAQGDVAALGL60
originating from the MMP23 protein. Similarly, the ion
at m/z 1825.6 could be ascribed to amino acid sequence
59FRYNSKDRKSQPMGL73 of ZA2G_HUMAN and/or
to 4283VSEDPINDGEWHRVTA4298 peptide of PGBM_
HUMAN. For two of the thirteen signals (m/z 2192.1
and m/z 3151.1), the resolving power of MALDI-RM and
the low mass difference between different amino acid
sequences identified by ESI MS/MS have not allowed
the specific contributes to MALDI-LM peak to be
unambiguously distinguished. urinary levels of AQP1 and PLIN2 were observed to be
correlated with the tumour size and stage but not with
grade [17]. However, despite these promising studies, none
was successfully able to predict RCC aggressiveness, and,
up to now, tumour size and growth rate are still the most
used prognostic factors. In addition, currently there is
only limited literature addressing urine markers for RCC. This is noteworthy especially if we consider the benefits
related to the possibility of following localized tumours or
monitoring drug-based therapy results by simply analysing
tumour-specific markers in an easily accessible biofluid
such as kidney excretory product [20, 21]. In this study, we systematically investigated the urinary
peptidome of a large cohort of RCC patients in order to
highlight possible features varying according to tumor
characteristics: size, stage and grade. To preserve the
homogeneity of the samples, we included only clear cell
RCC. Statistical data elaboration was focused on data
derived from urine samples pre-fractionated using C8
magnetic beads followed by MALDI profiling analysis. Previously, the application of this high-throughput
approach allowed us to build clusters of urinary peptides
with high diagnostic performances [12].h Through this strategy, we have observed that the uri-
nary abundance of fifteen and twenty-six peptides var-
ies depending on size and stage, respectively (Additional
file 1: Table S1A, Additional file 2: Table S2A), and among
them eight were shared and showed a consistent correla-
tion trend. On the other hand, only few signals displayed
a significant alteration according to Fuhrman grading
system (Additional file 3: Table S3A). Urinary peptides varied depending on tumour sizei Fifteen peptides showed a statistically significant vari-
ation according to tumour size (Additional file 1:
Table S1A). Almost all of these peptides also have a
statistically different expression from control subjects
(Additional file 1: Table S1A, second column). Interest-
ingly, the alteration was negative for three of them (range
from −0.307 to −0.312) and positive for the other twelve
(range from 0.31 to 0.514). i
Six ions present in MALDI-LM profiles and not
in MALDI-RM spectra were matched with peptide
sequences identified in ESI–MS/MS (average Mass
Measurement errors for LM: 222 ± 411.1 ppm): the ion
at m/z 3571.1 was recognized as the fragment 362SQP Chinello et al. J Transl Med (2015) 13:332 Page 5 of 11 . 1 Box-plot of identified peptides (below 2500 m/z) that resulted statistically varied according to class (CTRLs vs ccRCC) pT or grade. The po
e significant correlation with tumor size, TNM stage and Fuhrman grade is reported Fig. 1 Box-plot of identified peptides (below 2500 m/z) that resulted statistically varied according to class (CTRLs vs ccRCC) pT or grade. The pos-
sible significant correlation with tumor size, TNM stage and Fuhrman grade is reported Chinello et al. J Transl Med (2015) 13:332 Page 6 of 11 g. 2 Box-plot of identified peptides (above 2500 m/z) that resulted statistically varied according to class (CTRLs vs ccRCC) pT or grade. The pos
le significant correlation with tumor size, TNM stage and Fuhrman grade is reported Fig. 2 Box-plot of identified peptides (above 2500 m/z) that resulted statistically varied according to class (CTRLs vs ccRCC) pT or grade. The pos-
sible significant correlation with tumor size, TNM stage and Fuhrman grade is reported Page 7 of 11 Chinello et al. J Transl Med (2015) 13:332 PPLPPPPPPAQETQEPSPILDSEETRAAAPQ395 of the
DREB protein; the ion at 3723.8 as the 51 STHGKFHGTV
KAENGKLVINGNPITIFQERDPSK84
fragment
origi-
nated from G3P_HUMAN; the ion at m/z 4355.1 as the
acetylated form of the 386EEKDIKPIIKDEKGRVGSGS-
GRNLWVSGLSSTTRATDLK424 peptide belonging to
the SAFB2 protein; the ion at m/z 4626.9 as the 868HEVK-
TIDMRFRIHCQEARISPMSHILNAKELVLNINKLE906
fragment from CC168 protein. The remaining nine of the
fifteen signals were also detected in MALDI-RM profiles,
strengthening the accuracy of identity assignment (aver-
age Mass Measurement errors for RM: 36.7 ± 81.3 ppm). In particular, the ion at m/z 1755.8 was identified as a
peptide deriving from A1AGx, which is common to both
A1AG1 and A1AG2 protein isoforms. Urinary peptides varied depending on tumour sizei Even if Furhman
grade is one of the most widely accepted histological
prognostic factors [22], it has to be considered that some
controversial aspects related to the low accuracy due to
the grade heterogeneity within the same tumor, and to
the interobserver and intraobserver variability in assign-
ing tumor grade are present [23]. Three signals were attributed to the same specific
amino acid sequences reported in our previous papers
[12, 18]. The ion at m/z 1912.1 was already identified as
an UROM fragment [18], the ion at m/z 2192.1 as two
possible peptides belonging to GP162_HUMAN and/
or KPB1_HUMAN [12], while the ion at m/z 2660.8 was
unambiguously identified as a fragment from FIBA, also
by MALDI MS/MS analysis [12]. Moreover, most of these peptides were higher or lower
represented (p < 0.05) in urine of RCC patients com-
pared to control subjects (Additional file 1: Table S1A,
Additional file 2: Table S2A, Additional file 3: Table S3A). Some of them have shown significantly varied urinary
concentration according to pT or grade (Additional file 2:
Table S2B and Additional file 3: Table S3B, Figs. 1, 2)
and also at early stages pT1a and pT1b (Additional file 4:
Table S4). Discussion Renal cell carcinoma, the most common type of human
kidney cancer, is increasing in incidence and it is the most
lethal genitourinary malignancy. Several studies aiming at
biomarker discovery in RCC have been reported in recent
years. Most of them deal with the search of markers for
early diagnosis, for prognosis and for the prediction of
patients’ response to therapy. Several proteins correlating
with the pT were observed when comparing tissue from
patients at different RCC progression stages [10]. A
significant association between TGFBI tissue expression
with this progressive neoplasm at different stages and with
its diameter was very recently reported [16]. Moreover, One of the major advantages of MALDI profiling
strategy is that signals do not need any prior
knowledge about their identity in order to allow their
use as biomarkers. Nevertheless, the identification
of endogenous peptides could increase the biological
insight, exploring the function and the regulation
of bioactive molecules and degradome products. Page 8 of 11 Chinello et al. J Transl Med (2015) 13:332 to controls, a positive variation and an increase of its
urinary fragment levels from pT1 to pT3 were observed. Furthermore, the lack of identity carries drawbacks
for their possible translation into clinical routine
laboratories. We could identify fifteen peptides whose
urinary expression was significantly varied with tumour
size, stage and grade (Additional file 5: Table S5, Figs. 1,
2). Two of the signals were recognized as fragments
belonging to highly abundant proteins in urine, FIBA
and UMOD. However, the other identified peptides
might be attributed to proteins playing a possible role in
tumorigenesis, progression and aggressiveness. Disintegrin and metalloproteinase 19 (ADAM19) is a
cell surface glycoprotein belonging to the ADAM fam-
ily. These proteins are known to be involved in cell
adhesion, fusion, and migration and they have a role in
cancer cell proliferation and progression [28]. ADAM19
was described as up-regulated in human brain tumours
and correlating with its invasiveness. Moreover, high
expression of ADAM19 was also associated with lung
and kidney inflammatory and fibrotic processes [29]. An overexpression of ADAM19 in endometrial carci-
noma and its correlation with the progression and prog-
nosis [30] as well as in renal cell carcinomas has been
reported [31]. No statistical difference in its abundance
was observed in ccRCC patients but an increment was
noticed in urine of ccRCC at Grade 4. However, the func-
tions of ADAM19 in cancers still remain to be elucidated. Discussion In particular, the A1AGx protein, known as a1-acid
glycoprotein (AGP), is the major member of the APP
family, and its serum concentrations increase during
acute-phase reactions [24]. In addition, an increased
APP response was observed to be associated with
reduced survival rate, independently from stage of
malignant disease, including lung, pancreatic, renal,
and colorectal cancer and lymphoma [24]. Serum AGP
levels in the patient group (esophagus, gastric, colorectal,
lung, hepatic, pancreatic carcinoma) did not show any
statistical difference according to tumour size, stage, and
clinical status [25]. However, patients with advanced or
recurrent disease and/or metastasis had higher serum
levels of AGP. These findings suggest that its serum levels
may not be necessarily related to disease progression
[25]. Likewise, we found an increase of the urinary levels
of the AGP fragment compared to controls, especially
in the early stage pT1a, that decline continuously from
pT1 to pT3. A negative variation with tumour size was
also observed. Alteration of the A1AG1 non-glycosylated
forms levels with the pT was also reported in tissue of
RCC patients [10]. The authors investigated tissue from 9
patients at pT1, pT2 and pT3 and they observed a down-
expression of this protein in patients at pT1 and pT2 but
not at pT3. Complement C1r subcomponent-like protein (C1RL)
causes the proteolytic cleavage of HP/haptoglobin. The
complement system is well known to be involved in many
immune complex-mediated kidney diseases: an excessive
activation of the alternative complement pathway is asso-
ciated with autosomal dominant polycystic kidney dis-
ease (ADPKD) progression. In fact, levels of the specific
complement components such as CFB, SERPING1 and
C9 were found to be increased while C1RL, CD55 and
CD59 levels were decreased in urine of ADPKD patients. We found higher urinary levels of CRL in RCC patients
than in control subjects that slowly decreased from G1
to G4. However, its role in cancer is not known and its
detection in tissue is still under investigation [32].i We observed a statistically significant increase of uri-
nary levels of a peptide at m/z 1826 in ccRCC patients
compared to control subjects negatively varying with
grade. The identity of this signal could have originated
either from PGBM or from ZAG proteins. NOTCH2 belongs to the Notch family, including
NOTCH1,
NOTCH2,
NOTCH3,
and
NOTCH4
receptors. Their extracellular domain after the binding
with specific ligands induce an alteration of the gene
expression. Discussion The clear role of the notch family in RCC
is still unknown. However, a down-regulation of the
expression of Notch receptors and of Notch signalling
was reported, thus suggesting a possible role in the
progression of renal cell carcinoma [26]. Recently, a
specific role for each of the four Notch receptors in
RCC has also been shown [27]. These authors showed
a reduced expression of Nocth1 correlating with an
increase of the Fuhrman grade and tumour size while
Notch3 and 4 receptors were directly correlated with
tumour size. Therefore, they suggested a possible role of
NOTCHs in tumorigenesis of RCC. Our data shows that
Notch2 is also involved in RCC progression. In fact, even
if the urinary NOTCH2 is reduced in patients compared Basement
membrane-specific
heparan
sulfate
proteoglycan core protein (PGBM), belonging to heparin
sulfate proteoglycan (HSPG) family, has an important role
in vascularization. It has been shown that heparanase is
highly expressed with a positive correlation with tumour
stage and poor prognosis in RCC [33]. Moreover, HSPGs
have been reported in many metastatic tumours, and
their expression was observed in a variety of malignant
tumours, showing correlations with malignant phenotype
[34]. Higher expression of heparanase in ccRCCs than
in non-ccRCCs correlating with stage was shown by
immunostaining and RT-PCR [35]. Furthermore, specific
silencing of heparanase mRNA expression (786-O
and Caki-2 cells) with small interfering RNA (siRNA)
inhibited the invasiveness capabilities of these cells
in vitro [35]. Elevated heparanase expression was also Chinello et al. J Transl Med (2015) 13:332 Page 9 of 11 Page 9 of 11 shown to be an independent indicator of disease-specific
survival [35, 36]. These findings suggest an important role
of heparanase in invasion and metastasis and silencing of
the gene could represent a potential therapeutic target in
ccRCCs. have more complex implications in cellular functions,
such as RNA processing and metabolism, that could
potentially affect various signalling pathways in cancer
[49]. We observed low urinary levels for SAFB2 in
RCC, patients especially at grade 4. Low SAFB protein
levels were also suggested as possible predictors of poor
prognosis of breast cancer [50].hi Zinc-alpha-2-glycoprotein (ZA2G, AZGP1, ZAG) is a
protein associated with lipid mobilization, a process that
is also regulated by mTOR signaling. Discussion A down expres-
sion of the ZA2G was observed in the tissue of patients
affected by hepatocellular carcinoma (HCC) [37] and
it was associated with a poor overall survival, worst
disease-free survival, relapse-free survival and distant
metastatic progression-free survival. Moreover, ZA2G
protein was also found to be lower in 57 patients with a
rising of prostate specific antigen after surgery and not in
the other 32 patients [38]. ZAG expression was inversely
correlated with Gleason pattern and correlated with a
favourable outcome. There is a critical need for trials aimed at better defining
the progression of renal masses as well as finding robust
indicators of patient outcomes, such as overall survival
and disease specific survival. These studies should include
either hypothesis generating or hypothesis testing of lab-
oratory tools, tissue based or circulating biomarkers [51]. Moreover, the American Urological Association guide-
lines state that patients undergoing follow-up for treated
or observed renal masses should undergo basic labora-
tory testing and, depending on the risk, imaging (US, CT
or MR) every three months in high risks or yearly in low
risks. However, potential adverse effects and cost should
also be take into account. Recent attention has been paid
to the cumulative radiation exposure of the population
attributable to the widespread and increasing use of CT
scanning. For MRI, which does not involve the use of
ionizing radiation, the prime adverse effect to consider is
the development of nephrogenic systemic fibrosis (NSF)
due to IV gadolinium administration. No prospective val-
idation currently exists for the use of common laboratory
parameters in the early detection of metastases or both
in the staging and monitoring of patients with renal cell
carcinoma following treatment for recurrence. Moreover,
the finding of a biological aggressiveness marker is also
useful considering its role in the active surveillance of the
appropriately selected small renal mass, limiting adverse
health outcomes. In this context, it is desirable to gain
easily accessible, not invasive molecular indicators, which
correlate with consolidated progression factors, such as
tumor size, staging and grade. Moreover, an understand-
ing of the complex molecular alterations involved in the
development and progression of RCC could enable devel-
opment of immunohistochemical and immunoenzymatic
diagnostic tools and also open the doors for experimental
targeted therapies. MMP23 is a member of matrix metalloproteinase fam-
ily (MMP23A, MMP21, MMP23B, MMP22) that partici-
pates in many aspects of tumour growth and metastasis. Discussion Altered MMP23 expression has been observed in pros-
tate adenocarcinoma, multiple myeloma, synovial sar-
coma and in colorectal cancers [39–43]. We observed
that its urinary levels are lower in RCC patients com-
pared to controls and have a negative alteration depend-
ing on tumour size and pT. Drebrin (DREB) has been recently reported to be over-
expressed in human metastatic colon adenocarcinoma
cell line HCT-116 [44]. We also detected an increase of
its urinary levels in ccRCC, especially at grade 4. A pos-
sible role in tumour cell migration and invasion for this
protein has been shown in glioma. A constant overex-
pression of drebrin in U87 cells caused an alteration in
cell morphology with an increased invasiveness while a
silencing of drebrin gene expression decreases the inva-
sion and the migration [45]. Glyceraldehyde-3-phosphate
dehydrogenase
(G3P)
is implicated in nuclear functions such as transcription,
RNA transport, DNA replication and apoptosis. Down-
regulation of its expression was observed in colorectal
cancer [46] while it was over-expressed in human cancer
cell lines [47]. Moreover, increased expression of GAPDH
enhanced aggressiveness and vascularization of non-
Hodgkin’s lymphoma [48].f Additional files Additional file 1: Table S1. Peptides with a statistically significant
(p < 0.05) urinary abundance (m/z area) according to the tumour mass
(cm). Up/down refer to the over/under representation of the peptide in
ccRCC patients compared to control subjects. 2. Husillos Alonso A, Carbonero Garcia M, Gonzalez Enguita C. Is there a role
for systemic targeted therapy after surgical treatment for metastases of
renal cell carcinoma? World J Nephrol. 2015;4(2):254–62. doi:10.5527/wjn. v4.i2.254. 2. Husillos Alonso A, Carbonero Garcia M, Gonzalez Enguita C. Is there a role
for systemic targeted therapy after surgical treatment for metastases of
renal cell carcinoma? World J Nephrol. 2015;4(2):254–62. doi:10.5527/wjn. v4.i2.254. Additional file 2: Table S2. A) Peptides with a statistically significant
(p < 0.05) urinary abundance (m/z area) according to the pT. B) Peptides
with a statistical significant (p < 0.05) urinary abundance (m/z area)
compared to CTRLs and at different pT (1 = pT1a; 2 = pT1b; 3 = pT2a
and 4 = pT > 2b). Up/down refer to the over/under representation of the
peptide in ccRCC patients compared to control subjects Additional file 2: Table S2. A) Peptides with a statistically significant
(p < 0.05) urinary abundance (m/z area) according to the pT. B) Peptides
with a statistical significant (p < 0.05) urinary abundance (m/z area)
compared to CTRLs and at different pT (1 = pT1a; 2 = pT1b; 3 = pT2a
and 4 = pT > 2b). Up/down refer to the over/under representation of the
peptide in ccRCC patients compared to control subjects 3. Escudier B, Eisen T, Porta C, Patard JJ, Khoo V, Algaba F, et al. Renal cell
carcinoma: ESMO Clinical Practice Guidelines for diagnosis, treatment
and follow-up. Ann Oncol Off J Eur Soc Med Oncol ESMO. 2012;23(Suppl
7):65–71. doi:10.1093/annonc/mds227. 3. Escudier B, Eisen T, Porta C, Patard JJ, Khoo V, Algaba F, et al. Renal cell
carcinoma: ESMO Clinical Practice Guidelines for diagnosis, treatment
and follow-up. Ann Oncol Off J Eur Soc Med Oncol ESMO. 2012;23(Suppl
7):65–71. doi:10.1093/annonc/mds227. 4. Czarnecka AM, Kornakiewicz A, Kukwa W, Szczylik C. Frontiers in clinical
and molecular diagnostics and staging of metastatic clear cell renal cell
carcinoma. Future Oncol. 2014;10(6):1095–111. doi:10.2217/fon.13.258. Additional file 3: Table S3. A) Peptides with a statistical significant
(p < 0.05) urinary abundance (m/z area) according to the Grade. Received: 17 July 2015 Accepted: 10 October 2015 Received: 17 July 2015 Accepted: 10 October 2015 in progression algorithms for risk stratification of ccRCC
patients. Moreover, their urinary changes could also be
useful to the “watch-and-wait approach” for monitoring
small renal masses and could find a possible role in
management follow-up strategies (e.g. residual tumors). References 1. Hosoya N, Sakumoto M, Nakamura Y, Narisawa T, Bilim V, Motoyama
T, et al. Proteomics identified nuclear N-myc downstream-regulated
gene 1 as a prognostic tissue biomarker candidate in renal cell car-
cinoma. Biochim Biophys Acta. 2013;1834(12):2630–9. doi:10.1016/j. bbapap.2013.08.009. Acknowledgements 16. Lebdai S, Verhoest G, Parikh H, Jacquet SF, Bensalah K, Chautard D, et al. Identification and validation of TGFBI as a promising prognosis marker
of clear cell renal cell carcinoma. Urol Oncol. 2015;33(2):68.e11–8. doi:10.1016/j.urolonc.2014.06.005. This work was supported by grants from the Italian Ministry of University
and Research: FIRB 2007 (Rete nazionale per lo studio del proteoma umano,
no.RBRN07BMCT_11), FAR 2012-2014 (ex 60 %), and in part by Fondazione
Gigi & Pupa Ferrari Onlus. The research leading to these results has received
funding also from the European Union’s Seventh Framework Programme
FP7/2007-2013 under grant agreement FP7-PEOPLE-2013-ITN-608332. 17. Morrissey JJ, Mobley J, Song J, Vetter J, Luo J, Bhayani S, et al. Urinary
concentrations of aquaporin-1 and perilipin-2 in patients with renal cell
carcinoma correlate with tumor size and stage but not grade. Urology. 2014;83(1):256.e9–14. doi:10.1016/j.urology.2013.09.026. Author details 1 Department of Health Science, School of Medicine, University of Milano-
Bicocca (UNIMIB), Via Cadore, 48, 20900 Monza, Italy. 2 Urology Unit,
Department of Specialistic Surgical Sciences, Ospedale Maggiore Policlinico
Foundation, Milan, Italy. 3 Department of Laboratory Medicine, Hospital
of Desio, Desio, Italy. 4 Department of Surgical Pathology, Cytology, Medical
Genetics and Nephropathology, Azienda Ospedaliera San Gerardo, Monza,
Italy. 5 Department of Medicine, Surgery and Dental Sciences, Pathology
Unit, IRCCS‑Policlinico Foundation, Mangiagalli and Regina Elena, University
of Milan, Milan, Italy. 6 Department of Informatics, Systems and Communica-
tion, University of Milano-Bicocca, Milan, Italy. 13. Laird A, O’Mahony FC, Nanda J, Riddick AC, O’Donnell M, Harrison DJ,
et al. Differential expression of prognostic proteomic markers in primary
tumour, venous tumour thrombus and metastatic renal cell cancer tis-
sue and correlation with patient outcome. PLoS One. 2013;8(4):e60483. doi:10.1371/journal.pone.0060483. 14. Ngo TC, Wood CG, Karam JA. Biomarkers of renal cell carcinoma. Urol
Oncol. 2014;32(3):243–51. doi:10.1016/j.urolonc.2013.07.011. 15. Tosco L, Van Poppel H, Frea B, Gregoraci G, Joniau S. Survival and impact
of clinical prognostic factors in surgically treated metastatic renal cell
carcinoma. Eur Urol. 2013;63(4):646–52. doi:10.1016/j.eururo.2012.09.037. Authors’ contributions CC, MC have made contribution to the design of the study, performed and
elaborated the analysis, written the draft of the manuscript. GDS, AJS have
been involved in data acquisition and revision of the manuscript. GM, IZ have
conceived the statistical analysis. FM have made a substantial contribution
to conception, design of the study, interpretation of the data, drafting of the
manuscript and critical revision. SS, BR, SB have contributed to collect sample
and provided clinical characterization of the cohort. AG, MG have been
involved in sample collection and classification, revision of the manuscript. All
authors read and approved the final manuscript. 10. Junker H, Venz S, Zimmermann U, Thiele A, Scharf C, Walther R. Stage-
related alterations in renal cell carcinoma–comprehensive quantita-
tive analysis by 2D-DIGE and protein network analysis. PLoS One. 2011;6(7):e21867. doi:10.1371/journal.pone.0021867. 11. Frantzi M, Metzger J, Banks RE, Husi H, Klein J, Dakna M, et al. Discovery
and validation of urinary biomarkers for detection of renal cell carcinoma. J Proteomics. 2014;98:44–58. doi:10.1016/j.jprot.2013.12.010. 12. Chinello C, Cazzaniga M, De Sio G, Smith AJ, Gianazza E, Grasso A, et al. Urinary signatures of renal cell carcinoma investigated by peptid-
omic approaches. PLoS One. 2014;9(9):e106684. doi:10.1371/journal. pone.0106684. juro.2013.08.074. Additional file 4: Table S4. Peptides with a statistically significant
(p > 0.05) urinary abundance (m/z area) between pT1a and pT1b. Up/
down refers to the over/under representation of the peptide in pT1b
compared to pT1a patients. Additional file 4: Table S4. Peptides with a statistically significant
(p > 0.05) urinary abundance (m/z area) between pT1a and pT1b. Up/
down refers to the over/under representation of the peptide in pT1b
compared to pT1a patients. 7. Tsivian M, Rampersaud EN Jr, del Pilar Laguna Pes M, Joniau S, Leveillee
RJ, Shingleton WB, et al. Small renal mass biopsy–how, what and when:
report from an international consensus panel. BJU Int. 2014;113(6):854–
63. doi:10.1111/bju.12470. Additional file 5: Table S5. Identification of MALDI signals varied
according to tumour size, pT or grade. LM = linear mode, RM = reflector
mode, PTM = possible hypothetical modification predicted by Mascot. Additional file 5: Table S5. Identification of MALDI signals varied
according to tumour size, pT or grade. LM = linear mode, RM = reflector
mode, PTM = possible hypothetical modification predicted by Mascot. 8. Sarto C, Valsecchi C, Magni F, Tremolada L, Arizzi C, Cordani N, et al. Expression of heat shock protein 27 in human renal cell carcinoma. Proteomics. 2004;4(8):2252–60. doi:10.1002/pmic.200300797. 9. Raimondo F, Salemi C, Chinello C, Fumagalli D, Morosi L, Rocco F, et al. Proteomic analysis in clear cell renal cell carcinoma: identification of dif-
ferentially expressed protein by 2-D DIGE. Mol BioSyst. 2012;8(4):1040–51. doi:10.1039/c2mb05390j. Conclusions In this study, we highlight a number of peptides whose
urinary expression is altered depending on tumor size,
pT and grade. Among them, several play a possible
role in tumorigenesis, progression and aggressiveness,
enriching the molecular scenario of RCC development. These results could be a useful starting point for
future studies aimed at verifying their urinary levels
by immunoenzymatic assays (i.e. ELISA test) and the
resulting possibility for some of them to be implemented SAFB1 (scaffold attachment factor B1) and a second
family member Scaffold attachment factor B2 (SAFB2)
are multifunctional proteins implicated in a variety
of cellular processes including cell growth, apoptosis
and stress response. A possible role for SAFBx as
tumour suppressors has also been suggested [49]. There is numerous evidence that SAFB1/SAFB2 have a
contribution in cancer progression. SAFB1/SAFB2 may Page 10 of 11 Page 10 of 11 Chinello et al. J Transl Med (2015) 13:332 Additional files B) Pep-
tides with a statistically significant (p < 0.05) urinary abundance (m/z area)
compared to CTRLs and at different Grade (1, 2, 3 and 4). Up/down refer to
the over/under representation of the peptide in ccRCC patients compared
to control subjects. Additional file 3: Table S3. A) Peptides with a statistical significant
(p < 0.05) urinary abundance (m/z area) according to the Grade. B) Pep-
tides with a statistically significant (p < 0.05) urinary abundance (m/z area)
compared to CTRLs and at different Grade (1, 2, 3 and 4). Up/down refer to
the over/under representation of the peptide in ccRCC patients compared
to control subjects. 5. Ha SB, Kwak C. Current status of renal biopsy for small renal masses. Korean J Urol. 2014;55(9):568–73. doi:10.4111/kju.2014.55.9.568. 6. Shuch B, Hanley JM, Lai JC, Vourganti S, Setodji CM, Dick AW, et al. Adverse health outcomes associated with surgical management
of the small renal mass. J Urol. 2014;191(2):301–8. doi:10.1016/j. juro.2013.08.074. 6. Shuch B, Hanley JM, Lai JC, Vourganti S, Setodji CM, Dick AW, et al. Adverse health outcomes associated with surgical management
of the small renal mass. J Urol. 2014;191(2):301–8. doi:10.1016/j. juro.2013.08.074. Competing interests
h
h
d
l
h g
The authors declare that they have no competing interests. The authors declare that they have no competing interests. Page 11 of 11 Chinello et al. J Transl Med (2015) 13:332 18. Bosso N, Chinello C, Picozzi SC, Gianazza E, Mainini V, Galbusera C, et al. Human urine biomarkers of renal cell carcinoma evaluated by ClinProt. Proteomics Clin Appl. 2008;2(7–8):1036–46. doi:10.1002/prca.200780139. 38. Mills J, Oliver A, Sherwin JC, Frydenberg M, Peters JS, Costello A, et al. Utility of RhoC and ZAG protein expression as biomarkers for prediction
of PSA failure following radical prostatectomy for high grade prostate
cancer. Pathology. 2012;44(6):513–8. doi:10.1097/PAT.0b013e3283581780. 19. Gianazza E, Chinello C, Mainini V, Cazzaniga M, Squeo V, Albo G, et al. Alterations of the serum peptidome in renal cell carcinoma discriminat-
ing benign and malignant kidney tumors. J Proteomics. 2012;76(Spec
No):125–40. doi:10.1016/j.jprot.2012.07.032. 39. Hegedus L, Cho H, Xie X, Eliceiri GL. Additional MDA-MB-231 breast can-
cer cell matrix metalloproteinases promote invasiveness. J Cell Physiol. 2008;216(2):480–5. doi:10.1002/jcp.21417. 20. Raimondo F, Corbetta S, Chinello C, Pitto M, Magni F. The urinary pro-
teome and peptidome of renal cell carcinoma patients: a comparison of
different techniques. Expert Rev Proteomics. 2014;11(4):503–14. doi:10.15
86/14789450.2014.926222. 40. Acevedo VD, Gangula RD, Freeman KW, Li R, Zhang Y, Wang F, et al. Induc-
ible FGFR-1 activation leads to irreversible prostate adenocarcinoma and
an epithelial-to-mesenchymal transition. Cancer Cell. 2007;12(6):559–71. doi:10.1016/j.ccr.2007.11.004. 21. Pastore AL, Palleschi G, Silvestri L, Moschese D, Ricci S, Petrozza V, et al. Serum and urine biomarkers for human renal cell Carcinoma. Dis markers. 2015;2015:251403. doi:10.1155/2015/251403. 41. Carrasco DR, Sukhdeo K, Protopopova M, Sinha R, Enos M, Carrasco DE, et al. The differentiation and stress response factor XBP-1 drives multiple myeloma
pathogenesis. Cancer Cell. 2007;11(4):349–60. doi:10.1016/j.ccr.2007.02.015.fi 22. Volpe A, Patard JJ. Prognostic factors in renal cell carcinoma. World J Urol. 2010;28(3):319–27. doi:10.1007/s00345-010-0540-8. 42. Haldar M, Hancock JD, Coffin CM, Lessnick SL, Capecchi MR. A conditional
mouse model of synovial sarcoma: insights into a myogenic origin. Cancer Cell. 2007;11(4):375–88. doi:10.1016/j.ccr.2007.01.016. 23. Ficarra V, Brunelli M, Cheng L, Kirkali Z, Lopez-Beltran A, Martignoni G,
et al. Prognostic and therapeutic impact of the histopathologic definition
of parenchymal epithelial renal tumors. Eur Urol. 2010;58(5):655–68. doi:10.1016/j.eururo.2010.08.001. 43. Nguyen HM, Galea CA, Schmunk G, Smith BJ, Edwards RA, Norton
RS, et al. Intracellular trafficking of the KV1.3 potassium channel is
regulated by the prodomain of a matrix metalloprotease. J Biol Chem. 2013;288(9):6451–64. doi:10.1074/jbc.M112.421495. 24. Submit your next manuscript to BioMed Central
and take full advantage of: Submit your next manuscript to BioMed Central
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Submit your manuscript at
www.biomedcentral.com/submit 36. Dall’Oglio MF, Ribeiro-Filho LA, Antunes AA, Crippa A, Nesrallah L,
Goncalves PD, et al. Microvascular tumor invasion, tumor size and
Fuhrman grade: a pathological triad for prognostic evaluation of renal
cell carcinoma. J Urol. 2007;178(2):425–8. doi:10.1016/j.juro.2007.03.128
(di
i
) • Convenient online submission Competing interests
h
h
d
l
h Yildirim A, Meral M, Kaynar H, Polat H, Ucar EY. Relationship between
serum levels of some acute-phase proteins and stage of disease and
performance status in patients with lung cancer. Med Sci Monit. 2007;13(4):CR195–200. 44. Lin Q, Tan HT, Lim TK, Khoo A, Lim KH, Chung MC. iTRAQ analysis of
colorectal cancer cell lines suggests Drebrin (DBN1) is overexpressed
during liver metastasis. Proteomics. 2014;14(11):1434–43. doi:10.1002/
pmic.201300462. 25. Hashimoto S, Asao T, Takahashi J, Yagihashi Y, Nishimura T, Saniabadi AR,
et al. alpha1-acid glycoprotein fucosylation as a marker of carcinoma
progression and prognosis. Cancer. 2004;101(12):2825–36. doi:10.1002/
cncr.20713. 45. Terakawa Y, Agnihotri S, Golbourn B, Nadi M, Sabha N, Smith CA, et al. The role of drebrin in glioma migration and invasion. Exp Cell Res. 2013;319(4):517–28. doi:10.1016/j.yexcr.2012.11.008. 26. Sun S, Du R, Gao J, Ning X, Xie H, Lin X, et al. Expression and clinical
significance of Notch receptors in human renal cell carcinoma. Pathology. 2009;41(4):335–41. doi:10.1080/00313020902885003. 46. Koshiyama A, Ichibangase T, Imai K. Comprehensive fluorogenic derivat-
ization-liquid chromatography/tandem mass spectrometry proteomic
analysis of colorectal cancer cell to identify biomarker candidate. Biomed
Chromatogr. 2013;27(4):440–50. doi:10.1002/bmc.2811. 27. Aparicio LM, Villaamil VM, Gallego GA, Cainzos IS, Campelo RG, Rubira LV,
et al. Expression of Notch1 to -4 and their ligands in renal cell carcinoma:
a tissue microarray study. Cancer Genomics Proteomics. 2011;8(2):93–101. g
47. Markovets AA, Herman D. Analysis of cancer metabolism with high-
throughput technologies. BMC Bioinformatics. 2011;12(Suppl 10):S8. doi:10.1186/1471-2105-12-S10-S8. 28. Mochizuki S, Okada Y. ADAMs in cancer cell proliferation and progression. Cancer Sci. 2007;98(5):621–8. doi:10.1111/j.1349-7006.2007.00434.x. 48. Chiche J, Pommier S, Beneteau M, Mondragon L, Meynet O, Zunino B,
et al. GAPDH enhances the aggressiveness and the vascularization of
non-Hodgkin’s B lymphomas via NF-kappaB-dependent induction of HIF-
1alpha. Leukemia. 2014; doi:10.1038/leu.2014.324. 29. Qi B, Newcomer RG, Sang QX. ADAM19/adamalysin 19 structure, func-
tion, and role as a putative target in tumors and inflammatory diseases. Curr Pharm Des. 2009;15(20):2336–48. 30. Ou Q, Zhang Y, Yu L, Dong Y, Zhu C, Liao Q. Expression and clinical sig-
nificance of ADAM19 in endometrial carcinoma. Beijing Da Xue Xue Bao. 2008;40(2):165–9. 49. Hong EA, Gautrey HL, Elliott DJ, Tyson-Capper AJ. SAFB1- and SAFB2-
mediated transcriptional repression: relevance to cancer. Biochem Soc
Trans. 2012;40(4):826–30. doi:10.1042/BST20120030. 31. Roemer A, Schwettmann L, Jung M, Roigas J, Kristiansen G, Schnorr D,
et al. Increased mRNA expression of ADAMs in renal cell carcinoma and
their association with clinical outcome. Oncol Rep. 2004;11(2):529–36. Competing interests
h
h
d
l
h 31. Roemer A, Schwettmann L, Jung M, Roigas J, Kristiansen G, Schnorr D,
et al. Increased mRNA expression of ADAMs in renal cell carcinoma and
their association with clinical outcome. Oncol Rep. 2004;11(2):529–36. 50. Hammerich-Hille S, Bardout VJ, Hilsenbeck SG, Osborne CK, Oester-
reich S. Low SAFB levels are associated with worse outcome in breast
cancer patients. Breast Cancer Res Treat. 2010;121(2):503–9. doi:10.1007/
s10549-008-0297-6. 32. http://www.proteinatlas.org. 51. Donat SM, Diaz M, Bishoff JT, Coleman JA, Dahm P, Derweesh IH, et al. Follow-up for Clinically Localized Renal Neoplasms: AUA Guideline. J Urol. 2013;190(2):407–16. doi:10.1016/j.juro.2013.04.121. 33. Mikami S, Oya M, Mizuno R, Kosaka T, Katsube K, Okada Y. Invasion and
metastasis of renal cell carcinoma. Med Mol Morphol. 2014;47(2):63–7. doi:10.1007/s00795-013-0064-6. 34. Vlodavsky I, Beckhove P, Lerner I, Pisano C, Meirovitz A, Ilan N, et al. Signifi-
cance of heparanase in cancer and inflammation. Cancer Microenviron. 2012;5(2):115–32. doi:10.1007/s12307-011-0082-7. 35. Mikami S, Oya M, Shimoda M, Mizuno R, Ishida M, Kosaka T, et al. Expression of heparanase in renal cell carcinomas: implications for
tumor invasion and prognosis. Clin Cancer Res. 2008;14(19):6055–61. doi:10.1158/1078-0432.CCR-08-0750. Submit your next manuscript to BioMed Central
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of zinc-alpha2-glycoprotein in hepatocellular carcinoma associates with
poor prognosis. J Transl Med. 2012;10:106. doi:10.1186/1479-5876-10-106. 37. Huang Y, Li LZ, Zhang CZ, Yi C, Liu LL, Zhou X, et al. Decreased expression
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poor prognosis. J Transl Med. 2012;10:106. doi:10.1186/1479-5876-10-106.
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Positive mental health literacy: development and validation of a measure among Norwegian adolescents
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BMC public health
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* Correspondence: hanne.n.bjornsen@ntnu.no
1Department of Public Health and Nursing, Norwegian University of Science
and Technology, Postbox 8905, 7491 Trondheim, Norway
2Center for Health Promotion Research, Norwegian University of Science and
Technology, Trondheim, Norway Open Access Hanne Nissen Bjørnsen1,2*, Mary˗Elizabeth Bradley Eilertsen1,2, Regine Ringdal1,2, Geir Arild Espnes1,2
and Unni Karin Moksnes1,2 Hanne Nissen Bjørnsen1,2*, Mary˗Elizabeth Bradley Eilertsen1,2, Regine Ringdal1,2, Geir Arild Espnes1,2
and Unni Karin Moksnes1,2 Abstract Background: Mental health literacy (MHL), or the knowledge and abilities necessary to benefit mental health, is a
significant determinant of mental health and has the potential to benefit both individual and public mental health. MHL and its measures have traditionally focused on knowledge and beliefs about mental -ill-health rather than on
mental health. No measures of MHL addressing knowledge of good or positive mental health have been identified. Aim: This study aimed to develop and validate an instrument measuring adolescents’ knowledge of how to obtain
and maintain good mental health and to evaluate the psychometric properties of the instrument. More specifically,
the factor structure, internal and construct validity, and test-retest reliability were assessed. Methods: The participants were Norwegian upper secondary school students aged 15–21 years. The development
and validation of the instrument entailed three phases: 1) item generation based on the basic psychological needs
theory (BPNT), focus group interviews, and a narrative literature review, 2) a pilot study (n = 479), and 3) test-retest
(n = 149), known-groups validity (n = 44), and scale construction, item reduction through principal component
analysis (PCA), and confirmatory factor analysis (CFA) for factor structure and psychometric properties assessment
(n = 1888). Results: Thirty-two items were initially generated, and 15 were selected for the pilot study. PCA identified cross-
loadings, and a one-factor solution was examined. After removing five problematic items, CFA yielded a satisfactory
fit for a 10-item one-factor model, referred to as the mental health-promoting knowledge (MHPK-10) measure. The
test-retest evaluation supported the stability of the measure. McDonald’s omega was 0.84, and known-groups
validity test indicated good construct validity. Conclusion: A valid and reliable one-dimensional instrument measuring knowledge of factors promoting good
mental health among adolescents was developed. The instrument has the potential to complement current
measures of MHL and may be useful when planning mental health promotion activities and evaluating public
mental health education initiatives in adolescents. Keywords: Mental health literacy, Adolescence, Measurement, Health promotion © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated Bjørnsen et al. BMC Public Health (2017) 17:717
DOI 10.1186/s12889-017-4733-6 Bjørnsen et al. BMC Public Health (2017) 17:717
DOI 10.1186/s12889-017-4733-6 Background mental health [6]. Thus, a gap remains between the recent
conceptualization of MHL [12] and available MHL mea-
sures. An instrument that rigorously measures the positive
aspect of MHL can help determine a population’s or
individual’s level of knowledge of factors promoting
mental health. Furthermore, it could be used to evaluate
interventions and educational initiatives to increase our
understanding of the positive aspects of MHL and its
associations with good mental health in adolescents. Mental health in the Norwegian adolescent population
has received considerable attention in recent years and
has emerged as a public health concern that needs to be
addressed [1]. Adolescence is considered an important
transitional period in life during which individuals are
particularly sensitive to contextual and surrounding in-
fluences; this unique state leads to challenges but also
opportunities for improving health [2]. A major challenge to developing a measure that as-
sesses knowledge of factors promoting mental health is
that individuals’ conceptions of what is needed to obtain
and maintain mental health are highly individualized. However, there are known commonalities of the factors
essential for obtaining and maintaining mental health. In
this study, the basic psychological needs theory (BPNT)
[17] was utilized to ground the measure in dimensions
that are theoretically known to be important to good
mental health and has been identified as an applicable
conceptual framework for studying health-related behav-
ior [18]. According to the BPNT, good mental health
can be predicted by three dimensions: competence,
autonomy and relatedness [17]. Competence refers to
experiencing mastery and effectiveness in managing
one’s environment. Autonomy refers to a sense of free
will or acting out of one’s own interests and values. Finally, relatedness addresses the desire to interact with,
feel a connection to, and care for other people [19]. Health literacy (HL) concerns adolescents’ capacity to
make sound health decisions in the context of their every-
day lives [3] and is considered critical for effective participa-
tion in health promotion [4]. HL is a multifaceted, complex
and evolving concept, and Sørensen et al. [5] has developed
a definition and conceptual model relevant for the further
work of conceptualization and measure development for
mental health literacy (MHL). MHL is a component of HL and is also an evolving
concept. MHL is considered a significant determinant of
mental health and has the potential to benefit both
individual and public mental health [6, 7]. Background MHL has
been conceptualized in different ways since the term was
first coined by Jorm and colleagues in 1997 [6, 8–10]. Traditionally, MHL and its measures have focused on
knowledge and beliefs about mental -ill-health rather
than on mental health [11]. However, in past years,
MHL has evolved from a focus on mental -ill-health and
risk factors to providing an asset for health that can be
strengthened through educational initiatives [7]. Today,
MHL broadly refers to the knowledge and abilities ne-
cessary to benefit mental health [9]. A recent definition
of MHL outlines four key components: The aim of the current study was to describe the
development of an instrument measuring adolescents’
understanding of how to obtain and maintain good men-
tal health (in this study referred to as mental health-
promoting knowledge or MHPK) to represent MHL and
to evaluate its psychometric properties. More specific-
ally, the aims were to evaluate the factor structure, in-
ternal and construct validity, and test-retest reliability of
the instrument. “(1) Understanding how to obtain and maintain good
mental health; (2) understanding mental disorders
and their treatments; (3) decreasing stigma related to
mental disorders; (4) enhancing help-seeking efficacy
(knowing when, where, and how to obtain good mental
health care and developing competencies needed for
self-care)” Abstract The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bjørnsen et al. BMC Public Health (2017) 17:717 Page 2 of 10 Page 2 of 10 Methods The instrument was referred to as the MHPK and was
developed in a three-step process (Fig. 1). Focus group interviews To explore and include adolescents’ perceptions of good
mental health, five focus group interviews were con-
ducted [22]. Adolescents aged 15–21 years from four
upper secondary schools in an urban area in mid-
Norway participated in the discussions in phase 1 (Fig. 1). Participants were recruited through the schools’ student
councils by self-selection. There were 6–10 participants
in each group, and both genders were represented, with
a preponderance of girls. Semi-structured interview
guides were developed in advance and used during the
discussions. Mental health, factors important for good
mental health, and items from the MHPK scale were dis-
cussed with the adolescents during an approximately
one-hour session. The focus group discussions were
transcribed, and adolescents’ collective perceptions of
items and factors important for good mental health were
extracted and used in scale development. No further
analysis of the focus group discussions was performed
for the purpose of scale development. Expert panel generated. Item generation was grounded in the three di-
mensions of BPNT and based on the narrative literature
review and focus group discussions with adolescents. With
the 32 items, all three dimensions of the BPNT were
covered by a minimum of 7 items each, and recurrence in
item proposals was observed. Hence, the decision was
made within the research group to begin working with the
pool of 32 items. generated. Item generation was grounded in the three di-
mensions of BPNT and based on the narrative literature
review and focus group discussions with adolescents. With
the 32 items, all three dimensions of the BPNT were
covered by a minimum of 7 items each, and recurrence in
item proposals was observed. Hence, the decision was
made within the research group to begin working with the
pool of 32 items. For content validation, an expert panel was invited to
participate in the study (n = 10). Three public health
nurses and six researchers within the field of health pro-
motion (N = 9) provided iterative feedback during item
development. Invitations to participate in the expert
panel were extended to authors’ associates with appro-
priate academic qualifications and professional expertise
in the field of mental health and school health services. The expert panel was asked to categorize items within
the dimensions of competence, relatedness and auton-
omy. The items were included only if they were catego-
rized in the same dimension and considered relevant by
>7 members of the expert panel. This categorization
eventually resulted in the inclusion of 15 items in the
pilot study. Phase 1: item generation This conceptualization advances previous perceptions
of MHL as merely knowledge of mental disorders and is
in line with the WHO’s definition of mental health,
which states that mental health is more than the absence
of mental disorders and includes wellbeing, optimal
functioning and coping [13]. Items were generated through a deductive approach using
BPNT as the theoretical foundation [17, 20]. BPNT was
applied during the development of the instrument meas-
uring the positive component of MHL, i.e., component
one in the recent definition, to identify the factors that
actively promote mental health. In addition, a review of
the literature on MHL and the seven rights of mental
health (identity, meaning, mastering, belonging, safety,
participation and sense of community) [21] were utilized
to conceptualize knowledge of good mental health when
generating relevant items. Thirty-two items were initially Several scales have been developed to capture the broad
scope of MHL [8, 14–16]. However, the existing measures
mainly address knowledge of the three latter components,
namely mental disorders, stigma and help-seeking behav-
iors; no studies address knowledge of good or positive Bjørnsen et al. BMC Public Health (2017) 17:717 Page 3 of 10 Fig. 1 Description of timeline and phases of development of the instrument measuring mental health-promoting knowledge (MHPK) Fig. 1 Description of timeline and phases of development of the instrument measuring mental health-promoting knowledge (MHPK) Fig. 1 Description of timeline and phases of development of the instrument measuring mental health-promoting knowl Procedure Principals gave informed consent for data collection at the
designated schools. Information regarding participation
was provided by the research team through the schools’
teachers, and parents and students received written infor-
mation letters. Furthermore, a short informational video
was available to all participants on the schools’ e-learning
platform (i.e., “it’s learning”). Teachers were responsible
for allocating time and administering the survey, including
reading aloud an information letter provided by the
research group that stated that participation was voluntary
and anonymous. Students aged 16 years or older gave
consent for participation by completing the questionnaire,
whereas written parental consent for students aged 15 was
obtained. The study was approved by the Regional
Committee for Medical and Health Research Ethics (REK
midt 2014/1996). Sample 1 Over a three-week period in August 2016, a cross-
sectional classroom survey was conducted at five upper
secondary schools in an urban area in mid-Norway. Three of these five schools also participated in Phase 1,
and one school also participated in the pilot study. p
p
p
y
The questionnaire was administered to 2145 of 3281
students (65.4%), and n = 2087 (97.3%) responded with
usable information (Fig. 2). The ages of the respondents
ranged from 15 to 21 years. Seventy-four (3.9%) respon-
dents did not report their age, and missing age values were
replaced with the mean age of the sample (M = 17.02,
SD = 1.04). The adolescent sample consisted of 51% girls
and 49% boys; 62% of the students were from the “general
studies” stream, whereas 38% of students were from “vo-
cational studies” stream. When asked about their parents’
education, 38% of the adolescents responded “do not
know”; 4.8% reported that their parents had received pri-
mary school education; 19%, upper secondary school;
21.7%, less than 4 years of university education; and
25.8%, more than 4 years of university education. Phase 2: pilot testing A questionnaire including the 15-item scale was piloted
at one upper secondary school in phase 2 (Fig. 1). Fol-
lowing informed consent from the principal to pilot the
questionnaire at the designated school, each teacher
chose whether they wanted to administer the survey to
their class. The questionnaire was then administered by
teachers over a two-week period; the teachers chose the
session for administering the questionnaire at their con-
venience. The questionnaire was then given to 490 of
1075 students (46%); n = 479 (98%) responded. Pilot
data were explored using Stata [23], and initial principal Bjørnsen et al. BMC Public Health (2017) 17:717 Page 4 of 10 Fig. 2 Flowchart of participants from sample 1: August 2016
student population component analyses (PCA) were performed. Based on
the focus group interviews, the expert panel comments
and the pilot study results, two reversed items were de-
leted (e.g., in PCA, negative items generated a separate
factor), one item was reworded, and two new items were
added. In addition, “don’t know” was added as a re-
sponse option. A 15-item scale was constructed and
evaluated in phase 3 of the present study. Statistics STATA version 14.2 (StataCorp. 2015, Stata Statistical
Software: Release 14, College Station, TX: StataCorp LP
[23]) and Microsoft Excel (2011, version 14.7.1) were
used for statistical analyses. y
The 15 items were initially analyzed using principal
component analysis (PCA) orthogonal rotation by default
to explore the factor structure and identify split loadings
to reduce items; 0.32 was set as the minimum factor load-
ing, and loadings >0.55 were considered good [24]. Data
were examined for normality, frequency and patterns of
missing data. Testing for normality revealed significant
kurtosis and skewed data (p-value <0.001). The data were
determined to have a non-normal distribution, and thus
Satorra-Bentler (robust to non-normality) was used as an
estimation method in the confirmatory factor analysis
(CFA) [25, 26]. CFA was performed to evaluate the model
fit of the factor structure based on the PCA and to identify
problematic items by inspecting modification indices
(MI). Two different models were estimated to find the
best fit: a 10-item version with a one- or three-factor
solution. The fit indices assessed with cut-off values in-
cluded the following: Chi-square test (χ2) to evaluate the
global model fit; the Comparative Fit Index (CFI) and
Tucker-Lewis Index (TLI), with values >0.90 considered
adequate (preferably >0.95) [26]; Root Mean Square Error
of Approximation (RMSEA), with cut-off values of <0.8
(preferably <0.5) [26]; and Standardized Root Mean square
Residual (SRMR), where values <0.10 were considered
acceptable [27]. Inter-item correlations and correlations be-
tween factors were evaluated using Pearson’s r. McDonald’s Sample 2: known-groups validity Fig. 2 Flowchart of participants from sample 1: August 2016
student population Fig. 2 Flowchart of participants from sample 1: August 2016
student population To further validate the instrument, third-year nursing
students from the Norwegian University of Science and
Technology (NTNU) were asked to respond to the Bjørnsen et al. BMC Public Health (2017) 17:717 Page 5 of 10 omega was calculated to evaluate the internal consistency
of the measure [28]. MHPK instrument to complete a known-groups validity
test. Third-year nursing students are expected to have
higher levels of knowledge of the factors promoting
mental health than adolescents aged 15–21 years, con-
sidering their educational background in mental health
and health promotion. The instrument was distributed
at the end of a regular lecture, and students who wanted
to answer (n = 44) returned the completed MHPK
before they left class, thereby forming a discretionary
sample. The Norwegian Social Science Service (NSD)
approved the inclusion of nursing students to test
known-groups validity. Known-groups validity To test the construct validity of the instrument, a
known-groups validity test was performed; specifically, a
two-tailed independent samples t-test was conducted to
evaluate the mean group differences between the adoles-
cent/student sample and the sample of nursing students. To evaluate the strength of the differences in mean
scores between the adolescents and nursing students,
the effect sizes were interpreted using Cohen’s d [29]. Measure Three weeks after the initial data collection, the instrument
was administered to a discretionary sample subgroup
(n = 219) of the original sample to evaluate the test-retest
reliability of the instrument using Pearson’s correlation co-
efficient, r. A test-retest correlation coefficient above 0.70
was considered acceptable. The MHPK scale was included as part of a questionnaire
covering mental health and school health services. The
MHPK measure consisted of 15 items representing
statements of factors important to positive mental
health; respondents were asked to rate each item on a
six-point scale ranging from 1, “completely wrong”, to 5,
“completely correct”, in addition to 0, “don’t know”. Missing values All items were examined for missing values. In total, 94.6%
of adolescents (n = 1786) responded to all items, 3.4% were
missing one or two items, and the remaining 2% were miss-
ing 3–14 items. Items were missing at random and were
evenly distributed across the scale, ranging from 0.9% to
2.5% on each item. Cases were deleted listwise. Principal component analysis y
Fifteen items were included in the PCA. Parallel analysis,
eigenvalues and scree plots were used to determine how
many factors should be retained after PCA. Two factors
had eigenvalues above 1 (4.8 and 1.2), and the scree plot
leveled off immediately after the two factors [24, 27]. Factor 1 explained 48% of the variance, while factor 2
accounted for 12% of the variance; the other factors ex-
plained very little of the variance in the 15 variables. PCA revealed five problematic items (items 1, 3, 4, 9
and 10), referring to a split loading >0.32 on two factors. Items were removed after evaluation of the split loadings
and careful consideration of the item content (Table 1). Removing the five initially problematic items resulted in
a 10-item one-factor solution in the PCA that explained
41% of the variance; the other factors explained <10% of
the total variance. The 10-item version was referred to
as the MHPK-10 (a copy of the MHPK-10 can be found
as Additional file 1). The results of bivariate correlations
showed significant inter-item correlations of the 10
items ranging from r = 0.29 to 0.52. Bjørnsen et al. BMC Public Health (2017) 17:717 Page 6 of 10 Table 1 Items, descriptive statistics and factor loadings in PCA
Items
Mean
Split factor loadings
15-item version:
n = 1786
Factor loadings 10-item version:
n = 1813
Intended theoretica
dimension
Factor1
Factor2
1. Having at least a good friend
0.50
0.56
Relatedness
2. Handling stressful situations in a good
manner
4.20
0.62
Competence
3. Having influence on your own day
0.60
0.37
Autonomy
4. Acting out of your own wishes
0.59
0.44
Autonomy
5. Believing in yourself
4.62
0.70
Competence
6. Having good sleep routines
4.18
0.63
Competence
7. Making decisions based on own will
4.39
0.59
Autonomy
8. Setting limits for your own actions
4.30
0.66
Autonomy
9. Being a good friend
0.67
0.36
Relatedness
10. Feeling safe at home
0.70
0.42
Relatedness
11. Feeling that you belong in a community
4.58
0.66
Relatedness
12. Mastering your own negative thoughts
4.20
0.72
Competence
13. Setting limits for what is OK for me
4.41
0.72
Autonomy
14. Feeling valuable regardless of your own
accomplishments
4.20
0.74
Relatedness
15. Confirmatory factor analysis of the final 10-item version McDonald’s omega (ω = 0.84) of the one-factor model
was high, indicating support for the internal consistency
of the one-factor solution based on the measure’s in-
ternal structure [28]. The final 10-item one-factor model
showed good to excellent factor loadings (0.57–0.72)
and a reasonably good fit to the data (Fig. 3). Inspection
of MIs indicated that item 8 covaried with several other
items, revealing a covariance MI of 75.9 with item 7. After removing item 8, all MIs were ≤20. However, as
the preset criteria for model fit were met, no modifica-
tions based on MIs were made [30]. Following the PCA, the 10-item version was assessed in
CFA to evaluate the factor structure. Two models were
tested. The one-factor model was based on the PCA and
referred to the concept of MHPK as one component of
MHL. The three-factor model was based on the theoret-
ical foundation of BPNT and its three dimensions for
item development. The model fit indices presented in Table 2 reveal a
slightly better fit for the three-factor model than for the
one-factor model; however, both models showed a
decent fit to the data. The correlations between factors
in the three-factor model were strong (0.82 to 0.97), and
the one-factor model representing the positive compo-
nent of MHL was theoretically preferred. A final 10-item
one-factor model was therefore estimated and exhibited
an adequate model fit (Fig. 3). Test-retest The test-retest coefficient for the 10-item version was
r = 0.74, indicating acceptable reliability of the instru-
ment according to the a priori established cut-off value
of 0.70. Principal component analysis Experiencing school mastery
4.10
0.68
Competence
Explained variance per factor
48%
12%
41%
BPNT was used for item development and included three dimensions: competence, relatedness and autonomy
Frequency N per item range was 1840–1871
Cases were deleted listwise Known-groups validity Table 2 Fit indices for CFA models. One- and three-factor solutions
for the 10-item version of the MHPK
MHPK 10-item version
Model
χ2
df
χ2 /df
CFI
RMSEA
SRMR
TLI
Single-factor
169.41a
35
4.84
0.946
0.046
0.035
0.930
Three-factor
114.25a
32
3.57
0.967
0.038
0.027
0.953
Note: All p values are statistically significant (p < 0.001)a; n = 1813 Table 2 Fit indices for CFA models. One- and three-factor solutions
for the 10-item version of the MHPK
MHPK 10-item version
Model
χ2
df
χ2 /df
CFI
RMSEA
SRMR
TLI
Single-factor
169.41a
35
4.84
0.946
0.046
0.035
0.930
Three-factor
114.25a
32
3.57
0.967
0.038
0.027
0.953
Note: All p values are statistically significant (p < 0.001)a; n = 1813 The results of the independent samples t-tests showed
that nursing students (M = 4.69, SD = 0.33) scored signifi-
cantly higher on the scale than adolescents (M = 4.51,
SD = 0.54), t = 2.2012, p = 0.0278. The difference in mean
scores between adolescents and nursing students was 0.18
and of medium strength (Cohen’s d = 0.40). The results
support the instrument’s construct validity by showing Bjørnsen et al. BMC Public Health (2017) 17:717 Page 7 of 10 Fig. 3 The mental health promoting-knowledge instrument (MHPK-10). Legend: McDonald’s omega, item loadings (standardized), and explained variance
(R2) for the one-factor model representing one component of MHL. n = 1813. Reliability coefficient = 0.874 distress and viewing mentally negative conditions as ill-
nesses toward a greater focus on resources contributing to
positive development and wellbeing [31]. Consequently,
the positive conditions and factors involved in mental
health promotion need to be assessed with relevant and
psychometrically sound measures, such as measures of
MHL. MHL has not been consistently conceptualized in
the literature, and thus no gold standard for measuring
the concept exists [14]. However, in the field of HL, com-
plex conceptual models have been developed [5]. These
models are useful to place MHL in a wider context, but it
is important to emphasize that we do need a domain-
specific approach for MHL to draw attention to a
neglected area [10]. Known-groups validity Mental health is an integral part of
health [13], thus MHL may be merged with HL in the fu-
ture, but for now, there is a need for a domain-specific ap-
proach for mental health to specifically meet the need for
tailored measures in the evaluation of MHL interventions
[10] and assessment of MHL levels in populations. The
MHPK-10 measure is a contribution to the field of MHL
and is based on the most recent conceptualization of
MHL [14, 16]. When discussing the relationship between
knowledge and health behavior in the final paragraph of
this discussion section, MHPK will be discussed in the
context of Sørensen et al.’s conceptual model of HL. that third-year nursing students scored significantly higher
than adolescents on the measure of factors promoting
good mental health, as expected. Descriptive statistics for the MHPK-10 The adolescent population was used to generate descrip-
tive statistics for the MHPK-10. The mean score was
4.51 (SD = 0.54 Minimum = 0, Maximum = 5, 95%
CI = 4.29–4.53). The results of the MHPK-10 showed
that 19.33% of the student population had an insufficient
level of knowledge of factors promoting good mental
health (a mean score < 4 was used as a preliminary cut--
off for an insufficient level of knowledge since values 4
and 5 identifies the correct answer to each statement;
however, this cut-off must be further evaluated). In
terms of analyses using the MHPK-10, we suggest using
mean
scores
and
allowing
two
missing
items
per
respondent. Discussion In this study, we successfully developed a valid and reli-
able instrument that measures adolescents’ knowledge of
factors promoting good mental health. In promoting men-
tal health, there has been a shift from a problem-focused
approach emphasizing the prevention of psychological Bjørnsen et al. BMC Public Health (2017) 17:717 Page 8 of 10 Page 8 of 10 Strengths and limitations One strength of the current study is the large sample
size and high response rate. A sound psychometric
evaluation was performed to assess the MHPK-10 meas-
ure; the findings contribute to the field by enabling
future use and call for further development and valid-
ation of the instrument. However, the results should be
interpreted with some caution. The focus groups may
have been subject to self-selection bias. The instrument
may therefore be overly influenced by females’ opinions
on mental health, given the preponderance of girls in
the focus groups. However, males were represented and
contributed opinions and experiences in all focus groups
and in the expert panel. For the survey, teachers served
as administrators of the questionnaire and thus may
have influenced which classes had the opportunity to
participate in the study by serving as gatekeepers for
student participation. Factor structure The PCA yielded support for a one-factor solution of the
10-item version of the MHPK reflecting component one
(understanding how to obtain and maintain good mental
health [7]) of the four components included in the defin-
ition of MHL. A three-factor structure was also evaluated
considering the three dimensions of BPNT utilized to gen-
erate the items. The fit indices showed a slightly better fit
for the three-factor model than for the one-factor model. However, the covariance between the three factors was
high, and a one-factor solution corresponded better with
the instrument as a measure of one component of MHL. Furthermore, the dimension relatedness in the three-
factor solution was problematic and consisted of only two
items. Thus, the instrument was constructed as a one-
factor model with good to excellent factor loadings and a
good overall McDonald’s omega value corresponding to
the intended component of MHL. Finally, the MHPK-10 score distributions showed little
variance in the mean scores and hence demonstrate a
possible ceiling effect, indicating that the measure in its
current form does not sufficiently discriminate among
adolescents with high MHPK-10 levels. This ceiling
effect may cause difficulties in establishing the discrim-
inant validity of the scale. Implications and future research Future research should further refine the MHPK-10 to
be less intuitive, thus yielding more variance in the item
responses and reduce ceiling effects. Further testing of
the scale is needed to evaluate the cut-off values for suf-
ficient knowledge of factors important for good mental
health. Further validation of the instrument across sam-
ples and age groups is also needed. The implications for
adolescent mental health may include that the MHPK-
10 identifies mental health promotion areas with low
levels of knowledge in populations. Public health practi-
tioners could subsequently target their mental health
education toward the aspects with an identified need in
particular populations. The MHPK-10 also has the
potential
to
be
used
to
evaluate
mental
health- Development of the instrument have less knowledge, namely, upper secondary school stu-
dents. However, when examining the actual variance of
the results, the differences in mean scores were small. Both groups had acceptable levels of knowledge. This
finding can be interpreted as variations within the ac-
cepted level of knowledge that could be of clinical and
practical relevance in mental health promotion. At this
point, the instrument should not be used solely to detect
whether a population has sufficient knowledge of factors
promoting health but rather to identify the fraction of the
population lacking this knowledge and areas in need of
public mental health education. p
The approach used to develop the instrument followed
scientifically accepted principles [20]. The major challenge
was developing items that detected knowledge within the
scope of MHL in health promotion without being overly
intuitive. Intuitive items might have led to artificially high
mean scores, subsequently missing adolescents with low
knowledge of factors promoting good mental health. The
results were skewed, which may be because the population
had high levels of knowledge or because the scale was too
intuitive. It is challenging to cover all aspects of MHPK
because individuals’ conceptions of what is needed to
strengthen mental health vary. However, the items were
based on a solid theoretical foundation, adolescents’ opin-
ions and acknowledged expertise to ensure a solid
grounding for the instrument. Combined with the empir-
ically determined seven mental health rights [21], substan-
tial groundwork was established to ensure that the
instrument had a theoretical and empirical foundation to
measure factors promoting good mental health. Thus, the
MHPK-10 is considered a solid starting point for further
development and validation of a measure assessing one
component of MHL: knowledge of how to obtain and
maintain good mental health. Funding
Th
d This study was funded by the Research Council of Norway, grant number
238331, “Health Promotion – Worthwhile? Reorienting the Community
Health Care Services”, and by NTNU Norwegian University of Science and
Technology. Conclusion This study found support for a valid and reliable 10-item
one-factor measure of adolescents’ knowledge of factors pro-
moting good mental health. A rigorous evaluation of the
scale’s psychometric properties was performed, and satisfac-
tory internal consistency and construct validity were estab-
lished. The MHPK-10 is a solid first step toward creating a Evaluation of validity and reliability According to the results, the reliability of the instrument
was acceptable. The MHPK-10 demonstrated good in-
ternal validity and test-retest reliability. The test-retest
analyses were used to assess the consistency and sensitiv-
ity of the measure over time. The known-groups validity
test showed that the instrument was able to differentiate
between those who were expected to have greater know-
ledge of factors promoting good mental health based on
their university education and those who were expected to Bjørnsen et al. BMC Public Health (2017) 17:717 Page 9 of 10 Page 9 of 10 promoting education initiatives aimed at increasing
knowledge
of
factors
promoting
mental
health
to
improve and better tailor these initiatives. As MHL is
considered an outcome of mental health promotion
actions, every item in the MHPK-10 is considered ap-
plicable and translatable into public health practice; i.e.,
mental health education can be developed to improve
knowledge of any of the items [32]. The MHPK-10 iden-
tified approximately 20% of the student population as
having inadequate knowledge of factors promoting good
mental health. This finding corresponds well with estab-
lished numbers on adolescents’ mental health status;
specifically, 15–20% of Norwegian adolescents report
mental health issues [1]. An important next step is to
study the relationship between the MHPK-10 and men-
tal health, in particular, how levels of knowledge of fac-
tors promoting mental health are related to self-reported
mental health and mental health-promoting behavior. sound measure of the positive aspects of MHL and has the
potential to complement current measures of MHL for use
in health promotion. The results are particularly applicable
for guiding the development, targeting and evaluation of
public mental health education initiatives, with the main goal
of building a foundation for good mental health, wellbeing
and productive future lives for adolescents. The MHPK-10
measure provides a novel contribution and requires further
refinement and validation. Abbreviations BPNT: Basic psychological needs theory; MHL: Mental health literacy;
MHPK: Mental health-promoting knowledge Authors’ contributions
d
f
d h HNB and RR performed the quantitative and qualitative data collection and
prepared the data for analysis. UKM, MEBE and GAE obtained funding and
supervised the project. HNB initiated the scale development, conducted the
literature review, organized the expert panel and test-retest analysis, performed
the data analysis and drafted the manuscript. All authors provided input on the
manuscript and read and approved the final version. Ethics approval and consent to participate The study was approved by the Regional Committee for Medical and Health
Research Ethics (REK midt 2014/1996). All participants were informed that
participation was voluntary and anonymous through written letters, a video
made by the research group that was available on the school’s e-learning
platform, and oral information provided by teachers in each class prior to
distributing the questionnaires. Students aged 16 years and older gave
consent for participation by completing the questionnaire, and written
parental consent for students aged 15 was obtained. Acknowledgments It is important to consider the difference between adoles-
cents’ knowledge of the factors that promote mental
health and their possession of the skills to apply that
knowledge. With respect to the process of applying the
knowledge detected by the MHPK-10, Sørensen et al.’s HL
model is a relevant model. Sørensen et al.’s model intro-
duces competencies, knowledge and motivation of how to
access, understand, appraise and apply health-related
information as central in HL [4, 5]. According to the
model, health-related knowledge empowers people to par-
ticipate in health-promoting activities in communities [5],
e.g., in a school setting. The MHPK-10 instrument mea-
sures knowledge of factors important to obtain and main-
tain good mental health on an individual level for use in a
public health perspective. Although knowledge of these
factors or mental health literacy does not necessarily lead
to mental health-promoting behavior, we argue that know-
ledge is as a necessary foundation for making purposeful
health-promoting decisions, in line with Sørensen et al.’s
conceptual model of HL [5]. HL is known to affect health
behavior and consequently health outcomes [4], and we
expect MHL to have similar impact. Although knowledge
does not necessarily mean skills, again, we argue that
knowledge is fundamental for building skills to apply
knowledge and a necessary starting point for promoting
mental health among adolescents. The authors thank Mr. Kyrre Svarva for greatly assisting the research process
and sharing his pearls of wisdom, and Professor Arild Blekesaune for helping
calculating omega. We would also like to show our gratitude to the
adolescents for generously contributing their time and sharing their
thoughts and experiences. We also thank the expert panel and public health
nurses in Trondheim municipality for their collaboration. Additional file Additional file 1: MHPK-10 instrument. A copy of the 10-item MHPK
instrument that measures adolescents’ knowledge of how to obtain and
maintain good mental health. (PDF 105 kb) Availability of data and materials The raw data supporting the findings in this manuscript can be found at the
NTNU Norwegian University of Science and Technology, Department of
Public Health and Nursing, Trondheim, Norway and are available from the
corresponding author on reasonable request. Competing interests
Th
h
d
l
h Competing interests
The authors declare that they have no competing interests. p
g
The authors declare that they have no competing interests. Page 10 of 10 Page 10 of 10 Bjørnsen et al. BMC Public Health (2017) 17:717 Received: 19 April 2017 Accepted: 11 September 2017 27. Mehmetoglu M, Jakobsen TG. Applied statistics using Stata: a guide for the
social sciences. London: Sage; 2017. 28. Crutzen R, Peters GY. Scale quality: alpha is an inadequate estimate and factor-
analytic evidence is needed first of all. Health Psychol Rev. 2017;11:242–7. References 29. Cohen J. Statistical power analysis for the behavioral sciences. 2nd e
Hillsdale: Lawrence Earlbaum Associates; 1998. 1. FHI Norwegian Public Health Institute. Mental disorders among children
and adolescents in Norway. 2014. https://www.fhi.no/en/op/public-health-
report-2014/health%2D-disease/mental-health-children-adolescents/. Accessed 30 Mar 2017. 1. FHI Norwegian Public Health Institute. Mental disorders among children
and adolescents in Norway. 2014. https://www.fhi.no/en/op/public-health-
report-2014/health%2D-disease/mental-health-children-adolescents/. Accessed 30 Mar 2017. 30. Bowen N. Requesting and using modification indices in Mplus. 2017. http://
ssw.unc.edu/sswsig/sites/default/files/
Modification%20Indices%20in%20Mplus%20%28NK%20Bowen%29.pdf. Accessed 20 Feb 2017. 30. Bowen N. Requesting and using modification indices in Mplus. 2017. http://
ssw.unc.edu/sswsig/sites/default/files/
Modification%20Indices%20in%20Mplus%20%28NK%20Bowen%29.pdf. Accessed 20 Feb 2017. 2. Mulye TP, Park MJ, Nelson CD, Adams SH, Irwin CE Jr, Brindis CD. Trends in
adolescent and young adult health in the United States. J Adolesc Health. 2009;45:8–24. 2. Mulye TP, Park MJ, Nelson CD, Adams SH, Irwin CE Jr, Brindis CD. Trends in
adolescent and young adult health in the United States. J Adolesc Health. 2009;45:8–24. 31. Sancassiani F, Pintus E, Holte A, Paulus P, Moro MF, Cossu G, et al. Enhancing the emotional and social skills of the youth to promote their
wellbeing and positive development: a systematic review of universal
school-based randomized controlled trials. Clin Pract Epidemiol Ment
Health. 2015;11:21–40. 3. Kickbusch I. Health literacy: an essential skill for the twenty-first century. Health Educ. 2008;108:101–4. 3. Kickbusch I. Health literacy: an essential skill for the twenty-first century. Health Educ. 2008;108:101–4. 4. Barry MM, D'Eath M, Sixsmith J. Interventions for improving population
health literacy: insights from a rapid review of the evidence. J Health
Commun. 2013;18:1507–22. 32. Paakkari O, Torppa M, Kannas L, Paakkari L. Subjective health literacy:
development of a brief instrument for school-aged children. Scand J Public
Health. 2016;44:751–7. 5. Sørensen K, Van den Broucke S, Fullam J, Doyle G, Pelikan J, Slonska Z, et al. Health literacy and public health: a systematic review and integration of
definitions and models. BMC Public Health. 2012;12:80. 6. Wei Y, McGrath PJ, Hayden J, Kutcher S. Mental health literacy measures
evaluating knowledge, attitudes and help-seeking: a scoping review. BMC
Psychiatry. 2015;15:291. 7. Kutcher S, Wei Y, Coniglio C. Mental health literacy: past, present, and
future. Can J Psychiatr. 2016;61:154–8. 8. Jorm AF, Korten AE, Jacomb PA, Christensen H, Rodgers B, Pollitt P. “Mental
health literacy”: a survey of the public’s ability to recognise mental disorders
and their beliefs about the effectiveness of treatment. Med J Aust. 1997;166:182–6. 8. References Jorm AF, Korten AE, Jacomb PA, Christensen H, Rodgers B, Pollitt P. “Mental
health literacy”: a survey of the public’s ability to recognise mental disorders
and their beliefs about the effectiveness of treatment. Med J Aust. 1997;166:182–6. 9. Jorm AF. Mental health literacy: empowering the community to take action
for better mental health. Am Psychol. 2012;67:231–43. y
10. Jorm AF. Why we need the concept of “mental health literacy”. Health
Commun. 2015;30:1166–8. 11. Chambers D, Murphy F, Keeley HS. All of us? An exploration of the concept
of mental health literacy based on young people’s responses to fictional
mental health vignettes. Ir J Psychol Med. 2015;32:129–36. 12. Kutcher S, Wei Y, Costa S, Gusmão R, Skokauskas N, Sourander A. Enhancing
mental health literacy in young people. Eur Child Adolesc Psychiatry. 2016;25:567–9. 13. World Health Organization. Mental health action plan 2013–2020. 2013. http://apps.who.int/iris/bitstream/10665/89966/1/9789241506021_eng.pdf. Accessed 16 Jan 2017. 14. Jung H, von Sternberg K, Davis K. Expanding a measure of mental health
literacy: development and validation of a multicomponent mental health
literacy measure. Psychiatry Res. 2016;243:278–86. 15. O’Connor M, Casey L. The mental health Literacy Scale (MHLS): a new scale-
based measure of mental health literacy. Psychiatry Res. 2015;229:511–6. 16. Kutcher S, Bagnell A, Wei Y. Mental health literacy in secondary schools: a
Canadian approach. Child Adolesc Psychiatr Clin N Am. 2015;24:233–44. 17. Deci E, Vansteenkiste M. Self-determination theory and basic need
satisfaction: understanding human development in positive psychology. Ricerche Psicol. 2004;27:23–40. 18. Ng JY, Ntoumanis N, Thøgersen-Ntoumani C, Deci EL, Ryan RM, Duda JL, et
al. Self-determination theory applied to health contexts: a meta-analysis. Perspect Psychol Sci. 2012;7:325–40. Publisher’s Note 25. Satorra A, Bentler PM. A scaled difference chi-square test statistic for
moment structure analysis. Psychometrika. 2001;66:507–14. 5. Satorra A, Bentler PM. A scaled difference chi-square test statistic Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. moment structure analysis. Psychometrika. 2001;66:507–14. 26. Van de Schoot R, Lugtig P, Hox J. A checklist for testing me 26. Van de Schoot R, Lugtig P, Hox J. A checklist for testing measurement
invariance. Eur J Dev Psychol. 2012;9:486–92. 26. Van de Schoot R, Lugtig P, Hox J. A checklist
invariance. Eur J Dev Psychol. 2012;9:486–92. invariance. Eur J Dev Psychol. 2012;9:486–92. Received: 19 April 2017 Accepted: 11 September 2017 Received: 19 April 2017 Accepted: 11 September 2017 Submit your next manuscript to BioMed Central
and we will help you at every step: Submit your next manuscript to BioMed Central
and we will help you at every step: 19. Deci EL, Ryan RM. The “what” and “why” of goal pursuits: human needs and
the self-determination of behavior. Psychol Inq. 2000;11:227–68. • We accept pre-submission inquiries
• Our selector tool helps you to find the most relevant journal
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Submit your manuscript at
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and we will help you at every step: 20. Hinkin TR. A brief tutorial on the development of measures for use in
survey questionnaires. Organ Res Methods. 1998;1:104–21. 21. Holte A. De syv psykiske helserettighetene [The seven mental health rights]. 2016. http://www.psykologforeningen.no/publikum/velkommen-til-
psykologhjelp/de-syv-psykiske-helserettighetene. Accessed 18 Jan 2017. 21. Holte A. De syv psykiske helserettighetene [The seven mental health rights]. 2016. http://www.psykologforeningen.no/publikum/velkommen-til-
psykologhjelp/de-syv-psykiske-helserettighetene. Accessed 18 Jan 2017. 22. Kitzinger J. Qualitative research. Introducing focus groups. BMJ. 1995;311:
299–302. 22. Kitzinger J. Qualitative research. Introducing focus groups. BMJ. 1995;311:
299–302. 23. StataCorp. Stata statistical software: Release 14. College Station: StataCorp
LP; 2015. 23. StataCorp. Stata statistical software: Release 14. College Station: StataCorp
LP; 2015. 24. Tabachnick BG, Fidell LS. Using multivariate statistics. 6th ed. Essex: Pearson
Education Limited; 2014.
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|
English
| null |
Mildly Higher Serum Prolactin Levels Are Directly Proportional to Cumulative Pregnancy Outcomes in in-vitro Fertilization/Intracytoplasmic Sperm Injection Cycles
|
Frontiers in endocrinology
| 2,020
|
cc-by
| 4,901
|
Mildly Higher Serum Prolactin Levels
Are Directly Proportional to
Cumulative Pregnancy Outcomes in
in-vitro Fertilization/Intracytoplasmic
Sperm Injection Cycles Duoduo Zhang 1, Xi Yuan 2, Jingran Zhen 1*, Zhengyi Sun 1, Chengyan Deng 1 and Qi Yu 1 Keywords: prolactin, GnRH agonist protocol, IVF, ICSI, cumulated clinical pregnancy rate, cumulated live birth rate Hyperprolactinemia has long been considered detrimental to fertility due to irregularity
of ovulation. Whether mild hyperprolactinemia should be corrected before initiating
an in-vitro fertilization/intracytoplasmic sperm injection cycle (IVF/ICSI) has not been
determined; this study aimed to examine how different levels of prolactin affect IVF
outcomes. A total of 3,009 patients with basal prolactin level <50 ng/mL undergoing
IVF/ICSI cycles for tubal or male factors were recruited in this study. Patients diagnosed
with anovulation owing to polycystic ovarian syndrome or hyperandrogenism were
ruled out. Pregnancy outcomes were compared between patients with basal prolactin
levels higher or lower than the median level of prolactin (16.05 ng/mL). Multifactor
analyses were carried out among four subgroups depending on different prolactin levels. Repeated-measures analysis of variance was used to explore the relationship between
the ascending trend of prolactin levels over ovarian stimulation and the corresponding
cumulative pregnancy outcomes. There were significantly higher numbers of oocytes
(9 vs. 8, P = 0.013) and embryos (6 vs. 5, P = 0.015) in patients with basal prolactin
higher than 16.05 ng/mL. Basal prolactin higher than 30 ng/mL was positively related to
cumulative clinical pregnancy, and a level higher than 40 ng/mL was a good indicator for
the cumulative live birth rate. Throughout ovarian stimulation, the prognosis of pregnancy
improved with increasing prolactin levels. Patients with better cumulated pregnancy
outcomes had significantly higher prolactin levels as well as a profoundly increasing
trend during the stimulating process than those who did not conceive. For patients
who underwent the gonadotropin-releasing hormone agonist long protocol IVF/ICSI
treatment, a slightly higher prolactin level during the controlled ovarian hyperstimulation
protocol was a positive indicator for cumulated pregnancy/live birth rates. Edited by:
John Lui Yovich,
Pivet Medical Center, Australia
Reviewed by:
Joaquin Llacer,
Instituto Bernabeu, Spain
Ludwig Wildt,
Innsbruck Medical University, Austria
*Correspondence:
Jingran Zhen
zhen_amy@126.com Edited by:
John Lui Yovich,
Pivet Medical Center, Australia Reviewed by:
Joaquin Llacer,
Instituto Bernabeu, Spain
Ludwig Wildt,
Innsbruck Medical University, Austria *Correspondence:
Jingran Zhen
zhen_amy@126.com *Correspondence:
Jingran Zhen
zhen_amy@126.com Specialty section:
This article was submitted to
Reproduction,
a section of the journal
Frontiers in Endocrinology
Received: 02 December 2019
Accepted: 17 July 2020
Published: 28 August 2020
Citation:
Zhang D, Yuan X, Zhen J, Sun Z,
Deng C and Yu Q (2020) Mildly Higher
Serum Prolactin Levels Are Directly
Proportional to Cumulative Pregnancy
Outcomes in in-vitro
Fertilization/Intracytoplasmic Sperm
Injection Cycles. Front. Endocrinol. 11:584. ORIGINAL RESEARCH
published: 28 August 2020
doi: 10.3389/fendo.2020.00584 ORIGINAL RESEARCH
published: 28 August 2020
doi: 10.3389/fendo.2020.00584 ORIGINAL RESEARCH
published: 28 August 2020
doi: 10.3389/fendo.2020.00584 doi: 10.3389/fendo.2020.00584 Specialty section:
This article was submitted to
Reproduction,
a section of the journal
Frontiers in Endocrinology Received: 02 December 2019
Accepted: 17 July 2020
Published: 28 August 2020
Citation: INTRODUCTION the menstrual cycle before pituitary downregulation by GnRHa,
which we marked as T0. On the 2nd day of the next menstrual
cycle patients started receiving recombinant human follicle-
stimulating hormone (rFSH; Gonal-F, Merck-Serono) at an
individualized dose adjusted based on patient ovarian response. Final oocyte maturation was triggered by intramuscular injection
of 250 µg recombinant human chorionic gonadotropin (hCG;
Ovitrelle, Merck-Serono) and the 2nd hormone sample taken
on that morning was defined as T1. The third hormone
samples were collected on the morning after administering
hCG, and that time was defined as T2. After that, oocytes
were retrieved by ultrasound-guided transvaginal aspiration
at around 36 h after hCG trigger. Intramuscular injection of
40 mg P was administered daily for luteal phase support. Embryo development was evaluated daily until the fresh transfer
of cleavage stage embryos (Day 3). Embryos were evaluated
following a standardized scoring system (4). After fresh embryo
transfer, the remaining embryos were cultured to blastocysts (Day
5 or 6) before vitrification. Frozen-thawed embryo transfer may
be applied to either artificial or natural cycles. Prolactin (PRL) is known as a stimulator of the proliferation
and differentiation of mammary cells for lactation. The
primary regulator for PRL pituitary secretion is dopamine
via hypothalamic inhibitory signals, and this constitutes the
pharmacological basis for hyperprolactinemia treatment (1). In addition, PRL, as a stress hormone, is actively involved
in
metabolism,
electrolyte
transport,
angiogenesis,
and
immunity (1). y ( )
Serum PRL is ordinarily under 25 ng/L; a level above the
normal upper limit is diagnosed as hyperprolactinemia as long
as the sample is obtained without excessive stress challenges
before venipuncture. Hyperprolactinemia is a well-established
cause of hypogonadotropic hypogonadism (2); PRL acts on
kisspeptin-1 neurons expressing the PRL receptor (PRL-R) and
is responsible for decreased kisspeptin-1 and GnRH secretion,
leading to anovulation (3). Dopamine agonists are widely used
for suppression of serum PRL and resumption of ovulation in
infertile women with hyperprolactinemia seeking to conceive
naturally. However, follicle genesis in women undergoing in-
vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI)
treatment depends on exogenous gonadotropins, instead of
endogenous ones, and luteal phase support is always ensured by
sufficient progesterone (P) administration. Is it really necessary to
suppress the slightly higher PRL? Or is there a proper PRL range
to optimize IVF outcomes? INTRODUCTION We hypothesize an isolating mildly
increasing PRL level if these women have no organic lesions
such as prolactinoma would not negatively affect cumulated IVF
pregnancy outcomes. Serum FSH, luteinizing hormone (LH), PRL, estrogen
(E2), and P levels were measured by the automated Elecsys
Immunoanalyzer (Beckmann, USA). The inter-assay coefficients
of variation were <5 and <10% for E2 and P and <8% for FSH,
LH, and PRL, respectively. Clinical pregnancy was defined as intrauterine pregnancy with
at least one fetus with a positive heartbeat at 6 weeks of gestation
or later. Live birth was defined as the delivery of a live-born child
at >28 weeks of gestation. The clinical pregnancy rate (CPR)
and live birth rate (LBR) referred to the cumulated outcome
after transferring all embryos from the studied stimulating cycle. Secondary outcomes included the number of oocytes retrieved,
mature oocytes, two-pronuclear zygotes, and embryos. Citation: August 2020 | Volume 11 | Article 584 Frontiers in Endocrinology | www.frontiersin.org 1 A Retrospective Cohort Study Zhang et al. Abbreviations: BMI, body mass index; CPR, clinical pregnancy rate; COH,
controlled ovarian hyperstimulation; FSH, follicle stimulating hormone; GnRHa,
gonadotropin releasing hormone agonist; hCG, human chorionic gonadotropin;
LBR, live birth rate; ICSI, intracytoplasmic sperm injection; IVF, in vitro
fertilization; PRL, prolactinl; rFSH, recombined human FSH. MATERIALS AND METHODS The data analysis was carried out using SPSS 24.0 statistical
analysis software (IBM Inc., USA). The normality of distribution
of continuous variables was assessed using the Kolmogorov-
Smirnov test (cutoffat P = 0.01). Descriptive statistics for
continuous variables are reported as the mean ± standard
deviation (SD). Categorical variables were described using
frequency distributions and are presented as frequency and
percentage (%). The t-test for independent samples or the Mann-
Whitney U-test were used as appropriate to compare continuous
variables by group. The chi-squared test was used to compare
categorical variables by group. Repeated-measures analysis of
variance was used for measuring repeated longitudinal data. A
logistic regression model of the two groups (PRL ≤16.05 vs. PRL > 16.05 ng/mL) was developed to additionally adjust for
age, body mass index (BMI), basal FSH, basal E2, and duration
of infertility. Odds ratios were estimated with 95% confidence
intervals. All tests were two-sided and considered significant
at P < 0.05. This retrospective study included all women who underwent
IVF/ICSI treatment for tubal or male infertility with the
gonadotropin-releasing hormone agonist (GnRHa) long protocol
at Peking Medical College Hospital (PUMCH) between 1st
July 2014 and 31st March 2018. Patients diagnosed with
anovulatory diseases like polycystic ovarian syndrome (PCOS) or
hyperandrogenism were not included. The study was approved
by the Ethics Committee of PUMCH (No. S-K601). Exclusion
criteria were: Patients with serum P level ≥1.5 ng/mL during
a controlled ovarian hyperstimulation protocol (COH), patients
undergoing a freeze-all strategy, egg-donating cycles, basal PRL
level ≥50 ng/mL, previous diagnosis of pituitary lesions, or
abnormal thyroid functions. All patients included had to have
used up all fresh or vitrified embryos generated from the
stimulating cycle by the time of the study in order to analyze the
cumulative pregnancy outcomes. Sexual hormone levels were tested at three individual times
for each patient. The first basal one was on the 2nd day of DISCUSSION In this retrospective study, we analyzed the relationship between
basal PRL levels, as well as their increasing tendency, and
pregnancy outcomes of IVF/ICSI treatments for tubal/male
factor infertility. Hyperprolactinemia has long been considered
detrimental to fertility due to its effect on blocking LH secretion,
leading to anovulation, or luteolysis (2). However, in IVF, oocyte
maturation is induced by hCG trigger, and sufficient luteal phase
support is guaranteed by progesterone supplements. Therefore,
IVF procedures provide an ideal opportunity to observe the
potential effect of PRL on reproduction in comparison to
suppression of gonadotropins. This study was designed to answer
two main questions: [1] Are cumulative pregnancy outcomes
better in women with higher basal PRL levels when it is under
50 ng/mL?; [2]. In cycles with better pregnancy outcomes, will
there be greater increase of PRL throughout ovarian stimulation
(basal state, hCG day, and the day after hCG triggering)? In this retrospective study, we analyzed the relationship between
basal PRL levels, as well as their increasing tendency, and
pregnancy outcomes of IVF/ICSI treatments for tubal/male
factor infertility. Hyperprolactinemia has long been considered
detrimental to fertility due to its effect on blocking LH secretion,
leading to anovulation, or luteolysis (2). However, in IVF, oocyte
maturation is induced by hCG trigger, and sufficient luteal phase
support is guaranteed by progesterone supplements. Therefore,
IVF procedures provide an ideal opportunity to observe the
potential effect of PRL on reproduction in comparison to
suppression of gonadotropins. This study was designed to answer
two main questions: [1] Are cumulative pregnancy outcomes
better in women with higher basal PRL levels when it is under
50 ng/mL?; [2]. In cycles with better pregnancy outcomes, will
there be greater increase of PRL throughout ovarian stimulation
(basal state, hCG day, and the day after hCG triggering)? We further divided all patients into five groups according
to different basal PRL levels: Group I with PRL 0–9.9 ng/ml,
Group II, 10–19.9 ng/ml; Group III, 20–29.9 ng/ml; Group IV,
30–39.9 ng/ml; and Group V, 40–49.9 ng/ml (Table 2). Nearly
half of the patients were distributed in Group II. Therefore, we
applied Group II as a dummy variable. Other factors including
age, basal FSH, rFSH starting dose, total consumption of rFSH,
basal E2, and BMI were entered into the multifactor analysis. It turned out the last two factors (basal E2 and BMI) were not
statistically significant. RESULTS A total of 3,009 patients fulfilling the criteria were recruited in
the study, of whom, 2098 underwent IVF cycles and 911 received August 2020 | Volume 11 | Article 584 Frontiers in Endocrinology | www.frontiersin.org 2 Zhang et al. A Retrospective Cohort Study FIGURE 1 | Flowchart regarding patients’ inclusion and exclusion. COH, controlled ovarian hyperstimulation; P, progesterone; PRL, prolactin. FIGURE 1 | Flowchart regarding patients’ inclusion and exclusion. COH, controlled ovarian hyperstimulation; P, progesterone; PRL, prolactin. clinical pregnancy and 1,381 cases with cumulated live birth. The PRL levels of patients with positive pregnancy outcomes
were significantly higher at all measurement points than those
of patients with negative results. Moreover, a sharper spike was
observed in groups with positive clinical pregnancy or live birth. ICSI (Figure 1). Their demographic characters were shown in
Table 1. To explore the relationship between basal PRL (T0) and
pregnancy outcomes, we divided patients into two groups by
median PRL level (≤16.05 vs. >16.05 ng/mL). The two groups
were compared in terms of baseline characteristics and pregnancy
outcomes (Table 1). Patients with basal PRL >16.05 ng/m had
slightly but significantly more oocytes retrieved, MII oocytes,
fertilization, and embryos (P < 0.05). No statistically significant
differences in cumulated CPR and LBR were detected between
the two groups. Frontiers in Endocrinology | www.frontiersin.org DISCUSSION The results revealed that a higher basal
PRL was related to a better rate of cumulated clinical pregnancy
and live birth. In order to analyze the fluctuation of PRL levels through
COH in the GnRHa long protocol cycle and to examine whether
the change in PRL level is related to IVF pregnancy outcomes,
we compared the PRL levels between patients with positive
and negative pregnancy results on T0 (basal status), T1 (end
of follicular stage), and T2 (early initiation of luteal phase)
(Tables 3, 4). There were 1,585 cases with positive cumulated y
y
gg
g
Around 85% of PRL molecules in circulation are 23
kDa monomers, which is the major bioactive form of PRL. Approximately a quarter of patients with hyperprolactinemia
are
shown
to
have
macroprolactinemia. Women
with August 2020 | Volume 11 | Article 584 3 A Retrospective Cohort Study Zhang et al. TABLE 1 | Comparisons of baseline characteristics and pregnancy outcomes between patients with PRL ≤16.05 or > 16.05 ng/mL. PRL ≤16.05 ng/mL (1506)
PRL > 16.05 ng/mL (1503)
P
Age (years)
35.215
4.391
34.613
4.257
0.079
BMI (kg/m2)
22.364
3.186
21.835
3.063
0.102
Duration of infertility (years)
4
2, 6
4
3, 6
0.978
Basal sexual hormone
FSH (IU/L)
7.130
5.770, 9.000
7.190
5.930, 9.010
0.343
LH (IU/L)
3.655
2.520, 5.093
3.840
2.730, 5.230
0.193
E2 (pg/mL)
45.910
34.985, 58.730
46.240
35.320, 60.260
0.344
rFSH starting dose (ampoule)
4
3, 4
4
3, 4
0.608
Oocytes retrieved
8
5, 11
9
5, 12
0.013
MII oocytes
6
3, 10
7
4, 10
0.008
Zygotes
5
3, 9
6
3, 10
0.012
Embryos
5
3, 9
6
3, 10
0.015
Cumulative CPR
50.1%
755/1506
53.9%
810/1503
0.097
Cumulative LBR
44.5%
670/1506
47.3%
711/1503
0.065
Continuous variables following the normal distribution are presented as the mean (SD); non-normal distribution parameters are presented as the median (quartile); categorical variables
are presented as percentages (with their frequencies). ons of baseline characteristics and pregnancy outcomes between patients with PRL ≤16.05 or > 16.05 ng/mL. Continuous variables following the normal distribution are presented as the mean (SD); non-normal distribution parameters are presented as the median (quartile); categorical variables
are presented as percentages (with their frequencies). TABLE 2 | Multifactor analysis of the relationship between basal PRL and pregnancy outcomes. Frontiers in Endocrinology | www.frontiersin.org DISCUSSION Time point
Group
Positive
Negative
P
for
outcomes
outcomes
comparison
(ng/mL)
(ng/mL)
11PRL (T1–T0) Cumulative clinical
pregnancy
13.297 (15.096) 17.416 (15.959) < 0.001
Cumulative live birth
13.719 (15.330) 17.528 (15.852) < 0.001
12PRL (T2–T1) Cumulative clinical
pregnancy
2.534 (9.962)
1.720 (10.565)
0.030
Cumulative live birth
2.572 (9.990)
1.555 (10.611)
0.007
Continuous variables following the normal distribution are presented as the mean (SD). level, and the CPR did improve compared to that in the controls
(10.1 vs. 27.2% P < 0.05) owing to the significantly increased
PRL (13). In addition to its effect on oogenesis and embryogenesis,
PRL also boosts other physiological reproductive activities. When
either PRL genes or PRL-receptor genes were knocked out, a
profound decrease in progesterone levels was noticed in the luteal
phase of mice (11); moreover, the corpus luteum underwent early
degradation 2 days after mouse intercourse (14). We revealed that
a dramatic surge of PRL after luteinization was associated with
better cumulated IVF/ICSI outcomes. This is consistent with the
promoting effect of PRL on luteal function (15). PRL stimulates
the long chain receptor in the luteinized cells to activate
the Jak2/STAT5 pathway and suppress 20-α-hydroxysteroid
dehydrogenase, subsequently spurring progesterone production. Meanwhile, multiple vascular endothelial growth factors are
secreted into the ovaries to accelerate the vascularization of the
corpus luteum when the PRL short chain receptor is stimulated. A human study by Daly et al. raised the concern that mid-
luteal PRL levels were the lowest (15.0 ± 11.7 ng/mL) in
women with early pregnancy loss compared to those who were
infertile or expected to conceive normally (16). Furthermore,
PRL acts on the adaptive immune system. PRL receptors are
widely expressed on the surface of CD4+ T cells and B cells. Once stimulated, inflammatory factors such as interleukin-2 and
interferon-gamma would be suppressed (17). This process might
allow an immune privilege status between the maternal-fetal
interface leading to a smoother pregnancy. associated with higher embryo quality (6). In our study, PRL
levels were noted to increase throughout the COH. Unlike
the high PRL levels because of ovarian stimulation, pre-
existing hyperprolactinemia before IVF/ICSI treatment puzzles
physicians the most. Doldi et al. prescribed cabergoline as
pretreatment to women with hyperprolactinemia until egg-
retrieval. Thus, the PRL levels were significantly lower than
those of the control group who did not receive cabergoline. DISCUSSION However, there was no improvement of CPR in patients treated
with cabergoline adding the effect of rFSH consumption (38.1 ±
18.2 vs. 43.9 ± 28.5 ampoule; P < 0.05), lower MII oocyte rate
(87.9 vs. 80.4%; P < 0.05), and fertilization rate (70.8 vs. 60.8%;
P < 0.03) (7). It is known that better IVF/ICSI outcomes are observed
in patients with higher PRL levels in either the basal state
or during COH. According to the present findings, the group
with basal PRL level > 16.05 ng/mL experienced a surge in the
numbers of oocytes, MII oocytes, zygotes, and embryos. Previous
research by Mendoza et al. discovered that higher basal PRL
levels are related to larger numbers of mature oocytes and good
quality embryos (8), suggesting that PRL plays a role in oocyte
maturation as well as embryonic development. Oogenesis is
a complicated process involving oocytes and the granular cell
cumulus actively exchanging signals within the circulating body
fluid. Nakamura et al. reported that PRL receptor-knocked-out
mice can only produce eggs with intact germinal vesicles (9). In contrast, higher mature rates were found when exogenous
PRL was added to pre-antral follicle cultures of the IVF system
(10). It could be a possible hypothesis that a certain PRL level
guarantees the accomplishment of meiosis. Moreover, in the PRL
receptor in deprived mice, there was a sharp decrease of the
fertilization rate; most of the zygotes underwent retardation,
and only 19% developed to blastocysts (11). PRL participates
in embryo implantation via BRCA1, a protein expressed on
the surface of the trophoblast cells. As the PRL concentration
gradually increased in the pre-antral follicle culture (0, 10, 20
mIU/mL), BRCA1 expression also increased (12). Although there
was no statistical significance, there was an increasing trend of the
implantation rate from 47.0% in the control population to 56.1%
when cultured with 20 mIU/mL PRL (12). Since PRL improved
oogenesis and embryonic development, some researchers have
tried to improve the IVF outcomes by prescribing bromocriptine
to patients with a history of recurrent implantation failure until
the initiation day of rFSH. Therefore, PRL rebounded to a higher g
g
y
The major limitation of our study is that no causal
relationship between PRL and IVF pregnancy outcomes could be
inferred due to the study’s retrospective nature. In multifactor
regression, we found that the pregnancy outcomes became
better as PRL increased. DISCUSSION Outcomes
Group
Basal PRL (ng/mL)
%
Frequency (3,009 in total)
P
OR
95% CI
Cumulative clinical pregnancy
I
0–9.9
13.5
407
0.047
0.858
0.683, 0.879
II
10–19.9
56.1
1,689
–
1.000
–
III
20–29.9
21.8
655
0.569
0.997
0.823, 1.207
IV
30–39.9
6.4
192
0.046
1.281
1.030, 1.764
V
40–49.9
2.2
66
0.039
1.639
1.247, 2.837
Cumulative live birth
I
0–9.9
13.5
407
0.047
0.871
0.691, 0.997
II
10–19.9
56.1
1,689
–
1.000
–
III
20–29.9
21.8
655
0.354
1.030
0.850, 1.247
IV
30–39.9
6.4
192
0.341
1.139
0.830, 1.562
V
40–49.9
2.2
66
0.008
1.916
1.115, 3.290
Categorical variables are presented as percentages (with their frequencies), OR, and 95% CI. The clinical pregnancy rate and live birth rate are additionally adjusted for age, bFSH, rFSH
starting dose, and total consumption of rFSH. TABLE 2 | Multifactor analysis of the relationship between basal PRL and pregnancy outcomes. TABLE 3 | Comparison of serum PRL levels between different pregnancy outcomes at different time points by repeated-measures analysis of variance. Pregnancy outcomes
T0 (ng/mL)
T1 (ng/mL)
T2 (ng/mL)
Cumulative clinical pregnancy
16.21 (12.34, 22.05)
32.7 (23.92, 43.86)
33.16 (24.38, 46.69)
No clinical pregnancy
15.85 (11.62, 21.15)
27.46 (19.29, 38.76)
30.12 (20.31, 42.06)
P-Value
0.011
< 0.001
< 0.001
Cumulative live birth
16.25 (12.34, 22.08)
33.06 (24.31, 43.82)
33.45 (24.61, 46.475)
No live birth
15.85 (11.74, 21.23)
27.84 (19.56, 39.47)
30.31 (20.67, 42.42)
P-Value
0.020
< 0.001
< 0.001
Non-normal distribution parameters are presented as median (quartile). Non-normal distribution parameters are presented as median (quartile). research should study macroprolactin and the proportion of
active PRL levels. macroprolactinemia may have no symptoms despite their
elevated serum PRL levels due to inactive macroprolactin (5). That is to say, some asymptomatic hyperprolactinemia may
be caused by macroprolactinemia; thus, such patients may not
need dopamine agonist administration before IVF treatment. However, macroprolactin was not measured in our study. Future Kamel et al. found that women who conceived had a
remarkable increase of PRL compared to women who did
not conceive, supporting the variation we found between T2
vs. T1 and T1 vs. T0. Additionally, higher PRL levels were August 2020 | Volume 11 | Article 584 Frontiers in Endocrinology | www.frontiersin.org 4 A Retrospective Cohort Study Zhang et al. TABLE 4 | Absolute difference of serum PRL between different time points. TABLE 4 | Absolute difference of serum PRL between different time points. Frontiers in Endocrinology | www.frontiersin.org August 2020 | Volume 11 | Article 584 REFERENCES 11. Ormandy CJ, Camus A, Barra J, Damotte D, Lucas B, Buteau H, et al. Null mutation of the prolactin receptor gene produces multiple reproductive
defects in the mouse. Genes Dev. (1997) 11:167–78. doi: 10.1101/gad. 11.2.167 1. Freeman ME, Kanyicska B, Lerant A, Nagy G. Prolactin: structure,
function, and regulation of secretion. Physiol Rev. (2000) 80:1523–
631. doi: 10.1152/physrev.2000.80.4.1523 1. Freeman ME, Kanyicska B, Lerant A, Nagy G. Prolactin: structure,
function, and regulation of secretion. Physiol Rev. (2000) 80:1523–
631. doi: 10.1152/physrev.2000.80.4.1523 12. Takeuchi M, Seki M, Furukawa E, Takahashi A, Saito K, Kobayashi
M, et al. Improvement of implantation potential in mouse blastocysts
derived from ivf by combined treatment with prolactin, epidermal
growth factor and 4-hydroxyestradiol. Mol Hum Reprod. (2017) 23:557–
70. doi: 10.1093/molehr/gax035 2. Melmed S, Casanueva FF, Hoffman AR, Kleinberg DL, Montori VM, Schlechte
JA, et al. Diagnosis and treatment of hyperprolactinemia: an endocrine
society clinical practice guideline. J Clin Endocrinol Metab. (2011) 96:273–
88. doi: 10.1210/jc.2010-1692 3. Kokay
IC,
Petersen
SL,
Grattan
DR. Identification
of
prolactin-
sensitive
gaba
and
kisspeptin
neurons
in
regions
of
the
rat
hypothalamus involved in the control of fertility. Endocrinology. (2011)
152:526–35. doi: 10.1210/en.2010-0668 13. Moride N, Kuwahara A, Yamashita M, Tanaka Y, Matsuzaki T, Yasui T, et
al. Does the bromocriptine-rebound method improve embryo quality? J Med
Invest. (2011) 58:63–66. doi: 10.2152/jmi.58.63 14. Bachelot A, Beaufaron J, Servel N, Kedzia C, Monget P, Kelly PA,
et al. Prolactin independent rescue of mouse corpus luteum life span:
identification of prolactin and luteinizing hormone target genes. Am
J Physiol Endocrinol Metab. (2009) 297:E676–84. doi: 10.1152/ajpendo. 91020.2008 4. Lundin K, Ahlstrom A. Quality control and standardization of embryo
morphology scoring and viability markers. Reprod Biomed Online. (2015)
31:459–71. doi: 10.1016/j.rbmo.2015.06.026 4. Lundin K, Ahlstrom A. Quality control and standardization of embryo
morphology scoring and viability markers. Reprod Biomed Online. (2015)
31:459–71. doi: 10.1016/j.rbmo.2015.06.026 5. Shimatsu
A,
Hattori
N. Macroprolactinemia:
diagnostic,
clinical,
and
pathogenic
significance. Clin
Dev
Immunol. (2012)
2012:167132. doi: 10.1155/2012/167132 15. Sangeeta Devi Y, Halperin J. Reproductive actions of prolactin mediated
through short and long receptor isoforms. Mol Cell Endocrinol. (2014)
382:400–10. doi: 10.1016/j.mce.2013.09.016 6. Kamel A, Halim AA, Shehata M, AlFarra S, El-Faissal Y, Ramadan W, et al. Changes in serum prolactin level during intracytoplasmic sperm injection,
and effect on clinical pregnancy rate: a prospective observational study. BMC
Pregnancy Childbirth. (2018) 18:141. doi: 10.1186/s12884-018-1783-4 16. DISCUSSION Nevertheless, the power of the test
may be compromised due to the significantly different number
of patients in each subgroup and the number of patients
with hyperprolactinemia decrease with increasing basal PRL
levels. Reasonably, the beneficial effect of PRL cannot continue
permanently and constantly rising, and there should be an
inflection point of PRL level beyond which, the advantageous
effect on IVF/ICSI pregnancy outcomes would become harmful. However, in clinical practice, physicians are prone to prescribe
dopamine agonists to patients with high PRL level > 50 ng/mL
before entering a cycle; thus, we could not recruit such patients. Consequently, the inflection point could not be illustrated by
our recruited sample. In this study, we targeted mainly at
tubal or male factor infertility. Particularly we avoided including
anovulation or endometriosis because these diseases possibly
interfere with ovarian reserve or HPO axis and, in turn, affect
the PRL status. For example, PCOS or hyperandrogenemia
was both sorted to anovulatory disorders in our center and
excluded. However, if a patient had not meet the full diagnosis
criteria of PCOS, but merely demonstrated either a polycystic
ovarian morphology or very mild hyperandrogenemia which
appear not to interfere with regular ovulation, she could still
be included as long as she was sorted as tubal or male
factor infertility. This could lead to a potential bias since
PCOS or hyperandrogenemia will slightly increase the PRL
level. Another drawback of this study is we merely employed August 2020 | Volume 11 | Article 584 Frontiers in Endocrinology | www.frontiersin.org August 2020 | Volume 11 | Article 584 Frontiers in Endocrinology | www.frontiersin.org 5 Zhang et al. A Retrospective Cohort Study AUTHOR CONTRIBUTIONS DZ analyzed the data and drafted the manuscript. XY designed
the study and revised the manuscript. DZ and XY did a major and
equal contribution to this work. ZS was responsible for the data
acquisition. CD analyzed and interpreted the data. QY edited the
manuscript. JZ provided final approval for the submitted version
and is responsible for the whole work. All authors have critically
reviewed and approved the final submitted version. ETHICS STATEMENT The studies involving human participants were reviewed and
approved by the Ethics Committee of PUMCH (No. S-K601). The
patients/participants provided their written informed consent to
participate in this study. In conclusion, for patients receiving IVF/ICSI treatment
with a basal PRL level within the range of 0–50 ng/mL, higher
PRL levels were associated with higher numbers of oocytes,
mature oocytes, zygotes, and embryos. Both the cumulative
CRP and LBR increased with increasing PRL levels. There
was a remarkable surge of PRL level from the basal status
to the next day after hCG injection. The beneficial effect
of PRL on pregnancy outcomes may be attributed to the
facilitation of oogenesis and embryonic development, as well
as the improvement of luteal function. Hence, in clinical
settings, when physicians encounter a patient with asymptomatic
hyperprolactinemia planning IVF/ICSI treatment, the serum
PRL level may be not suppressed to an extremely low level if
organic lesions are excluded. DATA AVAILABILITY STATEMENT basal FSH as the major ovarian reserve indicator. As we
know, ovarian reserve markers are closely related to the
number of oocytes retrieved as well as the CCPR and CLBR. However, our center has not initiated universal AMH test until
2019, and the data of AFC are not uniformly documented. Luckily we will have had enough AMH data to analyze in
foreseeable future. The datasets generated for this study are available on request to
the corresponding author. REFERENCES Daly DC, Maslar IA, Rosenberg SM, Tohan N, Riddick DH. Prolactin
production by luteal phase defect endometrium. Am J Obstet Gynecol. (1981)
140:587–91. doi: 10.1016/0002-9378(81)90237-4 17. Chavez-Rueda K, Hernandez J, Zenteno E, Leanos-Miranda A, Legorreta-
Haquet MV, Blanco-Favela F. Identification of prolactin as a novel
immunomodulator on the expression of co-stimulatory molecules and
cytokine secretions on t and b human lymphocytes. Clin Immunol. (2005)
116:182–91. doi: 10.1016/j.clim.2005.03.013 7. Doldi N, Papaleo E, De Santis L, Ferrari A. Treatment versus no
treatment
of
transient
hyperprolactinemia
in
patients
undergoing
intracytoplasmic sperm injection programs. Gynecol Endocrinol. (2000)
14:437–41. doi: 10.3109/09513590009167716 8. Mendoza
C,
Cremades
N,
Ruiz-Requena
E,
Martinez
F,
Ortega
E,
Bernabeu
S,
et
al. Relationship
between
fertilization
results
after
intracytoplasmic
sperm
injection,
and
intrafollicular
steroid,
pituitary hormone and cytokine concentrations. Hum Reprod. (1999)
14:628–35. doi: 10.1093/humrep/14.3.628 Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest. 9. Nakamura E, Otsuka F, Inagaki K, Miyoshi T, Yamanaka R, Tsukamoto
N, et al. A novel antagonistic effect of the bone morphogenetic protein
system on prolactin actions in regulating steroidogenesis by granulosa cells. Endocrinology. (2010) 151:5506–18. doi: 10.1210/en.2010-0265 Copyright © 2020 Zhang, Yuan, Zhen, Sun, Deng and Yu. This is an open-access
article distributed under the terms of the Creative Commons Attribution License (CC
BY). The use, distribution or reproduction in other forums is permitted, provided
the original author(s) and the copyright owner(s) are credited and that the original
publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these
terms. 10. Kiapekou E, Loutradis D, Mastorakos G, Bletsa R, Beretsos P, Zapanti E,
et al. Effect of prl on in vitro follicle growth, in vitro oocyte maturation,
fertilization and early embryonic development in mice. Cloning Stem Cells. (2009) 11:293–300. doi: 10.1089/clo.2008.0046 August 2020 | Volume 11 | Article 584 Frontiers in Endocrinology | www.frontiersin.org Frontiers in Endocrinology | www.frontiersin.org 6
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https://openalex.org/W2096861214
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http://www.scielo.br/pdf/rbf/v25n3/18668.pdf
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Portuguese
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Procedimentos para o armazenamento de sementes de Poncirus trifoliata (L.) Raf.
|
Revista brasileira de fruticultura
| 2,003
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cc-by
| 2,984
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1 (Trabalho 055/2003). Recebido: 31/03/2003. Aceito para publicação: 01/10/2003. Financiado pelo MAPA e CNPq.
2 Eng. Agr., D.S., Pesquisador da Embrapa Clima Temperado, Cx.P. 406, 96001-970, Pelotas, RS. Bolsista do CNPq. Tel. (53) 275 8100. E-mail
rpedroso@cpact.embrapa.br.
3 Eng. Agr., D.S., Pesquisador da Embrapa Clima Temperado. Tel. (53) 275 8100. E-mail: wbscivit@cpact.embrapa.br.
4 Bolsista DTI do CNPq. Tel. (53) 275 8100. E-mail: eradmann@hotmail.com. INTRODUÇÃO Diversos métodos têm sido propostos para o armazenamento
de sementes de ‘Trifoliata’, destacando-se o tratamento físico com água
quente, a secagem, o tratamento químico com fungicidas e a redução da
temperatura do ambiente de estocagem (Koller et al., 1993). Esses
fundamentam-se na redução da atividade metabólica da semente e da
velocidade de multiplicação dos microrganismos contaminantes
(Barbedo & Bilia, 1998). A despeito de já se dispor de informações
sobre o assunto, muitas dúvidas persistem sobre os procedimentos
mais adequados para a conservação das sementes por um período
prolongado. Frutas cítricas para o consumo in natura têm sido demandadas
em diversos países, inclusive no Brasil (Agrianual, 2003). O Estado do
Rio Grande do Sul dispõe de extensas áreas com condições de solo e de
clima propícias à citricultura e potencialmente adequadas para a produção
de frutos com a coloração acentuada exigida pelo mercado (Oliveira et
al., 2001). Em decorrência disso, vários programas governamentais têm
sido implementados no Estado para fomentar o desenvolvimento dessa
alternativa agrícola. Este trabalho teve por objetivo estabelecer uma metodologia
eficiente, prática e econômica para a conservação de sementes do porta-
enxerto ‘Trifoliata’. Atualmente, a legislação do Rio Grande do Sul exige que as
mudas certificadas de citros sejam produzidas em ambientes protegidos
contra vetores de doenças (Secretaria da Agricultura e do Abastecimento,
1998), denominados de viveiros-telado. Para maximizar a utilização da
infra-estrutura dos viveiros é necessário dispor-se de metodologia que
possibilite a conservação das sementes dos porta-enxertos, permitindo
a realização de semeaduras durante todo o ano e reduzindo as perdas
de sementes, as quais apresentam um custo elevado. METHODOLOGY FOR SEED STORAGE OF Poncirus trifoliata (L.) Raf. ABSTRACT - The aim of this work was to establish a methodology for seed storage of Poncirus trifoliata, that is the main citrus rootstock in Rio
Grande do Sul. The pre-treatments evaluated were: a) chemical - Benomyl (1,5 g a.i. kg-1 of seeds); b) thermal/chemical - water immersion at 52oC for
10 minutes, following application of Benomyl (1,5 g a.i. kg-1 of seeds). The storage conditions were: a) environment - storage in dark room under not
controlled temperature and air moisture; b) Refrigerator - 8oC and 60% of air moisture; c) dissector – not controlled temperature and 16% of air
moisture; d) dissector in refrigerator - 8oC and 16% of air moisture; e) Cold chamber - 4oC and 70% of air moisture. The ‘Trifoliata’ seeds are
recalcitrants, because they are highly sensible to storage. It was not observed effect of pre-treatments for seed conservation. The storage in cold
chamber was the best treatment, maintaining 57,1% of seed viability during 150 days. PROCEDIMENTOS PARA O ARMAZENAMENTO DE SEMENTES DE
Poncirus trifoliata (L.) Raf.1 DROSO DE OLIVEIRA2, WALKYRIA BUENO SCIVITTARO3, ELIZETE BEATRIZ RADMANN4 ROBERTO PEDROSO DE OLIVEIRA2, WALKYRIA BUENO SCIVITTARO3, ELIZETE BEA RESUMO - Objetivou-se estabelecer uma metodologia para o armazenamento de sementes de Poncirus trifoliata, que é o principal porta-enxerto de
citros utilizado no Rio Grande do Sul. Foram avaliados os efeitos de dois pré-tratamentos: a) químico - Benomyl (1,5 g i.a kg-1 de sementes); b)
térmico-químico - imersão em água a 52oC, por 10 minutos, seguida da aplicação de Benomyl (1,5 g i.a kg-1 de sementes). As condições de
armazenamento estudadas foram: a) ambiente - estocagem em sala escura sob condições de temperatura e de umidade relativa do ar não controladas;
b) geladeira - temperatura de 8oC e umidade relativa do ar de 60%; c) dessecador - temperatura não controlada e umidade relativa do ar de 16%; d)
dessecador em geladeira - temperatura de 8oC e umidade relativa do ar de 16%; e) câmara fria - temperatura de 4oC e umidade relativa do ar de 70%. Verificou-se que as sementes de ‘Trifoliata’ são recalcitrantes, sendo bastante sensíveis ao armazenamento. Não houve efeito dos pré-tratamentos
na conservação das sementes. A melhor condição de armazenamento foi em câmara fria, permitindo manter o poder germinativo das sementes em
57,1% aos 150 dias. Termos para indexação: câmara fria, dessecador, geladeira, porta-enxerto, tratamento químico, tratamento térmico 461 461 Rev. Bras. Frutic., Jaboticabal - SP, v. 25, n. 3, p. 461-463, dezembro 2003 PROCEDIMENTOS PARA O ARMAZENAMENTO DE SEMENTES DE Poncirus trifoliata (L.) Raf. emprego da imersão das sementes em água a 52oC por 10 minutos. O
uso exclusivo do tratamento químico foi suficiente para controlar o
desenvolvimento de microrganismos. Também não se verificou
dormência das sementes de ‘Trifoliata’, para a qual o tratamento térmico
teria importância (Koller et al., 1993). Após, procedeu-se ao armazenamento das sementes, que
variou quanto às condições de temperatura e de umidade relativa do ar,
segundo os tratamentos: a) ambiente - estocagem em sala escura sob
condições de temperatura e de umidade relativa do ar não controladas;
b) geladeira - temperatura de 8oC e umidade relativa do ar de 60%; c)
dessecador - temperatura não controlada e umidade relativa do ar de
16%; d) dessecador em geladeira - temperatura de 8oC e umidade relativa
do ar de 16%; e) câmara fria - temperatura de 4oC e umidade relativa do
ar de 70%. Os resultados da análise de variância indicaram efeito
significativo para os fatores tempo e condições de armazenamento. Conforme esperado, quanto maior o tempo de armazenamento, menor a
viabilidade das sementes. O armazenamento em câmara fria foi o método
mais eficiente na conservação das sementes de ‘Trifoliata’ (Tabela 2). Independentemente do tempo de armazenamento, obteve-se uma
porcentagem média de 47,9% de germinação, por volta de nove vezes
maior do que quando armazenada em geladeira. Além disso, foi o
tratamento que possibilitou o maior tempo de armazenamento, sendo
este de sete meses (Koller et al., 1993). No tempo zero e a cada 30 dias, durante oito meses, foram
realizados testes de germinação das sementes. Para tanto, inicialmente,
removeu-se de forma manual o tegumento das sementes, sendo, cada
grupo de 10 sementes, disposto em saco plástico transparente com
dimensões de 10 cm x 12 cm, contendo papel de filtro esterilizado em
autoclave. Este foi umedecido com água destilada previamente
esterilizada. Em seguida, as sementes, nos respectivos sacos plásticos,
foram colocadas em câmara de germinação a 25oC, na ausência de luz. O
porcentual de germinação foi avaliado, semanalmente, durante 40 dias,
considerando-se germinada as plântulas com desenvolvimento da parte
aérea e sistema radicular. Em função dos resultados obtidos, observa-se que a redução
da temperatura do ambiente de conservação é um dos fatores
determinantes na conservação das sementes. Isto ocorre por
proporcionar diminuição da atividade metabólica que se processa na
semente e da velocidade de multiplicação de microrganismos. RESULTADOS E DISCUSSÃO Independentemente do tempo e das condições de
armazenamento das sementes de ‘Trifoliata’, a germinação ocorreu, em
sua maioria, do quinto ao trigésimo dia após o início do teste de
germinação. No presente trabalho, as sementes sempre foram mantidas em
saquinhos que permitiram trocas de umidade da semente com o ambiente
em que foram armazenadas. Portanto, o armazenamento em geladeira e
câmara fria foi realizado em alta umidade, respectivamente a 60% e 70%,
enquanto que em dessecador, em baixa umidade (16%). A perda completa
do poder germinativo das sementes armazenadas em dessecador em
apenas 30 dias (Tabela 2) pode ser atribuída à sensibilidade das sementes
de ‘Trifoliata’ à diminuição da umidade. Saipari et al. (1998) também
observaram perda do poder germinativo de sementes desse cultivar ao Sob condições de temperatura e umidade do ar não controladas,
as sementes de ‘Trifoliata’ apresentaram perda da viabilidade após 30
dias de armazenamento. Button et al. (1971) também haviam verificado
este fato após 28 dias e, por isso, as sementes desse cultivar podem ser
classificadas como recalcitrantes. No presente trabalho, não houve efeito significativo do pré-
tratamento das sementes na porcentagem de germinação, ao longo do
tempo de armazenamento (Tabela 1), sendo, portanto, desnecessário o rminação de sementes do porta-enxerto Poncirus trifoliata (L.) Raf. seleção Davis A em função do pré-tratamento
azenamento. Pelotas, RS. TABELA 1 - Porcentagem de germinação de sementes do porta-enxerto Poncirus trifoliata (L.) Raf. seleção Davis A em função do pré-tratamento
e do tempo de armazenamento. Pelotas, RS. 1Para a análise de variância, os dados foram transformados para arco seno de (X/100)1/2. Médias seguidas de mesma letra nas colunas não diferem significativamente entre si pelo teste de Duncan (5%). 1Para a análise de variância, os dados foram transformados para arco seno de (X/100)1/2. Médias seguidas de mesma letra nas colunas não diferem significativamente entre si pelo teste de Duncan (5%). TABELA 2 - Porcentagem de germinação de sementes do porta-enxerto Poncirus trifoliata (L.) Raf. seleção Davis A em função das condições e do
tempo de armazenamento. Pelotas, RS. TABELA 2 - Porcentagem de germinação de sementes do porta-enxerto Poncirus trifoliata (L.) Raf. seleção Davis A em função das condições e do
tempo de armazenamento. Pelotas, RS. TABELA 2 - Porcentagem de germinação de sementes do porta-enxerto Poncirus trifoliata (L.) Raf. seleção Davis A em função das condições e do
tempo de armazenamento. Pelotas, RS. PROCEDIMENTOS PARA O ARMAZENAMENTO DE SEMENTES DE Poncirus trifoliata (L.) Raf. Platt &
Opitz (1973), Eshuys (1974) e Mumford & Panggabean (1982) já haviam
recomendado o uso de temperaturas de 2oC a 7oC para conservação de
sementes de ‘Trifoliata’. Koller et al. (1993) conseguiram conservar
sementes de ‘Trifoliata’, com germinação entre 75% e 80%, por um
período de até um ano, sob temperaturas de 2oC a 5oC. Os tratamentos foram dispostos em delineamento inteiramente
ao acaso em parcelas subdivididas com três repetições, considerando-
se como parcelas as combinações dos fatores pré-tratamento e
condições de armazenamento das sementes e como subparcelas, o tempo
de armazenamento destas. As unidades experimentais foram constituídas
por grupos de 10 sementes, dispostas em sacos de papel manteiga. Para
a análise de variância, os dados foram transformados para arco seno de
(X/100)1/2. Outro fator fundamental para a conservação de sementes de
‘Trifoliata’ consiste no nível de umidade do ar existente no ambiente de
armazenamento (Koller et al. 1993). Por isso, são observadas diferenças
significativas em experimentos de vários autores, utilizando mesma
temperatura. Segundo Saipari et al. (1998), as sementes de ‘Trifoliata’
apresentam, em média, 52% de umidade no momento da colheita e 25%
após a secagem ao ar. Estas são tão recalcitrantes, que Mumford &
Panggabean (1982) observaram perda de 5% do poder germinativo
simplesmente em decorrência do processo de secagem ao ar. MATERIAL E MÉTODOS O experimento foi realizado, sob condições de laboratório, no
período de abril a dezembro de 2002, em Pelotas, RS. As sementes de
Poncirus trifoliata (L.) Raf. seleção Davis A foram removidas de frutos
completamente maduros, colhidos no final do mês de março do mesmo
ano. A remoção da muscilagem foi realizada manualmente, por meio da
lavagem das sementes com água corrente. Após, essas foram secas à
sombra, sobre papel-toalha, por 72 horas. O ‘Trifoliata’ [Poncirus trifoliata (L.) Raf.] é o principal porta-
enxerto utilizado no Rio Grande do Sul, principalmente por sua maior
tolerância ao frio e por conferir alta qualidade à fruta (Oliveira et al.,
2001). Além disso, é resistente à gomose de Phytophthora e ao vírus da
tristeza, muito embora seja suscetível ao viróide do exocorte e à clorose
de ferro em solos calcários e com pH igual ou superior a neutralidade
(Castle, 1987). Na seqüência, aplicaram-se às sementes dois pré-tratamentos:
a) químico, que consistiu na aplicação de Benomyl, na concentração de
1,5 g i.a kg-1 de sementes; b) térmico-químico, compreendendo a imersão
das sementes em água a 52oC, por 10 minutos, sob agitação constante,
seguida da aplicação de Benomyl, em concentração igual a do tratamento
anterior. As sementes oriundas de ambos os pré-tratamentos foram
selecionadas, eliminando-se as aparentemente mal formadas e as
danificadas, distribuídas em grupos de 10 e acondicionadas em sacos
de papel manteiga (10 cm x 10 cm). As sementes de ‘Trifoliata’ e de seus híbridos são mais
sensíveis ao armazenamento do que a dos demais porta-enxertos
utilizados na produção de mudas de citros (Mumford & Panggabean,
1982; Saipari et al., 1998), podendo ser consideradas recalcitrantes. Normalmente, quando armazenadas sob condições naturais, apresentam
uma perda significativa do poder germinativo em poucos dias (Button
et al., 1971). Rev. Bras. Frutic., Jaboticabal - SP, v. 25, n. 3, p. 461-463, dezembro 2003 Bras. Frutic., Jaboticabal - SP, v. 25, n. 3, p. 461-463, dezembro 20 462 PROCEDIMENTOS PARA O ARMAZENAMENTO DE SEMENTES DE Poncirus trifoliata (L.) Raf. TABELA 1 - Porcentagem de germinação de sementes do porta-enxerto Poncirus trifoliata (L.) Raf. seleção Davis A em função do pré-tratamento
e do tempo de armazenamento. Pelotas, RS. RESULTADOS E DISCUSSÃO TABELA 2 - Porcentagem de germinação de sementes do porta-enxerto Poncirus trifoliata (L.) Raf. seleção Davis A em função das condições e do
tempo de armazenamento. Pelotas, RS. 1Para a análise de variância, os dados foram transformados para arco seno de (X/100)1/2. Médias seguidas de mesma letra nas colunas não diferem significativamente entre si pelo teste de Duncan (5%). A 2 - Porcentagem de germinação de sementes do porta-enxerto Poncirus trifoliata (L.) Raf. seleção Davis A em funç
tempo de armazenamento. Pelotas, RS. rminação de sementes do porta-enxerto Poncirus trifoliata (L.) Raf. seleção Davis A em função das condições e do
amento. Pelotas, RS. Rev. Bras. Frutic., Jaboticabal - SP, v. 25, n. 3, p. 461-463, dezembro 2003 463 R. P. de OLIVEIRA et al. BARBEDO, C.J.; BILIA, D.A.C. Evolution of research on recalcitrant
seeds. Scientia agricola, Piracicaba, v.55, p.121-125, 1998. serem secas em ambiente com sílica a 9% de umidade relativa do ar. Segundo Nakagawa & Honjo (1979), as sementes de ‘Trifoliata’ somente
germinam quando mantidas a uma umidade superior a 20%, sendo ideal
secar as sementes até 28% (Koller et al., 1993). Outros porta-enxertos
de citros, com sementes menos recalcitrantes, como os limoeiros
verdadeiros, o ‘Cravo’, o ‘Galego’ e o ‘Rugoso Nacional’ apresentam
tolerância para menor umidade das sementes para o armazenamento
(6% a 8%) (Mumford & Panggabean, 1982). Desta forma, existe um nível
crítico de umidade para cada espécie, que precisa ser melhor estudado,
no qual o conteúdo de água é suficiente para proporcionar as reações
metabólicas essenciais à manutenção da viabilidade das sementes e
para evitar o desenvolvimento de microrganismos. serem secas em ambiente com sílica a 9% de umidade relativa do ar. Segundo Nakagawa & Honjo (1979), as sementes de ‘Trifoliata’ somente
germinam quando mantidas a uma umidade superior a 20%, sendo ideal
secar as sementes até 28% (Koller et al., 1993). Outros porta-enxertos
de citros, com sementes menos recalcitrantes, como os limoeiros
verdadeiros, o ‘Cravo’, o ‘Galego’ e o ‘Rugoso Nacional’ apresentam
tolerância para menor umidade das sementes para o armazenamento
(6% a 8%) (Mumford & Panggabean, 1982). Desta forma, existe um nível
crítico de umidade para cada espécie, que precisa ser melhor estudado,
no qual o conteúdo de água é suficiente para proporcionar as reações
metabólicas essenciais à manutenção da viabilidade das sementes e
para evitar o desenvolvimento de microrganismos. BUTTON, J.; BORNMAN, C.H.; HACKLAND, B.A. RESULTADOS E DISCUSSÃO Effect of some pre-
sowing treatments on the germination of Poncirus trifoliata and
Troyer citrange seeds. Citrus and Sub-tropical Fruit Journal,
Johannesburg, v.451, p.9-11, 1971. CASTLE, W.S. Citrus rootstocks. In: ROM, R.C.; CARLSON, R.F. (Ed.). Rootstocks for fruit crops. New York: Wiley, 1987. p.361-399. ESHUYS, W.A. Loss of viability of citrus and Poncirus trifoliata seed
during storage. Citrus and Sub-tropical Fruit Journal,
Johannesburg, v.489, p.5-7, 1974. KOLLER, O.L.; STUKER, H.; VERONA, L.A.F.; SOPRANO, E. Efeito da
umidade da semente, da temperatura de estocagem e da duração de
estocagem sobre a germinação de Poncirus trifoliata e de outros
porta-enxertos de citros. Revista Brasileira de Fruticultura, Cruz
das Almas, v.15, n.1, p.27-33, 1993. Em geral, durante o armazenamento, as sementes apresentaram
um progressivo ressecamento do tegumento, que passou a apresentar
trincas, havendo desprendimento do embrião ao se remover o
tegumento, principalmente naquelas armazenadas em dessecador. Esses
sintomas são evidências da perda de umidade pelas sementes. Esse
comportamento aconteceu somente nos últimos meses das sementes
armazenadas em câmara fria. As sementes com essas características
apodreceram durante o teste de germinação. MUMFORD, P.M.; PANGGABEAN, G. A comparison of the effects of
dry storage on seeds of citrus species. Seed Science and
Technology Zurich v10 n 2 p 257 266 1982 Technology, Zurich, v.10, n.2, p.257-266, 1982. NAKAGAWA, Y.; HONJO, H. Studies on the freezing hardness and
drought tolerance of citrus seeds. Bulletin of the Fruit Tree
Research Station, Series A, n.6, p.27-35, 1979. Os resultados obtidos permitiram ampliar o tempo de
armazenamento de sementes de ‘Trifoliata’, porém, existe a necessidade
de maiores estudos nessa linha de pesquisa, principalmente no que se
refere ao estabelecimento de níveis de tolerância das sementes à perda
de umidade e à baixa temperatura. Research Station, Series A, n.6, p.27-35, 1979. OLIVEIRA, R.P.; SCIVITTARO, W.B.; BORGES, R.S.; NAKASU, B.H. Mudas de citros. Pelotas: Embrapa Clima Temperado, 2001. 32p. (Sistemas de produção, 1). PLATT, R.G.; OPITZ, K.W. The propagation of citrus. In: REUTHER,
W. (Ed.). The citrus industry. Berkeley: University of California,
1973. v.3, p.1-47. Rev. Bras. Frutic., Jaboticabal - SP, v. 25, n. 3, p. 461-463, dezembro 2003 CONCLUSÕES 1) Não há efeito do pré-tratamento térmico na conservação
das sementes. SAIPARI, E.; GOSWAMI, A.M.; DADLANI, M. Effect of seed drying
on germination behavior in citrus. Scientia Horticulturae,
Amsterdam, v.73, p.185-190, 1998. 2) As sementes devem ser armazenadas em câmara fria. SECRETARIA DA AGRICULTURA E DO ABASTECIMENTO. Departamento de Produção Vegetal. Comissão Estadual de Sementes
e Mudas do Estado do Rio Grande do Sul. Normas e padrões de
produção de mudas de fruteiras para o Estado do Rio Grande do
Sul. Porto Alegre, 1998. 100p. REFERÊNCIAS BIBLIOGRÁFICAS AGRIANUAL 2003: anuário da agricultura brasileira. São Paulo: FNP
Consultoria & AgroInformativos, 2003. 544p. AGRIANUAL 2003: anuário da agricultura brasileira. São Paulo: FNP
Consultoria & AgroInformativos, 2003. 544p.
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Human Papillomavirus Type 16 Disables the Increased Natural Killer Cells in Early Lesions of the Cervix
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Journal of immunology research
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cc-by
| 6,782
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Hindawi
Journal of Immunology Research
Volume 2019, Article ID 9182979, 8 pages
https://doi.org/10.1155/2019/9182979 Hindawi
Journal of Immunology Research
Volume 2019, Article ID 9182979, 8 pages
https://doi.org/10.1155/2019/9182979 Jiexin Zhang,1 Shanshan Jin,2 Xiao Li,3 Lenan Liu,4 Lei Xi,3 Fang Wang
,1
and Shichang Zhang
1 1Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing Medical University, 210029 Nanjing, China
2Department of Oncology, The First Affiliated Hospital of Nanjing Medical University, 210029 Nanjing, China
3Department of Pathology, The First Affiliated Hospital of Nanjing Medical University, 210029 Nanjing, China
4Department of Obstetrics, The First Affiliated Hospital of Nanjing Medical University, 210029 Nanjing, China orrespondence should be addressed to Fang Wang; wangfang@njmu.edu.cn and Shichang Zhang; zsc78@yeah.n Received 31 January 2019; Revised 19 March 2019; Accepted 3 April 2019; Published 28 April 2019 Guest Editor: Hadi M. Yassine Guest Editor: Hadi M. Yassine Copyright © 2019 Jiexin Zhang et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The mechanism for pathogenesis of human papillomavirus (HPV) in the cervix has been investigated intensively. However,
detailed differences in the distribution and function of innate immune cells between high-risk HPV types, especially during the
chronic inflammation phase, have not been described fully. In this study, histologic pathology results of 245 women with HPV
type 16 only (HPV16+) or type 18 only (HPV18+) were analyzed retrospectively from January 2015 to November 2016. More
severe lesions of the cervix were observed in HPV16+ women compared with those in HPV18+ women. In total, 212 cervical
brush specimens were collected from women suffering from chronic inflammation, HPV16+, or HPV18+ from December 2016
to December 2018. Flow cytometry analysis showed that abundant NK cells along with aberrant Treg cells were found in the
HPV16-infected cervix. Quantitative real-time PCR demonstrated that higher expression levels of IFN-γ but muted IL-2 and
KLRG-1 expression was detected in the cervix of patients with HPV16+ compared to HPV18+, which were further confirmed
using 20 paraffin sections of cervical conization tissue. The ex vivo cytotoxicity experiment showed that the cytotoxicity of NK
cells was significantly decreased in the cervix of HPV16+ patients compared with that of HPV18+ patients. Collectively, our
results suggested that HPV16 disables the increased NK cells in the early lesion of the cervix, indicating that the local immune
system of the cervix is hyporesponsive to HPV16 infection and this may explain its bias for malignant transformation. 1. Introduction A recent worldwide
comprehensive survey indicated that up to half of cervical Journal of Immunology Research 2 using RNeasy FFPE Kits (QIAGEN) as per the manufac-
turer’s instructions. cells, have also been shown in the crosstalk between cervical
cancer cells and the virus [8, 9]. Nevertheless, there is no
report so far focusing on the profiles of these infiltrated
immune cells in tissues infected with different HPV types. The purpose of this study was to compare the distribution
and functional status of innate NK cells in tissue infected
with two high-risk HPV types (16 and 18) and to discuss
the possible mechanisms of HPV-original disease from a dif-
ferent perspective. The reverse transcription reaction from 1μg of RNA
template was carried out using a First Strand cDNA Synthesis
Kit (TOYOBO, Japan). Quantitative real-time PCR was per-
formed using SYBR Green Real-time PCR Master Mix
(TOYOBO, Japan) and detected by the LightCycler 480 II
real-time PCR system (Roche, USA). The expression level
was analyzed and normalized to β-actin in the cDNA sam-
ples. The fold change of gene expression was calculated using
the 2-ΔΔCT method. The primer sequences are listed in
Table 1. 3. Results 2.3. HPV DNA Detection and Type Identification. HPV DNA
detection and type identification were performed using a
cobas® x480 nuclear acid extraction system and a cobas®
z480 analyzer via a cobas® 4800 system liquid cytology prep-
aration kit according to the manufacturer’s instructions
(Roche, USA). Specimens with HPV16 or HPV18 were,
respectively, preserved as the HPV16+ group or HPV18+
group. Specimens derived from women with chronic inflam-
mation and HPV negative were used as controls. 3.1. Overall Histologic Pathology Profiles of HPV Type-
Infected Women. We retrospectively analyzed the histologic
pathology results of 245 HPV16- or HPV18-infected women
from January 2015 to November 2016. As shown in Table 2,
more severe lesions (≥CIN 2) were statistically presented
in women with HPV16 infection when compared with
HPV18+ women. 3.1. Overall Histologic Pathology Profiles of HPV Type-
Infected Women. We retrospectively analyzed the histologic
pathology results of 245 HPV16- or HPV18-infected women
from January 2015 to November 2016. As shown in Table 2,
more severe lesions (≥CIN 2) were statistically presented
in women with HPV16 infection when compared with
HPV18+ women. 3.2. NK Cell Infiltration in a HPV16-Infected Cervix. Both
HPV16 and HPV18 are categorized into high-risk HPV types
that have a strong probability to cause cervical invasive carci-
noma. We wondered whether the distribution patterns of
CD56+CD16+ representing NK cells were similar between
these two types of virus. In total, 48 cervical brush specimens
including 16 chronic inflammation controls, 16 HPV16+
cases, and 16 HPV18+ cases were collected. As shown in
Figure 1(a), compared to chronic inflammation control,
CD56+CD16+ NK cells were upregulated in both HPV16+
and HPV18+ cervical surfaces. Interestingly, the ratio was
even more abundant in the HPV16+ group than that in the
HPV18+ group (CD56+CD16+ NK cells 2 13 ± 0 38% vs. 0 53 ± 0 23%; p < 0 01). Cell smear inspection also showed
a relatively high density of fried egg-like NK cells in the
HPV16+ group. 2.4. Fluorescence-Activated Cell Sorting (FACS). Preserved
samples were washed three times with PBS before passing
through a 40μm strainer filter twice (STEMCELL Technolo-
gies, Canada). Remaining cells were conjugated to CD3,
CD4, CD56/CD16 (Beckman Coulter), and either CD16 or
CD25 antibodies in combination (Biolegend, USA) and sub-
jected to flow cytometry (CD56/16 with Beckman, others
with BD FACSCalibur, USA). The FACS results show the
percentages of each indicated immune cell type. 2.5. RNA Extraction and Quantitative Real-Time PCR. 2. Materials and Methods 2.1. Patient Characteristics. Eligible patients were married
women aged between 25 and 65 without histologically or
cytologically confirmed other diseases originating from the
reproductive system (including the vagina, uterus, and
ovary) but with cervical chronic inflammation only. This
research was authorized by the Ethical Committee of the
First Affiliated Hospital of Nanjing Medical University
(Nanjing, China). 2.6. Ex Vivo Immune Cell Cytotoxicity Experiments. The cer-
vical cancer cell line HeLa cells were labeled with CFSE
(0.5 μM, Invitrogen). CD56+CD16+ NK cells were isolated
from cervical brush specimens of women infected with
HPV16 or HPV18 via FACS (BD FACSAria II, USA). NK
cells were cocultured with CFSE-HeLa in designated ratios
in 24-well plates and incubated at a 37°C incubator for 4-6
hours. Cells were harvested, stained with 7-AAD (BD Phar-
mingen, USA), and subjected to flow cytometry (BD FACS-
Calibur, USA). Data were analyzed using the FlowJo 6.0
software (Tree Star). 2.2. Sample Collection. We used a speculum to expose the cer-
vical canal, and the coherent mass of tissue was cleared with a
cotton swab. A cervical brush was inserted into the cervical
canal to approximately 1-1.5 cm and was gently rotated in
the same direction for 5 times. Then, the cervical brush was
slowly extracted, and the exfoliated cells were preserved in a
Roche® sample collection tube for type detection or in 1640
cell culture medium containing 10% FBS. In total, 212 cervi-
cal brush specimens were collected for flow cytometry analy-
sis, ex vivo cytotoxicity detection, and quantitative real-time
PCR from December 2016 to December 2018. 2.7. Statistical Analysis. Statistical analyses were performed
using PRISM 7.01 (GraphPad Software Inc., San Diego,
CA). Data are presented as the means ± SD. All data were
analyzed using Student’s t-test, where p < 0 05 was consid-
ered statistically significant. The Bonferroni correction was
used for multiple comparisons. 1. Introduction cases is caused by HPV type 16 (HPV16), and HPV16 and
HPV type 18 (HPV18) together lead to up to 80% [4, 5]. Studies have provided solid evidence on the molecular
mechanisms by which HPV infiltrates local immune cells. NK cells are important biological barriers which are resident
in the cervix. They can identify virus-infected or virus-
transformed cells rapidly through pathways that do not
require presensitization and kill them. It has been reported
that NK cells emerge at an early stage in HPV-infected
lesions [6]. However, HPV can force activate NK cells to lose
their membrane receptors thus leading to their malfunction
and this can lead to carcinogenesis [7]. A reversal of the
CD4+/CD8+ ratio and a marker increase in the number of
Treg cells, which are the representative immunosuppressive cases is caused by HPV type 16 (HPV16), and HPV16 and
HPV type 18 (HPV18) together lead to up to 80% [4, 5]. HPV type 18 (HPV18) together lead to up to 80% [4, 5]. Studies have provided solid evidence on the molecular
mechanisms by which HPV infiltrates local immune cells. NK cells are important biological barriers which are resident
in the cervix. They can identify virus-infected or virus-
transformed cells rapidly through pathways that do not
require presensitization and kill them. It has been reported
that NK cells emerge at an early stage in HPV-infected
lesions [6]. However, HPV can force activate NK cells to lose
their membrane receptors thus leading to their malfunction
and this can lead to carcinogenesis [7]. A reversal of the
CD4+/CD8+ ratio and a marker increase in the number of
Treg cells, which are the representative immunosuppressive Based on the most recent data, the immune system of most
people infected with HPV needs at least three years in order
to eliminate the virus. Apart from an inadequate humoral
immune response, suppression of immune cells in the local
cervical environment is another major reason for these statis-
tics [1]. HPV invades vaginal squamous epithelium as well as
undifferentiated basal cells within the cervical canal epithe-
lium transitional zone [2]. So far, more than 100 HPV types
have been identified, among which 15 types (such as HPV
types 16, 18, 31, and 45) are considered high-risk types which
are more likely to be carcinogenic [3]. 3. Results The
total RNA of cervical brush specimens was extracted using
a TRIzol Reagent (Grand Island, USA) according to the man-
ufacturer’s instructions. Twenty paraffin sections of cervical
conization tissue were subjected to quantitative real-time
PCR. The unstained sections were deparaffinized using a
standard xylene and ethanol procedure. RNA was extracted The second batch of cervical brush specimens consisted
of 25 chronic inflammation controls, 22 HPV16+ cases, and Journal of Immunology Research 3 Table 1: Quantitative real-time PCR primers used in this study. Gene name
Primer
IFN-γ
F: gagtgtggagaccatcaagg
R: cgacagttcagccatcactt
IL-2
F: gccacagaactgaaacatct
R: gccttacctttagttccagaa
IL-6
F: agatttgagagtagtgaggaa
R: actgtctttgagcctgtctt
GM-CSF
F: gccactacaagcagcactg
R: tgtctgcctcctctctgga
CCL-3
F: taactcttcctcccttctcc
R: tggacccctcaggcactca
CCL-5
F: atcctccctcttcttctcct
R: ttcaggttcaaggactctcc
KLRG-1
F: tcaactccttttctgtgcatg
R: catctatcaaagtctgacctt
CD56
F: atgatgggtgaagagaaccg
R: aatgagatgtgtgtgtgtgc
CD27
F: gccttcagatgtgccctat
R: cagtgggtagagagagtcc
DNAM-1
F: gaagtcccatctctaccagt
R: agcttaaactctagtctttgg
β-Actin
F: tcatgaagtgtgacgtggacat
R: ctcaggaggagcaatgatcttg infection could. Other tested cytokines such as IL-4, GM-
CSF, CCL-3, and CCL-5 showed no significant difference
between infections with the two HPV types. To verify these
changes in cervical brush specimens, we again examined
women who were pathologically diagnosed with cervical
chronic inflammation and collected paraffin sections of cer-
vical conization tissue from those infected with HPV16 (10
cases) or HPV18 (10 cases). As shown in Figure 1(c), the
trends of the IFN-γ and IL-2 expression difference between
the two groups were identical. Taken together, the HPV16+
cervix is characterized by the increased population of NK cell
proportion and a distinguished cervical microenvironment. 3.3. NK Cells Exert a Discorded Effect in a HPV16+ Cervix. We also tested four typical cell membrane markers of NK
cells in the second batch of specimens. KLRG-1 is upregu-
lated in cells designated as vibrant NK cells possessing a cyto-
toxicity effect. But its expression was dull in the HPV16+
group (Figure 2(a)), indicating that it fails to respond when
the virus is mature and proceed to the next biological cycle
at the cervical surface. This result was also confirmed in the
paraffin sections (Figure 2(b)). CD56 transcription levels of
both HPV groups were similar or slightly higher than control
(Figure 2(a)). This does not agree with the result from flow
cytometry studies (Figure 1(a)). Considering that the com-
bined CD56/CD16 antibody used in Figure 1 was designed
for clinical use only, we chose another different commercial
CD16 antibody. 3. Results On the contrary, HPV18 15 HPV18+ cases that were collected to evaluate six soluble
cytokines that represented the NK cell-involved immune
status in the cervical microenvironment. As shown in
Figure 1(b), IFN-γ transcription level was relatively higher
in the HPV16+ group than that in the HPV18+ group, which
was consistent with elevated numbers of local NK cells. But
despite the viral types, HPV infection induced a decreasing
trend of IFN-γ transcription at the cervical surface. IL-2 is
an important cytokine in microbial infection and bridges
the body’s innate and adaptive immune systems. We found
that IL-2 levels were significantly lower in the HPV16+ group
indicating that the local immune response failed to be further
stimulated by virus infection. On the contrary, HPV18 3. Results The flow cytometry analysis of the third
batch of cervical brush specimens (16 cases in each group)
showed that CD16+ cells were markedly increased in
HPV16+ group (HPV16+0 693 ± 0 507% vs. control 0 043 ±
0 037% and HPV18+0 077 ± 0 083%; Figure 2(c)). Table 2: Characteristics of HPV16 or HPV18 infections correlated
to the severity cervical lesions. Table 2: Characteristics of HPV16 or HPV18 infections correlated
to the severity cervical lesions. Characteristics
HPV16+
HPV18+
P value
Average age (years)
40 0 ± 9 7
40 8 ± 9 6
0.8055
Histological diagnosis
<0.0001
Inflammation
54 (26.6)
27 (64.3)
CIN 1
22 (10.8)
8 (19.0)
CIN 2
29 (14.3)
3 (7.1)
CIN 3
98 (48.3)
4 (9.5)
Total
203
42
CIN: cervical intraepithelial neoplasia; HPV: human papillomavirus. g
Treg cells play an important role in “self-checking” the
regulatory immune response, and there is a trade-off
between them and NK cells which is believed to have a great
contribution towards HPV-induced carcinogenesis [10]. To
investigate whether increased infiltration of NK cells implied
Treg recession, 48 cervical brush specimens were analyzed
by flow cytometry. As expected, the CD4+CD25+ Treg cell
ratio in the HPV16+ group was the lowest among the three
groups (HPV16+1 30 ± 0 28% vs. control 6 70 ± 0 85% and
HPV18+4 64 ± 0 73%; Figure 2(d)). The cytotoxicity of NK
cells extracted from 3 HPV16+ women and 3 HPV18+
women was determined. As shown in Figure 3, the ratio of
CFSE+7-AAD+-labeled cancer cells failed to further elevate
when ten-fold ex vivo NK cells were added for coculture,
indicating that infiltrated NK cells do not exert an equivalent
function to eliminate transformed cells. 15 HPV18+ cases that were collected to evaluate six soluble
cytokines that represented the NK cell-involved immune
status in the cervical microenvironment. As shown in
Figure 1(b), IFN-γ transcription level was relatively higher
in the HPV16+ group than that in the HPV18+ group, which
was consistent with elevated numbers of local NK cells. But
despite the viral types, HPV infection induced a decreasing
trend of IFN-γ transcription at the cervical surface. IL-2 is
an important cytokine in microbial infection and bridges
the body’s innate and adaptive immune systems. We found
that IL-2 levels were significantly lower in the HPV16+ group
indicating that the local immune response failed to be further
stimulated by virus infection. 4. Discussion The aim of this study was to prospectively study the distribu-
tion patterns of infiltrated immune cells, in particular NK
cells, and HPV16- and HPV18-infected cervical surface
microenvironment and to elucidate the possible high-risk
HPV pathogenic mechanism from a new prospective by
highlighting the functional cell discrepancies between these
two types. We found that CD16+ NK cells were abundant
in the HPV16+ group of women with more IFN-γ secretion Journal of Immunology Research 4 Control
HPV 16+
HPV18+
0
1
2
3
CD3−CD56+CD16+NK cells(%)
⁎⁎
⁎⁎
103
102
101
100
Control
CD3–FITC
B1
0.0%
0.0%
0.0%
100.0%
B2
B4
B3
CD16+56-PE
103
102
101
100
HPV16+
CD3–FITC
B1
2.1%
0.1%
0.0%
97.8%
B2
B4
B3
CD16+56-PE
103
103
102
101
100
102
101
100
103
102
101
100
103
102
101
100
HPV18+
CD3–FITC
B1
0.0%
0.0%
0.0%
99.9%
B2
B4
B3
CD16+56-PE
(a)
Control
HPV 16+
HPV 18+
0
4
8
12
IL-2
Relative mRNA fold change
⁎⁎
⁎⁎
Control
HPV 16+
HPV 18+
0
1
2
3
4
GM-CSF
Relative mRNA fold hange
Control
HPV 16+
HPV 18+
0
1
2
3
4
5
IL-6
Relative mRNA fold change
Control
HPV 16+
HPV 18+
0
2
4
6
8
CCL-3
Relative mRNA fold change
Control
HPV 16+
HPV 18+
0
2
4
6
CCL-5
Relative mRNA fold change
Control
HPV 16+
HPV 18+
0
0.5
1.0
1.5
IL-2
Relative mRNA fold change
⁎⁎
⁎⁎
⁎⁎
(b)
HPV 16+
HPV 18+
0.0
0.5
1.0
1.5
2.0
42.0
42.5
43.0
IL-2
Relative mRNA fold change
Relative mRNA fold change
⁎⁎
HPV 16+
HPV 18+
0.0
0.75
0.5
0.95
0.85
1.05
IL-2
⁎⁎
(c)
ure 1: The differences of NK cells between HPV16- and HPV18-infected cervixes. (a) Distribution of CD56+CD16+ NK cells was anal
he cervical brush specimens by FACS. (b) Several soluble cytokines that represent NK cell involvement in the immune status w
estigated in the cervical brush specimens by quantitative real-time PCR. (c) IFN-γ and IL-2 expressions were measured in the cerv
ization tissue by quantitative real-time PCR. ∗P < 0 05; ∗∗P < 0 01. 4. Discussion Journal of Immunology Resea Control
HPV 16+
HPV18+
0
1
2
3
CD3−CD56+CD16+NK cells(%)
⁎⁎
⁎⁎
103
102
101
100
Control
CD3–FITC
B1
0.0%
0.0%
0.0%
100.0%
B2
B4
B3
CD16+56-PE
103
102
101
100
HPV16+
CD3–FITC
B1
2.1%
0.1%
0.0%
97.8%
B2
B4
B3
CD16+56-PE
103
103
102
101
100
102
101
100
103
102
101
100
103
102
101
100
HPV18+
CD3–FITC
B1
0.0%
0.0%
0.0%
99.9%
B2
B4
B3
CD16+56-PE
(a) 0
1
2
3
CD3−CD56+CD16+NK cells(%)
103
102
101
100
Control
CD3–FITC
B1
0.0%
0.0%
0.0%
100.0%
B2
B4
B3
CD16 56 PE
103
102
101
100
HPV16+
CD3–FITC
B1
2.1%
0.1%
0.0%
97.8%
B2
B4
B3
CD16+56-PE
103
103
102
101
100
102
101
100
103
102
101
100
103
102
101
100
HPV18+
CD3–FITC
B1
0.0%
0.0%
0.0%
99.9%
B2
B4
B3
CD16+56-PE (a)
Control
HPV 16+
HPV 18+
0
4
8
12
IL-2
Relative mRNA fold change
⁎⁎
⁎⁎
Control
HPV 16+
HPV 18+
0
1
2
3
4
GM-CSF
Relative mRNA fold hange
Control
HPV 16+
HPV 18+
0
1
2
3
4
5
IL-6
Relative mRNA fold change
Control
HPV 16+
HPV 18+
0
2
4
6
8
CCL-3
Relative mRNA fold change
Control
HPV 16+
HPV 18+
0
2
4
6
CCL-5
Relative mRNA fold change
Control
HPV 16+
HPV 18+
0
0.5
1.0
1.5
IL-2
Relative mRNA fold change
⁎⁎
⁎⁎
⁎⁎
(b)
1 05
IL-2 (a)
Control
HPV 16+
HPV 18+
0
4
8
12
IL-2
Relative mRNA fold change
⁎⁎
⁎⁎
Control
HPV 16+
HPV 18+
0
1
2
3
4
GM-CSF
Relative mRNA fold hange
HPV 18+ Control
HPV 16+
HPV 18+
0
4
8
12
IL-2
Relative mRNA fold change
⁎⁎
⁎⁎
Control
HPV 16+
HPV 18+
0
1
2
3
4
GM-CSF
Relative mRNA fold hange
Control
HPV 16+
HPV 18+
0
0.5
1.0
1.5
IL-2
Relative mRNA fold change
⁎⁎
⁎⁎
⁎⁎ Control
HPV 16+
HPV 18+
0
1
2
3
4
5
IL-6
Relative mRNA fold change
Control
HPV 16+
HPV 18+
0
2
4
6
8
CCL-3
Relative mRNA fold change
Control
HPV 16+
HPV 18+
0
2
4
6
CCL-5
Relative mRNA fold change
(b) Relative mRNA fold chang (b) Relative mRNA fold change
HPV 16+
HPV 18+
0.0
0.75
0.5
0.95
0.85
1.05
IL-2
⁎⁎ HPV 16+
HPV 18+
0.0
0.5
1.0
1.5
2.0
42.0
42.5
43.0
IL-2
Relative mRNA fold change
⁎⁎
(c) Figure 1: The differences of NK cells between HPV16- and HPV18-infected cervixes. 4. Discussion Control
HPV 16+
HPV 18+
0
5
10
15
KLRG-1
Relative mRNA fold change
Control
HPV 16+
HPV 18+
0
2
4
6
8
DNAM-1
Relative mRNA fold change
Control
HPV 16+
HPV 18+
0
4
8
12
CD56
Relative mRNA fold change
Control
HPV 16+
HPV 18+
0
1
2
3
4
CD27
Relative mRNA fold change
⁎⁎
⁎⁎
(a) HPV 16+
HPV 18 +
0.0
0.5
1.0
1.5
2.0
44.0
44.5
KLRG-1
Relative mRNA fold change
⁎⁎
(b) Relative mRNA fold change (b) (b)
103
104
102
101
100
100
101
102
103
104
Control
HPV 16+
HPV18+
0.0
0.5
1.0
1.5
CD3-CD16+NKcells (%)
⁎⁎
⁎⁎
103
104
102
101
100
103
104
102
101
100
100
101
102
103
104
100
101
102
103
104
Control
CD3 FITC, CD16 PE subset
0.067
CD3–FITC
CD16–PE
HPV16+
CD3 FITC, CD16 PE subset
0.073
CD3–FITC
CD16–PE
HPV18+
CD3 FITC, CD16 PE subset
0.020
CD3–FITC
CD16–PE
( ) 103
104
102
101
100
100
101
102
103
104
+
103
104
102
101
100
101
102
103
104
100
101
102
103
104
Control
FITC, CD16 PE subset
0.067
CD3–FITC
HPV16+
CD3 FITC, CD16 PE subset
0.073
CD3–FITC
CD16–PE
HPV18+
CD3 FITC, CD16 PE subset
0.020
CD3–FITC
CD16–PE
(c) Control
HPV 16+
HPV18+
0.0
0.5
1.0
1.5
CD3-CD16+NKcells (%)
⁎⁎
⁎⁎ CD3-CD16+NKcells (%) (c) (c)
101
102
103
104
100
104
HPV18+
CD4 FITC, CD25APC subset
4.64
CD4-FITC
CD25-APC
Contro lH
PV 16+
HPV18+
0
2
4
6
8
CD4+CD25+ Treg cells (%)
⁎⁎
⁎⁎
103
104
102
101
100
(d) 101
102
103
104
100
100
101
102
103
104
101
102
103
104
100
Control
CD4 FITC, CD25APC subset
6.70
CD4-FITC
CD25-APC
CD4 FITC, CD25APC subset
1.30
CD4-FITC
CD25-APC
HPV18+
HPV16+
CD4 FITC, CD25APC subset
4.64
CD4-FITC
CD25-APC
Contro lH
PV 16+
HPV18+
0
2
4
6
8
CD4+CD25+ Treg cells (%)
⁎⁎
⁎⁎
103
104
102
101
100
103
104
102
101
100
103
104
102
101
100
(d) 101
102
103
104
100
103
104
101
102
103
104
100
ol
APC subset
TC
CD4 FITC, CD25APC subset
1.30
CD4-FITC
CD25-APC
HPV18+
HPV16+
CD4 FITC, CD25APC subset
4.64
CD4-FITC
CD25-APC
103
104
102
101
100
103
104
102
101
100
(d) (d) Figure 2: The different effects of NK cells between HPV16- and HPV18-infected cervixes. 4. Discussion (a) Distribution of CD56+CD16+ NK cells was analyzed
in the cervical brush specimens by FACS. (b) Several soluble cytokines that represent NK cell involvement in the immune status were
investigated in the cervical brush specimens by quantitative real-time PCR. (c) IFN-γ and IL-2 expressions were measured in the cervical
conization tissue by quantitative real-time PCR. ∗P < 0 05; ∗∗P < 0 01. Journal of Immunology Research 5 Control
HPV 16+
HPV 18+
0
5
10
15
KLRG-1
Relative mRNA fold change
Control
HPV 16+
HPV 18+
0
2
4
6
8
DNAM-1
Relative mRNA fold change
Control
HPV 16+
HPV 18+
0
4
8
12
CD56
Relative mRNA fold change
Control
HPV 16+
HPV 18+
0
1
2
3
4
CD27
Relative mRNA fold change
⁎⁎
⁎⁎
(a)
HPV 16+
HPV 18 +
0.0
0.5
1.0
1.5
2.0
44.0
44.5
KLRG-1
Relative mRNA fold change
⁎⁎
(b)
gy observed. 4. Discussion CD16 is a vital membrane protein found on NK
ll
h
i d
HPV VLP
d
i
f ll
d b
results
imply
that
the
chronic
inflammation-induced
ib d
d
d
h
i
i
i
i
l NK
ll
Control
HPV 16+
HPV 18+
0
5
10
15
KLRG-1
Relative mRNA fold change
Control
HPV 16+
HPV 18+
0
2
4
6
8
DNAM-1
Relative mRNA fold change
Control
HPV 16+
HPV 18+
0
4
8
12
CD56
Relative mRNA fold change
Control
HPV 16+
HPV 18+
0
1
2
3
4
CD27
Relative mRNA fold change
⁎⁎
⁎⁎
(a)
HPV 16+
HPV 18 +
0.0
0.5
1.0
1.5
2.0
44.0
44.5
KLRG-1
Relative mRNA fold change
⁎⁎
(b)
103
104
102
101
100
100
101
102
103
104
Control
HPV 16+
HPV18+
0.0
0.5
1.0
1.5
CD3-CD16+NKcells (%)
⁎⁎
⁎⁎
103
104
102
101
100
103
104
102
101
100
100
101
102
103
104
100
101
102
103
104
Control
CD3 FITC, CD16 PE subset
0.067
CD3–FITC
CD16–PE
HPV16+
CD3 FITC, CD16 PE subset
0.073
CD3–FITC
CD16–PE
HPV18+
CD3 FITC, CD16 PE subset
0.020
CD3–FITC
CD16–PE
(c)
101
102
103
104
100
100
101
102
103
104
101
102
103
104
100
Control
CD4 FITC, CD25APC subset
6.70
CD4-FITC
CD25-APC
CD4 FITC, CD25APC subset
1.30
CD4-FITC
CD25-APC
HPV18+
HPV16+
CD4 FITC, CD25APC subset
4.64
CD4-FITC
CD25-APC
Contro lH
PV 16+
HPV18+
0
2
4
6
8
CD4+CD25+ Treg cells (%)
⁎⁎
⁎⁎
103
104
102
101
100
103
104
102
101
100
103
104
102
101
100
(d)
Figure 2: The different effects of NK cells between HPV16- and HPV18-infected cervixes. (a) Four typical cell membrane markers of NK cells
were evaluated in the cervical brush specimens by quantitative real-time PCR. (b) KLRG-1 expression was measured in the cervical conization
tissue by quantitative real-time PCR. (c) Distribution of CD16+ NK cells was analyzed in the cervical brush specimens by FACS. (d)
Distribution of CD4+CD25+ Treg cells was analyzed in the cervical brush specimens by FACS. ∗P < 0 05; ∗∗P < 0 01. 4. Discussion (a) Four typical cell membrane markers of NK cells
were evaluated in the cervical brush specimens by quantitative real-time PCR. (b) KLRG-1 expression was measured in the cervical conization
tissue by quantitative real-time PCR. (c) Distribution of CD16+ NK cells was analyzed in the cervical brush specimens by FACS. (d)
Distribution of CD4+CD25+ Treg cells was analyzed in the cervical brush specimens by FACS. ∗P < 0 05; ∗∗P < 0 01. observed. CD16 is a vital membrane protein found on NK
cells that can induce HPV-VLP endocytosis followed by
degranulation and cytokine secretion (such as IFN-γ and
tumor necrosis factor (TNF)) [6, 11]. Engagement of CD16
on NK cells results in its ADCC activation [12, 13]. There results
imply
that
the
chronic
inflammation-induced
antibody-dependent pathway is active in cervical NK cells. results
imply
that
the
chronic
inflammation-induced
antibody-dependent pathway is active in cervical NK cells. However, these increased NK cells still failed to eliminate
the mature HPV virus under circumstances that Treg cells
were locally restricted. It is probably due to inadequate IL-2 However, these increased NK cells still failed to eliminate
the mature HPV virus under circumstances that Treg cells
were locally restricted. 4. Discussion We also found
that HPV infection decreased IFN-γ transcription by
immune cells, and this was consistent with other studies. It
has been reported that the early gene products, E6 and E7,
regulate local cytokine and chemokine expression, for
instance the downregulation of IFN-γ and other components
related to the signal transduction pathway, against virus
infection [20]. was low in Eastern China. A future work will be focusing
on evaluating other candidate cytokines (which in the pres-
ent study showed a trend of change but with no statistical sig-
nificance) such as IL-6, GM-CSF, CCL-5, and membrane
protein DNAM-1 in HPV infection; (3) in the mixing of
specimens for flow cytometry analysis, according to Hubert’s
study [21], the number of NK cells in cervical biopsies was
too low to score, and they could only be counted in the
stroma. Therefore, 4 specimens of different patients were
mixed to produce a sample for flow cytometry analysis. Each
group was repeated independently for four times. g
p
p
p
y
One of the cervical cancer-preventive HPV vaccines—
Cervarix™—is currently authorized for use in Europe and
the United States. It is composed of viral components from
the later period of gene expression, such as product L1 of
HPV16 and HPV18 and an adjuvant, AS04. Although it has
certain positive effects on the prevention of viral reinfection,
its low concentration in the cervical environment is still an
annoying problem and cannot be ignored. More importantly,
it is far away from the curing the disease [22]. Another
medicinal product used against high-risk HPV types in
Canada is a quadrivalent HPV vaccine (Gardasil, Merck)
which was recently proved to be 30-50% effective against
lesions caused by all HPV types [23]. Over the past decade,
many researchers have devoted time and effort into develop-
ing a therapeutic vaccine. Some have made gratifying results
in animal models, but these vaccine applications are still
confined to the laboratory. Clinical observations also suggest
that antibodies in patients with persistent infection are mostly
negative. Therefore, it is questionable whether humoral Our study has some limitations: (1) cytokines were tested
using quantitative real-time PCR, and this can be problemat-
ical. Some researchers collected fluid by cervicovaginal lavage
in order to detect secretory proteins by ELISA. This is only
feasible if vaginal diseases are first excluded; (2) the number
of specimens collected was small. 4. Discussion 103
104
102
101
100
4
100
101
102
103
104
HPV18+
Q2
Q1
3.47
Q3
96.4
0.10
Q4
0.060
CFSE
7–AAD
T E
1 0 103
104
102
101
100
103
104
102
101
100
100
101
102
103
104
Control
Q2
Q1
4.15
Q3
95.8
0
Q4
0
CFSE
7–AAD
100
101
102
103
104
Q2
Q1
4.23
Q3
95.8
0
Q4
0.020
CFSE
7–AAD
HPV16+
7 AAD
T:E = 1:1 103
104
102
101
100
04
100
101
102
103
104
Q2
Q1
1.43
Q3
98.6
0
Q4
0
CFSE
7–AAD
T:E = 1:0 103
104
102
101
100
103
104
102
101
100
CFSE
100
101
102
103
104
100
101
102
103
104
Q2
Q1
6.82
Q3
93.2
0
Q4
0
CFSE
7–AAD
T:E = 1:10
Q2
Q1
4.99
Q3
95.0
0
Q4
0.014
CFSE
7–AAD 103
104
102
101
100
4
100
101
102
103
104
Q2
Q1
7.20
Q3
92.5
0.25
Q4
0.069
CFSE
7–AAD
1:1
1:10
0
2
4
6
8
T:E ratio
Cytotoxicity of NK cells (%)
Control
HPV 16+
HPV 18+
⁎⁎
⁎⁎
⁎
⁎ Figure 3: The cytotoxicity of NK cells derived from HPV16+ or HPV18+ women using HeLa cells labeled with CFSE. CD56+CD16+ NK cells
were isolated from cervical brush specimens of women infected with either HPV16 or HPV18, respectively, via FACS. NK cells were
cocultured with CFSE-HeLa in designated ratios in 24-well plates and incubated at a 37°C incubator for 4-6 hours. Cells were harvested
and stained with 7-AAD. CFSE+7-AAD+ cells were examined by FACS. ∗P < 0 05; ∗∗P < 0 01. production which is reported to positively regulate NK cell
cytotoxic function and downstream differentiation [14], as
well as virus-induced KLRG-1 expression. KLRG-1 belongs
to the C-lectin superfamily and is known to be an inhibitory
receptor of NK cells. KLRG-1- NK cells proliferate well, but
they lack a mature phenotype [15]. Nevertheless, recent
researches have emphasized that “NK cell education” which
is part of its functional modification is mainly conducted
via the ITIM/SHP1/SHP2/SHIP signaling pathway [16]. KLRG-1 is associated with the recruitment of SHIP and the
KLRG/SHIP disturbance renders NK cells hyporesponsive
and uneducated [17, 18]. Since effector CD8+ T cells also
express KLRG-1, we cannot rule out a role for exhausted
CD8+ T cells in an HPV-infected cervix [19]. 4. Discussion It is probably due to inadequate IL-2 Journal of Immunology Research 6 103
104
102
101
100
103
104
102
101
100
103
104
102
101
100
103
104
102
101
100
103
104
102
101
100
103
104
102
101
100
103
104
102
101
100
100
101
102
103
104
Control
Q2
Q1
4.15
Q3
95.8
0
Q4
0
CFSE
7–AAD
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
Q2
Q1
6.82
Q3
93.2
0
Q4
0
CFSE
7–AAD
100
101
102
103
104
Q2
Q1
4.23
Q3
95.8
0
Q4
0.020
CFSE
7–AAD
100
101
102
103
104
HPV18+
HPV16+
Q2
Q1
3.47
Q3
96.4
0.10
Q4
0.060
CFSE
7–AAD
100
101
102
103
104
Q2
Q1
1.43
Q3
98.6
0
Q4
0
CFSE
7–AAD
T:E = 1:1
T:E = 1:0
T:E = 1:10
Q2
Q1
4.99
Q3
95.0
0
Q4
0.014
CFSE
7–AAD
Q2
Q1
7.20
Q3
92.5
0.25
Q4
0.069
CFSE
7–AAD
1:1
1:10
0
2
4
6
8
T:E ratio
Cytotoxicity of NK cells (%)
Control
HPV 16+
HPV 18+
⁎⁎
⁎⁎
⁎
⁎
Figure 3: The cytotoxicity of NK cells derived from HPV16+ or HPV18+ women using HeLa cells labeled with CFSE. CD56+CD16+ NK cells
were isolated from cervical brush specimens of women infected with either HPV16 or HPV18, respectively, via FACS. NK cells were
cocultured with CFSE-HeLa in designated ratios in 24-well plates and incubated at a 37°C incubator for 4-6 hours. Cells were harvested
and stained with 7-AAD. CFSE+7-AAD+ cells were examined by FACS. ∗P < 0 05; ∗∗P < 0 01. 4. Discussion We enrolled women
infected with either HPV16 or HPV18, but a large proportion
of HPV women had a combined infection along with other
low-risk types. In particular, the incidence of HPV18 itself 7 Journal of Immunology Research Province (No. BK20151029), and the Key Laboratory for
the Laboratory Medicine of Jiangsu Province of China
(No. ZDXKB2016005). The authors would like to thank
Dr. Dev Sooranna, Imperial College London, for editing
the manuscript. Province (No. BK20151029), and the Key Laboratory for
the Laboratory Medicine of Jiangsu Province of China
(No. ZDXKB2016005). The authors would like to thank
Dr. Dev Sooranna, Imperial College London, for editing
the manuscript. immune status could be used as an indicator of regression for
any infection. The efficacy of therapeutic vaccines is closely
related to the T cell response, Th1 cytokine secretion, local
infiltration of CD4+ and CD8+ T cells, and the function of
NK cells in the cervix [24]. Based on this study, we raise
another question that will such therapeutic vaccines possess
equal capability for killing cervical cancer cells previously
infected by either HPV16 or HPV18? We believe that drugs/-
vaccines targeting specific HPV type will be a new promising
direction for HPV treatment. References [1] H. R. Mcmurray, D. Nguyen, T. F. Westbrook, and D. J. Mcance, “Biology of human papillomaviruses,” International
Journal of Experimental Pathology, vol. 82, no. 1, pp. 15–33,
2001. Conflicts of Interest We declare that we do not have any commercial or associative
interests that represent a conflict of interest in connection
with the work submitted. [12] J. H. W. Pahl, A. Cerwenka, and J. Ni, “Memory-like NK cells:
remembering a previous activation by cytokines and NK cell
receptors,” Frontiers in Immunology, vol. 9, p. 2796, 2018. [13] S. Jegaskanda, H. A. Vanderven, H. X. Tan et al., “Influenza
virus infection enhances antibody-mediated NK cell functions
via type I interferon-dependent pathways,” Journal of Virology,
vol. 93, no. 5, 2018. Data Availability [10] J. Li, X. Dong, L. Zhao et al., “Natural killer cells regulate
Th1/Treg and Th17/Treg balance in chlamydial lung infec-
tion,” Journal of Cellular and Molecular Medicine, vol. 20,
no. 7, pp. 1339–1351, 2016. All data supporting the results reported in the article are
generated and archived in the facilities of the Department
of Laboratory Medicine, the First Affiliated Hospital of
Nanjing Medical University. [11] K. A. Holder, J. Lajoie, and M. D. Grant, “Natural killer cells
adapt to cytomegalovirus along a functionally static pheno-
typic spectrum in human immunodeficiency virus infection,”
Frontiers in Immunology, vol. 9, p. 2494, 2018. Abbreviations HPV:
Human papillomavirus
CIN:
Cervical intraepithelial neoplasia
NK cell:
Natural killer cell
IFN-γ:
Interferon-γ
IL:
Interleukin
GM-CSF:
Granulocyte-macrophage colony-stimulating
factor
CCL:
CC-chemokine ligand
KLRG-1:
Killer cell lectin-like receptor subfamily G
member 1
DNAM-1: DNAX accessory molecule-1
ADCC:
Antibody-dependent cellular cytotoxicity
ITIM:
Immunoreceptor tyrosine-based inhibitory motif
SHP:
SH2-domain-containing protein tyrosine
phosphatase
SHIP:
SH2-domain-containing inositol-5-phosphatase
Treg:
Regulatory T cells. [6] V. M. Renoux, B. Bisig, I. Langers et al., “Human papillomavi-
rus entry into NK cells requires CD16 expression and triggers
cytotoxic activity and cytokine secretion,” European Journal of
Immunology, vol. 41, no. 11, pp. 3240–3252, 2011. [7] T. Garcia-Iglesias, A. del Toro-Arreola, B. Albarran-Somoza
et al., “Low NKp30, NKp46 and NKG2D expression and
reduced cytotoxic activity on NK cells in cervical cancer and
precursor lesions,” BMC Cancer, vol. 9, 2009. [8] J. H. Cui, K. R. Lin, S. H. Yuan et al., “TCR repertoire as a novel
indicator for immune monitoring and prognosis assessment of
patients with cervical cancer,” Frontiers in Immunology, vol. 9,
p. 2729, 2018. [9] J. Rotman, C. H. Mom, E. S. Jordanova, T. D. de Gruijl, and
G. G. Kenter, “DURVIT': a phase-I trial of single low-dose dur-
valumab (Medi4736) intratumourally injected in cervical can-
cer: safety, toxicity and effect on the primary tumour- and
lymph node microenvironment,” BMC Cancer, vol. 18, 2018. 5. Conclusions [2] V. Cogliano, R. Baan, K. Straif, Y. Grosse, B. Secretan, and F. E. Ghissassi, “Carcinogenicity of human papillomaviruses,” The
Lancet Oncology, vol. 6, no. 4, p. 204, 2005. In summary, our data demonstrated that the number of
NK cells was increased, but their cytotoxic function was
abnormal in an HPV16-infected cervix. This is the first study
emphasizing the unique immune profiles of the cervical
microenvironment between two high-risk HPV types. The
involved mechanisms may partially reveal the reason why
HPV16 is the most likely to cause cervical cancer and may
provide new potential strategies for its clinical management. [3] R. D. Burk, Z. Chen, and K. Van Doorslaer, “Human papillo-
maviruses: genetic basis of carcinogenicity,” Public Health
Genomics, vol. 12, no. 5-6, pp. 281–290, 2009. [4] WHO/ICO Information Centre on HPV and Cervical Cancer,
“HPV and cervical cancer in the 2007 report,” Vaccine, vol. 25,
Supplement 3, pp. C1–230, 2007. [5] P. E. Castle, M. H. Stoler, T. C. Wright Jr., A. Sharma, T. L. Wright, and C. M. Behrens, “Performance of carcinogenic
human papillomavirus (HPV) testing and HPV16 or HPV18
genotyping for cervical cancer screening of women aged 25
years and older: a subanalysis of the ATHENA study,” The
Lancet Oncology, vol. 12, no. 9, pp. 880–890, 2011. Acknowledgments This work was supported by grants from the National
Natural Science Foundation of China (Nos. 81501817 and
81671836), the Natural Science Youth Foundation of Jiangsu [14] K. M. Mirjačić Martinović, A. M. Vuletić, N. L. Babović, R. R. Džodić, G. M. Konjević, and V. B. Jurišić, “Attenuated in vitro Journal of Immunology Research 8 effects of IFN-α, IL-2 and IL-12 on functional and receptor
characteristics of peripheral blood lymphocytes in metastatic
melanoma patients,” Cytokine, vol. 96, pp. 30–40, 2017. [15] N. D. Huntington, H. Tabarias, K. Fairfax et al., “NK cell
maturation and peripheral homeostasis is associated with
KLRG1 up-regulation,” Journal of Immunology, vol. 178, no. 8,
pp. 4764–4770, 2007. [16] Y. He and Z. Tian, “NK cell education via nonclassical MHC
and non-MHC ligands,” Cellular & Molecular Immunology,
vol. 14, no. 4, pp. 321–330, 2017. [17] J. W. Wang, J. M. Howson, T. Ghansah et al., “Influence of
SHIP on the NK repertoire and allogeneic bone marrow trans-
plantation,” Science, vol. 295, no. 5562, pp. 2094–2097, 2002. [18] J. A. Wahle, K. H. T. Paraiso, R. D. Kendig et al., “Inappropri-
ate recruitment and activity by the Src homology region 2
domain-containing phosphatase 1 (SHP1) is responsible for
receptor dominance in the SHIP-deficient NK cell,” The Jour-
nal of Immunology, vol. 179, no. 12, pp. 8009–8015, 2007. [19] E. J. Wherry and M. Kurachi, “Molecular and cellular insights
into T cell exhaustion,” Nature Reviews Immunology, vol. 15,
no. 8, pp. 486–499, 2015. [20] V. Bordignon, E. di Domenico, E. Trento et al., “How human
papillomavirus replication and immune evasion strategies take
advantage of the host DNA damage repair machinery,”
Viruses, vol. 9, no. 12, p. 390, 2017. [21] P. Hubert, J. Doyen, X. Capelle et al., “Local applications of
GM-CSF induce the recruitment of immune cells in cervical
low-grade squamous intraepithelial lesions,” American Journal
of Reproductive Immunology, vol. 64, no. 2, pp. 126–136, 2010. [22] A. Monie, C. F. Hung, and T. C. Wu, “Preventive and thera-
peutic HPV vaccines,” Current Opinion in Investigational
Drugs, vol. 8, no. 12, pp. 1038–1050, 2007. [23] C. H. Righolt, S. Bozat-Emre, and S. M. Mahmud, “Effective-
ness of school-based and high-risk human papillomavirus
vaccination programs against cervical dysplasia in Manitoba,
Canada,” International Journal of Cancer, 2019. [24] R. K. Sharma, A. K. Srivastava, E. S. Acknowledgments Yolcu et al., “SA-4-1BBL as
the immunomodulatory component of a HPV-16 E7 protein
based vaccine shows robust therapeutic efficacy in a mouse
cervical cancer model,” Vaccine, vol. 28, no. 36, pp. 5794–
5802, 2010.
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Time to reconcile research findings and clinical practice on upper limb neurorehabilitation
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Frontiers in neurology
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TYPE Review
PUBLISHED 19 July 2022
DOI 10.3389/fneur.2022.939748 TYPE Review
PUBLISHED 19 July 2022
DOI 10.3389/fneur.2022.939748 Time to reconcile research
findings and clinical practice on
upper limb neurorehabilitation OPEN ACCESS
EDITED BY
Mariella Pazzaglia,
Sapienza University of Rome, Italy
REVIEWED BY
Jonathan Sanching Tsay,
University of California, Berkeley,
United States
Andrea Manca,
University of Sassari, Italy
*CORRESPONDENCE
Leonardo Boccuni
lboccuni@guttmann.com
SPECIALTY SECTION
This article was submitted to
Neurorehabilitation,
a section of the journal
Frontiers in Neurology
RECEIVED 09 May 2022
ACCEPTED 27 June 2022
PUBLISHED 19 July 2022 OPEN ACCESS
EDITED BY
Mariella Pazzaglia,
Sapienza University of Rome, Italy
REVIEWED BY
Jonathan Sanching Tsay,
University of California, Berkeley,
United States
Andrea Manca,
University of Sassari, Italy
*CORRESPONDENCE
Leonardo Boccuni
lboccuni@guttmann.com
SPECIALTY SECTION
This article was submitted to
Neurorehabilitation,
a section of the journal
Frontiers in Neurology
RECEIVED 09 May 2022
ACCEPTED 27 June 2022
PUBLISHED 19 July 2022 Leonardo Boccuni1,2,3*, Lucio Marinelli4,5, Carlo Trompetto4,6,
Alvaro Pascual-Leone1,7,8 and José María Tormos Muñoz1,2,3 1Institut Guttmann, Institut Universitari de Neurorehabilitació adscrit a la UAB, Badalona, Spain,
2Universitat Autònoma de Barcelona, Bellaterra, Spain, 3Fundació Institut d’Investigació en Ciències
de la Salut Germans Trias i Pujol, Badalona, Spain, 4Department of Neuroscience, Rehabilitation,
Ophthalmology, Genetics, Maternal and Child Health, University of Genova, Genova, Italy, 5IRCCS
Ospedale Policlinico San Martino, Department of Neuroscience, Division of Clinical
Neurophysiology, Genova, Italy, 6IRCCS Ospedale Policlinico San Martino, Department of
Neuroscience, Division of Neurorehabilitation, Genova, Italy, 7Hinda and Arthur Marcus Institute for
Aging Research and Deanna and Sidney Wolk Center for Memory Health, Hebrew SeniorLife,
Boston, MA, United States, 8Department of Neurology and Harvard Medical School, Boston, MA,
United States The problem: In the field of upper limb neurorehabilitation, the translation from
research findings to clinical practice remains troublesome. Patients are not
receiving treatments based on the best available evidence. There are certainly
multiple reasons to account for this issue, including the power of habit over
innovation, subjective beliefs over objective results. We need to take a step
forward, by looking at most important results from randomized controlled
trials, and then identify key active ingredients that determined the success
of interventions. On the other hand, we need to recognize those specific
categories of patients having the greatest benefit from each intervention, and
why. The aim is to reach the ability to design a neurorehabilitation program
based on motor learning principles with established clinical efcacy and
tailored for specific patient’s needs. CITATION
Boccuni L, Marinelli L, Trompetto C,
Pascual-Leone A and Tormos
Muñoz JM (2022) Time to reconcile
research findings and clinical practice
on upper limb neurorehabilitation. Front. Neurol. 13:939748. Introduction To make an example with a reaching task, performance will
show higher improvements for frequently reached targets
with stable conditions, in comparison to rarely reached
target (use-dependent learning), or when there is excessive
variability of conditions (reinforcement learning). The person
being trained is aware of the goals of the training based on
instructions received (explicit strategies), but unaware of the
kinematics and automatic adjustments underlying movement
performance (implicit recalibration). A broader classification
of motor learning principles related to neurorehabilitation
have been proposed by Maier et al. in 2019, specifically by
pointing out the following 15 principles: Repetitive practice,
spaced practice, dosage, task-specific practice, goal-oriented
practice, variable practice, increasing difficulty, multisensory
stimulation, rhythmic cueing, explicit feedback/knowledge of
results, implicit feedback/knowledge of performance, modulate
effector selection, action observation/embodied practice, motor
imagery, and social interaction (15). Given their practical
utility to describe neurorehabilitation interventions, we will
refer to these principles throughout the manuscript. Regarding
neuroplastic mechanisms, we should at least distinguish two
models of brain reorganization after stroke, the interhemispheric
inhibition model and vicariation model, the former taking
place after small lesions causing mild to moderate motor
impairment, the latter observed after larger lesions causing
moderate to severe motor impairment (16). The key difference
is the competitive vs. cooperative role of the contralesional
hemisphere. As a consequence, depending on the severity
of motor impairment, therapeutic approaches may prefer to
target more uni- or bihemispheric activation, such as when
performing unilateral vs. bilateral arm training (17, 18). To
date, we are far from having assimilated the knowledge of
active ingredients and motor learning principles within each
intervention. Coming back to the initial example, for most
therapists and clinicians CIMT is somehow synonymous of
“the glove”; this is because, in the original CIMT trial, patients
had to wear a mitt on the unaffected hand to limit its use
during the day, and to remind patients to accomplish functional
tasks with the affected hand (19). This vision is reductionist
and misleading, considering that the original authors have
shown that wearing a mitt is ineffective when tested as isolated
ingredient (20); in contrast, other key components of CIMT There is a translational gap between research findings and
clinical practice in the field of upper limb neurorehabilitation. The latest decades have seen an exponential growth of evidence,
that has not been followed by an adequate evolution of
treatments offered to patients. Time to reconcile research
findings and clinical practice on
upper limb neurorehabilitation doi: 10.3389/fneur.2022.939748 COPYRIGHT
© 2022 Boccuni, Marinelli, Trompetto,
Pascual-Leone and Tormos Muñoz. This is an open-access article
distributed under the terms of the
Creative Commons Attribution License
(CC BY). The use, distribution or
reproduction in other forums is
permitted, provided the original
author(s) and the copyright owner(s)
are credited and that the original
publication in this journal is cited, in
accordance with accepted academic
practice. No use, distribution or
reproduction is permitted which does
not comply with these terms. Proposed solutions:
The objective of the present manuscript is to
facilitate the translation of research findings to clinical practice. Starting
from a literature review of selected neurorehabilitation approaches, for
each intervention the following elements were highlighted: definition of
active ingredients; identification of underlying motor learning principles and
neural mechanisms of recovery; inferences from research findings; and
recommendations for clinical practice. Furthermore, we included a dedicated
chapter on the importance of a comprehensive assessment (objective
impairments and patient’s perspective) to design personalized and efective
neurorehabilitation interventions. Conclusions: It’s time to reconcile research findings with clinical practice. Evidence from literature is consistently showing that neurological patients
improve upper limb function, when core strategies based on motor learning
principles are applied. To this end, practical take-home messages in the
concluding section are provided, focusing on the importance of graded task
practice, high number of repetitions, interventions tailored to patient’s goals
and expectations, solutions to increase and distribute therapy beyond the
formal patient-therapist session, and how to integrate diferent interventions Frontiers in Neurology Frontiers in Neurology 01 frontiersin.org Boccuni et al. 10.3389/fneur.2022.939748 10.3389/fneur.2022.939748 to maximize upper limb motor outcomes. We hope that this manuscript will
serve as starting point to fill the gap between theory and practice in upper limb
neurorehabilitation, and as a practical tool to leverage the positive impact of
clinicians on patients’ recovery. stroke, neurorehabilitation, upper limb, motor learning, personalized medicine Introduction One example is Constraint-
Induced Movement Therapy (CIMT), an intensive, motor-
learning based intervention that has demonstrated the highest
evidence level for the improvement of upper limb motor
function (1), with results successfully replicated in several
randomized controlled trials (2). Despite two decades of
consistent findings, CIMT is rarely applied in most clinical
centers, whereas other interventions like Neurodevelopmental
Treatment (NDT, also known as Bobath concept) are popular
among therapists, irrespective that research evidence has shown
equal or even inferior effectiveness for NDT, in comparison
with other approaches (1, 3, 4). There are likely multiple
reasons to account for this. Sometimes adoption of a certain
intervention in the clinical practice flow is challenging because
of time, resources, training, and other requirements. Other
times, the scientific evidence is difficult to apply to the individual
patient since studies often focus on group average effects
effectively disregarding – rather than leveraging – individual
variability. The kind of personalized, optimal effective treatment
we all desire and that our patients deserve requires overcoming
these challenges. A
first
step
toward
evidence-based
practice
is
the
identification,
within
each
intervention,
of
those
core
elements (active ingredients) that are essential for treatment
effectiveness
(5). For
motor
training
approaches,
active
ingredients could be for instance relative to the organization
of task practice (number of repetitions, variability, global
vs. selective movement practice), the provision of a certain
type of feedback (implicit vs. explicit feedback), and so on. The following step is the identification of motor learning
principles and neuroplastic mechanisms underlying recovery,
to interpret the theoretical basis and the concrete results
of any neurorehabilitation approach. We could start by
considering four general motor learning principles: use-
dependent learning (6–8), reinforcement learning (9, 10),
explicit strategies (11, 12), and implicit recalibration (13, 14). Frontiers in Neurology frontiersin.org 02 Boccuni et al. 10.3389/fneur.2022.939748 10.3389/fneur.2022.939748 10.3389/fneur.2022.939748 techniques for mobilization and tone reduction, the inclusion
of bilateral arm training to accomplish tasks, and the use
of assistive devices such as orthotics and functional electrical
stimulation (25). have shown consistent effectiveness, but unfortunately are still
largely neglected. Introduction Therefore, the first objective of the present review was
to define each intervention based on key active ingredients,
underlying motor learning principles and neural mechanisms
likely taking place; the second objective was to draw inferences
from research findings, to support our recommendations for
clinical practice, familiarize clinicians with the methodology
of interpreting evidence, and stimulate further reading and
critical appraisal of scientific literature (21). To this end,
each chapter is organized with the headers “Intervention,”
“Rationale,” “Inferences” and “Recommendations” (see also
Table 1). Furthermore, a dedicated chapter on comprehensive
assessment of neurological patients is provided, as cornerstone
requirement to design a personalized and effective upper limb
neurorehabilitation treatment. Finally, we conclude with take-
home messages and remarks with direct implications for both
clinical practice and research. Intervention CIMT is the most widely investigated motor training
approach for upper limb neurorehabilitation, and one of the
most effective interventions to date (1, 2, 23). Being originally
developed for intensive rehabilitation of patients with mild to
moderate motor impairment (19) [bare minimum: 10◦wrist
extension, 10◦thumb abduction, and 10◦extension any two
other digits (24)], CIMT has been investigated in a number of
subsequent trials, although with different doses of therapy (2),
and recently adapted to be applicable also for those patients with
moderate to severe motor impairment (25). According to the
original authors, CIMT is a composite intervention that includes
three main elements: restrain of the unaffected hand for the
90% of waking hours, task-oriented training for up to 6 h/day,
and a transfer package to foster adherence to the intervention
and behavioral changes in the long term (use of logbook, diary,
home practice) (5). Within task-oriented training we can further
distinguish two components, i.e., task practice and shaping. Task
practice is the training of an activity of daily living for about
15–20 min, like preparing a meal, while shaping is the repetitive
performance of components of functional tasks, such as reaching
and grasping movements (26). A narrative review of Page et al. illustrates practical challenges and opportunities of CIMT, and
provides explicit examples to facilitate the implementation of
CIMT in a clinical setting (27). Notably, in the adaptation of
CIMT for severely impaired patients (“expanded CIMT”), three
important ingredients have been the adoption of preparatory Rationale CIMT is a composite motor training approach, where
we could identify the systematic application of the following
eight motor learning principles: repetitive practice, dosage,
task-specific practice, goal-oriented practice, variable practice,
increasing difficulty, explicit feedback/knowledge of results, and
modulate effector selection. The neural basis of the original
CIMT is the interhemispheric competition model, applicable
for healthy individuals and patients with relatively small lesions
causing mild to moderate impairment (16). According to
this model, unimanual activity is the result of the activation
of the contralateral hemisphere that sends efferent input to
the spinal cord, while at the same time inhibiting the other
hemisphere through transcallosal pathways (28). Consequently,
the tendency of using only the unaffected arm to accomplish
tasks may lead to activation of the unaffected hemisphere and
over-inhibition of the affected hemisphere, with negative impact
on motor recovery (learned non-use). Therefore, CIMT was
designed to restrain the use of the unaffected arm and promote
unimanual training of the affected arm. On the other hand,
when expanding CIMT to be applicable for severely impaired
patients, the vicariation model may be more suitable (16, 29). To summarize, in case of large lesions causing substantial
disruption of the corticospinal tract, the brain compensates
the lack of resources from primary sensorimotor cortex by
recruiting alternative ipsilesional and contralesional pathways
to perform unimanual activities. In fact, there are several
descending pathways that could subserve motor recovery,
arising from parietofrontal regions of both hemispheres and
forming direct and indirect (with the interpolation of reticular
formation) connections with primary motorneurons of the
affected arm (30, 31). Given the cooperative role of the
contralesional hemisphere, and the objective difficulty on
achieving a functional task with the sole use of the affected
arm, the expanded CIMT protocol considered the inclusion of
bilateral arm training, less restrictions of the unaffected arm, and
systematic adoption of assistive devices (25). Inferences To understand the relative importance of each component
on the final motor outcome, it’s useful to compare the
effectiveness of CIMT vs. conventional therapy, and the
effectiveness of the original CIMT trial vs. modified CIMT
versions, generally characterized by reduced time of affected-
arm therapy and unaffected-arm restrain, as compared to the
original version. Despite the heterogeneity of study designs, Frontiers in Neurology 03 frontiersin.org Boccuni et al. 10.3389/fneur.2022.939748 TABLE 1 Summary of upper limb neurorehabilitation interventions according to key active ingredients, rationale and recommendations for
clinical practice TABLE 1 Summary of upper limb neurorehabilitation interventions according to key active ingredients, rationale and recommendations for
clinical practice. clinical practice. Interventions
Key active
ingredients
Key motor
learning principles
and neural
mechanisms
Recommendations
Motor training approaches
CIMT
Shaping
Task practice
Explicit learning
Repetitive practice
Goal-oriented practice
Task-specific practice
Interhemispheric
inhibition model
Indicated for mild to
moderately impaired
patients; consider
strategies for
independent and
distributed practice; use
of a mitt is not
mandatory
Expanded CIMT
(adaptated for severely
impaired patients)
McCabe et al. (22)
(intensive rehabilitation
for severely impaired
patients)
Shaping
Task practice
Inclusion of orthotics,
FES and bilateral
training to
accomplish tasks
Explicit learning
Repetitive practice
Goal-oriented practice
Task specific practice
Vicaration model
Indicated for severely
impaired patients;
consider devices to assist
movement execution;
include both unilateral
and bilateral arm
training
Bobath Concept
Movement quality
Afferent input to
improve motor control,
body schema and
motor learning
Facilitation techniques
(handling,
environmental
adaptation,
verbal cueing)
Explicit learning
(implicit feedback/
knowledge of
performance)
Implicit learning
(sensorimotor
integration)
Task specific practice
Suitable for any level of
disability, but currently
equally or less effective
than other approaches. Consider repetitive
practice (high number of
repetitions) to increase
the effectiveness of the
intervention. Interventions assisting movement execution
Robotic therapy
Repetitive movement
performance
Explicit learning
Repetitive practice
Particularly useful to
assist shoulder and elbow
movements (reaching)
Functional electrical
stimulation
Repetitive movement
performance
Explicit learning
Repetitive practice
Sensorimotor integration
Particularly useful to
assist finger movements
(grasping)
splint/orthotics
Repetitive movement
performance
Explicit learning
Repetitive practice
Particularly useful for
wrist and thumb
functional positioning
Interventions providing feedback and multisensory stimulation
Virtual reality
Motivation, engagement
Visual and
auditory stimuli
Explicit learning
Explicit feedback
Multisensory stimulation
Particularly useful to
increase motivation,
avoid boredom and
promote independent
practice. Consider the
provision of cues about
movement quality
(implicit feedback)
(Continued) Frontiers in Neurology 04 frontiersin.org 10.3389/fneur.2022.939748 Boccuni et al. Inferences 10.3389/fneur.2022.939748 value, to keep the patient motivated to challenge him-herself
on subsequent trials (5, 26). Complex functional tasks (for
instance, drinking water from a bottle) are initially broken
down into simpler motor components (arm reaching, hand
grasping, elbow flexion/extension, etc), until the level that can be
practiced effectively and repeatedly by the patient; as the patient
progresses, components are gradually practiced in combination,
until the whole functional task can be accomplished. The
level of difficulty is also adjusted by modulating the required
number of repetitions, speed, and accuracy. The selection of
the task to be trained is based upon three criteria: (a) being
focused on the specific motor impairment shown by the patient
(which joints/muscles are involved? which movements are
compromised?); (b) being judged by therapists as having the
potential to improve the motor outcome of the patient (is
there a potential for recovery?); (c) being the task chosen by
the patient among a set of similar tasks, in order to meet
patient preferences and involve him/her in the decision making
process of selecting a task (26). In recent years, high-intensity
upper limb rehabilitation trials have consistently reported
clinically meaningful motor improvements by applying shaping
principles, such as decomposition of functional activities in
simpler motor tasks, systematic performance of repetitions and
provision of feedback, tasks being specific to motor impairments
and toward patient’s preferences and goals, and progressive
increase of task difficulty in order to meet patient’s capabilities
and approximate, as much as possible, the functional activity to
be trained (22, 38, 39). Restrain of the unaffected arm has been investigated as an
isolated intervention, namely “forced use therapy,” but failed
to produce any additional benefit on top of conventional
therapy, for self-perceived amount and quality of arm-hand
use (2). The same authors of the original CIMT compared
groups of chronic stroke patients receiving comparable doses
of motor therapy (shaping), but different levels of arm-hand
restrain, and found no between-group differences in motor
outcome (20). According to Taub et al., “there is nothing
talismanic about use of a sling, a protective safety mitt, or
other constraining device on the less-affected [upper limb]”
(32). The point is not wearing a physical restraint, but
rather finding a way to counteract the tendency of using
exclusively the less-affected arm for all daily activities, which
inevitably leads to learned non-use; in this perspective, shaping
is in itself already a form of restraint (33). Inferences Besides CIMT,
a recent meta-analysis (21 studies, 842 subjects) indicated
higher improvements for motor impairment, and a trend
toward higher functional outcomes, for bilateral arm training
as compared to unilateral arm training (34). Such findings
challenge the clinical relevance of unilateral training of the
affected arm by restraining the unaffected arm, especially if
we consider that most activities of daily living require bilateral
arm use, and that restraining the use of the unaffected arm
is impractical for more severely affected patients. In fact,
the expanded CIMT protocol, adapted for those patients
with moderate to severe motor impairment, also considered
bimanual arm training as part of the intervention, and a
reduced time wearing the mitt (25). Moreover, there is
accumulating evidence that contralateral arm training, i.e.,
training of the unaffected arm because of inability to elicit
movements on the affected arm, could be beneficial on both
the functional and neural domain (35, 36). The mechanism
of cross-education (improvement of motor function in the
untrained arm following contralateral arm training) may explain
this counterintuitive phenomenon. In a recent consensus paper,
neurophysiologists and clinicians agreed on that a slight increase
in interhemispheric imbalance is acceptable, if function of
the affected side is increased following training of the sound
limb (37). An important finding for CIMT and other high-intensity
approaches, is the difference in outcomes depending on the
stage of recovery. Notably, high intensities of therapy seem more
consistently beneficial in the chronic phase (6, 8, 24), than in
the acute/sub-acute phase after stroke (27, 28). One possible
explanation of such conflicting results is that blocked schedules
of intensive training in the early phases post stroke may
generate unwanted overload, detrimental for motor recovery
and learning (29). It has been suggested that distributing therapy
in short sessions throughout the day (spaced practice) might
optimize motor learning (28, 30) and even the likelihood of a
favorable outcome (31) in the early phases after stroke. Shaping is perhaps the most interesting element of CIMT,
surely as a key factor that determined the superiority of any
CIMT intervention over conventional therapy, but also because
it could be easily implemented in any motor intervention. Shaping can be defined as a structured motor skill training,
typically organized in ten 30-s trials for each motor function
to be trained, with explicit feedback provided at the end
of each trial (5, 26). Inferences TABLE 1 Continued
Interventions
Key active
ingredients
Key motor
learning principles
and neural
mechanisms
Recommendations
Action observation
Immediately after action
observation, patients
have to perform the
same movements with
the affected limb
Explicit learning
Action observation
Mirror neuron network
Indicated for severely
impaired patients. Useful
as additional therapy
time in preparation for
motor training
Mirror therapy
Large mirror
Unilateral arm
performance
(unaffected side)
No object manipulation
Explicit learning
Action observation
Indicated for severely
impaired patients. Useful
as additional therapy
time
Motor imagery
PRACTICE principles
Explicit learning
Mental practice
Indicated for severely
impaired patients. Useful
as additional therapy
time in preparation for
motor training
Non-motor interventions priming neuroplasticity
Non-invasive brain
stimulation
Stimulation parameters
and localization of
targets
Modulate neural activity
Prime motor learning
acquisition
Interhemispheric
inhibition model
Vicariation model
Consider different
protocols depending on
the level of impairment
and underlying
neural mechanisms. Consider performing
motor training during
(tDCS) or after
(TMS) neuromodulation
Somatosensory electrical
stimulation
More than one motor
point at the same time,
high frequency and
intensity near motor
threshold
Increased activation of
sensorimotor areas
Prime motor learning
acquisition
Train patients to use the
stimulator
independently, ideally in
the 2 h preceding
therapy. Consider
providing stimulation
concomitant with
therapy
Aerobic training
Exercise intensity
Performance
immediately after motor
training session
Prime motor learning
consolidation
Consider short high
intensity interval
training protocols
immediately after the
motor training session
patients receiving CIMT consistently showed better outcomes
in terms of arm-hand activities and self-reported amount
and quality of arm-hand use, than conventional therapy (2). However, there were no significant differences in effect sizes
between original and modified CIMT versions (2). Notably,
restraint of the unaffected arm and shaping techniques were
the two factors applied by any CIMT intervention, but not
included in the conventional therapy approaches. Therefore,
we can already focus our attention specifically on these
two elements. restraint of the unaffected arm and shaping techniques were
the two factors applied by any CIMT intervention, but not
included in the conventional therapy approaches. Therefore,
we can already focus our attention specifically on these
two elements. patients receiving CIMT consistently showed better outcomes
in terms of arm-hand activities and self-reported amount
and quality of arm-hand use, than conventional therapy (2). However, there were no significant differences in effect sizes
between original and modified CIMT versions (2). Notably, Frontiers in Neurology frontiersin.org 05 frontiersin.org Boccuni et al. Boccuni et al. Inferences Movements are organized as discrete
repetitions, so that it’s possible to train the same pattern of
actions consistently and monitor progressions by measuring the
number of repetitions performed, the time taken, the distance
covered and/or the level of spatial/temporal accuracy while
attaining a specific goal. The best performance within each
trial is recorded and presented to the patient as reference Frontiers in Neurology Rationale There
are
two
motor
learning
principles
that
are
systematically applied within NDT. The first one is task-
specific practice: by practicing functional tasks patients focus
on learning how to overcome specific limitations in activities
of daily living. The second one is the provision of implicit
feedback, which could be defined as attention upon movement
quality (15). Indeed, a considerable portion of the treatment is
dedicated to the improvement of movement quality, by making
patients aware of correct movement patterns vs. compensatory
and maladaptive movements. A third element is represented
by the neural basis underlying “the influence of afferent input”
“therapeutic handling,” and “environmental adaptations.” One
hypothesis is a multilevel sensorimotor integration (spinal,
subcortical, cortical) that is necessary for the initial phases
of motor learning acquisition at both conscious (cortical,
explicit strategy) and unconscious (spinal, subcortical, implicit
recalibration) levels. Recommendations 1. CIMT is an effective upper limb neurorehabilitation
approach
that
should
be
embraced
by
clinicians
and
adapted to optimally fit with specific patient’s needs and
resource constraints. 2. Currently, the design of high doses, spaced practice
interventions in the acute phase represent a challenge for
healthcare providers, and a relatively unexplored, yet promising
field of research. Being practically unfeasible for therapists to
be present with patients the whole day, soon wearable/portable
assistive devices will likely play a key role to achieve high doses,
low intensity therapy regimens. Frontiers in Neurology 06 frontiersin.org 10.3389/fneur.2022.939748 10.3389/fneur.2022.939748 Boccuni et al. Interventions Robotics assist movement execution by providing anti-
gravity support and/or by exerting external forces to move
joints in a specific direction. Functional electrical stimulation
generates movement by triggering internal forces (muscle
contraction), with the application of electricity flowing in
between pairs of electrodes placed over the muscle belly,
at intensities above the motor threshold. For both robotics
and functional electrical stimulation, it’s preferable that the
patient is actively engaged as much as possible in movement
performance, with assistive devices set to provide the least
amount of assistance to accomplish a certain task. If the
patient is unable to perform any movement, they may be
used to provide passive mobilization and preserve mobility,
to maintain/improve muscle trophism (for functional electrical
stimulation), and to normalize muscle tone (45–47). Orthotics
provide joint stability in a functional position, which in turn
may allow better movement execution and/or increased volume
of repetitions for adjacent structures, or at least prevent soft
tissue changes secondary to prolonged immobility in vicious
postures (contractures, muscle stiffness, joint deformities etc.). Active ingredients are the following: for robotics, we should
distinguish between end-effectors vs. exoskeletons, the former
leaving more freedom of movement and thus more suitable
for mild to moderate impairments, the latter taking control
over multi-joint coordination, which is helpful for moderate
to severely impaired patients. Other elements to consider is
whether the robot allows 2D vs. 3D movements, to which
extent it is possible to train proximal and distal components
at the same time, or to interact with physical objects. For
FES, active ingredients are stimulation parameters, in particular
the possibility of eliciting sustained muscle contraction reliably Bobath concept the publication of core characteristics of the Bobath Concept
(40), and the development of intensive rehabilitation programs
based on the Bobath Concept, that led to positive and clinically
meaningful improvements of upper limb motor impairment in
the subacute phase after stroke (44). Intervention Bobath
Concept,
or
Neurodevelopmental
Treatment
(NDT), is one of the most popular treatment approaches in
neurorehabilitation (4). NDT has been defined as a conceptual
framework with the following paramount characteristics: first,
the attention upon quality of movement; second, the influence
of afferent input on motor control, body schema and motor
learning; and finally, the use of facilitation techniques, which
includes therapeutic handling, environmental adaptations and
verbal cueing (40). Recommendations We believe that a clearer definition of the intervention
protocol, an update of NDT fundamentals based on scientific
evidence, and a broader consideration of motor learning
principles, with higher emphasis on the volume and intensity
of the treatment, might help improve the reproducibility and
efficacy of NDT. Inferences Several systematic reviews, comparing NDT vs. other
treatment approaches, have provided substantial evidence that
NDT have comparable or even unfavorable effects on several
upper limb motor outcomes (1, 3, 4). One reason could
be that focusing excessively on activities of daily living and
movement quality might come at the expenses of the number
of repetitions performed, which negatively impact core motor
learning principles like repetitive practice and dosage. Another
point of criticism is that NDT historically advised clinicians
against engaging patients in resistance training because it
may increase muscle tone (41), despite evidence showing that
resistance training improves strength and could be beneficial
for motor function, without any increase in spasticity (41–43). Finally, there is scant literature documenting the content and
theoretical foundations of the Bobath Concept, which is typically
taught orally in dedicated courses, thus making NDT prone to
detrimental heterogeneity in the interpretation and application
of the techniques (4). Recently, promising advancements were Frontiers in Neurology frontiersin.org 07 10.3389/fneur.2022.939748 10.3389/fneur.2022.939748 Boccuni et al. robotics, vs. functional electrical stimulation (FES) (22). Despite
being applied on severely impaired patients in the chronic
phase after stroke, clinically meaningful improvements in motor
impairment were detected for all intervention groups, without
any statistical between-group difference. Another large (n =
127) although less extensive (36 h in 12 weeks) multicentre
rehabilitation trial did not find any clinically meaningful
difference between stroke patients receiving equal amounts of
intensive robotic and therapist-patient therapy (51). Systematic
meta-analysis on the effectiveness of robotic therapy (which
included the two aforementioned studies) confirmed that there
was no clinically meaningful improvement of motor outcomes,
as compared to equal amounts of other interventions (52). Similarly, a systematic review indicated that FES therapy was
not superior to other training modalities for the improvements
of upper limb motor function (53). Therefore, if the patient
performs 100 repetitions of reaching to grasping movements,
motor improvements should be rather similar, no matter
whether the patient received the assistance from a therapist,
a robot, a FES equipment, a combination of these three
types of interventions. Notably, most effective interventions
considered a composite program, where robotic and/or FES
interventions were always performed with various degrees
of personnel assistance, and integrated on top of therapist-
patient sessions (22, 25, 38, 39, 44). Inferences Equal effectiveness
between technological and human interventions should be
seen as confirmation that motor learning principles, like
intensity of training and task specificity, work uniformly
across different types of approaches. Therefore, the choice
of an intervention over another should depend on factors
such as cost-effectiveness, personnel availability, and the
opportunity to provide additional training to the patient
in group sessions, independently from the therapist, or
even remotely. without causing discomfort and pain (48). Furthermore, the
possibility of triggering muscle contraction may be preferrable
to cyclic stimulation, because of the coupling between electrical
stimulation, movement execution and intention to move (49). For orthotics, the possibility to adapt and personalize the
device is fundamental to ensure functionality and patient’s
adherence. Ideal solutions are functional hand splinting made
of thermoplastic material, by molding the orthosis directly on
patient’s hand. Rationale A common goal of assistive devices is the improvement
of functionality, either active functionality (better movement
quality, higher number of repetitions) and passive functionality
(prevention of secondary complications from non-use and
immobility). In case of active functionality, the underlying
motor learning principle is repetitive task practice enhanced
by the adoption of assistive devices. Other motor learning
principles may be present, depending on the overall content
of the motor training program, the guidance of the therapist
and
the
visual/auditory/tactile/proprioceptive
feedback
directly associated with the device. Finally, if devices are
wearable/portable, this could lead to the application of the
principle of spaced practice/distributed practice throughout the
day, and increased total dosage of therapy delivered. Recommendations Therapists
should
consider
implementing
several
assistive
devices
for
the
rehabilitation
of
moderate
to
severe
upper
limb
paresis,
within
their
own
treatment
plan,
to
help
patients
perform
functional
movements,
but
also
to
give
“extra
hands”
to
the
therapist
and
leave
more
space
for
movement
guidance,
motivation,
and coaching. Inferences Feedback from a motor act refers to the knowledge
of the final result (explicit feedback) or to the knowledge
of
performance/movement
quality
(implicit
feedback)
(15). It’s worth noting that the terms explicit and implicit
also refer to learning mechanisms, with explicit learning
being
aware/intentional
and
implicit
learning
being
unaware/unintentional
(58);
therefore,
the
terms
differ
substantially depending on the argument. Explicit and implicit
feedback both subtends explicit learning, with the focus
purposely directed toward the end results of a movement
(target reached), or how well the movement was performed
(coordination, fluency, lack of compensatory movements). A practical example is a patient training forward reaching:
explicit feedback consists of the successful grasp of a physical
object (a bottle on the table) and positive encouragements from
the therapist for task completion; implicit feedback consists
on verbal and physical guidance to perform the movement
without excessive trunk flexion or shoulder elevation. Feedback
is an integral part of learning and has been developed in
different forms in any motor training program. There are
interventions that have been specifically designed to modulate
feedback, such as VR, action observation, mirror therapy and
motor imagery. Inferences non-immersive VR experience (55);
the therapeutic content of the exercise, i.e., being specifically
designed for neurorehabilitation purposes (56); the type of
feedback being provided (explicit and/or implicit feedback)
(57); and the integration of multisensory stimuli (visual,
auditory, haptic) (15). that needs to be generated. Elbow/wrist/hand movements into
flexion/extension can be elicited by contracting one muscle at a
time, eventually providing enough stability to other districts; one
example is the application of electrical stimulation of extensor
digitorum communis for finger extension, in combination with
stabilization of the wrist in neutral position (for instance, with
and orthosis). The same cannot be applied so easily to the
shoulder, because any movement requires the coordination
of a higher number of muscles at the same time, and the
control of higher degrees of freedom; for instance, shoulder
flexion involves the coordinated activation of the deltoid,
rotator cuffmuscles, trapezius, serratus anterior and enough
core strength. Rationale Motor learning principles that fit well with VR are goal-
oriented practice, explicit feedback/knowledge of performance,
and multisensory stimulation. Furthermore, because of patient’s
engagement (by gamification of therapy) and provision of
automatic feedback, VR could significantly improve dosage and
distribution of training. Finally,
if
robotics
have
predominantly
a
proximal
application at the level of the shoulder and the elbow,
and FES is effective for finger movements generated by
forearm muscles, functional hand splinting at the level
of the wrist and the hand may help stabilize the wrist
at
the
desired
angle,
and
recreate
a
functional
shape
of the hand as done by intrinsic hand muscles, with a
smooth palmar concavity and thumb in opposition, while
leaving fingers free to move. All these assistive devices
are not mutually exclusive, but rather complementary and
have been used simultaneously in composite upper limb
neurorehabilitation trials (25, 39) to perform whole arm
functional
movements,
such
as
combined
reaching
and
grasping tasks. Interventions providing feedback
and multisensory stimulation: Virtual
reality, action observation, mirror
therapy and motor imagery One modality is to provide enhanced knowledge of
result
and/or
performance
during
motor
skill
training,
so that the patient can better learn from errors; this
strategy
is
used
in
VR,
for
those
patients
capable
of
performing a functional task (independently or with the
use of assistive devices). Furthermore, an enriched and
immersive VR experience, with the provision of different
sources of stimuli (visual, auditory haptic) can improve
patient’s engagement and motivation, which in turn has Inferences Patients with moderate to severe motor impairments
require physical assistance to perform active-assisted motor
training of the affected limb. Being the distribution of motor
impairments rather evenly distributed at the whole upper limb,
usually patients require both proximal (trunk, scapula, shoulder,
elbow) and distal (wrist, hand, fingers) assistance at the same
time (50). That said, we should specify that some interventions have
more practical applicability for proximal vs. distal upper limb
components (22). Assistive devices that showed positive effect
on upper limb motor impairment were shoulder-elbow-wrist
robotics and wrist-hand-finger FES applications (1). Proximal
use of robotics is in line with the fact that most exoskeletons
may support as needed the weight of the arm. In general,
all antigravity support systems allow patients to perform a
wider range of movements, higher number of repetitions,
and better movement quality, especially when supervised by
a therapist and/or equipped with sensors for feedback on
movement performance. A predominantly distal use of FES,
on the other hand, is the result of the fact that electrical
stimulation is more capable of generating effective movements
at the level of wrist and finger into flexion/extension, and to
some extent also elbow flexion/extension, than of producing
shoulder/trunk movements. The main two variables to consider
here are the minimum number of muscles necessary to
produce the required movement, and the amount of force In the simplest form, the patient may use the unaffected
arm to support the affected arm, or the therapist may
set the scapula in the proper orientation, support the
weight of the arm and orient the wrist and the hand
in order to perform functional activities, such as reaching
and grasping (40, 44). Technological devices may be used
as complementary interventions to perform active-assisted
arm-hand training, with the therapist having more freedom
to provide verbal/physical guidance for correct movement
execution, or even without the constant presence of the
therapist, to perform additional therapy on top of therapist-
patient sessions. The most extensive upper limb rehabilitation trial provided
300 h of therapy in 5 weeks (5 h/day), and compared the
effectiveness of therapist-patient motor learning training, vs. Frontiers in Neurology 08 frontiersin.org 10.3389/fneur.2022.939748 10.3389/fneur.2022.939748 Boccuni et al. equipment, such as a helmet with a screen inside or gloves
fitted with sensors” (54). Active ingredients that likely play
a role are immersive vs. Rationale An alternative approach to VR is to provide patients
with
a
completely
fictitious
experience
of
a
functional
movement, regardless of the actual movement performed/not
performed, thus reducing feedback of their own body state. The fictitious experience can be externally (action observation,
mirror therapy) or internally (motor imagery) generated;
the goal is to activate the mirror neuron system, which has
overlapping pattern of activation during action execution,
action observation and motor imagery, thus represents a
potential way to trigger motor learning and neuroplasticity
without even producing a motor action (67); which is
particularly
appealing
for
those
patients
with
moderate
to severe motor impairment (68). Besides motor learning
mechanisms specifically pertaining to these interventions
(action observation/embodied practice, motor imagery) another
motor learning principle to consider is dosage, because of the
possibility to train unassisted/unsupervised, thus increasing the
overall volume of therapy performed. Recommendations Movement quality is a cornerstone ingredient of many
interventions,
by
helping
patients
discriminate
between
correct and incorrect movements, thus preventing maladaptive
learning. Intrinsic feedback gives information about movement
quality, and seems to be superior to extrinsic feedback for
long-lasting recovery of normal motor patterns (57); notably,
to decrease the risk of over-reliance on feedback, a faded
schedule should be considered (57). Unfortunately, most VR
devices provide a great amount of extrinsic feedback, with
intrinsic feedback totally absent or visually presented in the
form of mini-avatar in the corner of the screen. In this form,
the provision of intrinsic feedback depends upon constant
guidance by the therapist; however, if we agree that the greatest
advantage of VR is to provide therapist-free additional therapy
time, developers of new VR applications should focus on
solutions to train the patient independently, with automatic
provision of adequate intrinsic feedback, preferably delivered as
auditory/haptic feedback, rather than visual (15). As a technical
note, wearable VR glasses, integrated with positional sensors
of different body segments, might be an ideal starting point in
this direction. Action observation, mirror therapy and
motor imagery Several systematic reviews have been already published,
overall confirming the effectiveness of these interventions. In
particular, a Cochrane review concluded that action observation
determines a statistically significant, albeit not clinically
meaningful, improvement in upper limb motor function and
dependence on activities of daily living, the largest effect being
reported for hand function (69). Another Cochrane review
found overall moderate-quality evidence that mirror therapy
has a positive effect on both upper limb motor function, motor Intervention Virtual reality (VR) has been defined as “the computer-
generated
simulation
of
a
three-dimensional
image
or
environment that can be interacted with in a seemingly
real or physical way by a person using special electronic Frontiers in Neurology frontiersin.org 09 10.3389/fneur.2022.939748 Boccuni et al. being played, and guide patient in the subsequent task oriented
training, for a total of 30–40 min of therapy per day (61, 62). positive effects on performing higher amounts of therapy, at
sustained attention. A
systematic
Cochrane
review
comparing
VR
vs. conventional
therapy
found
no
statistically
significant
differences on upper limb motor function (59). However,
when VR was provided as additional therapy on top of
conventional therapy, results were significantly better than
conventional therapy in isolation (59). A recent three-arm
randomized controlled trial compared high-dose VR, vs. high-dose conventional therapy, vs. conventional therapy; for
upper limb motor function, comparable results were found
between high-dose VR vs. high-dose conventional therapy, and
higher gains in favor of both high-dose groups, as compared to
conventional therapy (38). Therefore, one clear advantage of VR
is the opportunity to increase the volume of therapy, while at the
same time enhancing patient’s engagement and sensorimotor
integration (60). Mirror therapy consists of placing a mirror vertically at
the patient’s midline, so that the unaffected side is reflected,
thus creating a visual illusion that the image in the mirror
corresponds to the affected side. Average training intensity
consists of 30 min per day, 5 days a week, for 4 weeks, typically
provided as additional therapy to motor training (63). There
is a tendency toward better results when using a large mirror,
performing unilateral movements with the unaffected arm,
without object manipulation (64). Motor
imagery
is
the
mental
rehearsal
of
physical
movements (65). Mental practice is structured as a 30-min
session, where the patient listens to an audio file in a dedicated,
quiet room; the audio file comprises 5 min of progressive
relaxation at the beginning and at the end of the session, and
20 min of guided motor imagery (65). Principles of mental
practice are part-whole practice with progressive difficulty,
repetitive and goal-focused practice, client- and impairment-
centered, and with emphasis on task accomplishment (66). training Non-invasive brain stimulation (NIBS), somatosensory
electrical stimulation and aerobic training are examples of
promising
interventions
targeting
biochemical,
synaptic
and neuroplastic mechanisms underlying motor learning,
although
they
do
not
consist
of
any
formal
motor
skill
practice,
neither
active
nor
imaginative. They
are
generally
provided
closely
to a
motor
training session,
to
enhance
motor
learning
acquisition
(immediately
before or during practice) and consolidation (immediately
after practice). 2) 2) The interplay between neuromodulation and motor activity. Motor activity performed either immediately before, during
or after neuromodulation can potentiate its effectiveness, but
also have undesired consequences, such as the abolishment
or even the reversal of the neuroplastic effects we were
initially aiming at (75). Therefore, enough rest time (a
few minutes) should be allocated before and after the
stimulation protocol. Inferences Action observation, mirror therapy and motor imagery
are
effective
interventions
requiring
minimal
resources,
that could be considered as additional therapy to prime
motor
learning
acquisition
before
the
formal
patient-
therapist
session
of
motor
skill
training,
while
virtual
reality could be used to increase the amount of active
motor training of the affected arm, possibly administered
independently from the therapist and in a stimulating,
playful way. Despite
the
great
potential
to
enhance
the
effectiveness
of
neurorehabilitation
interventions,
there
is a wide variability in response to non-invasive brain
stimulation (75). Here we reported four major sources of
variability, and provide suggestions on how to account
for them: 1) Severity of motor impairment. Patients with different
levels of motor impairments have quantitative, but also
qualitative
differences
in
terms
of
underlying
neural
correlates. Quantitatively, the larger the lesion size at the
level of the corticospinal tract, the more severe the motor
impairment (76, 77). To recap, there are two distinct
models of neuroplastic changes following brain lesions: the
interhemispheric competition model, where the activation
of the unaffected hemisphere over-inhibit the activity of
the affected hemisphere, and the vicariation model, where
the activation of the unaffected hemisphere is beneficial to
partially compensate for the loss of function at the level of
the unaffected hemisphere (16, 78). The interhemispheric
competition model is valid for patients with relatively small
lesions resulting in mild to moderate motor impairments,
while the vicariation model is applicable for patients
with larger lesions and more severe motor impairments. As a consequence, it is necessary to apply different
neuromodulation strategies, depending on the level of motor
impairment (16, 79). Interventions Action observation typically consists of watching few
minutes of video tapes about upper limb functional tasks,
performed by healthy subjects; the patient is aware that he/she
will practice the same task immediately afterwards, and therapist
is constantly present to ensure patient’s attention to the video Frontiers in Neurology 10 frontiersin.org 10.3389/fneur.2022.939748 Boccuni et al. upper
limb
neurorehabilitation,
the
goal
is
to
prime
motor
learning
acquisition,
typically
with
excitation
of
the
affected
sensorimotor
cortex
and/or
inhibition
of
competitive brain areas before (TMS) or during (TCS)
motor training. impairment and activities of daily living, though effect size varies
depending on the control condition (63). Finally, systematic
reviews indicated effectiveness of motor imagery on improving
upper limb motor function (1, 23, 70). Intervention Non-invasive
brain
stimulation
(NIBS)
refers
to
interventions to modulate neural excitability via transcranial
stimulation (71). There are two main NIBS modalities,
Transcranial Magnetic Stimulation (TMS) and Transcranial
Current Stimulation (TCS). Active ingredients are the following:
stimulation target, stimulation parameters (intensity, frequency,
stimulation protocol), duration, concurrent motor training. International guidelines have been published as reference for
researchers and clinicians (72, 73). 3) Stimulation parameters. Slight modification of stimulation
parameters can have relevant and somehow counterintuitive
consequences. For instance, increasing the duration of
the stimulation protocol reverses the effect of intermittent
theta burst stimulation from facilitatory to inhibitory, while
continuous theta burst stimulation switches from inhibitory
to facilitatory (80, 81). Moreover, increasing stimulation
intensity (82) or frequency (83) does not necessarily lead
to enhanced effectiveness. Therefore, stimulation parameters
should be carefully selected based on evidence of efficacy and
within safety recommendations (72). 3) Stimulation parameters. Slight modification of stimulation
parameters can have relevant and somehow counterintuitive
consequences. For instance, increasing the duration of
the stimulation protocol reverses the effect of intermittent
theta burst stimulation from facilitatory to inhibitory, while
continuous theta burst stimulation switches from inhibitory
to facilitatory (80, 81). Moreover, increasing stimulation
intensity (82) or frequency (83) does not necessarily lead
to enhanced effectiveness. Therefore, stimulation parameters
should be carefully selected based on evidence of efficacy and
within safety recommendations (72). Frontiers in Neurology Rationale NIBS aims at modulating cortical excitability, either by
inhibiting or facilitating neural discharge (74). Regarding Frontiers in Neurology frontiersin.org 11 Boccuni et al. 10.3389/fneur.2022.939748 10.3389/fneur.2022.939748 activation of sensorimotor areas before or during motor
training (100). 4) Aftereffect duration. Long-lasting effects upon cessation
of
the
stimulation
could
have
important
therapeutic
consequences,
as
intensive
motor
training
could
be
performed in a context of optimal neural state. However,
many stimulation protocols have short-lasting effects (<
30–60 min); as mentioned previously, concomitant motor
practice or extending the duration of the stimulation
are unreliable strategies. One emerging solution is the
performance of several stimulation trains within the
same day (spaced stimulation) (75, 84). For instance,
the application of two trains of continuous theta burst
stimulation, with a rest period of 10–15 min in between,
has shown long-lasting effects of at least 2 (and up to 10) h
(85, 86); four trains of continuous theta burst stimulation
(at zero, 15, 60 and 75 min) up to 32 h (87); and finally,
eight trains in 2 days produced a clinically meaningful
improvement of at least 3 weeks (88). Notably, the outcomes
of the studies were the amplitude of motor evoked potentials,
saccadic eye latency and visuospatial neglect. Future
studies should investigate similar paradigms for motor
rehabilitation interventions. Recommendations Regarding upper limb neurorehabilitation, there is currently
contrasting evidence on the effectiveness of non-invasive brain
stimulation, with some reviews in favor (89, 90) and others
against (91, 92) its added value on top of conventional
therapy. Further advances in the field might arise by controlling
for factors causing variability in treatment response, and
by exploring approaches capable of long-lasting and reliable
neuroplastic changes. Inferences From a theoretical standpoint, it’s important to clarify
that electrical stimulation should be considered as a pure
somatosensory
intervention
only
when
the
stimulation
is
above
the
sensory
threshold,
but
below
the
motor
threshold. In contrast, functional electrical stimulation is
a motor intervention, as it requires intensities well above
the motor threshold to assist the execution of arm-hand
movements. It is unclear whether electrical stimulation at
or above the motor threshold, resulting in a visible muscle
twitch but no overt movement, should be classified as
somatosensory or motor interventions. However, from a
clinical perspective, the use of electrical stimulation near
the motor threshold level could represent an appealing
solution to prime neuroplasticity (101), while not having a
practical interference with other motor training interventions
performed at the same time, such as motor skill training
and
motor
imagery
(102). Indeed,
peripheral
electrical
stimulation induces increased excitability of the corticospinal
tract
(101),
especially
when
delivered
synchronously
at
more
than
one
motor
point
(103),
at
high
frequency
(≥30 Hz) and at intensities above the motor threshold,
sufficient to produce visible muscle twitch or contraction
(104). Notably, in healthy individuals the application of
electrical stimulation concomitant to motor skill training
resulted into higher motor learning indexes, as compared
to consecutive sessions of electrical stimulation and motor
training (105). Intervention The American College of Sports Medicine (ACSM) defines
aerobic exercise as any activity recruiting large muscle
groups, maintained continuously and rhythmic in nature
(106). Active ingredients could be defined according to the
following variables: training modality (cyclic movements vs. discrete repetitions), training intensity, duration, inter-session
intervals and overall intensity and volume of the program, Recommendations There
are
still
several
unexplored
yet
promising
applications of electrical stimulation, namely using near-
motor threshold intensities, at more than one motor point,
and concomitant to other training regimens, that could
increase the overall effectiveness of therapy on motor learning
and recovery. Intervention The effect of somatosensory electrical stimulation on upper
limb motor function after stroke has been investigated in a
number of randomized controlled trials (93–98). Typically,
electrical stimulation was administered for 120 min, before the
motor intervention, with low frequencies (10 Hz) and intensity
above the sensory threshold, but below the motor threshold
(99). Only one systematic review (5 studies, 95 subjects)
investigated specifically somatosensory electrical stimulation on
upper limb motor performance after stroke, demonstrating
statistically significant better motor performance in favor of
repeated peripheral sensory stimulation (99). Inferences Motor memory encoding and consolidation are two
consecutive phases of the motor learning process. In healthy
individuals,
aerobic
exercise
enhances
motor
memory
consolidation, but not acquisition, with strongest effects
shown for high intensity exercises (107). Aerobic training
performed immediately after motor skill training improves
sleep-dependent consolidation (107), and leads to higher effects
on long-term retention than when performed immediately
before motor skill training, or when aerobic training is
not performed (108). In line with these findings, patients
in the chronic phase after stroke showed better retention
when 15 min of high intensity interval training (HIIT)
was performed immediately after motor skill training, as
compared when motor skill training was followed by a
period of rest (109). HIIT consists of repeated, short bouts
of exercise at elevated effort, interspersed by periods of
low-intensity exercise or rest. In the rehabilitation field,
the main attractiveness for HIIT is that results, even for
deconditioned patients, in cumulatively more time of exercise
at high intensity and lower perceived effort, compared to
moderate-intensity
continuous
aerobic
training
(110). A
recent review indicated the potentialities of HIIT, as safe and
effective intervention to promote cardiovascular, functional
and neuroplastic outcomes post stroke, but at the same
time highlighted that larger randomized trials are needed to
confirm findings from pilot studies (111). It’s important to
highlight that there are no specific safety issues for HIIT, as
compared to moderate intensity continuous cardiovascular
exercise (111). Figure 1A illustrates a schematic of the proposed workflow. The first step is to determine objective deficits, for instance
by using validated clinical assessments covering the ICF
domains of body function and structure (impairments) and
activity (limitations) (117). The second step is to consider
patient’s perspective to quantify real-life perceived functionality
(validated questionnaires) and to determine goals for treatment. However, sometimes it’s difficult for patients to identify their
own functional goals (27). To facilitate the decision-making
process, it’s recommended to use semi-structured interviews
such as for the Canadian Occupational Performance Measure
(118), and SMART principles for patient-centered goal setting
(119). Finally, there is growing interest in the development of
wearable sensors and remote assessments to automatically and
reliably monitor real-life functional use of the affected arm (120–
122). One of the greatest advantages of tele-assessment is that
it may be diriment to interpret the potential gap between what
is objectively measured in the clinic, and how patient perceives
arm functionality (123, 124). Rationale The objective of somatosensory electrical stimulation
is to prime motor learning acquisition, by increasing the Frontiers in Neurology 12 frontiersin.org Boccuni et al. 10.3389/fneur.2022.939748 intensity, the higher and enduring the therapeutic gains
(111, 114). in terms of frequency of sessions per week and total number
of weeks. intensity, the higher and enduring the therapeutic gains
(111, 114). Inferences Figure 1B
provides
a
practical
example
of
clinical
assessments
to
determine
objective
upper
limb
deficits
and select appropriate treatment strategies. The criteria for
selecting each assessment measure where the following: (1)
well-established psychometric properties (validity, reliability,
sensitivity to change); (2) clinical utility, meaning the ability to
already direct therapists toward concrete therapeutic goals; (3)
easiness of use, in terms of training and equipment required,
time to perform the assessment, and potential contraindications. For instance, the Stroke Upper Limb Capacity Scale (SULCS) is
a clinical assessment of upper limb activity, with excellent inter-
rater reliability and high responsiveness to change (125–127);
there is minimum training and equipment required; tasks are
organized hierarchically, so that therapists may decide to start
from the beginning or from the end of the assessment, and to Figure 1B
provides
a
practical
example
of
clinical
assessments
to
determine
objective
upper
limb
deficits
and select appropriate treatment strategies. The criteria for
selecting each assessment measure where the following: (1)
well-established psychometric properties (validity, reliability,
sensitivity to change); (2) clinical utility, meaning the ability to
already direct therapists toward concrete therapeutic goals; (3)
easiness of use, in terms of training and equipment required,
time to perform the assessment, and potential contraindications. Rationale The
main
goal
of
aerobic
training,
when
performed
immediately
after
motor
skill
training,
is
to
enhance
motor
learning
consolidation
(107–
109). Personalized, patient-centered goal setting is perceived
important by stroke survivors and caregivers, but this practice
is
often
lacking
(115). Tailoring
neurorehabilitation
to
individual needs and preferences, monitoring progressions,
and maintaining a positive and proactive attitude toward
realistic
goals,
is
important
to
improve
motivation,
adherence,
effectiveness
and
patient’s
satisfaction
of
the
treatment received (116). The foundation of personalized
interventions is a comprehensive assessment of both objective
impairment and patient’s perspective, and the definition
of shared goals and expectations between the patient and
the therapist. Funding The authors affiliated to Institut Guttmann disclosed
receipt of the following financial supports for the research,
authorship, and publication of this article: Programa Joan
Ribas Araquistain de Investigación, Innovación Terapéutica
en Prehabilitación, Rehabilitación, Abordaje integral de las
secuelas de Tumores cerebrales from Fundaciò Joan Ribas
Araquistain (Reference Project 2020.330), Assaig controlat
aleatori de l’efecte potenciador de l’estimulació transcranial
de soroll aleatori (tRNS) en la rehabilitació cognitiva dels
pacients amb lesió cerebral traumàtica from Fundaciò La Maratò
De TV3 Convocatòria d’ajuts projectes de recerca en Ictus
i Lesions Medullars i Cerebrals traumàtiques 2017 (reference
project 201735.10), BBHI, Barcelona Brain Health Initiative
from Fundaciò Bancària La Caixa. - Before/during motor task practice: consider motor imagery
and somatosensory electrical stimulation to prime motor
learning acquisition. For motor imagery, consider the
development of audio tracks that patients can listen to while
resting in a quiet room. If you are using a FES device
during the patient-therapist session, consider the possibility
of modulating the intensity of the stimulation above the
motor threshold for movement production, but remaining
between the sensory and motor threshold during rest periods. Recommendations There is international consensus that, once the patient
is medically stable and cardiopulmonary exercise testing
has been performed to rule out risks of adverse events,
an
aerobic
training
program
should
be
implemented
in
the
general
rehabilitation
program
(112,
113);
the
beneficial
effects
on
cardiovascular
status,
functional
recovery
and
motor
learning
are
directly
associated
with the intensity of the exercise so that, the higher the Frontiers in Neurology 13 frontiersin.org Boccuni et al. 10.3389/fneur.2022.939748 FIGURE 1
(A) Schematic representation of comprehensive upper limb neurorehabilitation assessment; (B) example of assessment algorithm for objective
upper limb neurological deficits, developed and applied at Guttmann Institute (Barcelona, Spain). 9-HPT: 9-Hole Peg Test; Arm-A: Arm Activity
Measure, section A; BBT: Box and Block Test; CIMT: Constraint Induced Movement Therapy; eCIMT: expanded CIMT; FM-UE: Fugl-Meyer Upper
Extremity assessment; NIBS: Non-Invasive Brain Stimulation; SULCS: Stroke Upper Limb Capacity Scale. FIGURE 1
(A) Schematic representation of comprehensive upper limb neurorehabilitation assessment; (B) example of assessment algorithm for objective
upper limb neurological deficits, developed and applied at Guttmann Institute (Barcelona, Spain). 9-HPT: 9-Hole Peg Test; Arm-A: Arm Activity
Measure, section A; BBT: Box and Block Test; CIMT: Constraint Induced Movement Therapy; eCIMT: expanded CIMT; FM-UE: Fugl-Meyer Upper
Extremity assessment; NIBS: Non-Invasive Brain Stimulation; SULCS: Stroke Upper Limb Capacity Scale. progress until upper limb functionality have been determined
(no need to perform all the tasks), which is time-saving and
avoids patient’s frustration or boredom for too difficult or too
easy tasks; finally, tasks cover common functional activities,
like drinking from a glass or fastening buttons, which could
already indicate goals for therapy. For these reasons, SULCS was considered as quick and useful entry evaluation; SULCS
score may be used as indication for further assessments (either
recommended and complementary) and therapeutic options. The present assessment algorithm has been developed at the
Guttmann Institute (Barcelona, Spain) by physiotherapists
and occupational therapists, and is currently being used Frontiers in Neurology 14 frontiersin.org Boccuni et al. 10.3389/fneur.2022.939748 10.3389/fneur.2022.939748 in the clinic as structured pipeline to evaluate upper limb
neurological deficits. - Robotics and FES: developers should consider the creation
of devices with both proximal and distal effectors, or
hybrid devices (robotic/proximal and FES/distal) for the
performance of reaching and grasping motor training. - Robotics and FES: developers should consider the creation
of devices with both proximal and distal effectors, or
hybrid devices (robotic/proximal and FES/distal) for the
performance of reaching and grasping motor training. Conclusions and future perspectives - Virtual
reality:
developers
should
prioritize
the
automatization for the provision of intrinsic feedback,
preferably as haptic/auditory feedback. Upper limb neurorehabilitation is a difficult field of
intervention, that keeps challenging specialized healthcare
providers to find tangible solutions to improve patients’ motor
outcomes and quality of life. The significant advancements of
the last years are the result of a collective, international effort
from thousands of passionate researchers and clinicians. It’s
now essential to put research findings into practice, to provide
patients with the best opportunities of regaining functionality
and independency. - For any intervention: clinicians, technological developers
and researchers should find therapeutic solutions that allow
patients to train unassisted and unsupervised. The addition
of independent therapy time on top of the formal patient-
therapist session may represent the leading strategy to
provide high volume of training, distributed throughout the
day, and economically sustainable in the long term. - Personalized
interventions:
perform
a
comprehensive
evaluation of both objective neurological deficits and
patient’s perspective to develop tailored therapeutic goals
and personalized interventions. For
the
sake
of
synthesis,
we
try
to
provide
a
list
of
specific
recommendations
for
clinical
practice,
technological developments, and future research on upper
limb neurorehabilitation: - Patient-therapist session: consider two main phases: shaping
and task practice. Shaping has the main goal of performing
a great number of repetitions in a short time frame, typically
singular components of a functional task, preferably trained
in an engaging, variable, and challenging way; enough time
should be dedicated to this section, at least half of the total
therapy time. Task practice should be performed as the
final part of the session, to capitalize what was done in the
previous phase for the relearning of a whole arm functional
activity. A third phase that was not mentioned previously
in this article but nonetheless should be considered,
is preparation. Preparation means daily briefing, body
awareness and physical techniques to ensure proper stability
of the proximal segments and mobility of the distal
components, before shaping and task practice; preparation
might require more time at the beginning of the rehabilitation
program but should be gradually reduced as soon as the
patient progresses, and in any case contained within one
third (or less) of the total therapy time, to prioritize
motor practice. Author contributions LB and JT conceived the original idea and drafted the first
version of the manuscript. All authors provided substancial
contributions and approved the submitted version. References Rehabilitation of stroke patients with plegic hands: randomized controlled trial of
expanded constraint-induced movement therapy. Restor Neurol Neurosci. (2018)
36:225–44. doi: 10.3233/RNN-170792 7. Dissociable Use-Dependent Processes for Volitional Goal-Directed Reaching. Available online at: https://royalsocietypublishing.org/doi/full/10.1098/rspb.2022. 0415 (accessed June 7, 2022). 26. Taub E, Crago JE, Burgio LD, Groomes TE, Cook EW, DeLuca SC, et al. An operant approach to rehabilitation medicine: overcoming learned nonuse by
shaping. J Exp Anal Behav. (1994) 61:281–93. doi: 10.1901/jeab.1994.61-281 8. Verstynen T, Sabes PN. How each movement changes the next: an
experimental and theoretical study of fast adaptive priors in reaching. J Neurosci. (2011) 31:10050–9. doi: 10.1523/JNEUROSCI.6525-10.2011 27. Page SJ, Boe S, Levine P. What are the ‘ingredients’ of modified constraint-
induced therapy? An evidence-based review, recipe, and recommendations. Restor
Neurol Neurosci. (2013) 31:299–309. doi: 10.3233/RNN-120264 9. Therrien AS, Wolpert DM, Bastian AJ. Effective reinforcement learning
following cerebellar damage requires a balance between exploration and motor
noise. Brain J Neurol. (2016) 139:101–14. doi: 10.1093/brain/awv329 10. Therrien AS, Wolpert DM, Bastian AJ. Increasing motor noise impairs
reinforcement learning in healthy individuals. eNeuro. (2018) 5:ENEURO.0050-
18.2018. doi: 10.1523/ENEURO.0050-18.2018 28. Nowak DA, Grefkes C, Ameli M, Fink GR. Interhemispheric competition
after stroke: brain stimulation to enhance recovery of function of the affected hand. Neurorehabil Neural Repair. (2009) 23:641–56. doi: 10.1177/1545968309336661 11. Taylor JA, Ivry RB. Implicit and explicit processes in motor learning. In: Prinz
W, Beisert M, Herwig A, editors. Action Science. Berkeley, CA: The MIT Press
(2013). p. 63–87. 29. Lotze M, Beutling W, Loibl M, Domin M, Platz T, Schminke U, et al. Contralesional motor cortex activation depends on ipsilesional corticospinal tract
integrity in well-recovered subcortical stroke patients. Neurorehabil Neural Repair. (2012) 26:594–603. doi: 10.1177/1545968311427706 12. Taylor JA, Krakauer JW, Ivry RB. Explicit and implicit contributions
to learning in a sensorimotor adaptation task. J Neurosci. (2014) 34:3023–
32. doi: 10.1523/JNEUROSCI.3619-13.2014 30. Lemon RN. Descending pathways in motor control. Annu Rev Neurosci. (2008) 31:195–218. doi: 10.1146/annurev.neuro.31.060407.125547 31. Baker
SN,
Zaaimi
B,
Fisher
KM,
Edgley
SA,
Soteropoulos
DS. Pathways
mediating
functional
recovery. Prog
Brain
Res. (2015)
218:389–412. doi: 10.1016/bs.pbr.2014.12.010 13. Mazzoni
P,
Krakauer
JW. An
implicit
plan
overrides
an
explicit
strategy
during
visuomotor
adaptation. J
Neurosci. (2006)
26:3642–5. doi: 10.1523/JNEUROSCI.5317-05.2006 14. Tsay JS, Kim HE, Haith AM, Ivry RB. Proprioceptive Re-alignment drives
Implicit Sensorimotor Adaptation. (2021). Available online at: https://www.biorxiv. org/content/10.1101/2021.12.21.473747v1 (accessed June 7, 2022). 32. Taub E, Uswatte G, Morris DM. References 1. Veerbeek
JM,
van
Wegen
E,
van
Peppen
R,
van
der
Wees
PJ,
Hendriks E, Rietberg M, et al. What is the evidence for physical therapy
poststroke? A
systematic
review
and
meta-analysis. PLoS
ONE. (2014)
9:e87987. doi: 10.1371/journal.pone.0087987 1. Veerbeek
JM,
van
Wegen
E,
van
Peppen
R,
van
der
Wees
PJ,
Hendriks E, Rietberg M, et al. What is the evidence for physical therapy
poststroke? A
systematic
review
and
meta-analysis. PLoS
ONE. (2014)
9:e87987. doi: 10.1371/journal.pone.0087987 20. Uswatte G, Taub E, Morris D, Barman J, Crago J. Contribution of the shaping
and restraint components of constraint-induced movement therapy to treatment
outcome. NeuroRehabilitation. (2006) 21:147–56. doi: 10.3233/NRE-2006-21206 20. Uswatte G, Taub E, Morris D, Barman J, Crago J. Contribution of the shaping
and restraint components of constraint-induced movement therapy to treatment
outcome. NeuroRehabilitation. (2006) 21:147–56. doi: 10.3233/NRE-2006-21206 21. Tsay
JS,
Winstein
CJ. Five
features
to
look
for
in
early-phase
clinical
intervention
studies. Neurorehabil
Neural
Repair. (2021)
35:3–9. doi: 10.1177/1545968320975439 2. Kwakkel G, Veerbeek JM, van Wegen EEH, Wolf SL. Constraint-
induced movement
therapy
after
stroke. Lancet
Neurol. (2015)
14:224–
34. doi: 10.1016/S1474-4422(14)70160-7 22. McCabe J, Monkiewicz M, Holcomb J, Pundik S, Daly JJ. Comparison
of
robotics,
functional
electrical
stimulation,
and
motor
learning
methods
for
treatment
of
persistent
upper
extremity
dysfunction
after
stroke:
a
randomized
controlled
trial. Arch
Phys
Med
Rehabil. (2015)
96:981–90. doi: 10.1016/j.apmr.2014.10.022 3. Paci M. Physiotherapy based on the Bobath concept for adults with post-
stroke hemiplegia: a review of effectiveness studies. J Rehabil Med. (2003) 35:2–
7. doi: 10.1080/16501970306106 4. Kollen BJ, Lennon S, Lyons B, Wheatley-Smith L, Scheper M, Buurke JH, et
al. The effectiveness of the bobath concept in stroke rehabilitation. Stroke. (2009)
40:e89–97. doi: 10.1161/STROKEAHA.108.533828 23. Pollock A, Farmer SE, Brady MC, Langhorne P, Mead GE, Mehrholz J, et al. Interventions for improving upper limb function after stroke. Cochrane Database
Syst Rev. (2014) 2014:CD010820. doi: 10.1002/14651858.CD010820.pub2 5. Morris DM, Taub E, Mark VW. Constraint-induced movement therapy:
characterizing the intervention protocol. Eur Medicophysica. (2006) 42:257–68. 24. Taub E, Uswatte G, Pidikiti R. Constraint-induced movement therapy: a new
family of techniques with broad application to physical rehabilitation–a clinical
review. J Rehabil Res Dev. (1999) 36:237–51. 6. Marinovic
W,
Poh
E,
de
Rugy
A,
Carroll
TJ. Action
history
influences subsequent movement via two distinct processes. Elife. (2017)
6:e26713. doi: 10.7554/eLife.26713 25. Uswatte G, Taub E, Bowman MH, Delgado A, Bryson C, Morris DM, et al. Conflict of interest The authors declare that the research was conducted in
the absence of any commercial or financial relationships
that
could
be
construed
as
a
potential
conflict
of interest. - After
motor
task
practice:
consider
aerobic
training
to
enhance
motor
learning
consolidation,
and
for
cardiovascular benefits. - After
motor
task
practice:
consider
aerobic
training
to
enhance
motor
learning
consolidation,
and
for
cardiovascular benefits. Frontiers in Neurology frontiersin.org 15 frontiersin.org 10.3389/fneur.2022.939748 10.3389/fneur.2022.939748 Boccuni et al. Publisher’s note organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed
or endorsed by the publisher. All claims expressed in this article are solely those of the
authors and do not necessarily represent those of their affiliated References Mental practice: applying motor PRACTICE and
neuroplasticity principles to increase upper extremity function. Stroke. (2014)
45:3454–60. doi: 10.1161/STROKEAHA.114.004313 67. Buccino G, Solodkin A, Small SL. Functions of the mirror neuron
system: implications for neurorehabilitation. Cogn Behav Neurol. (2006) 19:55–
63. doi: 10.1097/00146965-200603000-00007 46. Gobbo M, Gaffurini P, Vacchi L, Lazzarini S, Villafane J, Orizio C, et al. Hand passive mobilization performed with robotic assistance: acute effects on
upper limb perfusion and spasticity in stroke survivors. BioMed Res Int. (2017)
2017:1–6. doi: 10.1155/2017/2796815 68. Sharma
N,
Pomeroy
VM,
Baron
JC. Motor
imagery:
a
backdoor
to
the
motor
system
after
stroke? Stroke. (2006)
37:1941–
52. doi: 10.1161/01.STR.0000226902.43357.fc 47. Rayegani S, Shojaee H, Sedighipour L, Soroush MR, Baghbani M, Amirani
OB. The effect of electrical passive cycling on spasticity in war veterans with spinal
cord injury. Front Neurol. (2011) 2:39 doi: 10.3389/fneur.2011.00039 69. Borges LR, Fernandes AB, Melo LP, Guerra RO, Campos TF. Action
observation for upper limb rehabilitation after stroke. Cochrane Database Syst Rev. (2018) 10:CD011887. doi: 10.1002/14651858.CD011887.pub2 48. Ward AR, Oliver WG, Buccella D. Wrist extensor torque production
and discomfort associated with low-frequency and burst-modulated kilohertz-
frequency currents. Phys Ther. (2006) 86:1360–7. doi: 10.2522/ptj.20050300 70. Machado TC, Carregosa AA, Santos MS, Ribeiro NM da S, Melo A. Efficacy
of motor imagery additional to motor-based therapy in the recovery of motor
function of the upper limb in post-stroke individuals: a systematic review. Top
Stroke Rehabil. (2019) 26:548–53. doi: 10.1080/10749357.2019.1627716 49. Marquez-Chin C, Popovic MR. Functional electrical stimulation therapy for
restoration of motor function after spinal cord injury and stroke: a review. Biomed
Eng OnLine. (2020) 19:34. doi: 10.1186/s12938-020-00773-4 50. Beebe JA, Lang CE. Absence of a proximal to distal gradient of motor deficits
in the upper extremity early after stroke. Clin Neurophysiol. (2008) 119:2074–
85. doi: 10.1016/j.clinph.2008.04.293 71. Boes
AD,
Kelly
MS,
Trapp
NT,
Stern
AP,
Press
DZ,
Pascual-
Leone
A. Noninvasive
brain
stimulation:
challenges
and
opportunities
for
a
new
clinical
specialty. J
Neuropsychiatry
Clin
Neurosci. (2018)
30:173–9. doi: 10.1176/appi.neuropsych.17110262 51. Lo AC, Guarino PD, Richards LG, Haselkorn JK, Wittenberg GF,
Federman DG, et al. Robot-assisted therapy for long-term upper-limb impairment
after stroke. N Engl J Med. (2010) 362:1772–83. doi: 10.1056/NEJMoa09
11341 72. Rossi S, Antal A, Bestmann S, Bikson M, Brewer C, Brockmöller J, et al. Safety
and recommendations for TMS use in healthy subjects and patient populations,
with updates on training, ethical and regulatory issues: expert guidelines. Clin
Neurophysiol. References Adamovich
SV,
Fluet
GG,
Tunik
E,
Merians
AS. Sensorimotor
training
in
virtual
reality:
a
review. NeuroRehabilitation. (2009)
25:29. doi: 10.3233/NRE-2009-0497 39. Ward NS, Brander F, Kelly K. Intensive upper limb neurorehabilitation in
chronic stroke: outcomes from the queen square programme. J Neurol Neurosurg
Psychiatry. (2019) 90:498–506. doi: 10.1136/jnnp-2018-319954 61. Franceschini M, Ceravolo MG, Agosti M, Cavallini P, Bonassi S. Dall’Armi V,
et al. Clinical relevance of action observation in upper-limb stroke rehabilitation:
a possible role in recovery of functional dexterity. A randomized clinical trial. Neurorehabil Neural Repair. (2012) 26:456–62. doi: 10.1177/1545968311427406 40. Michielsen M, Vaughan-Graham J, Holland A, Magri A, Suzuki M. The
bobath concept – a model to illustrate clinical practice. Disabil Rehabil. (2019)
41:2080–92. doi: 10.1080/09638288.2017.1417496 62. Kim CH, Bang DH. Action observation training enhances upper extremity
function in subacute stroke survivor with moderate impairment: a double-
blind, randomized controlled pilot trial. J Korean Soc Phys Med. (2016) 11:133–
40. doi: 10.13066/kspm.2016.11.1.133 41. Miller
GJT,
Light
KE. Strength
training
in
spastic
hemiparesis:
should
it
be
avoided? NeuroRehabilitation. (1997)
9:17–28. doi: 10.1016/S1053-8135(97)00011-5 63. Thieme H, Morkisch N, Mehrholz J, Pohl M, Behrens J, Borgetto B, et al. Mirror therapy for improving motor function after stroke. Cochrane Database Syst
Rev. (2018) 7:CD008449. doi: 10.1002/14651858.CD008449.pub3 42. Riolo L, Fisher K. Is there evidence that strength training could help improve
muscle function and other outcomes without reinforcing abnormal movement
patterns or increasing reflex activity in a man who has had a stroke? Phys Ther. (2003) 83:844–51. doi: 10.1093/ptj/83.9.844 64. Morkisch N, Thieme H, Dohle C. How to perform mirror therapy after
stroke? Evidence from a meta-analysis. Restor Neurol Neurosci. (2019) 37:421–
35. doi: 10.3233/RNN-190935 43. Ada L, Dorsch S, Canning CG. Strengthening interventions increase strength
and improve activity after stroke: a systematic review. Aust J Physiother. (2006)
52:241–8. doi: 10.1016/S0004-9514(06)70003-4 65. Peters HT, Page SJ. Integrating mental practice with task-specific training
and behavioral supports in poststroke rehabilitation: evidence, components,
and augmentative opportunities. Phys Med Rehabil Clin N Am. (2015) 26:715–
27. doi: 10.1016/j.pmr.2015.06.004 44. Meyer S, Verheyden G, Kempeneers K, Michielsen M. Arm-hand boost
therapy during inpatient stroke rehabilitation: a pilot randomized controlled trial. medRxiv. (2021) 12:652042. doi: 10.3389/fneur.2021.652042 45. Stein C, Fritsch CG, Robinson C, Sbruzzi G, Plentz RDM. Effects of
electrical stimulation in spastic muscles after stroke. Stroke. (2015) 46:2197–
205. doi: 10.1161/STROKEAHA.115.009633 66. Page SJ, Peters H. References Improved motor recovery after
stroke
and
massive
cortical
reorganization
following
constraint-induced
movement
therapy. Phys
Med
Rehabil
Clin
N
Am. (2003)
14:S77–91,
ix. doi: 10.1016/S1047-9651(02)00052-9 15. Maier M, Ballester BR, Verschure PFMJ. Principles of neurorehabilitation
after stroke based on motor learning and brain plasticity mechanisms. Front Syst
Neurosci. (2019) 13:74. doi: 10.3389/fnsys.2019.00074 33. Mark VW, Taub E. Constraint-induced movement therapy for chronic stroke
hemiparesis and other disabilities. Restor Neurol Neurosci. (2004) 22:317–36. 16. Di Pino G, Pellegrino G, Assenza G, Capone F, Ferreri F, Formica D, et al. Modulation of brain plasticity in stroke: a novel model for neurorehabilitation. Nat
Rev Neurol. (2014) 10:597–608. doi: 10.1038/nrneurol.2014.162 34. Chen PM, Kwong PWH, Lai CKY, Ng SSM. Comparison of bilateral and
unilateral upper limb training in people with stroke: a systematic review and
meta-analysis. PLoS ONE. (2019) 14:e0216357. doi: 10.1371/journal.pone.0216357 17. McCombe Waller S, Whitall J. Bilateral arm training: why and who benefits? NeuroRehabilitation. (2008) 23:29–41. doi: 10.3233/NRE-2008-23104 35. Ehrensberger M, Simpson D, Broderick P, Monaghan K. Cross-education of
strength has a positive impact on post-stroke rehabilitation: a systematic literature
review. Top Stroke Rehabil. (2016) 23:126–35. doi: 10.1080/10749357.2015.1112062 18. Renner CIE, Brendel C, Hummelsheim H. Bilateral arm training vs. unilateral arm training for severely affected patients with stroke: exploratory single-
blinded randomized controlled trial. Arch Phys Med Rehabil. (2020) 101:1120–
30. doi: 10.1016/j.apmr.2020.02.007 36. Russell W, Pritchard-Wiart L, Manns PJ. Clinician perspectives on
cross-education in stroke rehabilitation. Disabil Rehabil. (2018) 40:2644–
9. doi: 10.1080/09638288.2017.1356382 19. Wolf SL, Winstein CJ, Miller JP, Taub E, Uswatte G, Morris D, et al. Effect of constraint-induced movement therapy on upper extremity function 3
to 9 months after stroke: the EXCITE randomized clinical trial. JAMA. (2006)
296:2095–104. doi: 10.1001/jama.296.17.2095 37. Manca A, Hortobágyi T, Carroll TJ, Enoka RM, Farthing JP, Gandevia
SC, et al. Contralateral effects of unilateral strength and skill training: modified
delphi consensus to establish key aspects of cross-education. Sports Med. (2021)
51:11–20. doi: 10.1007/s40279-020-01377-7 Frontiers in Neurology frontiersin.org 16 Boccuni et al. Boccuni et al. 10.3389/fneur.2022.939748 59. Laver KE, Lange B, George S, Deutsch JE, Saposnik G, Crotty
M. Virtual reality for stroke rehabilitation. Cochrane Database Syst Rev. 11:CD008349. doi: 10.1002/14651858.CD008349 38. Krakauer JW, Kitago T, Goldsmith J, Ahmad O, Roy P, Stein J,
et
al. Comparing
a
novel
neuroanimation
experience
to
conventional
therapy for high-dose intensive upper-limb training in subacute stroke:
the
SMARTS2
randomized
trial. Neurorehabil
Neural
Repair. (2021)
20:15459683211000730. doi: 10.1177/15459683211000730 60. References (2021) 132:269–306. doi: 10.1016/j.clinph.2020.10.003 52. Veerbeek JM, Langbroek-Amersfoort AC, van Wegen EEH, Meskers CGM,
Kwakkel G. Effects of robot-assisted therapy for the upper limb after stroke: a
systematic review and meta-analysis. Neurorehabil Neural Repair. (2017) 31:107–
21. doi: 10.1177/1545968316666957 73. Groppa
S,
Oliviero
A,
Eisen
A,
Quartarone
A,
Cohen
LG,
Mall
V,
et
al. A
practical
guide
to
diagnostic
transcranial
magnetic
stimulation:
report
of
an
IFCN
committee. Clin
Neurophysiol. (2012)
123:858–82. doi: 10.1016/j.clinph.2012.01.010 53. Eraifej J, Clark W, France B, Desando S, Moore D. Effectiveness of upper
limb functional electrical stimulation after stroke for the improvement of activities
of daily living and motor function: a systematic review and meta-analysis. Syst Rev. (2017) 6:40. doi: 10.1186/s13643-017-0435-5 74. Wagner
T,
Valero-Cabre
A,
Pascual-Leone
A. Non-invasive
human
brain
stimulation. Annu
Rev
Biomed
Eng. (2007)
9:527–
65. doi: 10.1146/annurev.bioeng.9.061206.133100 54. Home : Oxford English Dictionary. Available online at: https://www.oed.com/
(accessed June 9, 2022). 75. Huang YZ, Lu MK, Antal A, Classen J, Nitsche M, Ziemann U, et al. Plasticity
induced by non-invasive transcranial brain stimulation: a position paper. Clin
Neurophysiol. (2017) 128:2318–29. doi: 10.1016/j.clinph.2017.09.007 55. Jin M, Pei J, Bai Z, Zhang J, He T, Xu X, et al. Effects of virtual
reality in improving upper extremity function after stroke: a systematic review
and meta-analysis of randomized controlled trials. Clin Rehabil. (2022) 36:573–
96. doi: 10.1177/02692155211066534 76. Boccuni L, Meyer S. D’cruz N, Kessner SS, Marinelli L, Trompetto C, et al. Premotor dorsal white matter integrity for the prediction of upper limb motor
impairment after stroke. Sci Rep. (2019) 9:19712. doi: 10.1038/s41598-019-56334-w 56. Maier M, Rubio Ballester B, DuffA, Duarte Oller E, Verschure PFMJ. Effect of specific over nonspecific vr-based rehabilitation on poststroke motor
recovery: a systematic meta-analysis. Neurorehabil Neural Repair. (2019) 33:112–
29. doi: 10.1177/1545968318820169 77. Zhu LL, Lindenberg R, Alexander MP, Schlaug G. Lesion load of the
corticospinal tract predicts motor impairment in chronic stroke. Stroke. (2010)
41:910–5. doi: 10.1161/STROKEAHA.109.577023 78. Grefkes C, Fink GR. Connectivity-based approaches in stroke and recovery
of function. Lancet Neurol. (2014) 13:206–16. doi: 10.1016/S1474-4422(13)70264-3 57. Cirstea MC, Levin MF. Improvement of arm movement patterns and
endpoint control depends on type of feedback during practice in stroke survivors. Neurorehabil Neural Repair. (2007) 21:398–411. doi: 10.1177/15459683062
98414 79. Plow EB, Cunningham DA, Varnerin N, Machado A. Rethinking
stimulation
of
the
brain
in
stroke
rehabilitation:
why
higher
motor
areas
might
be
better
alternatives
for
patients
with
greater
impairments. Neuroscientist. References Guiraud T, Nigam A, Gremeaux V, Meyer P, Juneau M, Bosquet L. High-
intensity interval training in cardiac rehabilitation. Sports Med Auckl NZ. (2012)
42:587–605. doi: 10.2165/11631910-000000000-00000 111. Crozier J, Roig M, Eng JJ, MacKay-Lyons M, Fung J, Ploughman M, et al. High-intensity interval training after stroke: an opportunity to promote functional
recovery, cardiovascular health, and neuroplasticity. Neurorehabil Neural Repair. (2018) 32:543–56. doi: 10.1177/1545968318766663 91. Graef P, Dadalt MLR, Rodrigués DAM da S, Stein C, Pagnussat A de
S. Transcranial magnetic stimulation combined with upper-limb training for
improving function after stroke: a systematic review and meta-analysis. J Neurol
Sci. (2016) 369:149–58. doi: 10.1016/j.jns.2016.08.016 112. Billinger
Sandra
A,
Arena
Ross,
Bernhardt
Julie,
Eng
Janice
J,
Franklin
Barry
A,
Johnson
Cheryl
Mortag,
et
al. Physical
activity
and
exercise
recommendations
for
stroke
survivors. Stroke. (2014)
45:2532–53. doi: 10.1161/STR.0000000000000022 92. Elsner B, Kugler J, Pohl M, Mehrholz J. Transcranial direct current
stimulation (tDCS) for improving activities of daily living, and physical and
cognitive functioning, in people after stroke. Cochrane Database Syst Rev. (2020)
11:CD009645. doi: 10.1002/14651858.CD009645.pub4 113. MacKay-Lyons M, Billinger SA, Eng JJ, Dromerick A, Giacomantonio
N, Hafer-Macko C, et al. Aerobic exercise recommendations to optimize best
practices in care after stroke: AEROBICS 2019 update. Phys Ther. (2020) 100:149–
56. doi: 10.1093/ptj/pzz153 93. Conforto AB, Kaelin-Lang A, Cohen LG. Increase in hand muscle strength
of stroke patients after somatosensory stimulation. Ann Neurol. (2002) 51:122–
5. doi: 10.1002/ana.10070 94. Conforto
AB,
Ferreiro
KN,
Tomasi
C,
dos
Santos
RL,
Moreira
VL, Marie SKN, et al. Effects of somatosensory stimulation on motor
function after subacute stroke. Neurorehabil Neural Repair. (2010) 24:263–
72. doi: 10.1177/1545968309349946 114. Ploughman
M,
Kelly
LP. Four
birds
with
one
stone? Reparative,
neuroplastic,
cardiorespiratory,
and
metabolic
benefits
of
aerobic
exercise
poststroke. Curr
Opin
Neurol. (2016)
29:684–
92. doi: 10.1097/WCO.0000000000000383 95. Carrico C, Chelette KC, Westgate PM, Salmon-Powell E, Nichols L,
Sawaki L. Randomized trial of peripheral nerve stimulation to enhance modified
constraint-induced therapy after stroke. Am J Phys Med Rehabil. (2016) 95:397–
406. doi: 10.1097/PHM.0000000000000476 115. Lloyd A, Bannigan K, Sugavanam T, Freeman J. Experiences of stroke
survivors, their families and unpaid carers in goal setting within stroke
rehabilitation: a systematic review of qualitative evidence. JBI Database Syst Rev
Implement Rep. (2018) 16:1418–53. doi: 10.11124/JBISRIR-2017-003499 96. Carrico C, Chelette KC, Westgate PM, Powell E, Nichols L, Fleischer
A, et al. References (2015)
21:225–40. doi:
10.1177/107385841
4537381 58. Seger
CA. Implicit
learning. Psychol
Bull. (1994)
115:163–
96. doi: 10.1037/0033-2909.115.2.163 Frontiers in Neurology 17 frontiersin.org Boccuni et al. Boccuni et al. 10.3389/fneur.2022.939748 80. Gamboa OL, Antal A, Moliadze V, Paulus W. Simply longer is not better:
reversal of theta burst after-effect with prolonged stimulation. Exp Brain Res. (2010)
204:181–7. doi: 10.1007/s00221-010-2293-4 80. Gamboa OL, Antal A, Moliadze V, Paulus W. Simply longer is not better:
reversal of theta burst after-effect with prolonged stimulation. Exp Brain Res. (2010)
204:181–7. doi: 10.1007/s00221-010-2293-4 100. Veldman MP, Maurits NM, Zijdewind I, Maffiuletti NA, van Middelkoop S,
Mizelle JC, et al. Somatosensory electrical stimulation improves skill acquisition,
consolidation, and transfer by increasing sensorimotor activity and connectivity. J
Neurophysiol. (2018) 120:281–90. doi: 10.1152/jn.00860.2017 100. Veldman MP, Maurits NM, Zijdewind I, Maffiuletti NA, van Middelkoop S,
Mizelle JC, et al. Somatosensory electrical stimulation improves skill acquisition,
consolidation, and transfer by increasing sensorimotor activity and connectivity. J
Neurophysiol. (2018) 120:281–90. doi: 10.1152/jn.00860.2017 81. McCalley DM, Lench DH, Doolittle JD, Imperatore JP, Hoffman M, Hanlon
CA. Determining the optimal pulse number for theta burst induced change in
cortical excitability. Sci Rep. (2021) 11:8726. doi: 10.1038/s41598-021-87916-2 101. Chipchase LS, Schabrun SM, Hodges PW. Peripheral electrical stimulation
to induce cortical plasticity: a systematic review of stimulus parameters. Clin
Neurophysiol. (2011) 122:456–63. doi: 10.1016/j.clinph.2010.07.025 82. Chung SW, Rogasch NC, Hoy KE, Sullivan CM, Cash RFH, Fitzgerald
PB. Impact of different intensities of intermittent theta burst stimulation on the
cortical properties during TMS-EEG and working memory performance. Hum
Brain Mapp. (2018) 39:783–802. doi: 10.1002/hbm.23882 102. Carson RG, Buick AR. Neuromuscular electrical stimulation-promoted
plasticity of the human brain. J Physiol. (2019) 599:2375–99. doi: 10.1113/JP278298 102. Carson RG, Buick AR. Neuromuscular electrical stimulation-promoted
plasticity of the human brain. J Physiol. (2019) 599:2375–99. doi: 10.1113/JP278298 103. Ridding
MC,
Uy
J. Changes
in
motor
cortical
excitability
induced
by
paired
associative
stimulation. Clin
Neurophysiol. (2003)
114:1437–44. doi: 10.1016/S1388-2457(03)00115-9 83. Goldsworthy MR, Pitcher JB, Ridding MC, A. comparison of two different
continuous theta burst stimulation paradigms applied to the human primary
motor cortex. Clin Neurophysiol. (2012) 123:2256–63. doi: 10.1016/j.clinph.2012. 05.001 104. Chipchase LS, Schabrun SM, Hodges PW. Corticospinal excitability is
dependent on the parameters of peripheral electric stimulation: a preliminary
study. Arch Phys Med Rehabil. (2011) 92:1423–30. doi: 10.1016/j.apmr.2011.01.011 84. Goldsworthy MR, Pitcher JB, Ridding MC. Spaced non-invasive brain
stimulation: prospects for inducing long-lasting human cortical plasticity. Neurorehabil Neural Repair. (2015) 29:714–21. References doi: 10.1177/1545968314562649 105. Carvalho S, French M, Thibaut A, Lima W, Simis M, Leite J, et al. Median
nerve stimulation induced motor learning in healthy adults: a study of timing of
stimulation and type of learning. Eur J Neurosci. (2018) doi: 10.1111/ejn.13990 85. Nyffeler T, Wurtz P, Lüscher HR, Hess CW, Senn W, Pflugshaupt T, et al. Extending lifetime of plastic changes in the human brain. Eur J Neurosci. (2006)
24:2961–6. doi: 10.1111/j.1460-9568.2006.05154.x 106. Garber CE, Blissmer B, Deschenes MR, Franklin BA, Lamonte MJ, Lee IM,
et al. American college of sports medicine position stand. Quantity and quality of
exercise for developing and maintaining cardiorespiratory, musculoskeletal, and
neuromotor fitness in apparently healthy adults: guidance for prescribing exercise. Med Sci Sports Exerc. (2011) 43:1334–59. doi: 10.1249/MSS.0b013e318213fefb 86. Goldsworthy MR, Pitcher JB, Ridding MC. The application of spaced theta
burst protocols induces long-lasting neuroplastic changes in the human motor
cortex. Eur J Neurosci. (2012) 35:125–34. doi: 10.1111/j.1460-9568.2011.07924.x 107. Wanner
P,
Cheng
FH,
Steib
S. Effects
of
acute
cardiovascular
exercise
on
motor
memory
encoding
and
consolidation:
a
systematic
review
with
meta-analysis. Neurosci
Biobehav
Rev. (2020)
116:365–
81. doi: 10.1016/j.neubiorev.2020.06.018 87. Nyffeler T, Cazzoli D, Hess CW, Müri RM. One session of repeated parietal
theta burst stimulation trains induces long-lasting improvement of visual neglect. Stroke. (2009) 40:2791–6. doi: 10.1161/STROKEAHA.109.552323 88. Cazzoli D, Müri RM, Schumacher R, von Arx S, Chaves S, Gutbrod K, et
al. Theta burst stimulation reduces disability during the activities of daily living in
spatial neglect. Brain J Neurol. (2012) 135:3426–39. doi: 10.1093/brain/aws182 108. Roig M, Skriver K, Lundbye-Jensen J, Kiens B, Nielsen JB. A
single
bout
of
exercise
improves
motor
memory. PLoS
ONE. (2012)
7:e44594. doi: 10.1371/journal.pone.0044594 89. Zhang L, Xing G, Fan Y, Guo Z, Chen H, Mu Q. Short- and long-term effects
of repetitive transcranial magnetic stimulation on upper limb motor function
after stroke: a systematic review and meta-analysis. Clin Rehabil. (2017) 31:1137–
53. doi: 10.1177/0269215517692386 109. Nepveu JF, Thiel A, Tang A, Fung J, Lundbye-Jensen J, Boyd LA,
et al. A single bout of high-intensity interval training improves motor skill
retention in individuals with stroke. Neurorehabil Neural Repair. (2017) 31:726–
35. doi: 10.1177/1545968317718269 90. Xiang H, Sun J, Tang X, Zeng K, Wu X. The effect and optimal parameters of
repetitive transcranial magnetic stimulation on motor recovery in stroke patients: a
systematic review and meta-analysis of randomized controlled trials. Clin Rehabil. (2019) 33:847–64. doi: 10.1177/0269215519829897 110. References Nerve stimulation enhances task-oriented training in chronic,
severe motor deficit after stroke: a randomized trial. Stroke. (2016) 47:1879–
84. doi: 10.1161/STROKEAHA.116.012671 116. Sugavanam T, Mead G, Bulley C, Donaghy M, van Wijck F. The effects and
experiences of goal setting in stroke rehabilitation - a systematic review. Disabil
Rehabil. (2013) 35:177–90. doi: 10.3109/09638288.2012.690501 117. World Health Organization. International Classification of Functioning,
Disability and Health: ICF. Geneva: World Health Organization (2001). p. 299. 97. Celnik
P,
Hummel
F,
Harris-Love
M,
Wolk
R,
Cohen
LG. Somatosensory
stimulation
enhances
the
effects
of
training
functional
hand tasks in patients with chronic stroke. Arch Phys Med Rehabil. (2007)
88:1369–76. doi: 10.1016/j.apmr.2007.08.001 118. Law M, Baptiste S, McColl M, Opzoomer A, Polatajko H, Pollock N. The Canadian occupational performance measure: an outcome measure for
occupational therapy. Can J Occup Ther Rev Can Ergother. (1990) 57:82–
7. doi: 10.1177/000841749005700207 98. McDonnell MN, Hillier SL, Miles TS, Thompson PD, Ridding MC. Influence
of combined afferent stimulation and task-specific training following stroke: a
pilot randomized controlled trial. Neurorehabil Neural Repair. (2007) 21:435–
43. doi: 10.1177/1545968307300437 98. McDonnell MN, Hillier SL, Miles TS, Thompson PD, Ridding MC. Influence
of combined afferent stimulation and task-specific training following stroke: a
pilot randomized controlled trial. Neurorehabil Neural Repair. (2007) 21:435–
43. doi: 10.1177/1545968307300437 119. Bovend’Eerdt TJ, Botell RE, Wade DT. Writing SMART rehabilitation goals
and achieving goal attainment scaling: a practical guide. Clin Rehabil. (2009)
23:352–61. doi: 10.1177/0269215508101741 119. Bovend’Eerdt TJ, Botell RE, Wade DT. Writing SMART rehabilitation goals
and achieving goal attainment scaling: a practical guide. Clin Rehabil. (2009)
23:352–61. doi: 10.1177/0269215508101741 99. Conforto AB, Dos Anjos SM, Bernardo WM, Silva AA da, Conti J, Machado
AG, et al. Repetitive peripheral sensory stimulation and upper limb performance in
stroke: a systematic review and meta-analysis. Neurorehabil Neural Repair. (2018)
32:863–71. doi: 10.1177/1545968318798943 99. Conforto AB, Dos Anjos SM, Bernardo WM, Silva AA da, Conti J, Machado
AG, et al. Repetitive peripheral sensory stimulation and upper limb performance in
stroke: a systematic review and meta-analysis. Neurorehabil Neural Repair. (2018)
32:863–71. doi: 10.1177/1545968318798943 120. Patel S, Park H, Bonato P, Chan L, Rodgers M. A review of wearable sensors
and systems with application in rehabilitation. J NeuroEngineering Rehabil. (2012)
9:21. doi: 10.1186/1743-0003-9-21 Frontiers in Neurology 18 frontiersin.org Boccuni et al. 10.3389/fneur.2022.939748 10.3389/fneur.2022.939748 125. Roorda
LD,
Houwink
A,
Smits
W,
Molenaar
IW,
Geurts
AC. 123. Essers B, Meyer S, De Bruyn N, Van Gils A, Boccuni L, Tedesco
Triccas L, et al. Mismatch between observed and perceived upper limb function:
an eye-catching phenomenon after stroke. Disabil Rehabil. (2018) 41:1545–
51. doi: 10.1080/09638288.2018.1442504 124. Essers B, Coremans M, Veerbeek J, Luft A, Verheyden G. Daily life
upper limb activity for patients with match and mismatch between observed
function and perceived activity in the chronic phase post stroke. Sensors. (2021)
21:5917. doi: 10.3390/s21175917 122. Reinkensmeyer DJ, Burdet E, Casadio M, Krakauer JW, Kwakkel
G, Lang CE, et al. Computational neurorehabilitation: modeling plasticity
and
learning
to
predict
recovery.
J
NeuroEngineering
Rehabil.
(2016)
13:42. doi: 10.1186/s12984-016-0148-3 121. Tsay JS, Lee AS, Ivry RB, Avraham G. Moving Outside the Lab: The Viability
of Conducting Sensorimotor Learning Studies Online. (2021). Available online at:
https://www.biorxiv.org/content/10.1101/2021.01.30.181370v2 (accessed June 9,
2022). Boccuni et al. References Measuring
upper
limb
capacity
in
poststroke
patients:
development,
fit
of
the
monotone
homogeneity
model,
unidimensionality,
fit
of
the
double
monotonicity
model,
differential
item
functioning,
internal
consistency, and feasibility of the stroke upper limb capacity scale, SULCS. Arch
Phys
Med
Rehabil. (2011)
92:214–27. doi:
10.1016/j.apmr.2010. 10.034 121. Tsay JS, Lee AS, Ivry RB, Avraham G. Moving Outside the Lab: The Viability
of Conducting Sensorimotor Learning Studies Online. (2021). Available online at:
https://www.biorxiv.org/content/10.1101/2021.01.30.181370v2 (accessed June 9,
2022). 121. Tsay JS, Lee AS, Ivry RB, Avraham G. Moving Outside the Lab: The Viability
of Conducting Sensorimotor Learning Studies Online. (2021). Available online at:
https://www.biorxiv.org/content/10.1101/2021.01.30.181370v2 (accessed June 9,
2022). 126. Houwink
A,
Roorda
LD,
Smits
W,
Molenaar
IW,
Geurts
AC. Measuring
upper
limb
capacity
in
patients
after
stroke:
reliability
and
validity
of
the
stroke
upper
limb
capacity
scale. Arch
Phys
Med
Rehabil. (2011)
92:1418–22. doi:
10.1016/j.apmr.2011. 03.028 127. Knutson JS, Friedl AS, Hansen KM, Hisel TZ, Harley MY. Convergent
validity
and
responsiveness
of
the
stroke
upper
limb
capacity
scale. Arch Phys Med Rehabil. (2019) 100:140–143.e1. doi: 10.1016/j.apmr.2018. 07.433 19 Frontiers in Neurology frontiersin.org
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الاتجاهات الحدیثة فی البحث الجغرافی استخدام طریقة دلفی(Delphi Method) فی بحوث الجغرافیة التطبیقیة
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Abstract: This work focused on three important human pathogens; Escherichia coli, Salmonella typhi
and Klebsiella pneumonae. Results showed that there is several virulence factors in this bacteria
such as capsule, enzymes, motility and fimbriae. Escherichia coli included fimbriae type I, II and
III . Salmonella typhi contained only type II and III. While Klebsiella pneumonae included type I
and III. Probiotic preparations from Lactobacillus acidophilus appeared to affect the bacterial
growth and adhesion. Bacterial growth was inhibited by using stock lactic acid bacteria filtrate
and 50:50 diluted filtrate. Higher inhibition zones were recorded during the use of stock filtrate of
the probiotic on the pathogenic bacterial isolates. Bacterial adhesion to epithelial cells was
inhibited also by using the probiotic. In the case of Escherichia coli, the adhesion was reduced
from 59-61 to 24-21 and 33-30 bacterium/cell by using the stock and diluted probiotic
respectively. In the case of Salmonella typhi reduction of bacterial adherence was also observed
from 55-53 to 11-13 and 16-14bacterium/cell by using the stock and diluted probiotic
respectively. While in the case of Klebsiella pneumonae from 44-46 to 8-9 and 14-10
bacterium/cell by using the stock and diluted probiotic respectively. This results explained that the
bacterial adhesion is a crucial step in the colonization and pathogenesis of bacteria, which can be
inhibited by using probiotic preparations. Inhibitory effects of Probiotic on growth and adhesion of some
gram negative pathogenic bacteria Ziad M. Al-Khozai
College of science
Al-Qadisyia University Abstract: :ا ال
ركز هذا عملذع ى ذال ذنو ع ذك ي ية مهمذ مرلذ ملهاذ عو يذ هذEscherichia coli
Salmonella typhi
Klebsiella pneumonae
ع احت عن ئج ح ك ء هاه عبة مهم ى ال عمدمد من ىك مع عضذه ستذ من م ذع علح ضذ
إل زمل ت عحهك ألهد ب ع احت عن ئج ح ك ء Escherichia coli
ى ال عهد ب من عنكي أل ل ع ذ ع عذ ف ذ
حمن ح كتSalmonella typhi
ى ال عنكىمن ع عذ ع ذ ف عمذKlebsiella pneumonae
قذد ح ذكت ى ذال عهذد ب مذن
عنكي أل ل ع عذ قذ كذاعي يمنذت عدر تذ يذprobiotic
علحضذه مذن ية همذ Lactobacillus acidophilus
قذد ذه
ت ب ً ى ال لك ع ص ق عبة هم علهاذم ذ ع عنلذك عبة مذهم تذب ت بمنذت ي تذ اد ر مذ عنلذك عبة مذهم عرذا ع ذن
عمضذ
عكحظت ئج مشذ ير ىنذد تذ اد عه مذ علا ذ05:05
ف قذد ك ذت عك ذه منذ لت ع بذم عكحظذت ي تذ اد ر مذ ية همذ
ح مض عح مبLAB
غمه علا ى ذال ألننذ ا علهاذم عليذ ادم ذ عدر تذ كذاعي تذب منحظذ ع بذم عح ذع ى ذال
ق ي م ع ص ق عبة هم علهام ى ال عان مذ عننئمذ ي تذ ادprobiotic
ذ ح ذ ية همذEscherichia coli
ا ضذت
يب عانم عل صق من05
-
16
ية هم /خ م ذعال16
-
12
05
-
00
ية هم /خ م ىند ت اد علح كل علهكذز مذن عه مذ
probiotic
) علح كل علا منت ى ال ع ك ع ذ ح عذSalmonella typhi
كذ س ا ذ ف ذ عىذد خ عانمذ عل صذق
من00
-
00
ية هم /خ م ذعال60
-
66
62
-
61
ية هم /خ م ىند ت اد علح كل علهكذز مذن عه مذ علح ذكل علا ذ منذت
ى ال ع ك ع كاعي ي عنيبKlebsiella pneumonae
قد ك س ا ف عىد خ عانم عل صق من22
-
21
ية هم /خ م
ذعال8
-
5
65
-
62
ية هم /خ م ىند تذ اد علح ذكل علهكذز مذن عه مذ علح ذكل علا ذ منذت ى ذال ع ذك ع هذاه عن ذ ئج
تكا عهلم ع صذ ق عبة همذ كانذك عت تذم ذ ىل مذ ستذ مل ر سمه اذم ع ذ ملةذن ع ت ذب ي تذ اد مي حضذه ت
probiotic :ا
ركز هذا عملذع ى ذال ذنو ع ذك ي ية مهمذ مرلذ ملهاذ عو يذ هذEscherichia coli
Salmonella typhi
Klebsiella pneumonae
ع احت عن ئج ح ك ء هاه عبة مهم ى ال عمدمد من ىك مع عضذه ستذ من م ذع علح ضذ
إل زمل ت عحهك ألهد ب ع احت عن ئج ح ك ء Escherichia coli
ى ال عهد ب من عنكي أل ل ع ذ ع عذ ف ذ
حمن ح كتSalmonella typhi
ى ال عنكىمن ع عذ ع ذ ف عمذKlebsiella pneumonae
قذد ح ذكت ى ذال عهذد ب مذن
عنكي أل ل ع عذ قذ كذاعي يمنذت عدر تذ يذprobiotic
علحضذه مذن ية همذ Lactobacillus acidophilus
قذد ذه
ت ب ً ى ال لك ع ص ق عبة هم علهاذم ذ ع عنلذك عبة مذهم تذب ت بمنذت ي تذ اد ر مذ عنلذك عبة مذهم عرذا ع ذن
عمضذ
عكحظت ئج مشذ ير ىنذد تذ اد عه مذ علا ذ05:05
ف قذد ك ذت عك ذه منذ لت ع بذم عكحظذت ي تذ اد ر مذ ية همذ
ح مض عح مبLAB
غمه علا ى ذال ألننذ ا علهاذم عليذ ادم ذ عدر تذ كذاعي تذب منحظذ ع بذم عح ذع ى ذال
ق ي م ع ص ق عبة هم علهام ى ال عان مذ عننئمذ ي تذ ادprobiotic
ذ ح ذ ية همذEscherichia coli
ا ضذت
يب عانم عل صق من05
-
16
ية هم /خ م ذعال16
-
12
05
-
00
ية هم /خ م ىند ت اد علح كل علهكذز مذن عه مذ
probiotic
) علح كل علا منت ى ال ع ك ع ذ ح عذSalmonella typhi
كذ س ا ذ ف ذ عىذد خ عانمذ عل صذق
من00
-
00
ية هم /خ م ذعال60
-
66
62
-
61
ية هم /خ م ىند ت اد علح كل علهكذز مذن عه مذ علح ذكل علا ذ منذت
ى ال ع ك ع كاعي ي عنيبKlebsiella pneumonae
قد ك س ا ف عىد خ عانم عل صق من22
-
21
ية هم /خ م
ذعال8
-
5
65
-
62
ية هم /خ م ىند تذ اد علح ذكل علهكذز مذن عه مذ علح ذكل علا ذ منذت ى ذال ع ذك ع هذاه عن ذ ئج
تكا عهلم ع صذ ق عبة همذ كانذك عت تذم ذ ىل مذ ستذ مل ر سمه اذم ع ذ ملةذن ع ت ذب ي تذ اد مي حضذه ت
probiotic Journal of Kerbala University , Vol. 7 No.1 Scientific. 2009
Inhibitory effects of Probiotic on growth and adhesion of some
gram negative pathogenic bacteria ournal of Kerbala University , Vol. 7 No.1 Scientific. 2009 Microbiology and biochemical test: Microbiology and biochemical test: Three important human pathogens include Escherichia coli , Salmonella typhi and Klebsiella
pneumonae were chosen in this work. Three bacterial isolates for each species ,were obtained from
the biology department of college of science at Al-Qadisyia university. These isolates grown on
culture media, then biochemical and microbiological aspects were documented, included capsule,
hemolysis, lipase, gelatinase, oxidase, motility and catalase according to ( 10,11, 12 ). Fimbriae or Colonization factor antigen (CFA): Fimbriae or Colonization factor antigen (CFA): Fimbriae type I, II and III were screened according to by determination of agglutination of tanned
group A RBCs and bacterial cells in the presence of D-manose ( 3) . Probiotic and Bacterial adhesion test Fimbriae type I, II and III were screened according to by determination of agglutination of tanned
group A RBCs and bacterial cells in the presence of D-manose ( 3) . This test included some steps as follow:
Preparation of the probiotic This test included some steps as follow:
Preparation of the probiotic MRS broth was inoculated with 1% of LAB culture, then incubated anaerobically at 37C˚ for 24
hours. After incubation the culture was centrifuged at 6000 rpm for 15 minutes, the supernatant was
obtained, after adjusting the pH of the filtrate to 6.5 by using NaOH, it was filtered through
Millipore filter unit (0.22). This filtrate was used in the next steps (14,15). Preparation of bacterial suspension: Ten milliliter of nutrient broth medium was inoculated with bacterial growth, the culture was
then incubated at 37C˚ for over night (O.D.600 about 0.4) giving (1*109) cell/ml. Cultures of bacteria
were washed twice with PBS (phosphate buffer saline) and centrifuged at 1000g for 20 minutes and
resuspended in PBS(14,13). Journal of Kerbala University , Vol. 7 No.1 Scientific. 2009 rigid, straight, filamentous appendages on bacterial surface and are often no more than 4 to 7 nm in
diameter and from 0.2 up to 20 nm length. Fimbriae composed from protein called pilin, the
filamentous nature of fimbriae may mediate the adhesion by adhesins associated with fimbriae (3,4,
5).Probiotics (prebiotics) is a dietary adjuvant that beneficially affects the host physiology by
modulating mucosal and systemic immunity, as well as improving nutritional and microbial balance
in human intestinal tract. Currently probiotic preparations include different species of LAB (Lactic
acid bacteria) mainly(6) . The therapeutic effects of lactic acid bacteria include ; improvement of
nutritional quality of food and feed, metabolic stimuli of vitamin synthesis, and enzyme production,
stabilization of gut microflora and competitive exclusion of enteric pathogens ,enhancing the innate
host defenses by production of antimicrobial substances, reduction of serum cholesterol by
assimilation mechanism, decrease risk of colon cancer by detoxification of carcinogens ,and tumor
suppression by modulation of cell mediated immunity (7). LAB making large proportion from
normal flora of gut and vigina. And demonstrate a wide spectrum of antimicrobial characteristics,
including acid and bile resistance, antimicrobial systems(ex: bacteriocin, lactic acid, peroxide) and
adhesion to various types of pathogens ( 8 ). Lactobacillus acidophilus has superior capability of
producing lactic acid which is antimicrobial and helps the body protection from harmful bacteria
adhering the intestinal mucosa. This bacteria can inhibit the activities of adherence and
proliferation of pathogenic bacteria by several ways, such as decreasing luminal pH , rendering
specific nutrients unavailable to pathogens, decreasing the radix potential of the luminal
environment, and producing hydrogen peroxide under anaerobic conditions and producing
inhibitory compounds such as bacteriocin (6, 7). This work was carried in an attempt to investigate
the inhibitory rule of probiotic on bacterial growth and adhesion. Introduction : The ability of successful pathogens to survive in an immunologically hostile environment is
provided by large armamentarium of virulence mechanisms, which includes bacterial evade,
neutralize or counter the host defense systems, but also manipulate host homeostasis and normal
cell functions(1, 2, 3). Adhesion of bacteria to human tissue surfaces and implanted biomaterial
surfaces is an important step in the pathogenesis and infection. Fimbriae (or pili) are a group of 34 Probiotic sensitivity assay This assay was curried by using three bacterial strains for each species and two concentrations of
the LAB (lactic acid bacteria) filtrate against the bacterial strains, concentrations are stock filtrate or
(concentration-1)and 1:1 dilution with normal saline (concentration-2), result was carried by
measuring the minimum inhibition zone by making a 5mm discs by using cock porer (12, 14,15). Preparation of epithelial cell for probiotic assay : 35 Adhesion Test: A mixture of 0.2 ml of the bacterial suspension, 0.2 ml of the epithelial cell suspension and 0.1
ml of PBS was incubated at 37 C˚ for one hour. Unattached bacteria to uroepithelial cells were
removed by centrifugation in PBS at 1000g for 10 minutes. The final pellet was resuspended in PBS
then a drop of it was put onto a microscope slid, air dried fixed with methanol: acetic acid (3:1) and
stained with methylen blue. The adherent bacteria to epithelial cells were observed by compound
light microscope. Control of only epithelial cells was included (14). Journal of Kerbala University , Vol. 7 No.1 Scientific. 2009 Uroepithelial cells were collected from the urine of some healthy females by centrifugation at
1000g for 10 minutes the washed three times with PBS and centrifuged at 100g for 10 minutes
before resuspending in PBS. Results: Results demonstrate that the bacteria contained some types of fimbriae. Type III fimbriae was
found in all bacteria under test, while type II was recorded in Salmonella typhi only and type I
found in Escherichia coli and Klebsiella pneumonae while missing in Salmonella typhi as explain
in table (1). Table-1: Illustrate the fimbriae types of the bacteria. Fimbriae type III
Fimbriae type II
Fimbriae type I
Bacteria
+
-
+
Escherichia coli
+
+
-
Salmonella typhi
+
-
+
Klebsiella pneumonae Table-1: Illustrate the fimbriae types of the bacteria. The bacterial inhibition zone were estimated by using three isolates and two concentrations of the
LAB probiotic, results showed that both Klebsiella pneumonae and Salmonella typhi were more
sensitive than Escherichia coli especially when treated with stock filtrate or concentration (con. 1)of LAB filtrate ,as explained in table( 2). Table-2: Illustrate the bacterial sensitivity to the probiotic filtrate of the LAB . Bacteria
zone of inhibition ( mm)
Isolate number 1
Isolate number 2
Isolate number 3
Con. 1
Con. 2
Con.1
Con.2
Con.1
Con.2
Escherichia coli
9
8
11.5
10
12.5
11
Salmonella typhi
13.5
11
14
13.5
13
11.5
Klebsiella pneumonae
14.5
12
13.5
13
12.5
11.5
Con.1= concentration 1 of LAB (stock bacterial filtrate)
Con.2= concentration 2 of LAB (1:1 of bacterial filtrate: normal saline) Table-2: Illustrate the bacterial sensitivity to the probiotic filtrate of the LAB . zone of inhibition ( mm) Results of the effect of the probiotic reveled that, the bacterial strains adhesion was reduced by
using the same concentrations of the LAB in comparison to control, Escherichia coli adhesion to
Uroepithelial cells was reduced from 59-61 bacterium/ cell without probiotic to 33-30 and 24-21
bacterium/ cell by using con.2 and 1 of the probiotic respectively as explained in figure (1). 36 Journal of Kerbala University , Vol. 7 No.1 Scientific. 2009 Discussion: Although both motile and nonmotile species form biofilms, in motile species, the ability to move
using flagella or pili is generally required for efficient cell-to-surface attachment (15,16). The
fimbriae and capsule mediates the interactions between the bacterium and it's immediate
environment. It's importance in promoting the formation of biofilms and stimulate interspecies
coaggregation, thereby enhancing the bacterial colonization, and are an essential virulence factors
(15,17). Species under study appeared to have several types of fimbriae, these results are similar
agreed with the results of previous investigators. Enteropathogenic E. coli appear to have
predilection of adherence to the host cells is thought to involve bundle-forming pili (4). While S. typhi capsular polysaccharide and fimbriae, offer the protection from environmental insults and host
non specific immune response facilitating the invasion, by mediating the interaction between the
bacteria and the mucus surrounding host epithelial cells (2,16). In K. pneumonae, it has been shown
that expression of a polysaccharide capsule is essential for the colonization of the large intestine of
mice(4, 15). One interpretation of these data is that capsule and fimbriae is required for initial steps
of colonization by interacting with the mucus layers and this step is vital for successful colonization
in vivo(3). The concomitant increase in adhesion expression would have the net effect of enhancing
bacteria-epithelial cells interactions essential for long-term colonization ( 17 ).Inhibitory effects
were documented in this work by using the Lactic acid bacteria filtrate , these effects due to
secretion of inhibitory substances by this bacteria.Lactic acid bacteria have an inhibitory effect
against the gram negative and positive bacteria. Some investigators stated a high inhibitory effects
of LAB against the enteropathogenic bacteria (6). While others documented a significant inhibition
of LAB on Proteus mirabilis , proposing the effect to the presence of active antimicrobial secretions
such as lactocidin , plantaracin and acidophilin, these results agreed with the results obtained in this
work (9). The obtained data reflect a remarkable decrease in the numbers of all tested adhered
bacterial cells to the epithelium cells, these results come in comformity with results onbtaned by
some investigators . This was due to the effect of inhibitory substances found in the filtrate of LAB
isolates and the acidic pH which affect the growth of the gram negative bacteria by altering some
surface structures (18,19). Journal of Kerbala University , Vol. 7 No.1 Scientific. 2009 9-8
14-10
44-46
0
5
10
15
20
25
30
35
40
45
bacteria /cell
con.1
con.2
control
Figure-3 : Illustrate the inhibition of adhesion of Klebsiella pneumonae on the Uroepithelial cells. Figure-3 : Illustrate the inhibition of adhesion of Klebsiella pneumonae on the Uroepithelial cells. Journal of Kerbala University , Vol. 7 No.1 Scientific. 2009 24-21
33-30
59-61
0
10
20
30
40
50
60
bacteria /cell
con.1
con.2
control
Figure-1 : Illustrate the inhibition of adhesion of Escherichia coli on the Uroepithelial cells. -1 : Illustrate the inhibition of adhesion of Escherichia coli on the Uroepithelial cells. Adhesion of Salmonella typhi to Uroepithelial cells was reduced from 55-53 bacterium/ cell
without probiotic to 16-14 and 11-13 bacterium/ cell by using con.2 and 1 of the probiotic
respectively as explained in figure (2). Adhesion of Salmonella typhi to Uroepithelial cells was reduced from 55-53 bacterium/ cell
without probiotic to 16-14 and 11-13 bacterium/ cell by using con.2 and 1 of the probiotic
respectively as explained in figure (2). 11-13
16-14
55-53
0
10
20
30
40
50
60
bacteria /cell
con.1
con.2
control
Figure-2 : Illustrate the inhibition of adhesion of Salmonella typhi on the Uroepithelial cells. Also the adhesion of Klebsiella pneumonae to Uroepithelial cells was reduced from 44-46
bacterium/ cell without probiotic to 14-10 and 8-9 bacterium/ cell by using con.2 and 1 of the
probiotic respectively as explained in figure (3). 2 : Illustrate the inhibition of adhesion of Salmonella typhi on the Uroepithelial cells. Also the adhesion of Klebsiella pneumonae to Uroepithelial cells was reduced from 44-46
bacterium/ cell without probiotic to 14-10 and 8-9 bacterium/ cell by using con.2 and 1 of the
probiotic respectively as explained in figure (3). 37 Journal of Kerbala University , Vol. 7 No.1 Scientific. 2009 Discussion: (20) investigated the effect of Lactobacillus casei on E coli and found
that the inhibitory effect was not caused by bacteriophage of hydrogen peroxide but due to the
aggregation of E. coli an LAB . (14) reported that precoating of LAB strains reduced the binding of
uropathogenic coagulase negative Staphylococci and E. coli to 8 bacteria / cell . while (9) observed
a clear reduction in adhesion of Proteus mirabilis after the treatment with LAB filtrates reaching to
3-8 bacteria/ cell. Others found that the biosurfactant surlactin as released by lactobacillus isolates
may open the way to the development of anti-adhesive biologic coating against Enterococcus
faecalis , they reported a decrease in the number of adhering Enterococcus reaching to
approximately 70% ( 21,22 ). 38 References : 1- Brooks; Geo F.; Butel; Jane S.; Morse Stephan. A. C.(2001). Medical Microbiology. 26/E. Lange. Medical book. Me Graw: Hill. VSA. 1- Brooks; Geo F.; Butel; Jane S.; Morse Stephan. A. C.(2001). Medical Microbiology. 26/E. Lange. Medical book. Me Graw: Hill. VSA. 2- Russell, W.; and Herwald H. (2005). Concepts in bacterial virulence. J. Contributions of
Microbiology. Vol 12 .Karger. Switzerland. 2- Russell, W.; and Herwald H. (2005). Concepts in bacterial virulence. J. Contributions of
Microbiology. Vol 12 .Karger. Switzerland. 2- Russell, W.; and Herwald H. (2005). Concepts in bacterial virulence. J. Contributions of
Microbiology. Vol 12 .Karger. Switzerland. 3- An, Y.H. ;and Friedman R. J. (2000). Handbook of bacterial adhesion , principles, methods and
applications . Humana press. USA. 3- An, Y.H. ;and Friedman R. J. (2000). Handbook of bacterial adhesion , principles, methods and
applications . Humana press. USA. 4- Groisman, E.A. (2001). Principles of bacterial pathogenesis. Academic Press. University of
California. USA. 4- Groisman, E.A. (2001). Principles of bacterial pathogenesis. Academic Press. University of
California. USA. Anderson. Roy M.(1993). Medical Microbiology. Mosby. London. 5- Anderson. Roy M.(1993). Medical Microbiology. Mosby. London. 6 -Gibson, G.R. and Roberfroid, M.R.(2008). Handbook of Prebiotics. CRC. Press .USA. y
(
)
gy
y
6 -Gibson, G.R. and Roberfroid, M.R.(2008). Handbook of Prebiotics. CRC. Press .USA. (
)
7-Maitijasic, B. B.; and Rogelj, I. (1999). Bacteriocinogenic activity of lactobacilli isolated from
cheese and baby faeces. J. food technology and biotechnology. Vol. 37(2). 7-Maitijasic, B. B.; and Rogelj, I. (1999). Bacteriocinogenic activity of lactobacilli isolated from
cheese and baby faeces. J. food technology and biotechnology. Vol. 37(2). 8- Messen, W.; and De vuyst, L. (2002). Inhibitory substances produced by lactobacilli isolated
from sourdoughs – a review. J. Food microbiology. Vol.72(1-2). 8- Messen, W.; and De vuyst, L. (2002). Inhibitory substances produced by lactobacilli isolated
from sourdoughs – a review. J. Food microbiology. Vol.72(1-2). 9- Al-Jeboury, G. H. (2005). Probiotic Effect on Adhesion property of Proteus mirabilis. M.Sc. thesis in biotechnology. College of science/ Al-Nahrain university. 10- Macfaddin J.F. (2000). Biochemical tests for the identification of medical bacteria. 3rd ed. The
Williams and Wilkins-Baltimor USA. 11- Baron. Ellen Jo; Peterson Tance. R.; Finegold. Syndey. M.(1994). Diagnostic microbiology 9/E
Mosby. USA. 12- Collee, Gerald.; Fraser Andrew G.; Marmion barrie P.; Simmons. Antony.(1996). Practical
Medical Microbiology. 14/E Churchill Livingston. USA. 13- Iwahi, T. Abc, Y. and Tsuchiya, K. (1982). Virulence of E. References : coli in a sending urinary tract
infection in mice. J. Medical Microbiology. 15: 303-316. 14- Wary, S. K.; S. I H.ull.; S. I. Cook; R. C. Barrish; and Hall, R. A. (1986). Identification and
characterization of a Uroepithelial cell adhesion from a uropathogenic isolate Proteus mirabilis. J. Inflammatory immunology. Vol. 54 (1). 15-Lewus, C.B.; Kaiser, A. and Monyville, T. J. (1991). Inhibition of food borne bacterial
pathogens by bacteriocins from lactic acid bacteria isolated from meat. J. Applied environmental
microbiology., 57:1683-1688. gy
16- Romeo, T. (2008). Bacterial biofilms. J. Microbiology and Immunology. Vol.322. Springer
USA. 17- Wilson, M. ( 2002). Bacterial adhesion to host tissues, mechanisms and consequences . Advances in molecular and cellular microbiology Vol.1. Cambridge university press.UK. 18- Mims.C.A.; Palfire J.HL.;Roitt I.M.;Derek W.; Rosamurd W.; and Roy, M.A.(1995). Medical
Microbiology. Mosby, USA. 19- Chan, R.C.; Bruce A. W.; and Reid, G. A.. (1984). Adherence of cervical, vaginal and distal
urethral normal microflora to human Uroepithelial cells and the inhibition of adherence of gram-
negative uropathogenes by competitive exclusion. J. Urology. Vol. 131. 20- Blomberg, L. H.; A. Conway. (1993). Inhibition of adhesion of E. coli k88 to piglet ideal
mucous by Lactobacillus spp. J. Applied environmental microbiology. Vol.59. 21-Velraeds, M. M.; Van Der Mei, H. C.; Reid G.; and Busscher, H. J. (1996). Inhibition initial
adhesion of uropathogenic Enterococcus faecalis by biosurfictants from Lactobacillus isolates. J. Applied environmental microbiology. Vol.62. 22-Lamond, R.J. (2004).Bacterial Invasion of host cells .J. Advances in molecular and cellular
microbiology Vol. 5. Cambridge university press. UK. 39
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Impact of performance in a mandatory postgraduate surgical examination on selection into specialty training
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D. S. G. Scrimgeour1,3,5, J. Cleland1, A. J. Lee2, G. Griffiths4, A. J. McKinley3, C. Marx6
and P A Brennan5 1Centre For Healthcare Education Research and Innovation and 2Department of Medical Statistics, University of Aberdeen, and 3Department of
Colorectal Surgery, Aberdeen Royal Infirmary, Aberdeen, 4Department of Vascular Surgery, Ninewells Hospital, Dundee, 5Intercollegiate Committee
for Basic Surgical Examinations, and 6Royal College of Surgeons of England, London, UK
Correspondence to: Mr D. S. G. Scrimgeour, Centre For Healthcare Education Research and Innovation, University of Aberdeen, Aberdeen AB25 2ZD,
UK (e-mail: duncan.scrimgeour@nhs.net) Background: The Intercollegiate Membership of the Royal College of Surgeons (MRCS) examination is
undertaken by large numbers of trainees in the UK and internationally as a mandatory step within surgical
training. Unlike some high-stakes medical examinations, the MRCS is yet to be validated. A quantitative
study was undertaken to assess its predictive validity by investigating the relationship between MRCS
(Parts A and B) and national selection interview scores for general and vascular surgery in the UK. Background: The Intercollegiate Membership of the Royal College of Surgeons (MRCS) examination is
undertaken by large numbers of trainees in the UK and internationally as a mandatory step within surgical
training. Unlike some high-stakes medical examinations, the MRCS is yet to be validated. A quantitative
study was undertaken to assess its predictive validity by investigating the relationship between MRCS
(Parts A and B) and national selection interview scores for general and vascular surgery in the UK. Methods: Pearson correlation coefficients were used to examine the linear relationship between each
assessment, and linear regression analyses were employed to identify potential independent predictors of
the national selection score. All UK medical graduates who attempted the interview in 2011–2015 were
included. Methods: Pearson correlation coefficients were used to examine the linear relationship between each
assessment, and linear regression analyses were employed to identify potential independent predictors of
the national selection score. All UK medical graduates who attempted the interview in 2011–2015 were
included. Results: Some 84⋅4 per cent of the candidates (1231 of 1458) were matched with MRCS data. There was
a significant positive correlation between the first attempt score at Part B of the MRCS examination and
the national selection score (r = 0⋅38, P < 0⋅001). In multivariable analysis, 17 per cent of variance in the
national selection first attempt score was explained by the Part B MRCS score and number of attempts
(change in R2 value of 0⋅10 and 0⋅07 respectively; P < 0⋅001). D. S. G. Scrimgeour1,3,5, J. Cleland1, A. J. Lee2, G. Griffiths4, A. J. McKinley3, C. Marx6
and P A Brennan5 Candidates who required more than two
attempts at Part B were predicted to score 8⋅1 per cent less than equally matched candidates who passed
at their first attempt. Conclusion: This study supports validity of the MRCS examination, and indicates its predictive valu
regarding entry into specialist training. Funding information
Royal College of Surgeons of England
Royal College of Surgeons of Ireland
Royal College of Surgeons of Edinburgh
Royal College of Physicians and Surgeons of Glasgow
Presented at the Association of Surgeons of Great Britain and Ireland 2017 International Surgical Congress, Glasgow,
UK, May 2017 and the Scottish Medical Education Conference, Edinburgh, May 2017 Royal College of Surgeons of Edinburgh Royal College of Physicians and Surgeons of Glasgow newly qualified doctors enter the 2-year Foundation
Programme. Most doctors will continue their training
in one of three broad specialist areas: general practice,
medicine or surgery. To be considered for higher medical
or surgical training, UK doctors must also complete core
training and pass the relevant postgraduate examination,
MRCS or Membership of the Royal Colleges of Physi-
cians (MRCP). Overviews of the content of the MRCS © 2017 The Authors. BJS Open published by John Wiley & Sons Ltd on behalf of BJS Society Ltd
BJS Open 2017; 1: 67–74
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited. Original article Presented at the Association of Surgeons of Great Britain and Ireland 2017 International Surgical Congress, Glasgow,
UK, May 2017 and the Scottish Medical Education Conference, Edinburgh, May 2017 BJS Open 2017; 1: 67–74 Paper accepted 24 May 2017
Published online 29 August 2017 in Wiley Online Library (www.bjsopen.com). DOI: 10.1002/bjs5.7 Impact of performance in a mandatory postgraduate surgical
examination on selection into specialty training Scrimgeour1,3,5, J. Cleland1, A. J. Lee2, G. Griffiths4, A. J. McKinley3, C. Marx6
Brennan5 D. S. G. Scrimgeour1,3,5, J. Cleland1, A. J. Lee2, G. Griffiths4, A. J. McKinley3, C. M
and P. A. Brennan5 Methods examination and the specialty training selection process
for general and vascular surgery have been described
elsewhere2,3. The MRCS examination is designed to safe-
guard patients and ensure high standards for practising sur-
geons. It is a prerequisite for progression to higher specialty
training in the UK, a mandatory examination for all aspir-
ing surgeons who wish to train or work in the UK and a
means for overseas candidates to improve their opportuni-
ties in their home country2,4. examination and the specialty training selection process
for general and vascular surgery have been described
elsewhere2,3. The MRCS examination is designed to safe-
guard patients and ensure high standards for practising sur-
geons. It is a prerequisite for progression to higher specialty
training in the UK, a mandatory examination for all aspir-
ing surgeons who wish to train or work in the UK and a
means for overseas candidates to improve their opportuni-
ties in their home country2,4. In the absence of a specific ethics committee responsible for
postgraduate surgical examinations, both the ICBSE and its
Internal Quality Assurance subcommittee, which monitors
standards, approved the study. The sample included all UK medical graduates who had
attempted Part B of the MRCS examination from its insti-
tution in October 2008 to May 2015. Data were extracted
by the lead administrator from the intercollegiate MRCS
database held by the Royal College of Surgeons (RCS) of
England. Each candidate’s Part A score was merged with
their Part B score to create a complete MRCS history,
which included the self-declared demographics of sex, eth-
nicity, first language and date of birth. The Intercollegiate Committee for Basic Surgical Exam-
inations (ICBSE), which is responsible for the continued
development, quality assurance and standards of MRCS in
the UK, produces an annual report highlighting examina-
tion reliability5. To date, there has been no analysis of the
predictive validity of the MRCS examination, and it is not
known whether performance at this examination predicts
outcomes as a surgeon in training or beyond. Candidates were anonymized by a unique identifier
before data were released for analysis. This identifier cor-
responded to each candidate’s General Medical Council
number, which could be accessed only by the RCS lead
administrator and not by the research team. Introduction The Intercollegiate Membership of the Royal College of
Surgeons (MRCS) examination is one of the most widely
offered postgraduate surgical examinations in the world,
with up to 6000 doctors in the UK and overseas taking
the examination each year1,2. An overview of the medical
education and surgical training pathway in the UK is
provided in Fig. 1. Upon graduating from medical school, D. S. G. Scrimgeour, J. Cleland, A. J. Lee, G. Griffiths, A. J. McKinley, C. Marx and P. A. Brennan 68 Foundation
Year 1
Foundation
Year 2
Core/specialty
surgical training
Year 1
Competitive
entry into
surgical, medical
or GP training
MRCS examination
(Parts A and B)
National
selection
interview
FRCS
Competitive
entry after
medical school
Core/specialty
surgical training
Year 2
Higher specialty
training
Year 3–7/8
Consultant
Fig. 1 Surgical training pathway in the UK. MRCS, Membership of the Royal College of Surgeons; FRCS, Fellowship of the Royal
College of Surgeons; GP, general practice Core/specialty
surgical training
Year 1 Fig. 1 Surgical training pathway in the UK. MRCS, Membership of the Royal College of Surgeons; FRCS, Fellowship of the Royal
College of Surgeons; GP, general practice © 2017 The Authors.
BJS Open published by John Wiley & Sons Ltd on behalf of BJS Society Ltd www.bjsopen.com © 2017 The Authors.
BJS Open published by John Wiley & Sons Ltd on behalf of BJS Society Ltd Methods Given that MRCS is a prerequisite for progression to
higher specialty training in the UK, it is important to deter-
mine the relationship between MRCS examination scores
and outcomes in the higher specialty training national
selection process. The aim of this study was to examine
the predictive validity of MRCS in relation to outcomes in
this selection process, and, specifically, whether the results
of this mandatory examination can predict how candidates
will perform at the national selection interview for a large
surgical specialty, general surgery (which included vascular
surgery in the UK until 2013). All scores (recorded as percentages) for candidates who
had attempted the national selection interview for general
and vascular surgery since its origin in May 2011 were
then cross-linked with the MRCS database. The national
selection first attempt score was used as the main outcome
variable. As the Part B examination underwent changes, three
periods were considered: October 2008 to February 2010, BJS Open 2017; 1: 67–74 BJS Open 2017; 1: 67–74 Performance in a postgraduate surgical examination and selection into specialty training 69 Interviewed from May 2011 to May 2015
for the general and vascular surgery
national selection process
n = 1458
Excluded n = 318
Non-UK medical graduate n = 305
No GMC number n = 9
Duplicate name n = 4
Old MRCS examination
(Parts 1, 2 and 3)
n = 136
New MRCS examination
(Parts A and B)
n = 777
Matched with
national selection
scores
n = 774
Excluded
n = 457
Could not be matched n = 227
Part A missing
n = 3
n = 1231
n = 913
Fig. 2 Flow diagram of surgical trainees in the study. GMC, General Medical Council; MRCS, Membership of the Royal College of
Surgeons nterviewed from May 2011 to May 2015
for the general and vascular surgery
national selection process
n = 1458 Could not be matched n = 227 Part A missing
n = 3 Fig. 2 Flow diagram of surgical trainees in the study. GMC, General Medical Council; MRCS, Membership of the Royal College of
Surgeons May 2010 to October 2012, and February 2013 to present. Each of these periods was considered as one of the variables
of interest. May 2010 to October 2012, and February 2013 to present. Each of these periods was considered as one of the variables
of interest. national selection first attempt score. Methods The magnitude of
correlation was in accordance with Cohen’s guidelines7
(r = 0⋅01–0⋅29, low or weak correlation; r = 0⋅30–0⋅49,
moderate correlation; r ≥0⋅50, strong correlation). An
independent-samples t test or ANOVA was used to exam-
ine the relationship between MRCS and national selection
scores and demographic variables, dependent on the num-
ber of categories being compared. As part of national selection, candidates are divided into
three groups at the portfolio station based on how many
years they are from graduation: less than 5 years, 5–7 years
and more than 7 years. Years from graduation was also
considered as a variable of interest. As older doctors (aged at least 29 years at graduation from
medical school) have been shown to have problems with
their Annual Review of Competence Progression com-
pared with younger colleagues6, this age-related variable
was also considered in the analyses. Linear regression analysis was used to identify potential
independent predictors of the national selection score. All
potential predictors with P < 0⋅100 in univariable analysis
were entered simultaneously into the regression models. Any variable with P > 0⋅050 in the full model was subse-
quently removed until only statistically significant predic-
tors remained in each model. Candidates were categorized based on their total num-
ber of attempts required to pass each part of the MRCS
examination (1, 2, or 3 or more attempts). Stepwise linear regression analysis was used to measure
the change in R2 when single predictors were added to the
model in order to assess the magnitude of the contribution
of new predictors in explaining variance in national selec-
tion score. © 2017 The Authors.
BJS Open published by John Wiley & Sons Ltd on behalf of BJS Society Ltd www.bjsopen.com Statistical analysis Table 1 National selection descriptive statistics and the relationship between demographic variables and national selection first attempt
score for general and vascular surgery n descriptive statistics and the relationship between demographic variables and national selection first attempt
ular surgery Table 1 National selection descriptive statistics and the relationship between demographic variables and national selection first attempt
score for general and vascular surgery Values in parentheses are *percentages and †mean(s.d.). n.a., Not applicable; MRCS, Membership of the Royal College of Surgeons. ‡ANOVA, except §t
test © 2017 The Authors.
BJS Open published by John Wiley & Sons Ltd on behalf of BJS Society Ltd www.bjsopen.com Statistical analysis All analyses were conducted using SPSS® version 24.0
(IBM, Armonk, New York, USA). Pearson correlation
coefficients were used to examine the linear relationship
between both parts of the MRCS examination and the There were no violations of the assumptions for any of
the regression models and no collinearity was evident in
the data. BJS Open 2017; 1: 67–74 BJS Open 2017; 1: 67–74 © 2017 The Authors. BJS Open published by John Wiley & Sons Ltd on behalf of BJS Society Ltd D. S. G. Scrimgeour, J. Cleland, A. J. Lee, G. Griffiths, A. J. McKinley, C. Marx and P. A. Brennan 70 Table 1 National selection descriptive statistics and the relationship between demographic variables and national selection first attempt
score for general and vascular surgery
National selection
descriptive statistics (n = 774)*
National selection
first attempt score (%)†
Test statistic‡
d.f. P
Age (years)†
29⋅18(2⋅68)
Sex
0⋅25§
754
0⋅804
M
440 (57⋅0)
73⋅27(9⋅19)
F
332 (43⋅0)
73⋅43(8⋅01)
Missing
2
Ethnicity
6⋅22
4
< 0⋅001
White British
353 (59⋅0)
74⋅92(7⋅52)
Asian
156 (26⋅1)
71⋅32(8⋅14)
Black
20 (3⋅3)
72⋅09(9⋅67)
Other
69 (11⋅5)
71⋅43(10⋅33)
Missing
176
First language
2⋅70§
609
0⋅007
English
571 (93⋅5)
73⋅72(8⋅11)
Not English
40 (6⋅5)
70⋅10(9⋅37)
Missing
163
Mature medical graduate
1⋅26§
770
0⋅209
No (age < 29 years)
734 (95⋅1)
73⋅42(8⋅67)
Yes (≥29 years)
38 (4⋅9)
71⋅61(8⋅99)
Missing
2
No. of Part A MRCS attempts
4⋅64
2
0⋅010
1
598 (77⋅3)
73⋅85(8⋅69)
2
90 (11⋅6)
71⋅14(8⋅45)
≥3
86 (11⋅1)
72⋅24(8⋅59)
No. of Part B MRCS attempts
27⋅04
2
< 0⋅001
1
581 (75⋅1)
74⋅50(7⋅93)
2
150 (19⋅4)
71⋅01(10⋅10)
≥3
43 (5⋅6)
66⋅10(8⋅33)
Years from graduation
1⋅64
2
0⋅196
< 5
668 (86⋅4)
73⋅49(8⋅61)
5–7
92 (11⋅9)
72⋅86(9⋅31)
> 7
13 (1⋅7)
69⋅32(8⋅07)
Missing
1
Part B examination date
1⋅20
2
0⋅301
October 2008 to February 2010
111 (14⋅4)
73⋅14(11⋅07)
May 2010 to October 2012
423 (54⋅7)
73⋅02(8⋅41)
February 2013 to present
239 (30⋅9)
74⋅09(7⋅91)
Missing
1
Values in parentheses are *percentages and †mean(s.d.). n.a., Not applicable; MRCS, Membership of the Royal College of Surgeons. ‡ANOVA, except §t
test. Discussion Several groups have assessed the predictive validity of other
high-stakes medical examinations, including the United
States Medical Licensing Examination® (USMLE®)8–10,
the Quebec Licensing Examination11, the Medical Coun-
cil of Canada Qualifying Examination12–14, the MRCP
examination15–17 and Membership of the Royal College of
General Practitioners examination15. Performance in these
examinations predicts performance in clinical practice and
other medical assessment processes11,18,19. For example,
each part of the MRCP examination predicts the next17,
and candidates who score higher in all parts of the MRCP
examination do better in workplace-based assessments
than those who underperform16. Similarly, USMLE®
scores predict performance in the American Board of
Surgery In-Training Examination among general surgery
residents20, and higher USMLE® scores have been asso-
ciated with higher pass rates in the general surgery board
examination10. This may explain why surgical programme
directors in the USA regard USMLE® performance as the
most important factor for preliminary screening of general
surgery residents21. *Maximum predicted first attempt score, calculated using the reference
variables (a white candidate who passed Part B Membership of the Royal
College of Surgeons (MRCS) at the first attempt is expected to score
69⋅67 per cent at the national selection interview for general and vascular
surgery). †A candidate who just achieved the pass mark would have a
value of zero. Model: R2 = 0⋅18, n = 598. Part B (581 of 774, 75⋅1 per cent) of the MRCS exami-
nation at the first attempt. There were significant, weak
correlations between Part A first and passing attempt scores
and the national selection first attempt score (r = 0⋅19 and
0⋅20 respectively; P < 0⋅001). The correlations between
Part B first and passing attempt scores and the national
selection score were stronger (r = 0⋅38 and 0⋅30 respec-
tively; P < 0⋅001) (Table 2). The relationship between demographic factors and the
number of attempts at each part of the MRCS examination,
and how they affected the national selection first attempt
score, are presented in Table 1.i In keeping with these observations, the present study has
identified that performance in Part B of the MRCS exami-
nation predicts 17 per cent of the variance in national selec-
tion first attempt score for general and vascular surgery. Performance in a postgraduate surgical examination and selection into specialty training Performance in a postgraduate surgical examination and selection into specialty training 71 Table 2 Correlations between Membership of the Royal College
of Surgeons examination scores and national selection first
attempt score for general and vascular surgery 2 d.f., P < 0⋅001), ethnicity (t = 6⋅22, 4 d.f., P < 0⋅001) and
first language (t = 2⋅70, 609 d.f., P = 0⋅007). As these vari-
ables were all below the 10 per cent significance level in
univariable analysis, they were included in the multivari-
able analysis to identify independent predictors of national
selection score for general and vascular surgery (Table 3). The Part B MRCS passing attempt score and the number
of attempts required to pass Part B were significant predic-
tors of the general and vascular surgery national selection
first attempt score (change in R2 value of 0⋅10 and 0⋅07
respectively; P < 0⋅001). The model also predicted that
candidates who required three or more attempts at Part B
of the examination would obtain a national selection score
8⋅1 per cent lower than equally matched candidates who
passed Part B at their first attempt. Ethnicity had much
less influence on national selection score than number of
attempts (change in R2 = 0⋅01, P = 0⋅038), although Asian
candidates were predicted to perform slightly worse than
white candidates (national selection first attempt score
for general and vascular surgery: 67⋅9 versus 69⋅7 per cent
respectively; P = 0⋅019). Values are Pearson correlation coefficients; all are significant at P < 0⋅001
level. Table 3 Multivariable linear regression analysis of predictors of
national selection first attempt score for general and vascular
surgery Unstandardized coefficients
B
s.e. P
Constant*
69⋅67
0⋅97
< 0⋅001
How well candidates passed Part
B MRCS (% above pass mark)†
0⋅28
0⋅04
< 0⋅001
No. of Part B MRCS attempts
1
Reference
2
−2⋅93
0⋅79
< 0⋅001
≥3
−8⋅11
1⋅31
< 0⋅001
Ethnicity
White British
Reference
Asian
−1⋅77
0⋅75
0⋅019
Black
−1⋅08
1⋅74
0⋅536
Other
−1⋅30
1⋅02
0⋅201 © 2017 The Authors.
BJS Open published by John Wiley & Sons Ltd on behalf of BJS Society Ltd www.bjsopen.com Results The majority of national selection candidates were: white
British (353 of 598, 59⋅0 per cent), men (440 of 772, 57⋅0
per cent), spoke English as their first language (571 of 611,
93⋅5 per cent) and graduated from medical school before
the age of 29 years (734 of 772, 95⋅1 per cent). Only 13
(1⋅7 per cent) of 773 candidates attempted the national
selection interview for the first time more than 7 years after
graduating from medical school. More than half (423 of
773, 54⋅7 per cent) of the UK graduates in the cohort had
passed the Part B MRCS examination between May 2010
and October 2012 (Table 1). A total of 1231 (84⋅4 per cent) of 1458 candidates
long-listed for the general and vascular surgery national
selection process in the UK from May 2011 to May 2015
were matched with MRCS data (Fig. 2). Some 454 candi-
dates could not be included: 305 were non-UK graduates,
nine had no GMC number available, four were duplicate
entries and 136 had sat the MRCS examination in place
before 2008. Of the remaining 777 candidates, three had
incomplete Part A data available and were also excluded, so
that 774 candidates were matched on MRCS and national
selection data, and entered into the analysis. Approximately three-quarters of national selection can-
didates had passed Part A (598 of 774, 77⋅3 per cent) and BJS Open 2017; 1: 67–74 © 2017 The Authors. BJS Open published by John Wiley & Sons Ltd on behalf of BJS Society Ltd Discussion For example,
McDougle and colleagues23 compared graduates who ini-
tially failed Step 1 of the USMLE® with those who passed it
first time, finding that the relative risk of not being specialty
board certified was 2⋅2. Multiple attempts at the Medical
College Admission Test (MCAT) have also been associated
with an increased risk of failing Step 2 of the USMLE®24,
whereas multiple attempts at the Professional and Lin-
guistic Assessments Board (PLAB) test have been shown
to be independently predictive of unsatisfactory training
performance25. The relationship between the number of
attempts required to pass a postgraduate UK medical exam-
ination and performance in other medical assessments is
unknown, but one study17 found that as the number of
attempts at each part of the MRCP examination increased,
final passing score decreased. The present results add to
the evidence that candidates who require more attempts to
pass high-stakes postgraduate medical examinations are of
poorer calibre than those who require fewer attempts. This study also has implications for policy and prac-
tice. In keeping with other studies22–25, these results sug-
gest that candidates who require multiple attempts at
a postgraduate medical examination are more likely to
struggle with other medical assessment processes (in this
case, assessment via a selection process). This information
may identify doctors who will require additional support
through their training and guide national bodies when con-
sidering borderline candidates or appeals. The study has
implications for the number of times UK candidates should
be encouraged to attempt the MRCS examination. If those
requiring more than two attempts at Part B are unlikely to
be offered a national training number in general or vascular
surgery, this is important information for those setting out
on their postgraduate medical career. The strengths of this study are the size of the study
population and that the data are from multiple cohorts
and various versions of the MRCS examination. Discussion In
addition, candidates who required more than two attempts
at Part B were predicted to score substantially less at In univariable analysis, there were significant differences
in national selection first attempt score between the vari-
ables: number of attempts at Part A MRCS (t = 4⋅64, 2 d.f.,
P = 0⋅010), number of attempts at Part B MRCS (t = 27⋅04, BJS Open 2017; 1: 67–74 © 2017 The Authors. BJS Open published by John Wiley & Sons Ltd on behalf of BJS Society Ltd D. S. G. Scrimgeour, J. Cleland, A. J. Lee, G. Griffiths, A. J. McKinley, C. Marx and P. A. Brennan D. S. G. Scrimgeour, J. Cleland, A. J. Lee, G. Griffiths, A. J. McKinley, C. Marx and P. A. Brennan 72 in the present study is explained by Part B of the MRCS
examination, there may be an argument for using the (cur-
rently mandatory) Part B examination as part of the selec-
tion criteria for entry into general and vascular specialty
training in the UK. Given the small effect sizes and the
moderate positive correlation found, it would seem sensible
for other candidate factors to be considered in conjunction
with Part B performance. It is also important to acknowl-
edge that the association between MRCS performance and
national selection score could be due to an inherent prob-
lem with the MRCS examination and the assessment pro-
cess for national selection. The demographic of examiners,
for example, is likely to be broadly similar; thus, the same
unconscious bias that may occur at Part B of the MRCS
examination may also apply at national selection. the national selection interview than those who passed at
their first attempt. This difference in score would have
been below previous years’ minimum appointable scores,
suggesting that candidates who require more than two
attempts at Part B are unlikely to be offered a national
training number in general or vascular surgery. Although many candidates take several attempts to pass
high-stakes medical examinations5,17, most studies have
focused on the relationship between examination scores
and future performance without distinguishing between
candidates on the basis of number of attempts needed to
pass, despite evidence of a relationship between number of
attempts and performance indicators15,18,22. © 2017 The Authors.
BJS Open published by John Wiley & Sons Ltd on behalf of BJS Society Ltd www.bjsopen.com © 2017 The Authors.
BJS Open published by John Wiley & Sons Ltd on behalf of BJS Society Ltd Discussion Although
the examination is undertaken by more international than
UK candidates, the present study focused on UK med-
ical graduates for three reasons: UK candidates are the
most homogeneous group that take the MRCS examina-
tion; the examination has been designed to assess trainees
who have been through the UK training system and who
are likely to continue their surgical training in the UK; and
there was an available outcome measure for this group as
the UK national selection process for entry into general
and vascular specialty training has been shown to be reli-
able and fair28. Future research might usefully look at the
whole population of those who sit the MRCS examination
to compare MRCS performance across different groups. Other surgical specialty selection committees could carry
out similar studies, to examine whether the MRCS exam-
ination can predict how candidates will perform in the
respective national selection interviews. The study was lim-
ited by missing data for self-declared first language (163 of Although ethnicity was an independent predictor of the
national selection score in multivariable analysis in the
present study, it accounted for only 1 per cent of the
variance in score, and nearly one-quarter of ethnicity data
were missing from the cohort. Reassuringly, the other
non-modifiable variables (sex, age and first language) had
no effect on national selection performance and together
with the ethnicity data provides further evidence that the
national selection process for general and vascular surgery
in the UK is both fair and unbiased. Examination performance is not currently used as part
of the selection criteria for entering specialty training in
the UK. In a recent survey26 of US residency programme
directors spanning all specialties, 93 per cent cited the
USMLE® Step 1 score as the most important factor in
selection. USMLE® scores have been shown to be related
to MCAT performance, with one study27 finding that 17⋅7
per cent of the variance in Step 1 of the USMLE® score was
explained by MCAT performance. Given that 17 per cent
of the variance in the national selection first attempt score BJS Open 2017; 1: 67–74 Performance in a postgraduate surgical examination and selection into specialty training 73 7 Hemphill JF. Interpreting the magnitudes of correlation
coefficients. Am Psychol 2003; 58: 78–79. 774, 21⋅1 per cent) and ethnicity (176 of 774, 22⋅7 per cent),
which reduced the total number of candidates included in
the multivariable analysis. Discussion Although the rate of missing data
is similar to that (15–20 per cent) seen in other educational
studies29, future research should aim to gather a more com-
plete data set. 8 McCaskill QE, Kirk JJ, Barata DM, Wludyka PS, Zenni EA,
Chiu TT. USMLE Step 1 scores as a significant predictor of
future board passage in pediatrics. Ambul Pediatr 2007; 7:
192–195. 9 Hojat M, Paskin DL, Callahan CA, Nasca TJ, Louis DZ,
Veloski J et al. Postgraduate training components of
postgraduate competence: analyses of thirty years of
longitudinal data. Med Educ 2007; 41: 982–989. p
This study has examined only the relationship between
MRCS examination performance and national selection
performance for general and vascular surgery. To assess the
predictive validity of the MRCS examination further it will
be necessary to analyse the relationship between MRCS
and other surgical training outcomes. Further research
should therefore focus on the relationship between MRCS
examination results and performances in clinical practice
and the postgraduate exit examination that awards the
diploma of Fellowship of the Royal College of Surgeons
(FRCS). 10 Sutton E, Richardson JD, Ziegler C, Bond J, Burke-Poole
M, McMasters KM et al. Is USMLE Step 1 score a valid
predictor of success in surgical residency? Am J Surg 2014;
208: 1029–1034. 11 Tamblyn R, Abrahamowicz M, Brailovsky C, Maison PG,
Lescop J, Norcini J et al. Association between licensing
examination scores and resource use and quality of care in
primary care practice. JAMA 1998; 280: 989–996. 12 Brailovsky C, Rainsberry P, Allen T, Slade S. Predicting
Family Medicine Specialty Certification Status Using
Standardized Measures for a Sample of International Medical
Graduates Engaged in a Practice-Ready Assessment Pathway to
Provisional Licensure. Medical Council of Canada, College of
Family Physicians of Canada and Canadian Post-MD
Education Registry document. http://mcc.ca/wp-content/
uploads/reports-predictive-validity-journal-article.pdf
[accessed 1 February 2017]. © 2017 The Authors.
BJS Open published by John Wiley & Sons Ltd on behalf of BJS Society Ltd Acknowledgements The authors acknowledge I. Targett from the Royal
College of Surgeons of England for his help with data
collection, and both L. Smith and G. Ayre from the Inter-
collegiate Committee for Basic Surgical Examinations for
their support during this project. This study was funded
by the Royal Colleges of Surgeons of England, Ireland
and Edinburgh, and the Royal College of Physicians and
Surgeons of Glasgow. 13 13 Tamblyn R, Abrahamowicz M, Dauphinee D, Wenghofer E,
Jacques A, Klass D et al. Physician scores on a national
clinical skills examination as predictors of complaints to
medical regulatory authorities. JAMA 2007; 298: 993–1001. Disclosure: The authors declare no conflict of interest. 14 Wenghofer E, Klass D, Abrahamowicz M, Dauphinee D,
Jacques A, Smee S et al. Doctor scores on national qualifying
examinations predict quality of care in future practice. Med
Educ 2009; 43: 1166–1173. References 1 Brennan PA, Scrimgeour DS, Patel S, Patel R, Grif G, Croke
DT et al. Changing Objective Structured Clinical
Examinations stations at lunchtime during all day
postgraduate surgery examinations improves examiner
morale and stress. J Surg Educ 2017; [Epub ahead of print]. 15 Wakeford R, Denney M, Ludka-Stempien K, Dacre J,
McManus IC. Cross-comparison of MRCGP & MRCP(UK)
in a database linkage study of 2284 candidates taking both
examinations: assessment of validity and differential
performance by ethnicity. BMC Med Educ 2015; 15: 1–12. 2 Brennan PA, Sherman KP. The MRCS examination – an
update on the latest facts and figures. Br J Oral Maxillofac
Surg 2014; 52: 881–883. 16 Levy JB, Mohanaruban A, Smith D. The relationship
between performance in work place based assessments,
MRCP exam and outcome from core medical training. Med
Educ 2011; 45(Suppl 2): 5. 3 Rahbour G, Hogben K. How to Succeed in ST3 General
Surgery National Recruitment; 2014. http://careers.bmj.com/
careers/advice/view-article.html?id=20016482 [accessed 4
January 2017]. 17 McManus I, Ludka K. Resitting a high-stakes postgraduate
medical examination on multiple occasions: nonlinear
multilevel modelling of performance in the MRCP(UK)
examinations. BMC Med 2012; 10: 1–19. 4 Brennan PA, Smith L, Sherman KP. ICBSE: who are we
and where are we going? Bull R Coll Surg Engl 2014; 96:
288–289. 18 Tamblyn R, Abrahamowicz M, Dauphinee WD, Hanley JA,
Norcini J, Girard N et al. Association between licensure
examination scores and practice in primary care. JAMA 2002;
288: 3019–3026. 5 Brennan PA, Smith L. Intercollegiate Committee for Basic
Surgical Examinations. 2015/16 Annual Report; 2016. http://
www.intercollegiatemrcs.org.uk/new/pdf/Annual-Report_
2015-16.pdf [accessed 2 January 2017]. 19 Norcini JJ, Boulet JR, Opalek A, Dauphinee WD. The
relationship between licensing examination performance and
the outcomes of care by international medical school
graduates. Acad Med 2014; 89: 1157–1162. 6 Pyne Y, Ben-Shlomo Y. Older doctors and progression
through specialty training in the UK: a cohort analysis of
General Medical Council data. BMJ Open 2015; 5: 1–5. BJS Open 2017; 1: 67–74 © 2017 The Authors. BJS Open published by John Wiley & Sons Ltd on behalf of BJS Society Ltd www.bjsopen.com D. S. G. Scrimgeour, J. Cleland, A. J. Lee, G. Griffiths, A. J. McKinley, C. Marx and P. A. Brennan 74 diverse medical student population. J Natl Med Assoc 2005;
97: 1258–1262. 20 Spurlock DR, Holden C, Hartranft T. © 2017 The Authors.
BJS Open published by John Wiley & Sons Ltd on behalf of BJS Society Ltd BJS Open 2017; 1: 67–74 References Using United States
Medical Licensing Examination (USMLE) examination
results to predict later in-training examination performance
among general surgery residents. J Surg Educ 2010; 67:
452–456. 25 Tiffin PA, Illing J, Kasim AS, McLachlan JC. Annual Review
of Competence Progression (ARCP) performance of doctors
who passed Professional and Linguistic Assessments Board
(PLAB) tests compared with UK medical graduates: national
data linkage study. BMJ 2014; 348: g2622. 21 Makdisi G, Takeuchi T, Rodriguez J, Rucinski J, Wise L. How we select our residents – a survey of selection criteria
in general surgery residents. J Surg Educ 2011; 68:
67–72. 26 National Resident Matching Program (NRMP), Data
Release and Research Committee. Results of the 2016 NRMP
Program Director Survey. http://www.nrmp.org/wp-content/
uploads/2016/09/NRMP-2016-Program-Director-Survey
.pdf [accessed 6 May 2017]. 22 Dewhurst NG, McManus C, Mollon J, Dacre JE, Vale AJ. Performance in the MRCP(UK) examination 2003–4:
analysis of pass rates of UK graduates in relation to
self-declared ethnicity and gender. BMC Med 2007;
5: 1–8. 27 Gauer JL, Wolff JM, Jackson JB. Do MCAT scores predict
USMLE scores? An analysis on 5 years of medical student
data. Med Educ Online 2016; 21: 1–7. 23 McDougle L, Mavis BE, Jeffe DB, Roberts NK, Thomas L,
Andriole DA. Academic and professional career outcomes of
medical school graduates who failed USMLE Step 1 on the
first attempt. Adv Health Sci Educ Theory Pract 2013; 18:
279–289. 28 Lamont P, Griffiths G, Cochrane L. National selection into
general surgery: a pilot study. Ann R Coll Surg Engl 2011; 93:
1–10. 29 Enders CK. Using the expectation maximization algorithm
to estimate coefficient alpha for scales with item-level
missing data. Psychol Methods 2003; 8: 322–337. 24 Ogunyemi D, Taylor-Harris DS. Factors that correlate with
the US medical licensure examination Step-2 scores in a BJS Open 2017; 1: 67–74 BJS Open 2017; 1: 67–74 www.bjsopen.com www.bjsopen.com Graphical Abstract The contents of this page will be used as part of the graphical abstract of HTML only. It will not be published as part of
main article. Foundation
Year 1
Foundation
Year 2
Core/specialty
surgical training
Year 1
Competitive
entry into
surgical, medical
or GP training
MRCS examination
(Parts A and B)
National
selection
interview
FRCS
Competitive
entry after
medical school
Core/specialty
surgical training
Year 2
Higher specialty
training
Year 3–7/8
Consultant
Performance in the Intercollegiate Membership of the Royal College of Surgeons (MRCS) examination predicts general and vascular
surgery national selection first attempt score. This study, the first of its kind for the MRCS, supports both MRCS validity and its predictive
validity. Performance in the Intercollegiate Membership of the Royal College of Surgeons (MRCS) examination predicts general and vascular
surgery national selection first attempt score. This study, the first of its kind for the MRCS, supports both MRCS validity and its predictive
validity.
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Longitudinal evolution of 68 Ga-Pentixafor uptake in the remote myocardium early after acute myocardial infarction and its association with left ventricular remodelling
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Longitudinal evolution of 68 Ga-Pentixafor uptake in
the remote myocardium early after acute myocardial
infarction and its association with left ventricular
remodelling
Ping Wu
Department of Nuclear Medicine, First Hospital of Shanxi Medical University; Collaborative Innovation
Center for Molecular Imaging of Precision Medicine, Shanxi Medical University
Li Xu
Department of Nuclear Medicine, First Hospital of Shanxi Medical University; Collaborative Innovation
Center for Molecular Imaging of Precision Medicine, Shanxi Medical University
Qi Wang
Department of Biochemistry and Molecular Biology, School of Basic Medicine, Shanxi Medical
University
Xiaofang Ma
Department of Nuclear Medicine, First Hospital of Shanxi Medical University
Xinzhu Wang
Department of Biochemistry and Molecular Biology, School of Basic Medicine, Shanxi Medical
University
Hongliang Wang
Department of Nuclear Medicine, First Hospital of Shanxi Medical University; Collaborative Innovation
Center for Molecular Imaging of Precision Medicine, Shanxi Medical University
Sheng He
Department of Radiology, First Hospital of Shanxi Medical University
Huibin Ru
Department of Nuclear Medicine, First Hospital of Shanxi Medical University; Collaborative Innovation
Center for Molecular Imaging of Precision Medicine, Shanxi Medical University
Yuting Zhao
Department of Nuclear Medicine, First Hospital of Shanxi Medical University
Yuxin Xiao
Department of Nuclear Medicine, First Hospital of Shanxi Medical University
Jingying Zhang
Department of Nuclear Medicine, First Hospital of Shanxi Medical University
Xinchao Wang
Collaborative Innovation Center for Molecular Imaging of Precision Medicine, Shanxi Medical University
Page 1/19
Shaohui An
Collaborative Innovation Center for Molecular Imaging of Precision Medicine, Shanxi Medical University;
Shanghai United Imaging Healthcare Co., Ltd.
Marcus Hacker
Division of Nuclear Medicine, Department of Biomedical Imaging and Image-Guided Therapy, Medical
University of Vienna
Xiang Li
Division of Nuclear Medicine, Department of Biomedical Imaging and Image-Guided Therapy, Medical
University of Vienna
Xiaoli Zhang
Laboratory for Molecular Imaging, Department of Nuclear Medicine, Beijing Anzhen Hospital, Capital
Medical University
Yuetao Wang
Department of Nuclear Medicine, The Third Affiliated Hospital of Soochow University
Minfu Yang
Department of Nuclear Medicine, Beijing Chaoyang Hospital, Capital Medical University
Zhifang Wu
Department of Nuclear Medicine, First Hospital of Shanxi Medical University; Collaborative Innovation
Center for Molecular Imaging of Precision Medicine, Shanxi Medical University
Sijin Li ( lisjnm123@163.com )
Department of Nuclear Medicine, First Hospital of Shanxi Medical University; Collaborative Innovation
Center for Molecular Imaging of Precision Medicine, Shanxi Medical University https://orcid.org/00000003-0361-1248
Research Article
Keywords: 68Ga-Pentixafor, Remote myocardium, Inflammation, Acute myocardial infarction, 18F-FDG
Posted Date: November 4th, 2022
DOI: https://doi.org/10.21203/rs.3.rs-2195805/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License.
Read Full License
Page 2/19
Abstract
Purpose
Previous studies have initially reported accompanying elevated 18F-FDG inflammatory signal in the
remote area and its prognostic value after acute myocardial infarction (AMI). Non-invasive
characterization of the accompanying inflammation in the remote myocardium may be of potency in
guiding future targeted theranostics. In this study, we sought to focus on the longitudinal evolution of
68Ga-Pentixafor signals
in the remote myocardium following AMI.
Methods Twelve AMI rats and six Sham rats serially underwent 68Ga-Pentixafor imaging at pre-operation,
and 5, 7, 14 days post-operation. Maximum and mean standard uptake value (SUV) and
target(myocardium)-to-background ratio (TBR) were assessed to indicate the uptake intensity. Gated 18FFDG imaging and immunofluorescent staining were performed to obtain cardiac function and responses
of pro-inflammatory and reparative macrophages, respectively.
Results The uptake of 68Ga-Pentixafor in the infarcted myocardium peaked on day 5 (P < 0.001), retained
at day 7 (P < 0.01), and recovered at day 14 after AMI (P > 0.05), paralleling with the rise-fall proinflammatory M1 macrophages (P < 0.05). Correlated with the peak signal in the infarct territory, 68GaPentixafor uptake in the remote myocardium on day 5 early after AMI significantly increased (AMI vs.
Sham: SUVmean, SUVmax, and TBRmean: all P < 0.05), and strongly correlated with contemporaneous
EDV and/or ESV (SUVmean and TBRmean: both P < 0.05). The transitory remote signal recovered as of
day 7 post-AMI (AMI vs. Sham: P > 0.05).
Conclusions Corresponding with the peaked 68Ga-Pentixafor signal in the infarct area, the signal in the
remote region also elevated accordingly and led to left ventricular remodelling early after AMI, which was
attributed to the early surge of pro-inflammatory response. Further studies are warranted in the remote
myocardium to clarify the post-inflammation mechanism and the prognostic value.
Introduction
Acute myocardial infarction (AMI) is one of the leading causes of morbidity and mortality worldwide [1].
Escalated inflammatory cascade response is recognized as a major injury mechanism [2]. Preclinical
studies have shown severe inflammatory response in the infarcted territory affect the remote area, which
may contribute to adverse left ventricular (LV) remodelling and heart failure [3–4]. Thus, non-invasive and
dynamic characteristic assessment of the accompanying inflammation in the remote myocardium is
essentially critical in guiding future targeted theranostics.
18F-fluorodeoxyglucose (FDG)
imaging is widely used in clinical practice for the assessment of
myocardial viability and cardiac function, when pretreated with heparin, it can also be used to assess
myocardial inflammation [5–7]. Given controversial observations of previous studies regarding 18F-FDG
Page 3/19
inflammation in the remote region and the unexpected physiological uptake in the myocardium, other
specific inflammatory molecular probes are emerging [7–8].
Most recently, limited preclinical and clinical studies indicated the value of 68Ga-Pentixafor in the infarct
myocardium predicting cardiac function following AMI, which non-invasively visualize chemokine
receptor-4 (CXCR4) expressed by a wide range of leukocyte subtypes [9–11]. However, its dynamic
evolution in the remote myocardium post AMI and the inflammatory cellular basis are yet to be elucidated
currently. In this study, we sought to spy on these questions based on serial cardiac PET scans and
histologic methods in AMI rats.
Methods
Animal model and experimental protocols
A total of twelve AMI models and six Sham models were successfully established. Male Sprague-Dawley
rats (10–13 weeks old, 280–330 g) were used. Rats were anesthetized with 2% isoflurane gas and
artificially ventilated (9 ml/kg body weight, 75–85 stroke/min) with an animal ventilator. After left
thoracotomy was performed, the left anterior descending coronary artery was permanently ligated by 7 −
0 poly suture to induce AMI models. Infarction was visually assessed by regional cardiac cyanosis. The
Sham-operated controls underwent all surgical procedures except the coronary artery ligated.
Experimental protocols are depicted in Fig. 1. All animal procedures were approved by the Animal Care
Committee of the Shanxi Medical University. All experiments were carried out in accordance with the
Guide for the Care and Use of Laboratory Animals (NIH, 8th Edition, 2011).
In Vivo Cardiac Pet Imaging
Radiosynthesis of 68Ga-Pentixafor and 18F-FDG
68
Ga-Pentixafor was synthesized by the following steps. Firstly, 68Ga was eluted from 68Ge/68Ga
generators, concentrated using a cation exchange cartridge, mixed with a solution of Pentixafor, and then
heated at 100°C (PH ~ 5) for 15 minutes. 68Ga-Pentixafor was purified using a C18 solid phase extraction
cartridge, and then removed using ethanol/water. Lastly, chemical and radiochemical purity of 68GaPentixafor was analyzed by high-performance liquid chromatograph (HPLC) system [12]. 18F-FDG was
synthesized as previously described [13].
In -vivo Imaging
68
Ga-Pentixafor and 18F-FDG PET scans were performed pre-operation and 5, 7, 14 days post-operation
on a dedicated small-animal microPET/CT scanner (Inviscan, IRIS PET/CT, France). The anesthetization
was conducted with isoflurane and remained over the entire imaging. Rats were placed supine within the
Page 4/19
scanner. Heart rate, temperature and respiration were monitored continuously. 68Ga-Pentixafor imaging
was performed without special preparation. 18F-FDG imaging was performed 12 hours after 68GaPentixafor imaging at each time-point, with rats fasted no less than 4 h. Additionally, the electrode pads
were attached to simultaneously acquire gated cardiac function parameters. 68Ga-Pentixafor (5.5 ± 1.7
MBq) or 18F-FDG (6.5 ± 2.1MBq) were administered as a 0.10 to 0.15 ml bolus via the tail vein. A 10 min
static scan was acquired at 50 min after injection. All PET images were reconstructed using the 3DOSEM/MAP algorithm.
Imaging Analysis
Two experienced physicians in cardiac PET imaging visually assessed the PET/CT images and were in
consensus for the image interpretation. Tracer accumulation were measured with the Osirix workstation
(OsiriX version 3.5.1 64-bit; OsiriX Imaging Software). Briefly, 10 ~ 15mm2 of regions of interest (ROIs)
were placed on infarcted (apical anterolateral wall) and remote (inferior wall) myocardium to obtain
mean and maximum standardized uptake value (SUVmean and SUVmax), respectively (Fig. 2). For
background assessment, SUVmean of upper liver (2.0cm2) were measured (Fig. 2C). All ROIs were drawn
on trans-axial PET images, and then checked on trans-saggital and trans-coronal sections. At least 3
layers of SUV were measured and then averaged as final. The target-to-background ratio (TBR) of 68GaPentixafor was calculated to correct the heterogenous background among rats by dividing SUV of
myocardium by SUVmean of liver. The inflammatory volume was calculated by manually sketching the
continuous area with increased signals layer by layer in OsiriX software, with a reference from the absent
area in 18F-FDG imaging. Cardiac functional analysis of left ventricular ejection fraction (LVEF), enddiastolic volume (EDV), and end-systolic volume (ESV) were obtained by PMOD software (version 4.1).
Ex-vivo Immunohistological Staining
At least three rats in AMI group and one rat in Sham group were sacrificed at each time point. The hearts
were harvested, fixed in 4% paraformaldehyde solution, and cut into 4µm paraffin sections. All sections
were stained with Hematoxylin–eosin (HE) to assess morphological changes and infarcted size. The
infarct size was determined by three different slices from the same tissue.
To illustrate PET signal, the dynamic evolution of polarized M1/M2-macrophages was semi-quantified by
immunofluorescence double staining. CD68 staining (green) was used to identify macrophages, and
further iNOS/CD206 counterstaining(red) to identify M1/M2-macrophages. DAPI staining (blue) was to
identify nuclei. Briefly, sections were deparaffinized and rehydrated through a series of xylene and graded
alcohols. Then, antigen retrieval was performed by EDTA buffer (PH = 9). After blocking (5% serum in
PBS) for 30 min at room temperature, sections were incubated overnight with the primary antibody
including mouse-anti CD68 (Invitrogen, 1:100), rat-anti iNOS (Invitrogen, 1:100), and rabbit-anti CD206
(Invitrogen, 1:100) at 4°C. The secondary antibody incubated for 1 h at room temperature with anti-mouse
Page 5/19
IgG Alexa Fluor™ 488, anti-rat IgG Alexa Fluor™ Plus 594, and anti-rabbit IgG Alexa Fluor™ Plus 555
(Invitrogen, 1:500). Immunofluorescence microscopy was used to visualize sectional images. ImageJ
software (version 1.8.0 National Institutes of Health, Bethesda, MD, USA) was used to analyze M1/M2macrophages positivity in percentage and the infarcted size.
Statistical Analysis
Statistical analysis was performed using SPSS statistics 25 (IBM Corp.). Kolmogorov–Smirnov testing
was explored to observe the data normality. Continuous variables are expressed as mean ± SD, or as
median and range. Comparisons between AMI and Sham groups at individual time points were
performed with Student’s t-test or the Mann-Whitney U-test. Relations between parameters were analyzed
using Pearson correlation analysis. A P value < 0.05 was considered statistically significant.
Results
In-vivo PET imaging
The longitudinal cardiac function by 18F-FDG imaging
For the cardiac function, as illustrated in Fig. 3 and Table 1, LVEF was significantly dropped at day 5 early
after AMI versus the Sham group (45.34 ± 8.14% vs. 64.10 ± 13.08%, P = 0.028), and deteriorated over 14
days (34.12 ± 3.87% vs. 61.88 ± 6.32%, P = 0.003). EDV progressively decreased in the AMI group, with a
significant drop at day 7 after AMI versus the Sham group (291.39 ± 63.53 µl vs. 364.27 ± 17.20 µl, P =
0.037). ESV significantly elevated in AMI group around one week after AMI (day 5: 173.03 ± 38.94 µl vs.
125.40 ± 27.94 µl, P = 0.034;day 7: 148.75 ± 21.87 µl vs. 106.83 ± 10.38 µl, P = 0.008).
Table 1
Comparison of cardiac function between groups
preOP
pOP + 5d
pOP + 7d
pOP + 14d
AMI
sham
AMI
sham
AMI
sham
AMI
sham
LVEF(%)
64.30 ±
8.77
59.90 ±
6.58
45.34 ±
8.14*
64.10 ±
13.08
47.34 ±
9.72**
70.63 ±
2.89
34.12 ±
3.87**
61.88 ±
6.32
EDV(µl)
314.47
± 35.86
323.65
± 42.99
317.09
± 65.72
361.09
± 48.28
291.39 ±
63.53*
364.27
± 17.20
242.94
± 83.12
396.31
± 85.26
ESV(µl)
108.61
± 19.65
121.17
± 6.18
173.03
± 38.94*
125.40
± 27.94
148.75 ±
21.87**
106.83
± 10.38
156.93
± 44.57
147.65
± 12.16
AMI vs Sham: * P < 0.05, ** P < 0.01, *** P < 0.001. LVEF, left ventricular ejection fraction; EDV, enddiastolic volume; ESV, end-systolic volume; preOP, pre-operation; pOP, post-operation; AMI, acute
myocardial infarction.
Page 6/19
The Longitudinal Evolution Of Ga-pentixafor Imaging
The evolution of 68Ga-Pentixafor uptake was shown in Fig. 4 and Table 2. For the infarcted myocardium,
both SUV and TBR of 68Ga-Pentixafor peaked on day 5 after AMI (AMI vs. Sham: P < 0.001), retained on
day 7 (P < 0.01), and gradually declined until 14 days (P > 0.05). The volume of the inflammatory area
was gradually reduced after 5 days after AMI. Both SUV (SUVmean: r = 0.815, P < 0.001; SUVmax: r =
0.711, P = 0.001) and TBR (TBRmean: r = 0.731, P = 0.001; TBRmax: r = 0.613, P = 0.007) in the infarct
area was positively correlated with the inflammatory volume.
Table 2
Longitudinal evolution of
preOP
Infarct
Remote
pOP + 5d
Infarct
Remote
pOP + 7d
Infarct
Remote
pOP + 14d
Infarct
Remote
68
Ga-Pentixafor uptake in the infarct and remote region
SUVmean
SUVmax
TBRmean
TBRmax
AMI
0.10 ± 0.04
0.15 ± 0.05
0.30 ± 0.10
0.47 ± 0.14
sham
0.08 ± 0.02
0.14 ± 0.04
0.21 ± 0.07
0.35 ± 0.09
AMI
0.09 ± 0.03
0.15 ± 0.06
0.27 ± 0.08
0.46 ± 0.17
sham
0.09 ± 0.03
0.15 ± 0.05
0.23 ± 0.10
0.40 ± 0.12
AMI
0.50 ± 0.07***
0.72 ± 0.13***
1.16 ± 0.36***
1.70 ± 0.59***
sham
0.08 ± 0.04
0.15 ± 0.04
0.22 ± 0.06
0.41 ± 0.07
AMI
0.14 ± 0.03*
0.20 ± 0.04*
0.32 ± 0.09*
0.47 ± 0.17
sham
0.07 ± 0.04
0.11 ± 0.04
0.20 ± 0.09
0.32 ± 0.14
AMI
0.21 ± 0.04**
0.35 ± 0.12**
0.59 ± 0.14**
0.97 ± 0.23**
sham
0.08 ± 0.05
0.12 ± 0.05
0.25 ± 0.10
0.40 ± 0.11
AMI
0.10 ± 0.04
0.13 ± 0.05
0.27 ± 0.07
0.37 ± 0.08
sham
0.08 ± 0.02
0.13 ± 0.05
0.27 ± 0.06
0.45 ± 0.17
AMI
0.17 ± 0.02
0.22 ± 0.02
0.39 ± 0.09
0.53 ± 0.18
sham
0.08 ± 0.03
0.15 ± 0.04
0.28 ± 0.03
0.51 ± 0.06
AMI
0.14 ± 0.04
0.17 ± 0.04
0.33 ± 0.11
0.40 ± 0.11
sham
0.07 ± 0.04
0.18 ± 0.07
0.23 ± 0.07
0.64 ± 0.28
AMI vs Sham: * P < 0.05, ** P < 0.01, *** P < 0.001. preOP, pre-operation; pOP, post-operation; SUV,
standard uptake value; TBR, target background ratio; AMI, acute myocardial infarction.
For the remote myocardium, significantly elevated signals of 68Ga-Pentixafor on day 5 (SUVmean: AMI
vs. Sham: 0.14 ± 0.03 vs. 0.07 ± 0.04, P = 0.005; SUVmax: AMI vs. Sham: 0.20 ± 0.04 vs. 0.11 ± 0.04, P =
0.002; TBRmean: AMI vs. Sham: 0.32 ± 0.09 vs. 0.20 ± 0.09, P = 0.041) after AMI were observed,
Page 7/19
correlating with the contemporaneous SUVmean (r = 0.688, P = 0.04) and TBRmean (r = 0.868, P = 0.002)
in the infarct territory. Signals in the remote myocardium began to gradually recover from day 7 (AMI vs.
Sham: P > 0.05) and return to baseline levels by day 14. Of note, SUVmean in the remote region of 68GaPentixafor on day 5 after AMI negatively correlated with contemporaneous EDV (r = -0.826, P = 0.006) and
ESV (r = -0.821, P = 0.007), additionally, TBRmean in the remote region was also correlated with
contemporaneous EDV (r = -0.667, P = 0.049). No correlation of the distal signal intensity was found with
the inflammation area, LVEF, as well as LV volume at other time pint (P > 0.05). No difference of 68GaPentixafor uptake was detected on serial imaging in Sham group.
Associations Between Macrophages And Ga-pentixafor Signals
HE staining were used to observe morphological changes of cardiomyocytes. AMI groups showed
disorderly myocardial fibers with myocardial cells necrosis and inflammatory cell infiltration in the
infarction region (Fig. 5). The infarcted area determined by HE staining ranged from 10–20%.
Immunofluorescence images are shown in Fig. 6A, Macrophage infiltration in the infarcted myocardium
was dramatically increased and peaked approximately at day 5 after AMI, with pro-inflammatory M1macrophages predominating (Fig. 6B). As pro-inflammatory M1-macrophages decreased, the proportion
of M2-macrophages progressively increased over 14 days (Fig. 6C).
There were strong correlations between 68Ga-Pentixafor signals and percentage of M1/M2 macrophages
(M1: P < 0.05; M2: P < 0.01). The tendency of 68Ga-Pentixafor evolution was consistent with the rise-fall
M1 macrophages (SUVmean: r = 0.87, P = 0.002; SUVmax: r = 0.921, P < 0.001; TBRmean: r = 0.78, P =
0.013; TBRmax: r = 0.829, P = 0.006). Furthermore, the inflammatory area of 68Ga-Pentixafor was
correlated with both M1 and M2 macrophages (M1: r = 0.811, P = 0.008; M2: r = -0.88, P = 0.002).
Discussion
Previous molecular imaging studies have exemplified that persistent pro-inflammatory response after
AMI can contribute to adverse LV remodelling and heart failure [4]. Our study demonstrated that the 68GaPentixafor signal in both the infarcted and remote regions elevated significantly at 5 days after AMI. In
addition, an association of the remote signals and LV remodeling was initially observed in our
experiments. Ex-vivo tissue work-up confirmed a similar evolutionary trend of 68Ga-Pentixafor signals and
pro-inflammatory M1-macrophages in the infarcted area.
The combination of CXCR4 and its ligands, alpha-chemokine CXCL12 and migration inhibitory factor
(MIF) play a pivotal role in cell migration (“homing”), recruitment, adhesion, growth and proliferation,
involving in broad-spectrum diseases like hematopoietic and solid tumors, infected and non-infected
inflammations, and et al [14]. CXCR4 is thus a promising target in the theranostics [9, 15]. More currently,
preclinical and clinical studies proved the corresponding novel PET tracer 68Ga-Pentixafor can sensitively
Page 8/19
and non-invasively visualize the expression of CXCR4 in lymphoma [16], multiple myeloma [17], and
leukemia [18]. Limited studies implicated atherosclerosis [10] and myocardial infarction [11]. Meanwhile,
the corresponding radio-targeted therapeutic probes like LY2510924, 177Lu- and 90Y-pentixather are in
active exploration [19–20].
The orchestrated recruitment transition from proinflammatory to reparative cell may mitigate AMI injury,
promote repair, and improve prognosis [2, 21], in vivo non-invasive monitoring of these processes is
important for selecting the appropriate timing and intervention protocol. The CANTOS trial and a large
body of clinical trials followed in patients with AMI have proved this [21–22]. Currently, the application of
68
Ga-Pentixafor in AMI is in the most initial stage. To the best of our knowledge, only single-digit relevant
original articles have been published currently. Studies remain pending regarding the dynamic evolution
of CXCR4-inflammatory response, clinical significance of the radioactive signal, the interaction between
organs, as well as the therapeutic guidance potentials.
Regarding the prognostic value of 68Ga-Pentixafor signal in the myocardial infarction area, there are
inconsistencies in the animal and clinical studies currently. For example, Reiter et al. revealed in their
study concluding 13 patients 2–13 days post-AMI that the higher the 68Ga-Pentixafor signal, the smaller
the scar volumes at follow-up [23]. Yet, Hess et al. demonstrated that persistently high 68Ga-Pentixafor
signals in the infarcted zone within 3 days predict a higher incidence of acute LV rupture and chronic
contractile dysfunction [9]. Preliminary intervention trials found that an opportune inflammation blocking
(early after infarction) produce a positive outcome [9], whereas blocking at an inappropriate timing
(depletion of macrophages prior to AMI) will aggravate a poor prognosis [24]. These have raised the
demand for longitudinally in vivo inflammatory characterization, the success of inflammation-targeted
therapy largely depends on the appropriate target cells and timing.
Integrated from previous results, the 68Ga-Pentixafor signals of the infarcted myocardium surged
immediately after surgery, peaked approximately 3 days, and decreased visibly after 7 days [25–26], the
persistency of 68Ga-Pentixafor signals after AMI is shorter than 18F-FDG, the latter can persist to at least
14 days after AMI [8]. The evolution of 68Ga-Pentixafor signal observed in our study was comparable to
previous results, with the following updates. Firstly, previous studies used the remote region as a
background for uptake correction of interindividual heterogeneity, however, this is probably not the
optimal choice. According to previous studies, severe inflammatory response may affect the remote area
[3] and even the kidney [25], whereas 68Ga-Pentixafor uptake in the liver is relatively stable [27]. Therefore,
in the current study, the liver was used as the site for background correction, which may objectively reflect
the evolution of inflammatory signals in the non-infarcted area. Secondly, the 68Ga-Pentixafor signals
almost recovered to baseline on day 7 post-infarction in previous studies. Yet, in our study, although the
68
Ga-Pentixafor signal in the infarct area was reduced on day 7 versus day 5 post-AMI, it was still
significantly higher than that in the Sham group, which was consistent with the ex-vivo autoradiography
results on day 7 by Hess et al. They concluded that the higher sensitivity and lack of background signal
with autoradiography result in the positive results [9]. Besides, the correction using signals in the remote
Page 9/19
area might be another speculation. Our results may further justify the correct choice of using the liver as a
correction background [25–27]. Third, previous studies have mostly set time points of 3 days, 7 days, and
6 weeks post-AMI for assessment, we set 2 extra time points at 5 days and 14 days after AMI, filling the
gap of 68Ga-Pentixafor evolution in previous studies. Regarding the clinical translation in future, the
extended time window may implicate more feasibilities in patients monitoring after AMI. Furthermore, the
back-to-baseline signal at 14 days after AMI can be considered as the end of the early inflammatory
signal after AMI in rodents, which has not been clarified in previous 68Ga-Pentixafor studies. Fourth, Hess
et al. found the early infarct CXCR4 signals at 1 or 3 days after AMI correlated well with subsequent LVEF,
and the correlation at 3 days started to get weakened [9]. It can be speculated that the correlation will
further weaken with time. The temporal bias of monitoring (relatively later) might be an explanation of
the absent correlation between 68Ga-Pentixafor signals post-operation and LVEF in the present study.
Regarding the prognostic value of remote area, previous 18F-FDG inflammatory imaging studies indicated
controversial 18F-FDG signals elevated in the distal myocardium [3, 7–8]. Wollenweber et al. detected
increased 18F-FDG uptake in the non-infarcted area, spleen and bone marrow of AMI patients [7].
However, a recent study by Xi et al. in infarcted pigs did not find a meaningful increase in 18F-FDG uptake
in the non-infarcted region [8]. In a mice and clinical study by Werner et al, 68Ga-Pentixafor signals in the
infarct and remote myocardium were both correlated with signals in the kidney, moreover, the remote
signal in AMI patients within 4 days of reperfusion was the only independent predictor of renal events
[25]. However, none of aforementioned studies focus on the dynamic evolution and predictive value of
68Ga-Pentixafor signal
in the remote region. Our study preliminarily displayed the longitudinal 68Ga-
Pentixafor evolution in the remote area after AMI, and observed the capacity of 68Ga-Pentixafor in
detecting the unobtrusively increased inflammation in the remote myocardium early after AMI, as well as
its potential value in predicting cardiac remodelling, which lays the foundation for further prognostic
studies in the future.
As for the predictive mechanisms of the signals in the remote region, firstly, spreading of the severe
inflammatory response from the infarcted area to the non-infarcted area was concerned an established
mechanism [3]. Secondly, abnormal first-pass perfusion [28] or reduced microcirculatory reserve (ie.
coronary flow reserve) [29] were also identified in the non-infarcted area following revascularization in
AMI patients. Our previous clinical observational research also identified the presence of coronary
microvascular disease (CMVD) in the non-obstructed coronary territory in patients with obstructive
coronary artery disease [30]. Furthermore, amplification of extracellular matrix, upregulated matrix
metalloproteinases, systolic dysfunction, and thinning of the ventricular wall were also observed in the
remote myocardium after AMI, all of which may be associated with poor prognosis.
Regarding the type of inflammatory cells targeted by 68Ga-Pentixafor, an in-vitro study confirmed a broad
68
Ga-Pentixafor accumulation in monocytes/macrophages and lymphocytes, and for the former,
comparable uptake was observed in M1- and M2-polarized macrophages [31]. Our in-vivo studies clarified
a strong rise-fall parallel between 68Ga-Pentixafor accumulation and infiltrated M1-macrophages in
Page 10/19
infarcted territory, despite there was also a correlation between M2-macrophages and 68Ga-Pentixafor
accumulation. Thus, here it can be speculated that pro-inflammatory M1-macrophages attributed more to
the subsequent cardiac dysfunction or adverse remodelling. Regarding the observed profitably predictive
value of 68Ga-Pentixafor in AMI patients by Reiter et al. [23], the relatively later monitoring time-frame
possibly reflected mainly reparative inflammatory cells, exactly as the significantly increased M2macrophages percent around day 7–14 after AMI we observed. The controversial correspondence
between macrophage phenotype and 68Ga-Pentixafor in-vivo and ex-vivo may attribute to multi-factors
including species characteristics, diverse assessing time point, and heterogeneity among individuals,
which warrant further studies.
Given broad spectrum of leukocytes identified by 68Ga-Pentixafor, the research for more specific
radiotracers is ongoing. So far, large amount of neuro-inflammatory research reflected 18F-GE-180 that
targets translocator protein (TSPO) as a novel better PET radiotracer than 11C-R-PK11195, which
markedly identify M1-macrophages [32]. Over recent years, novel promising tracer such as 68GamCXCL12 [33], 68Ga-DOTATATE [34], and 68Ga-DOTA-ECL1i allosterically binding to CCR2 performed a
potentially valid assessment in cardiovascular inflammation [35]. More work is needed to validate the
value of these novel radiotracers in guiding theranostics of cardiovascular disease.
Limitations
Certain noticeable limitations of our study need to be acknowledged. The first major limitation concerns
the small sample size. Given the limited animal number in the current study, conclusions should be drawn
prudently. Further verifications need to be confirmed in large samples. Second, immunohistology was not
conducted to confirm additional leukocyte subpopulations beyond M1/M2 macrophages and the
evolution of the macrophages in the remote myocardium, and our work has not been performed to
explain the prognostic mechanism, which will be replenished in future work. Third, we did not observe
long-term change of 68Ga-Pentixafor signals. For the infarcted area, Hess et al. have shown that early
68
Ga-Pentixafor signal can predict chronic contractile dysfunction at 6 weeks [9]. For the remote
myocardium, due to non-significant difference in signal between the AMI and Sham groups at day 7 and
day 14, further assessment was not necessarily conducted. Forth, we used gated 18F-FDG metabolic
imaging than the gold criteria CMRI to evaluate the cardiac function, which may trigger questioning.
However, prior comparative study has proved an excellent agreement of cardiac function between gated
18
F-FDG and CMRI [28], additionally, we eliminated the outlier values in the parameters of cardiac
function, which may compensate for the deficiency.
Conclusion
Corresponding with the peaked 68Ga-Pentixafor signal in the infarct area, the signal in the remote region
also elevated accordingly and lead to left ventricular remodelling early after AMI, which was attributed to
Page 11/19
the early surge of pro-inflammatory response. Further studies are warranted in the remote myocardium to
clarify the post-inflammation mechanism and the prognostic value.
Declarations
Acknowledgment None
Funding This work was funded by the Young Scientists Fund of the National Natural Science Foundation
of China (Grant Nos. 82001873, 81900274).
Data availability statement The dataset generated and analyzed during this study is available from the
corresponding author on reasonable request.
Author’s contributions
Ping Wu and Li Xu: image interpreting and post-process, data analyzing, and manuscript drafting; Qi
Wang, Xiaofang Ma, Xinzhu Wang and Sheng He: model construction, histology
analyzing and manuscript embellishing; Hongliang Wang, Zhifang Wu, Shaohui An and Huibin Ru:
Preparation of the tracer, image acquisition and reconstruction; Yuting Zhao, Yuxin Xiao, Jingying
Zhang and Xinchao Wang: data collection and statistics analyzing; Minfu Yang, Yuetao Wang, Xiaoli
Zhang, Marcus Hacker and Xiang Li: enhancing the intellectual content; Sijin Li: conception and design,
critically revising the manuscript.
Compliance with ethical standards
Conflict of interest The authors declare no conflict of interest.
Ethical approval All animal procedures were approved by the Animal Care Committee of the Shanxi
Medical University. All experiments were carried out in accordance with the Guide for the Care and Use of
Laboratory Animals (NIH, 8th Edition, 2011).
Consent to participate/publication All authors have agreed to participate and publish the current paper.
References
1. Virani SS, Alonso A, Aparicio HJ, et al. Heart Disease and Stroke Statistics-2021 Update: A Report
From the American Heart Association. Circulation. 2021;143(8): e254-e743.
2. Ong SB, Hernández-Reséndiz S, Crespo-Avilan GE, et al. Inflammation following acute myocardial
infarction: Multiple players, dynamic roles, and novel therapeutic opportunities. Pharmacol Ther.
2018;186:73-87.
3. Lee WW, Marinelli B, van der Laan AM, et al. PET/MRI of inflammation in myocardial infarction. J Am
Coll Cardiol. 2012;59(2):153-163.
Page 12/19
4. Westman PC, Lipinski MJ, Luger D, et al. Inflammation as a Driver of Adverse Left Ventricular
Remodeling After Acute Myocardial Infarction. J Am Coll Cardiol. 2016;67(17):2050-2060.
5. Celiker-Guler E, Ruddy TD, Wells RG. Acquisition, Processing, and Interpretation of PET 18F-FDG
Viability and Inflammation Studies. Curr Cardiol Rep. 2021;23(9):124.
6. Fischer M, Zacherl MJ, Weckbach L, et al. Cardiac 18F-FDG Positron Emission Tomography: An
Accurate Tool to Monitor In vivo Metabolic and Functional Alterations in Murine Myocardial
Infarction. Front Cardiovasc Med. 2021;8:656742.
7. Wollenweber T, Roentgen P, Schäfer A, et al. Characterizing the inflammatory tissue response to acute
myocardial infarction by clinical multimodality noninvasive imaging. Circ Cardiovasc Imaging.
2014;7(5):811-818.
8. Xi XY, Zhang F, Wang J, et al. Functional significance of post-myocardial infarction inflammation
evaluated by 18F-fluorodeoxyglucose imaging in swine model. J Nucl Cardiol. 2020;27(2):519-531.
9. Hess A, Derlin T, Koenig T, et al. Molecular imaging-guided repair after acute myocardial infarction by
targeting the chemokine receptor CXCR4. Eur Heart J. 2020;41(37):3564-3575.
10. Li X, Yu W, Wollenweber T, et al. [68Ga]Pentixafor PET/MR imaging of chemokine receptor 4
expression in the human carotid artery. Eur J Nucl Med Mol Imaging. 2019;46(8):1616-1625.
11. Thackeray JT, Derlin T, Haghikia A, et al. Molecular Imaging of the Chemokine Receptor CXCR4 After
Acute Myocardial Infarction. JACC Cardiovasc Imaging. 2015;8(12):1417-1426.
12. Pan D, Xu YP, Yang RH, et al. A new (68)Ga-labeled BBN peptide with a hydrophilic linker for GRPRtargeted tumor imaging. Amino Acids. 2014;46(6):1481-1489.
13. Krasikova R. Synthesis modules and automation in F-18 labeling. Ernst Schering Res Found
Workshop. 2007;(62):289-316.
14. Döring Y, Pawig L, Weber C, Noels H. The CXCL12/CXCR4 chemokine ligand/receptor axis in
cardiovascular disease. Front Physiol. 2014;5:212.
15. Zhou W, Guo S, Liu M, Burow ME, Wang G. Targeting CXCL12/CXCR4 Axis in Tumor Immunotherapy.
Curr Med Chem. 2019;26(17):3026-3041.
16. Chen Z, Xue Q, Huang C, Yao S, Miao W. Burkitt Lymphoma/Leukemia Presented on 68Ga-Pentixafor
and 18F-FDG PET/CT. Clin Nucl Med. 2022;47(1):98-100.
17. Pan Q, Cao X, Luo Y, Li J, Feng J, Li F. Chemokine receptor-4 targeted PET/CT with 68Ga-Pentixafor in
assessment of newly diagnosed multiple myeloma: comparison to 18F-FDG PET/CT. Eur J Nucl Med
Mol Imaging. 2020;47(3):537-546.
18. Herhaus P, Habringer S, Philipp-Abbrederis K, et al. Targeted positron emission tomography imaging
of CXCR4 expression in patients with acute myeloid leukemia. Haematologica. 2016;101(8):932-940.
19. Galsky MD, Vogelzang NJ, Conkling P, et al. A phase I trial of LY2510924, a CXCR4 peptide
antagonist, in patients with advanced cancer [published correction appears in Clin Cancer Res. 2014
Aug 15;20(16):4414]. Clin Cancer Res. 2014;20(13):3581-3588.
Page 13/19
20. Herrmann K, Schottelius M, Lapa C, et al. First-in-Human Experience of CXCR4-Directed
Endoradiotherapy with 177Lu- and 90Y-Labeled Pentixather in Advanced-Stage Multiple Myeloma
with Extensive Intra- and Extramedullary Disease. J Nucl Med. 2016;57(2):248-251.
21. Huang S, Frangogiannis NG. Anti-inflammatory therapies in myocardial infarction: failures, hopes
and challenges. Br J Pharmacol. 2018;175(9):1377-1400.
22. Ridker PM, Everett BM, Thuren T, et al. Antiinflammatory Therapy with Canakinumab for
Atherosclerotic Disease. N Engl J Med. 2017;377(12):1119-1131.
23. Reiter T, Kircher M, Schirbel A, et al. Imaging of C-X-C Motif Chemokine Receptor CXCR4 Expression
After Myocardial Infarction With [68Ga]Pentixafor-PET/CT in Correlation With Cardiac MRI. JACC
Cardiovasc Imaging. 2018;11(10):1541-1543.
24. Hess A, Borchert T, Ross TL, Bengel FM, Thackeray JT. Characterizing the transition from immune
response to tissue repair after myocardial infarction by multiparametric imaging. Basic Res Cardiol.
2022;117(1):14.
25. Werner RA, Hess A, Koenig T, et al. Molecular imaging of inflammation crosstalk along the cardiorenal axis following acute myocardial infarction. Theranostics. 2021;11(16):7984-7994.
26. Thackeray JT, Derlin T, Haghikia A, et al. Molecular Imaging of the Chemokine Receptor CXCR4 After
Acute Myocardial Infarction. JACC Cardiovasc Imaging. 2015;8(12):1417-1426.
27. Herrmann K, Lapa C, Wester HJ, et al. Biodistribution and radiation dosimetry for the chemokine
receptor CXCR4-targeting probe 68Ga-pentixafor. J Nucl Med. 2015;56(3):410-416.
28. Bethke A, Shanmuganathan L, Andersen GØ, et al. Microvascular perfusion in infarcted and remote
myocardium after successful primary PCI: angiographic and CMR findings. Eur Radiol.
2019;29(2):941-950.
29. Van Herck PL, Paelinck BP, Haine SE, et al. Impaired coronary flow reserve after a recent myocardial
infarction: correlation with infarct size and extent of microvascular obstruction. Int J Cardiol.
2013;167(2):351-356.
30. Wu P, Guo XS, Zhang X, et al. Value of absolute quantification of myocardial perfusion by PET in
detecting coronary microvascular disease in patients with non-obstructive coronaries. Zhonghua Xin
Xue Guan Bing Za Zhi. 2020;48(3):205-210.
31. Borchert T, Beitar L, Langer LBN, et al. Dissecting the target leukocyte subpopulations of clinically
relevant inflammation radiopharmaceuticals. J Nucl Cardiol. 2021;28(4):1636-1645.
32. Boutin H, Murray K, Pradillo J, et al. 18F-GE-180: a novel TSPO radiotracer compared to 11C-RPK11195 in a preclinical model of stroke. Eur J Nucl Med Mol Imaging. 2015;42(3):503-511.
33. Jones MA, MacCuaig WM, Frickenstein AN, et al. Molecular Imaging of Inflammatory Disease.
Biomedicines. 2021;9(2):152.
34. Thackeray JT, Bankstahl JP, Wang Y, et al. Targeting post-infarct inflammation by PET imaging:
comparison of (68)Ga-citrate and (68)Ga-DOTATATE with (18)F-FDG in a mouse model. Eur J Nucl
Med Mol Imaging. 2015;42(2):317-327.
Page 14/19
35. Heo GS, Kopecky B, Sultan D, et al. Molecular Imaging Visualizes Recruitment of Inflammatory
Monocytes and Macrophages to the Injured Heart. Circ Res. 2019;124(6):881-890.
Figures
Figure 1
Working flow of the study. AMI, acute myocardial infarction; preOP, pre-operation; pOP, post-operation; S,
sacrifice.
Figure 2
The delineation of ROIs. ROIs drawn for infarcted (A) and remote (B) myocardium and liver(C).
Page 15/19
Figure 3
Longitudinal evolution of LVEF post-operation. The AMI and Sham group are illustrated in orange and
blue, respectively. AMI vs Sham: * P < 0.05, ** P < 0.01, *** P < 0.001. AMI, acute myocardial infarction;
preOP, pre-operation; pOP, post-operation.
Page 16/19
Figure 4
Longitudinal evolution of 68Ga-Pentixafor uptake in the infarct(A-D) and remote(E-H) area. AMI vs sham:
* P < 0.05, ** P < 0.01, *** P < 0.001. AMI, acute myocardial infarction; preOP, pre-operation; pOP, postoperation.
Page 17/19
Figure 5
HE staining of myocardial tissue in Sham (A) and AMI (B) rats 3 days after operation.
Page 18/19
Figure 6
Longitudinal evolution of M1/M2-macrophages. (A) Typical immunofluorescent double-stained pictures
of M1/M2-macrophages in the AMI and Sham groups pre- and post-surgery. (B) Percent of M1macrophages in AMI group. (C) Percent of M2-macrophages in AMI group. iNOS+/CD68+ and
CD206+/CD68+ represent M1 and M2, respectively.
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English
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Retrieval of atmospheric CO&lt;sub&gt;2&lt;/sub&gt; vertical profiles from ground-based near-infrared spectra
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Atmospheric measurement techniques
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cc-by
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Atmos. Meas. Tech., 14, 3087–3118, 2021
https://doi.org/10.5194/amt-14-3087-2021
© Author(s) 2021. This work is distributed under
the Creative Commons Attribution 4.0 License. Atmos. Meas. Tech., 14, 3087–3118, 2021
https://doi.org/10.5194/amt-14-3087-2021
© Author(s) 2021. This work is distributed under
the Creative Commons Attribution 4.0 License. Correspondence: Sébastien Roche (sebastien.roche@mail.utoronto.ca) Correspondence: Sébastien Roche (sebastien.roche@mail.utoronto.ca) Received: 26 October 2020 – Discussion started: 13 November 2020
Revised: 7 March 2021 – Accepted: 22 March 2021 – Published: 28 April 2021 Received: 26 October 2020 – Discussion started: 13 November 2020
Revised: 7 March 2021 – Accepted: 22 March 2021 – Published: 28 April 2021 Abstract. We evaluate vertical profile retrievals of CO2 from
0.02 cm−1 resolution ground-based near-infrared solar ab-
sorption spectra with the GFIT2 algorithm, using improved
spectroscopic line lists and line shapes. With these improve-
ments, CO2 profiles were obtained from sequential retrievals
in five spectral windows with different vertical sensitivities
using synthetic and real spectra. A sensitivity study using
synthetic spectra shows that the leading source of uncertainty
in the retrieved CO2 profiles is the error in the a priori tem-
perature profile, even with 3-hourly reanalysis a priori pro-
files. A 2 ◦C error in the temperature profile in the lower tro-
posphere between 0.6 and 0.85 atm causes deviations in the
retrieved CO2 profiles that are larger than the typical verti-
cal variations of CO2. To distinguish the effect of errors in
the a priori meteorology and trace gas concentration profiles
from those in the instrument alignment and spectroscopic pa-
rameters, we retrieve CO2 profiles from atmospheric spectra
while using an a priori profile built from coincident AirCore,
radiosonde, and surface in situ measurements at the Lamont,
Oklahoma (USA), Total Carbon Column Observing Network
station. In those cases, the deviations in retrieved CO2 pro-
files are also larger than typical vertical variations of CO2,
suggesting that remaining errors in the forward model limit
the accuracy of the retrieved profiles. Implementing a tem-
perature retrieval or correction and quantifying and model- ing an imperfect instrument alignment are critical to improve
CO2 profile retrievals. Without significant advances in mod-
eling imperfect instrument alignment, and improvements in
the accuracy of the temperature profile, the CO2 profile re-
trieval with GFIT2 presents no clear advantage over scaling
retrievals for the purpose of ascertaining the total column. 1
Introduction Carbon dioxide (CO2) is the most abundant well-mixed
greenhouse gas in the atmosphere and the main driver of
the increase in global mean surface temperatures since the
start of the industrial era (Ciais et al., 2013; Myhre et al.,
2013). A yearly global carbon budget has been produced by
the Global Carbon Project since 2012 (Friedlingstein et al.,
2019; Le Quéré et al., 2013, 2014, 2015b, a, 2016, 2018b,
a). It presents current knowledge of CO2 emissions to inform
policies that aim to reduce the emissions of greenhouse gases
into the atmosphere. The project uses ensembles of mod-
els and inventories, as well as CO2 surface measurements,
to estimate different components of the global emissions of
CO2. It also uses CO2 fluxes obtained from atmospheric in-
versions (Chevallier et al., 2005; van der Laan-Luijkx et al.,
2017; Rödenbeck et al., 2003; Saeki and Patra, 2017) as a Retrieval of atmospheric CO2 vertical profiles from
ground-based near-infrared spectra Sébastien Roche1, Kimberly Strong1, Debra Wunch1, Joseph Mendonca2, Colm Sweeney3, Bianca Baier4,3,
Sébastien C. Biraud5, Joshua L. Laughner6, Geoffrey C. Toon7, and Brian J. Connor8 1Department of Physics, University of Toronto, Toronto, ON, Canada
2Climate Research Division, Environment and Climate Change Canada, Toronto, ON, Canada
3NOAA Global Monitoring Laboratory, Boulder, CO, USA
4Cooperative Institute for Research in Environmental Sciences, University of Colorado, Boulder, CO, USA
5Lawrence Berkeley National Laboratory, Berkeley, CA, USA
6Division of Geological and Planetary Sciences, California Institute of Technology, Pasadena, CA, USA
7Jet Propulsion Laboratory, California Institute of Technology, Pasadena, CA, USA
8BC Consulting Limited, Martinborough, New Zealand S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra 3088 tude levels simultaneously and therefore the retrieved profile
shape is unchanged from the a priori profile shape. Techni-
cally, GFIT handles the scaling retrieval by weakly constrain-
ing the fitted VSF factor. The approach is equivalent to per-
forming an optimal estimation of the VSF, assigning a value
of unity to the a priori VSF and a value of 106 as its expected
range of variability. XCO2 can be retrieved with a 2σ pre-
cision and accuracy of 0.8 ppm (Wunch et al., 2010). GFIT
minimizes the spectral fit residuals: the difference between
the measured and calculated spectra. The measurement noise
is not required to be accurately known; all retrievals from
TCCON CO2 windows use an assumed signal-to-noise ratio
(SNR) of ∼200. This assumption has only a small effect on
the result because for CO2 the absorption line depths and the
spectral fitting residuals far exceed the measurement noise. semi-independent validation tool for these estimates; most of
the CO2 measurements used in these inversions come from
surface networks. Since 2014, the project makes mention of
the potential of inversions using space-based measurements
of total column CO2 to provide additional constraints on
sources and sinks of CO2. Column-averaged dry-air mole fractions of CO2 (XCO2),
are retrieved from solar absorption spectra measured from
space by the Atmospheric InfraRed Sounder (AIRS, Au-
mann et al., 2003), the Greenhouse gases Observing SATel-
lite (GOSAT and GOSAT-2) (Kuze et al., 2009, 2016; Naka-
jima et al., 2012), the Orbiting Carbon Observatory (OCO-2
and OCO-3) (Crisp, 2008, 2015; Eldering et al., 2019), and
Tansat (Liu et al., 2018). CO2 fluxes obtained from inver-
sions assimilating OCO-2 observations over land are now
becoming as reliable as those obtained from inversions using
surface air sampling networks (Chevallier et al., 2019). Mea-
surements of XCO2 by satellites can be made with unprece-
dented spatial coverage. Inversions using CO2 total columns
over land are less sensitive to transport errors than inversions
using surface CO2 (Basu et al., 2018; Rayner and O’Brien,
2001), which require accurate modeling of the planetary
boundary layer height and vertical mixing, both of which
are a major source of uncertainty in inversions (Parazoo et
al., 2012). S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra However, even small (< 1 ppm) spatially coherent
biases in column measurements can have a large impact on
inversions assimilating XCO2 (Chevallier et al., 2007), and
efforts must be made to characterize and minimize such bi-
ases (Kiel et al., 2019; O’Dell et al., 2018). Even though TCCON XCO2 observations are precise and
accurate, they explicitly lack information about the vertical
distribution of CO2 in the atmosphere, which is of interest for
the validation of satellite measurements and model simula-
tions and could improve the ability of atmospheric inversions
to resolve emissions at regional scales (Keppel-Aleks et al.,
2011). The most precise and accurate sources of information
on CO2 vertical profiles are provided by air samples collected
at different altitudes using weather balloons or aircraft, but
these observations are sparse in space and time. Aircraft ver-
tical profiles are used as validation tools for inversion studies
(Peters et al., 2007; Stephens et al., 2007; Pickett-Heaps et
al., 2011), which requires them to remain independent from
the inversion systems (Chevallier et al., 2019). Obtaining re-
liable CO2 profile information from ground-based direct-sun
measurements could significantly augment the number of ob-
servations available for verification and assimilation in atmo-
spheric inversions, and would allow TCCON to be used for
validation of thermal infrared satellite products, e.g., from
AIRS and GOSAT/2, and vertically resolved NIR GOSAT
and OCO-2 experimental products. Vertical profile informa-
tion derived from ground-based absorption spectra cannot be
as accurate as aircraft-based vertical profiles, and would also
be spatially sparse, but would provide a higher temporal sam-
pling. The Total Carbon Column Observing Network (TCCON)
is a ground-based network of high-resolution (0.02 cm−1)
ground-based Fourier transform infrared (FTIR) spectrome-
ters that record shortwave IR (SWIR) solar absorption spec-
tra (Wunch et al., 2011b). TCCON produces retrievals of
XCO2 which are widely used to validate satellite observa-
tions and to study the carbon cycle (Wunch et al., 2011a,
2017; Keppel-Aleks et al., 2012, 2013). New versions of the
TCCON retrieval algorithm (GGG) are released every few
years, and each new version is designed to improve the qual-
ity of the data. https://doi.org/10.5194/amt-14-3087-2021 Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra GFIT2 was first developed using the
GGG2014 version of the GGG suite (Wunch et al., 2015),
which uses a Voigt line shape to compute absorption coef-
ficients. In this study, we use the GGG2020 version, which
will be released in early 2021. This version of the code im-
plements quadratic speed-dependent Voigt line shapes with
line mixing (qSDV+LM) for CO2 (Mendonca et al., 2016)
and CH4 (Mendonca et al., 2017) bands, and qSDV line
shapes for O2 in the band centered at 1.27 µm (Mendonca et
al., 2019). The line mixing coefficients are derived with the
first-order Rosenkranz approximation (Rosenkranz, 1975). This leads to significantly better spectral fits, especially in
the strong CO2 band centered at 2.06 µm, and smaller vari-
ations of gas amount with air mass. Other improvements to
the forward model include (1) updates to the spectroscopic
line list (Toon, 2015) (2) a solar-gas stretch fitted to ac-
count for Doppler-driven differences between solar and tel-
luric wavenumber scales (in GGG2014 only the stretch in
the telluric wavenumber scale was fitted), and (3) improved
a priori profiles as described in Sect. 2.2. Figure 1. Contributions of different absorbing gases to the calcu-
lated transmittance spectrum on a dry winter day at a solar zenith
angle of 60.6◦for each of the spectral windows used to retrieve
CO2. the window centered at 7885 cm−1; see Table 1) divided by
0.2095 (Wunch et al., 2011b). OCO-2/3 and GOSAT/2 use
two windows comparable to the TCCON1 and Strong win-
dows to retrieve CO2 and use the O2 A-band (centered near
13158 cm−1). This study assesses the quality of CO2 profile retrievals
with GFIT2 implemented in GGG2020. Section 2 describes
the retrieval algorithm and our methodology. Section 3
presents a sensitivity study using synthetic spectra, followed
by retrievals using real measured spectra. Finally, Sect. 4
presents a summary of the results and conclusions. A qualitative representation of the vertical sensitivity
due to the range of different line opacities is presented
in Fig. 2, which shows the normalized CO2 Jacobian
for typical absorption lines in the Strong window (cen-
tered at 4852.87 cm−1), the “Weak1” window (centered
at 6074 cm−1), and the TCCON1 window (centered at
6220 cm−1). S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra CO2 profile retrievals from ground-based SWIR spectra
have been calculated using the band centered at 1.6 µm with a
Voigt line shape (Kuai et al., 2012), and in the band centered
at 2.06 µm with the PROFFIT optimal estimation software
package (Hase et al., 2004) fitted with a Voigt line shape with
line mixing (Dohe, 2013). In our approach, we use the GFIT2
software package initially described by Connor et al. (2016),
which is a profile retrieval algorithm based on the GGG soft-
ware suite, but modified such that it allows the profile shape
to vary during the retrieval process. Instead of retrieving a
single VSF value that scales the whole a priori profile, a VSF
value is retrieved for each atmospheric level. The algorithm
thus has much more freedom to fit the observed spectra but
is also more sensitive to uncertainties in the forward model
calculations such as errors in the atmospheric temperature GGG2014 (Wunch et al., 2015) is the current version of
the GGG software used by TCCON to transform measured
interferograms into spectra, and then to retrieve trace gas
mixing ratios from those spectra. Central to this process is
GFIT, a non-linear least-squares spectral fitting algorithm. A forward model computes an atmospheric transmittance
spectrum for a given observation geometry using a priori
knowledge of atmospheric conditions and assuming a per-
fectly aligned instrument. An inverse method then compares
the measured spectrum with the resulting calculation and ad-
justs the retrieved parameters to obtain the best fit. In GFIT,
these parameters include volume mixing ratio scaling fac-
tors (VSFs) for the different fitted gases. GFIT performs pro-
file scaling retrievals: for each retrieved trace gas, a single
VSF scales the entire a priori concentration profile at all alti- https://doi.org/10.5194/amt-14-3087-2021 Atmos. Meas. Tech., 14, 3087–3118, 2021 S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra 3089 Figure 1. Contributions of different absorbing gases to the calcu-
lated transmittance spectrum on a dry winter day at a solar zenith
angle of 60.6◦for each of the spectral windows used to retrieve
CO2. profile, spectroscopic errors, and instrument misalignment,
for example. Connor et al. (2016) showed that CO2 profile retrievals
in the CO2 band centered at 1.6 µm are very sensitive to er-
rors in spectroscopy. 2
Methods In this study, GFIT2 is used to retrieve CO2 profiles from the
two original TCCON retrieval windows and three new win-
dows that possess a large range of opacities, and therefore
vertical sensitivities. These windows are presented in Table 1
and Fig. 1. The TCCON1 window (centered at 6220 cm−1)
and TCCON2 window (centered at 6339.5 cm−1) are used to
derive XCO2 in the public TCCON data products, because
the spectral absorption line opacities are close to 1 and are
therefore equally sensitive at most altitudes. The CO2 line
intensities in the weak windows are 10 times smaller than
in the standard TCCON windows, providing more sensitiv-
ity to CO2 variations aloft. The CO2 lines in the “Strong”
window are 15 times stronger than those in the standard TC-
CON windows, providing more sensitivity to CO2 variations
near the surface. All windows have an average lower-state
energy (E′′) of roughly 240 cm−1, rendering the retrieved to-
tal column of CO2 highly independent of the assumed tem-
perature (< 0.1 % K−1). The derivation of XCO2 as calcu-
lated in GGG is described in Appendix A. XCO2 is the ratio
of the CO2 column to the column of dry air, and the col-
umn of dry air is expressed as the retrieved O2 column (from S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra The strong saturated lines of the Strong window
are more sensitive to levels below 5 km than in the TCCON1
window, but the Strong window also contains lines of inter-
mediate absorption strength that provide more uniform sensi-
tivity up to ∼10 km, and that extend the window’s sensitivity
to up to 30–40 km. The saturated lines in the Strong win-
dow correspond to the 20013–00001 band, while the lines
of intermediate strength around 4820 cm−1 come from the
R branch of the 21113–01101 band. The TCCON1 window
has more uniform sensitivity up to ∼10–15 km and contains
weak lines which contain information on CO2 above 15 km. The Weak1 window is less sensitive below 10 km and has
more uniform sensitivity between 10–20 km. Figure 2 also
shows little to no sensitivity to levels above ∼30 km in all
windows. 2.1
Retrieval algorithm The GFIT2 retrieval algorithm is described in detail in Ap-
pendix B and follows the formulation of Rodgers (2000). Currently, GGG has no option to simultaneously retrieve in-
formation about a gas from spectral windows that are not
contiguous in wavenumber. Therefore, we retrieve trace gas
information from each window separately. We see no ad- https://doi.org/10.5194/amt-14-3087-2021 Atmos. Meas. Tech., 14, 3087–3118, 2021 S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra 3090 Table 1. CO2 spectral windows used with GFIT2. Interfering absorbers labeled “solar” are due to absorption by heavy metal ions (e.g., Fe,
Si, Ca, Ni) in the solar photosphere. Also shown are the strength-weighted averages of the lower-state energy (E′′) and of the line strengths
(S) over all the CO2 lines in each window. The column of O2, retrieved with scaling retrievals from the O2 window, is used to compute
XCO2. Table 1. CO2 spectral windows used with GFIT2. Interfering absorbers labeled “solar” are due to absorption by heavy metal ions (e.g., Fe,
Si, Ca, Ni) in the solar photosphere. Also shown are the strength-weighted averages of the lower-state energy (E′′) and of the line strengths
(S) over all the CO2 lines in each window. The column of O2, retrieved with scaling retrievals from the O2 window, is used to compute
XCO2. Window
Center
Center
Width
Primary interfering
E′′
S
name
(µm)
(cm−1)
(cm−1)
absorbers
(cm−1)
(cm−1 (molecule cm−2)−1)
×10−23
TCCON1
1.61
6220
80
solar, H2O
245.3
1.14
TCCON2
1.58
6339.5
85
solar, H2O
254.6
1.14
Weak1
1.65
6074
70.8
CH4, solar, H2O
223.5
0.118
Weak2
1.54
6499.1
69.8
solar, H2O, HDO
229.3
0.130
Strong
2.06
4852.87
86.26
H2O, 13CO2, solar
243.8
17.8
O2
1.27
7885
240
solar, H2O, HF, CO2
203.4
0.00518
2. CO2 absorption lines (black line) overlaid on heat maps of the CO2 Jacobian for lines of (a) the Strong window, (b) the Weak1
and (c) the TCCON1 window. The color bar represents the normalized Jacobian where 1 corresponds to the maximum amongst all
Jacobians from the five CO2 windows. Lines of the Weak2 and TCCON2 windows are not shown as they look like the Weak1 and
1 windows, respectively. Figure 2. CO2 absorption lines (black line) overlaid on heat maps of the CO2 Jacobian for lines of (a) the Strong window, (b) the Weak1
window, and (c) the TCCON1 window. The color bar represents the normalized Jacobian where 1 corresponds to the maximum amongst all
the CO2 Jacobians from the five CO2 windows. Lines of the Weak2 and TCCON2 windows are not shown as they look like the Weak1 and
TCCON1 windows, respectively. species profiles (non-12C16O2 species included in Fig. 1, ex-
cept for “solar” and “other”; other CO2 isotopologues are
only retrieved as interfering species in the Strong window). https://doi.org/10.5194/amt-14-3087-2021 S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra etrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra Table 2. Components of the state vector in GFIT2 profile retrievals. These are all the retrieved parameters. Table 3. AirCore launch dates and number of coincident spectra
within ±1 h of the AirCore last sampling time and within ±1.5 h of
the closest a priori time. The range of solar zenith angles covered
by the coincident spectra is also shown. Launch date
Coincident
Solar zenith
spectra
angles (◦)
14 January 2012
65
60.6–73.8
15 January 2012
48
65.6–77.9
23 July 2013
44
20.8–36.5
26 February 2014
61
46.6–59.0
27 February 2014
41
46.2–53.3
17 September 2014
48
37.9–51.1
19 October 2016
31
47.1–50.3
11 April 2017
33
31.2–39.2 of the mass of the atmosphere. In Sect. 3.2, AirCore profiles
from the v20181101 dataset were used as “truth” to assess
the quality of GFIT2 profile retrievals. We used all AirCore
profiles measured over the Lamont TCCON station that had
coincident ground-based measurements within ±1 h of the
AirCore landing and within ±1.5 h of the closest a priori
time. All figures showing profiles use the average of profiles
retrieved from the coincident spectra. The launch dates of
the eight AirCore profiles used are presented in Table 3. An
iMet-1 radiosonde carried by the same balloon as the Air-
Core provides in situ temperature and relative humidity pro-
files. luric wavenumber scales. A zero-level offset is also retrieved
in the Strong window that makes use of saturated lines. In principle, a CO2 profile retrieval should have little sen-
sitivity to errors in the a priori CO2 profile (differences from
the true profile) since it can adjust for differences between
measured and calculated spectra caused by erroneous prior
profile shapes (Connor et al., 2016). However, the retrieval
may also conflate errors due to other sources, such as incor-
rect spectroscopic parameters, incorrect modeling of the in-
strument line shape, or errors in the a priori meteorology and
profiles of interfering species, with these errors in the a priori
CO2 profile. Instead of the diagonal prior covariance used in Sect. 3.1,
a more realistic CO2 prior covariance matrix was built for re-
trievals with real spectra in Sect. 3.2. S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra The difference between
GGG2020 a priori CO2 profiles and aircraft profiles (Biraud
et al., 2013) over Lamont from NOAA’s ObsPack (Sweeney
et al., 2017) between 500 and 5000 m were computed for 382
aircraft profiles and for each month between 2008 and 2016. The mean difference profile plus 1 standard deviation of the
month with the largest differences, August, was used to build
the diagonal of the a priori covariance matrix. The a priori
CO2 uncertainty can be expressed as S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra Aside from the retrieved gases, other fitted parameters are
part of the state vector. Orthogonal continuum basis func-
tions are used to fit the shape of a spectrum’s continuum, with
different orders of curvature. An overall frequency stretch is
retrieved for all lines, and a second “solar-gas” stretch is re-
trieved to correct for differences between the solar and tel- species profiles (non-12C16O2 species included in Fig. 1, ex-
cept for “solar” and “other”; other CO2 isotopologues are
only retrieved as interfering species in the Strong window). Aside from the retrieved gases, other fitted parameters are
part of the state vector. Orthogonal continuum basis func-
tions are used to fit the shape of a spectrum’s continuum, with
different orders of curvature. An overall frequency stretch is
retrieved for all lines, and a second “solar-gas” stretch is re-
trieved to correct for differences between the solar and tel- vantage to fitting non-contiguous windows simultaneously,
rather than separately, and then combining the results. In TC-
CON post-processing, the total columns retrieved from dif-
ferent retrieval windows (CO2 from the TCCON1 and TC-
CON2 windows, for example) are averaged after removing
window-dependent multiplicative biases, using retrieval er-
rors as weights. Table 2 summarizes the components of the
state vector used in GFIT2. A total of 51 VSFs are retrieved
(one for each atmospheric level) for the primary target gas,
while only 1 VSF is retrieved for each of the interfering https://doi.org/10.5194/amt-14-3087-2021 Atmos. Meas. Tech., 14, 3087–3118, 2021 S. Roche et al.: Retrieval of atmospheric CO2 vertical pro
Table 2. Components of the state vector in GFIT2 profile retrievals. These are all the retrieved parameters. State vector parameter
Number of elements
Main target gas (CO2)
51 (number of atmospheric
levels)
Interfering species
3–6 (scaling retrievals)
Continuum basis functions
N (5 in the Strong window, 3
in the other windows)
Continuum level
1
Continuum tilt
1
Continuum curvature
N −2
Frequency stretch
1
Solar-gas stretch
1
Zero-level offset
0 (1 in the Strong window) 3091 S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra
Table 2 Components of the state vector in GFIT2 profile retrievals
Table 3 AirCore launch dates and number of coinc DOFs = tr(A). DOFs = tr(A). DOFs = tr(A). The DOFs can be divided into the CO2 profile DOFs and
the DOFs corresponding to the rest of the state vector ele-
ments. The profile DOFs can be interpreted as the number of
independent pieces of information that improve the retrieved
CO2 profiles compared to the a priori profile. The DOFs are
shown in Figs. 4–7 and 9–10. Since the AirCore profiles do not extend down to the sur-
face or above about 25 km, other sources are used to com-
plete the “true” CO2 profile. The TCCON spectrometer used
in this study is located at the US Department of Energy At-
mospheric Radiation Measurement program (ARM) central
facility in Lamont, Oklahoma. The facility hosts a suite of in-
struments for remote and in situ measurements of the atmo-
sphere. When available within 5 h of the last AirCore sam-
pling time, surface CO2 and CH4 measurements from preci-
sion gas systems were used (Biraud and Moyes, 2001). When
they were not available, measurements from discrete flask
samples were used (on 23 July 2013, 27 February 2014, and
17 September 2014) (Biraud et al., 2002). Surface pressure,
temperature, and relative humidity were obtained from in situ
measurements at the Lamont central facility. S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra 3092 Figure 3. A priori uncertainty profiles for each of the 10 dates pre-
sented in Table 3. These are defined by Eq. (1). Since σ is defined
on an altitude grid, it varies slightly with pressure. obtained from an age climatology derived from a chemical
Lagrangian model (McKenna, 2002) of the stratosphere us-
ing equivalent latitude to account for air motion. The strato-
spheric priors also account for turbulent mixing with age
spectra (Andrews et al., 2001). A seasonal cycle parametriza-
tion is then applied, and the resulting CO2 profiles are cor-
rected to match the CO2 latitudinal gradients observed by the
High-Performance Instrumented Airborne Platform for En-
vironmental Research (HIAPER) Pole-to-Pole Observations
(HIPPO) (Wofsy, 2011) and by the Atmospheric Tomogra-
phy (ATom) mission (Wofsy et al., 2018). obtained from an age climatology derived from a chemical
Lagrangian model (McKenna, 2002) of the stratosphere us-
ing equivalent latitude to account for air motion. The strato-
spheric priors also account for turbulent mixing with age
spectra (Andrews et al., 2001). A seasonal cycle parametriza-
tion is then applied, and the resulting CO2 profiles are cor-
rected to match the CO2 latitudinal gradients observed by the
High-Performance Instrumented Airborne Platform for En-
vironmental Research (HIAPER) Pole-to-Pole Observations
(HIPPO) (Wofsy, 2011) and by the Atmospheric Tomogra-
phy (ATom) mission (Wofsy et al., 2018). 2.2
Datasets CO2 and CH4 a priori profiles were built by combining the
balloon-borne AirCore (Karion et al., 2010) profiles with sur-
face in situ measurements, adding the GGG2020 a priori pro-
file above the maximum altitude sampled by AirCore. These
composite profiles will be referred to as “truth”. The CH4
profile is included because CH4 is an interfering gas in the
Weak1 window. AirCore is a sampling system that consists
of a long, coiled stainless-steel tube initially filled with a dry
calibrated gas. As a balloon carries it up, the fill gas evacu-
ates. When the AirCore descends from the stratosphere, am-
bient air enters the tube through the open end. Upon landing,
the AirCore is quickly retrieved for subsequent laboratory
analysis, wherein the sample is pushed through a continu-
ous gas analyzer. The first gases to come out were the last
to enter, and vice versa, allowing the preserved atmospheric
trace gas concentration profiles to be derived. This method
has precision similar to, or better than, discrete gas flask sam-
ples, with a repeatability of 0.07 ppm for CO2 concentrations
(Karion et al., 2010). The balloons reach ∼30 km altitude,
with profiles retrieved to ∼25km, and therefore sample 98 % σ i = 3.99e−0.92xi + 0.98,
(1) (1) where x is the altitude of the ith atmospheric level in kilome-
ters. The a priori covariance is expressed as (2) zi,j = xi,
(2)
(1z)i,j =
zi,j −zT
i,j
,
(3)
(Sa)i,j =
σ T σ
i,j × e−
(1z)i,j
h
,
(4) (3) (4) where z is a matrix with each row containing the altitude pro-
file, 1z is the matrix of absolute altitude differences between
each level, Sa is the a priori covariance matrix, and h is the
length scale of interlayer correlations. The length scale was https://doi.org/10.5194/amt-14-3087-2021 Atmos. Meas. Tech., 14, 3087–3118, 2021 S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra 2.3
Information content and degrees of freedom Figure 3. A priori uncertainty profiles for each of the 10 dates pre-
sented in Table 3. These are defined by Eq. (1). Since σ is defined
on an altitude grid, it varies slightly with pressure. The information content in the profile retrieval can be quan-
tified using the averaging kernel matrix A (Rodgers, 2000). The information content H is defined as H = −1
2 ln(|I −A|),
(5) (5) set to 2 km based on the width of the rows of correlation ma-
trices built from the ensemble of aircraft vertical profiles. where “ln” is the natural logarithm and |I −A| is the deter-
minant of the difference between the identity matrix and the
averaging kernel matrix. The degrees of freedom for signal
(DOFs) can be expressed as where “ln” is the natural logarithm and |I −A| is the deter-
minant of the difference between the identity matrix and the
averaging kernel matrix. The degrees of freedom for signal
(DOFs) can be expressed as The vertical grid used in the retrievals presented in this
study has 51 levels between 0 and 70 km, and the spacing be-
tween levels increases with altitude (see Appendix A). Fig-
ure 3 shows the a priori uncertainty as a function of pressure
for each of the eight a priori states used to process the Lam-
ont spectra presented in Table 3. DOFs = tr(A). (6) (6) 3.1.1
Perturbed CO2 profile With a perturbed CO2 prior profile, the algorithm can retrieve
the true profile shape very well in all windows, even with an
a priori profile vastly different from the truth as shown in
Fig. 4. In Fig. 4a, when using the same prior that generated
the synthetic spectrum, the retrieved profiles do not align ex-
actly with the prior profile. This is due to small imperfections
in the synthetic spectra, but these result in differences of less
than 1 ppm at any altitude. In Fig. 4c the standard GGG2020
a priori profile is used as the a priori profile, while the “true”
CO2 profile used to generate the synthetic spectrum was built
from a composite “true” profile as described in Sect. 2.2. In each window the retrieved profile is within 2 ppm of the
truth. In Fig. 4e a constant CO2 profile with 380 ppm at all
levels is used as the a priori profile. Again, the retrieved pro-
files are within 2 ppm of the truth except at the bottom and
top of the profile where most of the information comes from
the a priori profile. This self-consistency test shows that the
GFIT2 algorithm works as expected and can accurately re-
trieve CO2 when the a priori CO2 profile is the only source
of uncertainty. 3.1.2
Perturbed H2O profile Over the course of a day, the water vapor profile can vary
by 40 % and the temperature profile can vary by more than
10 ◦C in the lowest troposphere, and therefore 3-hourly a
priori meteorological information could differ from the true
atmospheric state by several degrees Celsius for tempera-
ture and by 10 % for water vapor. In Sect. 3.1.2, we perturb
the a priori H2O profile, the main interfering absorber. In
Sect. 3.1.3, we perturb the temperature profile, as the inten-
sity and width of all absorption lines depend on temperature. Finally, in Sect. 3.1.3 we perturb spectroscopic line parame-
ters themselves to within their uncertainties. Figure 5 shows the effect of a +10 % perturbation to the H2O
vapor profile below 5 km for a dry winter day and a wet sum-
mer day. It leads to 2 ppm deviations from the CO2 a priori
profile in the Strong window under dry conditions and up to
15 ppm under wet conditions. In both cases, the deviations
from the truth in the CO2 profiles retrieved from the other
windows were within 2 ppm. S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra 3093 S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra in that parameter will dominate the variability in the retrieved
CO2 profiles regardless of the retrieval constraints. in Lamont, Oklahoma (USA). If the forward model were per-
fect and the a priori state equal to the true state of the atmo-
sphere, the retrieved scale factor at each level would be equal
to 1. However, errors in the forward model (including spec-
troscopy, a priori meteorological information, radiative trans-
fer, and instrument line shape) cause the retrieved scale fac-
tors to deviate from 1. To isolate the effect of instrument mis-
alignment and errors in spectroscopic parameters from errors
in a priori meteorology, we build a priori profiles of H2O,
temperature, CO2, and CH4 using in situ measurements. In
Sect. 3.2 we also use an a priori covariance matrix with off-
diagonal elements based on comparisons between the a priori
profile and aircraft profiles, as described in Sect. 2.2. 3.1
Synthetic spectra In this section, we attempt to identify the main sources of
error in the retrieved CO2 profiles. To do this we use syn-
thetic spectra that are calculated with GFIT’s forward model
for a given set of inputs (atmospheric conditions and spec-
troscopic parameters). These “perfect” synthetic spectra are
then used as measurements to be fitted in retrievals with one
perturbed input. Thus, when the perturbed input is not the
a priori CO2 profile itself, the a priori CO2 profile is the
“truth”. In Sect. 3.1.1, we look at the ability of the retrieval
algorithm to retrieve CO2 when it is the only unknown. 3
Results In Sect. 3.1, we investigate the sensitivity of the profile re-
trievals to different sources of error using synthetic spectra
produced by running the GGG forward model with a given
set of atmospheric conditions. The resulting spectra were
then used as input to the profile retrieval algorithm using
the same set of atmospheric conditions, except for a pertur-
bation in either the CO2, temperature, or H2O profiles, or
in the spectroscopic parameters of CO2 lines (air- and self-
broadened half-width coefficients and their temperature de-
pendence). In these retrievals, the SNR of the spectrum to
be fitted is set to 1000 and the CO2 a priori covariance ma-
trix is diagonal with 5 % (∼20 ppm) uncertainty at all levels. No noise is added to the calculated spectra, but the assumed
1000 : 1 SNR is used to build the measurement covariance
matrix and affects the relative weight of the measurement and
the a priori profile. The weak prior constraint and high SNR
serve to highlight the sources of variability in the retrieved
profiles. GGG2020 uses 3-hourly a priori profiles of the atmo-
spheric state. For each spectrum in the retrievals, GGG uses
the nearest a priori profile in time. The a priori meteorol-
ogy and H2O profiles are obtained from analyses of the
Global Modeling and Assimilation Office (GMAO) God-
dard Earth Observing System Version 5 Forward Process-
ing for Instrument Teams (GEOS5-FPIT) (Lucchesi, 2015). The CO2 a priori profiles are constructed from the desea-
sonalized NOAA Mauna Loa and Samoa flask data (Dlugo-
kencky et al., 2019) by determining the transport lag between
the measurement site and each level of the a priori profile
(Laughner et al., 2021a, b). In the troposphere, this is done
with an age-of-air formula and an effective latitude that ac-
counts for synoptic motion of air. In the stratosphere, this is In Sect. 3.2, CO2 profile retrievals are tested with atmo-
spheric solar absorption spectra measured at the TCCON site Atmos. Meas. Tech., 14, 3087–3118, 2021 https://doi.org/10.5194/amt-14-3087-2021 3.1.4
Perturbed line parameters Figure 4. The left-hand panels show CO2 profiles retrieved using
synthetic spectra. In (a), we use the AirCore profile, which was
used to generate the synthetic spectra, as the a priori profile. In (c),
we use the GGG2020 a priori CO2 profile as the a priori profile. In (e), we use a constant CO2 a priori profile. Panels (b), (d), and
(f) show the difference between the retrieved profiles and AirCore,
corresponding to (a), (c), and (e) respectively. The line list used by GGG is a compilation of different ver-
sions of the HITRAN line lists (Gordon et al., 2017; Roth-
man et al., 2005, 2009, 2013; Toon, 2015; Toon et al., 2016). GGG2020 has the option to use either the qSDV+LM line
shape or the Voigt line shape for some windows and gases
(Mendonca et al., 2016, 2017, 2019). The reference line lists
and the uncertainties on air- and self-broadened Lorentz half-
width coefficients, as well as their temperature dependence,
are summarized in Table 4. The qSDV+LM line shape is only
implemented for the CO2 lines of the two TCCON windows
and the Strong window, for the CH4 lines of the Weak1 win-
dow, and for the O2 lines of the oxygen window centered at
7885 cm−1. The qSDV+LM line shape is not implemented
for the CO2 lines of the Weak1 and Weak2 windows, but
these weak lines are minimally affected by line mixing, and
they lack laboratory measurements of speed-dependent line
parameters. The effect of errors in the half-width coefficients
on the retrieved CO2 profiles was tested by increasing both
the self- and air-broadened Lorentz half-width coefficients
by 0.1 % for all CO2 lines as shown in Fig. 7a. This per-
turbation corresponds to the median uncertainty of these pa-
rameters in the Strong and TCCON windows as shown in
Table 4. This caused deviations of up to 10 ppm in the Strong
window, 5 ppm in the TCCON windows, and 2 ppm in the
Weak windows. Similar deviations are obtained by perturb-
ing the temperature dependence of the half-width coefficients
by −1 % as shown in Fig. 7b. In this case, the deviations ap-
pear symmetric with the a priori profile compared to Fig. 7a. The shape of deviations in both cases is similar; it is also amplitude amongst the five windows, within 4 ppm, and the
deviation at ∼0.2 atm is ∼20 ppm. S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra 3094 p
g
p
Figure 5. The left-hand panels show CO2 profiles retrieved using
synthetic spectra. 10 % is added to the H2O profile below 5 km for
(a) dry conditions on 14 January 2012 and for (c) wet conditions on
23 July 2013. Panels (b) and (d) show the difference between the
retrieved profiles and AirCore, corresponding to (a) and (c) respec-
tively. Figure 4. The left-hand panels show CO2 profiles retrieved using
synthetic spectra. In (a), we use the AirCore profile, which was
used to generate the synthetic spectra, as the a priori profile. In (c),
we use the GGG2020 a priori CO2 profile as the a priori profile. In (e), we use a constant CO2 a priori profile. Panels (b), (d), and
(f) show the difference between the retrieved profiles and AirCore,
corresponding to (a), (c), and (e) respectively. Figure 5. The left-hand panels show CO2 profiles retrieved using Figure 5. The left-hand panels show CO2 profiles retrieved using
synthetic spectra. 10 % is added to the H2O profile below 5 km for
(a) dry conditions on 14 January 2012 and for (c) wet conditions on
23 July 2013. Panels (b) and (d) show the difference between the
retrieved profiles and AirCore, corresponding to (a) and (c) respec-
tively. Figure 5. The left-hand panels show CO2 profiles retrieved using
synthetic spectra. 10 % is added to the H2O profile below 5 km for
(a) dry conditions on 14 January 2012 and for (c) wet conditions on
23 July 2013. Panels (b) and (d) show the difference between the
retrieved profiles and AirCore, corresponding to (a) and (c) respec-
tively. 3.1.3
Perturbed temperature profile A +5 ◦C perturbation to the temperature profile below 5 km
(0.5 atm < P < 1.0 atm), as in Fig. 6a, leads to deviations
from the truth in the retrieved CO2 profiles of up to 50 ppm
in the Weak and TCCON windows, and up to 100 ppm in
the Strong window. In that case the fit residuals can ex-
ceed 1 % in the Strong window and 0.5 % in the TCCON
windows. For the retrievals used to obtain the profiles in
Fig. 6a the SNR was set to 100 in the Strong window, 200
in the TCCON windows, and 1000 in the Weak windows. In Fig. 6c and e the SNR is set to 1000 in all windows. In Fig. 6c, a +2 ◦C perturbation is applied between 8 and
13 km (0.2 atm < P < 0.35 atm). The amplitude of deviations
in the TCCON windows and in the Strong window is close
to 50 ppm at ∼0.9 atm and 100 ppm at ∼0.2 atm. In the
two Weak windows, the deviation amplitude is ∼10 ppm at
∼0.9 atm and ∼20 ppm at 0.2 atm. In Fig. 6e, a +2 ◦C per-
turbation is applied above 15 km. In the Strong window, the
resulting deviation at pressures > 0.6 atm has the smallest The total retrieval random error for the retrievals presented
in this section is ∼4.5 % (∼18 ppm), and the contribution
of random noise is ∼0.8 % (∼3 ppm); see Appendix D for
definitions of total and measurement noise errors. When the
deviations from the truth are larger than the a priori uncer-
tainty (∼20 ppm), it means the perturbation applied has a
severe effect on the retrieval. Of course this can be mitigated
by using a stronger a priori constraint or a measurement co-
variance matrix that reflects expected systematic errors, and
not just random noise, but always at the cost of reduced sen-
sitivity to CO2 too. The goal here is to estimate the relative
effect of different kinds of expected systematic errors on re-
trieved profile shapes. Stronger constraints can only reduce
the amplitude of the deviations from the truth, but the same
structures would remain. When the perturbation to a param-
eter other than CO2 results in deviations from the truth much
larger than those presented in Sect. 3.1.1, it means that errors https://doi.org/10.5194/amt-14-3087-2021 Atmos. Meas. Tech., 14, 3087–3118, 2021 Atmos. Meas. Tech., 14, 3087–3118, 2021 S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra The left-hand panels show CO2 profiles retrieved using
synthetic spectra for (a) +5 ◦C added to the a priori temperature
profile below 5 km, (c) +2 ◦C between 8 and 13 km, and (e) +2 ◦C
above 15 km. Panels (b), (d), and (f) show the difference between
the retrieved profiles and AirCore profile, corresponding to (a), (c),
and (e) respectively. Note the difference in the horizontal axis range
between the panels. Here 5 km corresponds to ∼0.55 atm, 8–13 km
to ∼0.36–0.17 atm and 15 km to ∼0.125 atm. Figure 7. The left-hand panels show CO2 profiles retrieved using
synthetic spectra. In (a) the air- and self-broadened half-width co-
efficients of all CO2 lines is increased by 0.1 %. In (c) the temper-
ature dependence of these coefficients is decreased by 1 %. In (e),
the synthetic spectrum used as “measurement” is generated with the
speed-dependent Voigt line shape with line mixing, but profiles are
retrieved using a Voigt line shape. Panels (b), (d), and (f) show the
difference between the retrieved profiles and AirCore, correspond-
ing to (a), (c), and (e) respectively. similar to the shape obtained in Fig. 6 from perturbing the
temperature profiles. This is because all those perturbations
ultimately lead to an altered line width and all cause residu-
als patterns that cannot be distinguished from each other, as
illustrated in Fig. 8. This implies that errors in the a priori
temperature profile, water vapor profile, and spectroscopic
widths are difficult to disentangle in the current GFIT2 pro-
file retrieval. A simultaneous temperature (hence pressure)
and CO2 profile retrieval would be necessary to overcome
these issues. the Weak windows, we can expect deviations from the truth
10 times larger than in Fig. 7, within ∼10–20 ppm. In Connor et al. (2016), the authors used a Voigt line shape. Figure 7e shows the effect of fitting with a Voigt line shape
a synthetic spectrum that was generated using qSDV+LM. In that case the fit residuals in the Strong window can ex-
ceed 1 % and the residuals in the TCCON windows can ex-
ceed 0.5 %. For these retrievals, the SNR is set to 100 in the
Strong window, 200 in the TCCON windows, and 1000 in
the Weak windows. The profiles retrieved from the Strong
window present deviations from the truth within 60 ppm. S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra 3095 p
p
g
p
Figure 6. The left-hand panels show CO2 profiles retrieved using
synthetic spectra for (a) +5 ◦C added to the a priori temperature
profile below 5 km, (c) +2 ◦C between 8 and 13 km, and (e) +2 ◦C
above 15 km. Panels (b), (d), and (f) show the difference between
the retrieved profiles and AirCore profile, corresponding to (a), (c),
and (e) respectively. Note the difference in the horizontal axis range
between the panels. Here 5 km corresponds to ∼0.55 atm, 8–13 km
to ∼0.36–0.17 atm and 15 km to ∼0.125 atm. Figure 7. The left-hand panels show CO2 profiles retrieved using
synthetic spectra. In (a) the air- and self-broadened half-width co-
efficients of all CO2 lines is increased by 0.1 %. In (c) the temper-
ature dependence of these coefficients is decreased by 1 %. In (e),
the synthetic spectrum used as “measurement” is generated with the
speed-dependent Voigt line shape with line mixing, but profiles are
retrieved using a Voigt line shape. Panels (b), (d), and (f) show the
difference between the retrieved profiles and AirCore, correspond-
ing to (a), (c), and (e) respectively. Figure 7. The left-hand panels show CO2 profiles retrieved using
synthetic spectra. In (a) the air- and self-broadened half-width co-
efficients of all CO2 lines is increased by 0.1 %. In (c) the temper-
ature dependence of these coefficients is decreased by 1 %. In (e),
the synthetic spectrum used as “measurement” is generated with the
speed-dependent Voigt line shape with line mixing, but profiles are
retrieved using a Voigt line shape. Panels (b), (d), and (f) show the
difference between the retrieved profiles and AirCore, correspond-
ing to (a), (c), and (e) respectively. Figure 6. The left-hand panels show CO2 profiles retrieved using
synthetic spectra for (a) +5 ◦C added to the a priori temperature
profile below 5 km, (c) +2 ◦C between 8 and 13 km, and (e) +2 ◦C
above 15 km. Panels (b), (d), and (f) show the difference between
the retrieved profiles and AirCore profile, corresponding to (a), (c),
and (e) respectively. Note the difference in the horizontal axis range
between the panels. Here 5 km corresponds to ∼0.55 atm, 8–13 km
to ∼0.36–0.17 atm and 15 km to ∼0.125 atm. Figure 6. 3.1.4
Perturbed line parameters In the TCCON windows,
the deviation at pressures > 0.6 atm is reduced to ∼10 ppm
while the deviation at pressures > 0.6 atm is comparable to
that in Fig. 6b. In the two Weak windows, the deviation at
∼0.9 atm is unchanged when to compared to Fig. 6b and the
deviation at ∼0.2 atm is reduced from ∼15 to ∼10 ppm. pp
From the results in Sect. 3.1.1, 3.1.2, and 3.1.3, we ob-
serve that CO2 profile retrievals do not need accurate prior
knowledge of the CO2 profile but require accurate knowl-
edge of the prior temperature and water vapor profiles. More-
over, these results suggest that errors in the temperature pro-
file are the main source of deviations from the truth in re-
trieved CO2 profiles. Retrievals using the two Weak windows
are the least affected by biases in the prior temperature and
water vapor profiles. The need for accurate a priori water va-
por profile could be alleviated by retrieving H2O profiles si-
multaneously with CO2 profiles, but this was not tested with
GFIT2, which currently can only retrieve the main target gas
in a window with profile retrievals. In addition, H2O profile
retrievals would also be affected by temperature errors. Atmos. Meas. Tech., 14, 3087–3118, 2021 https://doi.org/10.5194/amt-14-3087-2021 S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra 3096 Table 4. The 1σ relative errors of the air- and self-broadened Lorentz half-width coefficients (b) and of their temperature dependence (n). The values from Benner et al. (2016) and Devi et al. (2007a, b) use the median 1σ uncertainty for the whole band, from the Appendix or
supplemental files of these studies. The values for the Voigt line shape use the error codes reported in the HITRAN2016 line list (Gordon et
al 2017) Table 4. The 1σ relative errors of the air- and self-broadened Lorentz half-width coefficients (b) and of their temperature dependence (n). The values from Benner et al. (2016) and Devi et al. (2007a, b) use the median 1σ uncertainty for the whole band, from the Appendix or
supplemental files of these studies. The values for the Voigt line shape use the error codes reported in the HITRAN2016 line list (Gordon et
al., 2017). Line shape
Window (band)
b (air)
n (air)
b (self)
n (self)
Reference
(%)
(%)
(%)
(%)
Voigt
TCCON1
≥1 and < 2
–
≥1 and < 2
–
Toth et al. (2008)
TCCON2
Weak1
From < 10
Lamouroux et al. (2015),
Weak2
to < 1
Gordon et al. (2017)
Strong
qSDV+LM
TCCON1 (30013–00001)
0.13
–
0.07
Devi et al. (2007a)
TCCON2 (30012–00001)
0.14
0.07
Devi et al. (2007b)
Strong (20013–00001)
0.03
0.12
0.09
0.33
Benner et al. (2016)
Strong (21113–01101)
0.25
1.47
0.49
2.27 trieved from the TCCON1 window observed by Connor et
al. (2016) when fitting real spectra could be entirely due to
the use of the Voigt line shape. In all panels of Fig. 8 except panels (c) and (g), all the
residual features correspond to CO2 absorption lines. In
Fig. 8c, with a perturbation to the a priori temperature pro-
file, there is an added contribution of temperature errors on
interfering species. Furthermore, the residuals in Fig. 8g re-
sult from a combination of errors in the a priori meteorology
and trace gas profiles but are dominated by temperature er-
rors. Perturbations in the temperature profile, CO2 profile, or
CO2 line width coefficients all cause residuals with the same
shape because they all affect the width of CO2 lines. S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra It is
not possible to de-weight the effect of any of those errors by
adjusting the measurement error without also losing the abil-
ity to correct for residuals caused by CO2 errors. Residuals
caused by realistic temperature errors as shown in Fig. 8c are
of the same magnitude as those caused by unrealistically high
errors in the a priori CO2 profile shape as shown in Fig. 8d. g
p
The effect of the errors in the a priori water vapor and tem-
perature profiles and in the spectroscopic parameters cannot
be mitigated by adjusting the measurement covariance, for
example by using a variable SNR. Figure 8 shows an exam-
ple of spectral residuals from fits to synthetic spectra from
the Strong window using scaling retrievals, but with differ-
ent perturbations applied. Showing residuals from scaling re-
trievals reveals systematic features that the profile retrieval
will attempt to suppress. Figure 8b presents residuals from
fitting a synthetic spectrum using the same a priori profile
that was used to generate the synthetic spectrum. It shows
small (< 0.05 %) residuals, caused by the use of a constant
ILS across the window for a faster convolution of the spec-
trum with the ILS. The corresponding profiles are shown in
Fig. 4a. In Fig. 8c, a 2 ◦C offset is applied to the a priori
temperature profile between 8 and 13 km before fitting the
synthetic spectrum. In Fig. 8d, a constant a priori CO2 pro-
file is used to fit a synthetic spectrum that was generated with
an AirCore CO2 profile as an a priori profile. In Fig. 8e, the
air- and self-broadened Lorentz half-width coefficients are
increased by 0.1 % compared to the parameters used to gen-
erate the synthetic spectrum. In Fig. 8f, the temperature de-
pendence of the air- and self-broadened Lorentz half-width
coefficients is decreased by 1 % compared to the parame-
ters used to generate the synthetic spectrum. In Fig. 8g, the
GGG2020 a priori meteorology and trace gas profiles are
used as a priori profiles instead of the a priori profiles con-
structed with AirCore profiles used to generate the synthetic
spectrum. S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra In
the two TCCON windows, the deviations from the truth are
within 30 ppm. In the Weak1 window, the deviations from the
truth are within 10 ppm, because qSDV+LM was not used to
calculate the CO2 line absorptions themselves, but only for
the relatively strong CH4 lines in that window. In the Weak2
window, there is no difference between the two line lists or
line shape, and thus the retrieved profile does not differ from
the a priori profile. Therefore, even if we assume perfect a
priori meteorology, the deviations in the CO2 profiles re- A factor of 10 increase in the perturbations applied to the
width coefficients or their temperature dependence also leads
to a factor of 10 increase in the amplitude of deviations in the
retrieved CO2 profiles. Figure 7a and b use perturbations cor-
responding to uncertainties in the line parameters when using
qSDV+LM for the TCCON windows and the Strong window. The same perturbations were applied for all five windows. However, in the Weak1 and Weak2 windows, these pertur-
bations are 10 times smaller than realistic uncertainties as
reported in Table 4 for the Voigt line shape. Therefore, for https://doi.org/10.5194/amt-14-3087-2021 Atmos. Meas. Tech., 14, 3087–3118, 2021 Atmos. Meas. Tech., 14, 3087–3118, 2021 3.1.5
Synthetic spectra: discussion The other panels show residuals from fits to a synthetic
spectrum, using the same inputs used to generate the synthetic spectrum except for (b) no perturbation; (c) +2 ◦C perturbation to the a priori
temperature between 8 and 13 km; (d) a CO2 prior profile set to 380 ppm at all levels, corresponding to ∼15 ppm offset from the unperturbed
prior; (e) air- and self-broadened Lorentz half-width coefficients increased by 0.1 %; (f) temperature dependence of the half-width coefficients
decreased by 1 %; and (g) using the a priori profiles that would be used by TCCON operational processing, instead of that constructed from
in situ measurements, resulting in a combination of different errors in the a priori profiles such as H2O, temperature, and CO2. Note the
vertical scale of panels (b), (e), and (f) is 5 times smaller than that of panels (c), (d), and (g). In Sect. 3.2, GFIT2 is tested with real spectra using an a
priori profile built from in situ measurements. In that case,
the deviations from the truth in the retrieved CO2 profile
caused by errors in the a priori meteorology (temperature,
pressure, and water vapor profiles) are minimized, and the re-
maining deviations are caused by errors in the spectroscopic
line parameters, in the radiative transfer, in the instrument
line shape, or in the pointing of the sun tracker. to derive information about the vertical distribution of CO2,
even with ill-defined a priori CO2 profiles. Panels (a) and (b)
of Fig. 7 show that profile information could still be retrieved
to within ∼5 ppm given realistic errors in line width parame-
ters. But as shown with Fig. 6, a temperature retrieval, or cor-
rection, is critical to producing reliable CO2 profile retrievals. This study does not show the effect of typical instrument mis-
alignment errors on the retrieved profiles. GFIT2 currently
has no capacity to fit the instrument line shape (ILS) of a
misaligned instrument given specific angular and shear mis-
alignments and instead always assumes a perfect ILS. This is
an area of future development for the program. The effect of
an error in the instrument’s internal field of view and the ef-
fect of a zero-level offset are presented in Appendix F; both
should lead to minor deviations from the truth, within less
than 3 ppm. to derive information about the vertical distribution of CO2,
even with ill-defined a priori CO2 profiles. 3.1.5
Synthetic spectra: discussion For retrievals on synthetic spectra, the “measurement” SNR
is set to 1000, which is high compared to most solar spec-
tra measured by TCCON. So in profiles retrieved from real
spectra, we can expect a greater influence of the a priori CO2
profile: the deviations will be smaller, and the degrees of free-
dom for signal will be lower than those shown in the figures
of Sect. 3.1. This is not a desirable outcome; the a priori CO2
covariance is meant to nudge the retrieval such that the so-
lution lies close to realistic ensembles of CO2 profiles, not
to constrain deviations caused by temperature errors. Tun-
ing the a priori or measurement covariances is not the right
approach until profile deviations caused by typical errors in
spectroscopy or meteorology are smaller than typical verti-
cal variations in CO2 profiles. Figure 4 shows that the profile
retrieval algorithm works well and could be a powerful tool Atmos. Meas. Tech., 14, 3087–3118, 2021 https://doi.org/10.5194/amt-14-3087-2021 : Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra 3097 S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra
3097 S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra
3097
Figure 8. Panel (a) shows an example of calculated lines in the Strong CO2 window. The other panels show residuals from fits to a synthetic
spectrum, using the same inputs used to generate the synthetic spectrum except for (b) no perturbation; (c) +2 ◦C perturbation to the a priori
temperature between 8 and 13 km; (d) a CO2 prior profile set to 380 ppm at all levels, corresponding to ∼15 ppm offset from the unperturbed
prior; (e) air- and self-broadened Lorentz half-width coefficients increased by 0.1 %; (f) temperature dependence of the half-width coefficients
decreased by 1 %; and (g) using the a priori profiles that would be used by TCCON operational processing, instead of that constructed from
in situ measurements, resulting in a combination of different errors in the a priori profiles such as H2O, temperature, and CO2. Note the
vertical scale of panels (b), (e), and (f) is 5 times smaller than that of panels (c), (d), and (g). Figure 8. Panel (a) shows an example of calculated lines in the Strong CO2 window. 3.1.5
Synthetic spectra: discussion Panels (a) and (b)
of Fig. 7 show that profile information could still be retrieved
to within ∼5 ppm given realistic errors in line width parame-
ters. But as shown with Fig. 6, a temperature retrieval, or cor-
rection, is critical to producing reliable CO2 profile retrievals. This study does not show the effect of typical instrument mis-
alignment errors on the retrieved profiles. GFIT2 currently
has no capacity to fit the instrument line shape (ILS) of a
misaligned instrument given specific angular and shear mis-
alignments and instead always assumes a perfect ILS. This is
an area of future development for the program. The effect of
an error in the instrument’s internal field of view and the ef-
fect of a zero-level offset are presented in Appendix F; both
should lead to minor deviations from the truth, within less
than 3 ppm. 3.2
Real spectra The points represent the 51 levels of the vertical grid. The DOFs for
each retrieval window are indicated in (b) and (d). Figure 9. CO2 profiles retrieved from spectra measured at the La-
mont TCCON site on 14 January 2012, at 61–74◦solar zenith an-
gle, coincident with AirCore measurements using (a) the AirCore
“truth” as a priori profiles and (c) the GGG2020 a priori profiles. In (b) and (d) the difference of the retrieved profiles minus the Air-
Core profile is shown, corresponding to (a) and (c), respectively. The points represent the 51 levels of the vertical grid. The DOFs for
each retrieval window are indicated in (b) and (d). Figure 10. Same as Fig. 9 but for spectra measured on 11 April
2017 at 28–39◦solar zenith angle. Even with ideal prior knowledge of the meteorology and
trace gas profiles, the CO2 deviations from the truth can be as
large as 50 ppm as shown in Figs. 9a and 10a. When synthetic
spectra were perturbed with realistic errors in line width pa-
rameters, profile deviations remained within 5 ppm for pro-
files retrieved from the Strong window and within 10 ppm for
the TCCON windows. This suggests that the main cause of
deviations in Figs. 9a and 10a is not due to errors in spec-
troscopic parameters. The assumption that there is no con-
tribution from temperature errors in the radiosonde profile is
supported by the CO2 profile deviation being smallest in the
Strong window, which is the most sensitive to temperature
errors. Although the effect of typical perturbations in the in-
strument field of view, zero-level offset, and spectroscopic
parameters is relatively small compared to the effect of tem-
perature errors, the cumulative effect of these errors could
explain the deviations from the truth in Figs. 9a and 10a. than the random noise in the measurement, the root mean
square is a conservative estimate of the noise. In Sect. 3.2.1,
we present CO2 profiles retrieved from real spectra and we
attempt to isolate the effect of errors in instrument line shape,
in spectroscopic parameters, and in pointing, from the effect
of errors in meteorology. In Sect. 3.2.2, we present an anal-
ysis of the information content and altitude sensitivity of the
retrieval. Finally, in Sect. 3.2.3, we compare XCO2 derived
from the scaling retrieval to XCO2 derived from the profile
retrieval. 3.2
Real spectra Here the algorithm is tested with real spectra measured at
Lamont as described in Sect. 2.2. A scaling retrieval is per-
formed before each profile retrieval and the root mean square
of the residuals from the scaling retrieval is used as measure-
ment uncertainty for the profile retrieval. Since the residuals
from the scaling retrieval include systematic features larger https://doi.org/10.5194/amt-14-3087-2021 Atmos. Meas. Tech., 14, 3087–3118, 2021 S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra 3098
S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra
Figure 9. CO2 profiles retrieved from spectra measured at the La-
mont TCCON site on 14 January 2012, at 61–74◦solar zenith an-
gle, coincident with AirCore measurements using (a) the AirCore
“truth” as a priori profiles and (c) the GGG2020 a priori profiles. In (b) and (d) the difference of the retrieved profiles minus the Air-
Core profile is shown, corresponding to (a) and (c), respectively. Figure 10. Same as Fig. 9 but for spectra measured on 11 April
2017 at 28–39◦solar zenith angle. Even with ideal prior knowledge of the meteorology and 3098 vertical profiles from ground-based near-infrared spectra
Figure 10. Same as Fig. 9 but for spectra measured on 11 April
2017 at 28–39◦solar zenith angle. Figure 9. CO2 profiles retrieved from spectra measured at the La-
mont TCCON site on 14 January 2012, at 61–74◦solar zenith an-
gle, coincident with AirCore measurements using (a) the AirCore
“truth” as a priori profiles and (c) the GGG2020 a priori profiles. In (b) and (d) the difference of the retrieved profiles minus the Air-
Core profile is shown, corresponding to (a) and (c), respectively. The points represent the 51 levels of the vertical grid. The DOFs for
each retrieval window are indicated in (b) and (d). Figure 9. CO2 profiles retrieved from spectra measured at the La- Figure 9. CO2 profiles retrieved from spectra measured at the La-
mont TCCON site on 14 January 2012, at 61–74◦solar zenith an-
gle, coincident with AirCore measurements using (a) the AirCore
“truth” as a priori profiles and (c) the GGG2020 a priori profiles. In (b) and (d) the difference of the retrieved profiles minus the Air-
Core profile is shown, corresponding to (a) and (c), respectively. S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra Here, the vertical representation is not a concern. Using
51 vertical levels only affects the speed of the retrieval. The
retrieved profiles can then be reduced to a number of partial
columns corresponding to the DOFs. This was not done here
because it is evident that large deviations due to temperature
errors could easily bias the resulting partial columns. The
reduction into a subset of layers also requires an arbitrary
choice: in Fig. 13 the altitude ranges were set such that the
DOFs of the first two partial columns would be roughly close
to 1 in each window. We could also have chosen two regions
with approximately equal DOFs from 0–7 and 7–70 km. The
partial column averaging kernels overlap with each other, so
the partial columns are not completely uncorrelated even if
their respective DOFs are higher than 1. The DOFs are not
exactly independent pieces of information, as it is impossible
to obtain independent partial column amounts from direct-
sun measurements on the ground (see Appendix C), but an ar-
bitrary criterion can be defined to identify distinct layers, for
example if the peaks in their partial column averaging kernels
are separated by a given fraction of their widths in altitude. the a priori covariance introduce inter-layer correlations that
reduce large differences between levels over a given length
scale (see Sect. 2.2). Retrievals on real spectra after applying
a +5 ◦C offset to the radiosonde temperature profile below
5 km lead to a +100 ppm offset at ∼0.9 atm. The CO2 pro-
files in Fig. 10c differ less with those in Fig. 10a than do
the profiles in Fig. 9a and c. In Fig. 11, the difference be-
tween the GGG2020 and radiosonde temperature profile on
11 April 2017 is ∼3 ◦C for the first two levels above the sur-
face, but the average difference between 0.85 and 0.6 atm is
−0.15 ◦C compared to −1.05 ◦C on 14 January 2012. In aircraft profiles over Lamont between 2008 and 2018
from NOAA’s ObsPack, the steepest vertical gradients in
CO2 profiles are ∼5 ppm km−1 between the surface and
∼3 km. In its current state, CO2 profile retrieval with GFIT2
cannot distinguish these vertical variations from CO2 devia-
tions caused by errors in the forward model, even with very
accurate a priori meteorology. 3.2.1
Profiles p
g
Figure 11 shows the difference between the GGG2020 a
priori temperature profile, used in Figs. 9c and 10c, and the
radiosonde temperature profile used in Figs. 9a and 10a. In
both cases, we replace the a priori surface temperature with
the measured surface temperature. On 14 January 2012, the
radiosonde temperature profile is about 1 ◦C higher than the
GGG2020 a priori profile at pressures < 0.6 atm. The shape
of the Strong window CO2 profile deviations in Fig. 9c is
consistent with the sensitivity tests using synthetic spectra in
Sect. 3.1.3. In Fig. 6a, a +5 ◦C offset below 5 km results in
+500 ppm CO2 error at ∼0.9 atm, while in Fig. 10, a −1 ◦C
offset in the lower troposphere leads to a −50 ppm error at
∼0.9 atm. The deviations are smoother in Figs. 9 and 10
than in Fig. 6 because the SNR of real spectra is between
200 and 500 instead of 1000, and because of the smooth-
ing effect of the off-diagonal elements of the a priori co-
variance used in this section. The off-diagonal elements of Figures 9 and 10 show CO2 profiles retrieved from real spec-
tra measured from Lamont, OK, on 14 January 2012 and
11 April 2017, respectively. In each figure, panel (a) shows
profiles retrieved using in situ profiles (the “truth”) as the
a priori profiles. In those cases, we assume that deviations
from the truth caused by errors in a priori meteorology (pres-
sure, temperature, and water vapor profiles) are minimized,
and the remaining deviations can be attributed to the combi-
nation of instrument misalignment (ILS), pointing errors, or
errors in spectroscopic parameters. Panel (c) shows profiles
retrieved using the GGG2020 a priori profiles. A first com-
plication for obtaining a satisfactory CO2 profile retrieval is
that the a priori CO2 profiles in GGG2020 already compare
well with in situ profiles, typically within 5 ppm over Lam-
ont. In Figs. 9c and 10c, the profile that most closely matches
the AirCore is the a priori profile. https://doi.org/10.5194/amt-14-3087-2021 Atmos. Meas. Tech., 14, 3087–3118, 2021 S. Roche et al.: Retrieval of atmospheric CO2 vertical profi
Figure 11. Temperature profile difference for the GGG2020 a priori
minus radiosonde on 14 January 2012 and 11 April 2017. The ra-
diosonde profile is included in the a priori profiles used in panel (a)
of Figs. S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra 3099 Table 5. Shannon information content (H), degrees of freedom for
signal (DOFs) for the CO2 profile, and ratio of residuals (RR) aver-
aged over all 492 profile retrievals from near-infrared TCCON spec-
tra measured at Lamont and coincident within ±1 h of the AirCore
last sampling time. The standard deviation is also shown. Window
H
DOFs
RR
name
TCCON1
5.4 ± 0.6
2.7 ± 0.2
0.988 ± 0.014
TCCON2
5.4 ± 0.6
2.7 ± 0.2
0.992 ± 0.009
Weak1
2.3 ± 0.7
1.7 ± 0.3
0.996 ± 0.002
Weak2
2.5 ± 0.9
1.8 ± 0.4
0.994 ± 0.008
Strong
6.8 ± 1.0
3.0 ± 0.4
0.957 ± 0.038 Figure 11. Temperature profile difference for the GGG2020 a priori
minus radiosonde on 14 January 2012 and 11 April 2017. The ra-
diosonde profile is included in the a priori profiles used in panel (a)
of Figs. 9 and 10, and the GGG2020 a priori profiles are used in
panel (b) of Figs. 9 and 10. In situ temperature measurements are
used for both cases at the surface. The dashed line marks the average
difference, with the value indicated in the legend. subtracted to XCO2 obtained from profile retrievals for each
window. Figure 13 shows the sums of the rows of the partial col-
umn averaging kernel matrix over different altitude ranges. The sum from 0 to 70 km is the total column averaging ker-
nel (see Appendix C). The total column averaging kernel is
close to 1 at all levels in all windows, indicating good sensi-
tivity to changes in the CO2 total column. The partial column
kernels show that most of this sensitivity comes from alti-
tudes below 15 km. That the total column averaging kernel is
close to 1 at all levels is not inconsistent with the large de-
viations we observe in the retrieved CO2 profiles. If the total
column averaging kernel is exactly one at each level, adding
N molecules of CO2 anywhere in the atmosphere will lead
to N more molecules in the retrieved total column. However,
in the presence of a priori temperature errors, for example,
the retrieved value can be biased. The averaging kernel indi-
cates that without the effect of these errors, the CO2 profile
retrieval would have excellent sensitivity to CO2 and would
be able to provide information about CO2 in two distinct lay-
ers. S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra Typical errors in the a priori
temperature profiles will prevent operational use of CO2 pro-
file retrieval without a scheme for retrieving or correcting the
temperature profiles. Atmos. Meas. Tech., 14, 3087–3118, 2021 3.2.1
Profiles 9 and 10, and the GGG2020 a priori profiles are used in
panel (b) of Figs. 9 and 10. In situ temperature measurements are
used for both cases at the surface. The dashed line marks the average
difference, with the value indicated in the legend. S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra 3.2.3
XCO2 The XCO2 derived from profile and scaling retrievals using
the GGG2020 a priori profiles was compared to XCO2 de-
rived from the CO2 profile built from the AirCore CO2 pro-
file, in situ surface measurements of CO2, and the GGG2020
a priori CO2 above the maximum altitude sampled by the
AirCore. The results are shown in Fig. 14 for the 8 days we
have AirCore profiles that are coincident with measurements
at the Lamont TCCON station. Despite the large deviations
observed in retrieved profiles, the XCO2 derived from profile
retrievals compares well to the AirCore XCO2, but it does not
present a clear improvement over the XCO2 derived from the
scaling retrievals. The effect of temperature errors on XCO2
derived from scaling and profile retrievals is relatively small
because the spectral windows utilize the entire (fundamental)
band. Across a wide window, the residuals due to tempera-
ture errors show alternating positive and negative residuals,
because of the different temperature sensitivities of absorp-
tion lines. Collectively, these lines have a small net tempera-
ture sensitivity. The scaling retrieval, which can only add or
remove CO2 at all levels simultaneously, is limited in its abil-
ity to fit out such residuals across a wide window by adjust-
ing the CO2 scale factor. For profile retrievals, although large
deviations are observed in the retrieved profile, they compen-
sate each other when deriving the total column. These devia-
tions compensate due to the wide windows including a range
of spectral lines with different temperature sensitivities. If a
narrow window over only a few lines were used instead, we
would expect more localized errors in the retrieved CO2 pro-
files, and total columns sensitive to temperature errors. Figure 13. Sum of the rows of the partial column averaging kernel
matrix over different altitude ranges as indicated by the legend, for
each of the five CO2 windows. The sum between 0–70 km is the
total column averaging kernel. The numbers in each panel are the
DOFs corresponding to each of the altitude ranges. Additional analysis of the vertical sensitivity of the retrieval
is presented in Appendix D, as well as a decomposition of the
retrieval error into the interference, measurement noise, and
smoothing errors as shown in Fig. D8. The interference error
is the smallest (< 0.5 %) contribution but does not include
the effect of temperature errors. S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra 3100 Figure 12. Shannon information content (top left), degrees of freedom for signal for the CO2 profile (top right), and ratio of residuals (bottom
left), and profile minus scaling retrieval XCO2 (bottom right) for all Lamont spectra coincident within ±1 h of the AirCore last sampling
time for AirCores launched on the dates indicated on the right. Each new date is marked by a vertical dashed line. Figure 12. Shannon information content (top left), degrees of freedom for signal for the CO2 profile (top right), and ratio of residuals (bottom
left), and profile minus scaling retrieval XCO2 (bottom right) for all Lamont spectra coincident within ±1 h of the AirCore last sampling
time for AirCores launched on the dates indicated on the right. Each new date is marked by a vertical dashed line. Figure 13. Sum of the rows of the partial column averaging kernel
matrix over different altitude ranges as indicated by the legend, for
each of the five CO2 windows. The sum between 0–70 km is the
total column averaging kernel. The numbers in each panel are the
DOFs corresponding to each of the altitude ranges. https://doi.org/10.5194/amt-14-3087-2021 3.2.2
Information content and averaging kernel Table 5 presents the average values of the Shannon infor-
mation content, H, and of the CO2 profile DOFs, from all
profile retrievals performed on Lamont spectra when using
the GGG2020 a priori profiles. It also includes the ratio of
residuals (RR) of the spectral fits (see Appendix B, Eq. B10),
which represents the residuals of the profile retrievals as a
fraction of the residuals of the scaling retrievals. The same
quantities are plotted in Fig. 12 for each spectrum. The RR
is always smaller than 1 because the profile retrieval has
more freedom to adjust the calculated spectrum and so can
never produce larger residuals than scaling retrievals. Fig-
ure 12 also shows XCO2 obtained from the scaling retrievals https://doi.org/10.5194/amt-14-3087-2021 Atmos. Meas. Tech., 14, 3087–3118, 2021 4
Summary and conclusions In this study we investigated the use of CO2 profile re-
trievals from near-infrared solar absorption spectra measured
by TCCON. The performance of CO2 profile retrieval was
reassessed after improvements were implemented in the for-
ward model of GGG. Retrievals were performed using five
CO2 windows with significantly different optical opacities. In this study we investigated the use of CO2 profile re-
trievals from near-infrared solar absorption spectra measured
by TCCON. The performance of CO2 profile retrieval was
reassessed after improvements were implemented in the for-
ward model of GGG. Retrievals were performed using five
CO2 windows with significantly different optical opacities. We first use retrievals on synthetic spectra to check the
self-consistency. Typical errors in the a priori H2O profile,
which is retrieved with a scaling retrieval, caused limited de-
viations from the truth in the CO2 profile, within 5–10 ppm
in the Strong window, and within 2 ppm in the other win-
dows. Perturbing the CO2 air- and self-broadened Lorentz
half-width coefficients and their temperature dependence to
within their estimated uncertainties led to CO2 deviations
from the truth of less than 5 ppm. The implementation of a
non-Voigt line shape is a significant improvement to CO2
profile retrievals; errors in spectroscopic parameters are no
longer the leading source of uncertainty in retrieved profiles. We observed deviations from the truth of up to 100 ppm in
profiles retrieved with typical temperature errors. The tem-
perature profile is an important retrieval input but is not re-
trieved; thus spectral residuals caused by errors in the a priori
temperature profile are free to be suppressed by adjustments
to the CO2 scale factors. The implementation of a temper-
ature profile retrieval, or correction, is critical to improve
CO2 profile retrieval results. In GGG2020, 3-hourly a pri-
ori temperature profiles are used, but temperatures can still
vary by several degrees between 3-hourly profiles and can
still be wrong even without any time mismatch. Temperature
could be retrieved from CO2 windows and from windows
with temperature-sensitive water vapor absorption lines. Figure 14. XCO2 derived from scaling (dashed lines and squares)
and profile (solid lines and circles) retrievals for each CO2 window
when using the GGG2020 a priori profiles, compared to XCO2 de-
rived from smoothed AirCore profiles (see Appendix C). The black
dotted line marks the 1-to-1 line. 4
Summary and conclusions When comparing with scaling
retrievals, the AirCore profile is smoothed using the total column
averaging kernel of the scaling retrieval, and when comparing to
profile retrievals the AirCore profile is smoothed using the averag-
ing kernel matrix of the profile retrieval. The legend indicates the
slopes and squared Pearson correlation coefficients of fits to lines
passing through the origin, assuming that in the absence of CO2 the
retrieval would return a CO2 value of zero. S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra rections to reduce the effect of systematic imperfections in
the forward model. rections to reduce the effect of systematic imperfections in
the forward model. 3.2.3
XCO2 The measurement noise error
decreases from ∼1 % at the surface to ∼0.2 % at pressures
less than 0.6 atm (> 5 km), and the smoothing error domi-
nates and decreases roughly from ∼3 % at the surface to 1 %
at the top of the atmosphere. Atmos. Meas. Tech., 14, 3087–3118, 2021 https://doi.org/10.5194/amt-14-3087-2021 S. Roche et al.: Retrieval of atmospheric CO2 vertical pro
Figure 14. XCO2 derived from scaling (dashed lines and squares)
and profile (solid lines and circles) retrievals for each CO2 window
when using the GGG2020 a priori profiles, compared to XCO2 de-
rived from smoothed AirCore profiles (see Appendix C). The black
dotted line marks the 1-to-1 line. When comparing with scaling
retrievals, the AirCore profile is smoothed using the total column
averaging kernel of the scaling retrieval, and when comparing to
profile retrievals the AirCore profile is smoothed using the averag-
ing kernel matrix of the profile retrieval. The legend indicates the
slopes and squared Pearson correlation coefficients of fits to lines
passing through the origin, assuming that in the absence of CO2 the
retrieval would return a CO2 value of zero. S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra 3101 S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra Atmos. Meas. Tech., 14, 3087–3118, 2021 https://doi.org/10.5194/amt-14-3087-2021 https://doi.org/10.5194/amt-14-3087-2021 3.2.4
Real spectra: discussion Profile retrievals that use real spectra and an a priori profile
built from coincident in situ measurements show CO2 pro-
file deviations up to 40–50 ppm. Even when the errors due to
the a priori meteorology are minimized, deviations from the
truth due to instrument misalignment, radiative transfer, sun-
tracker pointing, or uncertainties in line parameters are larger
than the steepest vertical CO2 gradients (∼5 ppm km−1) ob-
served in the ensemble of aircraft profiles from NOAA’s Ob-
sPack. We then perform retrievals with atmospheric TCCON
spectra collected at the Lamont site, which were coincident
with AirCore profiles, including radiosonde profiles of tem-
perature and relative humidity; these were considered as the
true state of the atmosphere. When running retrievals with
the truth as the a priori profiles, the deviations due to errors
in the a priori meteorology are minimized and the resulting
deviations are caused by instrument misalignment, errors in
spectroscopy, or sun tracker pointing. We observed CO2 de-
viations of up to 40 ppm in that case. Even with ideal knowl-
edge of the a priori meteorology, the CO2 deviations are
larger than the largest expected vertical CO2 variations and
no useful profile information can be inferred from the pro-
file retrieval. Stricter alignment requirements, which can be
challenging to achieve in practice, or the ability to model an
imperfect instrument line shape are needed to improve profile
retrieval results. The sensitivity study of Sect. 3.1 could then When performing retrievals on the same spectra but re-
placing the AirCore a priori profile with a standard a pri-
ori profile, small errors in the a priori temperature profile
cause large deviations in the retrieved CO2 profile. Despite
the large deviations in the retrieved profiles, the retrieval still
shows high sensitivity to XCO2 but does not present a clear
improvement over XCO2 obtained from scaling retrievals. Introducing a temperature retrieval or correction, as well as
the ability to model an imperfect instrument line shape, is
the best avenue to improve the CO2 profile retrieval results. Appendix E presents an attempt at applying empirical cor- https://doi.org/10.5194/amt-14-3087-2021 Atmos. Meas. Tech., 14, 3087–3118, 2021 3102 Atmos. Meas. Tech., 14, 3087–3118, 2021 Appendix A: Vertical columns The total col-
umn of CO2 is columnCO2 =
N
X
i=1
sfi × vmri × di × vpi,
(A4) The expected χ2 of the maximum a posteriori probability
solution should be (A4) χ2 ˆx −x
=
ˆx −x
T
KT S−1
y K + S−1
a
ˆx −x
≈n,
(B3) (B3) where sf is the retrieved scaling factor and vmr is the a pri-
ori CO2 wet mole fraction (molecules of CO2 per molecules
of air). In the forward model, the retrieval grid is not verti-
cal, but along the slant path from the instrument towards the
sun, the scaling factors retrieved for the slant layers are used
with the corresponding vertical layers to compute the vertical
column. The a priori profiles used by GFIT are built on the
prescribed altitude grid directly above the site. This should
contribute to an unknown error, largest at high solar zenith
angles when the projection of the sun ray on the ground can
reach a few hundred kilometers; in that case the a priori slant
profiles of temperature and H2O could be significantly dif-
ferent from the vertical profile directly above the instrument. where n is the number of state vector elements. A solution
is accepted when the ratio of the squared state update to the
estimated variance is a negligible fraction of the expected χ2: 1x
KT S−1
y (y −f (xi)) + S−1
a (xa −xi)
≪n. (B4) (B4) In an algorithm, “≪n” must use a specific limit, and in
GFIT2, “< n/10” was used. If the inequality check is made
with a parameter that is too large, like “< n”, the algorithm
may take fewer iterations to converge but will take the same
steps at each iteration, often leading to a retrieved profile
closer to the a priori profile. The inequality check should be
done with a small enough fraction of n that reducing it further
does not significantly affect the solution. In an algorithm, “≪n” must use a specific limit, and in
GFIT2, “< n/10” was used. If the inequality check is made
with a parameter that is too large, like “< n”, the algorithm
may take fewer iterations to converge but will take the same
steps at each iteration, often leading to a retrieved profile
closer to the a priori profile. et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectr A method to combine the profiles obtained from sequen-
tial retrievals in different spectral windows still needs to be
developed. Alternatively, the ability to perform simultaneous
retrievals using multiple spectral windows could be imple-
mented in GFIT2. be extended to assess the effect of specific misalignments on
the retrieved profiles. In these retrievals, we used a full a priori covariance ma-
trix, with off-diagonal elements, based on comparisons be-
tween the GGG2020 a priori and aircraft vertical profiles
from NOAA’s ObsPack over the Lamont TCCON site. Be-
fore tuning the a priori covariance and considering stronger
regularizations, it must be shown that CO2 deviations caused
by typical errors in the a priori meteorology are smaller than
typical variability in real CO2 profiles. Because it is more
computationally expensive, and because it requires stronger
constraints on the a priori statistics than scaling retrievals, a
profile retrieval must present clear advantages over a scaling
retrieval to justify its operational use. And with each new im-
provement to the CO2 a priori profiles, requirements for pro-
file retrieval to be better than scaling retrieval become more
stringent. Atmos. Meas. Tech., 14, 3087–3118, 2021 S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra 3103 Appendix A: Vertical columns To find the state vector with maximum a posteriori probabil-
ity given a measurement, the cost function J is minimized, The vertical grid for the retrievals presented in this study has
51 levels from 0 to 70 km, with spacing increasing with alti-
tude and following: J = (y −f (x))T S−1
y (y −f (x))
+ (xa −x)T R(xa −x),
(B1) zi = i × (0.4 + 0.02 × i),
(A1) (A1) (B1) by iteratively solving for the state update 1x in the least
square problem: where zi is the altitude in kilometers of the ith level. Each
level is associated with an effective vertical path distance vp:
KT
i S−1
y Ki + R + γ D
1x = KT
i S−1
y (y −f (xi))
+ R(xa −xi). (B2) vpi ≈0.4 + 0.04 × i. (A2) (A2) + R(xa −xi). (B2) (B2) The total column of air in molecules per square meter can be
obtained as The total column of air in molecules per square meter can be
obtained as Here, y is the measured transmittance spectrum, f is the for-
ward model that computes a transmittance spectrum from the
state vector x, Sy is the measurement covariance matrix, xa
is the a priori state vector, and the regularization matrix R is
taken to be the inverse of the a priori covariance matrix Sa. K is the Jacobian matrix, and each column of K contains the
derivative of the spectrum with respect to an element of the
state vector, K = ∂f (x)
∂x . The Levenberg–Marquardt parame-
ter γ is applied to a scaling matrix D, which is also taken to
be S−1
a . The Levenberg–Marquardt parameter affects the size
of the state update so that smaller steps may be taken when
the linearization of the forward model is not satisfactory. columnAir =
N
X
i=1
vpi × di,
(A3) (A3) where d is the air number density in molecules of air per
cubic meter. N is the number of atmospheric levels. If the
prescribed grid contains layers below the altitude of the site
considered, their effective vertical path will be 0. The layer
containing the site altitude will be truncated. Appendix C: Averaging kernel This is the relative change in the cost function produced by
a state vector update when using the forward model and a
linear approximation of the forward model. The Levenberg–
Marquardt parameter is then adjusted as follows: This is the relative change in the cost function produced by
a state vector update when using the forward model and a
linear approximation of the forward model. The Levenberg–
Marquardt parameter is then adjusted as follows: The state vectors of GFIT and GFIT2 contain scaling factors
to be applied to a priori mole fractions. The averaging kernel
matrix is – r > 0.75. The linearization of the forward model is sat-
isfactory and γ is reduced to allow larger steps. A =
KT S−1
y K + S−1
a
−1
KT S−1
y K. (C1) (C1) – γ = γ
2 – γ = γ
2 It is a change in the retrieved state for a change in the state
vector elements. It is a change in the retrieved state for a change in the state
vector elements. – r ≥0.25. Intermediate case, make no change to γ and
reset the number of consecutive divergences. – r ≥0.25. Intermediate case, make no change to γ and
reset the number of consecutive divergences. (ASF)i,j = δ ˆxi
δxj
(C2) (C2) – ndiv = 0 – ndiv = 0 – ndiv = 0 Even though the averaging kernel is dimensionless, its units
can be written as, for example, “ppm per ppm” to indicate
that it is the change at a given level for a change at a different
level. – r < 0.25. The linearization of the forward model is not
satisfactory, increment the number of consecutive diver-
gences, γ is increased to take smaller steps. To obtain the averaging kernel in ppm per ppm, – ndiv = ndiv + 1 – if γ = 0 then γ =1
– if γ > 0 then γ = 10γ (Avmr)i,j = (ASF)i,j
vmri
vmrj
,
(C3) (C3) If ndiv reaches some specified maximum number, there will
not be another iteration. When r < 0.25, it means that the
linearization of the forward model is not good enough. In
GFIT2, this was not allowed to happen more than twice in
a row. S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra Jpred = (y −f (xi) −K1x)T S−1
y (y −f (xi) −K1x)
+ (xa −xi −1x)T S−1
a (xa −xi −1x),
(B7) where ynoise is the measurement uncertainty, and N is the
number of spectral points. Profile retrievals from real spectra
are presented in Sect. 3 where the root mean square of the
residuals from a scaling retrieval is used as ynoise. In that case
Eq. (B10) is the average ratio of residuals (RR) between the
profile and scaling retrieval. (B7) where Jold is the cost function using the state vector at the
beginning of the ith iteration, Jnew is the cost function using
the updated state vector at the end of the ith iteration, and
Jpred is the cost function using the state vector update and
the linear approximation: The retrieval covariance matrix is The retrieval covariance matrix is ˆS =
KT S−1
y K + S−1
a
−1
. (B11) (B11) f (x + 1x) ∼= f (x) + K1x. (B8) (B8) The square root of its diagonal elements is used as the uncer-
tainty in the retrieved scaling factors. The ratio r is then evaluated: The ratio r is then evaluated: r = Jnew −Jold
Jpred −Jold
. (B9) r = Jnew −Jold
Jpred −Jold
. (B9) Appendix A: Vertical columns The inequality check should be
done with a small enough fraction of n that reducing it further
does not significantly affect the solution. The column-averaged dry-air mole fraction of CO2
(XCO2) is the ratio of the column of CO2 to the column of
dry air, where the column of dry air is expressed as the col-
umn of O2 divided by 0.2095 (Wunch et al., 2011b): If convergence is not reached in the ith iteration, an al-
gorithm determines if the Levenberg–Marquardt parameter
needs to be adjusted for the next iteration (Fletcher, 1971). Three different cost functions are used: XCO2 = 0.2095 × columnCO2
columnO2
,
(A5) (A5) Jold = (y −f (xi))T S−1
y (y −f (xi))
+ (xa −xi)T S−1
a (xa −xi),
(B5)
Jnew = (y −f (xi + 1x))T S−1
y (y −f (xi + 1x))
+ (xa −xi −1x)T S−1
a (xa −xi −1x),
(B6) where the O2 column is retrieved from a spectral window
centered at 7885 cm−1. For the official TCCON products,
columnCO2 is a weighted average of the columns retrieved
from the TCCON1 and TCCON2 windows. (B5) (B6) Atmos. Meas. Tech., 14, 3087–3118, 2021 Atmos. Meas. Tech., 14, 3087–3118, 2021 https://doi.org/10.5194/amt-14-3087-2021 3104 S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra The sin-
gular vectors all show an increasing number of oscillations
with decreasing singular value. In each window, the first sin-
gular vector is close to a uniform weighting at all altitudes
and has 3 to 10 times more sensitivity than the second pat-
tern. The singular vector in panel (d) has a structure like that
of the CO2 profile deviations observed in the sensitivity tests
of Sect. 3.1. ˆ fraction of the largest singular value in parenthesis. The sin-
gular vectors all show an increasing number of oscillations
with decreasing singular value. In each window, the first sin-
gular vector is close to a uniform weighting at all altitudes
and has 3 to 10 times more sensitivity than the second pat-
tern. The singular vector in panel (d) has a structure like that
of the CO2 profile deviations observed in the sensitivity tests
of Sect. 3.1. ˆ ctot
a =
nlev
X
i=1
cai. (C8) ctot
a =
nlev
X
i=1
cai. (C8) S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra 3105 Figure D1. Right singular vector of the Jacobian associated with the
eight largest singular values for profile retrievals from the Strong
CO2 window on 14 January 2012. The singular values are shown
above each panel, and the singular value normalized to the largest
singular value is shown in parenthesis. The averaging kernel matrix would ideally be an identity
matrix, meaning that adding N molecules in the jth layer
would lead to N more molecules retrieved in that layer. How-
ever, adding N molecules in the jth layer will lead to an in-
crease in the width of CO2 absorption lines of a spectrum
observed from the ground. As illustrated for CO2 in Fig. 2,
each wavenumber is affected by the CO2 concentration over
a range of altitudes, because the spectrum observed on the
ground is the product of all the spectra that would be ob-
served at each altitude. Even if that change in line widths
was the only change in the spectrum and could be fitted per-
fectly, it would be impossible to exactly attribute that change
to a specific level. Although the total column averaging ker-
nel could be exactly 1 at each level, the averaging kernel ma-
trix can never be exactly the identity matrix for direct-sun
measurements from the ground. Figure D1. Right singular vector of the Jacobian associated with the
eight largest singular values for profile retrievals from the Strong
CO2 window on 14 January 2012. The singular values are shown
above each panel, and the singular value normalized to the largest
singular value is shown in parenthesis. Figure D2. Same as Fig. D2 but for the TCCON1 window. The column averaging kernel matrix can be used to de-
grade higher-resolution profiles before comparing them to
retrieved profiles (Rodgers and Connor, 2003). cs = Acol (c −ca) + ca,
(C6) (C6) where cs is the smoothed partial column profile, c is the par-
tial column profile to be smoothed, and ca is the a priori par-
tial column profile. Or, using the total column averaging ker-
nel, Figure D2. Same as Fig. D2 but for the TCCON1 window. ctot
s = ctot
a + aT (c −ca),
(C7) (C7) where aT is the transpose of a, and the “tot” superscript in-
dicates a total column: fraction of the largest singular value in parenthesis. Appendix C: Averaging kernel Increasing γ will lead to a smaller step for the state
vector update, increasing the chance that the linearization of
the forward model at the next step will be better and r ≥0.25. where vmr is the a priori mole fraction at the ith and jth
levels and the partial column averaging kernel matrix in
molecules cm−2 per molecules cm−2 is (Acol)i,j = (ASF)i,j
vmri × di × spi
vmrj × dj × spj
,
(C4) (C4) where sp denotes the widths of the slant layers along the sun
ray that correspond to the altitude levels of the prescribed
vertical grid. The total column averaging kernel vector can
be obtained from the partial column averaging kernel matrix: In GFIT2 r > 0.75 in most cases, and if γ is not initially
set to 0 it will tend towards zero until the convergence crite-
rion is met; thus the initial value of γ was set to 0. However,
the increase of the parameter is often triggered when fitting
noisier spectra and can give the algorithm a chance to con-
verge when it would otherwise need more iterations or fail
without γ . aj =
nlev
X
i=1
(Acol)i,j. (C5) (C5) After the last iteration, the goodness of the retrieval is
checked by evaluating the reduced χ2 of the spectral residu-
als. It represents the change in the total column (molecules cm−2)
caused by a change in the partial column of the jth layer. It should ideally be equal to 1 at each level, meaning that
adding N target molecules anywhere in the atmosphere will
lead to N more molecules in the retrieved total column. χ2
red (y −f (x)) = 1
N
N
X
i=1
yi −f (x)i
ynoise
2
,
(B10) (B10) (B10) Atmos. Meas. Tech., 14, 3087–3118, 2021 Atmos. Meas. Tech., 14, 3087–3118, 2021 Atmos. Meas. Tech., 14, 3087–3118, 2021 https://doi.org/10.5194/amt-14-3087-2021 S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra Si = AxeSa,eAT
xe, where Sa,e is the part of the a priori covariance matrix that
corresponds to “extra” state vector elements other than CO2
scale factors. With N total state vector elements and nlev at-
mospheric levels, Sa,e has dimensions (N-nlev,N-nlev). Axe
is the subset of the averaging kernel matrix that character-
izes the smoothing effect of the extra state vector elements
on the CO2 profiles, with dimensions (nlev,N-nlev). The in-
terference error is the smallest contribution to the total error
and most of the error comes from the smoothing effect of
the a priori CO2 covariance, followed by the contribution of
measurement noise which oscillates between ∼10 %–25 %
of the a priori CO2 uncertainty. If temperature were retrieved,
for example with a temperature offset or with a scale factor
added to the extra state vector elements, we would expect the
interference error to increase. Figure D4. Same as Fig. D1 but for the Weak1 window. responding eigenvalue. The error patterns of SN associated
with the four largest eigenvalues are shown in Fig. D6, and
those of Sm are shown in Fig. D7. In both cases, the largest
error pattern peaks at the surface and falls to 0 at ∼0.9 atm;
these peaks in the error patterns correspond to a minimum in
the singular vectors of the CO2 Jacobian. The large errors in
the retrieved CO2 profiles are explained by the larger a pri-
ori uncertainty in the lower troposphere, and by the relatively
larger effect of errors at wavenumbers strongly weighted at
low altitudes. This is because “sensitivity” is determined by
the Jacobian; the retrieval will simply preferentially adjust
CO2 at levels where a given change in CO2 causes a larger
change in radiance. At pressures larger than ∼0.9 atm, the
error patterns of SN represent vertical scales that cannot be
resolved in the retrieval, with a vertical scale of 0.3 atm or
less. Appendix E: Empirical corrections In Sect. 3 we saw that CO2 profile retrievals have high sensi-
tivity to CO2 in the absence of errors in the a priori meteorol-
ogy and systematic errors in instrument line shape. Here we
investigate the possibility of empirically removing the effect
of those errors by de-weighting systematic spectral fitting
residuals using empirical orthogonal functions (EOFs). EOFs
have been used, for example, with retrievals from GOSAT
and OCO-2 measurements (O’Dell et al., 2018). The uncertainty in the retrieved CO2 profile is taken to be
the square root of the diagonal elements of ˆS even though the
retrieval covariance is not diagonal. It is presented in Fig. D8
as a percentage of the a priori uncertainties. The retrieval er-
ror is always smaller than the a priori covariance by construc-
tion in optimal estimation, so this alone gives no indication
of a successful retrieval. But the retrieval is more sensitive
to altitudes where the retrieval uncertainty is a smaller frac-
tion of the a priori uncertainty. The error from the diagonal
of SN and Sm is also shown. In addition to SN, the smooth-
ing contribution from state vector elements other than CO2
scale factors is shown as Si, the interference error covariance S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra 3106 Figure D5. Same as Fig. D1 but for the Weak2 window. Figure D3. Same as Fig. D1 but for the TCCON2 window. Figure D3. Same as Fig. D1 but for the TCCON2 window. Figure D5. Same as Fig. D1 but for the Weak2 window. Appendix D: Information content and error analysis The retrieval covariance matrix ˆS can be expressed as a
sum of the null space covariance SN and the measurement
noise covariance Sm (Rodgers, 1990): The singular value decomposition of the CO2 Jacobian ma-
trix can provide information on the relative precision with
which different vertical patterns are measured. The Jacobian
matrix K is decomposed into SN =
S−1
a
+ KT S−1
y K
−1
S−1
a
S−1
a
+ KT S−1
y K
−1
,
(D2)
Sm =
S−1
a
+ KT S−1
y K
−1
KT S−1
y
× K
S−1
a
+ KT S−1
y K
−1
. (D3) (D2) K(nmp,nlev) = U(nmp,nlev)L(nlev,nlev)
× VT (nlev,nlev),
(D1) (D1) × K
S−1
a
+ KT S−1
y K
−1
. (D3) where nmp is the number of measured spectral points, nlev
is the number of atmospheric levels, U is the matrix of left
singular vectors, L is the diagonal matrix of singular values,
and VT is the transpose of the matrix of right singular vec-
tors. The right singular vectors of K associated with the eight
largest singular values are shown in Figs. D1 to D5 for each
CO2 band on 14 January 2012. The right singular vectors rep-
resent independently measured vertical patterns with a preci-
sion indicated by their corresponding singular values shown
above each panel. The singular values are also shown as a (D3) The error patterns of these matrices hold information on ver-
tical structures in the CO2 profiles that the retrieval cannot
resolve, due to the smoothing effect of the a priori covariance
matrix Sa in the case of SN, and due to the effect of measure-
ment noise in the case of Sm, as the measurement error co-
variance matrix Sy only represents random errors in the mea-
sured radiances. The error patterns of a matrix are defined as
its eigenvectors multiplied by the square root of their cor- https://doi.org/10.5194/amt-14-3087-2021 Atmos. Meas. Tech., 14, 3087–3118, 2021 (Rodgers and Connor, 2003): (Rodgers and Connor, 2003): Figure D4. Same as Fig. D1 but for the Weak1 window. Si = AxeSa,eAT
xe,
(D4) (D4) S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra 3107 Figure D6. The four largest error patterns of the null space covariance matrix for a Lamont spectrum measured on 14 January 2012. gest error patterns of the null space covariance matrix for a Lamont spectrum measured on 14 January 2012. Figure D6. The four largest error patterns of the null space covariance matrix for a Lamont spectrum measur Figure D7. Same as Fig. D6 but for the measurement noise covariance matrix. Figure D7. Same as Fig. D6 but for the measurement noise covariance matrix. Figure D8. Panel (a) shows the square root of the diagonal elements
of the retrieval total error covariance matrix ˆS, the null space covari-
ance matrix SN, the interference error covariance matrix Si, and the
measurement noise error covariance matrix Sm expressed as a frac-
tion of the a priori uncertainty σ a. Each line is the average from
the set of 8 days with AirCore measurements over Lamont, and the
bands indicate the standard deviation. Panel (b) shows the a priori
uncertainty. We use a linear combination of left singular vectors. Each
singular vector is associated with a scaling factor. The scal-
ing factor is part of the state vector and adjusted during
the retrieval using 100 % uncertainty. Before each inversion
step, the spectrum “c” calculated with the forward model be-
comes: c = c +
N
X
i=1
aiui,
(E1) (E1) where N is the number of EOFs to use, ordered with decreas-
ing singular value. The first EOF, associated with the highest
singular value, is like the scaled average residual from all the
spectral residuals in the matrix M. Our implementation dif-
fers from that described by O’Dell et al. (2018) in that here
the EOFs are derived from a set of residuals obtained using
scaling retrievals, and not using profile retrievals. Since they
are meant to remove systematic errors in the calculated spec-
tra before the retrieval adjusts the CO2 scaling factors, the
EOFs should be derived from a large set of residuals obtained
with scaling retrievals to have a significant effect on the pro-
file retrieval. If they are derived from residuals obtained with
profile retrievals, these mainly include systematic error pat-
terns corresponding to interfering species, which are not the
main source of deviations in retrieved CO2 profiles. E1
Empirical orthogonal functions To reduce the effect of systematic residuals on retrieved pro-
files, EOFs of the spectral fitting residuals were derived to
find and remove systematic patterns in the residuals related to
temperature errors, instrument line shape, and other effects. The residuals divided by air mass from a set of retrievals cov-
ering a wide range of observational conditions are stored in
a matrix M(m,n), with n the number of spectra and m the
number of spectral points. Then a singular value decompo-
sition is performed on this matrix. The columns of the ma-
trix of left singular vectors are orthogonal basis vectors of Atmos. Meas. Tech., 14, 3087–3118, 2021 https://doi.org/10.5194/amt-14-3087-2021 E2
Results One year of measurements from the East Trout Lake (SK,
Canada) TCCON station were processed in three ways: with
scaling retrievals, with profile retrievals, and with profile re-
trievals including the first EOF derived from residuals ob-
tained with the scaling retrievals. The residuals used to de-
rive the EOFs are filtered such that spectra that would not
pass the TCCON quality checks are not included. To avoid
isolated spectra with large residuals having a disproportion-
ate impact on the singular value decomposition of the ma-
trix of residuals, all the spectra are ordered by increasing so-
lar zenith angle and filtered based on the root mean square
of the residuals: the 500-point rolling median is computed,
and the median of the 500-point rolling standard deviation
is used as an estimate of the standard deviation σ; then only
spectra within 1σ of the rolling median for all windows are
used to derive the EOFs. The matrix of residuals resulting
from this filtering includes 42 037 out of 64 245 total spec-
tra. XCO2 was retrieved from each window separately. The
statistics on the retrieved XCO2 error are shown in Table E1
for each retrieval type and for each window. In all win-
dows but the Strong window, the changes in XCO2 error
between the different retrieval methods are small, less than
0.05 ppm. This is 8 times smaller than the reported TCCON
1σ single-measurement precision of 0.4 ppm. However, the
mean XCO2 error is ∼55 % larger in the strong window with
profile retrievals compared to scaling retrievals. Figure E1. Fraction of the variability in the spectral residuals ac-
counted for by each empirical orthogonal function in each CO2 win-
dow. The EOF numbers are shown in decreasing order of singular
value. Panel (b) highlights the blue rectangle inside panel (a). error patterns may be attributed to systematic errors for the
first EOF, such as errors in spectroscopy, or in the instrument
line shape, or a persistent bias in meteorology. The error pat-
terns can also correspond to errors in the a priori meteorol-
ogy. The temporal frequency of each error pattern is con-
tained in the corresponding right singular vector. The right
singular vectors could help diagnose, for example, biases in a
priori temperature profiles on different timescales. E2
Results The right
singular vectors can also be used to find correlations between
each spectral residual pattern and other quantities measured
in time, such as differences between a priori and measured
meteorology. Figures E2 to E6 show quantities derived from each type
of retrieval for an example day and for each window. In each
window, the profile retrieval with the first EOF appears as an
intermediate case between the profile retrieval and the scal-
ing retrieval. In each case, the root mean square of the resid-
uals is smaller for profile retrieval with the first EOF, but the
XCO2 error is not necessarily smaller. If the residual patterns corresponding to CO2 lines have
the same shape as residuals caused by errors in the a priori
CO2 profile shape, adjustments to the CO2 scaling factors
will compete with adjustments to the EOF scaling factors in
the retrieval. Because higher-order EOFs are associated with
residuals with different time periodicity, they can also intro-
duce errors that do not exist in calculated spectra. We chose
to only include the first EOF, which represents residual pat-
terns common to most spectra. The leading EOF can explain
40 % to 52 % of the variability in the residuals, depending on
the window, as shown in Fig. E1. The fraction of variability
is obtained as the singular value of a given EOF divided by
the sum of all singular values. The first 10 EOFs in each win-
dow are above the noise level of singular values and account
for over 90 % of the variability in the residuals. y
In Fig. E7a, XCO2 differences are shown between profile
and scaling retrievals, and between profile retrievals includ-
ing the first EOF and scaling retrievals in Fig. E7b. We have
seen that differences in XCO2 error between the different re-
trieval types are within 0.05 ppm. However, differences in
XCO2 between profile and scaling retrievals can be several
times larger than the XCO2 error, indicating different sources
of bias between profile and scaling retrievals. In the Weak1
window, the median of the XCO2 absolute differences are
∼4 times larger than the median XCO2 error and ∼3 times
larger in the Strong window. In the TCCON1, TCCON2,
and Weak2 windows, the median of the XCO2 absolute dif-
ference is smaller than the median XCO2 error. S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra 3108 S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra 3108
S. Roche et al.: Retrieval of atmospheric CO
Figure E1. Fraction of the variability in the spectral residuals ac-
counted for by each empirical orthogonal function in each CO2 win-
dow. The EOF numbers are shown in decreasing order of singular
value. Panel (b) highlights the blue rectangle inside panel (a). S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra Atmos. Meas. Tech., 14, 3087–3118, 2021 S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra When
using scaling retrieval residuals, each EOF includes different
error patterns corresponding to CO2 absorption lines. These where N is the number of EOFs to use, ordered with decreas-
ing singular value. The first EOF, associated with the highest
singular value, is like the scaled average residual from all the
spectral residuals in the matrix M. Our implementation dif-
fers from that described by O’Dell et al. (2018) in that here
the EOFs are derived from a set of residuals obtained using
scaling retrievals, and not using profile retrievals. Since they
are meant to remove systematic errors in the calculated spec-
tra before the retrieval adjusts the CO2 scaling factors, the
EOFs should be derived from a large set of residuals obtained
with scaling retrievals to have a significant effect on the pro-
file retrieval. If they are derived from residuals obtained with
profile retrievals, these mainly include systematic error pat-
terns corresponding to interfering species, which are not the
main source of deviations in retrieved CO2 profiles. When
using scaling retrieval residuals, each EOF includes different
error patterns corresponding to CO2 absorption lines. These Figure D8. Panel (a) shows the square root of the diagonal elements
of the retrieval total error covariance matrix ˆS, the null space covari-
ance matrix SN, the interference error covariance matrix Si, and the
measurement noise error covariance matrix Sm expressed as a frac-
tion of the a priori uncertainty σ a. Each line is the average from
the set of 8 days with AirCore measurements over Lamont, and the
bands indicate the standard deviation. Panel (b) shows the a priori
uncertainty. Figure D8. Panel (a) shows the square root of the diagonal elements
of the retrieval total error covariance matrix ˆS, the null space covari-
ance matrix SN, the interference error covariance matrix Si, and the
measurement noise error covariance matrix Sm expressed as a frac-
tion of the a priori uncertainty σ a. Each line is the average from
the set of 8 days with AirCore measurements over Lamont, and the
bands indicate the standard deviation. Panel (b) shows the a priori
uncertainty. the residuals and those associated with the largest singular
values represent the main patterns in the residuals, while the
corresponding right singular vectors can provide information
on the temporal frequency of these patterns. https://doi.org/10.5194/amt-14-3087-2021 Atmos. Meas. Tech., 14, 3087–3118, 2021 S. S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra 3109 Figure E2. Quantities derived from retrievals on East Trout Lake
measurements on 29 March 2018 for the Strong window. The re-
trieval type is indicated by the legend. Panel (a) shows the column-
integrated CO2 scale factor. Panel (b) shows XCO2 and panel (c)
shows the XCO2 error. Panel (d) shows the root mean square of the
residuals as a fraction of the continuum level. Figure E3. Same as Fig. E2 but for the TCCON1 window. Figure E2. Quantities derived from retrievals on East Trout Lake
measurements on 29 March 2018 for the Strong window. The re-
trieval type is indicated by the legend. Panel (a) shows the column-
integrated CO2 scale factor. Panel (b) shows XCO2 and panel (c)
shows the XCO2 error. Panel (d) shows the root mean square of the
residuals as a fraction of the continuum level. Figure E3. Same as Fig. E2 but for the TCCON1 window. Table E2. Squared Pearson correlation coefficient for XCO2 be-
tween the scaling and profile retrievals (SCL–PRF), and between
the profile retrieval with the first EOF and the profile retrieval
(EOF–PRF). EOF in a profile retrieval reduces the XCO2 differences be-
tween the scaling and profile retrievals, but the XCO2 of the
profile retrieval with EOF is more strongly correlated with
the XCO2 of the profile retrieval than that of the scaling re-
trieval as shown in Table E2. R2
SCL–PRF
EOF–PRF
Strong
0.9368
0.9929
Weak1
0.9633
0.9951
Weak2
0.9586
0.9814
TCCON1
0.9922
0.9995
TCCON2
0.9931
0.9999
Atmos. Meas. Tech., 14, 3087–3118, 2021 R2
SCL–PRF
EOF–PRF
Strong
0.9368
0.9929
Weak1
0.9633
0.9951
Weak2
0.9586
0.9814
TCCON1
0.9922
0.9995
TCCON2
0.9931
0.9999 When compared to preliminary data from aircraft mea-
surements, the deviations in the CO2 profiles obtained with
profile retrievals are larger than the vertical variations in the
aircraft measurement. When the retrieved profiles present Atmos. Meas. Tech., 14, 3087–3118, 2021 Atmos. Meas. Tech., 14, 3087–3118, 2021 https://doi.org/10.5194/amt-14-3087-2021 https://doi.org/10.5194/amt-14-3087-2021 E2
Results In all but
the Weak1 window, the XCO2 differences are 25 % to 35 %
smaller between August and November than for the rest of
the year. In Fig. E7b, the XCO2 differences between the pro-
file retrievals with EOF and the scaling retrievals are smaller
and more consistent between windows than in Fig. E7a. And
the median of the XCO2 absolute differences is smaller than
the median XCO2 error in all windows. Including the leading Atmos. Meas. Tech., 14, 3087–3118, 2021 https://doi.org/10.5194/amt-14-3087-2021 Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra
310
ble E1. Statistics on the retrieved XCO2 error for one year of measurements at the East Trout Lake TCCON station. “SD” indicates th
andard deviation. XCO2 error
Scaling retrieval
Profile retrieval
Profile retrieval with
(ppm)
the first EOF
Window
Mean
Median
SD
Mean
Median
SD
Mean
Median
SD
Strong
0.51
0.38
0.37
0.79
0.63
0.60
0.78
0.61
0.59
Weak1
0.89
0.64
0.68
0.91
0.67
0.66
0.90
0.66
0.66
Weak2
0.80
0.56
0.64
0.81
0.61
0.56
0.80
0.61
0.56
TCCON1
0.74
0.48
0.66
0.79
0.51
0.70
0.79
0.51
0.70
TCCON2
0.69
0.45
0.61
0.74
0.47
0.66
0.74
0.47
0.66
gure E2. Quantities derived from retrievals on East Trout Lake
easurements on 29 March 2018 for the Strong window. The re-
eval type is indicated by the legend. Panel (a) shows the column-
egrated CO2 scale factor. Panel (b) shows XCO2 and panel (c)
ows the XCO2 error. Panel (d) shows the root mean square of the
siduals as a fraction of the continuum level. OF in a profile retrieval reduces the XCO2 differences be-
Figure E3. Same as Fig. E2 but for the TCCON1 window. Table E2. Squared Pearson correlation coefficient for XCO2 b
tween the scaling and profile retrievals (SCL–PRF), and betwee
the profile retrieval with the first EOF and the profile retriev
(EOF–PRF). S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra https://doi.org/10.5194/amt-14-3087-2021 S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra In panel (b), the XCO2 obtained from the scaling retrieval is subtracted from the XCO2 obtained from the profile retrieval with EOF. In
panel (c), the XCO2 error from the scaling retrieval is shown, with the median values as dashed lines. In the top two panels, the horizontal
dashed lines show the median values of absolute differences in XCO2. Figure F1. Zero-level offset retrieved from the Strong CO2 window
for the Lamont spectra coincident within ±1 h of the last AirCore
sampling time and within ±1.5 h of the closest a priori time on each
of the days indicated by the legend. The dashed lines mark the me-
dian value for each date. S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra 3110 p
2
p
g
p
Figure E4. Same as Fig. E2 but for the TCCON2 window. Figure E6. Same as Fig. E2 but for the Weak2 window. Figure E6. Same as Fig. E2 but for the Weak2 window. Figure E6. Same as Fig. E2 but for the Weak2 window. Figure E4. Same as Fig. E2 but for the TCCON2 window. Figure E4. Same as Fig. E2 but for the TCCON2 window. Figure E6. Same as Fig. E2 but for the Weak2 window. Figure E4. Same as Fig. E2 but for the TCCON2 window. large deviations typical of temperature errors like that in
Fig. 9b, the CO2 profile obtained from profile retrieval with
the first EOF reduces the amplitude of the deviations, but the
shape persists. This is expected as the first EOF represents
the average residuals, which should not include residual fea-
tures caused by temperature errors, unless the temperature
errors were always biased in the same way. We would expect
the first EOF to reduce deviations like that in Fig. 9a. In such
cases, the CO2 profiles obtained from profile retrieval with
the first EOF are smoother than profile retrievals but present
no clear advantage over scaling retrievals. Figure E5. Same as Fig. E2 but for the Weak1 window. Figure E5. Same as Fig. E2 but for the Weak1 window. Atmos. Meas. Tech., 14, 3087–3118, 2021 https://doi.org/10.5194/amt-14-3087-2021 https://doi.org/10.5194/amt-14-3087-2021 S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra 3111 Figure E7. In panel (a), the XCO2 obtained from the scaling retrieval is subtracted from the XCO2 obtained from the profile retrieval. In panel (b), the XCO2 obtained from the scaling retrieval is subtracted from the XCO2 obtained from the profile retrieval with EOF. In
panel (c), the XCO2 error from the scaling retrieval is shown, with the median values as dashed lines. In the top two panels, the horizontal
dashed lines show the median values of absolute differences in XCO2. Figure E7. In panel (a), the XCO2 obtained from the scaling retrieval is subtracted from the XCO2 obtained from the profile retrieval. https://doi.org/10.5194/amt-14-3087-2021 Appendix F: Synthetic spectra, perturbed field of view
and zero-level offset The difference in the retrieved profiles with and without the added
offset is shown for each window and for all the days with AirCore
profiles over Lamont. In the Strong window, where the offset is re-
trieved, the differences are less than 0.001 ppm. S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra 3112 3112
S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra
Figure F2. The left-hand panels show CO2 profiles retrieved using
synthetic spectra. In (a), we use the AirCore profile, which was used
to generate the synthetic spectra, as the a priori profile. In (c), the
internal field of view is perturbed by +7 %, increasing the width of
the ILS. In (e), the zero-level offset is perturbed by +0.002 and is
not retrieved in the Strong window. Panels (b), (d), and (f) show the
difference between the retrieved profiles and AirCore, correspond-
ing to (a), (c), and (e) respectively. Figure F3. Using real Lamont spectra with the AirCore profile as
the a priori profile, the zero-level offset was first retrieved from the
Strong window and then added in the Weak and TCCON windows. The difference in the retrieved profiles with and without the added
offset is shown for each window and for all the days with AirCore
profiles over Lamont. In the Strong window, where the offset is re-
trieved, the differences are less than 0.001 ppm. O2 vertical profiles from ground-based near-infrared spectra
Figure F3. Using real Lamont spectra with the AirCore profile as
the a priori profile, the zero-level offset was first retrieved from the
Strong window and then added in the Weak and TCCON windows. The difference in the retrieved profiles with and without the added
offset is shown for each window and for all the days with AirCore
profiles over Lamont. In the Strong window, where the offset is re-
trieved, the differences are less than 0.001 ppm. Figure F2. The left-hand panels show CO2 profiles retrieved using
synthetic spectra. In (a), we use the AirCore profile, which was used
to generate the synthetic spectra, as the a priori profile. In (c), the
internal field of view is perturbed by +7 %, increasing the width of
the ILS. Appendix F: Synthetic spectra, perturbed field of view
and zero-level offset The saturated lines of the Strong window allow a zero-level
offset to be fit. Figure F1 shows the zero-level offset retrieved
from the Strong window using real spectra for each of the
days with Lamont data used in Sect. 3. The median abso-
lute value is at most 0.001 on 23 July 2013. The effect of
a zero-level offset on retrieved profiles was tested with syn-
thetic spectra. Figure F2a and b are the same as Fig. 4a and b
and show profiles retrieved from synthetic spectra in the ref-
erence case, when no perturbation is applied. Figure F2e and
f show the effect of a +0.002 perturbation to the zero-level
offset, without retrieving it in the Strong window. This has a
large effect in the profile retrieved from the Strong window,
with deviations from the truth within 30 ppm, and a smaller
effect in the other bands with deviations up to 10 ppm. Figure F1. Zero-level offset retrieved from the Strong CO2 window
for the Lamont spectra coincident within ±1 h of the last AirCore
sampling time and within ±1.5 h of the closest a priori time on each
of the days indicated by the legend. The dashed lines mark the me-
dian value for each date. In Fig. F2c and d we also consider the effect of one type of
ILS error by perturbing the internal field of view by +7 %,
which leads to a widening of the ILS. The unperturbed in-
ternal field of view of the spectrometer is 2.4 mrad. The de-
viations from the truth are within 1 ppm for P > 0.5 atm and
within 3 ppm for P < 0.5 atm. 3 ppm at all altitudes as shown in Fig. F3 using days with
AirCore profiles at Lamont. This sensitivity test shows the effect of zero-level offsets
will not be a major source of variability in the retrieved pro-
files. If the zero-level offset retrieved from the Strong win-
dow is added to the TCCON and Weak windows before the
retrieval, the change in the retrieved profiles is less than Atmos. Meas. Tech., 14, 3087–3118, 2021 https://doi.org/10.5194/amt-14-3087-2021 O2 vertical profiles from ground-based near-infrared spectra
Figure F3. Using real Lamont spectra with the AirCore profile as
the a priori profile, the zero-level offset was first retrieved from the
Strong window and then added in the Weak and TCCON windows. https://doi.org/10.5194/amt-14-3087-2021 Atmos. Meas. Tech., 14, 3087–3118, 2021 References Code availability. It is our intention to make the profile retrieval
code available as part of a future version of GGG and hence publicly
available through TCCON; until then it can be made available upon
request to the corresponding author. Andrews, A. E., Boering, K. A., Daube, B. C., Wofsy, S. C.,
Loewenstein, M., Jost, H., Podolske, J. R., Webster, C. R.,
Herman, R. L., Scott, D. C., Flesch, G. J., Moyer, E. J.,
Elkins, J. W., Dutton, G. S., Hurst, D. F., Moore, F. L., Ray,
E. A., Romashkin, P. A., and Strahan, S. E.: Mean ages of
stratospheric air derived from in situ observations of CO2,
CH4, and N2O, J. Geophys. Res.-Atmos., 106, 32295–32314,
https://doi.org/10.1029/2001JD000465, 2001. Data availability. The data used in this study consist of synthetic
spectra generated with GGG2020 and measured spectra from
the Lamont and East Trout Lake TCCON stations. Scaling re-
trieval products from those sites using the GGG2014 algorithm
(https://doi.org/10.14291/tccon.ggg2014.lamont01.r1/1255070,
Wennberg et al., 2016; https://doi.org/10.14291/tccon.ggg2014. easttroutlake01.r1,
Wunch
et
al.,
2018)
are
available
pub-
licly through CaltechDATA (https://tccondata.org, last access:
13 November 2020). Measured solar absorption spectra can be
obtained by contacting the TCCON site PIs. Aumann, H. H., Chahine, M. T., Gautier, C., Goldberg, M. D.,
Kalnay, E., McMillin, L. M., Revercomb, H., Rosenkranz,
P. W., Smith, W. L., Staelin, D. H., Strow, L. L., and
Susskind, J.: AIRS/AMSU/HSB on the aqua mission: De-
sign, science objectives, data products, and processing sys-
tems, IEEE Trans. Geosci. Remote Sens., 41, 253–263,
https://doi.org/10.1109/TGRS.2002.808356, 2003. Basu, S., Baker, D. F., Chevallier, F., Patra, P. K., Liu, J., and
Miller, J. B.: The impact of transport model differences on
CO2 surface flux estimates from OCO-2 retrievals of col-
umn average CO2, Atmos. Chem. Phys., 18, 7189–7215,
https://doi.org/10.5194/acp-18-7189-2018, 2018. Author contributions. SR analyzed the data and prepared the pa-
per with detailed feedback from all co-authors. JM implemented
qSDV+LM into GFIT and provided guidance for its use with
GFIT2. BJC wrote the initial GFIT2 algorithm and SR implemented
changes relevant to this study. GCT is the author of GGG and pro-
vided support and significant conceptual input with DW, KS, and
BJC. JLL and SR developed the code to generate the GGG2020 a
priori profiles. The East Trout Lake solar spectra were provided by
DW and JM. CS and BB provided the AirCore data. SCB provided
the Lamont surface and aircraft measurements. Benner, D. C., Devi, V. References M., Sung, K., Brown, L. R., Miller, C. E.,
Payne, V. H., Drouin, B. J., Yu, S., Crawford, T. J., Mantz, A. W., Smith, M. A. H., and Gamache, R. R.: Line parameters in-
cluding temperature dependences of air- and self-broadened line
shapes of 12C16O2: 2.06-µm region, J. Mol. Spectrosc., 326, 21–
47, https://doi.org/10.1016/j.jms.2016.02.012, 2016. Biraud, S. and Moyes, A.: Atmospheric Radiation Measurement
(ARM) user facility. Updated hourly. Precision Carbon Dioxide
Mixing Ratio System (PGS). 2001-04-11 to 2019-07-22, South-
ern Great Plains (SGP) Central Facility, Lamont, OK (C1), ARM
Data Center, https://doi.org/10.5439/1025262, 2001. Competing interests. The authors declare that they have no conflict
of interest. Biraud, S., Moyes, A., and Ermold, B.: Atmospheric Radiation
Measurement (ARM) user facility. Updated hourly. Flask Sam-
plers for Carbon Cycle Gases and Isotopes (GHGFLASK). 2002-04-02 to 2019-05-23, Southern Great Plains (SGP)
Central
Facility,
Lamont,
OK
(C1),
ARM
Data
Center,
https://doi.org/10.5439/1482660, 2002. Acknowledgements. The GEOS-5-FPIT data used in this study
have been provided by the Global Modeling Assimilation Office
(GMAO) at NASA Goddard Space Flight Center. The Lamont solar
spectra were provided by Coleen Roehl and Paul Wennberg from
the California Institute of Technology. The Lamont TCCON site
operates with funding from NASA. In situ observations and verti-
cal profiles collected over the Southern Great Plains were supported
by the Office of Biological and Environmental Research of the US
Department of Energy under contract no. DE-AC02-05CH11231
as part of the Atmospheric Radiation Measurement (ARM) Pro-
gram, ARM Aerial Facility (AAF), and Terrestrial Ecosystem Sci-
ence (TES) Program. The GNU parallel package (Tange, 2011) has
been used to run tests with GFIT2. Biraud, S. C., Torn, M. S., Smith, J. R., Sweeney, C., Riley, W. J.,
and Tans, P. P.: A multi-year record of airborne CO2 observations
in the US Southern Great Plains, Atmos. Meas. Tech., 6, 751–
763, https://doi.org/10.5194/amt-6-751-2013, 2013. Chevallier, F., Fisher, M., Peylin, P., Serrar, S., Bousquet, P.,
Bréon, F. M., Chédin, A., and Ciais, P.: Inferring CO2 sources
and sinks from satellite observations: Method and applica-
tion to TOVS data, J. Geophys. Res.-Atmos., 110, 1–13,
https://doi.org/10.1029/2005JD006390, 2005. Chevallier, F., Bréon, F. M., and Rayner, P. J.: Contribution of
the Orbiting Carbon Observatory to the estimation of CO2
sources and sinks: Theoretical study in a variational data as-
similation framework, J. Geophys. Res.-Atmos., 112, D09307,
https://doi.org/10.1029/2006JD007375, 2007. Financial support. This research has been supported by the Natu-
ral Sciences and Engineering Research Council of Canada (grant
no. 433842-2012) and the Canadian Space Agency (grant no. 45-
7014551). Chevallier, F., Remaud, M., O’Dell, C. W., Baker, D., Peylin, P.,
and Cozic, A.: Objective evaluation of surface- and satellite-
driven carbon dioxide atmospheric inversions, Atmos. Chem. Phys., 19, 14233–14251, https://doi.org/10.5194/acp-19-14233-
2019, 2019. Review statement. This paper was edited by Frank Hase and re-
viewed by two anonymous referees. Ciais, P., Sabine, C., Bala, G., Bopp, L., Brovkin, V., Canadell, J.,
Chhabra, A., DeFries, R., Galloway, J., Heimann, M., Jones, C., Appendix F: Synthetic spectra, perturbed field of view
and zero-level offset In (e), the zero-level offset is perturbed by +0.002 and is
not retrieved in the Strong window. Panels (b), (d), and (f) show the
difference between the retrieved profiles and AirCore, correspond-
ing to (a), (c), and (e) respectively. Figure F3. Using real Lamont spectra with the AirCore profile as
the a priori profile, the zero-level offset was first retrieved from the
Strong window and then added in the Weak and TCCON windows. The difference in the retrieved profiles with and without the added
offset is shown for each window and for all the days with AirCore
profiles over Lamont. In the Strong window, where the offset is re-
trieved, the differences are less than 0.001 ppm. Figure F2. The left-hand panels show CO2 profiles retrieved using
synthetic spectra. In (a), we use the AirCore profile, which was used
to generate the synthetic spectra, as the a priori profile. In (c), the
internal field of view is perturbed by +7 %, increasing the width of
the ILS. In (e), the zero-level offset is perturbed by +0.002 and is
not retrieved in the Strong window. Panels (b), (d), and (f) show the
difference between the retrieved profiles and AirCore, correspond-
ing to (a), (c), and (e) respectively. Atmos. Meas. Tech., 14, 3087–3118, 2021 3113 S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra N., Moazzen-Ahmadi, N., Müller, H. S. Crisp, D.: Measuring atmospheric carbon dioxide from space
with
the
Orbiting
Carbon
Observatory-2
(OCO-2),
in:
Earth
Observing
Systems
XX,
edited
by:
Butler,
J. J.,
Xiong, X. (Jack), and Gu, X., SPIE., vol. 9607, 960702,
https://doi.org/10.1117/12.2187291, 2015. P., Naumenko, O. V., Nikitin, A. V., Polyansky, O. L., Rey, M., Devi, V. M., Benner, D. C., Brown, L. R., Miller, C. E., and Toth, R. A.: Line mixing and speed dependence in CO2 at 6227.9 cm−1:
Constrained multispectrum analysis of intensities and line shapes
in the 30013 ←00001 band, J. Mol. Spectrosc., 245, 52–80,
https://doi.org/10.1016/j.jms.2007.05.015, 2007a. Hase, F., Hannigan, J. W., Coffey, M. T., Goldman, A., Höpfner,
M., Jones, N. B., Rinsland, C. P., and Wood, S. W.: Intercompar-
ison of retrieval codes used for the analysis of high-resolution,
ground-based FTIR measurements, J. Quant. Spectrosc. Radiat. Transf., 87, 25–52, https://doi.org/10.1016/j.jqsrt.2003.12.008,
2004. Devi, V. M., Benner, D. C., Brown, L. R., Miller, C. E., and Toth,
R. A.: Line mixing and speed dependence in CO2 at 6348 cm−1:
Positions, intensities, and air- and self-broadening derived with
constrained multispectrum analysis, J. Mol. Spectrosc., 242, 90–
117, https://doi.org/10.1016/j.jms.2007.02.018, 2007b. Karion,
A.,
Sweeney,
C.,
Tans,
P.,
and
Newberger,
T.:
AirCore:
An
innovative
atmospheric
sampling
sys-
tem,
J. Atmos. Ocean. Technol.,
27,
1839–1853,
https://doi.org/10.1175/2010JTECHA1448.1, 2010. Dlugokencky, E. J., Mund, J. W., Crotwell, A. M., Crotwell, M. J., and Thoning, K. W.: Atmospheric Carbon Dioxide Dry Air
Mole Fractions from the NOAA ESRL Carbon Cycle Coopera-
tive Global Air Sampling Network, 1968–2018, Version: 2019-
07, https://doi.org/10.15138/wkgj-f215, 2019. Keppel-Aleks, G., Wennberg, P. O., and Schneider, T.: Sources
of variations in total column carbon dioxide, Atmos. Chem. Phys.,
11,
3581–3593,
https://doi.org/10.5194/acp-11-3581-
2011, 2011. Keppel-Aleks, G., Wennberg, P. O., Washenfelder, R. A., Wunch,
D., Schneider, T., Toon, G. C., Andres, R. J., Blavier, J.-F., Con-
nor, B., Davis, K. J., Desai, A. R., Messerschmidt, J., Notholt,
J., Roehl, C. M., Sherlock, V., Stephens, B. B., Vay, S. A., and
Wofsy, S. C.: The imprint of surface fluxes and transport on vari-
ations in total column carbon dioxide, Biogeosciences, 9, 875–
891, https://doi.org/10.5194/bg-9-875-2012, 2012. Dohe, S.: Measurements of atmospheric CO2 columns using
ground-based FTIR spectra, PhD thesis, Karlsruhe Institute of
Technology, Karlsruhe, Germany, 161 pp., 2013. Eldering, A., Taylor, T. E., O’Dell, C. W., and Pavlick, R.:
The OCO-3 mission: measurement objectives and expected
performance based on 1 year of simulated data, Atmos. Meas. S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra 3114 Tian, H., Tilbrook, B., Tubiello, F. N., van der Werf, G. R., Wilt-
shire, A. J., and Zaehle, S.: Global Carbon Budget 2019, Earth
Syst. Sci. Data, 11, 1783–1838, https://doi.org/10.5194/essd-11-
1783-2019, 2019. Quéré, C. Le, Myneni, R., Piao, S., and Thornton, P.: Carbon
and Other Biogeochemical Cycles, in: Climate Change 2013 –
The Physical Science Basis, edited by: Intergovernmental Panel
on Climate Change, Cambridge University Press, Cambridge,
United Kingdom and New York, NY, USA, 465–570, 2013. Quéré, C. Le, Myneni, R., Piao, S., and Thornton, P.: Carbon
and Other Biogeochemical Cycles, in: Climate Change 2013 –
The Physical Science Basis, edited by: Intergovernmental Panel
on Climate Change, Cambridge University Press, Cambridge,
United Kingdom and New York, NY, USA, 465–570, 2013. Gordon, I. E., Rothman, L. S., Hill, C., Kochanov, R. V., Tan, Y.,
Bernath, P. F., Birk, M., Boudon, V., Campargue, A., Chance, K. V., Drouin, B. J., Flaud, J.-M., Gamache, R. R., Hodges, J. T.,
Jacquemart, D., Perevalov, V. I., Perrin, A., Shine, K. P., Smith,
M.-A. H., Tennyson, J., Toon, G. C., Tran, H., Tyuterev, V. G.,
Barbe, A., Császár, A. G., Devi, V. M., Furtenbacher, T., Har-
rison, J. J., Hartmann, J.-M., Jolly, A., Johnson, T. J., Karman,
T., Kleiner, I., Kyuberis, A. A., Loos, J., Lyulin, O. M., Massie,
S. T., Mikhailenko, S. N., Moazzen-Ahmadi, N., Müller, H. S. P., Naumenko, O. V., Nikitin, A. V., Polyansky, O. L., Rey, M.,
Rotger, M., Sharpe, S. W., Sung, K., Starikova, E., Tashkun, S. A., Auwera, J. Vander, Wagner, G., Wilzewski, J., Wcisło, P.,
Yu, S., and Zak, E. J.: The HITRAN2016 molecular spectro-
scopic database, J. Quant. Spectrosc. Radiat. Transf., 203, 3–69,
https://doi.org/10.1016/j.jqsrt.2017.06.038, 2017. Connor, B. J., Sherlock, V., Toon, G., Wunch, D., and Wennberg,
P. O.: GFIT2: an experimental algorithm for vertical profile re-
trieval from near-IR spectra, Atmos. Meas. Tech., 9, 3513–3525,
https://doi.org/10.5194/amt-9-3513-2016, 2016. Jacquemart, D., Perevalov, V. I., Perrin, A., Shine, K. P., Smith, Barbe, A., Császár, A. G., Devi, V. M., Furtenbacher, T., Har- Crisp, D.: NASA Orbiting Carbon Observatory: measuring the col-
umn averaged carbon dioxide mole fraction from space, J. Appl. Remote Sens., 2, 023508, https://doi.org/10.1117/1.2898457,
2008. rison, J. J., Hartmann, J.-M., Jolly, A., Johnson, T. J., Karman, T., Kleiner, I., Kyuberis, A. A., Loos, J., Lyulin, O. M., Massie, S. T., Mikhailenko, S. https://doi.org/10.5194/amt-14-3087-2021 Atmos. Meas. Tech., 14, 3087–3118, 2021 Atmos. Meas. Tech., 14, 3087–3118, 2021 S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra R., van Heuven, S., Van-
demark, D., Viovy, N., Wiltshire, A., Zaehle, S., and Zeng, N.:
Global Carbon Budget 2015, Earth Syst. Sci. Data, 7, 349–396,
https://doi.org/10.5194/essd-7-349-2015, 2015b. Laughner, J. L., Andrews, A., Roche, S., Kiel, M., Toon, G. C.,
Wunch, D., ACE-FTS team, ATom team, AirCore team, and
Wennberg, P. O.: Revised formulation of the TCCON priors in
GGG2020, in preparation, 2021a. p
g
Le Quéré, C., Andrew, R. M., Canadell, J. G., Sitch, S., Kors-
bakken, J. I., Peters, G. P., Manning, A. C., Boden, T. A., Tans,
P. P., Houghton, R. A., Keeling, R. F., Alin, S., Andrews, O. D.,
Anthoni, P., Barbero, L., Bopp, L., Chevallier, F., Chini, L. P.,
Ciais, P., Currie, K., Delire, C., Doney, S. C., Friedlingstein, P.,
Gkritzalis, T., Harris, I., Hauck, J., Haverd, V., Hoppema, M.,
Klein Goldewijk, K., Jain, A. K., Kato, E., Körtzinger, A., Land-
schützer, P., Lefèvre, N., Lenton, A., Lienert, S., Lombardozzi,
D., Melton, J. R., Metzl, N., Millero, F., Monteiro, P. M. S.,
Munro, D. R., Nabel, J. E. M. S., Nakaoka, S., O’Brien, K.,
Olsen, A., Omar, A. M., Ono, T., Pierrot, D., Poulter, B., Rö-
denbeck, C., Salisbury, J., Schuster, U., Schwinger, J., Séférian,
R., Skjelvan, I., Stocker, B. D., Sutton, A. J., Takahashi, T., Tian,
H., Tilbrook, B., van der Laan-Luijkx, I. T., van der Werf, G. R., Viovy, N., Walker, A. P., Wiltshire, A. J., and Zaehle, S.:
Global Carbon Budget 2016, Earth Syst. Sci. Data, 8, 605–649,
https://doi.org/10.5194/essd-8-605-2016, 2016. Laughner, J., Andrews, A., Roche, S., Kiel, M., and Toon,
G.:
ginput
v1.0.10:
GGG2020
prior
profile
software
(Version
1.0.10),
CaltechDATA
[Computer
software],
https://doi.org/10.22002/D1.1944, 2021b. Le Quéré, C., Andres, R. J., Boden, T., Conway, T., Houghton, R. A., House, J. I., Marland, G., Peters, G. P., van der Werf, G. R.,
Ahlström, A., Andrew, R. M., Bopp, L., Canadell, J. G., Ciais,
P., Doney, S. C., Enright, C., Friedlingstein, P., Huntingford, C.,
Jain, A. K., Jourdain, C., Kato, E., Keeling, R. F., Klein Gold-
ewijk, K., Levis, S., Levy, P., Lomas, M., Poulter, B., Raupach,
M. R., Schwinger, J., Sitch, S., Stocker, B. D., Viovy, N., Zaehle,
S., and Zeng, N.: The global carbon budget 1959–2011, Earth
Syst. Sci. Data, 5, 165–185, https://doi.org/10.5194/essd-5-165-
2013, 2013. Le Quéré, C., Peters, G. P., Andres, R. J., Andrew, R. M., Boden,
T. A., Ciais, P., Friedlingstein, P., Houghton, R. S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra A., Marland, G.,
Moriarty, R., Sitch, S., Tans, P., Arneth, A., Arvanitis, A., Bakker,
D. C. E., Bopp, L., Canadell, J. G., Chini, L. P., Doney, S. C.,
Harper, A., Harris, I., House, J. I., Jain, A. K., Jones, S. D., Kato,
E., Keeling, R. F., Klein Goldewijk, K., Körtzinger, A., Koven,
C., Lefèvre, N., Maignan, F., Omar, A., Ono, T., Park, G.-H.,
Pfeil, B., Poulter, B., Raupach, M. R., Regnier, P., Rödenbeck, C.,
Saito, S., Schwinger, J., Segschneider, J., Stocker, B. D., Taka-
hashi, T., Tilbrook, B., van Heuven, S., Viovy, N., Wanninkhof,
R., Wiltshire, A., and Zaehle, S.: Global carbon budget 2013,
Earth Syst. Sci. Data, 6, 235–263, https://doi.org/10.5194/essd-
6-235-2014, 2014. Le Quéré, C., Andrew, R. M., Friedlingstein, P., Sitch, S., Pongratz,
J., Manning, A. C., Korsbakken, J. I., Peters, G. P., Canadell,
J. G., Jackson, R. B., Boden, T. A., Tans, P. P., Andrews, O. D., Arora, V. K., Bakker, D. C. E., Barbero, L., Becker, M.,
Betts, R. A., Bopp, L., Chevallier, F., Chini, L. P., Ciais, P.,
Cosca, C. E., Cross, J., Currie, K., Gasser, T., Harris, I., Hauck,
J., Haverd, V., Houghton, R. A., Hunt, C. W., Hurtt, G., Ily-
ina, T., Jain, A. K., Kato, E., Kautz, M., Keeling, R. F., Klein
Goldewijk, K., Körtzinger, A., Landschützer, P., Lefèvre, N.,
Lenton, A., Lienert, S., Lima, I., Lombardozzi, D., Metzl, N.,
Millero, F., Monteiro, P. M. S., Munro, D. R., Nabel, J. E. M. S., Nakaoka, S., Nojiri, Y., Padin, X. A., Peregon, A., Pfeil, B.,
Pierrot, D., Poulter, B., Rehder, G., Reimer, J., Rödenbeck, C.,
Schwinger, J., Séférian, R., Skjelvan, I., Stocker, B. D., Tian, H.,
Tilbrook, B., Tubiello, F. N., van der Laan-Luijkx, I. T., van der
Werf, G. R., van Heuven, S., Viovy, N., Vuichard, N., Walker,
A. P., Watson, A. J., Wiltshire, A. J., Zaehle, S., and Zhu, D.:
Global Carbon Budget 2017, Earth Syst. Sci. Data, 10, 405–448,
https://doi.org/10.5194/essd-10-405-2018, 2018a. C. E., Cross, J., Currie, K., Gasser, T., Harris, I., Hauck, Lenton, A., Lienert, S., Lima, I., Lombardozzi, D., Metzl, N., Le Quéré, C., Moriarty, R., Andrew, R. M., Peters, G. P., Ciais, P.,
Friedlingstein, P., Jones, S. D., Sitch, S., Tans, P., Arneth, A.,
Boden, T. A., Bopp, L., Bozec, Y., Canadell, J. G., Chini, L. P.,
Chevallier, F., Cosca, C. E., Harris, I., Hoppema, M., Houghton,
R. A., House, J. I., Jain, A. S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra 3115 Boden, T. A., Houghton, R. A., House, J. I., Keeling, R. F., Tans,
P., Arneth, A., Bakker, D. C. E., Barbero, L., Bopp, L., Chang,
J., Chevallier, F., Chini, L. P., Ciais, P., Fader, M., Feely, R. A.,
Gkritzalis, T., Harris, I., Hauck, J., Ilyina, T., Jain, A. K., Kato,
E., Kitidis, V., Klein Goldewijk, K., Koven, C., Landschützer,
P., Lauvset, S. K., Lefèvre, N., Lenton, A., Lima, I. D., Metzl,
N., Millero, F., Munro, D. R., Murata, A., Nabel, J. E. M. S.,
Nakaoka, S., Nojiri, Y., O’Brien, K., Olsen, A., Ono, T., Pérez,
F. F., Pfeil, B., Pierrot, D., Poulter, B., Rehder, G., Rödenbeck,
C., Saito, S., Schuster, U., Schwinger, J., Séférian, R., Steinhoff,
T., Stocker, B. D., Sutton, A. J., Takahashi, T., Tilbrook, B., van
der Laan-Luijkx, I. T., van der Werf, G. R., van Heuven, S., Van-
demark, D., Viovy, N., Wiltshire, A., Zaehle, S., and Zeng, N.:
Global Carbon Budget 2015, Earth Syst. Sci. Data, 7, 349–396,
https://doi.org/10.5194/essd-7-349-2015, 2015b. Boden, T. A., Houghton, R. A., House, J. I., Keeling, R. F., Tans, Kuze, A., Suto, H., Shiomi, K., Kawakami, S., Tanaka, M., Ueda,
Y., Deguchi, A., Yoshida, J., Yamamoto, Y., Kataoka, F., Tay-
lor, T. E., and Buijs, H. L.: Update on GOSAT TANSO-
FTS performance, operations, and data products after more
than 6 years in space, Atmos. Meas. Tech., 9, 2445–2461,
https://doi.org/10.5194/amt-9-2445-2016, 2016. P., Arneth, A., Bakker, D. C. E., Barbero, L., Bopp, L., Chang, J., Chevallier, F., Chini, L. P., Ciais, P., Fader, M., Feely, R. A.,
Gkritzalis, T., Harris, I., Hauck, J., Ilyina, T., Jain, A. K., Kato, P., Lauvset, S. K., Lefèvre, N., Lenton, A., Lima, I. D., Metzl, p
g
Lamouroux, J., Régalia, L., Thomas, X., Vander Auwera, J.,
Gamache, R. R., and Hartmann, J.-M.: CO2 line-mixing database
and software update and its tests in the 2.1 µm and 4.3 µm
regions, J. Quant. Spectrosc. Radiat. Transf., 151, 88–96,
https://doi.org/10.1016/j.jqsrt.2014.09.017, 2015. N., Millero, F., Munro, D. R., Murata, A., Nabel, J. E. M. S., Nakaoka, S., Nojiri, Y., O’Brien, K., Olsen, A., Ono, T., Pérez, , Saito, S., Schuster, U., Schwinger, J., Séférian, R., Steinhoff, T., Stocker, B. D., Sutton, A. J., Takahashi, T., Tilbrook, B., van
der Laan-Luijkx, I. T., van der Werf, G. S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra Tech., 12, 2341–2370, https://doi.org/10.5194/amt-12-
2341-2019, 2019. Keppel-Aleks, G., Wennberg, P. O., O’Dell, C. W., and Wunch,
D.: Towards constraints on fossil fuel emissions from total
column carbon dioxide, Atmos. Chem. Phys., 13, 4349–4357,
https://doi.org/10.5194/acp-13-4349-2013, 2013. Fletcher, R.: A modified Marquardt subroutine for non-linear least
squares, Report R67, Atomic Energy Research Establishment,
Harwell, Berkshire, 1971. Friedlingstein, P., Jones, M. W., O’Sullivan, M., Andrew, R. M.,
Hauck, J., Peters, G. P., Peters, W., Pongratz, J., Sitch, S., Le
Quéré, C., Bakker, D. C. E., Canadell, J. G., Ciais, P., Jack-
son, R. B., Anthoni, P., Barbero, L., Bastos, A., Bastrikov, V.,
Becker, M., Bopp, L., Buitenhuis, E., Chandra, N., Chevallier,
F., Chini, L. P., Currie, K. I., Feely, R. A., Gehlen, M., Gilfillan,
D., Gkritzalis, T., Goll, D. S., Gruber, N., Gutekunst, S., Har-
ris, I., Haverd, V., Houghton, R. A., Hurtt, G., Ilyina, T., Jain,
A. K., Joetzjer, E., Kaplan, J. O., Kato, E., Klein Goldewijk, K.,
Korsbakken, J. I., Landschützer, P., Lauvset, S. K., Lefèvre, N.,
Lenton, A., Lienert, S., Lombardozzi, D., Marland, G., McGuire,
P. C., Melton, J. R., Metzl, N., Munro, D. R., Nabel, J. E. M. S.,
Nakaoka, S.-I., Neill, C., Omar, A. M., Ono, T., Peregon, A.,
Pierrot, D., Poulter, B., Rehder, G., Resplandy, L., Robertson, E.,
Rödenbeck, C., Séférian, R., Schwinger, J., Smith, N., Tans, P. P., Kiel, M., O’Dell, C. W., Fisher, B., Eldering, A., Nassar, R.,
MacDonald, C. G., and Wennberg, P. O.: How bias correction
goes wrong: measurement of XCO2 affected by erroneous sur-
face pressure estimates, Atmos. Meas. Tech., 12, 2241–2259,
https://doi.org/10.5194/amt-12-2241-2019, 2019. Kuai, L., Wunch, D., Shia, R. L., Connor, B., Miller, C., and Yung,
Y.: Vertically constrained CO2 retrievals from TCCON measure-
ments, J. Quant. Spectrosc. Radiat. Transf., 113, 1753–1761,
https://doi.org/10.1016/j.jqsrt.2012.04.024, 2012. Kuze, A., Suto, H., Nakajima, M., and Hamazaki, T.: Ther-
mal and near infrared sensor for carbon observation Fourier-
transform spectrometer on the Greenhouse Gases Observing
Satellite for greenhouse gases monitoring, Appl. Opt., 48, 6716–
6733, https://doi.org/10.1364/AO.48.006716, 2009. https://doi.org/10.5194/amt-14-3087-2021 https://doi.org/10.5194/amt-14-3087-2021 Atmos. Meas. Tech., 14, 3087–3118, 2021 S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra 3116 Lombardozzi, D., Metzl, N., Munro, D. R., Nabel, J. E. M. S.,
Nakaoka, S., Neill, C., Olsen, A., Ono, T., Patra, P., Peregon,
A., Peters, W., Peylin, P., Pfeil, B., Pierrot, D., Poulter, B., Re-
hder, G., Resplandy, L., Robertson, E., Rocher, M., Rödenbeck,
C., Schuster, U., Schwinger, J., Séférian, R., Skjelvan, I., Stein-
hoff, T., Sutton, A., Tans, P. P., Tian, H., Tilbrook, B., Tubiello,
F. N., van der Laan-Luijkx, I. T., van der Werf, G. R., Viovy, N.,
Walker, A. P., Wiltshire, A. J., Wright, R., Zaehle, S., and Zheng,
B.: Global Carbon Budget 2018, Earth Syst. Sci. Data, 10, 2141–
2194, https://doi.org/10.5194/essd-10-2141-2018, 2018b. Chevallier, F., Crowell, S. M. R., Feng, L., Palmer, P. I., Dubey,
M., García, O. E., Griffith, D. W. T., Hase, F., Iraci, L. T., Kivi, Chevallier, F., Crowell, S. M. R., Feng, L., Palmer, P. I., Dubey,
M., García, O. E., Griffith, D. W. T., Hase, F., Iraci, L. T., Kivi,
R., Morino, I., Notholt, J., Ohyama, H., Petri, C., Roehl, C. M.,
Sha, M. K., Strong, K., Sussmann, R., Te, Y., Uchino, O., and Ve-
lazco, V. A.: Improved retrievals of carbon dioxide from Orbiting
Carbon Observatory-2 with the version 8 ACOS algorithm, At-
mos. Meas. Tech., 11, 6539–6576, https://doi.org/10.5194/amt-
11-6539-2018, 2018. Parazoo, N. C., Denning, A. S., Kawa, S. R., Pawson, S., and
Lokupitiya, R.: CO2 flux estimation errors associated with moist
atmospheric processes, Atmos. Chem. Phys., 12, 6405–6416,
https://doi.org/10.5194/acp-12-6405-2012, 2012. Liu, Y., Wang, J., Yao, L., Chen, X., Cai, Z., Yang, D., Yin,
Z., Gu, S., Tian, L., Lu, N., and Lyu, D.: The TanSat mis-
sion: preliminary global observations, Sci. Bull., 63, 1200–1207,
https://doi.org/10.1016/j.scib.2018.08.004, 2018. Peters, W., Jacobson, A. R., Sweeney, C., Andrews, A. E., Con-
way, T. J., Masarie, K., Miller, J. B., Bruhwiler, L. M. P., Petron,
G., Hirsch, A. I., Worthy, D. E. J., van der Werf, G. R., Ran-
derson, J. T., Wennberg, P. O., Krol, M. C., and Tans, P. P.:
An atmospheric perspective on North American carbon dioxide
exchange: CarbonTracker, Proc. Natl. Acad. Sci., 104, 18925–
18930, https://doi.org/10.1073/pnas.0708986104, 2007. Lucchesi, R.: File Specification for GEOS-5 FP-IT (forward pro-
cessing for instrument teams), Tech. rep., NASA Goddard Space
Flight Center, Greenbelt, MD, USA, available at: https://gmao. gsfc.nasa.gov/pubs/docs/Lucchesi865.pdf (last access: 13 Octo-
ber 2020), 2015. Pickett-Heaps, C. A., Rayner, P. J., Law, R. M., Ciais, P., Patra,
P. S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra K., Bousquet, P., Peylin, P., Maksyutov, S., Marshall, J., Rö-
denbeck, C., Langenfelds, R. L., Steele, L. P., Francey, R. J.,
Tans, P., and Sweeney, C.: Atmospheric CO2 inversion valida-
tion using vertical profile measurements: Analysis of four in-
dependent inversion models, J. Geophys. Res., 116, D12305,
https://doi.org/10.1029/2010JD014887, 2011. McKenna, D. S., Konopka, P., Grooß, J.-U., Günther, G., Müller,
R., Spang, R., Offermann, D., and Orsolini, Y.: A new Chemi-
cal Lagrangian Model of the Stratosphere (CLaMS) 1. Formu-
lation of advection and mixing, J. Geophys. Res., 107, 4309,
https://doi.org/10.1029/2000jd000114, 2002. Mendonca, J., Strong, K., Toon, G. C., Wunch, D., Sung,
K., Deutscher, N. M., Griffith, D. W. T., and Franklin,
J. E.:
Improving
atmospheric
CO2
retrievals
using
line
mixing and speed-dependence when fitting high-resolution
ground-based solar spectra, J. Mol. Spectrosc., 323, 15–27,
https://doi.org/10.1016/j.jms.2016.01.007, 2016. Rayner, P. J., and O’Brien, D. M.: The utility of remotely sensed
CO2 concentration data in surface source inversions, Geophys. Res. Lett., 28, 175–178, https://doi.org/10.1029/2000GL011912,
2001. Rödenbeck, C., Houweling, S., Gloor, M., and Heimann, M.: CO2
flux history 1982–2001 inferred from atmospheric data using a
global inversion of atmospheric transport, Atmos. Chem. Phys.,
3, 1919–1964, https://doi.org/10.5194/acp-3-1919-2003, 2003. Mendonca, J., Strong, K., Sung, K., Devi, V. M., Toon, G. C.,
Wunch, D., and Franklin, J. E.: Using high-resolution laboratory
and ground-based solar spectra to assess CH4 absorption coeffi-
cient calculations, J. Quant. Spectrosc. Radiat. Transf., 190, 48–
59, https://doi.org/10.1016/j.jqsrt.2016.12.013, 2017. Rodgers, C. D.: Characterization and error analysis of profiles re-
trieved from remote sounding measurements, J. Geophys. Res.,
95,
5587–5595,
https://doi.org/10.1029/JD095iD05p05587,
1990. Mendonca, J., Strong, K., Wunch, D., Toon, G. C., Long, D. A., Hodges, J. T., Sironneau, V. T., and Franklin, J. E.: Us-
ing a speed-dependent Voigt line shape to retrieve O2 from
Total Carbon Column Observing Network solar spectra to im-
prove measurements of XCO2, Atmos. Meas. Tech., 12, 35–50,
https://doi.org/10.5194/amt-12-35-2019, 2019. Rodgers, C. D.: Inverse Methods for Atmospheric Sounding: The-
ory and Practice, World Scientific, Singapore, 2000. Rodgers, C. D. and Connor, B. J.: Intercomparison of remote
sounding instruments, J. Geophys. Res.-Atmos., 108, 4116,
https://doi.org/10.1029/2002JD002299, 2003. S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra K., Johannessen, T., Kato, E., Keel-
ing, R. F., Kitidis, V., Klein Goldewijk, K., Koven, C., Landa,
C. S., Landschützer, P., Lenton, A., Lima, I. D., Marland, G.,
Mathis, J. T., Metzl, N., Nojiri, Y., Olsen, A., Ono, T., Peng, S.,
Peters, W., Pfeil, B., Poulter, B., Raupach, M. R., Regnier, P., Rö-
denbeck, C., Saito, S., Salisbury, J. E., Schuster, U., Schwinger,
J., Séférian, R., Segschneider, J., Steinhoff, T., Stocker, B. D.,
Sutton, A. J., Takahashi, T., Tilbrook, B., van der Werf, G. R.,
Viovy, N., Wang, Y.-P., Wanninkhof, R., Wiltshire, A., and Zeng,
N.: Global carbon budget 2014, Earth Syst. Sci. Data, 7, 47–85,
https://doi.org/10.5194/essd-7-47-2015, 2015a. Millero, F., Monteiro, P. M. S., Munro, D. R., Nabel, J. E. M. Nakaoka, S., Nojiri, Y., Padin, X. A., Peregon, A., Pfeil, B., Pierrot, D., Poulter, B., Rehder, G., Reimer, J., Rödenbeck, C., winger, J., Séférian, R., Skjelvan, I., Stocker, B. D., Tian Tilbrook, B., Tubiello, F. N., van der Laan-Luijkx, I. T., van der Le Quéré, C., Andrew, R. M., Friedlingstein, P., Sitch, S., Hauck,
J., Pongratz, J., Pickers, P. A., Korsbakken, J. I., Peters, G. P.,
Canadell, J. G., Arneth, A., Arora, V. K., Barbero, L., Bastos,
A., Bopp, L., Chevallier, F., Chini, L. P., Ciais, P., Doney, S. C.,
Gkritzalis, T., Goll, D. S., Harris, I., Haverd, V., Hoffman, F. M.,
Hoppema, M., Houghton, R. A., Hurtt, G., Ilyina, T., Jain, A. K., Johannessen, T., Jones, C. D., Kato, E., Keeling, R. F., Gold-
ewijk, K. K., Landschützer, P., Lefèvre, N., Lienert, S., Liu, Z., Le Quéré, C., Moriarty, R., Andrew, R. M., Canadell, J. G., Sitch, S.,
Korsbakken, J. I., Friedlingstein, P., Peters, G. P., Andres, R. J., https://doi.org/10.5194/amt-14-3087-2021 Atmos. Meas. Tech., 14, 3087–3118, 2021 S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra J., Meinardi, S., Miller, D. O., Montzka, S. A., Moore, F. L., Morgan, E. J., Murphy, D. M., Murray, L. T., Stephens, B. B., Gurney, K. R., Tans, P. P., Sweeney, C., Pe-
ters, W., Bruhwiler, L., Ciais, P., Ramonet, M., Bousquet, P.,
Nakazawa, T., Aoki, S., Machida, T., Inoue, G., Vinnichenko,
N., Lloyd, J., Jordan, A., Heimann, M., Shibistova, O., Langen-
felds, R. L., Steele, L. P., Francey, R. J., and Denning, A. S.:
Weak Northern and Strong Tropical Land Carbon Uptake from
Vertical Profiles of Atmospheric CO2, Science, 316, 1732–1735,
https://doi.org/10.1126/science.1137004, 2007. A., Moore, F. L., Morgan, E. J., Murphy, D. M., Murray, L. T., Nault, B. A., Neuman, J. A., Newman, P. A., Nicely, J. M., Pan, X., Paplawsky, W., Peischl, J., Prather, M. J., Price, D. J., Ray, E., Reeves, J. M., Richardson, M., Rollins, A. W., Rosenlof, K. H., Ryerson, T. B., Scheuer, E., Schill, G. P., Schroder, J. C.,
S h
J P S Cl i
J M
S
d S D
S
h
B B H., Ryerson, T. B., Scheuer, E., Schill, G. P., Schroder, J. C.,
Schwarz, J. P., St.Clair, J. M., Steenrod, S. D., Stephens, B. B.,
Strode, S. A., Sweeney, C., Tanner, D., Teng, A. P., Thames, A. B., Thompson, C. R., Ullmann, K., Veres, P. R., Vizenor, N.,
Wagner, N. L., Watt, A., Weber, R., Weinzierl, B., Wennberg,
P. O., Williamson, C. J., Wilson, J. C., Wolfe, G. M., Woods,
C. T., and Zeng, L. H.: ATom: Merged Atmospheric Chemistry,
Trace Gases, and Aerosols, ORNL DAAC, Oak Ridge, Ten-
USA h
//d i
/10 3334/ORNLDAAC/1581 2018 ,
y
,
,
,
,
,
,
,
,
Schwarz, J. P., St.Clair, J. M., Steenrod, S. D., Stephens, B. B.,
Strode, S. A., Sweeney, C., Tanner, D., Teng, A. P., Thames, A. y
Schwarz, J. P., St.Clair, J. M., Steenrod, S. D., Stephens, B. B., arz, J. P., St.Clair, J. M., Steenrod, S. D., Stephens, B. Strode, S. A., Sweeney, C., Tanner, D., Teng, A. P., Thames, A. Sweeney, C., Higgs, J., Wolter, S., Crotwell, A., Neff, D., Dlu-
gokencky, E., Lang, P., Novelli, P., Mund, J., Moglia, E., and
Crotwell, M.: Earth System Research Laboratory Carbon Cycle
and Greenhouse Gases Group Flask-Air Sample Measurements
of CO2, CH4, CO, N2O, H2, and SF6 from the Aircraft Program,
1992–Present, Version 1, NOAA National Centers for Environ-
mental Information, https://doi.org/10.7289/V5N58JMF, 2017. S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra 3117 V. M., Fally, S., Flaud, J. M., Gamache, R. R., Goldman,
A., Jacquemart, D., Kleiner, I., Lacome, N., Lafferty, W. J.,
Mandin, J. Y., Massie, S. T., Mikhailenko, S. N., Miller, C. E., Moazzen-Ahmadi, N., Naumenko, O. V., Nikitin, A. V., Or-
phal, J., Perevalov, V. I., Perrin, A., Predoi-Cross, A., Rinsland,
C. P., Rotger, M., Šimeˇcková, M., Smith, M. A. H., Sung, K.,
Tashkun, S. A., Tennyson, J., Toth, R. A., Vandaele, A. C., and
Vander Auwera, J.: The HITRAN 2008 molecular spectroscopic
database, J. Quant. Spectrosc. Radiat. Transf., 110, 533–572,
https://doi.org/10.1016/j.jqsrt.2009.02.013, 2009. H. F., Liu, Y., He, W., Chen, H., Masarie, K. A., Krol,
M. C., and Peters, W.: The CarbonTracker Data Assimila-
tion Shell (CTDAS) v1.0: implementation and global car-
bon balance 2001–2015, Geosci. Model Dev., 10, 2785–2800,
https://doi.org/10.5194/gmd-10-2785-2017, 2017. Wennberg, P. O., Wunch, D., Roehl, C. M., Blavier, J.-F.,
Toon, G. C., and Allen, N. T.: TCCON data from Lam-
ont (US), Release GGG2014.R1 [Data set], CaltechDATA,
https://doi.org/10.14291/tccon.ggg2014.lamont01.r1/1255070,
2016. Rothman, L. S., Gordon, I. E., Babikov, Y., Barbe, A., Chris Benner,
D., Bernath, P. F., Birk, M., Bizzocchi, L., Boudon, V., Brown,
L. R., Campargue, A., Chance, K., Cohen, E. A., Coudert, L. H.,
Devi, V. M., Drouin, B. J., Fayt, A., Flaud, J. M., Gamache, R. R.,
Harrison, J. J., Hartmann, J. M., Hill, C., Hodges, J. T., Jacque-
mart, D., Jolly, A., Lamouroux, J., Le Roy, R. J., Li, G., Long,
D. A., Lyulin, O. M., Mackie, C. J., Massie, S. T., Mikhailenko,
S., Müller, H. S. P., Naumenko, O. V., Nikitin, A. V., Orphal, J.,
Perevalov, V., Perrin, A., Polovtseva, E. R., Richard, C., Smith,
M. A. H., Starikova, E., Sung, K., Tashkun, S., Tennyson, J.,
Toon, G. C., Tyuterev, V. G., and Wagner, G.: The HITRAN2012
molecular spectroscopic database, J. Quant. Spectrosc. Radiat. Transf., 130, 4–50, https://doi.org/10.1016/j.jqsrt.2013.07.002,
2013. Wofsy, S. C.: HIAPER Pole-to-Pole Observations (HIPPO):
fine-grained,
global-scale
measurements
of
climatically
important atmospheric gases and aerosols, Philos. Trans. R. Soc. A
Math. Phys. Eng. Sci.,
369,
2073–2086,
https://doi.org/10.1098/rsta.2010.0313, 2011. Wofsy, S. C., Afshar, S., Allen, H. M., Apel, E., Asher, E. C., Bar-
letta, B., Bent, J., Bian, H., Biggs, B. C., Blake, D. R., Blake,
N., Bourgeois, I., Brock, C. A., Brune, W. H., Budney, J. W.,
Bui, T. P., Butler, A., Campuzano-Jost, P., Chang, C. S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra Myhre, G., Shindell, D., Bréon, F., Collins, W., Fuglestvedt, J.,
Huang, J., Koch, D., Lamarque, J., Lee, D., Mendoza, B., Naka-
jima, T., Robock, A., Stephens, G., Takemura, T., and Zhan,
H.: Anthropogenic and Natural Radiative Forcing, in: Climate
Change 2013 – The Physical Science Basis, edited by: Inter-
governmental Panel on Climate Change, Cambridge University
Press, Cambridge, United Kingdom and New York, NY, USA,
659–740, 2013. Rosenkranz, P.: Shape of the 5 mm oxygen band in the at-
mosphere,
IEEE
Trans. Antennas
Propag.,
23,
498–506,
https://doi.org/10.1109/TAP.1975.1141119, 1975. Rothman, L. S., Jacquemart, D., Barbe, A., Benner, D. C., Birk,
M., Brown, L. R., Carleer, M. R., Chackerian, C., Chance,
K., Coudert, L. H., Dana, V., Devi, V. M., Flaud, J. M.,
Gamache, R. R., Goldman, A., Hartmann, J. M., Jucks, K. W., Maki, A. G., Mandin, J. Y., Massie, S. T., Orphal, J.,
Perrin, A., Rinsland, C. P., Smith, M. A. H., Tennyson, J.,
Tolchenov, R. N., Toth, R. A., Vander Auwera, J., Varanasi, P.,
and Wagner, G.: The HITRAN 2004 molecular spectroscopic
database, J. Quant. Spectrosc. Radiat. Transf., 96, 139–204,
https://doi.org/10.1016/j.jqsrt.2004.10.008, 2005. Nakajima, M., Kuze, A., and Suto, H.: The current status of
GOSAT and the concept of GOSAT-2, in: Sensors, Sys-
tems, and Next-Generation Satellites XVI, edited by: Mey-
nart, R., Neeck, S. P., and Shimoda, H., SPIE., 8533, 21–30,
https://doi.org/10.1117/12.974954, 2012. O’Dell, C. W., Eldering, A., Wennberg, P. O., Crisp, D., Gunson,
M. R., Fisher, B., Frankenberg, C., Kiel, M., Lindqvist, H., Man-
drake, L., Merrelli, A., Natraj, V., Nelson, R. R., Osterman, G. B.,
Payne, V. H., Taylor, T. E., Wunch, D., Drouin, B. J., Oyafuso,
F., Chang, A., McDuffie, J., Smyth, M., Baker, D. F., Basu, S., Rothman, L. S., Gordon, I. E., Barbe, A., Benner, D. C., Bernath,
P. F., Birk, M., Boudon, V., Brown, L. R., Campargue, A.,
Champion, J. P., Chance, K., Coudert, L. H., Dana, V., Devi, https://doi.org/10.5194/amt-14-3087-2021 Atmos. Meas. Tech., 14, 3087–3118, 2021 S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra S., Chin,
M., Commane, R., Correa, G., Crounse, J. D., Cullis, P. D.,
Daube, B. C., Day, D. A., Dean-Day, J. M., Dibb, J. E., Di-
gangi, J. P., Diskin, G. S., Dollner, M., Elkins, J. W., Erdesz,
F., Fiore, A. M., Flynn, C. M., Froyd, K., Gesler, D. W., Hall,
S. R., Hanisco, T. F., Hannun, R. A., Hills, A. J., Hintsa, E. J., Hoffman, A., Hornbrook, R. S., Huey, L. G., Hughes, S.,
Jimenez, J. L., Johnson, B. J., Katich, J. M., Keeling, R., Kim,
M. J., Kupc, A., Lait, L. R., Lamarque, J.-F., Liu, J., McKain,
K., McLaughlin, R. J., Meinardi, S., Miller, D. O., Montzka, S. A., Moore, F. L., Morgan, E. J., Murphy, D. M., Murray, L. T.,
Nault, B. A., Neuman, J. A., Newman, P. A., Nicely, J. M., Pan,
X., Paplawsky, W., Peischl, J., Prather, M. J., Price, D. J., Ray,
E., Reeves, J. M., Richardson, M., Rollins, A. W., Rosenlof, K. H., Ryerson, T. B., Scheuer, E., Schill, G. P., Schroder, J. C.,
Schwarz, J. P., St.Clair, J. M., Steenrod, S. D., Stephens, B. B.,
Strode, S. A., Sweeney, C., Tanner, D., Teng, A. P., Thames, A. B., Thompson, C. R., Ullmann, K., Veres, P. R., Vizenor, N.,
Wagner, N. L., Watt, A., Weber, R., Weinzierl, B., Wennberg,
P. O., Williamson, C. J., Wilson, J. C., Wolfe, G. M., Woods,
C. T., and Zeng, L. H.: ATom: Merged Atmospheric Chemistry,
Trace Gases, and Aerosols, ORNL DAAC, Oak Ridge, Ten-
nessee, USA, https://doi.org/10.3334/ORNLDAAC/1581, 2018. Commane, R., Correa, G., Crounse, J. D., Cullis, P. D., M. A. H., Starikova, E., Sung, K., Tashkun, S., Tennyson, J.,
Toon, G. C., Tyuterev, V. G., and Wagner, G.: The HITRAN2012
molecular spectroscopic database, J. Quant. Spectrosc. Radiat. Transf., 130, 4–50, https://doi.org/10.1016/j.jqsrt.2013.07.002,
2013. F., Fiore, A. M., Flynn, C. M., Froyd, K., Gesler, D. W., Hall, S. R., Hanisco, T. F., Hannun, R. A., Hills, A. J., Hintsa, E. J., Hoffman, A., Hornbrook, R. S., Huey, L. G., Hughes, S., Saeki, T. and Patra, P. K.: Implications of overestimated anthro-
pogenic CO2 emissions on East Asian and global land CO2 flux
inversion, Geosci. Lett., 4, 9, https://doi.org/10.1186/s40562-
017-0074-7, 2017. Jimenez, J. L., Johnson, B. J., Katich, J. M., Keeling, R., Kim, M. J., Kupc, A., Lait, L. R., Lamarque, J.-F., Liu, J., McKain, McLaughlin, R. J., Meinardi, S., Miller, D. O., Montzk K., McLaughlin, R. https://doi.org/10.5194/amt-14-3087-2021 S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra schmidt, J., Miller, C. E., Morino, I., Notholt, J., Oyafuso, F. A., Rettinger, M., Robinson, J., Roehl, C. M., Salawitch, R. J., Sherlock, V., Strong, K., Sussmann, R., Tanaka, T., Thomp-
son, D. R., Uchino, O., Warneke, T., and Wofsy, S. C.: A
method for evaluating bias in global measurements of CO2 to-
tal columns from space, Atmos. Chem. Phys., 11, 12317–12337,
https://doi.org/10.5194/acp-11-12317-2011, 2011a. schmidt, J., Miller, C. E., Morino, I., Notholt, J., Oyafuso, F. A., Rettinger, M., Robinson, J., Roehl, C. M., Salawitch, R. J., Sherlock, V., Strong, K., Sussmann, R., Tanaka, T., Thomp-
son, D. R., Uchino, O., Warneke, T., and Wofsy, S. C.: A
method for evaluating bias in global measurements of CO2 to-
tal columns from space, Atmos. Chem. Phys., 11, 12317–12337,
https://doi.org/10.5194/acp-11-12317-2011, 2011a. Wunch, D., Mendonca, J., Colebatch, O., Allen, N. T., Blavier, J.-F.,
Roche, S., Hedelius, J., Neufeld, G., Springett, S., Worthy, D.,
Kessler, R., and Strong, K.: TCCON data from East Trout Lake,
SK (CA), Release GGG2014.R1 [Data set], CaltechDATA,
https://doi.org/10.14291/tccon.ggg2014.easttroutlake01.r1,
2018. Wunch, D., Toon, G. C., Blavier, J.-F. L., Washenfelder, R. A.,
Notholt, J., Connor, B. J., Griffith, D. W. T., Sherlock, V., and
Wennberg, P. O.: The Total Carbon Column Observing Network,
Philos. Trans. R. Soc. A Math. Phys. Eng. Sci., 369, 2087–2112,
https://doi.org/10.1098/rsta.2010.0240, 2011b. Wunch, D., Toon, G. C., Sherlock, V., Deutscher, N. M., Liu,
C., Feist, D. G., and Wennberg, P. O.: Documentation for the
2014 TCCON Data Release (Version GGG2014.R0), Caltech-
DATA, https://doi.org/10.14291/tccon.ggg2014.documentation. r0/1221662, 2015. Wunch, D., Wennberg, P. O., Osterman, G., Fisher, B., Naylor, B.,
Roehl, C. M., O’Dell, C., Mandrake, L., Viatte, C., Kiel, M.,
Griffith, D. W. T., Deutscher, N. M., Velazco, V. A., Notholt, J.,
Warneke, T., Petri, C., De Maziere, M., Sha, M. K., Sussmann,
R., Rettinger, M., Pollard, D., Robinson, J., Morino, I., Uchino,
O., Hase, F., Blumenstock, T., Feist, D. G., Arnold, S. G., Strong,
K., Mendonca, J., Kivi, R., Heikkinen, P., Iraci, L., Podolske,
J., Hillyard, P. W., Kawakami, S., Dubey, M. K., Parker, H. A.,
Sepulveda, E., García, O. E., Te, Y., Jeseck, P., Gunson, M. R.,
Crisp, D., and Eldering, A.: Comparisons of the Orbiting Carbon
Observatory-2 (OCO-2) XCO2 measurements with TCCON, At-
mos. Meas. Tech., 10, 2209–2238, https://doi.org/10.5194/amt-
10-2209-2017, 2017. Rettinger, M., Pollard, D., Robinson, J., Morino, I., Uc O., Hase, F., Blumenstock, T., Feist, D. G., Arnold, S. S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra Wunch, D., Toon, G. C., Wennberg, P. O., Wofsy, S. C., Stephens,
B. B., Fischer, M. L., Uchino, O., Abshire, J. B., Bernath, P.,
Biraud, S. C., Blavier, J.-F. L., Boone, C., Bowman, K. P., Brow-
ell, E. V., Campos, T., Connor, B. J., Daube, B. C., Deutscher,
N. M., Diao, M., Elkins, J. W., Gerbig, C., Gottlieb, E., Grif-
fith, D. W. T., Hurst, D. F., Jiménez, R., Keppel-Aleks, G., Kort,
E. A., Macatangay, R., Machida, T., Matsueda, H., Moore, F.,
Morino, I., Park, S., Robinson, J., Roehl, C. M., Sawa, Y., Sher-
lock, V., Sweeney, C., Tanaka, T., and Zondlo, M. A.: Cali-
bration of the Total Carbon Column Observing Network us-
ing aircraft profile data, Atmos. Meas. Tech., 3, 1351–1362,
https://doi.org/10.5194/amt-3-1351-2010, 2010. Tange,
O.:
GNU
Parallel
–
The
Command-Line
Power
Tool,
USENIX
Mag.,
36,
42–47,
Zenodo,
https://doi.org/10.5281/zenodo.16303, 2011. Toon, G. C.: Atmospheric Line List for the 2014 TCCON
Data Release (Version GGG2014.R0) [Data set], CaltechDATA,
https://doi.org/10.14291/tccon.ggg2014.atm.r0/1221656, 2015. Toon, G. C., Blavier, J.-F., Sung, K., Rothman, L. S., and E. Gor-
don, I.: HITRAN spectroscopy evaluation using solar occultation
FTIR spectra, J. Quant. Spectrosc. Radiat. Transf., 182, 324–336,
https://doi.org/10.1016/j.jqsrt.2016.05.021, 2016. Wunch, D., Wennberg, P. O., Toon, G. C., Connor, B. J., Fisher,
B., Osterman, G. B., Frankenberg, C., Mandrake, L., O’Dell, Toth, R. A., Brown, L. R., Miller, C. E., Malathy Devi, V., and
Benner, D. C.: Spectroscopic database of CO2 line parame-
ters: 4300–7000 cm−1, J. Quant. Spectrosc. Radiat. Transf., 109,
906–921, https://doi.org/10.1016/j.jqsrt.2007.12.004, 2008. Wunch, D., Wennberg, P. O., Toon, G. C., Connor, B. J., Fisher,
B., Osterman, G. B., Frankenberg, C., Mandrake, L., O’Dell,
C., Ahonen, P., Biraud, S. C., Castano, R., Cressie, N., Crisp,
D., Deutscher, N. M., Eldering, A., Fisher, M. L., Griffith, D. W. T., Gunson, M., Heikkinen, P., Keppel-Aleks, G., Kyrö,
E., Lindenmaier, R., Macatangay, R., Mendonca, J., Messer- C., Ahonen, P., Biraud, S. C., Castano, R., Cressie, N., Crisp,
D., Deutscher, N. M., Eldering, A., Fisher, M. L., Griffith, D. van der Laan-Luijkx, I. T., van der Velde, I. R., van der Veen,
E., Tsuruta, A., Stanislawska, K., Babenhauserheide, A., Zhang, https://doi.org/10.5194/amt-14-3087-2021 Atmos. Meas. Tech., 14, 3087–3118, 2021 S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra 3118 Atmos. Meas. Tech., 14, 3087–3118, 2021 S. Roche et al.: Retrieval of atmospheric CO2 vertical profiles from ground-based near-infrared spectra G., Strong, K., Mendonca, J., Kivi, R., Heikkinen, P., Iraci, L., Podolske, J., Hillyard, P. W., Kawakami, S., Dubey, M. K., Parker, H. A., pulveda, E., García, O. E., Te, Y., Jeseck, P., Gunson, M. R., Atmos. Meas. Tech., 14, 3087–3118, 2021
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https://zenodo.org/records/3131159/files/SimilarityMeasuresOfBipolarNeutrosophic.pdf
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Chinese
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Similarity measures of bipolar neutrosophic sets and their application to multiple criteria decision making
|
Zenodo (CERN European Organization for Nuclear Research)
| 2,019
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cc-by
| 9,375
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Neural Comput & Applic
DOI 10.1007/s00521-016-2479-1
SCI-Expanded Neural Comput & Applic
DOI 10.1007/s00521-016-2479-1
SCI-Expanded SCI-Expanded Neural Comput & Applic
DOI 10.1007/s00521-016-2479-1 ORIGINAL ARTICLE Similarity measures of bipolar neutrosophic sets
and their application to multiple criteria decision making Keywords Neutrosophic sets Bipolar neutrosophic set
Dice similarity measure Hybrid vector similarity
Decision making Keywords Neutrosophic sets Bipolar neutrosophic set
Dice similarity measure Hybrid vector similarity
Decision making & Vakkas Uluc¸ay
vulucay27@gmail.com
1
Gaziantep University, 27310 Gaziantep, Turkey
2
Department of Mathematics, Kilis 7 Aralık University,
79000 Kilis, Turkey Similarity measures of bipolar neutrosophic sets
and their application to multiple criteria decision making Vakkas Uluc¸ay1
• Irfan Deli2 • Mehmet S¸ahin1 Received: 5 March 2016 / Accepted: 6 July 2016
The Natural Computing Applications Forum 2016 Abstract In this paper, we introduced some similarity
measures for bipolar neutrosophic sets such as; Dice sim-
ilarity measure, weighted Dice similarity measure, Hybrid
vector similarity measure and weighted Hybrid vector
similarity measure. Also we examine the propositions of
the similarity measures. Furthermore, a multi-criteria
decision-making method for bipolar neutrosophic set is
developed based on these given similarity measures. Then,
a practical example is shown to verify the feasibility of the
new method. Finally, we compare the proposed method
with the existing methods in order to demonstrate the
practicality and effectiveness of the developed method in
this paper. many different areas, such as decision-making problems
[1, 2, 4, 7–11, 19, 23, 31, 37–39, 42, 52]. In additionally,
since the neutrosophic sets are hard to be applied in some
real problems because of the truth-membership degree,
indeterminacy-membership degree and falsity-membership
degree lie in 0; 1þ½, single-valued neutrosophic sets
introduced by Wang et al. [40]. Recently, Lee [21, 22] proposed notation of bipolar
fuzzy set and their operations based on fuzzy sets. A
bipolar fuzzy set has two completely independent parts,
which are positive membership degree Tþ ! ½0; 1 and
negative membership degree T ! ½1; 0. Also the
bipolar fuzzy models have been studied by many authors
including
theory
and
applications
in
[12, 17, 25, 34, 35, 50]. After the definition of Smaran-
dache’s neutrosophic set, neutrosophic sets and neutro-
sophic logic have been applied in many real applications
to handle uncertainty. The neutrosophic set uses one
single value in 0; 1þ½ to represent the truth-membership
degree, indeterminacy-membership degree and falsity-
membership degree of an element in the universe X. Then, Deli et al. [18] introduced the concept of bipolar
neutrosophic sets, as an extension of neutrosophic sets. In
the bipolar neutrosophic sets, the positive membership
degree TþðxÞ; IþðxÞ; FþðxÞ denotes the truth member-
ship, indeterminate membership and false membership of
an element x 2 X corresponding to a bipolar neutrosophic
set
A
and
the
negative
membership
degree
TðxÞ; IðxÞ; FðxÞ denotes the truth membership, inde-
terminate membership and false membership of an ele-
ment
x 2 X
to
some
implicit
counter-property
corresponding to a bipolar neutrosophic set A. where Tþ; Iþ; Fþ : E ! ½0; 1; T; I; F : X ! ½1; 0: The positive membership degree TþðxÞ; IþðxÞ; FþðxÞ
denotes the truth membership, indeterminate membership
and false membership of an element x 2 X corresponding
to a bipolar neutrosophic set A and the negative member-
ship degree TðxÞ; IðxÞ; FðxÞ denotes the truth mem-
bership, indeterminate membership and false membership
of an element x 2 X to some implicit counter-property
corresponding to a bipolar neutrosophic set A. Definition 4
[18] Let A1 ¼ hx; Tþ
1 ðxÞ; Iþ
1 ðxÞ; Fþ
1 ðxÞ;
T
1 ðxÞ;
I
1 ðxÞ; F
1 ðxÞi
and
A2 ¼ hx; Tþ
2 ðxÞ;
Iþ
2 ðxÞ;
Fþ
2 ðxÞ; T
2 ðxÞ; I
2 ðxÞ; F
2 ðxÞi be two BNSs in a universe of
discourse X. Then the following operations are defined as
follows: 1 Introduction As a generalization of classical sets, fuzzy set [51] and
intuitionistic fuzzy set [3], neutrosophic set was presented
by Smarandache [32, 33] to capture the incomplete, inde-
terminate and inconsistent information. The neutrosophic
set has three completely independent parts, which are truth-
membership degree, indeterminacy-membership degree
and falsity-membership degree; therefore, it is applied to Similarity measure is an important tool in constructing
multi-criteria decision-making methods in many areas such 12 3 Neural Comput & Applic membership function, respectively. They are respectively
defined by
TA : E ! ½0; 1;
IA : E ! ½0; 1;
FA : E ! ½0; 1
such that 0 TAðxÞ þ IAðxÞ þ FAðxÞ 3. membership function, respectively. They are respectively
defined by as medical diagnosis, pattern recognition, clustering anal-
ysis, decision making and so on. Similarity measures under
all sorts of fuzzy environments including single-valued
neutrosophic environments have been studied by many
researchers in [5, 6, 13, 14, 15, 20–24, 26–30, 43–49]. Also, Deli and S¸ubas’s [16] and S¸ahin et al. [36] presented
some similarity measures on bipolar neutrosophic sets
based on correlation coefficient similarity measure and
Jaccard vector similarity measure of neutrosophic set and
applied to a decision-making problem, respectively. This
paper is constructed as follows. In Sect. 2, some basic
definitions of neutrosophic sets and bipolar neutrosophic
sets are introduced. In Sect. 3, we propose some simi-
larity measures for bipolar neutrosophic sets such as;
Dice similarity measure, weighted Dice similarity mea-
sure, Hybrid vector similarity measure and weighted
Hybrid vector similarity measure and investigate their
several properties. In Sect. 4, a multi-criteria decision-
making method for bipolar neutrosophic set is developed
based on these given similarity measures and a practical
example is given. In Sect. 5, we compare the proposed
method with the existing methods in order to demonstrate
the practicality and effectiveness of the developed
method in this paper. In Sect. 6, the conclusions are
summarized. TA : E ! ½0; 1;
IA : E ! ½0; 1;
FA : E ! ½0; 1
such that 0 TAðxÞ þ IAðxÞ þ FAðxÞ 3. Definition 3
[18] A bipolar neutrosophic set(BNS) A in
X is defined as an object of the form A ¼ fhx; TþðxÞ; IþðxÞ; FþðxÞ; TðxÞ; IðxÞ; FðxÞi : x 2 Xg: where where 2 Preliminary 1. A1 ¼ A2 if and only if Tþ
1 ðxÞ ¼ Tþ
2 ðxÞ; Iþ
1 ðxÞ ¼ Iþ
2 ðxÞ;
Fþ
1 ðxÞ ¼ Fþ
2 ðxÞ and T
1 ðxÞ ¼ T
2 ðxÞ; I
1 ðxÞ ¼ I
2 ðxÞ;
F
1 ðxÞ ¼ F
2 ðxÞ: 1. A1 ¼ A2 if and only if Tþ
1 ðxÞ ¼ Tþ
2 ðxÞ; Iþ
1 ðxÞ ¼ Iþ
2 ðxÞ;
Fþ
1 ðxÞ ¼ Fþ
2 ðxÞ and T
1 ðxÞ ¼ T
2 ðxÞ; I
1 ðxÞ ¼ I
2 ðxÞ;
F
1 ðxÞ ¼ F
2 ðxÞ: 1. A1 ¼ A2 if and only if Tþ
1 ðxÞ ¼ Tþ
2 ðxÞ; Iþ
1 ðxÞ ¼ Iþ
2 ðxÞ;
Fþ
1 ðxÞ ¼ Fþ
2 ðxÞ and T
1 ðxÞ ¼ T
2 ðxÞ; I
1 ðxÞ ¼ I
2 ðxÞ;
F
1 ðxÞ ¼ F
2 ðxÞ: In the section, we give some concepts related to neutro-
sophic sets and bipolar neutrosophic sets. 2. A1 [ A2 ¼fhx; maxðTþ
1 ðxÞ; Tþ
2 ðxÞÞ;
Iþ
1 ðxÞþIþ
2 ðxÞ
2
; minðFþ
1 ðxÞ; Fþ
2 ðxÞÞ; minðT
1 ðxÞ; T
2 ðxÞÞ;
I
1 ðxÞþI
2 ðxÞ
2
; maxðF
1 ðxÞ; F
2 ðxÞÞig8x 2 X: 2. A1 [ A2 ¼fhx; maxðTþ
1 ðxÞ; Tþ
2 ðxÞÞ;
Iþ
1 ðxÞþIþ
2 ðxÞ
2
; minðFþ
1 ðxÞ; Fþ
2 ðxÞÞ; minðT
1 ðxÞ; T
2 ðxÞÞ;
I
1 ðxÞþI
2 ðxÞ
2
; maxðF
1 ðxÞ; F
2 ðxÞÞig8x 2 X: 2. A1 [ A2 ¼fhx; maxðTþ
1 ðxÞ; Tþ
2 ðxÞÞ;
Iþ
1 ðxÞþIþ
2 ðxÞ
2
; minðFþ
1 ðxÞ; Fþ
2 ðxÞÞ; minðT
1 ðxÞ; T
2 ðxÞÞ;
I
1 ðxÞþI
2 ðxÞ
2
; maxðF
1 ðxÞ; F
2 ðxÞÞig8x 2 X: Definition 1
[32] Let E be a universe. A neutrosophic set
A over E is defined by Definition 1
[32] Let E be a universe. A neutrosophic set
A over E is defined by Definition 1
[32] Let E be a universe. A neutrosophic set
A over E is defined by A ¼ fhx; ðTAðxÞ; IAðxÞ; FAðxÞÞi : x 2 Eg: A ¼ fhx; ðTAðxÞ; IAðxÞ; FAðxÞÞi : x 2 Eg: 3. 2 Preliminary A1 \ A2 ¼fhx; minðTþ
1 ðxÞ; Tþ
2 ðxÞÞ;
Iþ
1 ðxÞþIþ
2 ðxÞ
2
; maxðFþ
1 ðxÞ; Fþ
2 ðxÞÞ; maxðT
1 ðxÞ;
T
2 ðxÞÞ; I
1 ðxÞþI
2 ðxÞ
2
; minðF
1 ðxÞ; F
2 ðxÞÞig8x 2 X: 3. A1 \ A2 ¼fhx; minðTþ
1 ðxÞ; Tþ
2 ðxÞÞ;
Iþ
1 ðxÞþIþ
2 ðxÞ
2
; maxðFþ
1 ðxÞ; Fþ
2 ðxÞÞ; maxðT
1 ðxÞ;
T
2 ðxÞÞ; I
1 ðxÞþI
2 ðxÞ
2
; minðF
1 ðxÞ; F
2 ðxÞÞig8x 2 X: 3. A1 \ A2 ¼fhx; minðTþ
1 ðxÞ; Tþ
2 ðxÞÞ;
Iþ
1 ðxÞþIþ
2 ðxÞ
2
; maxðFþ
1 ðxÞ; Fþ
2 ðxÞÞ; maxðT
1 ðxÞ;
T
2 ðxÞÞ; I
1 ðxÞþI
2 ðxÞ
2
; minðF
1 ðxÞ; F
2 ðxÞÞig8x 2 X: where TAðxÞ, IAðxÞ and FAðxÞ are called truth-membership
function, indeterminacy-membership function and falsity-
membership function, respectively. They are, respectively,
defined by 2
4. Ac ¼ fhx; 1 Tþ
A ðxÞ; 1 Iþ
A ðxÞ; 1 Fþ
A ðxÞ; 1 T
A ðxÞ;
1 I
A ðxÞ; 1 F
A ðxÞig 2
4. Ac ¼ fhx; 1 Tþ
A ðxÞ; 1 Iþ
A ðxÞ; 1 Fþ
A ðxÞ; 1 T
A ðxÞ;
1 I
A ðxÞ; 1 F
A ðxÞig TA : E !0; 1þ½;
IA : E !0; 1þ½;
FA : E !0; 1þ½
such that 0 TAðxÞ þ IAðxÞ þ FAðxÞ 3þ. TA : E !0; 1þ½;
IA : E !0; 1þ½;
FA : E !0; 1þ½ 5. A1 A2 if and only if Tþ
1 ðxÞ Tþ
2 ðxÞ; Iþ
1 ðxÞ Iþ
2 ðxÞ;
Fþ
1 ðxÞ Fþ
2 ðxÞ
and
T
1 ðxÞ T
2 ðxÞ; I
1 ðxÞ
I
2 ðxÞ;
F
1 ðxÞ F
2 ðxÞ: 5. A1 A2 if and only if Tþ
1 ðxÞ Tþ
2 ðxÞ; Iþ
1 ðxÞ Iþ
2 ðxÞ;
Fþ
1 ðxÞ Fþ
2 ðxÞ
and
T
1 ðxÞ T
2 ðxÞ; I
1 ðxÞ
I
2 ðxÞ;
F
1 ðxÞ F
2 ðxÞ: ch that 0 TAðxÞ þ IAðxÞ þ FAðxÞ 3þ. Definition 2
[40] Let E be a universe. 2 Preliminary A single-valued
neutrosophic set (SVN-set) A over E is defined by Definition 2
[40] Let E be a universe. A single-valued
neutrosophic set (SVN-set) A over E is defined by Definition 2
[40] Let E be a universe. A single-valued
neutrosophic set (SVN-set) A over E is defined by
A ¼ fhx; ðTAðxÞ; IAðxÞ; FAðxÞÞi : x 2 Eg: Definition 5
[45] Let A ¼ hTAðxiÞ; IAðxiÞ; FAðxiÞi and
B ¼ hTBðxiÞ; IBðxiÞ; FBðxiÞi be two SVNSs in a universe of
discourse X ¼ ðx1; x2; . . .; xnÞ: Then Dice similarity mea-
sure between SVNSs A and B in the vector space is defined
as follows: A ¼ fhx; ðTAðxÞ; IAðxÞ; FAðxÞÞi : x 2 Eg: A ¼ fhx; ðTAðxÞ; IAðxÞ; FAðxÞÞi : x 2 Eg: where TAðxÞ, IAðxÞ and FAðxÞ are called truth-membership
function, indeterminacy-membership function and falsity- 12 123 Neural Comput & Applic DðA; BÞ ¼ 1
n
X
n
i¼1
2ðTAðxiÞTBðxiÞ þ IAðxiÞIBðxiÞ þ FAðxiÞFBðxiÞÞ
½ððTAÞ2ðxiÞ þ ðIAÞ2ðxiÞ þ ðFAÞ2ðxiÞÞ þ ððTBÞ2ðxiÞ þ ðIBÞ2ðxiÞ þ ðFBÞ2ðxiÞÞ
DðA; BÞ ¼ 1
n
X
n
i¼1
2ðTAðxiÞTBðxiÞ þ IAðxiÞIBðxiÞ þ FAðxiÞFBðxiÞÞ
½ððTAÞ2ðxiÞ þ ðIAÞ2ðxiÞ þ ðFAÞ2ðxiÞÞ þ ððTBÞ2ðxiÞ þ ðIBÞ2ðxiÞ þ ðFBÞ2ðxiÞÞ
It satisfies the following properties: It satisfies the following properties: 3 Similarity measures of bipolar neutrosophic sets 1. 0 DðA; BÞ 1; In this section, we introduce some similarity measures for
bipolar neutrosophic sets including Dice similarity mea-
sure, weighted Dice similarity measure, Hybrid vector
similarity measure and weighted Hybrid vector similarity
measure by extending the studies in [29, 45]. 2. DðA; BÞ ¼ DðB; AÞ; 3. DðA; BÞ ¼ 1 for A ¼ B i.e.TAðxiÞ ¼ TBðxiÞ; IAðxiÞ ¼
IBðxiÞ; FAðxiÞ ¼ FBðxiÞ
ði ¼ 1; 2. . .; nÞ 8 xiði ¼ 1; 2;
. . .; nÞ 2 X: Definition 6
[29] Let A ¼ hTAðxiÞ; IAðxiÞ; FAðxiÞi and
B ¼ hTBðxiÞ; IBðxiÞ; FBðxiÞi be two SVN-sets in a universe
of discourse X ¼ ðx1; x2; . . .; xnÞ: Then Definition
7
Let
A ¼ hTþ
A ðxiÞ; Iþ
A ðxiÞ;
Fþ
A ðxiÞ; T
A ðxiÞ;
I
A ðxiÞ; F
A ðxiÞi and B ¼ hTþ
B ðxiÞ; Iþ
B ðxiÞ; Fþ
B ðxiÞ; T
B ðxiÞ; EðX; YÞ ¼ k
2ðTAðxiÞTBðxiÞ þ IAðxiÞIBðxiÞ þ FAðxiÞFBðxiÞÞ
½ððTAÞ2ðxiÞ þ ðIAÞ2ðxiÞ þ ðFAÞ2ðxiÞÞ þ ððTBÞ2ðxiÞ þ ðIBÞ2ðxiÞ þ ðFBÞ2ðxiÞÞ
þ ð1 kÞ
TAðxiÞTBðxiÞ þ IAðxiÞIBðxiÞ þ FAðxiÞFBðxiÞ
½
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ððTAÞ2ðxiÞ þ ðIAÞ2ðxiÞ þ ðFAÞ2ðxiÞÞ
q
:
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ððTBÞ2ðxiÞ þ ðIBÞ2ðxiÞ þ ðFBÞ2ðxiÞÞ
q
! : EðX; YÞ ¼ k
2ðTAðxiÞTBðxiÞ þ IAðxiÞIBðxiÞ þ FAðxiÞFBðxiÞÞ
½ððTAÞ2ðxiÞ þ ðIAÞ2ðxiÞ þ ðFAÞ2ðxiÞÞ þ ððTBÞ2ðxiÞ þ ðIBÞ2ðxiÞ þ ðFBÞ2ðxiÞÞ
þ ð1 kÞ
TAðxiÞTBðxiÞ þ IAðxiÞIBðxiÞ þ FAðxiÞFBðxiÞ
½
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ððTAÞ2ðxiÞ þ ðIAÞ2ðxiÞ þ ðFAÞ2ðxiÞÞ
q
:
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ððTBÞ2ðxiÞ þ ðIBÞ2ðxiÞ þ ðFBÞ2ðxiÞÞ
q
! : EðX; YÞ ¼ k
2ðTAðxiÞTBðxiÞ þ IAðxiÞIBðxiÞ þ FAðxiÞFBðxiÞÞ
½ððTAÞ2ðxiÞ þ ðIAÞ2ðxiÞ þ ðFAÞ2ðxiÞÞ þ ððTBÞ2ðxiÞ þ ðIBÞ2ðxiÞ þ ðFBÞ2ðxiÞÞ
þ ð1 kÞ
TAðxiÞTBðxiÞ þ IAðxiÞIBðxiÞ þ FAðxiÞFBðxiÞ
½
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ððTAÞ2ðxiÞ þ ðIAÞ2ðxiÞ þ ðFAÞ2ðxiÞÞ
q
:
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ððTBÞ2ðxiÞ þ ðIBÞ2ðxiÞ þ ðFBÞ2ðxiÞÞ
q
! : EðX; YÞ ¼ k
2ðTAðxiÞTBðxiÞ þ IAðxiÞIBðxiÞ þ FAðxiÞFBðxiÞÞ
½ððTAÞ2ðxiÞ þ ðIAÞ2ðxiÞ þ ðFAÞ2ðxiÞÞ þ ððTBÞ2ðxiÞ þ ðIBÞ2ðxiÞ þ ðFBÞ2ðxiÞÞ
þ ð1 kÞ
TAðxiÞTBðxiÞ þ IAðxiÞIBðxiÞ þ FAðxiÞFBðxiÞ
½
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ððTAÞ2ðxiÞ þ ðIAÞ2ðxiÞ þ ðFAÞ2ðxiÞÞ
q
:
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ððTBÞ2ðxiÞ þ ðIBÞ2ðxiÞ þ ðFBÞ2ðxiÞÞ
q
! : It satisfies the following properties:
I
B ðxiÞ; F
B ðxiÞi b It satisfies the following properties: I
B ðxiÞ; F
B ðxiÞi be two BNSs in the set X ¼ fx1; x2; . . 3 Similarity measures of bipolar neutrosophic sets DwðA; BÞ ¼ 1 for A ¼ B i.e.,Tþ
A ðxiÞ ¼ Tþ
B ðxiÞ; Iþ
A ðxiÞ ¼
Iþ
B ðxiÞ; Fþ
A ðxiÞ
¼ Fþ
B ðxiÞ; T
A ðxiÞ
¼ T
B ðxiÞ; I
A ðxiÞ ¼
I
B ðxiÞ; F
A ðxiÞ ¼ F
B ðxiÞ ði ¼ 1; 2. . .; nÞ
8 xi ði ¼ 1;
2; . . .; nÞ 2 X: Proof 1. It is clear from Definition 8. DwðA; BÞ ¼
X
n
i¼1
wi
½ðTþ
A ðxiÞTþ
B ðxiÞ þ Iþ
A ðxiÞIþ
B ðxiÞ þ Fþ
A ðxiÞFþ
B ðxiÞÞðT
A ðxiÞT
B ðxiÞ þ I
A ðxiÞI
B ðxiÞ þ F
A ðxiÞF
B ðxiÞÞ
½ððTþ
A Þ2ðxiÞ þ ðIþ
A Þ2ðxiÞ þ ðFþ
A Þ2ðxiÞÞ þ ððTþ
B Þ2ðxiÞ þ ðIþ
B Þ2ðxiÞ þ ðFþ
B Þ2ðxiÞÞ
ððT
A Þ2ðxiÞ þ ðI
A Þ2ðxiÞ þ ðF
A Þ2ðxiÞÞ ððT
B Þ2ðxiÞ þ ðI
B Þ2ðxiÞ þ ðF
B Þ2ðxiÞÞ
0
B
@
1
C
A : DwðA; BÞ ¼
X
i¼1
wi
½ðTþ
A ðxiÞTþ
B ðxiÞ þ Iþ
A ðxiÞIþ
B ðxiÞ þ Fþ
A ðxiÞFþ
B ðxiÞÞðT
A ðxiÞT
B ðxiÞ þ I
A ðxiÞI
B ðxiÞ þ F
A ðxiÞF
B ðxiÞÞ
½ððTþ
A Þ2ðxiÞ þ ðIþ
A Þ2ðxiÞ þ ðFþ
A Þ2ðxiÞÞ þ ððTþ
B Þ2ðxiÞ þ ðIþ
B Þ2ðxiÞ þ ðFþ
B Þ2ðxiÞÞ
ððT
A Þ2ðxiÞ þ ðI
A Þ2ðxiÞ þ ðF
A Þ2ðxiÞÞ ððT
B Þ2ðxiÞ þ ðI
B Þ2ðxiÞ þ ðF
B Þ2ðxiÞÞ
0
B
@
1
C
A : Example 2
Suppose that A ¼ h0:8; 0:2; 0:5; 0:2; 0:3;
0:4i, B ¼ h0:6; 0:4; 0:3; 0:1; 0:2; 0:3i be two BNSs
in the set and
0 3 Then Example 2
Suppose that A ¼ h0:8; 0:2; 0:5; 0:2; 0:3;
0:4i, B ¼ h0:6; 0:4; 0:3; 0:1; 0:2; 0:3i be two BNSs
in the set and w ¼ 0:3. Then, 0:4i, B ¼ h0:6; 0:4; 0:3; 0:1; 0:2; 0:3i be two BNSs
in the set and w ¼ 0:3. 3 Similarity measures of bipolar neutrosophic sets DwðA; BÞ ¼ DwðB; AÞ; ½ðð0:5Þð0:7Þ þ ð0:2Þð0:1Þ þ ð0:6Þð0:3ÞÞðð0:4Þð0:2Þ þ ð0:3Þð0:2Þ þ ð0:7Þð0:1ÞÞ
½ðð0:5Þ2 þ ð0:2Þ2 þ ð0:6Þ2Þ þ ðð0:7Þ2 þ ð0:1Þ2 þ ð0:3Þ2Þ
ðð0:4Þ2 þ ð0:3Þ2 þ ð0:7Þ2Þ ðð0:2Þ2 þ ð0:2Þ2 þ ð0:1Þ2Þ
0
B
@
1
C
A
82 Definition
8
Let
A ¼ hTþ
A ðxiÞ; Iþ
A ðxiÞ;
Fþ
A ðxiÞ; T
A ðxiÞ;
I
A ðxiÞ; F
A ðxiÞi and B ¼ hTþ
B ðxiÞ; Iþ
B ðxiÞ; Fþ
B ðxiÞ; T
B ðxiÞ;
I
B ðxiÞ; F
B ðxiÞi be two BNSs in the set X ¼ fx1; x2; . . .; xng
and wi 2 ½0; 1 be the weight of each element xi for i ¼
1; 2; . . .; n such that Pn
i¼1 wi ¼ 1. Then, weighted Dice
similarity measure between BNS A and B, denoted
DwðA; BÞ, is defined as;
3. DwðA; BÞ ¼ 1 for A ¼ B i.e.,Tþ
A ðxiÞ ¼ Tþ
B ðxiÞ; Iþ
A ðxiÞ ¼
Iþ
B ðxiÞ; Fþ
A ðxiÞ
¼ Fþ
B ðxiÞ; T
A ðxiÞ
¼ T
B ðxiÞ; I
A ðxiÞ ¼
I
B ðxiÞ; F
A ðxiÞ ¼ F
B ðxiÞ ði ¼ 1; 2. . .; nÞ
8 xi ði ¼ 1;
2; . . .; nÞ 2 X:
Proof
1. It is clear from Definition 8. Definition
8
Let
A ¼ hTþ
A ðxiÞ; Iþ
A ðxiÞ;
Fþ
A ðxiÞ; T
A ðxiÞ;
I
A ðxiÞ; F
A ðxiÞi and B ¼ hTþ
B ðxiÞ; Iþ
B ðxiÞ; Fþ
B ðxiÞ; T
B ðxiÞ;
I
B ðxiÞ; F
B ðxiÞi be two BNSs in the set X ¼ fx1; x2; . . .; xng
and wi 2 ½0; 1 be the weight of each element xi for i ¼
1; 2; . . .; n such that Pn
i¼1 wi ¼ 1. Then, weighted Dice
similarity measure between BNS A and B, denoted
DwðA; BÞ, is defined as; 3. DwðA; BÞ ¼ 1 for A ¼ B i.e.,Tþ
A ðxiÞ ¼ Tþ
B ðxiÞ; Iþ
A ðxiÞ ¼
Iþ
B ðxiÞ; Fþ
A ðxiÞ
¼ Fþ
B ðxiÞ; T
A ðxiÞ
¼ T
B ðxiÞ; I
A ðxiÞ ¼
I
B ðxiÞ; F
A ðxiÞ ¼ F
B ðxiÞ ði ¼ 1; 2. . .; nÞ
8 xi ði ¼ 1;
2; . . .; nÞ 2 X: 3. 3 Similarity measures of bipolar neutrosophic sets .; xng:
Then, Dice similarity measure between BNS A and B,
denoted D(A, B), is defined as; I
B ðxiÞ; F
B ðxiÞi be two BNSs in the set X ¼ fx1; x2; . . .; xng:
Then, Dice similarity measure between BNS A and B,
denoted D(A, B), is defined as; 1. 0 DðA; BÞ 1;
2. DðA; BÞ ¼ DðB; AÞ;
3. DðA; BÞ ¼ 1 for A ¼ B i.e. Then, Dice similarity measure between BNS A and B,
denoted D(A, B), is defined as; 1. 0 DðA; BÞ 1;
2. DðA; BÞ ¼ DðB; AÞ;
3. DðA; BÞ ¼ 1 for A ¼ B i.e. DðA; BÞ ¼ 1
n
X
n
i¼1
½ðTþ
A ðxiÞTþ
B ðxiÞ þ Iþ
A ðxiÞIþ
B ðxiÞ þ Fþ
A ðxiÞFþ
B ðxiÞÞðT
A ðxiÞT
B ðxiÞ þ I
A ðxiÞI
B ðxiÞ þ F
A ðxiÞF
B ðxiÞÞ
½ððTþ
A Þ2ðxiÞ þ ðIþ
A Þ2ðxiÞ þ ðFþ
A Þ2ðxiÞÞ þ ððTþ
B Þ2ðxiÞ þ ðIþ
B Þ2ðxiÞ þ ðFþ
B Þ2ðxiÞÞ
ððT
A Þ2ðxiÞ þ ðI
A Þ2ðxiÞ þ ðF
A Þ2ðxiÞÞ ððT
B Þ2ðxiÞ þ ðI
B Þ2ðxiÞ þ ðF
B Þ2ðxiÞÞ
0
B
@
1
C
A DðA; BÞ ¼ 1
n
X
n
i¼1
½ðTþ
A ðxiÞTþ
B ðxiÞ þ Iþ
A ðxiÞIþ
B ðxiÞ þ Fþ
A ðxiÞFþ
B ðxiÞÞðT
A ðxiÞT
B ðxiÞ þ I
A ðxiÞI
B ðxiÞ þ F
A ðxiÞF
B ðxiÞÞ
½ððTþ
A Þ2ðxiÞ þ ðIþ
A Þ2ðxiÞ þ ðFþ
A Þ2ðxiÞÞ þ ððTþ
B Þ2ðxiÞ þ ðIþ
B Þ2ðxiÞ þ ðFþ
B Þ2ðxiÞÞ
ððT
A Þ2ðxiÞ þ ðI
A Þ2ðxiÞ þ ðF
A Þ2ðxiÞÞ ððT
B Þ2ðxiÞ þ ðI
B Þ2ðxiÞ þ ðF
B Þ2ðxiÞÞ
0
B
@
1
C
A 123 123 12 Neural Comput & Applic Example 1
Suppose that A ¼ h0:5; 0:2; 0:6; 0:4; 0:3;
0:7i, B ¼ h0:7; 0:1; 0:3; 0:2; 0:2; 0:1i be two BNSs
in the set. Then,
1. 0 DwðA; BÞ 1;
2. 3 Similarity measures of bipolar neutrosophic sets h
Definition 9
Let A ¼ hTþ
A ðxiÞ; Iþ
A ðxiÞ; Fþ
A ðxiÞ; T
A ðxiÞ; I
A
ðxiÞ; F
A ðxiÞi and B ¼ hTþ
B ðxiÞ; Iþ
B ðxiÞ; Fþ
B ðxiÞ; T
B ðxiÞ; I
B
ðxiÞ; F
B ðxiÞi be two BNSs in the set X ¼ fx1; x2; . . .; xng. Then, hybrid vector similarity measure between BNS A
and B, denoted HybV(A, B), is defined as; Definition 9
Let A ¼ hTþ
A ðxiÞ; Iþ
A ðxiÞ; Fþ
A ðxiÞ; T
A ðxiÞ; I
A
ðxiÞ; F
A ðxiÞi and B ¼ hTþ
B ðxiÞ; Iþ
B ðxiÞ; Fþ
B ðxiÞ; T
B ðxiÞ; I
B
ðxiÞ; F
B ðxiÞi be two BNSs in the set X ¼ fx1; x2; . . .; xng. Then, hybrid vector similarity measure between BNS A
and B, denoted HybV(A, B), is defined as; Definition 9
Let A ¼ hTþ
A ðxiÞ; Iþ
A ðxiÞ; Fþ
A ðxiÞ; T
A ðxiÞ; I
A
ðxiÞ; F
A ðxiÞi and B ¼ hTþ
B ðxiÞ; Iþ
B ðxiÞ; Fþ
B ðxiÞ; T
B ðxiÞ; I
B
ðxiÞ; F
B ðxiÞi be two BNSs in the set X ¼ fx1; x2; . . .; xng. Then, hybrid vector similarity measure between BNS A
and B, denoted HybV(A, B), is defined as; 3. Since Tþ
A ðxiÞ ¼ Tþ
B ðxiÞ; Iþ
A ðxiÞ ¼ Iþ
B ðxiÞ; Fþ
A ðxiÞ ¼ Fþ
B
ðxiÞ; T
A ðxiÞ ¼ T
B ðxiÞ; I
A ðxiÞ ¼ I
B ðxiÞ; F
A ðxiÞ ¼ F
B
ðxiÞ ði ¼ 1; 2. . .; nÞ 8 xi ði ¼ 1; 2; . . .; nÞ 2 X, we have
DwðA; BÞ ¼ 1. ð
Þ
The proof is completed. h
,
y
y
and B, denoted HybV(A, B), is defined as; ð
Þ
The proof is completed. ð
Þ
The proof is completed. h
y
and B, denoted HybV(A, ð
Þ
The proof is completed. 3 Similarity measures of bipolar neutrosophic sets Then, DwðA; BÞ ¼ 0:3
½ðð0:8Þð0:6Þ þ ð0:2Þð0:4Þ þ ð0:5Þð0:3ÞÞðð0:2Þð0:1Þ þ ð0:3Þð0:2Þ þ ð0:4Þð0:3ÞÞ
½ðð0:8Þ2 þ ð0:2Þ2 þ ð0:5Þ2Þ þ ðð0:6Þ2 þ ð0:4Þ2 þ ð0:3Þ2Þ
ðð0:2Þ2 þ ð0:3Þ2 þ ð0:4Þ2Þ ðð0:1Þ2 þ ð0:2Þ2 þ ð0:3Þ2Þ
0
B
@
1
C
A
¼ 0:1378 DwðA; BÞ ¼ 0:3
½ðð0:8Þð0:6Þ þ ð0:2Þð0:4Þ þ ð0:5Þð0:3ÞÞðð0:2Þð0:1Þ þ ð0:3Þð0:2Þ þ ð0:4Þð0:3ÞÞ
½ðð0:8Þ2 þ ð0:2Þ2 þ ð0:5Þ2Þ þ ðð0:6Þ2 þ ð0:4Þ2 þ ð0:3Þ2Þ
ðð0:2Þ2 þ ð0:3Þ2 þ ð0:4Þ2Þ ðð0:1Þ2 þ ð0:2Þ2 þ ð0:3Þ2Þ
0
B
@
1
C
A
¼ 0:1378 ð0:8Þð0:6Þ þ ð0:2Þð0:4Þ þ ð0:5Þð0:3ÞÞðð0:2Þð0:1Þ þ ð0:3Þð0:2Þ þ ð0:4Þð0:3ÞÞ
½ðð0:8Þ2 þ ð0:2Þ2 þ ð0:5Þ2Þ þ ðð0:6Þ2 þ ð0:4Þ2 þ ð0:3Þ2Þ
ðð0:2Þ2 þ ð0:3Þ2 þ ð0:4Þ2Þ ðð0:1Þ2 þ ð0:2Þ2 þ ð0:3Þ2Þ
1
C
A Proposition 1
Let DwðA; BÞ be a weighted Dice similarity
measure between BNSs A and B. Then, we have 123 123 Neural Comput & Applic 2. 2. 3 Similarity measures of bipolar neutrosophic sets DwðA; BÞ ¼
X
n
i¼1
wi
½ðTþ
A ðxiÞTþ
B ðxiÞ þ Iþ
A ðxiÞIþ
B ðxiÞ þ Fþ
A ðxiÞFþ
B ðxiÞÞðT
A ðxiÞT
B ðxiÞ þ I
A ðxiÞI
B ðxiÞ þ F
A ðxiÞF
B ðxiÞÞ
½ððTþ
A Þ2ðxiÞ þ ðIþ
A Þ2ðxiÞ þ ðFþ
A Þ2ðxiÞÞ þ ððTþ
B Þ2ðxiÞ þ ðIþ
B Þ2ðxiÞ þ ðFþ
B Þ2ðxiÞÞ
ððT
A Þ2ðxiÞ þ ðI
A Þ2ðxiÞ þ ðF
A Þ2ðxiÞÞ ððT
B Þ2ðxiÞ þ ðI
B Þ2ðxiÞ þ ðF
B Þ2ðxiÞÞ
0
B
@
1
C
A
¼
X
n
i¼1
wi
½ðTþ
B ðxiÞTþ
A ðxiÞ þ Iþ
B ðxiÞIþ
A ðxiÞ þ Fþ
B ðxiÞFþ
A ðxiÞÞðT
B ðxiÞT
A ðxiÞ þ I
B ðxiÞI
A ðxiÞ þ F
B ðxiÞF
A ðxiÞÞ
½ððTþ
B Þ2ðxiÞ þ ðIþ
B Þ2ðxiÞ þ ðFþ
B Þ2ðxiÞÞ þ ððTþ
A Þ2ðxiÞ þ ðIþ
A Þ2ðxiÞ þ ðFþ
A Þ2ðxiÞÞ
ððT
B Þ2ðxiÞ þ ðI
B Þ2ðxiÞ þ ðF
B Þ2ðxiÞÞ ððT
A Þ2ðxiÞ þ ðI
A Þ2ðxiÞ þ ðF
A Þ2ðxiÞÞ
0
B
@
1
C
A :
¼ DwðB; AÞ
½ðTþ
B ðxiÞTþ
A ðxiÞ þ Iþ
B ðxiÞIþ
A ðxiÞ þ Fþ
B ðxiÞFþ
A ðxiÞÞðT
B ðxiÞT
A ðxiÞ þ I
B ðxiÞI
A ðxiÞ þ F
B ðxiÞF
A ðxiÞÞ
½ððTþ
B Þ2ðxiÞ þ ðIþ
B Þ2ðxiÞ þ ðFþ
B Þ2ðxiÞÞ þ ððTþ
A Þ2ðxiÞ þ ðIþ
A Þ2ðxiÞ þ ðFþ
A Þ2ðxiÞÞ
ððT
B Þ2ðxiÞ þ ðI
B Þ2ðxiÞ þ ðF
B Þ2ðxiÞÞ ððT
A Þ2ðxiÞ þ ðI
A Þ2ðxiÞ þ ðF
A Þ2ðxiÞÞ
0
B
@
1
C
A 3. Since Tþ
A ðxiÞ ¼ Tþ
B ðxiÞ; Iþ
A ðxiÞ ¼ Iþ
B ðxiÞ; Fþ
A ðxiÞ ¼ Fþ
B
ðxiÞ; T
A ðxiÞ ¼ T
B ðxiÞ; I
A ðxiÞ ¼ I
B ðxiÞ; F
A ðxiÞ ¼ F
B
ðxiÞ ði ¼ 1; 2. . .; nÞ 8 xi ði ¼ 1; 2; . . .; nÞ 2 X, we have
DwðA; BÞ ¼ 1. The proof is completed. 3 Similarity measures of bipolar neutrosophic sets Then,
3. HybVwðA; BÞ ¼ 1 for
A ¼ B i.e.,Tþ
A ðxiÞ ¼
Tþ
B ðxiÞ;
Iþ
A ðxiÞ ¼ Iþ
B ðxiÞ; Fþ
A ðxiÞ ¼ Fþ
B ðxiÞ; T
A ðxiÞ ¼ T
B ðxiÞ;
I
A ðxiÞ ¼ I
B ðxiÞ; F
A ðxiÞ ¼ F
B ðxiÞði ¼ 1; 2. . .; nÞ 8 xi
ði ¼ 1; 2; . . .; nÞ 2 X: Example 3
Suppose that A ¼ h0:6; 0:3; 0:4; 0:1; 0:3;
0:4i, B ¼ h0:5; 0:2; 0:3; 0:4; 0:2; 0:5i be two BNSs
in the set. Then, HybVðA; BÞ ¼ ð0:4Þ 1
6
½ðð0:6Þð0:5Þ þ ð0:3Þð0:2Þ þ ð0:4Þð0:3ÞÞðð0:1Þð0:4Þ þ ð0:3Þð0:2Þ þ ð0:4Þð0:5ÞÞ
2½ðð0:6Þ2 þ ð0:3Þ2 þ ð0:4Þ2Þ þ ðð0:5Þ2 þ ð0:2Þ2 þ ð0:3Þ2Þ
ðð0:1Þ2 þ ð0:3Þ2 þ ð0:4Þ2Þ ðð0:4Þ2 þ ð0:2Þ2 þ ð0:5Þ2Þ
0
B
@
1
C
A
þ ð1 ð0:4ÞÞ 1
6
½ðð0:6Þð0:5Þ þ ð0:3Þð0:2Þ þ ð0:4Þð0:3ÞÞðð0:1Þð0:4Þ þ ð0:3Þð0:2Þ þ ð0:4Þð0:5ÞÞ
2½
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðð0:6Þ2 þ ð0:3Þ2 þ ð0:4Þ2Þ
q
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðð0:5Þ2 þ ð0:2Þ2 þ ð0:3Þ2Þ
q
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðð0:1Þ2 þ ð0:3Þ2 þ ð0:4Þ2Þ
q
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðð0:4Þ2 þ ð0:2Þ2 þ ð0:5Þ2Þ
q
0
B
B
B
@
1
C
C
C
A
= 0 1421 HybVðA; BÞ ¼ ð0:4Þ 1
6
½ðð0:6Þð0:5Þ þ ð0:3Þð0:2Þ þ ð0:4Þð0:3ÞÞðð0:1Þð0:4Þ þ ð0:3Þð0:2Þ þ ð0:4Þð0:5ÞÞ
2½ðð0:6Þ2 þ ð0:3Þ2 þ ð0:4Þ2Þ þ ðð0:5Þ2 þ ð0:2Þ2 þ ð0:3Þ2Þ
ðð0:1Þ2 þ ð0:3Þ2 þ ð0:4Þ2Þ ðð0:4Þ2 þ ð0:2Þ2 þ ð0:5Þ2Þ
0
B
@
1
C
A
þ ð1 ð0:4ÞÞ 1
6
½ðð0:6Þð0:5Þ þ ð0:3Þð0:2Þ þ ð0:4Þð0:3ÞÞðð0:1Þð0:4Þ þ ð0:3Þð0:2Þ þ ð0:4Þð0:5ÞÞ
2½
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðð0:6Þ2 þ ð0:3Þ2 þ ð0:4Þ2Þ
q
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðð0:5Þ2 þ ð0:2Þ2 þ ð0:3Þ2Þ
q
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðð0:1Þ2 þ ð0:3Þ2 þ ð0:4Þ2Þ
q
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðð0:4Þ2 þ ð0:2Þ2 þ ð0:5Þ2Þ
q
0
B
B
B
@
1
C
C
C
A
= 0.1421 = 0.1421 Definition
10
Let
A ¼ hTþ
A ðxiÞ; Iþ
A ðxiÞ; Fþ
A ðxiÞ; T
A ðxiÞ;
I
A ðxiÞ; F
A ðxiÞi and B ¼ hTþ
B ðxiÞ; Iþ
B ðxiÞ; Fþ
B ðxiÞ; T
B ðxiÞ;
I
B ðxiÞ; F
B ðxiÞi be two BNSs in the set X ¼ fx1; x2; . . .; xng
and wi 2 ½0; 1 be the weight of each element xi for i ¼
1; 2; . . .; n such that Pn
i¼1 wi ¼ 1. 4 BN-multi-criteria decision-making methods .; amÞ be the set of attributes, HybVwðA; BÞ ¼ k
X
n
i¼1
wi
½ðTþ
A ðxiÞTþ
B ðxiÞ þ Iþ
A ðxiÞIþ
B ðxiÞ þ Fþ
A ðxiÞFþ
B ðxiÞÞ ðT
A ðxiÞT
B ðxiÞ þ I
A ðxiÞI
B ðxiÞ þ F
A ðxiÞF
B ðxiÞÞ
2½ððTþ
A Þ2ðxiÞ þ ðIþ
A Þ2ðxiÞ þ ðFþ
A Þ2ðxiÞÞ þ ððTþ
B Þ2ðxiÞ þ ðIþ
B Þ2ðxiÞ þ ðFþ
B Þ2ðxiÞÞ
ððT
A Þ2ðxiÞ þ ðI
A Þ2ðxiÞ þ ðF
A Þ2ðxiÞÞ ððT
B Þ2ðxiÞ þ ðI
B Þ2ðxiÞ þ ðF
B Þ2ðxiÞÞ
0
B
@
1
C
A
þ ð1 kÞ
X
n
i¼1
wi
½ðTþ
A ðxiÞTþ
B ðxiÞ þ Iþ
A ðxiÞIþ
B ðxiÞ þ Fþ
A ðxiÞFþ
B ðxiÞÞ ðT
A ðxiÞT
B ðxiÞ þ I
A ðxiÞI
B ðxiÞ þ F
A ðxiÞF
B ðxiÞÞ
2½
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðTþ
A Þ2ðxiÞ þ ðIþ
A Þ2ðxiÞ þ ðFþ
A Þ2ðxiÞ
q
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðTþ
B Þ2ðxiÞ þ ðIþ
B Þ2ðxiÞ þ ðFþ
B Þ2ðxiÞ
q
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðT
A Þ2ðxiÞ þ ðI
A Þ2ðxiÞ þ ðF
A Þ2ðxiÞ
q
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðT
B Þ2ðxiÞ þ ðI
B Þ2ðxiÞ þ ðF
B Þ2ðxiÞ
q
0
B
B
B
@
1
C
C
C
A HybVwðA; BÞ ¼ k
X
n
i¼1
wi
½ðTþ
A ðxiÞTþ
B ðxiÞ þ Iþ
A ðxiÞIþ
B ðxiÞ þ Fþ
A ðxiÞFþ
B ðxiÞÞ ðT
A ðxiÞT
B ðxiÞ þ I
A ðxiÞI
B ðxiÞ þ F
A ðxiÞF
B ðxiÞÞ
2½ððTþ
A Þ2ðxiÞ þ ðIþ
A Þ2ðxiÞ þ ðFþ
A Þ2ðxiÞÞ þ ððTþ
B Þ2ðxiÞ þ ðIþ
B Þ2ðxiÞ þ ðFþ
B Þ2ðxiÞÞ
ððT
A Þ2ðxiÞ þ ðI
A Þ2ðxiÞ þ ðF
A Þ2ðxiÞÞ ððT
B Þ2ðxiÞ þ ðI
B Þ2ðxiÞ þ ðF
B Þ2ðxiÞÞ
0
B
@
1
C
A
þ ð1 kÞ
X
n
i¼1
wi
½ðTþ
A ðxiÞTþ
B ðxiÞ þ Iþ
A ðxiÞIþ
B ðxiÞ þ Fþ
A ðxiÞFþ
B ðxiÞÞ ðT
A ðxiÞT
B ðxiÞ þ I
A ðxiÞI
B ðxiÞ þ F
A ðxiÞF
B ðxiÞÞ
2½
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðTþ
A Þ2ðxiÞ þ ðIþ
A Þ2ðxiÞ þ ðFþ
A Þ2ðxiÞ
q
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðTþ
B Þ2ðxiÞ þ ðIþ
B Þ2ðxiÞ þ ðFþ
B Þ2ðxiÞ
q
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðT
A Þ2ðxiÞ þ ðI
A Þ2ðxiÞ þ ðF
A Þ2ðxiÞ
q
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðT
B Þ2ðxiÞ þ ðI
B Þ2ðxiÞ þ ðF
B Þ2ðxiÞ
q
0
B
B
B
@
1
C
C
C
A i 1
½ðTþ
A ðxiÞTþ
B ðxiÞ þ Iþ
A ðxiÞIþ
B ðxiÞ þ Fþ
A ðxiÞFþ
B ðxiÞÞ ðT
A ðxiÞT
B ðxiÞ þ I
A ðxiÞI
B ðxiÞ þ F
A ðxiÞF
B ðxiÞÞ
2½ððTþ
A Þ2ðxiÞ þ ðIþ
A Þ2ðxiÞ þ ðFþ
A Þ2ðxiÞÞ þ ððTþ
B Þ2ðxiÞ þ ðIþ
B Þ2ðxiÞ þ ðFþ
B Þ2ðxiÞÞ
ððT
A Þ2ðxiÞ þ ðI
A Þ2ðxiÞ þ ðF
A Þ2ðxiÞÞ ððT
B Þ2ðxiÞ þ ðI
B Þ2ðxiÞ þ ðF
B Þ2ðxiÞÞ
0
B
@
1
C
A
ð1 kÞ
X
n
i¼1
wi ½ðTþ
A ðxiÞTþ
B ðxiÞ þ Iþ
A ðxiÞIþ
B ðxiÞ þ Fþ
A ðxiÞFþ
B ðxiÞÞ ðT
A ðxiÞT
B ðxiÞ þ I
A ðxiÞI
B ðxiÞ þ F
A ðxiÞF
B ðxiÞÞ
2½
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðTþ
A Þ2ðxiÞ þ ðIþ
A Þ2ðxiÞ þ ðFþ
A Þ2ðxiÞ
q
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðTþ
B Þ2ðxiÞ þ ðIþ
B Þ2ðxiÞ þ ðFþ
B Þ2ðxiÞ
q
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðT
A Þ2ðxiÞ þ ðI
A Þ2ðxiÞ þ ðF
A Þ2ðxiÞ
q
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðT
B Þ2ðxiÞ þ ðI
B Þ2ðxiÞ þ ðF
B Þ2ðxiÞ
q
0
B
B
B
@
1
C
C
C
A ½ðTþ
A ðxiÞTþ
B ðxiÞ þ Iþ
A ðxiÞIþ
B ðxiÞ þ Fþ
A ðxiÞFþ
B ðxiÞÞ ðT
A ðxiÞT
B ðxiÞ þ I
A ðxiÞI
B ðxiÞ þ F
A ðxiÞF
B ðxiÞÞ
2½
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðTþ
A Þ2ðxiÞ þ ðIþ
A Þ2ðxiÞ þ ðFþ
A Þ2ðxiÞ
q
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðTþ
B Þ2ðxiÞ þ ðIþ
B Þ2ðxiÞ þ ðFþ
B Þ2ðxiÞ
q
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðT
A Þ2ðxiÞ þ ðI
A Þ2ðxiÞ þ ðF
A Þ2ðxiÞ
q
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðT
B Þ2ðxiÞ þ ðI
B Þ2ðxiÞ þ ðF
B Þ2ðxiÞ
q
1
C
C
C
A w ¼ ðw1; w2; . 3 Similarity measures of bipolar neutrosophic sets Then, weighted hybrid
vector similarity measure between BNS A and B, denoted
HybVwðA; BÞ, is defined as; 4 BN-multi-criteria decision-making methods In this section, we introduce applications of weighted
similarity measures in multi-criteria decision making
problems under bipolar neutrosophic environment. Definition 11
[36] Let U ¼ ðu1; u2; . . .; unÞ be a set of
alternatives, A ¼ ða1; a2; . . 3 Similarity measures of bipolar neutrosophic sets h HybVðA; BÞ ¼ k 1
n
X
n
i¼1
½ðTþ
A ðxiÞTþ
B ðxiÞ þ Iþ
A ðxiÞIþ
B ðxiÞ þ Fþ
A ðxiÞFþ
B ðxiÞÞðT
A ðxiÞT
B ðxiÞ þ I
A ðxiÞI
B ðxiÞ þ F
A ðxiÞF
B ðxiÞÞ
2½ððTþ
A Þ2ðxiÞ þ ðIþ
A Þ2ðxiÞ þ ðFþ
A Þ2ðxiÞÞ þ ððTþ
B Þ2ðxiÞ þ ðIþ
B Þ2ðxiÞ þ ðFþ
B Þ2ðxiÞÞ
ððT
A Þ2ðxiÞ þ ðI
A Þ2ðxiÞ þ ðF
A Þ2ðxiÞÞ ððT
B Þ2ðxiÞ þ ðI
B Þ2ðxiÞ þ ðF
B Þ2ðxiÞÞ
0
B
@
1
C
A
þ ð1 kÞ 1
n
X
n
i¼1
½ðTþ
A ðxiÞTþ
B ðxiÞ þ Iþ
A ðxiÞIþ
B ðxiÞ þ Fþ
A ðxiÞFþ
B ðxiÞÞðT
A ðxiÞT
B ðxiÞ þ I
A ðxiÞI
B ðxiÞ þ F
A ðxiÞF
B ðxiÞÞ
2½
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðTþ
A Þ2ðxiÞ þ ðIþ
A Þ2ðxiÞ þ ðFþ
A Þ2ðxiÞ
q
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðTþ
B Þ2ðxiÞ þ ðIþ
B Þ2ðxiÞ þ ðFþ
B Þ2ðxiÞ
q
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðT
A Þ2ðxiÞ þ ðI
A Þ2ðxiÞ þ ðF
A Þ2ðxiÞ
q
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðT
B Þ2ðxiÞ þ ðI
B Þ2ðxiÞ þ ðF
B Þ2ðxiÞ
q
0
B
B
B
@
1
C
C
C
A HybVðA; BÞ ¼ k 1
n
X
n
i¼1
½ðTþ
A ðxiÞTþ
B ðxiÞ þ Iþ
A ðxiÞIþ
B ðxiÞ þ Fþ
A ðxiÞFþ
B ðxiÞÞðT
A ðxiÞT
B ðxiÞ þ I
A ðxiÞI
B ðxiÞ þ F
A ðxiÞF
B ðxiÞÞ
2½ððTþ
A Þ2ðxiÞ þ ðIþ
A Þ2ðxiÞ þ ðFþ
A Þ2ðxiÞÞ þ ððTþ
B Þ2ðxiÞ þ ðIþ
B Þ2ðxiÞ þ ðFþ
B Þ2ðxiÞÞ
ððT
A Þ2ðxiÞ þ ðI
A Þ2ðxiÞ þ ðF
A Þ2ðxiÞÞ ððT
B Þ2ðxiÞ þ ðI
B Þ2ðxiÞ þ ðF
B Þ2ðxiÞÞ
0
B
@
1
C
A
þ ð1 kÞ 1
n
X
n
i¼1
½ðTþ
A ðxiÞTþ
B ðxiÞ þ Iþ
A ðxiÞIþ
B ðxiÞ þ Fþ
A ðxiÞFþ
B ðxiÞÞðT
A ðxiÞT
B ðxiÞ þ I
A ðxiÞI
B ðxiÞ þ F
A ðxiÞF
B ðxiÞÞ
2½
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðTþ
A Þ2ðxiÞ þ ðIþ
A Þ2ðxiÞ þ ðFþ
A Þ2ðxiÞ
q
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðTþ
B Þ2ðxiÞ þ ðIþ
B Þ2ðxiÞ þ ðFþ
B Þ2ðxiÞ
q
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðT
A Þ2ðxiÞ þ ðI
A Þ2ðxiÞ þ ðF
A Þ2ðxiÞ
q
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðT
B Þ2ðxiÞ þ ðI
B Þ2ðxiÞ þ ðF
B Þ2ðxiÞ
q
0
B
B
B
@
1
C
C
C
A i 1
½ðTþ
A ðxiÞTþ
B ðxiÞ þ Iþ
A ðxiÞIþ
B ðxiÞ þ Fþ
A ðxiÞFþ
B ðxiÞÞðT
A ðxiÞT
B ðxiÞ þ I
A ðxiÞI
B ðxiÞ þ F
A ðxiÞF
B ðxiÞÞ
2½
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðTþ
A Þ2ðxiÞ þ ðIþ
A Þ2ðxiÞ þ ðFþ
A Þ2ðxiÞ
q
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðTþ
B Þ2ðxiÞ þ ðIþ
B Þ2ðxiÞ þ ðFþ
B Þ2ðxiÞ
q
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðT
A Þ2ðxiÞ þ ðI
A Þ2ðxiÞ þ ðF
A Þ2ðxiÞ
q
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðT
B Þ2ðxiÞ þ ðI
B Þ2ðxiÞ þ ðF
B Þ2ðxiÞ
q
0
B
B
B
@
1
C
C
C
A
½ðTþ
A ðxiÞTþ
B ðxiÞ þ Iþ
A ðxiÞIþ
B ðxiÞ þ Fþ
A ðxiÞFþ
B ðxiÞÞðT
A ðxiÞT
B ðxiÞ þ I
A ðxiÞI
B ðxiÞ þ F
A ðxiÞF
B ðxiÞÞ
2½
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðTþ
A Þ2ðxiÞ þ ðIþ
A Þ2ðxiÞ þ ðFþ
A Þ2ðxiÞ
q
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðTþ
B Þ2ðxiÞ þ ðIþ
B Þ2ðxiÞ þ ðFþ
B Þ2ðxiÞ
q
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðT
A Þ2ðxiÞ þ ðI
A Þ2ðxiÞ þ ðF
A Þ2ðxiÞ
q
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðT
B Þ2ðxiÞ þ ðI
B Þ2ðxiÞ þ ðF
B Þ2ðxiÞ
q
0
B
B
B
@
1
C
C
C
A 123 123 12 Neural Comput & Applic Example 3
Suppose that A ¼ h0:6; 0:3; 0:4; 0:1; 0:3;
0:4i, B ¼ h0:5; 0:2; 0:3; 0:4; 0:2; 0:5i be two BNSs
in the set. Algorithm [bij]m×n =
⎛
⎜
⎜
⎜
⎝
a1
a2
· · ·
an
u1
b11
b12
· · ·
b1n
u2
b21
b22
· · ·
b2n
... ... ... ... ... um
bm1
bm2
· · ·
bmn
⎞
⎟
⎟
⎟
⎠ [bij]m×n =
⎛
⎜
⎜
⎜
⎝
a1
a2
· · ·
an
u1
b11
b12
· · ·
b1n
u2
b21
b22
· · ·
b2n
... ... ... ... ... um
bm1
bm2
· · ·
bmn
⎞
⎟
⎟
⎟
⎠ Step 1 Give the decision–making matrix ½bijmn; for
decision; Step 2 Compute the positive ideal (or negative ideal)
bipolar neutrosophic solution for ½bijmn;; Step 3 Calculate the weighted hybrid vector (or Dice)
similarity measure between positive ideal (or negative
ideal) bipolar neutrosophic solution b
j and bi ¼ ½bij1n
for all i ¼ 1; 2. . .; m and j ¼ 1; 2. . .; n as; is called an NB-multi-attribute decision-making matrix of
the decision maker. 4 BN-multi-criteria decision-making methods . .; wnÞT be the weight vector of the attributes
Cjðj ¼ 1; 2; . . .; nÞ such that wj 0 and Pn
j¼1 ¼ 1 and bij ¼
hTþ
ij ; Iþ
ij ; Fþ
ij ; T
ij ; I
ij ; F
ij i be the decision matrix whose
entries are the rating values of the alternatives. Then, Proposition 2
Let HybVwðA; BÞ be a weighted hybrid
vector similarity measure between bipolar neutrosophic
sets A and B. Then, we have 1. 0 HybVwðA; BÞ 1;
2. HybVwðA; BÞ ¼ HybVwðB; AÞ; 123 Neural Comput & Applic [bij]m×n =
⎛
⎜
⎜
⎜
⎝
a1
a2
· · ·
an
u1
b11
b12
· · ·
b1n
u2
b21
b22
· · ·
b2n
... ... ... ... ... um
bm1
bm2
· · ·
bmn
⎞
⎟
⎟
⎟
⎠
is called an NB-multi-attribute decision-making matrix of
the decision maker. Algorithm
Step 1 Give the decision–making matrix ½bijmn; for
decision;
Step 2 Compute the positive ideal (or negative ideal)
bipolar neutrosophic solution for ½bijmn;;
Step 3 Calculate the weighted hybrid vector (or Dice)
similarity measure between positive ideal (or negative
ideal) bipolar neutrosophic solution b
j and bi ¼ ½bij1n
for all i ¼ 1; 2. . .; m and j ¼ 1; 2. . .; n as; [bij]m×n =
⎛
⎜
⎜
⎜
⎝
a1
a2
· · ·
an
u1
b11
b12
· · ·
b1n
u2
b21
b22
· · ·
b2n
... ... ... ... ... um
bm1
bm2
· · ·
bmn
⎞
⎟
⎟
⎟
⎠
Algorithm
Step 1 Give the decision–making matrix ½bijmn; for
decision;
Step 2 Compute the positive ideal (or negative ideal)
bipolar neutrosophic solution for ½bijmn;;
Step 3 Calculate the weighted hybrid vector (or Dice) Algorithm HybVwðb
j ; biÞ ¼ k
X
n
j¼1
wj
½ððT
j ÞþðTijÞþ þ ðI
j ÞþðIijÞþ þ ðF
j ÞþðFijÞþÞððT
j ÞðTijÞ þ ðI
j ÞðIijÞ þ ðF
j ÞðFijÞÞ
2½ðððT
j ÞþÞ2 þ ððI
j ÞþÞ2 þ ððF
j ÞþÞ2Þ þ ððTþ
ij Þ2 þ ðIþ
ij Þ2 þ ðFþ
ij Þ2Þ
ðððT
j ÞÞ2 þ ððI
j ÞÞ2 þ ððF
j ÞÞ2Þ ððT
ij Þ2 þ ðI
ij Þ2 þ ðF
ij Þ2Þ
0
B
B
@
1
C
C
A
þ ð1 kÞ
X
n
j¼1
wj
½ððT
j ÞþðTijÞþ þ ðI
j ÞþðIijÞþ þ ðF
j ÞþðFijÞþÞðT
j ÞðTijÞ þ ðI
j ÞðIijÞ þ ðF
j ÞðFijÞÞ
2½
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ððT
j ÞþÞ2 þ ððI
j ÞþÞ2 þ ððF
j ÞþÞ2
q
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðTþ
ij Þ2 þ ðIþ
ij Þ2 þ ðFþ
ij Þ2
q
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ððT
j ÞÞ2 þ ððI
j ÞÞ2 þ ððF
j ÞÞ2
q
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðT
ij Þ2 þ ðI
ij Þ2 þ ðF
ij Þ2
q
0
B
B
B
@
1
C
C
C
A HybVwðb
j ; biÞ ¼ k
X
n
j¼1
wj
½ððT
j ÞþðTijÞþ þ ðI
j ÞþðIijÞþ þ ðF
j ÞþðFijÞþÞððT
j ÞðTijÞ þ ðI
j ÞðIijÞ þ ðF
j ÞðFijÞÞ
2½ðððT
j ÞþÞ2 þ ððI
j ÞþÞ2 þ ððF
j ÞþÞ2Þ þ ððTþ
ij Þ2 þ ðIþ
ij Þ2 þ ðFþ
ij Þ2Þ
ðððT
j ÞÞ2 þ ððI
j ÞÞ2 þ ððF
j ÞÞ2Þ ððT
ij Þ2 þ ðI
ij Þ2 þ ðF
ij Þ2Þ
0
B
B
@
1
C
C
A
þ ð1 kÞ
X
n
j¼1
wj
½ððT
j ÞþðTijÞþ þ ðI
j ÞþðIijÞþ þ ðF
j ÞþðFijÞþÞðT
j ÞðTijÞ þ ðI
j ÞðIijÞ þ ðF
j ÞðFijÞÞ
0
1
½ððT
j ÞþðTijÞþ þ ðI
j ÞþðIijÞþ þ ðF
j ÞþðFijÞþÞðT
j ÞðTijÞ þ ðI
j ÞðIijÞ þ ðF
j ÞðFijÞÞ
2½
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ððT
j ÞþÞ2 þ ððI
j ÞþÞ2 þ ððF
j ÞþÞ2
q
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðTþ
ij Þ2 þ ðIþ
ij Þ2 þ ðFþ
ij Þ2
q
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ððT
j ÞÞ2 þ ððI
j ÞÞ2 þ ððF
j ÞÞ2
q
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðT
ij Þ2 þ ðI
ij Þ2 þ ðF
ij Þ2
q
0
B
B
B
@ ð3Þ Step 4. Algorithm Determine the nonincreasing order of si ¼
HybVwðb
j ; biÞ and select the best alternative. Step 4. Determine the nonincreasing order of si ¼
HybVwðb
j ; biÞ and select the best alternative. Also; positive ideal bipolar neutrosophic solution u ¼
ðb
1; b
2; . . .; b
nÞ is the solution of decision matrix ½bijmn
where every component of has the following form: Example 4
Let us consider the decision-making problem
given in [41] for bipolar neutrosophic set. ‘‘Global envi-
ronmental concern is a reality, and an increasing attention
is focusing on the green production in various industries. A
car company is desirable to select the most appropriate
green supplier for one of the key elements in its manu-
facturing process.’’ [41]. After pre-evaluation, four sup-
pliers(alternatives) are taken into consideration, which are
denoted by u1, u2, u3 and u4. Three criteria are considered
including a1 is product quality; a2 is technology capability;
a3 is pollution control. Assume the weight vector of the b
j ¼ hmaxifTþ
ij g; minifIþ
ij g; minifFþ
ij g;
minifT
ij g; maxifI
ij g; maxifF
ij giðj ¼ 1; 2. . .; nÞ
ð1Þ and negative ideal bipolar neutrosophic solution u ¼
ðb
1; b
2; . . .; b
nÞ is the solution of decision matrix ½bijmn
where every component of has the following form: and negative ideal bipolar neutrosophic solution u ¼
ðb
1; b
2; . . .; b
nÞ is the solution of decision matrix ½bijmn
where every component of has the following form: b
j ¼ hminifTþ
ij g; maxifIþ
ij g; maxifFþ
ij g; maxifT
ij g; b
j ¼ hminifTþ
ij g; maxifIþ
ij g; maxifFþ
ij g; maxifT
ij g;
minifI
ij g; minifF
ij gi b
j ¼ hminifTþ
ij g; maxifIþ
ij g; maxifFþ
ij g; maxifT
ij g; minifI
ij g; minifF
ij gi
ð2Þ ð2Þ Now we give an algorithm as; Now we give an algorithm as; 12 123 12 Neural Comput & Applic decision-making problem with the bipolar neutrosophic
information given by S¸ahin et al. [36], Deli et al. [18], Deli
and S¸ubas¸ [16]. S¸ahin et al. [36] present a method by using
Jaccard vector similarity and weighted Jaccard vector
similarity measure and Deli and S¸ubas¸ [16] present a
method by using correlation measure based on multi-cri-
teria decision making for bipolar neutrosophic sets. Algorithm Step 1 The decision–making matrix ½bijmn is given by a
expert as; ⎛
⎜
⎜
⎝
a1
a2
a3
u1
0.4, 0.5, 0.3, −0.6, −0.4, −0.5
0.6, 0.1, 0.2, −0.4, −0.3, −0.2
0.8, 0.6, 0.5, −0.3, −0.2, −0.1
u2
0.6, 0.4, 0.2, −0.4, −0.5, −0.7
0.6, 0.2, 0.3, −0.5, −0.2, −0.3
0.7, 0.4, 0.5, −0.1, −0.3, −0.4
u2
0.7, 0.2, 0.4, −0.2, −0.6, −0.4
0.9, 0.3, 0.6, −0.2, −0.2, −0.5
0.6, 0.1, 0.5, −0.2, −0.4, −0.6
u4
0.8, 0.6, 0.5, −0.5, −0.3, −0.6
0.6, 0.4, 0.3, −0.1, −0.3, −0.4
0.9, 0.6, 0.4, −0.5, −0.3, −0.6
⎞
⎟
⎟
⎠ Step 2 The positive ideal bipolar neutrosophic solutions
for are computed as; u ¼ ½h0:8; 0:2; 0:2; 0:6; 0:3;
0:4i; h0:9; 0:1; 0:2; 0:5; 0:2; 0:2i; h0:9; 0:1; 0:4;
0:5; 0:2; 0:1i: Deli et al. [18] contains two major phrases. The method
firstly use score, certainty and accuracy functions to com-
pare the bipolar neutrosophic sets. Secondly, he use bipolar
neutrosophic weighted average operator and bipolar neu-
trosophic weighted geometric operator to aggregate the
bipolar neutrosophic information. The ranking results
obtained by different methods are summarized in Table 2. Step 3 The weighted hybrid vector similarity measure
between positive ideal (or negative ideal) bipolar
neutrosophic solution for alternative uj 2 U are com-
puted and selected the best alternative. In Table 2, there are some differences between the
ranking results obtained by the methods. The optimal
alternative is u3 obtained by the proposed methods except
the result obtained by the method of Deli et al.’s method
[18] and the proposed method HybVw with k ¼ 0:9. The
reason may be score, certainty and accuracy functions and
weighted
average
operator
and
bipolar
neutrosophic
weighted geometric operator in Deli et al.’s method [18]
and parameter k in the proposed method HybVw. Generally,
the proposed methods can effectively overcome the Step 4 Ranking the alternatives (Table 1). Algorithm Also, three criteria is W ¼ fw1; w2; w3gT ¼ f0:2; 0:5; 0:3gT. In
order to determine the decision information, an expert has
gathered the criteria values for the four possible alterna-
tives under bipolar neutrosophic environment. Then Table 1 Results for different
values of k 5 Comparative analysis and discussion In this subsection, a comparative study is presented to show
the flexibility and feasibility of the introduced NB-multi-
attribute decision-making method. Therefore, different
methods are used to solve the same NB-multi-attribute Table 1 Results for different
values of k
Similarity measure
Values
Measure value
Ranking order
HybVwðu ; uiÞ
k ¼ 0:25
HybVwðu ; u1Þ ¼ 0:24683
u3
u1
u4
u2
HybVwðu ; u2Þ ¼ 0:117780
HybVwðu ; u3Þ ¼ 0:27833
HybVwðu ; u4Þ ¼ 0:21136
HybVwðu ; uiÞ
k ¼ 0:3
HybVwðu ; u1Þ ¼ 0:27063
u3
u1
u4
u2
HybVwðu ; u2Þ ¼ 0:19497
HybVwðu ; u3Þ ¼ 0:30222
HybVwðu ; u4Þ ¼ 0:22904
HybVwðu ; uiÞ
k ¼ 0:6
HybVwðu ; u1Þ ¼ 0:41342
u3
u1
u4
u2
HybVwðu ; u2Þ ¼ 0:29803
HybVwðu ; u3Þ ¼ 0:44555
HybVwðu ; u4Þ ¼ 0:33510
HybVwðu ; uiÞ
k ¼ 0:9
HybVwðu ; u1Þ ¼ 0:55620
u1
u3
u4
u2
HybVwðu ; u2Þ ¼ 0:40109
HybVwðu ; u3Þ ¼ 0:54313
HybVwðu ; u4Þ ¼ 0:44116 123 123 Neural Comput & Applic Table 2 The ranking results of different methods
Methods
Ranking results
The proposed method HybVw with
k ¼ 0:25
u3
u1
u4
u2
The proposed method HybVw with
k ¼ 0:3
u3
u1
u4
u2
The proposed method HybVw with
k ¼ 0:6
u3
u1
u4
u2
The proposed method HybVw with
k ¼ 0:9
u1
u3
u4
u2
The proposed method Dwðu ; uiÞ
u3
u1
u4
u2
Deli and S¸ubas’s method [16]
u3
u1
u4
u2
Deli et al:0s method [18]
u2
u3
u4
u1
S¸ahin et al.0s method [36]
u3
u4
u2
u1 11. Broumi S, Bakali A, Talea M, Smarandache F (2016) Isolated
single valued neutrosophic graphs. neutrosophic Sets Syst
11:74–78 12. Balasubramanian A, Prasad KM, Arjunan K (2015) Bipolar
interval valued fuzzy subgroups of a group. Bull Math stat Res
3(3):234–239 13. Broumi S, Smarandache F (2013) Several similarity measures of
neutrosophic sets. Neutrosophic Sets Syst 1(1):54–62 14. Broumi S, Deli I, Smarandache F (2014) Distance and similarity
measures of interval neutrosophic soft sets. In: Critical review,
center for mathematics of uncertainty, Creighton University,
USA, vol 8, pp 14–31 15. Broumi S, Deli I (2016) Correlation measure for neutrosophic
refined sets and its application in medical diagnosis. Palest J Math
5(1):135–143 16. 5 Comparative analysis and discussion Deli I, Subas YA (2016) Multiple criteria decision making
method on single valued bipolar neutrosophic set based on cor-
relation coefficient similarity measure. In: International confer-
ence on mathematics and mathematics education (ICMME-
2016), Frat University, May 12–14, Elazg, Turkey decision-making problems which contain bipolar neutro-
sophic information. So, we think the proposed methods
developed in this paper is more suitable to handle this
application example. 17. Chen J, Li S, Ma S, Wang X (2014) m-Polar fuzzy sets: an
extension of bipolar fuzzy sets. Sci World J. doi:10.1155/2014/
416530 18. Deli I, Ali M, Smarandache F (2015) Bipolar neutrosophic sets
and their application based on multi-criteria decision making
problems. In:
2015
International
conference
on
advanced
mechatronic
systems
(ICAMechS). IEEE.(2015,
August),
pp 249–254 6 Conclusion This paper developed a multi-criteria decision-making
method for bipolar neutrosophic set is developed based on
these given similarity measures. To get the comprehensive
values, some similarity measures for bipolar neutrosophic
sets such as; Dice similarity measure, weighted Dice sim-
ilarity measure, Hybrid vector similarity measure and
weighted Hybrid vector similarity measure are introduced. In the future, it shall be significant to research some special
kinds of bipolar neutrosophic measures. 19. Deli I, Broumi S (2015) Neutrosophic soft matrices and NSM-
decision making. J Intell Fuzzy Syst 28:2233–2241 20. Karaaslan F (2016) Correlation coefficient between possibility
neutrosophic soft sets. Math Sci Lett 5(1):71–74 21. Lee KM (2000) Bipolar-valued fuzzy sets and their operations. In: Proceedings of international conference on intelligent tech-
nologies, Bangkok, Thailand, pp 307–312 22. Lee KJ (2009) Bipolar fuzzy subalgebras and bipolar fuzzy ideals
of BCK/BCI-algebras. Bull Malays Math Sci Soc 32(3):361–373 23. Ma YX, Wang JQ, Wang J, Wu XH (2016) An interval neutro-
sophic linguistic multi-criteria group decision–making method
and its application in selecting medical treatment options. Neural
Comput Appl. doi:10.1007/s00521-016-2203-1 24. Maji PK (2013) Neutrosophic soft set. Ann Fuzzy Math Inf
5(1):157–168 References 25. Majumder SK (2012) Bipolar valued fuzzy sets in -semigroups. Math Aeterna 2(3):203–213 1. Ali M, Smarandache F (2016) Complex neutrosophic set. Neural
Comput Appl 25:1–18 26. Majumdar P, Smanata SK (2014) On similarity and entropy of
neutrosophic sets. J Intell Fuzzy Syst 26(3):1245–1252 2. Ali M, Deli I, Smarandache F (2016) The theory of neutrosophic
cubic sets and their applications in pattern recognition. J Intell
Fuzzy Syst 30(4):1957–1963 27. Mondal K, Pramanik S (2015) Neutrosophic tangent similarity
measure and its application to multiple attribute decision making. Neutrosophic Sets Syst 9:85–92 3. Atanassov K (1986) Intuitionistic fuzzy sets. Fuzzy Sets Syst
20:87–96 28. Mukherjee A, Sarkar S (2014) Several similarity measures of
neutrosophic soft sets and its application in real life problems. Ann Pure Appl Math 7(1):1–6 4. Athar KA (2014) Neutrosophic multi-criteria decision making
method. New Math Nat Comput 10(02):143–162 29. Pramanik S, Biswas P, Giri BC (2015) Hybrid vector similarity
measures and their applications to multi-attribute decision mak-
ing under neutrosophic environment. Neural Comput Appl 1–14. doi:10.1007/s00521-015-2125-3 5. Aydog˘du A (2015) On similarity and entropy of single valued
neutrosophic sets. Gen Math Notes 29(1):67–74 6. Broumi S, Smarandache F (2013) Several similarity measures of
neutrosophic sets. Neutro Sophic Sets Syst 1(1):54–62 p
p
y
7. Broumi S (2013) Generalized neutrosophic soft set. Int J Comput
Scie Eng Inf Technol(IJCSEIT) 3/2:17–30 30. Pramanik S, Mondal K (2015) Cosine similarity measure of rough
neutrosophic sets and its application in medical diagnosis. Global
J Adv Res 2(1):212–220 8. Broumi S, Talea M, Bakali A, Smarandache F (2016) Single
valued neutrosophic graphs. J New Theory 10:86–101 31. Peng JJ, Wang JQ, Zhang HY, Chen XH (2014) An outranking
approach for multi-criteria decision–making problems with sim-
plified neutrosophic sets. Appl Soft Comput 25:336–346 9. Broumi S, Talea M, Bakali A, Smarandache F (2016) On bipolar
single valued neutrosophic graphs. J New Theory 11:84–102 32. Smarandache F (1998) A unifying field in logics neutrosophy:
neutrosophic probability, set and logic. American Research Press,
Rehoboth 10. Broumi S, Smarandache F, Talea M, Bakali A (2016) An intro-
duction to bipolar single valued neutrosophic graph theory. Appl
Mech Mater 841:184–191 12 3 Neural Comput & Applic and their application in multi-criteria decision–making problems. Int J Fuzzy Syst. doi:10.1007/s40815-016-0180-2 33. Smarandache F (2005) Neutrosophic set, a generalisation of the
intuitionistic fuzzy sets. Int J Pure Appl Math 24:287–297 34. References Santhi V, Shyamala G (2015) Notes On Bipolar-Valued Multi
Fuzzy Subgroups Of A Group. Int J Math Arch (IJMA). 6(6). ISSN 2229–5046 43. Xu X, Zhang L, Wan Q (2012) A variation coefficient similarity
measure and its application in emergency group decision making. Syst Eng Proc 5:119–124 44. Ye J (2014) Vector similarity measures of simplified neutro-
sophic sets and their application in multicriteria decision making. Int J Fuzzy Syst 16(2):204–215 35. Saeid AB (2009) Bipolar-valued fuzzy BCK/BCI-algebras. World Appl Sci J 7(11):1404–1411 36. S¸ahin M, Deli I, and Ulucay V (2016) Jaccard vector similarity
measure of bipolar neutrosophic set based on multi-criteria
decision making. In: International conference on natural science
and engineering (ICNASE’16), March 19–20, Kilis 45. Ye J (2014) Improved correlation coefficients of single valued
neutrosophic sets and interval neutrosophic sets for multiple
attribute decision making. J Intell Fuzzy Syst 27:24532462 46. Ye J, Zhang QS (2014) Single valued neutrosophic similarity
measures for multiple attribute decision making. Neutrosophic
Sets Syst 2:48–54 37. Tian ZP, Wang J, Wang JQ, Zhang HY (2016) Simplified neu-
trosophic
linguistic
multi-criteria
group
decision–making
approach to green product development. Group Decis Negot
1–31. doi:10.1007/s10726-016-9479-5 y
47. Ye S, Ye J (2014) Dice similarity measure between single valued
neutrosophic multisets and its application in medical diagnosis. Neutrosophic Sets Syst 6:48–52 38. Tian ZP, Wang J, Wang JQ, Zhang HY (2016) An improved
MULTIMOORA approach for multi-criteria decision–making
based on interdependent inputs of simplified neutrosophic lin-
guistic information. Neural Comput Appl. doi:10.1007/s00521-
016-2378-5 p
y
48. Ye J, Fu J (2016) Multi-period medical diagnosis method using a
single valued neutrosophic similarity measure based on tangent
function. Comput Methods Program Biomed 123:142–149 39. Tian ZP, Zhang HY, Wang J, Wang JQ, Chen XH (2015) Multi-
criteria decision–making method based on a cross-entropy with
interval neutrosophic sets. Int J Syst Sci. doi:10.1080/00207721. 1102359 49. Ye J (2015) Single-valued neutrosophic similarity measures
based on cotangent function and their application in the fault
diagnosis of steam turbine. Soft Comput 1–9. doi:10.1007/
s00500-015-1818-y 40. Wang H, Smarandache FYQ, Zhang Q, Sunderraman R (2010)
Single
valued
neutrosophic
sets. Multispace
Multistructure
4:410–413 50. Jun YB, Kang MS, Kim HS (2009) Bipolar fuzzy structures of
some types of ideals in hyper BCK-algebras. Sci Math Jpn
70:109–121 41. Wu J, Cao QW (2013) Same families of geometric aggregation
operators with intuitionistic trapezoidal fuzzy numbers. Appl
Math Model 37:318–327 51. Zadeh LA (1965) Fuzzy sets. References Inf Control 8:338–35 52. Zhang HY, Ji P, Wang JQ, Chen XH (2016) A neutrosophic
normal cloud and its application in decision–making. Cogn
Comput. doi:10.1007/s12559-016-9394-8 42. Wu XH, Wang JQ, Peng JJ, Chen XH (2016) Cross-entropy and
prioritized aggregation operator with simplified neutrosophic sets 123 View publication stats
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Helicobacter pylori Infection Promotes Gastric Premalignancies and Malignancies Lesions and Demotes Hyperplastic Polyps: A 5 Year Multicentric Study among Cameroonian Dyspeptic Patients
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Abstract Background: Helicobacter pylori infection is the most well-known risk factor for gastric mucosa abnormalities. However, some geographic regions with persistent high H. pylori infection rates do not suffer from high gastric mucosa
lesions incidence. The aim of the study was to establish the relationship between H. pylori infection and gastric
pathological features in Cameroon. Methods: We performed a retrospective study, collecting data from the University
Teaching Hospital and the Cameroon Pasteur institute on 1290 patients (mean age 46.31 ± 16.45 years, sex ratio 1.19:1)
for whom histological features of the gastric mucosa and H. pylori infection were investigated from 2014 to 2019. Data were extracted from the medical records; hospital computerized databases; or clinical charts of these patients and
reviewed according to gender and age of participants. The study was approved by the Ethical Committee of Medical
Sciences. Result: Approximately 3% (2.56%) of the sample population were with normal gastric mucosa whereas
chronic gastritis, atrophic gastritis, intestinal metaplasia, dysplasia, carcinoma, hyperplastic polyps and MALT lymphoma
was found in 75.35, 8.2, 7.7, 2.8, 9.3, 1.55 and 0.8% of cases respectively. Unlike hyperplasia (OR= 0.3838), infected
participants were in a high risk to develop gastric lesions with an odds ratio of 1.1775, 1.4866, 1.4415, 1.2088, 0.9408
and 0.9075 for gastritis, atrophic gastric, dysplasia, carcinoma, intestinal metaplasia and MALT lymphoma respectively. Conclusion: our finding showed that chronic gastritis, gastric premalignancies and malignancies are positively link to
Helicobacter pylori infection and that hyperplastic polyp is inversely associated with H. pylori infection in our milieu. Keywords: H pylori infection- Gastric precancerous and cancerous lesions- Cameroon. Asian Pac J Cancer Prev, 24 (1), 171-183 Infection due to this bacterium can be life-long in
the host unless it is eradicated (Amieva and El-Omar,
2008). Although 85% of infected patients have only mild
asymptomatic gastritis, gastric colonization with this
bacterium in some cases can cause several gastrointestinal
diseases such as, chronic gastritis, gastro-duodenal ulcers
and gastric malignancies. It is reported that 15% of
H. pylori infected patients can develop peptic ulcer disease
(PUD) and less than 1% can develop gastric cancer
(Amieva and El-Omar, 2008; Niyaz and Leonardo, 2005). DOI:10.31557/APJCP.2023.24.1.171
Degeneration of the Gastric Lining DOI:10.31557/APJCP.2023.24.1.171
Degeneration of the Gastric Lining RESEARCH ARTICLE Editorial Process: Submission:06/16/2022 Acceptance:01/08/2023 Helicobacter pylori Infection Promotes Gastric Premalignancies
and Malignancies Lesions and Demotes Hyperplastic Polyps:
A 5 Year Multicentric Study among Cameroonian Dyspeptic
Patients Ghislaine Florice Faujo Nintewoue1, Laure Brigitte Kouitcheu Mabeku2* Asian Pacific Journal of Cancer Prevention, Vol 24
171
1Department of Biochemistry, Faculty of Science, University of Dschang, Cameroon. 2Deparment of Microbiology, Faculty of
Sciences, University of Yaounde 1, Cameroon. *For Correspondence: laurebkouitcheu@yahoo.fr Abstract Many epidemiological surveys conducted in Africa
where incidence of infection is higher have revealed
that 70–90% of duodenal and gastric ulcer cases is
due to this bacterium (Alkout et al., 2000; Eyoum et
al., 2020).There are two form of gastric malignancies
or neoplasia; adenocarcinomas which represents
95% of the stomach cancers and Mucosa-associated
lymphoid tissue lymphoma (MALT lymphoma) which
accounts for up to 3 % (Freeman et al., 1972). Both the Participants and Baseline assessment The study involved the analysis of existing clinical
and laboratory data from all consecutive patients with
dyspepsia whose gastric biopsies samples were brought
for examination at the anatomo-cytopathology unit
of either CPC or CHU from 2014 to 2019. Data were
extracted from the medical records; hospital computerized
databases; or clinical charts of the patients and reviewed
according to a pre-established protocol including the
following demographics variables (sex and age). The
biopsy specimens were usually taken from the antrum and
other locations if required. Biopsy sample from patient
with more than one biopsy specimen collected were mixed
before processing, so one biopsy sample or mixed biopsy
sample were analyzed per participant and the number of
biopsy specimen analyzed was equal to the number of
participants. Only patients for whom histological features
of the gastric mucosa and H. pylori were investigated were
included in the study. All biopsies samples were analyzed
by a dedicated anatomo pathologist in the selected
health facilities. Samples were fixed overnight in a 10%
buffered formaldehyde solution, processed, embedded
in paraffin, cut and the slides were stained by the
Hematoxylin-Eosin (HE) method for histological features
detection. Histological features of the gastric mucosa
included: normal gastric mucosa that is gastric mucosa
without injuries, chronic inflammation, inflammatory
activity, gastric atrophy, intestinal metaplasia, dysplasia,
hyperplasia polyps, adenocarcinoma and MALT
lymphoma. All these variables were classified as absent
or present, and when present the intensity of inflammation
and the neutrophil activity were ranked as mild, moderate Several diagnostic methods have been developed
in order to detect Helicobacter pylori accurately. These
tests include non-invasive methods and invasive methods
which require upper gastrointestinal endoscopy to obtain
gastric biopsy samples (Kouitcheu et al., 2020). Among
these, histological examination is one of the most useful
invasive diagnostic tests for H. pylori infection (Kouitcheu
et al., 2020). Histology plays a pivotal role in detecting
H. pylori and it also provides more information about the
degree of inflammation and associated pathologies, such as
atrophic gastritis (AG), intestinal metaplasia (IM), MALT
lymphoma, hyperplasia and gastric cancer. y p
yp p
g
As expected in developing country, Cameroon is
an endemic area for H. pylori infection. Prevalence
of infection ranges from 92.2% among apparently
healthy children in the Southern region in 2004 to
64.34% among patients with gastro-duodenal disorders
aged 35 years and above in the Littoral region in 2018
(Kouitcheu et al., 2018). All these previous studies on
H. Introduction About 1
in 12 of all oncological deaths is attributable to gastric
cancer. Gastric cancer has the fifth highest incidence
among cancers, with 5.7% of all new cases attributable to
the disease and over a million new cases of gastric cancer
are diagnosed, worldwide each year (Bray et al., 2018). two form of gastric neoplasia are related to H. pylori
infection. In fact, Helicobacter pylori provokes a local
inflammation in almost all host, a continuous process
which increases the risk of developing atrophic gastritis,
intestinal metaplasia, dysplasia and non cardia gastric
adenocarcinoma (Correa, 2004). On the other hands,
long time gastric colonization by H. pylori induce local
humoral and T-cell response in the gastric mucosa, this
persistent stimulation of activated B cells by T cells lead to
the development of lymphoid follicles which predisposes
the development of MALT lymphoma (Katharina et al.,
2015). Hyperplastic polyps (HPs) result from hyper-
regenerative epithelium in response to an underlying
chronic inflammatory stimulus (Carmack et al., 2009). It
account for approximately 75 percent of gastric polyps
in geographic areas where H. pylori is common. Over
time, polyps may remain stable, increase in size, or
regress following H. pylori eradication (Ljubicic et al.,
1999). Polyps have malignant potential, malignancy
develops in hyperplastic polyps through a dysplasia/
carcinoma sequence (Ahm et al., 2014). Between 1 and
20 percent of hyperplastic polyps have been reported to
harbor foci of dysplasia (Ahm et al., 2014). The risk of
malignancy in hyperplastic polyps is increased in polyps
>1 cm (Ahm et al., 2014). Globally, gastric cancer is the
leading cause of infection-associated cancers. According
to GLOBOCAN 2018 data, gastric cancer is the third
leading cause of cancer deaths worldwide, following only
lung and colorectal cancer in overall mortality. About 1
in 12 of all oncological deaths is attributable to gastric
cancer. Gastric cancer has the fifth highest incidence
among cancers, with 5.7% of all new cases attributable to
the disease and over a million new cases of gastric cancer
are diagnosed, worldwide each year (Bray et al., 2018). cancerous lesions among population are not available in
the country to our knowledge. Therefore, this study was
investigated in a Cameroonian population characterised
by its high incidence of H. pylori infection in order to
visualise the morbidity associated with this bacterium. Setting and study design From March to May 2019, we performed a multicentre
retrospective study based on gastric endoscopy biopsy data
collected from 2014 to 2019 at the anatomo-cytopathology
unit of two reference health facilities located in Yaoundé,
the Centre region of Cameroon; the University Teaching
Hospital (CHU) and the Cameroon Pasteur institute
(CPC). The University Teaching Hospital is a tertiary
hospital. It is a large teaching hospital which offers full and
high-complexity service serving around 2,765,600 people
from its catchment area. Its anatomo cytopathology unit
receives patients referred from other hospitals in Yaoundé
and also from different parts of the country. The Cameroon
Pasteur Institute is a reference clinical and research Centre
of the country. It has the highest technical platform that
can support all types of clinical analysis. Its anatomo
cytopathology laboratory receives and analyses clinical
samples collected for oncology analysis all around the
country, because adequate technical platform for oncology
analysis are not widely available in Cameroon. Introduction Helicobacter pylori is a gram negative, spiral shaped
micro-organism that inhabits and colonizes the harsh
acidic environment of the gastric mucosa. More than 50%
of the world’s wide population is chronically infected by
this bacterium. However, a great discrepancy exists in
the prevalence of H. pylori worldwide; 20 to 50% of the
population seems to be infected in the developed countries
and up to 90% in the developing countries (Alkout et al.,
2000). This steep decline of H. pylori infection between
developed countries and developing ones is mainly due
to the improvement in personal hygiene and community
sanitation that prevent re-infection, since the prevalence
of this infection varies according to the socioeconomic
conditions and the living standard of the populations. This disparity may also be due to the widespread use of
H. pylori eradication therapy that reduced the prevalence
of this infection in developed countries. 171 Ghislaine Florice Faujo Nintewoue and Laure Brigitte Kouitcheu Mabeku two form of gastric neoplasia are related to H. pylori
infection. In fact, Helicobacter pylori provokes a local
inflammation in almost all host, a continuous process
which increases the risk of developing atrophic gastritis,
intestinal metaplasia, dysplasia and non cardia gastric
adenocarcinoma (Correa, 2004). On the other hands,
long time gastric colonization by H. pylori induce local
humoral and T-cell response in the gastric mucosa, this
persistent stimulation of activated B cells by T cells lead to
the development of lymphoid follicles which predisposes
the development of MALT lymphoma (Katharina et al.,
2015). Hyperplastic polyps (HPs) result from hyper-
regenerative epithelium in response to an underlying
chronic inflammatory stimulus (Carmack et al., 2009). It
account for approximately 75 percent of gastric polyps
in geographic areas where H. pylori is common. Over
time, polyps may remain stable, increase in size, or
regress following H. pylori eradication (Ljubicic et al.,
1999). Polyps have malignant potential, malignancy
develops in hyperplastic polyps through a dysplasia/
carcinoma sequence (Ahm et al., 2014). Between 1 and
20 percent of hyperplastic polyps have been reported to
harbor foci of dysplasia (Ahm et al., 2014). The risk of
malignancy in hyperplastic polyps is increased in polyps
>1 cm (Ahm et al., 2014). Globally, gastric cancer is the
leading cause of infection-associated cancers. According
to GLOBOCAN 2018 data, gastric cancer is the third
leading cause of cancer deaths worldwide, following only
lung and colorectal cancer in overall mortality. Asian Pacific Journal of Cancer Prevention, Vol 24 Statistical analysis pylori Infection According to Age and Sex of Participants
n, number; SD, Standard deviation; X2, Chi-square
Variables
Histological features
R square
(P value)
Nor (%)
n=33
CSG (%)
n=972
AG (%)
n=106
IM (%)
n=100
DYS (%)
n=36
GC (%)
n=120
MALT (%)
n= 10
HPs (%)
n=20
Sex
Female
18 (54.5)
523 (53.8)
62 (58.5)
60 (60)
14 (38.8)
75 (62.5)
2 (20.0)
10 (50)
Male
15 (45.5)
449 (46.2)
44 (41.5)
40 (40)
22 (61.2)
45 (37.5)
8 (80.0)
10 (50)
Total
number
(Prevalence)
33 (2.56)
972 (75.35)
106 (8.22)
100 (7.75)
36 (2.79)
120 (9.30)
10 (0.77)
20 (1.55)
Age (years)
Mean ± SD
40.17± 9.59
41.73±15.35
46.57±16.33
54.34±15.69
55.77± 19.51
56.55 ± 16.61
47.13± 19.81
52.25±19.65
0.012
(p=0.5879)
≤20
2 (6.1)
45 (4.6)
04 (3.8)
4 (4.0)
1 (2.8)
2 (1.7)
1 (10.0)
1 (5.0)
21-39
17 (51.5)
340 (35)
32 (30.2)
16 (16.0)
8 (22.2)
32 (26.7)
2 (20.0)
5 (25.0)
40-59
8 (24.2)
414 (42.6)
47 (44.3)
45 (45.0)
11 (30.5)
40 (33.3)
4 (40.0)
5 (25.0)
≥60
6 (18.2)
173 (17.8)
23 (21.7)
35 (35.0)
16 (44.5)
46 (38.3)
3 (30.0)
9 (45.0)
Total
number
(Prevalence)
33 (2.56)
972 (75.35)
106 (8.22)
100 (7.75)
36 (2.79)
120 (9.30)
10 (0.77)
20 (1.55)
Table 2. Statistical analysis The data collected throughout this study was coded
before being entered in the database and analysed using Variables
Number (%)
H. pylori positive (%)
H. pylori negative (%)
X2, (P value)
Gender, Sex ratio: 1.19/1
Female
702
309 (45.71)
279 (45.44)
0.9554
Male
588
367 (54.29)
335 (54.56)
Total (%)
1290
676 (52.40)
614 (47.6)
Age (years), (Mean ±SD)
46.31 ± 16.45)
48.46 ± 4.31
49.15 ± 4.07
≤20
55
29 (4.28)
26 (4.23)
0.4664, (0.9260)
20-39
425
221 (32.69)
204 (33.22)
40-59
484
250 (36.98)
234 (38.11)
>60
326
176 (26.03)
150 (24.42)
Total (%)
1290
676 (52.40)
614 (47.6)
Table 1. Characteristic and Distribution of H. pylori Infection According to Age and Sex of Participants
n, number; SD, Standard deviation; X2, Chi-square able 1. Characteristic and Distribution of H. pylori Infection According to Age and Sex of Participants Variables
Number (%)
H pylori positive (%)
H pylori negative (%)
X2 (
Table 1. Characteristic and Distribution of H. pylori Infection According to Age and Sex of Participants Variables
Number (%)
H. pylori positive (%)
H. pylori negative (%)
X2, (P value)
Gender, Sex ratio: 1.19/1
Female
702
309 (45.71)
279 (45.44)
0.9554
Male
588
367 (54.29)
335 (54.56)
Total (%)
1290
676 (52.40)
614 (47.6)
Age (years), (Mean ±SD)
46.31 ± 16.45)
48.46 ± 4.31
49.15 ± 4.07
≤20
55
29 (4.28)
26 (4.23)
0.4664, (0.9260)
20-39
425
221 (32.69)
204 (33.22)
40-59
484
250 (36.98)
234 (38.11)
>60
326
176 (26.03)
150 (24.42)
Total (%)
1290
676 (52.40)
614 (47.6)
Table 1. Characteristic and Distribution of H. Participants and Baseline assessment pylori prevalence in Cameroon were investigated
on a specific age group and used a non-invasive test
which is not reliable for a current infection. Moreover,
studies concerning the simultaneous evaluation of H. pylori colonisation assayed histologically (an accurate
invasive diagnostic test for current H. pylori infection) in
association to gastric histopathological features such as
hyperplastic polyps, MALT lymphoma, precancerous and 172 Asian Pacific Journal of Cancer Prevention, Vol 24
2 DOI:10.31557/APJCP.2023.24.1.171
Degeneration of the Gastric Lining or severe (Price, 1991). Chronic inflammation was
considered when high level of mononuclear leucocytes,
including lymphocytes, plasmocytes and macrophages
was found. Inflammatory activity was confirmed by
the detection of polymorphonuclear cells (neutrophils)
in the lamina propria, epithelium or lumen. H. pylori
infection was established though histology examination. Participants were considered H. pylori positive if the
bacteria could be detected histologically in any of the
Giemsa-stained slides. the SPSS 20 software package. The frequency of each
histological feature of the gastric mucosa according to
H. pylori infection was determined and compared to
that of normal mucosa taken as the reference group. Differences in the distribution of these variables according
to sociodemographic parameters were assessed by the
chi-square test and those with value P < 0.05 were
considered statistically significant. Odds ratios (OR) and
95% confidence intervals (95% CI) were calculated for
each gastric lesions in relation to the presence of H. pylori
infection using univariable and multivariable logistic
regression. Results Characteristic of the study population according to
H. pylori infection A total of 1290 dyspeptic participants were enrolled
in this study; 158 in 2014, 201 in 2015, 188 in 2016, 360
in 2017 and 383 in 2018. Among these participants, 1123
were from the CPC and 167 from the UHC. Their mean
age was 46.31 ± 16.45 years (range 08 – 89 years), and
the age groups from 20 to 39 (33.10%, 427/1290) and Ethical consideration This study was approved by the Institutional Review
Board of University Teaching Hospital (Approval no 703/
AR/CHUY/DG/DGA/CAPRC) and Cameroon Pasteur
institute CPC (Approval no 000378/2019/CPC/DG/DAF/
SRH) and the Ethical Committee of Medical Sciences
for the Centre Region of Cameroon (Approval no 0438 /
CRERSHC/ 2019). Nor, Normal mucosa; CSG, Chronic gastritis; AG, Atrophic Gastritis; IM, Intestinal metaplasia; DYS, Dysplasia; GC, Gastric cancer; MALT,
MALT l
h
HP
H
l
i Nor, Normal mucosa; CSG, Chronic gastritis; AG, Atrophic Gastritis; IM, Intestinal metaplasia; DYS, Dysplasia; GC, Gastric
MALT lymphoma; HPs, Hyperplasia. Asian Pacific Journal of Cancer Prevention, Vol 24
173 Asian Pacific Journal of Cancer Prevention, Vol 24
173 Statistical analysis Gastric Histological Features According to Age and Gender in the Sample Population Variables
Histological features
R square
(P value)
Nor (%)
n=33
CSG (%)
n=972
AG (%)
n=106
IM (%)
n=100
DYS (%)
n=36
GC (%)
n=120
MALT (%)
n= 10
HPs (%)
n=20
Sex
Female
18 (54.5)
523 (53.8)
62 (58.5)
60 (60)
14 (38.8)
75 (62.5)
2 (20.0)
10 (50)
Male
15 (45.5)
449 (46.2)
44 (41.5)
40 (40)
22 (61.2)
45 (37.5)
8 (80.0)
10 (50)
Total
number
(Prevalence)
33 (2.56)
972 (75.35)
106 (8.22)
100 (7.75)
36 (2.79)
120 (9.30)
10 (0.77)
20 (1.55)
Age (years)
Mean ± SD
40.17± 9.59
41.73±15.35
46.57±16.33
54.34±15.69
55.77± 19.51
56.55 ± 16.61
47.13± 19.81
52.25±19.65
0.012
(p=0.5879)
≤20
2 (6.1)
45 (4.6)
04 (3.8)
4 (4.0)
1 (2.8)
2 (1.7)
1 (10.0)
1 (5.0)
21-39
17 (51.5)
340 (35)
32 (30.2)
16 (16.0)
8 (22.2)
32 (26.7)
2 (20.0)
5 (25.0)
40-59
8 (24.2)
414 (42.6)
47 (44.3)
45 (45.0)
11 (30.5)
40 (33.3)
4 (40.0)
5 (25.0)
≥60
6 (18.2)
173 (17.8)
23 (21.7)
35 (35.0)
16 (44.5)
46 (38.3)
3 (30.0)
9 (45.0)
Total
number
(Prevalence)
33 (2.56)
972 (75.35)
106 (8.22)
100 (7.75)
36 (2.79)
120 (9.30)
10 (0.77)
20 (1.55)
Table 2. Gastric Histological Features According to Age and Gender in the Sample Population Variables
Histological features
Table 2. Gastric Histological Features According to Age and Gender in the Sample Population Ghislaine Florice Faujo Nintewoue and Laure Brigitte Kouitcheu Mabeku Table 3. Histological Features of the Gastric Lining According to H. pylori Status among the Study Population Taken
Subjects with Normal Gastric Lining as Reference Histological features
Number
H. pylori positive (%)
H. pylori negative (%)
OR (IC 95)
P value
Normal
33
03 (9.0)
30 (91.0)
Reference
Chronic gastritis
972
519 (53.4)
452 (46.6)
0.08709 (0.02775-0.259)
P< 0.0001*
Atrophic gastritis
106
65 (61.3)
41 (38.7)
0.06308 (0.01956-0.1996)
P< 0.0001*
Intestinal metaplasia
100
51 (51.0)
49 (49.0)
0.09601 (0.02967-0.3355)
P< 0.0001*
Dysplasia
36
22 (61.1)
14 (38.9)
0.06364 (0.01857-0.2355)
P< 0.0001*
Gastric cancer
120
68 (56.7)
52 (43.3)
0.07647 (0.02378-0.2599)
P< 0.0001*
MALT lymphoma
10
5 (50.0)
5 (50.0)
0.01 (0.02273-0.6297)
P=0.0103*
Hyperplasia
20
06 (30.0)
14 (70.0)
0.2333 (0.05909-0.947)
P=0.0666
Table 3. Histological Features of the Gastric Lining According to H. pylori Status among the Study Population Taken
Subjects with Normal Gastric Lining as Reference
OR, odd ratio; (95% CI), 95% confidence intervals. Statistical analysis Statistical test with subjects with normal mucosa as the reference group. *, Significant. Histological Features of the Gastric Lining According to H. pylori Status among the Study Population Take
with Normal Gastric Lining as Reference Frequency of gastric histological features among the
study population from 40 to 59 (37.52%, 484/1290) years old were the
most represented. Fifty four percent (54.42%, 702/1290)
versus 45.58% (588/1290) of participants were females,
given a sex ratio of 1.19:1 (Table 1). One thousand two hundred and ninety (1290) biopsy
sample or mixed biopsy sample were collected from the
overall dyspeptic participants recruited and examined for
histological features. Thirty three (33) of these gastric
specimen showed normal mucosa, given a proportion
of participant with gastric mucosa without injury of
2.56% (33/1290) among our sample population. The
mean age of participant showing normal gastric mucosa
was 40.17 ± 9.59 years with 20 to 39 years old (51.5%)
as the age group with the highest incidence and female
was the predominant gender (54.5%). The remaining
97.44% (1257/1290) of our sample population presented
histopathological features in their gastric mucosa (Table
2). Among the overall 1290 dyspeptic patients enrolled
in this study, 89 in 2014, 119 in 2015, 105 in 2016, 202
in 2017 and 161 in 2018 were H. pylori infected, giving
an infection prevalence of 56.33%, 59.20%, 55.85%,
56.11% and 42.04% respectively from 2014 to 2018, and
an overall prevalence of 52.40% in our sample population. Their mean age was 48.46 ± 4.31 years and 49.15 ± 4.07
for infected and non-infected patients respectively and
the age group 40 to 59 (36.98%) was the most affected. According to the gender, females were more affected by
H. pylori infection than males (54.29 versus 47.71%)
(Table 1). Chronic gastritis predominated and was found in
972 (75.35%) cases. The average age of patients with A i
P
ifi J
l f C
P
ti
V l 24
174
Variables
Number (%)
H. pylori positive (%)
H. pylori negative (%)
X2 (p value)
Inflammatory infiltrate
Yes
914 (94.03)
475 (91.34)
439 (97.12)
0.0002*
No
58 (5.97)
45 (8.66)
13 (2.88)
Total n(%)
972 (75.35)
520 (53.50)
452 (46.50)
Intensity of the inflammatory infiltrate
Mild
419 (43.10)
184 (38.7)
235 (53.5)
20.12 (< 0. Statistical analysis 0001)*
Moderate
32 (3.29)
19 (4.00)
13 (2.96)
Severe
463 (47.63)
272 (57.3)
191 (43.51)
Total n (%)
914 (94.03)
475 (51.96)
439 (48.04)
Neutrophil activity
Yes
352 (64.94)
212 (74.38)
140 (54.47)
(< 0.0001)*
No
190 (35.05)
73 (25.61)
117 (45.52)
Total n (%)
542 (42.01)
285 (52.58)
257 (47.42)
Intensity of neutrophil activity
Mild
257 (47.42)
148 (57.59)
109 (42.41)
3.22
Moderate
86 (15.87)
57 (66.28)
29 (33.72)
Severe
9 (1.66)
07 (77.77)
02 (22.22)
-0.1999
Total n (%)
352
212 (60.22)
140 (39.77)
Table 4. Frequency and Intensity of Inflammatory Infiltrate and Neutrophil Activity According to H. pylori Status
among the Study Population
n, Number; X2, Chi-square and Intensity of Inflammatory Infiltrate and Neutrophil Activity According to H. pylori Status
l ti quency and Intensity of Inflammatory Infiltrate and Neutrophil Activity According to H. pylori Status
tudy Population DOI:10.31557/APJCP.2023.24.1.171
Degeneration of the Gastric Lining DOI:10.31557/APJCP.2023.24.1.171 DOI:10.31557/APJCP.2023.24.1.171
Degeneration of the Gastric Lining
Variable
Total
Present
Absent
Univariate logistic regression
Multivariate logistic regression
OR (95% CI)
p value
OR (95% CI)
p value
Normal
Age ≥ 20
Yes
55
2 (6.06)
53 (4.22)
0.6808
0.6045
0.5771
0.4682
No
1235
31 (93.94)
1204 (95.78)
(0.1589- 2.9162)
(0.1307- 2.5487)
Gender
Female
702
18 (54.55)
684 (54.42)
0.9948
0.9882
0.9932
0.9849
Male
588
15 (45.45)
573 (45.58)
(0.4969- 1.9915)
(0.4920- 2.0051)
H. pylori status
Yes
676
3 (9.09)
673 (53.54)
0.0869
0.0001*
0.086
0.0001*
No
614
30 (90.91)
584 (46.46)
(0.0264- 0.2859)
(0.0261- 0.2833)
Chronic gastritis
Age ≥ 20
Yes
55
45 (81.82)
10 (18.18)
0.6684
0.2574
0.6792
0.2772
No
1235
927 (75.04)
308 (24.96)
(0.3329-1.3422)
(0.3380-1.3648)
Gender
Female
702
523 (74.5)
179 (25.5)
11.031 (0.8548-1.4235)
0.4507
10.995
0.4662
Male
588
449 (76.32)
139 (23.68)
(0.8518-1.4193)
H. pylori status
Yes
676
519 (76.78)
157 (23.22)
11.775 (0.9139 -1.5171)
0.2064
10.995
0.4662
No
613
452 (73.74)
161 (26.26)
(0.8518 -1.4193)
Atrophic gastritis
Age ≥ 20
Yes
55
4 (3.77)
51 (4.31)
11.478
0.7946
11.793
0.7559
No
1235
102 (96.23)
1133 (95.69)
(0.4067- 3.2398)
(0.4169- 3.3359)
Gender
Female
702
62 (58.49)
640 (54.05)
0.8349
0.3801
0.8331
0.375
Male
588
44 (41.51)
544 (45.95)
(0.5580- 1.2492)
(0.5565-1.2472)
H. Statistical analysis pylori status
Yes
676
65 (61.32)
611 (51.60)
14.866
0.0563*
14.918
0.0544*
No
614
41 (38.68)
573 (48.40)
(0.9894- 2.2337)
(0.9925- 2.2422)
Intestinal metaplasia
Age ≥ 20
Yes
55
4 (4.00)
51 (4.29)
10.725(0.3799-3.0281)
0.8948
10.596
0.9131
No
1235
96 (96,00)
1139 (95.71)
(0.3745-2.9979)
Gender
Female
702
60 (60.00)
642 (53.95)
0.781
0.2443
0.7816
0.2457
Male
588
40 (40.00)
548 (46.05)
(0.5152-1.1840)
(0.5156-1.1848)
H. pylori status
Yes
676
51 (51.00)
625 (52.52)
0.9408
0.7697
0.9429
0.7781
No
614
49 (49.00)
565 (47.48)
(0.6255-1.4151)
(0.6266-1.4190)
Dysplasia
Age ≥ 20
Yes
55
1 (2.78)
54 (4.31)
15.703
0.6589
28.19
0.6271
No
1235
35 (97.22)
1200 (95.69)
(0.2118-11.6444)
(0.6777-11.7263)
Table 5. Histological Features Adjusted to Socio-demographic Factors and H. pylori Status among the Study Population
using Univariate and Multivariate Logistic Regression Analysis Table 5. Histological Features Adjusted to Socio-demographic Factors and H. pylori Status among the Study Population
using Univariate and Multivariate Logistic Regression Analysis Asian Pacific Journal of Cancer Prevention, Vol 24
175 Ghislaine Florice Faujo Nintewoue and Laure Brigitte Kouitcheu Mabeku chronic gastritis was 41.73± 15.35 years, and the most
respectively (Table 4). Variable
Total
Present
Absent
Univariate logistic regression
Multivariate logistic regression
OR (95% CI)
p value
OR (95% CI)
p value
Dysplasia
Gender
Female
702
14 (38.89)
688 (54.86)
19.101
0.0618
0.6937
0.0644
Male
588
22 (61.11)
566 (45.14)
(0.9684-3.7675)
(0.4709-1.0221)
H. pylori status
Yes
676
22 (61.11)
654 (52.15)
14.415
0.2912
12.253
0.2945
No
614
14 (38.89)
600 (47.85)
(0.7309-2.8429)
(0.8381-1.7914)
Gastric cancer
Age ≥ 20
Yes
55
2 (1.67)
53 (4.53)
27.969
0.1569
28.19
0.1542
No
1235
118 (98.33)
1117 (95.47)
(0.6734-11.6166)
(0.6777-11.7263)
Gender
Female
702
75 (62.50)
627 (53.59)
0.6929
0.0632
0.6937
0.0644
Male
588
45 (37.50)
543 (46.41)
(0.4705-1.0203)
(0.4709-1.0221)
H. pylori status
Yes
676
68 (56.67)
608 ( 51.97)
1.2088
0.3267
12.253
0.2945
No
614
52 (43.33)
562 (48.03)
(0.8276-1.7655)
(0.8381-1.7914)
MALT lymphoma
Age ≥ 20
Yes
55
1 (10.00)
54 (4.22)
0.3964(0.0493- 3.1853)
0.3841
0.4136
0.4083
No
1235
9 (90.00)
1226 (95.78)
(0.0510-3.3518)
Gender
Female
702
2 (20.00)
700 (54.69)
48.198
0.0471*
47.914
0.0481*
Male
588
8 (80.00)
580 (45.31)
(1.0207-22.7606)
(1.0131-22.6620)
H. Frequency of histological features according to H. pylori
infection f
H. pylori was detected in 3 patients with normal gastric
mucosa, given an infection rate of 9% (3/33) among
patients with normal gastric mucosa. This difference
was significant when comparing to H. pylori negative
patients with normal mucosa (p= 0.0001). H. pylori was
detected in 519/972 (53.4 %) specimens of gastritis,
in 51/100 (51.0 %) specimens of intestinal metaplasia,
in 22/36 (61.1 %) specimens of dysplasia, in 68/120
(56.7 %) specimens of carcinoma, in 6/20 (30.0%)
specimen of hyperplasia, in 65/106 (61.3%) of atrophic
gastric and in 5/10 (50.0%) of lymphoma. Considering
participants with normal mucosa as the reference group,
H. pylori-positive participants had statistically significant
high prevalence for histological lesions such as gastritis
(p <0.0001), atrophy (p <0.0001), metaplasia (p <0.0001),
dysplasia (p <0.0001), carcinoma (p <0.0001) and MALT
(p=0.0337) (Table 4). Among patients with hyperplasia,
H. pylori-negative participants were highly affected by
this gastric lesion and the difference was not significative
compared to patient with normal mucosa (P=0.0666)
(Table 3). H. pylori infection and inflammatory infiltrate H. pylori was detected in 91.34% of biopsies samples
with inflammatory infiltrate versus 8.66% with no
inflammation. This difference was significant (P= 0.0002). Regarding the intensity of inflammation, 38.7%, 4% and
57.3% of specimens with mild, moderate, and marked
inflammation respectively were H. pylori positive, while
53.5, 2.96 and 43.51% showed respectively the same
characteristic among non-infected samples. This difference
was also significant (P <0.0001) (Table 4). H. pylori infection and neutrophil activity Frequency of histological features according to H. pylori
infection Frequency of histological features according to H. pylori
infection Neutrophil activity and the presence of H. pylori
were significantly associated (P <0.0001). H. pylori was
detected in 74.38% (212/285) of specimens showing
neutrophil activity, while in specimens with no neutrophil
activity, 25.61% and 45.52% were respectively H. pylori
positive and H. pylori negative. Among H. pylori positive
specimens, 57.59%, 66.28%, 77.77% were with mild,
moderate and markedly intense neutrophil activity,
while 42.41%, 33.72% and 22.22% of sample revealed
the respective characteristic in H. pylori negative ones. However, this difference was not significant (p = 0.1999)
(Table 4). DOI:10.31557/APJCP.2023.24.1.171
Degeneration of the Gastric Lining DOI:10.31557/APJCP.2023.24.1.171
Degeneration of the Gastric Lining dysplasia, and 1.2088 (0.8276-1.7655) times to develop
carcinoma. For intestinal metaplasia (OR= 0.9408, IC
95=0.6255-1.4151) and MALT lymphoma (OR = 0.9075
IC 95=0.2616- 3.1476), infected patients were slightly
affected than non-infected ones. This positive relationship
between H. pylori infection and histopathological features
of the gastric mucosa persisted even after adjustment with
confounding factors such as age and sex. dysplasia, and 1.2088 (0.8276-1.7655) times to develop
carcinoma. For intestinal metaplasia (OR= 0.9408, IC
95=0.6255-1.4151) and MALT lymphoma (OR = 0.9075
IC 95=0.2616- 3.1476), infected patients were slightly
affected than non-infected ones. This positive relationship
between H. pylori infection and histopathological features
of the gastric mucosa persisted even after adjustment with
confounding factors such as age and sex. gastritis. The most represented age of incidence was
40-59 years old for atrophic gastritis (47/106, 44.3%) and
intestinal metaplasia (45/100, 45%), and up to 60 years
old for dysplasia (16/36, 44.5%) and gastric carcinoma
(46/120, 38.3%). As gender is concerned, females were
predominantly affected among patients with atrophic
gastritis, intestinal metaplasia and gastric carcinoma; male
among those with dysplasia. For the others histological lesions, the prevalence of
hyperplastic polyps was 1.55% (20/1290) with 52.25
±19.65 years and up to 60 years old respectively as the
average age and the most represented age of incidence
(9/20, 45.0%) among patients with these type of gastric
lesion. Only ten (0.8%) cases of MALT lymphoma were
noticed. Their average age was 47.13 ±19.81 years old
with 40-59 years old as the most represented age of
incidence for this lesion (4/10, 40.0%). Females were
predominantly affected among patients with MALT
lymphoma, while no difference between the two sexes
was observed concerning those with hyperplasia (Table 2). Statistical analysis pylori status
Yes
676
5 (50.00)
671 (52.42)
0.9075
0.8784
0.8952
0.8619
No
614
5 (50.00)
609 (47.58)
(0.2616- 3.1476)
(0.2571- 3.1166)
Hyperplasia
Age ≥ 20
Yes
55
1 (5.00)
54 (4.25)
0.8437
0.8696
0.7931
0.8234
No
1235
19 (95.00)
1216 (95.75)
(0.1109-6.4186)
(0.1035-6.0745)
Gender
Female
702
10 (50.00)
692 (54.49)
11.972
0.6896
12.007
0.6856
Male
588
10 (50.00)
578 (45.51)
(0.4949-2.8964)
(0.4954-2.9104)
H. pylori status
Yes
676
6 (30.00)
670 (52.76)
0.3838
0.0512*
0.3822
0.0503*
No
614
14 (70.00)
600 (47.24)
(0.1466-1.0051)
(0.1459-1.0013)
N or n, number; OR, Odd ratio; (95% CI), 95% confidence intervals; *Significant. Table 5. Continued respectively (Table 4). respectively (Table 4). chronic gastritis was 41.73± 15.35 years, and the most
represented age of incidence of chronic gastritis was 40
to 59 years (42.6%) with female predominance (53.8%,
523/972) (Table 2). Nine hundred and fourteen (914)
versus 58 patients had inflammatory infiltrate. Among
inflammatory infiltrate, 419 (45.8%) were mild, 32 (3.6%)
were moderate, and 463 (50.6%) case were markedly
intense (Table 4). Neutrophil activity was noticed in
542 patients. Of these, 190 (35.05%) had no activity
while 257 (47.42%), 86 (15.87%) and 9 (1.66) were with
mild, moderate and markedly intense neutrophil activity chronic gastritis was 41.73± 15.35 years, and the most
represented age of incidence of chronic gastritis was 40
to 59 years (42.6%) with female predominance (53.8%,
523/972) (Table 2). Nine hundred and fourteen (914)
versus 58 patients had inflammatory infiltrate. Among
inflammatory infiltrate, 419 (45.8%) were mild, 32 (3.6%)
were moderate, and 463 (50.6%) case were markedly
intense (Table 4). Neutrophil activity was noticed in
542 patients. Of these, 190 (35.05%) had no activity
while 257 (47.42%), 86 (15.87%) and 9 (1.66) were with
mild, moderate and markedly intense neutrophil activity For the premalignant and malignant lesions: the
prevalence of atrophic gastritis was 8.2% (106/1290),
prevalence of intestinal metaplasia was 7.7% (100/1290),
prevalence of dysplasia was 2.8% (36/1290) and
prevalence of carcinoma was 9.3% (120/1290). The average age of patients with atrophic gastritis,
intestinal metaplasia, dysplasia and gastric carcinoma
was 46.57 ± 16.33, 54.34 ±15.69, 55.77 ±19.51 and
56.55 ± 16.61 respectively compared with 40.17 ± 9.59
and 41.73 ± 15.35 for normal mucosa and chronic 176 Asian Pacific Journal of Cancer Prevention, Vol 24 Discussion This observation
may simply reflect the high number of females in our
sample population due to the fact that women are more
prompt for clinical examination than men. However, the
results of some previous studies carried out in Cameroon
showed that because males are naturally more active and
less hygienic than females, they were associated with
a higher risk of acquiring H. pylori infection than the
females (Kouitcheu et al., 2018). It is generally believed that the development of gastric
cancer is a multistep process involving sequential changes
of the gastric mucosa from non-atrophic gastritis to
atrophic gastritis, intestinal metaplasia (IM), dysplasia also
known as precancerous or premalignant lesions and finally
carcinoma (Correa, 2004). Several studies demonstrated
this multistep process in the development of gastric cancer
has been reported: in a nationwide cohort study performed
in the Netherlands to evaluate the risk of gastric cancer
associated with the various premalignant gastric lesions, it
was revealed that, atrophic gastritis, intestinal metaplasia,
mild-moderate dysplasia, and severe dysplasia were
associated with annual incidences of gastric cancer of
0.1%, 0.25%, 0.6%, and 6.0%, respectively (De Vries et
al., 2008); in systematic reviews, the incidence of gastric
cancer associated with IM ranges from 0% to 10%, with
the variable range attributable to various sample sizes and
follow-up periods (Kim et al., 2016). Regarding histologic features of biopsy samples,
2.56% of this dyspeptic population was with normal
gastric mucosa and the remaining 97.44% presented
various pathological features (Table 2). Such observations
indicated that clinical signs of dyspepsia are commonly
correlated with gastric mucosa lesions or pathological
features. Chronic gastritis with the prevalence of 75.35% was
the most frequent histological lesion found among the
overall samples. This result is considerably lower than
that of Bravo et al. in Colombia who found the prevalence
of gastritis as 83.6% (Bravo et al., 2003), but similar
regarding the age group of 40 to 59 years as that with the
highest incidence of gastritis (Bravo et al., 2003). The
majority of biopsies with chronic gastritis were found to
be H. pylori infected (53.4%), a result that match reports
in the literature that this infection is a major cause of
gastritis (Gonzalez-Carbajal et al., 2005). Our results
were concordant with figures of 50.4% (Dooley et al.,
1989), 48% (Satoskar and Vora 1994), 46.5 % (Maitra and
Ghosh, 1991), and 45.6% (Parvez et al., 2015) of H. Discussion However, the prevalence we
found correlates with the results of some studies done
in other developing countries within and out of Africa;
50.6% in the North of South Africa (Venda) (Samie et al.,
2007) and about 58% in Guatemala (Dowsett et al., 1999)
among the examined individuals. a marked infiltrate of chronic inflammatory cells usually
indicates the presence of H. pylori and that the degree of
chronic inflammation is directly related to the intensity of
colonization by this bacterium (Abu-Ahmad et al., 2011). y
(
,
)
According to our data, neutrophil activity and the
presence of H. pylori were significantly associated
(P <0.0001), with a higher prevalence of infection
but non-significant in biopsies having marked
neutrophil activity (p = 0.1999). This coincides with
the findings of Tanko et al. (2008) and Sasa et al. (2002)
showing a positive relationship between the degree of H. pylori colonisation and the intensity of neutrophil activity. As a consequence of H. pylori interactions with the
epithelium, pro-inflammatory chemokines and cytokines,
including IL-8, IL-1β, tumour necrosis factor alpha
(TNFα), IL-6, IL-12, CCL2-5, CCL20, and CXCL1-3 are
up regulated in the infected gastric mucosa (Reymunde
et al., 1993). The presence of these chemokines leads to
the recruitment of immune cells (Reymunde et al., 1993). So, H. pylori infection leads invariably to an inflammatory
process in the stomach which is characterised by an
increase in the number of lymphocytes, macrophages and
plasmocytes in the lamina propria in variable degrees. This
inflammation may be accompanied by neutrophils which
contribute to gastritis by secreting inflammatory cytokines
and releasing tissue damaging factors from neutrophilic
granules such as neutrophil-derived reactive oxygen
radicals and proteases indicating inflammatory activity
(Evans et al., 1995; Tanko et al., 2008). It has been reported that in developing countries
most individuals were infected during childhood and
continued to be infected as adults, with a peak prevalence
of 80% just before 50 years old (Torres et al., 1998). In
the present study, the prevalence of infection according
to age exhibited a usual trend as reported in developing
countries. It increases with age, from less than 20 years
old, reaches a peak at 40 years old and remains almost the
same as you grow older. Prevalence of H. pylori infection was higher among
women than men (54.29 versus 45.71%). Discussion As reported in previous epidemiological investigations
on H. pylori infection, Cameroon is an endemic area of
infection by this pathogen (Kouitcheu et al., 2018). Also,
H. pylori clinical isolates circulating in Cameroon has been
shown to be highly resistant against antibiotics currently
used in the eradication of this pathogen (Kouitcheu et al.,
2019). Despite the high prevalence of H. pylori infection
in the Cameroonian population and the broad spectrum
of resistance of circulating strains, this is the first study
characterising the association between current H. pylori
infection assayed by histology and severe gastric lesions
in a large Cameroonian population who underwent upper
endoscopy for gastric complaints. Adjustment of the relationship between H. pylori
infection and histological features of the gastric mucosa
to confounding factors such as age and sex of the
participants was also evaluated using logistic regression
(Table 5). Age group less than 20 years old was taken as
reference since this age group was the least affected for
each histological feature (Table 5). Compared to infected
patients, non-infected patients were more protected from
gastric lesions (OR= 0.0869, IC 95 (0.0264- 0.2859). Unless for hyperplasia where non-infected patients
were more affected than infected ones (OR= 0.3838
(0.1466-1.0051), H. pylori infected patients were more
subjected to develop histopathological features compared
to non-infected ones. In fact H. pylori infected subjects
were 1.1775 (0.9139 -1.5171) times more prone to develop
gastritis, 1.4866 (0.9894- 2.2337) times to develop
atrophic gastric, 1.4415 (0.7309-2.8429) times to develop The prevalence of H. pylori infection was 52.40%
among the overall 1290 dyspeptic patients enrolled in
this study. A comparison with previous studies carried
out in Cameroon shows that this prevalence is lower
than the 92.2%, 79.3% and 64.34% reported in previous
studies performed in Cameroon using different methods
of H pylori detection (Kouitcheu et al., 2018). It is also
lower that the prevalence of 73.2% reported in a study
performed in the Littoral region of Cameroon using
histological examination (Eyoum et al., (2020). The
discrepancy between the present prevalence and the
previous ones may be due to a rise in the socioeconomic Asian Pacific Journal of Cancer Prevention, Vol 24
177 Ghislaine Florice Faujo Nintewoue and Laure Brigitte Kouitcheu Mabeku status and residential conditions of the target population
since the prevalence of infection parallels improvement
of economy and sanitary standards and is also related
to the risk factors exposition of the sample population
(Kouitcheu et al., 2019). Asian Pacific Journal of Cancer Prevention, Vol 24
8 Discussion There is a fact that helps to
understand the increase of mucosa pathological severity
with age. In fact, the degradation of the gastric mucosa
is a chronic process starting with a local inflammation
of the gastric mucosa, the continuity of this process
progressively leads to chronic superficial gastritis, atrophic
gastritis, intestinal metaplasia, dysplasia and finally gastric
adenocarcinoma (Correa, 1992; Correa, 2004). Since
this progression is related to chronic inflammation of the
gastric mucosa, there is an age-associated increase in the
prevalence of precancerous and cancerous states. Asian Pacific Journal of Cancer Prevention, Vol 24
179
Regarding gastric cancer, our finding is in accordance
with previous studies reporting that H. pylori is a risk
factor in the development of gastric cancer. In fact,
H. pylori has been found to increase the odds ratio of
stomach cancer by 5.9 fold within ten years of infection
(Prashanth and Adam, 2019). Also, a recent meta-analysis
showed that eradication of H. pylori resulted in 37%
reduction in the incidence of gastric cancer (Li et al., 2014)
and that H. pylori eradication is one of the most promising
approach in gastric cancer prevention (Li et al., 2014). Adenocarcinomas are divided into cardia and non-cardia
cancer based on their anatomical site. Non-cardia cancers
are brought about by chronic gastritis and inflammation
of the stomach lining and are associated with H. pylori
infection in 90% of cases (Ford et al., 2014). The
pathogenesis of cardia cancer remains unclear, although
two distinct aetiologies have been proposed: one is
associated with gastro-oesophageal reflux disease and
resembles oesophageal adenocarcinoma, and the other is
associated with H. pylori atrophic gastritis and resembles
non-cardia cancer (Mukaisho et al., 2015). Koreans
reported that H. pylori infection was associated with a Our data as gender is concern showed that dyspeptic
women were at a slightly higher risk of acquiring atrophic
gastritis, intestinal metaplasia and gastric carcinoma than
males and this is the inverse for dysplasia (Table 2). Such
observation may simply reflect the higher proportion of
female in our sample population since it is documented
that oestrogen is protective, as both delayed menopause
and increased fertility lower the risk of gastric cancer
(Derakhshan et al., 2009). Moreover, men currently have
been shown to have an increased risk of developing gastric
pathologies than women due to the fact that they are more
exposed to risk factor such as acquiring H. Discussion pylori infected subjects were
1.4866 (0.9894- 2.2337) times more prone to develop
atrophic gastritis, 0.9408 (0.6255-1.4151) times to develop
intestinal metaplasia, 1.4415 (0.7309-2.8429) times to
develop dysplasia, and 1.2088 (0.8276-1.7655) times to
develop carcinoma, suggesting that the presence of this
bacteria is a risk factor for these gastric lesions (Table
5). This positive relationship between H. pylori infection
and both precancerous and cancerous lesions persist even
after adjustment with confounding factors such as age and
sex. These results are in accordance with several studies
((Almouradi et al., 2013; Olmez et al., 2015; Uemura et
al., 2001) and also with the findings of the meta-analysis in
which the prevalence of atrophy and intestinal metaplasia
was higher in individuals infected by H. pylori than in
uninfected individuals (Marques-Silva et al., 2014). In
the current study, the probability to develop atrophy or
dysplasia was higher than metaplasia between H. pylori
(+) patients and H. pylori (-) ones. Such observation
indicates that bacterial factors seem to exert a higher
influence on the development of atrophy and dysplasia,
whereas environmental and host factors would play an
important role in the development of intestinal metaplasia
(Kim et al., 2008). In fact, it is reported that although the
risk developing gastric cancer is increased in patients with
gastric intestinal metaplasia, and that gastric intestinal
metaplasia (GIM) is an intermediate precancerous gastric
lesion in the gastric cancer cascade of chronic gastritis,
atrophic gastritis, intestinal metaplasia (IM), dysplasia,
and adenocarcinoma (Correa, 2004), the absolute risk
is modest and specific subsets of patients with gastric
intestinal metaplasia may be at higher risk for progression
(Correa, 1992). Such an observation may be also due to the
loss of current H. pylori infection in intestinal metaplasia
(Prashanth and Adam, 2019). i The distribution of gastric precancerous and cancerous
lesions according to the age and sex of participants showed
that the prevalence of these gastric lesions had an upward
trend with aging from chronic gastritis to precancerous and
cancer states, while for normal mucosa, it had a downward
trend with aging (Table 2). This observation is in accord
with previous studies reporting that severe gastric lesions
are predominantly pathologies of the middle aged and
elderly (Ebili et al., 2015), and those revealing that age
≥50 years is an independent risk factor for IM (Hirota
et al., 2006; Correa, 1992). Discussion pylori
associated gastritis detected in gastric specimens and less
than those showing H. pylori associated gastritis in 66.9%
from India (Akanda and Rahman, 2011), in 62.5% from
Pakistan (Dandin et al., 2012) and in 67% from Jordan
(Yakoob and Hussainy, 2010).li Prevalence of 9.3% was noticed in the current
study for carcinoma, 8.2%, 7.7% and 2.8% for atrophic
gastritis, intestinal metaplasia and dysplasia (Table 2). This prevalence follows the same tendency observed
in a previous study performed in Cameroon reporting
intestinal metaplasia rate of 6.3% (5/79) among patients
with chronic antral gastritis in Yaoundé, Cameroon, but
differ regarding the rate of atrophic gastritis of 74.7%
(Firmin et al., 2015). The limited number of samples
(79 patients) in the above previous study compared to the
current one could explain such a difference. The current
prevalence of gastric precursors is less or close than that
of some previous studies: prevalence of gastric IM of
7% (Sonnenberg et al., 2010) and 15% (Almouradi et al., When examining inflammatory infiltrate with respect
to H. pylori infection, we realised that biopsies with
inflammatory infiltrate (P= 0.0002) and mostly those
with marked inflammation (P <0.0001) were significantly
colonised with the bacterium. This finding is concordant
with that of previous studies reporting that the presence of Asian Pacific Journal of Cancer Prevention, Vol 24
78 DOI:10.31557/APJCP.2023.24.1.171
Degeneration of the Gastric Lining a statistically significant prevalence rate for atrophy (p
<0.0001), metaplasia (p <0.0001), dysplasia (p <0.0001)
and carcinoma (p <0.0001) among infected subjects
compared to infected subjects with normal mucosa
(Table 3). Moreover, H. pylori infected subjects were
1.4866 (0.9894- 2.2337) times more prone to develop
atrophic gastritis, 0.9408 (0.6255-1.4151) times to develop
intestinal metaplasia, 1.4415 (0.7309-2.8429) times to
develop dysplasia, and 1.2088 (0.8276-1.7655) times to
develop carcinoma, suggesting that the presence of this
bacteria is a risk factor for these gastric lesions (Table
5). This positive relationship between H. pylori infection
and both precancerous and cancerous lesions persist even
after adjustment with confounding factors such as age and
sex. These results are in accordance with several studies
((Almouradi et al., 2013; Olmez et al., 2015; Uemura et
al., 2001) and also with the findings of the meta-analysis in
which the prevalence of atrophy and intestinal metaplasia
was higher in individuals infected by H. pylori than in
uninfected individuals (Marques-Silva et al., 2014). Discussion In
the current study, the probability to develop atrophy or
dysplasia was higher than metaplasia between H. pylori
(+) patients and H. pylori (-) ones. Such observation
indicates that bacterial factors seem to exert a higher
influence on the development of atrophy and dysplasia,
whereas environmental and host factors would play an
important role in the development of intestinal metaplasia
(Kim et al., 2008). In fact, it is reported that although the
risk developing gastric cancer is increased in patients with
gastric intestinal metaplasia, and that gastric intestinal
metaplasia (GIM) is an intermediate precancerous gastric
lesion in the gastric cancer cascade of chronic gastritis,
atrophic gastritis, intestinal metaplasia (IM), dysplasia,
and adenocarcinoma (Correa, 2004), the absolute risk
is modest and specific subsets of patients with gastric
intestinal metaplasia may be at higher risk for progression
(Correa, 1992). Such an observation may be also due to the
loss of current H. pylori infection in intestinal metaplasia
(Prashanth and Adam, 2019). i 2013) was found in studies conducted among dyspeptic
patients in the United States respectively in 2010 and
2013 and 8% in Netherlands (DeVries et al., 2008). Also,
prevalence of 10.3% and 14.7%, 3% and 15%, 11.2%
and 21.6% respectively for atrophy and metaplasia was
found among dyspeptic patients from South Eastern Brazil
(Rodrigues et al., 2019), from the Southern region of Brazil
(Muller et al., 2007), and from the Brazilian Northeast
region (Motta et al., 2008). However, this prevalence of
precancerous states is far lower than that reported in the
meta-analysis by Marques-Silva et al. (2014) revealing
an atrophic gastritis prevalence of 33.4% and increased
to 42% in countries with high incidence of gastric cancer,
an intestinal metaplasia (IM) prevalence of 25% and
extensive IM of 13% in the worldwide population in
general. We think that the low prevalence of precancerous
lesions in our sample population compared to that in the
above meta-analysis may be due to the fact that our region
is an area with low incidence of gastric cancer. a statistically significant prevalence rate for atrophy (p
<0.0001), metaplasia (p <0.0001), dysplasia (p <0.0001)
and carcinoma (p <0.0001) among infected subjects
compared to infected subjects with normal mucosa
(Table 3). Moreover, H. Discussion However, this type of gastric lesions
should be diagnoses and management as some polyps
have malignant potential. The prevalence of hyperplastic
polyps (HPs) was 1.55% (20/1290) in our sample
population and 70.0% (14/20) of biopsies with this lesion
were H. pylori negative. This difference was significant
(P= 0.0503) (Table 5), indicating a negative association
between H. pylori infection and hyperplasia. These results
provide further evidence for the inverse association
between H. pylori infection and hyperplastic polyps. In fact, several studies had demonstrated that H. pylori
infection is inversely associated with the occurrence of
hyperplastic polyps (Carmack et al., 2009; Xinjuan et al.,
2020). The possible reasons for the negative association
between the infection and hyperplastic polyps remain
controversial. It has been suggested that the increased
prevalence of hyperplastic polyps is caused by IM of
gastric mucosa, rather than the decreased prevalence
of the infection (Carmack et al., 2009). However, this
explanation was not supported by other studies reporting
that hyperplastic polyps are widely found in geographic
areas where H. pylori is common and that polyps may
regress following H. pylori eradication (Ljubicic et al.,
1999; Kyburz and Muller, 2017). Prevalence rates of MALT lymphoma was 0.8% in
the sample population. This prevalence was significantly
elevated (p=0.0337) among H. pylori infected patients
compared to infected subjects with normal mucosa (Table
3). Moreover, infected patients were slightly more affected
by this type of gastric lesions compared to non-infected
ones (OR = 0.9075, IC 95 = 0.2616-3.1476), suggesting
a positive relationship between H. pylori infection and
MALT lymphoma (Table 5). H. pylori infection leads to
chronic gastritis which involves both T cells and B cells
arising from the mucosal associated lymphoid tissue
(MALT). The recognition of the H. pylori antigen by
the immune system leads to T cell activation, lymphoid
follicle formation, and B cell proliferation (Katharina
et al., 2015). It is thought that the H. pylori antigen-
presenting cells interact with CD4-expressing cells. This
CD4 cell then binds to a B cell in the marginal zone
causing hyperproliferation of B cells (D’Elios et al., 1999),
also known as a MALT lymphoma. The involvement of
Helicobacter pylori in MALT lymphoma is now well
established through the following observations; H. pylori
eradication allows lymphoma regression in 60% to 90% of
patients (Wotherspoon et al., 1993), H. Discussion However, there are contradictory
studies where the association of H. pylori with gastric
precancerous and cancerous lesions tended to be null
or inverse. In fact, Kato et al. (2006) documented no
difference in the presence of intestinal metaplasia among
the study group of children with and without H. pylori
infection; no child in a Brazilian cohort of 96 children with
H. pylori gastritis were found to have gastric intestinal
metaplasia (Carvalho et al., 2012). One explanation for the
geographic variation in the association between H. pylori
and gastric cancer could be the differences in the genetics
of H. pylori or host genetics in populations from different
geographic regions. It has been shown that the presence of
certain H. pylori genotype (vacA m1) and polymorphisms
of the host inflammatory cytokines (interleukin-1β, IL-10
and IL-17) were associated with the presence of gastric
precancerous lesions (Wroblewski et al., 2010). Bacterial
virulence factors such as cytotoxin-associated gene A
(CagA), vacuolating cytotoxin A (VacA) and the adhesion
protein BabA2 likely play an important role in determining
the outcome of H. pylori infection (Cover and Blanke,
2005). A longitudinal cohort study following 4655 healthy
asymptomatic subjects for 7.7 years, with gastric cancer
development in 45 patients showed that the risk of gastric
cancer increased progressively from H. pylori–negative/
cagA-negative to H. pylori–positive/cagA-negative, and
finally H. pylori–positive/cagA-positive (Ohata et al.,
2004). The average age of patients affected by MALT
lymphoma was 47.13 ±19.81 years old with 40-59 years
as the most represented age group of incidence for this
lesion type (4/10, 40.0%) and males were more affected
than women with a significant risk (P= 0.0471). Our results
coincide with literature reporting that approximately
70% of severe gastric lesions are diagnosed between
the ages of 55 and 84 years and that men have a 5- and
2-fold increased risk of cardia cancer and non-cardia
types, respectively (Sonnenberg and Genta, 2015). The
mechanism by which men are predisposed to developing
gastric cancer is likely multi-factorial. Currently, men
have been shown to have an increased risk of developing
gastric pathologies than women due to the fact that they
are more exposed to risk factor such as acquiring H. pylori
infection, poor eating habits, active or passive tobacco
consumption, drug abuse (Montalban et al., 1999). Gastric polyps are usually found incidentally on
upper gastrointestinal endoscopy and they are generally
asymptomatic. Discussion pylori infection,
poor eating habits, active or passive tobacco consumption,
drug abuse (Montalban et al., 1999). When the prevalence of precursor lesions and cancer
were evaluated in relation to H. pylori infection, we
found a positive relationship between H. pylori infection
and both precancerous and cancerous lesions. In fact,
H. pylori was detected in 61.3%, 51.0 %, 61.1 % and
56.7 % of specimens with atrophic gastritis, intestinal
metaplasia, dysplasia, and carcinoma respectively with Ghislaine Florice Faujo Nintewoue and Laure Brigitte Kouitcheu Mabeku 2.88-fold higher risk of gastric cardia adenocarcinoma
Bae and Kim (2016), and another meta-analysis reported
a positive association between H. pylori infection and
gastric cardia adenocarcinoma in geographic regions
with a high incidence of gastric cardia cancer (Cavaleiro-
Pinto et al., 2011). However, there are contradictory
studies where the association of H. pylori with gastric
precancerous and cancerous lesions tended to be null
or inverse. In fact, Kato et al. (2006) documented no
difference in the presence of intestinal metaplasia among
the study group of children with and without H. pylori
infection; no child in a Brazilian cohort of 96 children with
H. pylori gastritis were found to have gastric intestinal
metaplasia (Carvalho et al., 2012). One explanation for the
geographic variation in the association between H. pylori
and gastric cancer could be the differences in the genetics
of H. pylori or host genetics in populations from different
geographic regions. It has been shown that the presence of
certain H. pylori genotype (vacA m1) and polymorphisms
of the host inflammatory cytokines (interleukin-1β, IL-10
and IL-17) were associated with the presence of gastric
precancerous lesions (Wroblewski et al., 2010). Bacterial
virulence factors such as cytotoxin-associated gene A
(CagA), vacuolating cytotoxin A (VacA) and the adhesion
protein BabA2 likely play an important role in determining
the outcome of H. pylori infection (Cover and Blanke,
2005). A longitudinal cohort study following 4655 healthy
asymptomatic subjects for 7.7 years, with gastric cancer
development in 45 patients showed that the risk of gastric
cancer increased progressively from H. pylori–negative/
cagA-negative to H. pylori–positive/cagA-negative, and
finally H. pylori–positive/cagA-positive (Ohata et al.,
2004). (Kim et al., 2016). 2.88-fold higher risk of gastric cardia adenocarcinoma
Bae and Kim (2016), and another meta-analysis reported
a positive association between H. pylori infection and
gastric cardia adenocarcinoma in geographic regions
with a high incidence of gastric cardia cancer (Cavaleiro-
Pinto et al., 2011). Author Contribution Statement KMLB conceived of the study, designed the
experiments and supervised the work. FNGF extracted
data from the medical records, hospital computerized
databases and clinical charts of the patients. KMLB
provided the facilities for the study and drafted the
manuscript. All the authors read and approved the final
manuscript. Bray F, Ferlay J, Soerjomataram I, et al (2018). Global cancer
statistics 2018: GLOBOCAN estimates of incidence and
mortality worldwide for 36 cancers in 185 countries. CA
Cancer J Clin, 68, 394-424. Cammarota G, Montalto M, Tursi A, et al (1995). Helicobacter
pylori reinfection and rapid relapse of low-grade B-cell
gastric lymphoma. Lancet, 345, 192. Carmack SW, Centa RM, Schuler CM, Saboorian MH (2009). The current spectrum of gastric polyps: a 1 year national
study of over 120,000 patients. Am J Gastroenterol, 104,
1524. Conflict of interest The authors declare that they have no competing
interests. DOI:10.31557/APJCP.2023.24.1.171
Degeneration of the Gastric Lining the differences in ethnicity or differences in the prevalence
of H. pylori (Hooi et al., 2017). Board of University Teaching Hospital (Approval no 703/
AR/CHUY/DG/DGA/CAPRC) and Cameroon Pasteur
institute CPC (Approval no 000378/2019/CPC/DG/DAF/
SRH) and the Ethical Committee of Medical Sciences
for the Centre Region of Cameroon (Approval no 0438
/CRERSHC/ 2019). It should be noted that in this study 52.4% of patients
evaluated were currently H. pylori infected whereas gastric
pathological features was detected in 97.44% cases. So,
we believe that H pylori infection alone cannot explain
these overall gastric lesions and that there must be other
critical cofactors affecting the risk of H. pylori infection
in the infection-precancerous and cancer relationship such
as sex, age as well as life style. Another reason which
could explain such situation may be the underestimation
of the true risk of H. pylori infection in the development of
precancerous and cancerous lesions due to loss of H. pylori
as the mucosa undergoes malignant transformation. In fact,
although, there is evidence that H. pylori is frequently
found in gastric biopsy specimens from individuals with
atrophic gastritis, intestinal metaplasia and gastric cancer,
it is reported that with the development of advanced
gastric tumours, the bacteria can be lost from the stomach
(Prashanth and Adam, 2019) and that with the loss of
infection, patients with gastric cancer may be H. pylori
negative even though they have been infected in the past
(Karnes et al., 1991). i Availability of data The datasets used and/or analyzed during the current
study are available from the corresponding author on
reasonable request. References Abu-Ahmad NM, Odeh A, Sallal AKJ (2011). Prevalence of
Helicobacter pylori gastritis at the north of Jordan. Jordan
J Biol Sci, 4, 71-6. Ahm JY, Son DH, Choi KD, et al (2014). Neoplasms arising in
large gastric hyperplastic polyps: endoscopic and pathologic
features. Gastrointest Endosc, 80, 1005. In conclusion, our findings revealed that the prevalence
of H. pylori among our sample population is high as is
the case in most of the African countries. It was also
shown that degeneration of the gastric lining which
results in pathologies such as chronic gastritis, precursors
of malignancy, adenocarcinomas and MALT are mainly
caused by Helicobacter pylori and that hyperplastic polyp
is inversely associated with H. pylori infection. H. pylori
eradication, early detection and treatment of precursor
lesions could significantly impact these outcomes in our
milieu. Further studies have to be carried out in order
to find out the various strains which in our context is
responsible for this degeneration. Akanda MR, Rahman AN (2011). Comparative study of different
methods for detection of Helicobacter pylori in gastric
biopsies. Dinajpur Med Col J, 4, 1-6. Alkout AM, Blackwell CC, Weir DM (2000). Increased
inflammatory responses of persons of blood group O to
Helicobacter pylori. J Infect Dis, 181, 1364–9. Almouradi T, Hiatt T, Attar B (2013). Gastric intestinal metaplasia
in an underserved population in the USA: prevalence,
epidemiologic and clinical features. Gastroenterol Res
Pract, 2013, 4. Amieva MR, El-Omar EM (2008). Host-bacterial interactions in
Helicobacter pylori infection. Gastroenterology, 134, 306–3. Bae JM, Kim EH (2016). Helicobacter pylori infection and risk
of gastric cancer in korea: a quantitative systematic review. J Prev Med Public Health, 49, 197–204. Bravo LE, Cortes A, Carrascal E, et al (2003). Helicobacter
pylori: patología y prevalencia en biopsias gástricas en
Colombia. Colomb Med, 34, 124-34. Discussion pylori eradication
therapy is now considered as the first therapeutic approach
for low grade gastric mucosal lymphoma (Cammarota
et al., 1995), if a reinfection occurs, gastric mucosal
lymphoma reappears and evolves more rapidly because
neoplastic cells are already sensitized to H. pylori antigens The average age of patients with hyperplasia was 52.25
±19.65 compared to 40.17 ± 9.59 in those with normal
mucosa. Moreover, the most represented age of incidence
of hyperplasia (9/20, 45.0%) was up to 60 years old. This
finding agrees with other studies, reporting that older
age appeared to be associated with an increased risk for
hyperplasia polyps (Kyburz and Muller, 2017; Takeuchi
et al., 2017; Stolte et al., 1994). As gender is concerned, there was no difference
between the two sexes. This is in accordance with
studies from other geographical regions, according to
which gender has no significant influence on the risk of
hyperplastic polyps (Kyburz and Muller, 2017; Dore et al.,
2017). However, studies carried out among the western
and Chinese populations have shown that, females have an
increase risk in the development of gastric polyps (Dore
et al., 2017). These conflicting results may be attributed to 180 Asian Pacific Journal of Cancer Prevention, Vol 24 DOI:10.31557/APJCP.2023.24.1.171
Degeneration of the Gastric Lining DOI:10.31557/APJCP.2023.24.1.171
Degeneration of the Gastric Lining Acknowledgements We acknowledge the support of the staffs of the
University Teaching Hospital (CHU) and the Cameroon
Pasteur institute (CPC) of Cameroon for putting medical
records; hospital computerized databases; or clinical
charts of patients from their anatomo-cytopathology unit
to our disposal. Carvalho MA, Machado NC, Ortolan EVP, Rodrigues MAM
(2012). Upper gastrointestinal histopathological findings
in children and adolescents with non ulcer dyspepsia with
Helicobacter pylori infection. J Pediat Gastroenterol Nutr,
55, 523–9. Cavaleiro-Pinto M, Peleteiro B, Lunet N, Barros H (2011). Helicobacter pylori infection and gastric cardia cancer:
systematic review and meta-analysis. Cancer Causes
Control, 22, 375–87. Approved by scientific Body
No applicable. Approved by scientific Body
No applicable. Prevalence
of Helicobacter pylori Infection and Histologic Gastritis in
Asymptomatic Persons. N Engl J Med, 321, 1562-6. Kouitcheu MLB, Mohamadou BE, Kamden SC, Tchidjo M
(2020). Stool antigen testing, a reliable noninvasive method
of assessment of Helicobacter pylori infection among
patients with gastro-duodenal disorders in Cameroon. Dig
Dis Sci, 66, 511-20. Dore MP, Pes GM, Rocchi C, et al (2017). Are gastric
hyperplastic polyps an additional manifestation in celiac
disease? Results from a retrospective study. Medicine
(Baltimore), 96, 5923. Kouitcheu MLB, Noudjeu MI, Leundji H (2018). Potential risk
factors and prevalence of Helicobacter pylori infection
among adult patients with dyspepsia symptoms in Cameroon. BMC Infect Dis, 18, 278. Dowsett AS, Archila L, Segreto AV, et al (1999). Helicobacter
pylori infection in indigenous families of Central America:
Serostatus and oral and fingernail carriage. J Clin Microbiol,
37, 2456–60. Kyburz A, Muller A (2017). Helicobacter pylori and extragastric
diseases. Curr Top Microbiol Immunol, 400, 325-47. Ebili HO, Oluwasola AO, Akang EE, Ogunbiyi JO (2015). Clinico-pathological features of gastric carcinoma in Ibadan,
Nigeria 2000-2011. Niger Med J, 56, 126–31. Li WQ, Ma JL, Zhang L, et al (2014). Effects of Helicobacter
pylori treatment on gastric cancer incidence and mortality
in subgroups. J Natl Cancer Inst, 106. Evans Jr, Evans DJ, Takemura DG, et al (1995). Characterization
of a Helicobacter pylori neutrophil-activating protein. Infect
Immun, 63, 2213–20. Ljubicic N, Banic M, Kujundzic M, et al (1999). The effect
of eradicating Helicobacter pylori infection on the course
of adenomatous and hyperplastic gastric polyps. Eur J
GastroenteroL Hepatol, 11, 727. Eyoum BB, Eloumou BSF, Mohamadou BE, et al (2020). Prevalence of Helicobacter pylori Infection and Relevant
Endoscopic Features among Patients with Gastro-Duodenal
Disorders, a three years Cross Sectional Study in the Littoral
Region of Cameroon. Eur J Sci Res, 157, 247 – 57. Maitra TN, Ghosh S (1991). Gatritis and Helicobacter
(Camylobacter) Pylori Merely one more piece in the jigsaw
puzzle or the final answer?. Indian J Pathol Microbiol, 34,
67-79. Firmin A, Dominique NN, Félicien NE, Carole MN, Roger ND,
(2015). Helicobacter pylori and precancerous conditions of
the stomach: the frequency of infection in a cross-sectional
study of 79 consecutive patients with chronic antral gastritis
in Yaoundé, Cameroon. Pan Afr Med J, 20, 52. Marques-Silva L, Areia M, Elvas L, Dinis-Ribeiro M (2014). Prevalence of gastric pre-cancerous conditions: a systematic
review and meta-analysis. Eur J Gastroenterol Hepatol,
26, 378-87. Approved by scientific Body
No applicable. Correa P (1992). Human gastric carcinogenesis: a multistep
and multifactorial process —First American Cancer Society
Award lecture on cancer epidemiology and prevention,” Ethical consideration
This study was approved by the Institutional Review 181 Asian Pacific Journal of Cancer Prevention, Vol 24 Ghislaine Florice Faujo Nintewoue and Laure Brigitte Kouitcheu Mabeku Ghislaine Florice Faujo Nintewoue and Laure Brigitte Kouitcheu Mabeku in subjects with atrophic body gastritis. Gastroenterology,
101, 167-74. Cancer Res, 52, 6735–40. Cancer Res, 52, 6735–40. Cancer Res, 52, 6735–40. Cover TL, Blanke SR (2005). Helicobacter pylori VacA, a
paradigm for toxin multifunctionality. Nat Rev Microbiol,
3, 320–2. Katharina T, Kerstin W, Peter N, Alexander D (2015). Molecular
Pathogenesis of MALT Lymphoma. Gastroenterol Res Pract,
Article ID 102656:10 pages. Correa P (2004). The biological model of gastric carcinogenesis. IARC Sci Publ, 301. Kato S, Nakajima S, Nishino Y, et al (2006). Association
between gastric atrophy and Helicobacter pylori infection
in Japanese children: a retrospective multicenter study. Dig
Dis Sci, 51, 99–104. Dandin AS, Pawale J, Athanikar S (2012). Helicobacter pylori
associated gastritis. J Clin Diagn Res, 6, 211-4. D’Elios MM, Amedei A, Manghetti M, et al (1999). Impaired
T-cell regulation of B-cell growth in Helicobacter
pylori–related gastric low-grade MALT lymphoma. Gastroenterology, 117, 1105. Kim GH, Liang PS, Bang SJ, Hwang JH (2016). Screening and
surveillance for gastric cancer in the United States: is it
needed?. Gastrointest Endosc, 84, 18-28. Kim GH, Liang PS, Bang SJ, Hwang JH (2016). Screening and
surveillance for gastric cancer in the United States: is it g
needed?. Gastrointest Endosc, 84, 18-28. Kim N, Park YS, Cho SI, et al (2008). Prevalence and risk factors
of atrophic gastritis and intestinal metaplasia in a Korean
population without significant gastroduodenal disease. Helicobacter, 13, 245-55. Derakhshan MH, Liptrot S, Paul J, et al (2009). Oesophageal
and gastric intestinal-type adenocarcinomas show the same
male predominance due to a 17 year delayed development
in females. Gut, 58, 16-23. DeVries AC, van Grieken NCT, Looman CWN, et al
(2008). Gastric cancer risk in patients with premalignant
gastric lesions: a nationwide cohort study in the
Netherlands. Gas-troenterol, 134, 945–52. Kouitcheu MLB, Eyoum BB, Tepap ZC, Tali NLD, Leundji H
(2019). Broad Spectrum Resistance in Helicobacter pylori
isolated from gastric biopsies of patients with dyspepsia
in Cameroon and phenotypic detection of Efflux-mediated
antimicrobial resistance. BMC Infect Dis, 19, 3-11. Dooley CP, Cohen H, Fitzgibbons PL, et al (1989). DOI:10.31557/APJCP.2023.24.1.171
Degeneration of the Gastric Lining DOI:10.31557/APJCP.2023.24.1.171
Degeneration of the Gastric Lining pylori infection increases risk of gastric cancer. Int J Cancer,
109, 138-43. hyperplastic polyps inversely associated with current
Helicobacter pylori infection. Exp Ther Med, 19, 3143-9. hyperplastic polyps inversely associated with current
Helicobacter pylori infection. Exp Ther Med, 19, 3143-9. Olmez S, Aslan M, Erten R, Sayar S, Bayram I (2015). The
Prevalence of Gastric Intestinal Metaplasia and Distribution
of Helicobacter pylori Infection, Atrophy, Dysplasia, and
Cancer in Its Subtypes. Gastroenterol Res Pract, 2015,
434039. Yakoob MY, Hussainy AS (2010). Chronic gastritis and
Helicobacter pylori: A histopathological study of gastric
mucosal biopsies. J Coll Physicians Surg Pak, 20, 773-5. Parvez M, Dhiraj B, Ni Ku MB, et al (2015). Helicobacter
pylori Associated Gastritis in Northern Maharashtra,
India: A Histopathological Study of Gastric Mucosal
Biopsies. J Clin Diagn Res, 9, 4-6. This work is licensed under a Creative Commons Attribution-
Non Commercial 4.0 International License. This work is licensed under a Creative Commons Attribution-
Non Commercial 4.0 International License. Prashanth R, Adam B (2019). Epidemiology of gastric cancer:
global trends, risk factors and prevention. Gastroenterol
Rev, 14, 26–38. Price AB (1991). The Sydney System: histological division. J Gastroenterol Hepatol, 6, 209-22. Samie A, Obi CL, Barrett LJ, Powell SM, Guerrant RL (2007). Prevalence of Campylobacter species, Helicobacter pylori
and Arcobacter species in stool samples from the Venda
region, Limpopo, South Africa: studies using molecular
diagnostic methods. J Inf Secur, 54, 558–66. Sasa G. Milosav S. Vuka K (2002). The relationship between the
density of H. pylori colonization and the degree of gastritis
severity. Gastroenterol Hepatol, 21, 3-4. Satoskar A, Vora IM (1994). Incidence of Helicobacter pylori
associated gastritis in the urban population from India. Trop
Geogr Med, 46, 167-68. Sonnenberg A, Genta RM (2015). Prevalence of benign gastric
polyps in a large pathology database. Dig Liver Dis, 47,
164-9. Sonnenberg A, Lash RH, Genta RM (2010). A national study of
Helicobactor pylori infection in gastric biopsy specimens. Gastroenterology, 139, 1894-1901. Stolte M, Sticht T, Eidt S, Ebert D, Finkenzeller G (1994). Frequency, location, and age and sex distribution of various
types of gastric polyp. Endoscopy, 26, 659-65. Reymunde A, Deren J, Nachamkin I, Oppenheim D, Weinbaum
G (1993). Production of chemoattractant by Helicobacter
pylori. Dig Dis Sci, 38, 1697–1701. Rodrigues MF, Guerra MR, Alvarenga AVR, Souza DZO,
Costa RAVS (2019). Asian Pacific Journal of Cancer Prevention, Vol 24
183 Approved by scientific Body
No applicable. Montalban C, Boixeda D, Bellas C (1999). Helicobacter pylori
eradication in gastric mucosa-associated lymphoid tissue
lymphomas. Ann Intern Med, 124, 275. Freeman C, Berg JW, Cutler SJ (1972). Occurrence and
prognosis of extranodal lymphomas. Cancer, 29, 252. Ford AC, Forman D, Hunt RH, et al (2014). Helicobacter pylori
eradication therapy to prevent gastric cancer in healthy
asymptomatic infected individuals: systematic review and
meta-analysis of randomized controlled trials. BMJ, 348,
3174. Motta CR, Cunha MP, Queiroz DM, et al (2008). Gastric
precancerous lesions and Helicobacter pylori infection in
relatives of gastric cancer patients from Northeastern Brazil. Digestion, 78, 3-8. Mukaisho K, Nakayama T, Hagiwara T, et al (2015). Two distinct
etiologies of gastric cardia adenocarcinoma: interactions
among pH, Helicobacter pylori, and bile acids. Front
Microbiol, 6, 412. Gonzalez-Carbajal M, Sevilla LF, Gra B (2005). Histological
alterations of the gastric mucous and prevalence of
Helicobacter pylori in dyspeptic patients. Rev Panam
Infectol, 7, 8-15. Muller LB, Fagundes RB, Moraes CC, Rampazzo A (2007). Prevalence of Helicobacter pylori infection and gastric
cancer precursor lesions in patients with dyspepsia. Arq
Gastroenterol, 44, 93-8. Hirota WK, Zuckerman MJ, Adler DJ et al (2006). ASGE
guideline: The role of endoscopy in the surveillance of
premalignant conditions of the upper GI tract. Gastrointestinal
Endoscopy, 63, 570–80. Niyaz A and Leonardo AS (2005). Helicobacter pylori and
Gastro duodenal pathologies; New threats of the old friend. Biomed Central, 2005. Hooi JKY, Lai WY, Ng WK, et al (2017). Global prevalence of
Helicobacter pylori infection: Systematic review and meta-
analysis. Gastroenterology, 153, 420-9. Ohata H, Kitauchi S, Yoshimura N, et al (2004). Progression
of chronic atrophic gas-tritis associated with Helicobacter Karnes WE Jr, Samlo VIM, Siurala M, et al (1991). Positive serum
antibody and negative tissue staining for Helicobacter pylori 182 Asian Pacific Journal of Cancer Prevention, Vol 24
182 DOI:10.31557/APJCP.2023.24.1.171
Degeneration of the Gastric Lining Helicobacter pylori infection and
gastric cancer precursor lesions: prevalence and associated
factors in a reference laboratory in Southeastern Brazil. Arq
Gastroenterol, 56, 419-24. Uemura N, Okamoto S, Yamamoto S, et al (2001). Helicobacter
pylori infection and the development of gastric cancer. N
Engl J Med, 345, 784-9. Takeuchi C, Yamamichi N, Shimamoto T, et al (2017). Gastric
polyps diagnosed by double-contrast upper gastrointestinal
barium X-ray radiography mostly arise from the Helicobacter
pylori-negative stomach with low risk of gastric cancer in
Japan. Gastric Cancer, 20, 314-21. Tanko MN, Manasseh AN, Echejoh GO, et al (2008). Relation
between helicobacter pylori, inflammatory (neutrophil)
activity, chronic gastritis, gastric atrophy and intestinal
metaplasia. Nigerian J Clin Pract, 11, 270-4. Torres J, Leal-Herrera Y, Perez-Perez G, et al (1998). A
community-based seroepidemiologic study of Helicobacter
pylori infection in Mexico. SOJ Infect Dis, 178, 1089. Wotherspoon AC, Doglioni C, Diss TC, et al (1993). Regression
of primary low-grade B-cell gastric lymphoma of mucosa-
associated lymphoid tissue type after eradication of
Helicobacter pylori. Lancet, 342, 575–7. Wroblewski LE, Peek RM, Wilson KT (2010). Helicobacter
pylori and gastric cancer: Factors that modulate disease risk. Clin Microbiol Rev, 23, 713–39. Xinjuan Y, Zhengqiang W, Lili W, et al (2020). Gastric
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Альманах клинической медицины. 2019; 47 (6): 525–534. doi: 10.18786/2072-0505-2019-47-075 О б з о р О б з о р Хронический панкреатит
и сахарный диабет: обзор литературы Ломакина Е.Ю.1 • Таратина О.В.1 • Белоусова Е.А.1 особенностям, подходам к лечению. Причиной
эндокринной недостаточности поджелудоч-
ной железы при ХП выступает вторичное вос-
палительное
поражение
панкреатического
островкового аппарата. Объяснение особен-
ностям СД 3с следует искать в анатомо-физи-
ологических
взаимоотношениях
внешнесе-
креторного и внутрисекреторного аппарата
поджелудочной железы. Сегодня большинство
положений по панкреатогенному СД основа-
ны на эмпирическом подходе и носят скорее
декларативный характер, так как глубокие ме-
ханизмы патогенеза этого типа диабета и тем
более патогенетического лечения недостаточ-
но изучены. Тем не менее все больные ХП или
другой патологией поджелудочной железы
должны быть обследованы на наличие панкре-
атогенного СД. Обоснование. Достаточно давно ведется дис-
куссия о том, как связаны между собой хрони-
ческий панкреатит (ХП) и сахарный диабет (СД). В случае развития у одного пациента следует
ли их рассматривать как два независимых за-
болевания или одно из них – закономерное
следствие другого? Если верно последнее,
каковы механизмы развития СД при ХП? В су-
ществующих консенсусах специалистов-пан-
креатологов этому вопросу уделено недо-
статочно внимания, а основные положения
имеют невысокий уровень доказательности. В Российский консенсус по диагностике и ле-
чению ХП, принятый в 2016 г., положения о СД
не включены. В разрабатываемых отечествен-
ных рекомендациях и согласительных доку-
ментах необходимо предусмотреть выделение
панкреатогенного СД как самостоятельного
СД «другого типа» с учетом механизмов его
развития и клинических особенностей. Цель –
охарактеризовать состояние проблемы панк-
реатогенного СД, показать отличия этого типа
диабета от СД 1-го и 2-го типов с патогенети-
ческих и клинических позиций. Методы. Для
раскрытия темы в обзоре использованы дан-
ные метаанализов, систематических обзоров
и основные положения существующих кли-
нических рекомендаций и консенсусов, пред-
ставленные в PubMed и E-library. Результаты. По разным данным, СД 3с типа, или латентное
нарушение толерантности к глюкозе при ХП,
развивается у 25–80% больных ХП. У больных острым панкреатитом снижение толерантно-
сти к глюкозе выявляется в 40–60%, а после
острого панкреатита гипергликемия отмечает-
ся у 15–18%. При СД 1-го и 2-го типов часто раз-
вивается экскреторная недостаточность под-
желудочной железы, хотя данные о ее частоте
крайне противоречивы, что свидетельствует
о недостаточной изученности проблемы. Для
СД 3с типа характерна манифестация на позд-
них стадиях ХП, сочетание с экскреторной
недостаточностью
поджелудочной
железы,
лабильность течения со склонностью к гипог-
ликемии, отсутствие кетоацидоза. Группу наи-
более высокого риска составляют пациенты
с длительным течением ХП, с предшествующей
частичной резекцией поджелудочной железы
и больные с ранним формированием кальци-
фицирующего панкреатита, преимущественно
алкогольного генеза. Альманах клинической медицины. 2019; 47 (6): 525–534. doi: 10.18786/2072-0505-2019-47-075 Альманах клинической медицины. 2019; 47 (6): 525–534. doi: 10.18786/2072-0505-2019-47-075 имеет постоянный вектор от инсулинсекрети-
рующих клеток к глюкагонсекретирующим и да-
лее к соматостатинсекретирующим клеткам [14]. Наконец, имеются достоверные данные о связи
островков и протоков в ПЖ взрослого человека. В частности, при выделении островков человека
в них всегда обнаруживается определенное коли-
чество (20–60%) протоковых клеток [14]. Ломакина Екатерина
Юрьевна – мл. науч. сотр. отделения
гастроэнтерологии1
*
* 129110, г. Москва,
ул. Щепкина, 61/2,
Российская Федерация. Тел.: +7 (926) 525 93 97. E-mail: kate-den@bk.ru Ломакина Екатерина
Юрьевна – мл. науч. сотр. отделения
гастроэнтерологии1
*
* 129110, г. Москва,
ул. Щепкина, 61/2,
Российская Федерация. Тел.: +7 (926) 525 93 97. E-mail: kate-den@bk.ru
Таратина Олеся
Валериевна – канд. мед. наук, науч. сотр. отделения
гастроэнтерологии,
доцент кафедры
гастроэнтерологии
факультета
усовершенствования
врачей1; ORCID: https://
orcid.org/0000-0001-
6112-6609. E-mail:
taratina.o@gmail.com контролироваться диетой и пероральными саха-
роснижающими препаратами, в тяжелых случаях
необходима инсулинотерапия». В случае развития ХП и СД у одного пациен-
та следует ли их рассматривать как два независи-
мых заболевания или одно из них – закономерное
следствие другого, – этим вопросом продолжают
задаваться исследователи. Объяснение следует ис-
кать в анатомо-физиологических взаимоотноше-
ниях экскреторного и инкреторного аппарата ПЖ
[6–13]. В этой связи целью настоящего обзора стал
анализ современного состояния проблемы панкре-
атогенного СД, выявление отличий этого типа ди-
абета от СД 1-го и 2-го типов с патогенетических
и клинических позиций. Таратина Олеся
Валериевна – канд. мед. наук, науч. сотр. отделения
гастроэнтерологии,
доцент кафедры
гастроэнтерологии
факультета
усовершенствования
врачей1; ORCID: https://
orcid.org/0000-0001-
6112-6609. E-mail:
taratina.o@gmail.com Гормоны существенно влияют на внешнесе-
креторную часть ПЖ, хотя данные экспериментов
in vivo противоречивы. Согласно некоторым из
них, инсулин усиливает экзокринную панкреати-
ческую секрецию (базальную и холецистокинин-
и ацетилхолин-индуцированную) in vivo у крыс
[17–22], а при экспериментальном индуцирован-
ном диабете у овец восстанавливает сниженную
внешнюю секрецию до нормального уровня [23],
а также (в высоких дозах) повышает базальную се-
крецию амилазы [18, 19] и усиливает транскрип-
цию гена амилазы [24]. Однако в других работах
инсулин не оказывал никакого влияния на сни-
женную базальную панкреатическую секрецию
у диабетических кроликов [25] либо увеличивал
объем выделяемого сока ПЖ у крыс только при
введении в максимальной дозе, тогда как физио-
логические дозы инсулина не оказывали никако-
го эффекта [26]. В ряде исследований в ответ на
введение инсулина не было отмечено изменения
базальной секреции амилазы [17, 26], липазы [18,
26] и трипсина [19, 26]. 1 ГБУЗ МО «Московский
областной научно-ис-
следовательский кли-
нический институт им.
М.Ф. Владимирского»;
129110, г. Москва,
ул. Щепкина, 61/2,
Российская Федерация Альманах клинической медицины. 2019; 47 (6): 525–534. doi: 10.18786/2072-0505-2019-47-075 Можно сказать, сегодня
принята точка зрения, согласно которой инсулин
и панкреатический полипептид оказывают тро-
фическое действие на внешнесекреторную часть
ПЖ, стимулируя синтез белков, рост и дифферен-
цировку ацинарных клеток через рецептор инсу-
линоподобного фактора роста 1-го типа (IGF-1)
[27, 28], кроме того, инсулин стимулирует синтез
и секрецию амилазы и других панкреатических
ферментов [29]. Методы Для раскрытия темы использованы данные мета-
анализов, систематических обзоров и основных
положений существующих клинических реко-
мендаций и консенсусов из баз данных PubMed
и E-library. Поиск источников проводился по
ключевым словам: “chronic pancreatitis”, “diabetes”,
“pancreatogenic diabetes” / «хронический панкреа-
тит», «сахарный диабет», «панкреатогенный сахар-
ный диабет». Белоусова Елена
Александровна – д-р
мед. наук, профессор,
руководитель отделения
гастроэнтерологии,
заведующая кафедрой
гастроэнтерологии
факультета
усовершенствования
врачей1; ORCID: https://
orcid.org/0000-0003-
4523-3337. E-mail:
eabelous@yandex.ru Результаты Анатомо-физиологические взаимодействия
в поджелудочной железе Хронический панкреатит
и сахарный диабет: обзор литературы Вопрос об оптимальном
и рациональном медикаментозном лечении
панкреатогенного СД пока остается спорным,
поскольку доказательная база длительной эф-
фективности и безопасности для разных саха-
роснижающих препаратов при данной патоло-
гии отсутствует и соглашения в этой области не
достигнуто. Общие рекомендации по лечению,
представленные в ряде международных кон-
сенсусов, сводятся к осторожному назначению
инсулина. Заключение. Панкреатогенный СД
отличается от СД 1-го и 2-го типов по ряду па-
раметров: механизмам развития гиперглике-
мии, гормональному профилю, клиническим Ключевые слова: хронический панкреатит,
сахарный диабет, панкреатогенный сахарный
диабет Для цитирования: Ломакина ЕЮ, Таратина ОВ,
Белоусова ЕА. Хронический панкреатит и са-
харный диабет: обзор литературы. Альманах
клинической медицины. 2019;47(6):525–34. doi:
10.18786/2072-0505-2019-47-075. Поступила 11.11.2019; доработана 04.12.2019;
принята к публикации 05.12.2019; опубликована
онлайн 16.12.2019 С
а
р
в
д как связаны между собой ХП и СД, ведется доста-
точно давно [1–8], но в существующих консенсу-
сах этому вопросу уделено небольшое внимание,
а сформулированные положения имеют недоста-
точно высокий уровень доказательности [1–5]. На практике этот тип диабета расценивается и ле-
чится как СД 2-го типа, хотя механизмы его раз-
вития иные. Наиболее типичная рекомендация
в существующей литературе звучит следующим
образом: «На ранних стадиях СД при ХП может С
ахарный диабет (СД) и нарушение толе-
рантности к глюкозе достаточно часто
встречаются при заболеваниях поджелу-
дочной железы (ПЖ): остром и хрониче-
ском панкреатите (ХП), раке ПЖ, хирургических
вмешательствах на ПЖ, муковисцидозе (кистоз-
ном фиброзе). Однако до последнего времени нет
четкого понимания этой проблемы, включая ме-
ханизмы развития, классификацию, диагности-
ку и лечебные подходы. Дискуссия на тему о том, как связаны между собой ХП и СД, ведется доста-
точно давно [1–8], но в существующих консенсу-
сах этому вопросу уделено небольшое внимание,
а сформулированные положения имеют недоста-
точно высокий уровень доказательности [1–5]. На практике этот тип диабета расценивается и ле-
чится как СД 2-го типа, хотя механизмы его раз-
вития иные. Наиболее типичная рекомендация
в существующей литературе звучит следующим
образом: «На ранних стадиях СД при ХП может 525 Белоусова Елена
Александровна – д-р
мед. наук, профессор,
руководитель отделения
гастроэнтерологии,
заведующая кафедрой
гастроэнтерологии
факультета
усовершенствования
врачей1; ORCID: https://
orcid.org/0000-0003-
4523-3337. E-mail:
eabelous@yandex.ru Анатомо-физиологические взаимодействия
в поджелудочной железе В исследованиях in vitro и in vivo была показана тес-
ная связь эндокринной и экзокринной частей ПЖ
[6, 7, 11, 12, 14, 15]. Островки Лангерганса состав-
ляют лишь 1–3% массы паренхимы железы. Но они
расположены среди ацинусов, не окружены капсу-
лой и через них проходит более 15% панкреатиче-
ского кровотока, который направлен от островков
к экзокринной ткани (инсулоацинарная порталь-
ная система ПЖ) [16]. Прекапиллярные артерио-
лы, первыми достигающие островков, распадаются
на капилляры, которые образуют внутриостров-
ковые клубочки, а затем выходят из панкреатиче-
ских островков уже как эфферентные капилляры,
снабжающие кровью экзокринную ткань [11, 14,
16]. Помимо островков Лангерганса эндокринная
часть ПЖ представлена отдельными эндокринны-
ми клетками и небольшими ростками эндокрин-
ной ткани, расположенными в ацинарной части
паренхимы и в эпителии протоков [14]. В итоге
концентрация гормонов ПЖ в ее внешнесекретор-
ной ткани значительно выше, чем в общем крово-
токе, что обеспечивает паракринную и эндокрин-
ную регуляцию ацинарной секреции. Направление
кровотока в капиллярной внутриостровковой сети Наиболее выраженное трофическое и стимули-
рующее влияние инсулин оказывает на ацинарные
клетки, расположенные близко к островкам (пери-
инсулярные ацинусы) – так называемый гало-фе-
номен [11, 29]. Эти клетки имеют больший размер,
чем другие, содержат большее количество зимо-
генных гранул [11, 29]. У больных с полностью
утраченной функцией бета-клеток гистопатологи-
ческие изменения в экзокринной части ПЖ более
выражены, чем у пациентов с остаточной секреци-
ей инсулина [11, 28]. Эффекты введения глюкагона на внешнюю
панкреатическую секрецию у экспериментальных
животных и человека также варьируют в зависи-
мости от изучаемого вида и условий эксперимента. Обзор Обзор 526 Альманах клинической медицины. 2019; 47 (6): 525–534. doi: 10.18786/2072-0505-2019-47-075 В соответствии с существующей классифика-
цией Всемирной организации здравоохранения
[45, 46], существует несколько типов СД: Описано ингибирующее действие глюкагона на
стимулированную холецистокинином и други-
ми секретагогами экскреторную функцию ПЖ
in vivo у крыс [30], собак [31] и людей [32], а так-
же постпрандиально у человека [33]. При дли-
тельном введении глюкагона наблюдалась потеря
ацинусами крыс до 90% зимогенных гранул при
атрофии экзокринной части ПЖ [14]. Другие ав-
торы сообщают об отсутствии влияния глюкаго-
на на базальную внешнюю секрецию у крыс [34],
кроликов [35], собак [36] и человека [33], а также
на стимулированную секрецию у человека [37]. Опубликованы работы, в которых глюкагон уве-
личивал постпрандиальную внешнюю секрецию
у крыс [38] и собак [26]. В более поздней работе
введение глюкагона крысам снижало фермента-
тивную активность амилазы, липазы и трипсина
в соке ПЖ, но не оказывало влияния на объем се-
кретируемого сока [26]. Анатомо-физиологические взаимодействия
в поджелудочной железе •
СД 1-го типа, к которому относят иммуноопо-
средованный и идиопатический СД, сопрово-
ждающиеся деструкцией β-клеток и, как пра-
вило, развитием абсолютной недостаточности
инсулина; у
•
СД 2-го типа, при котором может преобладать
либо инсулинорезистентность, а недостаточ-
ность инсулина носить относительный харак-
тер, либо дефект секреции инсулина с рези-
стентностью к инсулину или без нее; у
у
•
гестационный диабет; •
другие специфические типы СД. В эту рубрику
включают различные генетические дефекты,
эндокринопатии, лекарственные, токсические
и инфекционные формы диабета, а также диа-
бет, развившийся вследствие заболеваний экзо-
кринной части ПЖ (СД тип 3с) [5, 6, 8, 10, 11,
13, 44, 47–50]. Соматостатин является отрицательным регуля-
тором функции пищеварительной системы, в том
числе экзокринной функции ПЖ: он подавляет как
базальную (до 50% от исходного уровня), так и сти-
мулированную панкреатическую секрецию [39, 40]. Предполагается, что соматостатин оказывает пря-
мой угнетающий эффект на ПЖ и опосредован-
ный через снижение выработки холецистокинина
[39]. В модели стрептозотоцин-индуцированного
мочеизнурения у крыс уровень соматостатина по-
вышен, экзокринная функция ПЖ снижена [41]. Сахарный диабет 3с типа может быть следстви-
ем острого и хронического панкреатита, травм,
опухолей, резекции ПЖ, гемохроматоза либо му-
ковисцидоза. Этот термин был введен в 2014 г. Американской диабетической ассоциацией (ADA)
[51] и принят в том числе Российским панкреато-
логическим клубом в консенсусе 2017 г. [5]. Снижение толерантности к глюкозе выявляется
у 40–60% больных острым панкреатитом, и при-
близительно у 15–18% пациентов после острого
панкреатита отмечается гипергликемия, которая
самопроизвольно разрешается через 4–6 месяцев
[11]. Преходящая гипергликемия развивается так-
же при обострении ХП, что связано с отеком ПЖ В целом сегодня считается, что глюкагон, со-
матостатин и панкреатический полипептид ин-
гибируют внешнюю секрецию ПЖ [11–14, 27, 41]. Однако подавляющее большинство исследований
по влиянию этих гормонов на экзокринную функ-
цию ПЖ были выполнены только на животных
моделях, а об их эффектах in vivo у человека сведе-
ний мало. К тому же базальная и стимулированная
секреция этих островковых гормонов различаются
при СД 1-го и 2-го типов [42]. Механизмы развития сахарного диабета при хроническом панкреатите;
ПЖ – поджелудочная железа
Диффузное воспаление
с вовлечением островков
Лангерганса
Развитие фиброза
и склероза ПЖ
Снижение кровотока
в островках Лангерганса
Снижение синтеза инсулина
Гипергликемия
Снижение
трофики
Снижение
кровотока
Снижение синтеза
ферментов ПЖ
Усиление
эндокринной
недостаточности ПЖ Диффузное воспаление
с вовлечением островков
Лангерганса Таким образом, взаимодействие между остров-
ками и ацинусами значимое и двунаправленное:
эндокринная ткань участвует в регуляции панкре-
атической секреции, а ацинарная часть обеспечи-
вает функционирование островкового аппарата за
счет кровотока. Альманах клинической медицины. 2019; 47 (6): 525–534. doi: 10.18786/2072-0505-2019-47-075 Альманах клинической медицины. 2019; 47 (6): 525–534. doi: 10.18786/2072-0505-2019-47-075 СД 1-го типа изолированно поражаются β-клет-
ки и снижается выработка только инсулина. Это
коренное отличие панкреатогенного СД от СД
1-го типа определяет разницу клинической кар-
тины и данных лабораторных тестов при этих за-
болеваниях. У больных СД 3с типа часто сохраня-
ется остаточная продукция инсулина, вследствие
чего реже возникают кетоацидоз и гиперосмоляр-
ные состояния, но поражение глюкагон-продуци-
рующих α-клеток и дефицит глюкагона приводят
к относительному гиперинсулинизму и частым
эпизодам гипогликемий [2, 41]. Подчеркнем: вы-
сокая концентрация глюкозы также стимулирует
формирование фиброза в ПЖ через активацию ее
звездчатых клеток, что может замкнуть порочный
круг [57] (см. рисунок). и ингибирующим влиянием трипсина (его содержа-
ние в крови при остром панкреатите и обострении
ХП повышается) на продукцию инсулина. По мере
разрешения панкреатической атаки уровень глю-
козы крови обычно нормализуется [11]. Согласно
D. Andersen и соавт. [52], клинические и лабора-
торные характеристики СД 3с типа отчасти сходны
с СД 1-го типа (часто развиваются гипогликеми-
ческие состояния, периферическая и печеночная
чувствительность к инсулину нормальная или сни-
жена), отчасти – с СД 2-го типа (редко развивает-
ся кетоацидоз). Уровень инсулина при СД 3с типа
может быть как в пределах нормы, так и низким,
уровень глюкагона – тоже «нормальным» или низ-
ким (при 1- и 2-м типах он нормальный или повы-
шен), характерны низкие уровни панкреатического
полипептида и глюкозозависимого инсулинотроп-
ного пептида (GIP); уровень глюкагоноподобного
пептида-1 (GLP-1) может варьировать. В патогенезе СД при ХП обсуждается роль кон-
ституционально обусловленной периферической
инсулинорезистентности, которая значительно
чаще встречается у людей с ожирением и гипер-
липидемией. Показано, что чем выше степень
ожирения, тем выше риск развития различных
осложнений ХП, включая нарушение толерантно-
сти к углеводам и СД [11]. Гипотеза об этиологиче-
ской роли аутоантител, формирующихся при ХП,
в развитии панкреатогенного СД не нашла своего
подтверждения. В панъевропейских клинических
рекомендациях (HaPanEU) по ХП [3] и в работе
N. Ewald и соавт. [2] были предложены диагности-
ческие критерии СД 3с типа. Истинную заболеваемость и распространен-
ность вторичного СД оценить сложно. Вместе
с тем недавний крупный обзор работ, посвящен-
ных этой теме, предполагает, что в западной попу-
ляции среди всех случаев сахарного диабета тип 3с
составляет от 5 до 10% [53]. Так, в немецком ретро-
спективном исследовании, включавшем 1868 па-
циентов, СД 3с типа составил 9,2% [54], при этом
более чем в половине случаев данную патологию
неверно классифицируют [2]. Анатомо-физиологические взаимодействия
в поджелудочной железе Снижение
трофики Снижение кровотока
в островках Лангерганса Снижение
кровотока Снижение синтеза
ферментов ПЖ Панкреатогенный сахарный диабет р
р
д
Нарушение толерантности к глюкозе и СД – до-
вольно частая проблема при заболеваниях ПЖ
и после оперативных вмешательств на ней [5, 43]. Причиной эндокринной недостаточности ПЖ
при ХП служит вторичное поражение панкреати-
ческого островкового аппарата (рисунок) [41, 44]. Механизмы развития сахарного диабета при хроническом панкреатите;
ПЖ – поджелудочная железа 527 Альманах клинической медицины. 2019; 47 (6): 525–534. doi: 10.18786/2072-0505-2019-47-075 Хронический панк-
реатит – наиболее частая причина СД 3с типа, при-
близительно 78,5% случаев СД 3с развивается при
ХП [54]. Оценочно при ХП «панкреатогенный» СД
или латентное нарушение толерантности к глюко-
зе возникают у 25–80% больных [10, 54, 55], при
этом устойчивое нарушение углеводного обмена
отмечено в среднем через 5 лет от начала основ-
ного заболевания, а в последующем наблюдается
линейная зависимость частоты развития СД при
ХП от времени [11]. Однако следует отметить, что
в ряде исследований диагноз СД 3с типа не доказан
какими-то специальными признаками, а устанав-
ливался только по сочетанию с ХП. Большие критерии: •
установленный диагноз ХП с экзокринной не-
достаточностью ПЖ; •
отсутствие аутоиммунных маркеров, ассоции-
рованных с СД 1-го типа. Малые критерии: •
отсутствие секреции панкреатического поли-
пептида; •
нарушение секреции инкретинов (например,
GLP-1); •
отсутствие выраженной инсулинорезистентно-
сти (по данным индекса HOMA-IR – Homeostasis
model assessment of insulin resistance); При ХП протоковое и тканевое давление в ПЖ
повышается, что ведет к нарушению панкреати-
ческой микроциркуляции и тканевой гипоксии,
которая, предположительно, способствует акти-
вации звездчатых клеток ПЖ, их пролиферации
и стимулирует секрецию ими коллагена 1-го типа,
что приводит к фиброзу ПЖ [56]. Это способству-
ет постепенному и прогрессирующему ухудше-
нию функции не только ацинарной, внешнесекре-
торной части, но и всего эндокринного аппарата
ПЖ, то есть не только β-клеток, но и других гор-
монпродуцирующих, в том числе α-клеток. При •
повреждение функции β-клеток (нарушено со-
отношение C-пептид / глюкоза либо по оцен-
ке HOMA-B – Homeostasis model assessment of
beta-cell); •
низкое содержание в сыворотке крови липидов,
жирорастворимых витаминов (А, D, Е и К). Для достоверного диагноза СД 3с типа необ-
ходимо наличие двух больших критериев, только
сочетания СД и ХП недостаточно. Именно отсут-
ствие второго критерия в ряде работ (исследование
аутоиммунных маркеров, ассоциированных с СД Обзор Обзор 528 Альманах клинической медицины. 2019; 47 (6): 525–534. doi: 10.18786/2072-0505-2019-47-075 1-го типа) может поставить диагноз СД 3с типа под
сомнение. Оценка изменения концентрации панк-
реатического полипептида в ответ на инсулин-ин-
дуцированную гипогликемию, инфузию секретина
или пробный завтрак с различными нутриентами
может помочь при дифференциальной диагности-
ке СД 3с и раннего СД 1-го и 2-го типов [2], однако
в настоящее время в России оценка малых крите-
риев в работе практического врача, специализи-
рованных отделений невыполнима и даже в боль-
шинстве НИИ мало реальна. Экзокринная недостаточность поджелудочной
железы при сахарном диабете Экзокринная недостаточность поджелудочной
железы при сахарном диабете железы при сахарном диабете
Обсуждается и обратная зависимость: наличие
экзокринной недостаточности ПЖ при установ-
ленном СД. Экзокринная недостаточность ПЖ
по данным обоих функциональных тестов – пря-
мого (секретин-панкреозиминового) и непрямого
(концентрация фекальной эластазы) – выявляется
у 50% пациентов с СД 1-го типа и 35% СД 2-го типа
[58]. В большинстве исследований экзокринной
функции ПЖ при СД регистрировали легкую или
умеренную степень внешнесекреторной недоста-
точности ПЖ, которая выражалась в относитель-
ном снижении продукции бикарбонатов и фер-
ментов, а тяжелая экзокринная недостаточность
ПЖ со стеатореей встречается сравнительно редко
[11]. Отмечалась обратная корреляция уровня фе-
кальной эластазы с длительностью диабета и со-
держанием гликированного гемоглобина и пря-
мая – с концентрацией С-пептида в смешанной
популяции больных СД [59] и в группе больных
СД 2-го типа [60]. При СД 2-го типа в 15–73% слу-
чаев нарушается синтез амилазы и трипсина и на-
блюдается обратная связь активности панкреати-
ческих ферментов с постпрандиальной гликемией
[60]. В целом при СД 1-го типа базальная и сти-
мулированная продукция протеолитических фер-
ментов нарушается относительно чаще, а уровень
фекальной эластазы-1 ниже, чем при СД 2-го типа,
но при развитии периферической нейропатии (ди-
абетической энтеропатии) и диареи у больных СД
2-го типа экзокринная недостаточность ПЖ была
установлена у всех пациентов, а продукция амила-
зы и бикарбонатов при введении различных стиму-
ляторов достигала всего лишь 40% от нормы [11]. Предполагается несколько механизмов развития
экзокринной недостаточности ПЖ при диабете: •
диабетическая ангиопатия, предположительно
способствующая развитию панкреатического
фиброза [13, 64]; •
диабетическая нейропатия и нарушение энте-
ропанкреатических рефлексов [1, 13, 64]; •
мутации гена HNF1B при диабете зрелого типа
у молодых (maturity onset diabetes of the young –
MODY) 5-го типа и другие моногенные заболе-
вания [64]. Ген HNF1B, в котором описано около
30 мутаций, кодирует гомеодоменсодержащий
транскрипционный
фактор,
вовлеченный
в раннее развитие почки, ПЖ, печени, половых
путей [65]. Связанные с HNF1B заболевания
представляют собой комбинацию почечных
(билатеральная ренальная кистозная гиподис-
плазия, почечные кисты, аномалии лоханки)
и внепочечных проявлений. Экстраренальный
фенотип включает СД (MODY5), экзокринную
недостаточность ПЖ, атрофию ПЖ, изменения
печеночных проб, разнообразные аномалии
половых путей у женщин и бесплодие у муж-
чин, в отдельных случаях – незначительную ум-
ственную отсталость [65]; •
одновременное
повреждение
экзокринной
и эндокринной части ПЖ при вирусных инфек-
циях [64]. Лечение пациентов с сочетанной эндо- Альманах клинической медицины. 2019; 47 (6): 525–534. doi: 10.18786/2072-0505-2019-47-075 наблюдается положительная корреляция меж-
ду остаточной функцией бета-клеток и концен-
трацией фекальной эластазы-1 [42, 61]; •
изменение инкреции или дисбаланс инсулина
и других островковых гормонов (глюкагона, со-
матостатина, панкреатического полипептида)
[42]; повышение уровня глюкагона было выяв-
лено при СД как 1-го, так и 2-го типа, что мо-
жет привести к атрофии ацинарного аппарата
[11–14, 27, 41]; •
аутоиммунные процессы, общие для СД
и ХП: в нескольких небольших исследовани-
ях в Японии было показано наличие антител
к различным антигенам экзокринной части ПЖ
при СД 1-го типа, например, антитела к панк-
реатическому цитокератину были выявлены
у 39%, антитела к липазе – у 73,5% и антитела
к лактоферрину или карбоангидразе – у 77%
всех пациентов [62, 63]; Альманах клинической медицины. 2019; 47 (6): 525–534. doi: 10.18786/2072-0505-2019-47-075 Альманах клинической медицины. 2019; 47 (6): 525–534. doi: 10.18786/2072-0505-2019-47-075 к снижению уровня инсулина и усугубляет ги-
пергликемию [2, 49, 67]. Установлено, что приме-
нение ферментных препаратов увеличивает син-
тез обоих инкретинов. Это приводит к усилению
постпрандиальной секреции инсулина. В настоя-
щее время созданы два типа лекарственных пре-
паратов, модулирующих эффекты GLP-1: ингиби-
тор фермента дипептидилпептидазы-4 (DPP-4),
разрушающей инкретины, и пролонгированный
DPP-4-резистентный аналог GLP-1. Данные пре-
параты с успехом применяются при СД 2-го типа. Обсуждалась возможность их применения при
панкреатогенном СД, поскольку эти препараты
усиливают глюкозозависимую секрецию инсули-
на, что ведет к увеличению массы β-клеток и улуч-
шению их функции без риска гипогликемии [67,
70]. Однако, как показали данные двух метаанали-
зов, у больных СД 2-го типа назначение этих пре-
паратов само по себе провоцирует развитие ХП
[67, 69]. Именно поэтому от их применения реко-
мендовано воздержаться до получения убедитель-
ных данных об эффективности и безопасности
при СД 3с типа [67, 69]. препаратов. Рекомендуется прием пищи неболь-
шими порциями 5–6 раз в сутки, коррекция
мальабсорбции, дефицита витаминов и микроэле-
ментов [66–68]. Необходима заместительная ферментная те-
рапия для компенсации экзокринной недоста-
точности ПЖ, развивающейся параллельно с СД
3с типа [67]. У пациентов с экзокринной недо-
статочностью ПЖ мальдигестия и последующая
мальабсорбция питательных веществ ухудшают
энтеральную секрецию инкретинов (GLP-1 и GIP),
которые важны для глюкозозависимой секреции
инсулина. Заместительная ферментная терапия не
только улучшает переваривание и всасывание ну-
триентов, но также повышает синтез инкретинов
и секрецию инсулина, что приводит к снижению
гипергликемии [49, 67]. Надо, однако, помнить, что
уменьшение симптомов мальабсорбции и маль-
дигестии улучшает усвоение нутриентов, в том
числе глюкозы, что может усилить гипергликемию
[44, 49, 68]. Эти два положения не являются про-
тиворечием, следует добиваться разумного балан-
са данных процессов подбором доз ферментных
препаратов. Однозначных подходов к медикамен-
тозному лечению СД 3-го типа в настоящее время
нет. Однако в ряде консенсусов [44, 49, 68] даны
рекомендации по лечению таких пациентов. С точ-
ки зрения патофизиологии панкреатогенного СД
показано лечение небольшими дозами инсулина,
так как имеется его абсолютный дефицит, обуслов-
ленный нарушением функции β-клеток [44, 49, 68]. Предписаны режимы и дозы, разработанные для
СД 1-го типа [44, 69]. Вместе с тем лабильность СД,
быстрый переход от гипергликемии к гипоглике-
мии определяют необходимость проявлять край-
нюю осторожность при лечении инсулином и осу-
ществлять постоянный контроль уровня глюкозы
в крови, в связи с чем таким пациентам рекоменду-
ется установка инсулиновой помпы [49, 70]. Лечение пациентов с сочетанной эндо- и экзокринной недостаточностью поджелудочной
железы •
дефицит инсулина, приводящий к снижению
трофики ПЖ, способствует ее атрофии [13, 28,
41]; Диетические ограничения и лечение основного
заболевания (ХП) у пациентов с панкреатоген-
ным СД обязательны. Нередко стихание атаки
ХП в сочетании с диетической коррекцией при-
водит к снижению гликемии без лекарственных •
дефицит
инсулина
уменьшает
секрецию
панкреатических ферментов, в частности, омакина Е.Ю., Таратина О.В., Белоусова Е.А. Хронический панкреатит и сахарный диабет: обзор литературы 529 Литература / References 12. Цымбалюк ВЮ, Кокуева ОВ, Новоселя НВ,
Цымбалюк ИЮ, Середа СА. Состояние угле-
водного обмена и инкретинового статуса
при
патологии
панкреатодуоденальной
зоны. Кубанский научный медицинский
вестник. 2010;(3–4):196–201. [Tsimbaljuk VJ,
Novoselja NV, Tsimbaljuk IJ, Sereda SA. [Condi-
tion of the carbohydrate metabolism and the
status of incretins at the pathology of pancre-
atoduodenal zone]. Kuban Scientific Medical
Bulletin. 2010;(3–4):196–201. Russian.] borodova EV, Belousova EA, Voskanyan SE, Vi-
nokurova LV, Grinevich VB, Darvin VV, Dubtso-
va EA, Dyuzheva TG, Egorov VI, Efanov MG,
Izrailov RE, Korobka VL, Kotiv BN, Kokhanen-
ko NY, Kucheryavyy YA, Livzan MA, Lyadov VK,
Nikolskaya KA, Osipenko MF, Pasechnikov VD,
Plotnikova EY, Sablin OA, Simanenkov VI, Ts-
virkun VV, Tsukanov VV, Shabunin AV, Bor-
din DS. [Russian consensus on exo- and en-
docrine pancreatic insufficiency after surgical
treatment]. Ter Arkh. 2018;90(8):13–26. Rus-
sian. doi: 10.26442/terarkh201890813-26.] borodova EV, Belousova EA, Voskanyan SE, Vi-
nokurova LV, Grinevich VB, Darvin VV, Dubtso-
va EA, Dyuzheva TG, Egorov VI, Efanov MG,
Izrailov RE, Korobka VL, Kotiv BN, Kokhanen-
ko NY, Kucheryavyy YA, Livzan MA, Lyadov VK,
Nikolskaya KA, Osipenko MF, Pasechnikov VD,
Plotnikova EY, Sablin OA, Simanenkov VI, Ts-
virkun VV, Tsukanov VV, Shabunin AV, Bor-
din DS. [Russian consensus on exo- and en-
docrine pancreatic insufficiency after surgical
treatment]. Ter Arkh. 2018;90(8):13–26. Rus-
sian. doi: 10.26442/terarkh201890813-26.] 1. el Newihi H, Dooley CP, Saad C, Staples J,
Zeidler A, Valenzuela JE. Impaired exocrine
pancreatic function in diabetics with diar-
rhea and peripheral neuropathy. Dig Dis Sci. 1988;33(6):705–10. doi: 10.1007/bf01540434. 2. Ewald N, Hardt PD. Diagnosis and treatment
of diabetes mellitus in chronic pancreatitis. World J Gastroenterol. 2013;19(42):7276–81. doi: 10.3748/wjg.v19.i42.7276. 3. Löhr JM, Dominguez-Munoz E, Rosendahl J,
Besselink M, Mayerle J, Lerch MM, Haas S,
Akisik F, Kartalis N, Iglesias-Garcia J, Keller J,
Boermeester M, Werner J, Dumonceau JM,
Fockens P, Drewes A, Ceyhan G, Lindkvist B,
Drenth J, Ewald N, Hardt P, de Madaria E, Witt H,
Schneider A, Manfredi R, Brøndum FJ, Rudolf S,
Bollen T, Bruno M; HaPanEU/UEG Working
Group. United European Gastroenterology
evidence-based guidelines for the diagnosis
and therapy of chronic pancreatitis (HaPanEU). United European Gastroenterol J. 2017;5(2):
153–99. doi: 10.1177/2050640616684695. 13. Губергриц НБ, Фоменко ПГ, Колкина ВЯ. Внешнесекреторная недостаточность под-
желудочной железы при сахарном диабе-
те. Российский журнал гастроэнтерологии,
гепатологии и колопроктологии. 2009;19(5):
61–7. [Gubergrits NB, Fomenko PG, Kolki-
na VYa. [Exocrine pancreatic insufficiency at
diabetes mellitus]. Russian Journal of Gas-
troenterology, Hepatology, Coloproctology. 2009;19(5):61–7. Russian.] 6. Литература / References Śliwińska-Mossoń M, Milnerowicz S, Milnerow-
icz H. Diabetes mellitus secondary to pancre-
atic diseases (type 3c): The effect of smoking
on the exocrine-endocrine interactions of the
pancreas. Diab Vasc Dis Res. 2018;15(3):243–
59. doi: 10.1177/1479164118764062. Schneider A, Manfredi R, Brøndum FJ, Rudolf S, 7. Blumenthal HT, Probstein JG, Berns AW. Inter-
relationship of diabetes mellitus and pancrea-
titis. Arch Surg. 1963;87:844–50. doi: 10.1001/
archsurg.1963.01310170130022. 14. Баранов СА, Нечаев ВМ. Поджелудочная
железа как единый функционально взаи-
мосвязанный орган. Медицинский совет. 2017;(11):148–51. doi: 10.21518/2079-701X-
2017-11-148-151. [Baranov SA, Nechaev VM. [Pancreas as a single functionally interrelated
organ]. Medical Council. 2017;(11):148–51. Russian. doi: 10.21518/2079-701X-2017-11-
148-151.] 4. Rickels MR, Bellin M, Toledo FG, Robert-
son RP, Andersen DK, Chari ST, Brand R,
Frulloni L, Anderson MA, Whitcomb DC;
PancreasFest Recommendation Conference
Participants. Detection, evaluation and treat-
ment of diabetes mellitus in chronic pancre-
atitis: recommendations from PancreasFest
2012. Pancreatology. 2013;13(4):336–42. doi:
10.1016/j.pan.2013.05.002. 8. Duggan SN, Ewald N, Kelleher L, Griffin O, Gib-
ney J, Conlon KC. The nutritional management
of type 3c (pancreatogenic) diabetes in chron-
ic pancreatitis. Eur J Clin Nutr. 2017;71(1):3–8. doi: 10.1038/ejcn.2016.127. 9. Malka D, Hammel P, Sauvanet A, Ru-
fat P, O'Toole D, Bardet P, Belghiti J, Berna-
des P, Ruszniewski P, Lévy P. Risk factors for
diabetes mellitus in chronic pancreatitis. Gastroenterology. 2000;119(5):1324–32. doi:
10.1053/gast.2000.19286. 15. Talukdar R, Sasikala M, Pavan Kumar P, Rao GV,
Pradeep R, Reddy DN. T-helper cell-me-
diated islet inflammation contributes to
β-cell dysfunction in chronic pancreatitis. Pancreas. 2016;45(3):434–42. doi: 10.1097/
MPA.0000000000000479. 5. Хатьков ИЕ, Маев ИВ, Абдулхаков СР, Алек-
сеенко СА, Алиханов РБ, Бакулин ИГ, Бакули-
на НВ, Барановский АЮ, Белобородова ЕВ,
Белоусова ЕА, Восканян СЭ, Винокурова ЛВ,
Гриневич ВБ, Дарвин ВВ, Дубцова ЕА, Дюже-
ва TГ, Егоров ВИ, Ефанов МГ, Израилов РЕ,
Коробка ВЛ, Котив БН, Коханенко НЮ, Ку-
черявый ЮА, Ливзан МА, Лядов ВК, Николь-
ская КА, Осипенко МФ, Пасечников ВД,
Плотникова ЕЮ, Саблин ОА, Симаненков ВИ,
Цвиркун ВВ, Цуканов ВВ, Шабунин АВ, Бор-
дин ДС; Профессиональное медицинское со-
общество «Панкреатологический клуб». Рос-
сийский консенсус по экзо- и эндокринной
недостаточности поджелудочной железы
после хирургического лечения. Терапевти-
ческий архив. 2018;(8):13–26. doi: 10.26442/
terarkh201890813-26. [Khatkov IE, Maev IV,
Abdulkhakov SR, Alekseenko SA, Alikhanov RB,
Bakulin IG, Bakulina NV, Baranovskiy AY, Belo- 10. Конфликт интересов Авторы декларируют отсутствие явных и потенциальных конфликтов
интересов, связанных с публикацией настоящей статьи. Финансирование Е.Ю. Ломакина – сбор и анализ литературы по базам данных, анализ
и интерпретация результатов, написание текста статьи; О.В. Таратина –
анализ
результатов,
написание
и
редактирование
текста;
Е.А. Белоусова – концепция и дизайн статьи, редактирование текста,
утверждение итогового варианта текста рукописи. Все авторы внесли
существенный вклад в проведение исследования и подготовку статьи,
прочли и одобрили финальную версию перед публикацией. Финансирование осуществлялось ГБУЗ МО МОНИКИ им. М.Ф. Вла
димирского за счет средств, выделяемых для проведения научно-ис-
следовательских работ. Заключение Существование типа сахарного диабета 3с в до-
статочной степени умозрительно, диагностиче-
ские критерии данного типа СД, в отличие от СД
1-го и 2-го типов, весьма условны. Лечение эмпи-
рическое и доказательной базы для выбора того
или иного метода не существует. В этой связи необ-
ходимы исследования механизмов развития панк-
реатогенного СД для разработки патогенетически
обоснованных методов лечения. Вместе с тем нельзя не признать, что тип 3с СД
отличается от СД 1-го и 2-го типов по ряду пара-
метров: механизмам развития гипергликемии,
гормональному профилю, клиническим особенно-
стям, подходам к лечению. Причиной эндокринной
недостаточности ПЖ при ХП выступает вторич-
ное воспалительное поражение панкреатического
островкового аппарата. Объяснение особенностям
СД 3с типа следует искать в анатомо-физиологи-
ческих взаимоотношениях внешнесекреторного
и внутрисекреторного аппарата ПЖ. Согласно
основным положениям существующих консенсу-
сов все больные ХП или другой патологией ПЖ
должны быть обязательно обследованы на наличие
панкреатогенного СД, но группу наиболее высоко-
го риска составляют пациенты с длительным тече-
нием ХП, с предшествующей частичной резекцией
ПЖ и больные с ранним формированием кальци-
фицирующего панкреатита, преимущественно ал-
когольного генеза. Применение метформина и других перораль-
ных сахароснижающих препаратов рекомендова-
но при невысокой гипергликемии на начальных
стадиях СД 3с типа (HbA1C ≤ 8%) или в случае со-
путствующей инсулинорезистентности [49, 67, 71]. Опубликованы данные, согласно которым терапия
метформином ассоциирована со снижением риска
рака ПЖ [10, 69, 71], что делает обоснованным на-
значение этого препарата при ХП. В лечении СД 2-го типа используются инкре-
тины, к которым относятся GLP-1 и GIP. При этом
типе диабета отмечено снижение их эффектов. При ХП синтез инкретинов может уменьшаться
вследствие нарушения переваривания и всасы-
вания нутриентов, что дополнительно приводит Обзор 530 Альманах клинической медицины. 2019; 47 (6): 525–534. doi: 10.18786/2072-0505-2019-47-075 Альманах клинической медицины. 2019; 47 (6): 525–534. doi: 10.18786/2072-0505-2019-47-075 Литература / References 20. Kanno T, Saito A. The potentiating influences
of insulin on pancreozymin-induced hyperpo-
larization and amylase release in the pancre-
atic acinar cell. J Physiol. 1976;261(3):505–21. doi: 10.1113/jphysiol.1976.sp011571. 33. Wettergren A, Schjoldager B, Mortensen PE,
Myhre J, Christiansen J, Holst JJ. Truncated
GLP-1 (proglucagon 78-107-amide) inhib-
its gastric and pancreatic functions in man. Dig Dis Sci. 1993;38(4):665–73. doi: 10.1007/
bf01316798. 45. World Health Organization. Definition, diag-
nosis and classification of diabetes mellitus
and its complications: report of a WHO con-
sultation. Part 1, Diagnosis and classification of
diabetes mellitus. World Health Organization;
1999 [Internet]. Available from: https://apps. who.int/iris/handle/10665/66040. 21. Singh J, Adeghate E. Effects of islet hormones
on nerve-mediated and acetylcholine-evoked
secretory responses in the isolated pancre-
as of normal and diabetic rats. Int J Mol Med. 1998;1(3):627–34. doi: 10.3892/ijmm.1.3.627. 22. Dermott JM, Gooya JM, Asefa B, Weiler SR,
Smith M, Keller JR. Inhibition of growth by
p205: a nuclear protein and putative tumor
suppressor expressed during myeloid cell dif-
ferentiation. Stem Cells. 2004;22(5):832–48. doi: 10.1634/stemcells.22-5-832. 34. Adler G. Effect of glucagon on the secretory
process in the rat exocrine pancreas. Cell Tis-
sue Res. 1977;182(2):193–204. doi: 10.1007/
bf00220588. 46. Дедов ИИ, Шестакова МВ, Майоров АЮ,
Викулова ОК, Галстян ГР, Кураева ТЛ, Пе-
теркова ВА, Смирнова ОМ, Старостина ЕГ,
Суркова ЕВ, Сухарева ОЮ, Токмакова АЮ,
Шамхалова МШ, Ярек-Мартынова ИР, Ар-
темова ЕВ, Бешлиева ДД, Бондаренко ОН,
Волеводз НН, Григорян ОР, Гомова ИС, Дже-
милова ЗН, Есаян РМ, Ибрагимова ЛИ, Ка-
лашников ВЮ, Кононенко ИВ, Лаптев ДН,
Липатов ДВ, Мотовилин ОГ, Никонова ТВ,
Роживанов РВ, Шестакова ЕА. Алгоритмы
специализированной медицинской помо-
щи больным сахарным диабетом. Под ред. И.И. Дедова, М.В. Шестаковой, А.Ю. Майо-
рова. 9-й вып. Сахарный диабет. 2019;22(S1):
1–144. doi: 10.14341/DM221S1. [Dedov II,
Shestakova MV, Mayorov AY, Vikulova OK, Gal-
styan GR, Kuraeva TL, Peterkova VA, Smirno-
va OM, Starostina EG, Surkova EV, Sukhare-
va OY, Tokmakova AY, Shamkhalova MS,
Jarek-Martynova IR, Artemova EV, Beshlie-
va DD, Bondarenko ON, Volevodz NN, Grigory-
an OR, Gomova IS, Dzhemilova ZN, Esayan RM,
Ibragimova LI, Kalashnikov VY, Kononenko IV,
Laptev DN, Lipatov DV, Motovilin OG, Nikono-
va TV, Rozhivanov RV, Shestakova EA. [Stand-
ards of specialized diabetes care]. Edited by
II Dedov, MV Shestakova, AYu Mayorov. 9th edi-
tion. Diabetes mellitus. 2019;22(S1):1–144. Russian. doi: 10.14341/DM221S1.] 35. Jarett L, Lacy PE. Effect of glucagon on the ac-
inar portion of the pancreas. Endocrinology. 1962;70:867–73. doi: 10.1210/endo-70-6-867. 23. Литература / References Pierzynowski SG, Podgurniak P, Mikołajczyk M,
Szczesny W. Insulin and the parasympathetic
dependence of pancreatic juice secretion in
healthy and alloxan diabetic sheep. Q J Exp
Physiol. 1986;71(3):401–7. doi: 10.1113/exp-
physiol.1986.sp002998. 36. Papp M, Fehér S, Varga B, Folly G. Humoral in-
fluences on local blood flow and external se-
cretion of the resting dog pancreas. Acta Med
Acad Sci Hung. 1977;34(4):185–8. 37. Dreiling DA, Janowitz HD, Haemmerli UP, Mar-
shall D. The effect of glucagon on the exocrine
pancreatic secretion of man. J Mt Sinai Hosp
N Y. 1958;25(3):240–3. 24. Yang YK, Zhu WY. [Effect of insulin on the func-
tion of pancreatic exocrine]. Sheng Li Xue Bao. 1995;47(3):238–44. Chinese. 25. Alvarez C, López MA. Effect of alloxan diabetes
on exocrine pancreatic secretion in the an-
esthetized rabbit. Int J Pancreatol. 1989;5(3):
229–38. doi: 10.1007/bf02924468. 38. Shaw HM, Heath TJ. The effect of glucagon on
the formation of pancreatic juice and bile in
the rat. Can J Physiol Pharmacol. 1973;51(1):
1–5. doi: 10.1139/y73-001. 26. Ferrer R, Medrano J, Diego M, Calpena R,
Graells L, Moltó M, Pérez T, Pérez F, Salido G. Effect of exogenous insulin and glucagon on
exocrine pancreatic secretion in rats in vivo. Int
J Pancreatol. 2000;28(1):67–75. doi: 10.1385/
IJGC:28:1:67. 39. Emoto T, Miyata M, Izukura M, Yumiba T, Mizu-
tani S, Sakamoto T, Matsuda H. Simultaneous
observation of endocrine and exocrine func-
tions of the pancreas responding to somato-
statin in man. Regul Pept. 1997;68(1):1–8. doi:
10.1016/s0167-0115(96)00125-5. 27. Logsdon CD. Stimulation of pancreatic ac-
inar cell growth by CCK, epidermal growth
factor, and insulin in vitro. Am J Physiol. 1986;251(4 Pt 1):G487–94. doi: 10.1152/ajp-
gi.1986.251.4.G487. 40. von der Ohe M, Layer P, Wollny C, Ensinck JW,
Peeters TL, Beglinger C, Goebell H. Somatosta-
tin 28 and coupling of human interdigestive
intestinal motility and pancreatic secretion. Gastroenterology. 1992;103(3):974–81. doi:
10.1016/0016-5085(92)90031-s. 28. Williams JA, Goldfine ID. The insulin-pancreatic
acinar axis. Diabetes. 1985;34(10):980–6. doi:
10.2337/diab.34.10.980. 41. Czakó L, Hegyi P, Rakonczay Z Jr, Wittmann T,
Otsuki M. Interactions between the endocrine
and exocrine pancreas and their clinical rele-
vance. Pancreatology. 2009;9(4):351–9. doi:
10.1159/000181169. 47. 47. Gao W, Zhou Y, Li Q, Zhou Q, Tan L, Song Y,
Zhao X, Yu M, Zheng S, Ye H, Zeng B, Lin Q,
Zhou J, Liu Y, Huang H, Zhang H, Hu X, Li Z,
Dai X, Chen R. Литература / References Hart PA, Bellin MD, Andersen DK, Bradley D,
Cruz-Monserrate Z, Forsmark CE, Goodarzi MO,
Habtezion A, Korc M, Kudva YC, Pandol SJ, Ya-
dav D, Chari ST; Consortium for the Study of
Chronic Pancreatitis, Diabetes, and Pancreatic
Cancer (CPDPC). Type 3c (pancreatogenic) di-
abetes mellitus secondary to chronic pancre-
atitis and pancreatic cancer. Lancet Gastroen-
terol Hepatol. 2016;1(3):226–37. doi: 10.1016/
S2468-1253(16)30106-6. 16. Murakami T, Hitomi S, Ohtsuka A, Tagu-
chi T, Fujita T. Pancreatic insulo-acinar por-
tal systems in humans, rats, and some
other mammals: scanning electron mi-
croscopy of vascular casts. Microsc Res
Tech. 1997;37(5–6):478–88. doi: 10.1002/
(SICI)1097-0029(19970601)37:5/6<478::AID-
JEMT10>3.0.CO;2-N. 11. Губергриц НБ, Беляева НВ. Экзо- и эндокрин-
ная функции поджелудочной железы: один
шаг от дуэта до дуэли. Сучасна гастроенте-
рологiя. 2006;30(4):18–30. [Gubergrits NB, Be-
lyayeva NV. [Exocrine and endocrine functions
of the pancreas: one step from duet to duel]. Modern Gastroenterology. 2006;30(4):18–30. Russian.] 17. Berg T, Johansen L, Brekke IB. Insulin potenti-
ates cholecystokinin (CCK)-induced secretion
of pancreatic kallikrein. Acta Physiol Scand. 1985;123(1):89–95. doi:
10.1111/j.1748-
1716.1985.tb07565.x. 18. Duan RD, Wicker C, Erlanson-Albertsson C. Ef-
fect of insulin administration on contents, se- 531 Ломакина Е.Ю., Таратина О.В., Белоусова Е.А. Хронический панкреатит и сахарный диабет: обзор литературы Альманах клинической медицины. 2019; 47 (6): 525–534. doi: 10.18786/2072-0505-2019-47-075 cretion, and synthesis of pancreatic lipase and
colipase in rats. Pancreas. 1991;6(5):595–602. doi: 10.1097/00006676-199109000-00014. 44. de-Madaria E, Abad-González A, Apari-
cio JR, Aparisi L, Boadas J, Boix E, de-Las-
Heras G, Domínguez-Muñoz E, Farré A,
Fernández-Cruz L, Gómez L, Iglesias-García J,
García-Malpartida K, Guarner L, Lariño-Noia J,
Lluís F, López A, Molero X, Moreno-Pérez O,
Navarro S, Palazón JM, Pérez-Mateo M, Sa-
bater L, Sastre Y, Vaquero EC, Martínez J. The
Spanish Pancreatic Club's recommendations
for the diagnosis and treatment of chron-
ic pancreatitis: part 2 (treatment). Pancre-
atology. 2013;13(1):18–28. doi: 10.1016/j. pan.2012.11.310. 31. Itoh H, Matsuyama T, Namba M, Watanabe N,
Komatsu R, Kono N, Tarui S. Effect of gluca-
gon-(1-21)-peptide
on
secretin-stimulated
pancreatic exocrine secretion in anesthe-
tized dogs. Life Sci. 1989;44(12):819–25. doi:
10.1016/0024-3205(89)90379-2. 19. Sofranková A. Effect of exogenous and en-
dogenous insulin on the secretory response
of the pancreas to the octapeptide of chole-
cystokinin (CCK8) in normal rats. Physiol Bohe-
moslov. 1984;33(5):391–8. 32. Harada H, Kochi F, Hanafusa E, Kobayashi T,
Oka H, Kimura I. Studies on the effect of gluca-
gon on human pancreatic secretion by analy-
sis of endoscopically obtained pure pancreatic
juice. Gastroenterol Jpn. 1985;20(1):28–36. doi:
10.1007/bf02774671. Литература / References Analysis of global gene expres-
sion profiles suggests a role of acute inflam-
mation in type 3C diabetes mellitus caused by
pancreatic ductal adenocarcinoma. Diabetolo-
gia. 2015;58(4):835–44. doi: 10.1007/s00125-
014-3481-8. 29. Keller J, Layer P. Acinar-islet interactions:
Pancreatic exocrine insufficiency in diabetes
mellitus. In: Johnson CD, Imrie CW, editors. Pancreatic disease. Basic science and clinical
management. Springer Verlag: London; 2004. p. 267–78. doi: 10.1007/978-1-85233-904-
3_21. 42. Henderson JR. Why are the islets of Langer-
hans? Lancet. 1969;2(7618):469–70. doi:
10.1016/s0140-6736(69)90171-8. 43. Ewald N, Bretzel RG. Diabetes mellitus sec-
ondary to pancreatic diseases (Type 3c) – are
we neglecting an important disease? Eur J In-
tern Med. 2013;24(3):203–6. doi: 10.1016/j. ejim.2012.12.017. 30. Biedzinski TM, Bataille D, Devaux MA, Sarles H. The effect of oxyntomodulin (glucagon-37)
and glucagon on exocrine pancreatic secre-
tion in the conscious rat. Peptides. 1987;8(6):
967–72. doi: 10.1016/0196-9781(87)90122-7. 48. Uc A, Andersen DK, Bellin MD, Bruce JI, Drew-
es AM, Engelhardt JF, Forsmark CE, Lerch MM,
Lowe ME, Neuschwander-Tetri BA, O'Keefe SJ,
Palermo TM, Pasricha P, Saluja AK, Singh VK, 532 Обзор Обзор Альманах клинической медицины. 2019; 47 (6): 525–534. doi: 10.18786/2072-0505-2019-47-075 66. Рекомендации Российской гастроэнтеро-
логической ассоциации по диагностике
и
лечению
хронического
панкреатита. Российский
журнал
гастроэнтерологии,
гепатологии, колопроктологии. 2013;23(1):
66–87. [Ivashkin VT, Maev IV, Okhlobystin AV,
Kucheryavyy YuA, Trukhmanov AS, Sheptu-
lin AA, Shifrin OS, Lapina TL, Osipenko MF,
Simanenkov VI, Khlynov IB, Alekseyenko SA,
Alekseyeva OP, Chikunova MV. [Guidelines of
the Russian gastroenterological association on
diagnostics and treatment of a chronic pan-
creatitis]. Russian Journal of Gastroenterology,
Hepatology, and Coloproctology. 2013;23(1):
66–87. Russian.] ra H, Otsuki M. High glucose activates rat pan-
creatic stellate cells through protein kinase C
and p38 mitogen-activated protein kinase
pathway. Pancreas. 2007;34(3):364–72. doi:
10.1097/MPA.0b013e31802f0531. Szigethy EM, Whitcomb DC, Yadav D, Con-
well DL. Chronic Pancreatitis in the 21st Centu-
ry – Research Challenges and Opportunities:
Summary of a National Institute of Diabetes
and Digestive and Kidney Diseases Workshop. Pancreas. 2016;45(10):1365–75. doi: 10.1097/
MPA.0000000000000713. 58. Hardt
PD,
Hauenschild
A,
Nalop
J,
Marzeion AM, Jaeger C, Teichmann J, Bret-
zel RG, Hollenhorst M, Kloer HU; S2453112/
S2453113 Study Group. High prevalence of
exocrine pancreatic insufficiency in diabetes
mellitus. A multicenter study screening fecal
elastase 1 concentrations in 1,021 diabetic pa-
tients. Pancreatology. 2003;3(5):395–402. doi:
10.1159/000073655. 49. Литература / References Magarini F, Albarello L, Alfieri S, Amodio A,
Andriulli A, Anti M, Arcidiacono P, Baiocchi L,
Balzano G, Benini L, Berretti D, Boraschi P, Bus-
carini E, Calculli L, Carroccio A, Campra D, Cele-
brano MR, Capurso G, Casadei R, Cavestro GM,
Chilovi F, Conigliaro R, Dall'Oglio L, De Ange-
lis C, De Boni M, De Pretis G, Di Priolo S, Di Se-
bastiano PL, Doglietto GB, Falconi M, Filauro M,
F i
i G F
ll
i L F i i A G i
E Gh
L 52. Andersen DK, Korc M, Petersen GM, Eibl G,
Li D, Rickels MR, Chari ST, Abbruzzese JL. Dia-
betes, Pancreatogenic Diabetes, and Pancre-
atic Cancer. Diabetes. 2017;66(5):1103–10. doi:
10.2337/db16-1477. 63. Taniguchi T, Okazaki K, Okamoto M, Seko S,
Tanaka J, Uchida K, Nagashima K, Kurose T,
Yamada Y, Chiba T, Seino Y. High prevalence of
autoantibodies against carbonic anhydrase II
and lactoferrin in type 1 diabetes: concept of
autoimmune exocrinopathy and endocrinop-
athy of the pancreas. Pancreas. 2003;27(1):26–
30. doi: 10.1097/00006676-200307000-00004. lis C, De Boni M, De Pretis G, Di Priolo S, Di Se-
bastiano PL, Doglietto GB, Falconi M, Filauro M, 53. Cui Y, Andersen DK. Pancreatogenic dia-
betes: special considerations for manage-
ment. Pancreatology. 2011;11(3):279–94. doi:
10.1159/000329188. Frieri G, Frulloni L, Fuini A, Gaia E, Ghezzo L, Gabbrielli A, Graziani R, Loriga P, Macarri G, Manes G, Manfredi R, Malesci A, Mariani A, 54. Ewald N, Kaufmann C, Raspe A, Kloer HU, Bret-
zel RG, Hardt PD. Prevalence of diabetes melli-
tus secondary to pancreatic diseases (type 3c). Diabetes Metab Res Rev. 2012;28(4):338–42. doi: 10.1002/dmrr.2260. Massucco P, Milani S, Mutignani M, Pasquali C, Pederzoli P, Pezzilli R, Pietrangeli M, Rocca R, Pederzoli P, Pezzilli R, Pietrangeli M, Rocca R,
Russello D, Siquini W, Traina M, Uomo G, Ven-
eroni L, Ventrucci M, Zilli M, Zamboni G. Italian
consensus guidelines for chronic pancreatitis. Dig Liver Dis. 2010;42 Suppl 6:S381–406. doi:
10.1016/S1590-8658(10)60682-2. Russello D, Siquini W, Traina M, Uomo G, Ven- 64. Chakraborty PP, Chowdhury S. A look inside
the pancreas: the “Endocrine-Exocrine Cross-
talk”. Endocrinol Metab Synd. 2015;4(1):160. doi: 10.4172/2161-1017.1000160. 55. Pan J, Xin L, Wang D, Liao Z, Lin JH, Li BR, Du TT,
Ye B, Zou WB, Chen H, Ji JT, Zheng ZH, Hu LH,
Li ZS. Risk Factors for diabetes mellitus in
chronic pancreatitis: a cohort of 2011 patients. Medicine (Baltimore). 2016;95(14):e3251. doi:
10.1097/MD.0000000000003251. 65. Литература / References Martínez J, Abad-González Á, Aparicio JR,
Aparisi L, Boadas J, Boix E, de las Heras G,
Domínguez-Muñoz
E,
Farré
A,
Fernán-
dez-Cruz L, Gómez L, Iglesias-García J,
García-Malpartida K, Guarner L, Lariño-Noia J,
Lluís F, López A, Molero X, Moreno-Pérez Ó,
Navarro S, Palazón JM, Pérez-Mateo M, Sabat-
er L, Sastre Y, Vaquero EC, De-Madaria E; Club
Español Pancreático (CEP). [Recommendations
of the Spanish Pancreatic Club on the diag-
nosis and treatment of chronic pancreatitis:
part 1 (diagnosis)]. Gastroenterol Hepatol. 2013;36(5):326–39. doi:
10.1016/j.gastro-
hep.2012.12.004. Spanish. 59. Ewald N, Raspe A, Kaufmann C, Bretzel RG,
Kloer HU, Hardt PD. Determinants of Exo-
crine Pancreatic Function as Measured by
Fecal Elastase-1 Concentrations (FEC) in Pa-
tients with Diabetes mellitus. Eur J Med Res. 2009;14(3):118–22. doi: 10.1186/2047-783x-
14-3-118. 67. Makuc J. Management of pancreatogenic dia-
betes: challenges and solutions. Diabetes Me-
tab Syndr Obes. 2016;9:311–5. doi: 10.2147/
DMSO.S99701. 68. Frulloni L, Falconi M, Gabbrielli A, Gaia E,
Graziani R, Pezzilli R, Uomo G, Andriulli A,
Balzano G, Benini L, Calculli L, Campra D,
Capurso G, Cavestro GM, De Angelis C, Ghez-
zo L, Manfredi R, Malesci A, Mariani A, Mutig-
nani M, Ventrucci M, Zamboni G, Amodio A,
Vantini I; Italian Association for the Study
of the Pancreas (AISP), Bassi C, Delle Fave G,
Frulloni L, Vantini I, Falconi M, Frulloni L, Gab-
brielli A, Graziani R, Pezzilli R, Capurso IV,
Cavestro GM, De Angelis C, Falconi M, Gaia E,
Ghezzo L, Gabbrielli A, Graziani R, Manfredi R,
Malesci A, Mariani A, Mutignani M, Pezzilli R,
Uomo G, Ventrucci M, Zamboni G, Vantini I,
Magarini F, Albarello L, Alfieri S, Amodio A,
Andriulli A, Anti M, Arcidiacono P, Baiocchi L,
Balzano G, Benini L, Berretti D, Boraschi P, Bus-
carini E, Calculli L, Carroccio A, Campra D, Cele-
brano MR, Capurso G, Casadei R, Cavestro GM,
Chilovi F, Conigliaro R, Dall'Oglio L, De Ange-
lis C, De Boni M, De Pretis G, Di Priolo S, Di Se-
bastiano PL, Doglietto GB, Falconi M, Filauro M,
Frieri G, Frulloni L, Fuini A, Gaia E, Ghezzo L,
Gabbrielli A, Graziani R, Loriga P, Macarri G,
Manes G, Manfredi R, Malesci A, Mariani A,
Massucco P, Milani S, Mutignani M, Pasquali C,
Pederzoli P, Pezzilli R, Pietrangeli M, Rocca R,
Russello D, Siquini W, Traina M, Uomo G, Ven-
eroni L, Ventrucci M, Zilli M, Zamboni G. Italian
consensus guidelines for chronic pancreatitis. Dig Liver Dis. Литература / References 2010;42 Suppl 6:S381–406. doi:
10.1016/S1590-8658(10)60682-2. 68. Frulloni L, Falconi M, Gabbrielli A, Gaia E,
Graziani R, Pezzilli R, Uomo G, Andriulli A,
Balzano G, Benini L, Calculli L, Campra D,
Capurso G, Cavestro GM, De Angelis C, Ghez-
zo L, Manfredi R, Malesci A, Mariani A, Mutig-
nani M, Ventrucci M, Zamboni G, Amodio A,
Vantini I; Italian Association for the Study
of the Pancreas (AISP), Bassi C, Delle Fave G,
Frulloni L, Vantini I, Falconi M, Frulloni L, Gab-
brielli A, Graziani R, Pezzilli R, Capurso IV,
Cavestro GM, De Angelis C, Falconi M, Gaia E,
Ghezzo L, Gabbrielli A, Graziani R, Manfredi R,
Malesci A, Mariani A, Mutignani M, Pezzilli R,
Uomo G Ventrucci M Zamboni G Vantini I 50. Губергриц НБ, Лукашевич ГМ, Голубова ОА,
Беляева НВ, Загоренко ЮА. Панкреатоген-
ный сахарный диабет. Российский журнал
гастроэнтерологии, гепатологии и коло-
проктологии. 2007;17(6):11–6. [Gubergrits NB,
Lukashevich GM, Golubova OA, Belyaeva YA,
Zagorenko YuA. [Pancreatogenic diabetes
mellitus]. Russian Journal of Gastroenterology,
Hepatology, Coloproctology. 2007;17(6):11–6. Russian.] 60. Terzin V, Várkonyi T, Szabolcs A, Lengyel C,
Takács T, Zsóri G, Stájer A, Palkó A, Wittmann T,
Pálinkás A, Czakó L. Prevalence of exocrine
pancreatic insufficiency in type 2 diabetes
mellitus with poor glycemic control. Pancre-
atology. 2014;14(5):356–60. doi: 10.1016/j. pan.2014.07.004. 61. Young AA, Cooper GJ, Carlo P, Rink TJ, Wang MW. Response to intravenous injections of amylin
and glucagon in fasted, fed, and hypoglycemic
rats. Am J Physiol. 1993;264(6 Pt 1):E943–50. doi: 10.1152/ajpendo.1993.264.6.E943. Cavestro GM, De Angelis C, Falconi M, Gaia E, Ghezzo L, Gabbrielli A, Graziani R, Manfredi R, 51. American Diabetes Association. Diagnosis and
classification of diabetes mellitus. Diabetes
Care. 2014;37 Suppl 1:S81–90. doi: 10.2337/
dc14-S081. Malesci A, Mariani A, Mutignani M, Pezzilli R, Uomo G, Ventrucci M, Zamboni G, Vantini I, 62. Panicot L, Mas E, Thivolet C, Lombardo D. Cir-
culating antibodies against an exocrine pan-
creatic enzyme in type 1 diabetes. Diabetes. 1999;48(12):2316–23. doi:
10.2337/diabe-
tes.48.12.2316. E.Yu. Lomakina1 • O.V. Taratina1 • E.A. Belousova1 E.Yu. Lomakina1 • O.V. Taratina1 • E.A. Belousova1 Background: For a long time there has been
a discussion about how chronic pancreatitis (CP)
and diabetes mellitus (DM) are related to each
other. If a patient has both conditions, should
they be viewed as two separate disorders, or one
of them is a plausible consequence of the other? If the latter is true, what are pathophysiological
mechanisms of DM in CP? Current consensus
documents by specialists in pancreatic diseases
pay little attention to this issue, and their main
statements have low level of evidence. The
Russian consensus on the diagnosis and treat-
ment of CP (2016) contains no statements on
DM. In the Russian guidelines and consensus
documents to be developed, it is necessary to
include provisions on the pancreatogenic DM
as an independent “other type DM’, with con-
sideration of its pathophysiological mechanisms
and clinical particulars. Aim: To characterize
the state-of-the-art in pancreatogenic DM, to
demonstrate its differences from DM types 1 and
2 from pathogenetic and clinical perspectives. Methods: The review is based on the results
of meta-analyses, systematic reviews and main
provisions of the existing clinical guidelines and
consensus documents available from PubMed
and E-library. Results: According to various
sources, Type 3c DM, or latent impaired glucose
tolerance in CP, can eventually develop in 25 to
80% patients with CP. Impaired glucose toler-
ance is found in 40 to 60% of patients with acute
pancreatitis, with persistent hyperglycemia after
acute episode seen in 15 to 18% of the patients. Exocrine pancreatic insufficiency is commonly
seen in Type 1 and Type 2 diabetic patients, al-
though the data on its prevalence are highly con-
tradictory indicating a lack of knowledge in the
field. Type 3c DM is characterized by its manifes-
tation at later stages of CP, concomitant excreto-
ry deficiency of the pancreas, brittle course with
proneness to hypoglycemia and no ketoacidosis. Ekaterina Yu. Lomakina – MD, Junior Research
Fellow, Department of Gastroenterology1
*
* 61/2 Shchepkina ul., Moscow, 129110, Russian
Federation. Tel.: +7 (926) 525 93 97. E-mail: kate-den@bk.ru The highest risk group includes patients with
longstanding CP, previous partial pancreatic re-
section and patients with early calcifying pan-
creatitis, mainly of the alcoholic origin. Литература / References Воевода МИ, Иванова АА, Шахтшнейдер ЕВ,
Овсянникова АК, Михайлова СВ, Астра-
кова КС, Воевода СМ, Рымар ОД. Молеку-
лярная генетика MODY. Терапевтический
архив. 2016;(4):117–24. doi: 10.17116/ter-
arkh2016884117-124. [Voevoda MI, Ivano-
va AA, Shakhtshneider EV, Ovsyannikova AK,
Mikhailova SV, Astrakova KS, Voevoda SM, Ry-
mar OD. [Molecular genetics of maturity-onset
diabetes of the young]. Terapevticheskiy arkh-
iv. 2016;(4):117–24. Russian. doi: 10.17116/ter-
arkh2016884117-124.] 69. Cui Y, Andersen DK. Diabetes and pancreatic
cancer. Endocr Relat Cancer. 2012;19(5):F9–26. doi: 10.1530/ERC-12-0105. 70. Meier JJ, Giese A. Diabetes associated with
pancreatic
diseases. Curr
Opin
Gastro-
enterol. 2015;31(5):400–6. doi:
10.1097/
MOG.0000000000000199. 56. Watanabe S, Nagashio Y, Asaumi H, Nomi-
yama Y, Taguchi M, Tashiro M, Kihara Y, Naka-
mura H, Otsuki M. Pressure activates rat pan-
creatic stellate cells. Am J Physiol Gastrointest
Liver Physiol. 2004;287(6):G1175–81. doi:
10.1152/ajpgi.00339.2004. 71. Gudipaty L, Rickels MR. Pancreatogenic (type
3c) diabetes. Pancreapedia: Exocrine Pan-
creas Knowledge Base. 2015. doi: 10.3998/
panc.2015.35. 57. Nomiyama Y, Tashiro M, Yamaguchi T,
Watanabe S, Taguchi M, Asaumi H, Nakamu- 533 Ломакина Е.Ю., Таратина О.В., Белоусова Е.А. Хронический панкреатит и сахарный диабет: обзор литературы Almanac of Clinical Medicine. 2019; 47 (6): 525–534. doi: 10.18786/2072-0505-2019-47-075 1 Moscow Regional Research and Clinical Institute
(MONIKI); 61/2 Shchepkina ul., Moscow, 129110,
Russian Federation E.Yu. Lomakina1 • O.V. Taratina1 • E.A. Belousova1 Optimal
and rational medical treatment of pancreato-
genic DM still remains disputable, while the
evidence base of the efficacy and safety of var-
ious anti-diabetic agents in this disease is lack-
ing, and no consensus on the issue has been yet
reached. General treatment guidelines given in
a number of international consensus documents
are limited to cautious insulin administration. Conclusion: Pancreatogenic DM differs from
Type 1 and Type 2 DM in a number of aspects,
namely, mechanisms of hyperglycemia, hormon-
al profiles, clinical particulars and treatment ap-
proaches. Endocrine pancreatic insufficiency in
CP is caused by secondary inflammatory injury of
the pancreatic islets. The key to specifics of Type
3c DM lies in anatomical and physiological in-
terplay of the exocrine and endocrine compart-
ments of the pancreas. At presents, most provi-
sions on pancreatogenic DM are empirical and
seem to be rather declarative, because intrinsic
mechanisms of this type of diabetes and more-
over its pathogenetically based treatment have
been poorly studied. Nevertheless, all patients
with CP or other pancreatic diseases should be
assessed for pancreatogenic DM. Olesya V. Taratina – MD, PhD, Research Fellow,
Department of Gastroenterology; Associate
Professor, Chair of Gastroenterology, Postgraduate
Training Faculty1; ORCID: https://orcid.org/0000-
0001-6112-6609. E-mail: taratina.o@gmail.com Elena A. Belousova – MD, PhD, Professor, Head of
Department of Gastroenterology, Head of Chair of
Gastroenterology, Postgraduate Training Faculty1;
ORCID: https://orcid.org/0000-0003-4523-3337. E-mail: eabelous@yandex.ru Key words: chronic pancreatitis, diabetes melli-
tus, pancreatogenic diabetes mellitus Key words: chronic pancreatitis, diabetes melli-
tus, pancreatogenic diabetes mellitus For citation: Lomakina EYu, Taratina OV, Belousova EA. Chronic pancreatitis and diabetes mellitus: a review
of the literature. Almanac of Clinical Medicine. 2019;47(6):525–34. doi: 10.18786/2072-0505-2019-47-
075. Received 11 November 2019; revised 4 December
2019; accepted 5 December 2019; published
16 December 2019 Received 11 November 2019; revised 4 December 2019; accepted 5 December 2019; published
16 December 2019 2019; accepted 5 December 2019; published
16 December 2019 Funding The study was performed with financial support from MONIKI from the research budget. Conflict of interests Conflict of interests The authors declare no conflict of interests. Authors' contributions E.Yu. Lomakina, literature search and analysis, analysis and interpretation of the results, text writing; O.V. Taratina, analysis
of the results, text writing and editing; E.A. Belousova, the paper concept and design, text editing, approval of the final
version of the manuscript. All the authors have contributed significantly to the study conduct and preparation of the paper,
have read and approved its final version before the publication. 534
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Correspondence: Marie Dumont (marie.dumont@meteo.fr) Correspondence: Marie Dumont (marie.dumont@meteo.fr) Received: 30 December 2020 – Discussion started: 9 March 2021
Revised: 5 October 2021 – Accepted: 11 October 2021 – Published: 30 November 2021 Received: 30 December 2020 – Discussion started: 9 March 2021
Revised: 5 October 2021 – Accepted: 11 October 2021 – Published: 30 November 2021 Abstract. In climate models, the snow albedo scheme gen-
erally calculates only a narrowband or broadband albedo,
which leads to significant uncertainties. Here, we present
the Versatile ALbedo calculation metHod based on spec-
trALLy fixed radiative vAriables (VALHALLA version 1.0)
to optimize spectral snow albedo calculation. For this op-
timization, the energy absorbed by the snowpack is cal-
culated by the spectral albedo model Two-streAm Radia-
tive TransfEr in Snow (TARTES) and the spectral irradi-
ance model Santa Barbara DISORT Atmospheric Radiative
Transfer (SBDART). This calculation takes into account the
spectral characteristics of the incident radiation and the op-
tical properties of the snow based on an analytical approx-
imation of the radiative transfer of snow. For this method,
30 wavelengths, called tie points (TPs), and 16 reference ir-
radiance profiles are calculated to incorporate the absorbed
energy and the reference irradiance. The absorbed energy is
then interpolated for each wavelength between two TPs with
adequate kernel functions derived from radiative transfer the-
ory for snow and the atmosphere. We show that the accuracy
of the absorbed energy calculation primarily depends on the
adaptation of the irradiance of the reference profile to that
of the simulation (absolute difference < 1 W m−2 for broad-
band absorbed energy and absolute difference < 0.005 for
broadband albedo). In addition to the performance in terms
of accuracy and calculation time, the method is adaptable to
any atmospheric input (broadband, narrowband) and is easily
adaptable for integration into a radiative scheme of a global
or regional climate model. 1
Introduction Solar irradiance is an essential source of energy to snow and
ice surfaces (Warren, 1982). Absorption of shortwave radia-
tion strongly depends upon the physical properties of snow
and atmospheric conditions. The albedo, defined as the frac-
tion of reflected solar radiation, is very high for fresh snow
and limits energy absorption by the snowpack. Darker or
old snow and glacial ice absorb more solar energy (Warren,
1982; Gardner and Sharp, 2010). The snowpack may also
contain light-absorbing particles (LAPs; McKenzie Skiles
and Painter, 2018), leading to a decrease in albedo (Warren,
1982; Picard et al., 2009; Gardner and Sharp, 2010; Libois
et al., 2013; Dumont et al., 2014). Also, the optical proper-
ties of snow and ice strongly vary with the wavelength (e.g. ice refraction index of Warren and Brandt, 2008). The snow
spectral albedo, defined as the fraction between reflected
and incident solar energy for a given wavelength (Grenfell
et al., 1994), is higher for near-ultraviolet (near-UV, 300–
400 nm), visible (400–700 nm), and near-infrared (near-IR,
750–1400 nm) spectra but is lower in the IR part of the so-
lar spectrum (Warren, 1982; Gardner and Sharp, 2010). The
changes in albedo with snow and ice properties play a major
role in the melt–albedo feedback (Cess et al., 1991). An in-
crease in temperature favours rapid metamorphism and melt-
ing of the snow cover, which leads to coarser snow grains and
a less reflective surface. More energy is then absorbed and
made available for heating the snowpack, enhancing snow
metamorphism and melting (e.g. Flanner and Zender, 2006). The solar zenith angle (SZA; valid for direct radiation) and Geosci. Model Dev., 14, 7329–7343, 2021
https://doi.org/10.5194/gmd-14-7329-2021
© Author(s) 2021. This work is distributed under
the Creative Commons Attribution 4.0 License. Geosci. Model Dev., 14, 7329–7343, 2021
https://doi.org/10.5194/gmd-14-7329-2021
© Author(s) 2021. This work is distributed under
the Creative Commons Attribution 4.0 License. A versatile method for computing optimized snow albedo from
spectrally fixed radiative variables: VALHALLA v1.0
Florent Veillon1,2, Marie Dumont2, Charles Amory1,3, and Mathieu Fructus2
1Laboratory of Climatology, Department of Geography, SPHERES, University of Liège, Liège, Belgium
2Université Grenoble Alpes, Université de Toulouse, Météo-France, CNRS, CNRM,
Centre d’Etudes de la Neige, 38000 Grenoble, France
3U i
i é G
bl Al
CNRS I
i
d
Gé
i
d l’E
i
38000 G
bl
F Florent Veillon1,2, Marie Dumont2, Charles Amory1,3, and Mathieu Fructus2
1Laboratory of Climatology, Department of Geography, SPHERES, University of Liège, Liège, Belgium
2Université Grenoble Alpes, Université de Toulouse, Météo-France, CNRS, CNRM,
Centre d’Etudes de la Neige, 38000 Grenoble, France
3 g
3Université Grenoble Alpes, CNRS, Institut des Géosciences de l’Environnement, 38000 Grenoble, France F. Veillon et al.: VALHALLA v1.0 The sensitivity of the albedo calculations
to the atmospheric and snow properties is also assessed. The
results are compared with reference albedo calculations at
different spectral resolutions and with other existing method-
ologies (van Dalum et al., 2019; Gardner and Sharp, 2010). Implementation considerations in climate and land models
are finally discussed. In addition to the above-mentioned requirements, accuracy
in the estimation of the energy absorbed at the snow surface
can be achieved through spectral calculation of the albedo
but remains numerically expensive. This also requires spec-
tral calculations of the solar irradiance that are not available
most of the time in climate models. This is usually over-
come in most global and regional climate models by com-
puting broadband or narrowband albedo to estimate the en-
ergy budget at the snow and ice surfaces (Gardner and Sharp,
2010; Kuipers Munneke et al., 2011). The broadband albedo
is defined as the ratio between total reflected and total in-
cident solar energy integrated across the entire solar spec-
trum, whereas the narrowband albedo is integrated over a
limited range of the solar spectrum. These integrations, how-
ever, lead to a bias in the calculation of the snowpack albedo,
which ultimately propagates in the computation of the sur-
face energy and mass budgets. gy
g
To overcome these uncertainties while maintaining an ad-
equate calculation time to remain competitive, new meth-
ods are developed. One of them, recently developed by
van Dalum et al. (2019), effectively couples a snow spec-
tral albedo model with a narrowband atmospheric radiation
scheme. This method (Spectral-to-NarrOWBand ALbedo
module; SNOWBAL) allows the coupling of the radiative
transfer model TARTES (Two-streAm Radiative TransfEr
in Snow, Libois et al., 2013) with the European Centre
for Medium-Range Weather Forecasts (ECMWF) radiation
McRad scheme based on the shortwave Rapid Radiation
Transfer Model (RRTMsw) embedded in RACMO2 (Mlawer
et al., 1997; Clough et al., 2005; Morcrette et al., 2008;
ECMWF, 2009). They used the first 12 of the 14 prede-
fined representative wavelengths (RWs, for every 14 bands of
RRTMsw) dependent on irradiance distribution and albedo
within a spectral band to calculate the narrowband albedo
and radiation absorption in each (sub)surface snow layer. To
determine the 12 RWs, a limited number of properties of the
atmosphere are selected using a look-up table (LUT). They
demonstrate that RWs primarily depend on the SZA, cloud
content, and water vapour. F. Veillon et al.: VALHALLA v1.0 atmospheric conditions (e.g. clouds, aerosols loads, water
vapour column) determine the amount of energy reaching the
surface of the snowpack. For example, clouds influence the
proportion of solar radiation reaching the surface and con-
tribute to total incident radiation by emitting longwave radi-
ation (Wetherald and Manabe, 1988; Schneider et al., 2019). For realistic estimates of the energy balance and melt over
snow and ice surfaces, accurate knowledge of a set of atmo-
spheric and snowpack properties is thus required (Picard et
al., 2012). snow and ice. The impact of snow properties on the RWs
is not accounted for in the LUTs since it is negligible in the
case tested in van Dalum et al. (2019). This might be a lim-
itation when the narrowbands of the atmospheric model are
too large. The use of this method with a model other than
RACMO2 will require a recalculation of the LUTs for a dif-
ferent set of narrowbands. The accuracy of the method is also
expected to increase if more narrowbands are available, re-
ducing the sub-band spectral variability. Here, we describe a novel method for accurately calcu-
lating the solar energy absorbed by the snowpack based on
the determination of spectrally fixed radiative variables. The
method is named VALHALLA for Versatile ALbedo calcula-
tion metHod based on spectrALLy fixed radiative vAriables
(version 1.0). This method maintains adequate accuracy of
absorbed energy values while reducing calculation time irre-
spective of the radiative transfer scheme used for the atmo-
sphere. While VALHALLA like SNOWBAL is a coupling
scheme, VALHALLA fulfils a different niche than SNOW-
BAL since it allows accurate calculation when only broad-
band atmospheric inputs are available and accounts for snow
property variations. SNOWBAL requires accurate snow ra-
diative transfer calculations for a limited number of wave-
lengths and an adequate representation of the atmosphere,
i.e. cloud content, water vapour, SZA, direct-to-diffuse irra-
diance ratio. VALHALLA requires accurate radiative trans-
fer calculations for both snow and the atmosphere for a lim-
ited number of wavelengths. The proposed method takes ad-
vantage of the spectral characteristics of incident radiation
and optical snow properties based on the analytical approx-
imation of the radiative transfer within the snowpack pro-
vided by Kokhanovsky and Zege (2004). The accuracy of the
methods is assessed using accurate calculation at a spectral
resolution of 1 nm. F. Veillon et al.: VALHALLA v1.0 This method is tested on differ-
ent types of snow and for clear-sky and cloudy atmospheric
conditions, and it represents broadband snow albedo with
low uncertainties (< 0.01). In van Dalum et al. (2020, 2021),
the SNOWBAL module is evaluated in RACMO2 over the
Greenland ice sheet. This method can therefore be used on
large surfaces while accurately representing the albedo of Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. 7330 2.2.1
Theoretical basis The
spectral
direct
albedo,
also
called
directional–
hemispherical reflectance, r, of a homogeneous, optically
infinite snowpack can be approximated by the following
relationship (Libois et al., 2013; Dumont et al., 2017;
Kokhanovsky et al., 2018): r(λ) = exp
−u(µ0)
s
64π
3ρiceSSA(1 −g)
2n(λ)B
λ
+ 3 ρice
ρLAP
cLAPCLAP
abs (λ)
! ,
(2) (2) where u(µ0) = 3
7(1 + 2µ0); µ0 is the cosine of the solar
zenith angle, n(λ) is the imaginary part of the ice refractive
index at the wavelength λ, cLAP is the light-absorbing parti-
cle concentration, ρLAP is the volumetric mass of the light-
absorbing particles, and CLAP
abs (λ) is the absorption cross sec-
tion of LAPs. SSA =
S
V ρice
,
(1) SSA =
S
V ρice
, (1) When neglecting the spectral variations due to the pres-
ence of LAPs in Eq. (2), the first-order spectral albedo varia-
tions can be approximated as with ρice, the volumetric mass of ice (917 kg m−3). We used two shape parameters that are relevant for the
optical properties of snow: the asymmetry parameter g (di-
mensionless) and the absorption enhancement parameter B
(dimensionless; Libois et al., 2013). g quantifies the amount
of radiation that is scattered forward for a snow grain, and
B quantifies the lengthening of photon paths inside a snow
grain due to internal multiple reflections. r(λ) ∼exp
−J
r
n(λ)
λ
! ,
(3) (3) where J = u(µ0)
q
128π
3ρiceSSA(1−g)B. J is constant with λ and
depends only on SZA and snow physical properties. where J = u(µ0)
q
128π
3ρiceSSA(1−g)B. J is constant with λ and
depends only on SZA and snow physical properties. In Tuzet et al. (2017) and later studies, TARTES was used
for calculations of radiative transfer with a spectral resolution
of 20 nm. This resolution is the best compromise between the
accuracy of radiation and calculation time, which is still very
important, and makes this model configuration computation-
ally expensive. The fraction of absorbed energy in the snowpack with re-
spect to the incoming energy, fp, is thus related to the spec-
tral albedo by the following relationship: (4) fp(λ) = 1 −r(λ). (4) For the atmosphere, we use the Beer–Lambert law to ex-
press the first-order spectral variations of the incoming solar
radiation. 2.2.1
Theoretical basis The Beer–Lambert law establishes a relationship
between the radiation transmitted through a given medium I
and the incident irradiance I0 at the wavelength λ. Let L be
the thickness of the media and ca the absorption coefficient. Then, F. Veillon et al.: VALHALLA v1.0 atmospheric layers and 40 radiation streams. The main in-
put parameters used in this study are the aerosol optical
depth (AOD), the cloud layer optical depth (τ), the bound-
ary layer aerosol type selector (IAER), and SZA. With these
parameters, SBDART calculated direct and diffuse irradiance
(W m−2) for each wavelength between 0.320 and 4.000 µm. This atmospheric radiative transfer model was chosen since
it provides accurate simulations of solar irradiance in snow-
covered areas (e.g. Tuzet et al., 2020) and offers a large num-
ber of parameters to set for the atmospheric properties. calculation of the absorbed radiation at the tie points can
be performed with any radiative transfer model. In the fol-
lowing, we selected the Two-streAm Radiative TransfEr in
Snow (TARTES; Libois et al., 2013) for the snowpack and
the Santa Barbara DISORT Atmospheric Radiative Transfer
(SBDART; Ricchiazzi et al., 1998) for the atmosphere. 2.1.1
Radiative transfer in snow, TARTES TARTES calculates spectral albedo in a multilayer snowpack
when the physical properties of each layer and the angu-
lar and spectral characteristics of the radiation are known
(Libois et al., 2014), and it is embedded in the detailed
snowpack model Crocus (e.g. Tuzet et al., 2017). TARTES
is based on the Kokhanovsky and Zege (2004) formalism
for weakly absorbing media to describe the single-scattering
properties of each layer and the delta-Eddington (Joseph et
al., 1976) approximation to solve the radiative transfer equa-
tion. TARTES represents the snowpack as a stack of hori-
zontal homogeneous layers. For calculation, physical proper-
ties of the snowpack (e.g. grain radius, grain shape, density,
thickness, type of LAPs, LAP concentration) and SZA for di-
rect radiation (for diffuse radiation, SZA is fixed at 53◦) are
used as inputs. The grain size is characterized by the snow
specific surface area (SSA; expressed in m2 kg−1, defined as
the ratio between the surface of the air–ice interface S and
the ice mass volume V ): 2
Method The VALHALLA method relies on accurate calculations of
the solar radiation absorbed by the snowpack for a small
number of selected wavelengths, named tie points (TPs) in
the following. The number of tie points is kept as small as
possible to limit the computing resources. Between these tie
points, the VALHALLA method interpolates the absorbed
radiation based on kernel functions that reflect the main
spectral variations of the absorbed radiation across the so-
lar spectrum. The general reasoning of the method consists
of assuming that the spectral variation between tie points
can be approximated using the refractive index of ice. The Geosci. Model Dev., 14, 7329–7343, 2021 https://doi.org/10.5194/gmd-14-7329-2021 7331 F. Veillon et al.: VALHALLA v1.0 F. Veillon et al.: VALHALLA v1.0 F. Veillon et al.: VALHALLA v1.0 and ca(λ) varies with the atmospheric profile, i.e. with the
aerosols properties, the properties of gas in the atmosphere
(water vapour, ozone, etc.), the solar zenith angle and the
cloud properties. Here we assume that the spectral variations
of the solar irradiance at the surface can be written as the broadband incident radiation is only available from the
atmospheric model. For each TP, the absorbed energy and irradiance are calcu-
lated using TARTES–SBDART and used for determining the
values of variables D and J. Between two tie points TPn and TPn+1, we assume that
the absorbed energy can be approximated by I(λ) ∼Eref(λ)exp
−D
r
n(λ)
λ
! ,
(6) (6) Eabs
λTPn+1
TPn
where Eref is the total incident energy at the wavelength λ
for a reference atmospheric profile and D is constant with
wavelength. In other words, this means that, for instance, for
a given location, we assume that changes in the atmospheric
solar irradiance with time can be modelled using Eq. (6); i.e. main spectral changes are driven by the water vapour (as-
suming that the refractive index of water vapour is close to
n(λ)).
= Cbi+1
bi
Eref
λTPn+1
TPn
exp
−DTPn+1
TPn
v
u
u
u
t
n
λTPn+1
TPn
λTPn+1
TPn
1 −exp
−J TPn+1
TPn
v
u
u
u
t
n
λTPn+1
TPn
λTPn+1
TPn
. (9) (9) As a consequence, we assume that the absorbed energy by
a snowpack for a given wavelength Eabs(λ) can be approxi-
mated by To determine these variables, which take into account all
snow and illumination properties, an optimization by the
least-square method is used. Indeed, D and J are mutually
dependent. Eabs(λ) ∼Eref(λ)exp
−D
r
n(λ)
λ
! fp(λ). (7) (7) In the context of optimization, variable D is written as 2.2.2
Interpolation method GTPn+1
TPn
= exp
−DTPn+1
TPn
s
n(TPn)
TPn
! ,
(10) The VALHALLA method is based on precise calculation of
the absorbed energy at the tie points (TPs) and on the interpo-
lation between these wavelengths based on the general shape
of the spectrum given in the equation above (Eq. 7). The
method calculates the absorbed energy since this is the vari-
able directly used in the energy budget of the snowpack. The
snow albedo can be directly diagnosed from the absorbed en-
ergy (e.g. Eq. 4). (10) with DTPn+1
TPn
= −log
GTPn+1
TPn
s
TPn
n(TPn),
(11) (11) The method uses a reference irradiance profile with a spec-
tral resolution of 1 nm, Eref. For each SZA value (varying be-
tween 10 and 80◦), a reference irradiance profile is calculated
with SBDART. In total, 16 reference profiles were used (one
set for clear-sky and partially cloudy conditions and the other
one for full overcast conditions; see Sect. 2.5.2). These pro-
files are used to calculate a coefficient C between the broad-
band reference irradiance Eref (integral of the reference pro-
file) and narrow or broadband irradiance Eexp,i given by an
atmospheric model for each narrow or broad spectral band i: and J is J TPn+1
TPn
= −log
1 −
EabsTPn
Cbi+1
bi
ErefTPnGTPn+1
TPn
s
TPn
n(TPn). (12) (12) The optimization is realized on the variable GTPn+1
TPn
and
uses absorbed energy Eabs and total irradiance Eref for
TPn+1. 1TPn+1
TPn
= EabsTPn+1 −Cbi+1
bi
ErefTPn+1GTPn+1
TPn
1 −exp Cbi+1
bi
=
Eexp,i
R bi+1
bi
Eref(λ)dλ
,
(8) (8) −J TPn+1
TPn
s
n(T Pn+1)
T Pn+1
!
(13) (13) where bi and bi+1 are the max and min wavelengths of the
bands in which the atmospheric model is providing the so-
lar incident radiation. Cbi+1
bi
thus represents the scaling factor
between the incident radiation provided by the atmospheric
model and the reference irradiance for each narrow or broad
spectral band of the atmospheric model. In the following, we
use bi = 320 nm and bi+1 = 4000 nm. Thus, we assume that Namely, an optimization method is used to solve Eq. (9). The optimization algorithm finds the value of GTPn+1
TPn
for
which 1TPn+1
TPn
is the closest to zero, with 1TPn+1
TPn
being the
difference between the left and right sides of Eq. (9). https://doi.org/10.5194/gmd-14-7329-2021 Geosci. 2.1.2
Radiative transfer in the atmosphere, SBDART The model Santa Barbara DISORT Atmospheric Radiative
Transfer (SBDART; Ricchiazzi et al., 1998) is used for ra-
diative transfer calculation in clear-sky and cloudy condi-
tions in the atmosphere. SBDART uses discrete ordinate ra-
diative transfer (DISORT; Stamnes et al., 1988) to solve the
radiative transfer equation in the atmosphere vertically ho-
mogeneously. This model is organized to permit up to 65 I(λ) = I0(λ)exp(−ca(λ)L),
(5) (5) https://doi.org/10.5194/gmd-14-7329-2021 Geosci. Model Dev., 14, 7329–7343, 2021 7332 https://doi.org/10.5194/gmd-14-7329-2021 2.2.3
Numerical settings selected after a principal component analysis of the spectral
absorbed energy. The principal component analysis aimed at
obtaining a list of representative parameters with the most
pronounced influence on the absorbed energy spectrum. For
each value of the identified parameter and for each wave-
length between 320 and 4000 nm, an irradiance profile is cal-
culated. 2.2.2
Interpolation method Model Dev., 14, 7329–7343, 2021 F. Veillon et al.: VALHALLA v1.0 7333 Figure 1. Spectral positions of TPs on an example of an absorbed energy profile for a snowpack without LAPs. Figure 1. Spectral positions of TPs on an example of an absorbed energy profile for a snowpack without LAP Figure 1. Spectral positions of TPs on an example of an absorbed energy profile for a snowpack without LAPs. Selection of the tie points The tie points, TPs, are the reference wavelengths for ab-
sorbed energy and total irradiance. For all types of snow and
cloud cover, a total of 30 TPs are selected as a compromise
between accuracy and computational time (Fig. 1). The TP
has been selected as the local maxima and minima of ab-
sorbed energy after several optimization tests (not shown). Reference irradiance profiles To account for a representative set of atmospheric conditions,
different reference irradiance profiles depending on SZA and
cloud cover are chosen. These profiles are calculated by SB-
DART simulations with a spectral resolution of 1 nm for two
cloud cover types. For simulations of clear-sky and partly
cloudy conditions, reference irradiance profiles with values
of τ equal to 0.5 are calculated. For simulations of full over-
cast conditions, these profiles are calculated with a value of
τ equal to 10 (Table 1). TARTES settings The main TARTES input parameters used in this study are
the surface specific area (SSA) of the first layer of the snow-
pack, the snow water equivalent (SWE) for each layer of the
snowpack, and the LAP concentration. We consider a snow-
pack with three layers of varying thickness and density (Ta-
ble 3). These layers represent at most the first 20 cm of the
snowpack, whose physical properties largely determine the
albedo of the snow. The principal parameters for albedo cal-
culation are the SSA of the first layer, the SWE of the first
layers, and the LAP concentration of two first layers of the
snowpack. We selected a wide range of SSA values (2 to
155 m2 kg−1) in order to cover most of the snow types found
on Earth (Domine et al., 2007). SWE gives the mass of snow
and is the product between thickness (t) and density (d). For
pure snow (without LAPs), the SWE values are provided for
the first three layers of the snowpack. For snow with LAPs,
the SWE and LAP concentration (for soot and dust) are pro-
vided for the first two layers of the snowpack. For layers 3
and 4, the values of all input parameters are fixed. The ranges
of LAP content for soot and dust have been selected to cover F. Veillon et al.: VALHALLA v1.0 Table 1. Atmospheric parameters of reference irradiance profiles. Each irradiance profile is calculated for eight values of SZA and two values
of τ. The other parameters are fixed in the SBDART model. Table 1. Atmospheric parameters of reference irradiance profiles. Each irradiance profile is calculated for eight values of SZA and two values
of τ. The other parameters are fixed in the SBDART model. Cloud cover conditions
Clear-sky and partially cloudy
Full overcast
Solar zenith angle (◦, SZA)
10; 20; 30; 40; 50; 60; 70; 80
Boundary layer aerosol type (IAER)
2 – urban
Aerosol optical depth (AOD)
0.07
Cloud layer optical depth (τ)
0.5
10
Integrated ozone concentration (atm-cm)
0.3
Integrated water vapour amount (g cm−2)
0.35
Surface altitude (km)
2.1
Optical depth of each stratospheric aerosol layer
0.013
Atmospheric profile
3 – mid-latitude winter Clear-sky and partially cloudy
Full overcast Table 2. Atmospheric parameters of simulations. Each irradiance profile is calculated for eight values of the SZA, five values of IAER,
three values of the AOD, and five values of τ (one for clear-sky conditions, three for partially cloudy conditions, and one for full overcast
conditions). The other parameters are fixed in the SBDART model. Parameter
Values
Solar zenith angle (◦, SZA)
10; 20; 30; 40; 50; 60; 70; 80
Boundary layer aerosol type (IAER)
0
1
2
3
4
no boundary layer
rural
urban
oceanic
tropospheric
Aerosol optical depth (AOD)
0.01
0.07
0.14
Cloud layer optical depth (τ)
0
0.1
0.5
0.9
10
Integrated ozone concentration (atm-cm)
0.3
Integrated water vapour amount (g cm−2)
0.35
Surface altitude (km)
2.1
Optical depth of each stratospheric aerosol layer
0.013
Atmospheric profile
3 – mid-latitude winter (AFGL standards) 3.1
Sensitivity of the absorbed energy to input
parameters the wide range of conditions that can be encountered in the
various regions of the world from almost pristine snow in
Antarctica to highly polluted snow (see e.g. Table 2 in Tuzet
et al., 2020, for soot and Sterle et al., 2013, for dust). As
recommended by Libois et al. (2014), we set the shape pa-
rameters B and g to 1.6 and 0.85. The refractive index from
Warren and Brandt (2008) was used. Figure 2 shows the sensitivity of the median error on the ab-
sorbed energy calculated by the method to the atmospheric
and snowpack physical properties. The calculated energy for
one simulation is compared to the reference absorbed energy,
calculated by TARTES–SBDART at 1 nm resolution, for the
same simulation and each SZA. Overall, the median error
on the broadband absorbed energy calculated for all simu-
lations decreases with increasing SZA. Concerning the at-
mospheric properties, the median error on absorbed energy
exhibits a stronger sensitivity to τ than to AOD. The me-
dian errors are small for values of τ equal to 0.1, 0.5, and 10
(absolute difference < 1 W m−2) but remain larger for values
equal to 0.0, 0.9, and 5.0 (e.g. median error = 3.6 W m−2 for
τ = 5 and SZA = 10◦). Errors are lower when using an ad-
equate reference irradiance profile (τ of simulation (τsimu)
equal to τ of reference (τref), τ = 0.5 and 10), and the calcu-
lated absorbed energy is therefore very sensitive to τ (median SBDART settings The main SBDART input parameters used in this study are
the aerosol optical depth (AOD), cloud layer optical depth
(τ), boundary layer aerosol type selector (IAER), and SZA
(Table 2). For the cloud properties, we used liquid water
droplets with a radius of 8 µm. These parameters have been https://doi.org/10.5194/gmd-14-7329-2021 Geosci. Model Dev., 14, 7329–7343, 2021 7334 3.2
Sensitivity to cloud cover conditions errors between 1.5 and −3.6 W m−2). Regarding AOD, the
median errors are small (absolute difference < 0.5 W m−2)
and show little change with τ. This demonstrates that AOD
exerts a very small influence on the median error and thus on
the calculation of the energy absorbed by the method. Con-
cerning the properties of the snow cover, the SSA value of
the first layer has little impact on the error of the absorbed
energy. For the different SSA values, the median errors are
small (absolute difference < 0.5 W m−2) and vary little de-
pending on the value studied. The presence of LAPs in the
snowpack leads to an increase in the median error (absolute
difference < 1 W m−2) compared to pure snow (absolute dif-
ference < 0.1 W m−2). Overall the method slightly overesti-
mates the energy absorbed by the snowpack (mostly positive
errors). The error is not very sensitive to the physical proper-
ties of the snowpack and to the AOD. However, the error is
very sensitive to τ of the simulations and thus to the τ chosen
for the reference profile. The sensitivity to cloud conditions
is investigated in more detail in the next section. Figure 2 shows the median errors on the broadband absorbed
energy for all the simulations. For each of them, the bias on
the broadband absorbed energy is shown in Fig. 3. Figure 3a
and b show the distribution of the biases in the broadband
absorbed energy and the broadband albedo as a function of
SZA and τ. These biases are determined as the difference be-
tween the absorbed energy calculated by VALHALLA and
TARTES–SBDART at 1 nm resolution. Overall, the broad-
band albedo biases vary little with SZA, and the biases of the
absorbed energy decrease with SZA. This is consistent with
higher absorbed energy for lower SZA (higher incoming ra-
diation and lower albedo). For simulations with a value of τ
equal to 0.5 and 10 as well as each SZA value, an adequate
reference irradiance profile is used (τsimu = τref). More than
75 % of the errors are positive, meaning that VALHALLA
overestimates the absorbed energy. The errors are low and
range between −1 and 1.5 W m−2 with a median error of
−0.76 and −0.95 W m−2 for τ equal to 0.5 and 10, respec-
tively. 3
Results In this section, we compare the simulated broadband ab-
sorbed energy resulting from VALHALLA for 30 TPs with
that obtained with TARTES–SBDART for the same spectral
range between 320 and 4000 nm. We first analyse the impact
of incident solar radiation, cloud cover conditions, and snow
properties on the errors in the estimated absorbed energy and
albedo. The efficiency of the method is then compared to the
TARTES–SBDART calculation for different spectral resolu-
tions ranging from 1 nm (reference simulations) to 100 nm. Geosci. Model Dev., 14, 7329–7343, 2021 https://doi.org/10.5194/gmd-14-7329-2021 7335 F. Veillon et al.: VALHALLA v1.0 Table 3. Snow properties of simulations. The spectral albedo is calculated for eight values of SZA and five values of SSA for the snowpack
first layer. For snow without LAPs, the SWE values are provided for the first three layers of the snowpack. For snow with LAPs, the SWE
and the light-absorbing particle concentration (for soot and dust) are provided for the first two layers of the snowpack. For layer 3, the values
of all input parameters, besides soot and dust contents, are constant. For layer 4, all input parameters are constant. Table 3. Snow properties of simulations. The spectral albedo is calculated for eight values of SZA and five values of SSA for the snowpack
first layer. For snow without LAPs, the SWE values are provided for the first three layers of the snowpack. For snow with LAPs, the SWE
and the light-absorbing particle concentration (for soot and dust) are provided for the first two layers of the snowpack. For layer 3, the values
of all input parameters, besides soot and dust contents, are constant. For layer 4, all input parameters are constant. Solar zenith angle (SZA, ◦)
10; 20; 30; 40; 50; 60; 70; 80
Layer 1
SSA (m2 kg−1)
2, 5, 42, 82, 155
SWE (kg m−2)
1
4
15
Thickness (t, m)
t: 0.01
t: 0.02
t: 0.05
Density (d, kg m3)
d: 100
d: 200
d: 300
Soot concentration (ng g−1)
0, 100, 200
Dust concentration (ng g−1)
0, 25 000, 50 000
Layer 2
SSA (m2 kg−1)
42
SWE (kg m−2)
1
4
15
Thickness (t, m)
t: 0.01
t: 0.02
t: 0.05
Density (d, kg m3)
d: 100
d: 200
d: 300
Soot concentration (ng g−1)
0, 100, 200
Dust concentration (ng g−1)
0, 25 000, 50 000
Layer 3
SSA (m2 kg−1)
42
SWE (kg m−2)
1
4
12.5
15
Thickness (t, m)
t: 0.01
t: 0.02
t: 0.05
t: 0.05
Density (d, kg m3)
d: 100
d: 200
d: 250
d: 300
Soot concentration (ng g−1)
0, 100
Dust concentration (ng g−1)
0, 25 000
Layer 4
SSA (m2 kg−1)
42
SWE (kg m−2)
600
Thickness (t, m)
t: 2
Density (d, kg m3)
d: 300
Soot concentration (ng g−1)
0
Dust concentration (ng g−1)
0 https://doi.org/10.5194/gmd-14-7329-2021 F. Veillon et al.: VALHALLA v1.0 7336 and 0.9, the reference irradiance profile used has a τ value
different from the simulations (τsimu ̸= τref). For those with
τ equal to 0 and 0.1, the biases are overall negative (for ap-
proximately 90 % of the biases) and vary between −4 and
0.5 W m−2. For τ equal to 0.9, the biases of the absorbed en-
ergy are positive (for more than 95 % of biases) and range
between −0.5 and 2.8 W m−2. The absolute error in the ab-
sorbed energy decreases with SZA for all τ values. rors for these two simulations are located at the wavelengths
at which absorption is maximal (between 1 and 1.5 µm). When the snowpack is absorbing a large amount of energy,
such as for low SSA, the biases in the spectral and broadband
absorbed energy increase. The biases in the absorbed energy
are therefore relatively sensitive to the SSA of the first layer
of the snowpack and thus remain very sensitive to the absorb-
ing properties of the snowpack. gy
Figure 3c and d show the spectral variation of the refer-
ence absorbed energy calculated by TARTES–SBDART and
that calculated by VALHALLA. The absorbed energy pro-
files presented are calculated for a simulation with two values
of τ (0 and 10) between 320 and 4000 nm at 1 nm resolution. The spectral error of the absorbed energy is also calculated
as the difference between the energy calculated by VAL-
HALLA and TARTES–SBDART. For the simulation with τ
equal to 0 (clear sky; Fig. 3c), the majority of the errors are
negative and are up to −9 Wm−2 µm−1. The higher errors
are located at the wavelengths at which the absorption is the
highest (between 1 and 1.5 µm). For the one with τ equal
to 10 (full overcast, Fig. 3d), the method represents the ab-
sorbed energy very well (errors close to 0 Wm−2 µm−1). The
use of an adequate reference irradiance profile (τsimu = τref)
thus leads to a decrease in the error on the spectral and broad-
band absorbed energy, despite a slight overestimation of the
energy absorbed by the method (positives errors). However,
when τsimu ̸= τref, the error on the absorbed energy is higher. When τ of the simulation is lower than τ of the reference pro-
file (τsimu < τref), the absorbed energy is underestimated by
the method (globally negative errors). 3.3
Sensitivity to snow physical properties Figure 4a and b show the distribution of the biases in broad-
band absorbed energy and albedo for varying SZA and SSA
of the first layer of the snowpack. Broadband energy biases
decrease with increasing SSA as the absolute absorbed en-
ergy is also decreasing. For an SSA equal to 2 m2 kg−1, the
biases vary between −2 and 4 W m−2 as opposed to a varia-
tion of −1.5 to 1.5 W m−2 for an SSA equal to 155 m2 kg−1. Figure 4c and d show the spectral variation of the refer-
ence absorbed energy calculated by TARTES–SBDART and
that calculated by the method. The absorbed energy pro-
files presented are calculated for a simulation with two ex-
treme SSA values (5 m2 kg−1, representative of old snow,
and 155 m2 kg−1 for new snow) between 320 and 4000 nm
at 1 nm resolution. For these two simulations, the spectral
errors of the absorbed energy are greater for an SSA value
equal to 5 m2 kg−1 (up to −10 Wm−2 µm−1), than for an
SSA of 155 m2 kg−1 (> −8 Wm−2 µm−1). The highest er- Figure 4a and b show the distribution of the biases in broad-
band absorbed energy and albedo for varying SZA and SSA
of the first layer of the snowpack. Broadband energy biases
decrease with increasing SSA as the absolute absorbed en-
ergy is also decreasing. For an SSA equal to 2 m2 kg−1, the
biases vary between −2 and 4 W m−2 as opposed to a varia-
tion of −1.5 to 1.5 W m−2 for an SSA equal to 155 m2 kg−1. Figure 4c and d show the spectral variation of the refer-
ence absorbed energy calculated by TARTES–SBDART and
that calculated by the method. The absorbed energy pro-
files presented are calculated for a simulation with two ex-
treme SSA values (5 m2 kg−1, representative of old snow,
and 155 m2 kg−1 for new snow) between 320 and 4000 nm
at 1 nm resolution. For these two simulations, the spectral
errors of the absorbed energy are greater for an SSA value
equal to 5 m2 kg−1 (up to −10 Wm−2 µm−1), than for an
SSA of 155 m2 kg−1 (> −8 Wm−2 µm−1). The highest er- 3.4
Sensitivity to LAPs Figure 5a and b show the distribution of the biases in broad-
band absorbed energy and albedo for various SZA and LAP
contents. Broadband energy biases increase with the pres-
ence of LAPs in the snowpack. However, for pure snow, the
biases are more negative than for snow with LAPs, and the
spread of the biases is greater (between −4 and 1.2 W m−2). For snow with dust or soot, the distribution of biases is very
similar (between −1 and 2 W m−2), whereas for snow with
a mix of dust and soot, the spread is larger (between −2 and
2.7 W m−2). Figure 5c and d show the spectral variation of the reference
absorbed energy calculated by TARTES–SBDART and that
calculated by VALHALLA. The absorbed energy profiles
presented are calculated for a simulation with two LAP con-
centrations contained in the snowpack (a snowpack which
contains 25 000 ng g−1 of dust and a snowpack which con-
tains a mix of 100 ng g−1 of soot and 50 000 ng g−1 of dust)
between 320 and 4000 nm at 1 nm resolution. With LAPs be-
ing highly absorbent at the beginning of the spectrum (be-
tween 0.3 and 0.8 µm; Warren, 1982), the highest spectral
absolute errors are consequently located in this wavelength
range. The method is indeed based on the ice refractive in-
dex (e.g. Eq. 9) and thus partly failed to reproduce changes
in the refractive index due to the presence of LAPs. For a
snowpack containing a mix of LAPs (Fig. 5d), the errors at
the beginning of the spectrum are higher than for a snow-
pack containing only dust (Fig. 5c). The presence of a mix of
LAPs in the snowpack generates errors of up to −30 Wm−2 µ
against maximum errors of −20 Wm−2 µ for the snowpack
containing only dust. The biases on the spectral and broad-
band energy increase with the amount of energy absorbed by
the snowpack. The biases on the absorbed energy are there-
fore very sensitive to the LAP content in the snowpack and
thus remain very sensitive to the absorbing properties of the
snowpack. F. Veillon et al.: VALHALLA v1.0 When τ of the simula-
tion is higher than τ of the reference profile τsimu > τref, the
absorbed energy is overestimated by the method (positives
errors). The biases of the absorbed energy are therefore very
sensitive to τ of the simulations and therefore to the optical
thickness chosen for the reference profile. 3.2
Sensitivity to cloud cover conditions For the simulations with a value of τ equal to 0, 0.1, https://doi.org/10.5194/gmd-14-7329-2021 Geosci. Model Dev., 14, 7329–7343, 2021 F. Veillon et al.: VALHALLA v1.0 3.5
Comparison to calculations with regular spectral
resolution In Fig. 6 we compare the broadband albedo bias obtained
with the VALHALLA methods to the bias obtained for spec-
tral resolution varying from 2 to 100 nm. The comparison
was performed using the simulations from Sect. 2.2.3. For
regular spectral resolution, the absolute bias generally in- Geosci. Model Dev., 14, 7329–7343, 2021 https://doi.org/10.5194/gmd-14-7329-2021 7337 F. Veillon et al.: VALHALLA v1.0 Figure 2. Median error of the broadband absorbed energy for varying SZA, the τ, the AOD, the SSA m2 kg−1, and LAP type. The median
error on the broadband absorbed energy is calculated with the ensemble of all the simulations described in Sect. 2.2.3 using 30 TPs with
TARTES–SBDART at 1 nm resolution as a reference. Figure 2. Median error of the broadband absorbed energy for varying SZA, the τ, the AOD, the SSA m2 kg−1, and LAP type. The median
error on the broadband absorbed energy is calculated with the ensemble of all the simulations described in Sect. 2.2.3 using 30 TPs with
TARTES–SBDART at 1 nm resolution as a reference. Figure 3. Bias on the broadband absorbed energy (a) and on the broadband albedo (b) as a function of the SZA and τ. The biases are
calculated for all the simulations described in Sect. 2.2.3 between the absorbed energy calculated by the method and the reference absorbed
energy calculated by TARTES–SBDART. The red lines indicate the median (same as in Fig. 2), the box shows the 25th to 75th percentiles, and
the whiskers show the 5th to 95th percentiles. Example of absorbed energy profiles for a snowpack without LAPs as a function of wavelength
calculated for an SZA of 30◦and a τ value of 0 (clear sky, c) as well as a τ value of 10 (full overcast, d). The black lines represent the absorbed
energy calculated by TARTES–SBDART at 1 nm resolution, and the red lines represent the absorbed energy calculated by VALHALLA. In
blue, the errors on the absorbed energy for these same simulations are shown as a function of wavelength. The green vertical lines represent
the TPs used in VALHALLA. BB error corresponds to the broadband error for the absorbed energy in panels (c) and (d). Figure 3. Bias on the broadband absorbed energy (a) and on the broadband albedo (b) as a function of the SZA and τ. The biases are
calculated for all the simulations described in Sect. 3.5
Comparison to calculations with regular spectral
resolution 2.2.3 between the absorbed energy calculated by the method and the reference absorbed
energy calculated by TARTES–SBDART. The red lines indicate the median (same as in Fig. 2), the box shows the 25th to 75th percentiles, and
the whiskers show the 5th to 95th percentiles. Example of absorbed energy profiles for a snowpack without LAPs as a function of wavelength
calculated for an SZA of 30◦and a τ value of 0 (clear sky, c) as well as a τ value of 10 (full overcast, d). The black lines represent the absorbed
energy calculated by TARTES–SBDART at 1 nm resolution, and the red lines represent the absorbed energy calculated by VALHALLA. In
blue, the errors on the absorbed energy for these same simulations are shown as a function of wavelength. The green vertical lines represent
the TPs used in VALHALLA. BB error corresponds to the broadband error for the absorbed energy in panels (c) and (d). https://doi.org/10.5194/gmd-14-7329-2021 Geosci. Model Dev., 14, 7329–7343, 2021 https://doi.org/10.5194/gmd-14-7329-2021 7338 F. Veillon et al.: VALHALLA v1.0 Figure 4. Bias on the broadband absorbed energy (a) and on the broadband albedo (b) as a function of the SZA and the SSA of the first
layer (m2 kg−1). The biases are calculated for all the simulations described in Sect. 2.5.3 and 2.5.4 between the absorbed energy calculated
by the method and the reference absorbed energy calculated by the TARTES–SBDART. The red lines indicate the median, the box shows
the 25th to 75th percentiles, and the whiskers show the 5th to 95th percentiles. Example of absorbed energy profiles for a snowpack without
LAPs as a function of wavelength calculated for an SZA of 30◦and an SSA value of 5 m2 kg−1 (old snow, c) as well as an SSA value of
155 m2 kg−1 (fresh snow, d). The black lines represent the absorbed energy calculated by TARTES–SBDART at 1 nm resolution, and the
red lines represent the absorbed energy calculated by VALHALLA. In blue, the errors on the absorbed energy for these same simulations are
shown as a function of wavelength. The green vertical lines represent the TPs used in VALHALLA. Figure 4. Bias on the broadband absorbed energy (a) and on the broadband albedo (b) as a function of the SZA and the SSA of the first
layer (m2 kg−1). The biases are calculated for all the simulations described in Sect. 2.5.3 and 2.5.4 between the absorbed energy calculated
by the method and the reference absorbed energy calculated by the TARTES–SBDART. The red lines indicate the median, the box shows
the 25th to 75th percentiles, and the whiskers show the 5th to 95th percentiles. Example of absorbed energy profiles for a snowpack without
LAPs as a function of wavelength calculated for an SZA of 30◦and an SSA value of 5 m2 kg−1 (old snow, c) as well as an SSA value of
155 m2 kg−1 (fresh snow, d). The black lines represent the absorbed energy calculated by TARTES–SBDART at 1 nm resolution, and the
red lines represent the absorbed energy calculated by VALHALLA. In blue, the errors on the absorbed energy for these same simulations are
shown as a function of wavelength. The green vertical lines represent the TPs used in VALHALLA. variables explaining the variations in absorbed energy using
spectrally fixed radiative variables. 4.1
On the accuracy of the method We have shown that the absorbed energy calculated by VAL-
HALLA is very sensitive to τ of the simulation and therefore
to the use of an adequate reference irradiance profile. The
use of a reference profile that is not adapted to the irradi- https://doi.org/10.5194/gmd-14-7329-2021 We determined 30 TPs,
corresponding to the local minima and maxima of the ab-
sorbed energy at which the exact calculation of the absorbed
energy is performed. In addition, we used 16 different ref-
erence irradiance profiles to interpolate between these TPs. We evaluated the accuracy of the method for several atmo-
spheric and snow properties that influence the amount of en-
ergy reaching the ground and snow albedo, such as τ, AOD,
SSA, and LAP content. We have shown that absorbed energy
and albedo errors due to the use of this method are small (ab-
solute difference < 1 W m−2 for broadband absorbed energy
and absolute difference < 0.005 for broadband albedo) and
correspond to a factor of 6 in terms of computation times
compared to calculations made at 20 nm resolution. creases with the spectral steps and tends to be more negative. This means that the bias on the absorbed energy tends to be
more positive when the spectral steps increase. We believe
that for low spectral resolution, the integration over the spec-
trum is missing the absorption bands, leading the integral to
be higher than for smaller spectral steps (see e.g. the spec-
trum in Figs. 3–5). The VALHALLA method presents biases
in the broadband albedo with an absolute difference lower
than 0.005, which is comparable to the bias obtained with
resolutions lower than or equal to 20 nm (reference resolu-
tion used at Météo France in research activities). The VAL-
HALLA method uses 30 wavelengths (30 TPs) when the cal-
culation at 20 nm resolution requires 184 wavelengths. Thus,
for the same bias in the broadband albedo, the VALHALLA
method uses 6 times fewer bands than a calculation at 20 nm
resolution. 4
Discussion The green vertical lines represent
the TPs used in VALHALLA. associated absolute error evolves as a function of the amount
of energy absorbed by the snowpack and is therefore driven
by the absorbing factors such as the SZA and the SSA. The
error on the absorbed energy therefore increases with a de-
crease in the solar angle and a decrease in the SSA value of
the first layer of the snowpack. Although the absolute error
decreases with SZA, the relative error generally increases for
high SZA, as can be seen in Figs. 3–5. For SZA higher than
85◦(not tested here), the broadband albedo might be interpo-
lated between the value at 85◦and 1. The choice of an ade-
quate reference irradiance profile for the simulation globally
determines the accuracy of the absorbed energy error calcu-
lated by VALHALLA. However, the choice of TPs is also a
determining factor in a good estimate of the energy absorbed
by the method. ance of the simulation (τsimu ̸= τref) leads to a clear increase
in the error on the absorbed energy. To reduce the uncer-
tainties resulting from the method, a preliminary calculation
of reference irradiance profiles adapted to each cloud con-
dition could be initialized. The reference profiles can also
be adapted to the cloud types (liquid water or ice droplets,
droplet radius) when this information is available together
with the solar radiation. Therefore, for all optical thickness
values used in the simulations, the irradiance in the method
can be adapted. The presence of LAPs in the snow cover
leads to an increase in errors on the absorbed energy, espe-
cially at the beginning of the spectrum where LAPs strongly
impact the absorption efficiency. The method fails to accu-
rately represent the absorbed energy between two TPs in the
visible range in the presence of LAPs since it is based on
the ice refractive index only. To reduce the uncertainties at
the beginning of the spectrum and thus reduce the broadband
error, it would be possible to increase the number of TPs at
the beginning of the spectrum. However, this would increase
the calculation time. The choice of the number of TPs is dis-
cussed later in this section. The other variables studied, such
as the SZA and the SSA, appear to be less influential. The 4
Discussion We presented the VALHALLA method for calculating ab-
sorbed energy and albedo based on a calculation of the main Geosci. Model Dev., 14, 7329–7343, 2021 https://doi.org/10.5194/gmd-14-7329-2021 F. Veillon et al.: VALHALLA v1.0 7339 Figure 5. Bias on the broadband absorbed energy (a) and on the broadband albedo (b) as a function of the SZA and the LAP content. The
biases are calculated for all the simulations described in Sect. 2.5.3 and 2.5.4 between the absorbed energy calculated by the method and the
reference absorbed energy calculated by TARTES–SBDART. The red lines indicate the median, the box shows the 25th to 75th percentiles,
and the whiskers show the 5th to 95th percentiles. Example of absorbed energy profiles for a snowpack with LAPs as a function of wavelength
calculated for an SZA of 30◦for a snowpack which contains dust (c) and a mix of soot and dust (d). The black lines represent the absorbed
energy calculated by TARTES–SBDART at 1 nm resolution, and the red lines represent the absorbed energy calculated by VALHALLA. In
blue, the errors on the absorbed energy for these same simulations are shown as a function of wavelength. The green vertical lines represent
the TPs used in VALHALLA. Figure 5. Bias on the broadband absorbed energy (a) and on the broadband albedo (b) as a function of the d absorbed energy (a) and on the broadband albedo (b) as a function of the SZA and the LAP content. The Figure 5. Bias on the broadband absorbed energy (a) and on the broadband albedo (b) as a function of the SZA and the LAP content. The
biases are calculated for all the simulations described in Sect. 2.5.3 and 2.5.4 between the absorbed energy calculated by the method and the
reference absorbed energy calculated by TARTES–SBDART. The red lines indicate the median, the box shows the 25th to 75th percentiles,
and the whiskers show the 5th to 95th percentiles. Example of absorbed energy profiles for a snowpack with LAPs as a function of wavelength
calculated for an SZA of 30◦for a snowpack which contains dust (c) and a mix of soot and dust (d). The black lines represent the absorbed
energy calculated by TARTES–SBDART at 1 nm resolution, and the red lines represent the absorbed energy calculated by VALHALLA. In
blue, the errors on the absorbed energy for these same simulations are shown as a function of wavelength. 4.2
Sensitivity to tie points The accuracy of the method is sensitive to the locations and
to the number of TPs. An increase or a decrease in the num-
ber of TPs could improve or alter the representation of the
absorbed energy. Using an overly large number of TPs leads https://doi.org/10.5194/gmd-14-7329-2021 Geosci. Model Dev., 14, 7329–7343, 2021 7340 F. Veillon et al.: VALHALLA v1.0 7340
F. Veillon et al.: VALHALLA v1.0
Figure 6. Errors on the broadband albedo for different constant spectral resolutions (left) and comparison with the errors of our method
(right). For these resolutions, the broadband albedo is computed by TARTES–SBDART and is compared to the one computed at 1 nm
resolution. The red lines indicate the median (same as in Fig. 2), the box shows the 25th to 75th percentiles, and the whiskers show the 5th
to 95th percentiles. The grey dotted lines correspond to an error of ±0.01 as in Gardner and Sharp (2010). 7340
F. Veillon et al.: VALHALLA v Figure 6. Errors on the broadband albedo for different constant spectral resolutions (left) and comparison with the errors of our method
(right). For these resolutions, the broadband albedo is computed by TARTES–SBDART and is compared to the one computed at 1 nm
resolution. The red lines indicate the median (same as in Fig. 2), the box shows the 25th to 75th percentiles, and the whiskers show the 5th
to 95th percentiles. The grey dotted lines correspond to an error of ±0.01 as in Gardner and Sharp (2010). cost of Gardner and Sharp (2010) is thus lower than the one
of VALHALLA. to a decrease in the calculated error but increases the calcu-
lation time, especially if the TP number is increased at the
beginning of the spectrum to compensate for the oscillations
of the absorbed energy when the snowpack contains LAPs. For a lower TP number, the oscillations at the beginning of
the spectrum due to LAPs are not well represented by the
method, and this leads to a significant increase in the error. With the use of 15 TPs, the error on the broadband albedo
increases globally by a factor of 10 to 15 for snow contain-
ing LAPs. With 10 TPs, the error increases by a factor of 25
and 50 for the same type of snow. The effect of LAPs on
the absorbed energy is therefore poorly represented when the
number of TPs is too low. 4.2
Sensitivity to tie points The use of 30 TPs is therefore a
good compromise between precision for snowpacks contain-
ing LAPs and calculation time. The SNOWBAL coupling scheme from van Dalum et
al. (2019) described in the Introduction of our study pro-
vides albedo calculation with an accuracy better than 0.01. Thanks to the physics of the snow radiative transfer model
TARTES, SNOWBAL accurately calculates the vertical dis-
tribution of the absorbed energy in the different snow layers. For VALHALLA, when using the method with TARTES or
with any other multilayer radiative transfer model for snow
(e.g. SNICAR; He et al., 2018), the vertical distribution of
the absorbed energy is calculated for the TPs but the vertical
profile of broadband absorbed energy is not directly avail-
able. This would require further development of the method. SNOWBAL used 14 representative wavelengths (RWs) for
which accurate snow radiative transfer calculations are per-
formed. The number of RWs depends on the number of nar-
rowbands available for the solar radiation. For VALHALLA,
accurate snow and atmosphere radiative transfer simulations
are performed for 30 TPs. When using 15 TPs, our method
fails to converge to a good representation of the broadband
albedo (increasing the error by a factor of 10 to 15). The use
of more TPs (30) is therefore necessary for an improved rep-
resentation of the broadband albedo. TPs and RWs are not
directly comparable, since the number of RWs depends on
the number of narrowbands available, and this is not the case
for the number of TPs. F. Veillon et al.: VALHALLA v1.0 functions derived from radiative transfer theory for snow and
the atmosphere. water content, direct-to-diffuse irradiance radiation, etc.). VALHALLA requires both snow and atmosphere radiative
transfer calculations for the TPs. This difference together
with the need for more than 15 TPs implies that the com-
putational cost of VALHALLA is higher than the compu-
tational cost of SNOWBAL. However, the accuracy of the
SNOWBAL methods depends on the number and range of
the narrowband solar radiation available. SNOWBAL accu-
racy increases when the sub-band spectral variability is re-
duced. Here, we used the VALHALLA method with broad-
band solar irradiance inputs, i.e. the worst case. The method
was also tested with narrowband solar radiation inputs (from
AROME, not shown; Seity et al., 2011), providing similar
accuracy for the absorbed energy as the one presented with
broadband inputs. water content, direct-to-diffuse irradiance radiation, etc.). VALHALLA requires both snow and atmosphere radiative
transfer calculations for the TPs. This difference together
with the need for more than 15 TPs implies that the com-
putational cost of VALHALLA is higher than the compu-
tational cost of SNOWBAL. However, the accuracy of the
SNOWBAL methods depends on the number and range of
the narrowband solar radiation available. SNOWBAL accu-
racy increases when the sub-band spectral variability is re-
duced. Here, we used the VALHALLA method with broad-
band solar irradiance inputs, i.e. the worst case. The method
was also tested with narrowband solar radiation inputs (from
AROME, not shown; Seity et al., 2011), providing similar
accuracy for the absorbed energy as the one presented with
broadband inputs. For the different properties of the atmosphere and snow
studied, the cloud layer optical depth (τ) and the LAP con-
tent of the snow cover are the main variables influencing the
calculation of the absorbed energy by the method. Indeed,
when the value of τ of the simulation is equal to that of the
reference irradiance profile, the method converges towards a
value of absorbed energy close to that calculated as a refer-
ence. On the other hand, when this value is not equal to that
of the reference profile, differences in absorbed energy are
noticeable at certain wavelengths. For snowpacks containing
LAPs, the method encounters difficulties in representing the
variation in absorbed energy at the beginning of the spectrum
and therefore generates significant differences in energy. 4.4
Implementation considerations The VALHALLA method therefore determines the ab-
sorbed energy for all wavelengths between 320 and 4000 nm
using 30 TPs. This number of TPs is necessary for a good
representation of the absorbed energy when the snow con-
tains LAPs. Despite an overestimation of the energy ab-
sorbed by the method, the results obtained with 30 TPs are
similar to the results of TARTES–SBDART at 20 nm. This
results in a reduction of the calculation time by a factor of 6
(30 TPs versus 180 wavelengths). In addition to the perfor-
mance in terms of calculation time, the method is versatile
and adaptable to any atmospheric input (broadband, narrow-
band). The VALHALLA method has been developed to provide ac-
curate calculation of the solar energy absorbed by the snow-
pack at low computational cost compared to full spectral cal-
culation. The VALHALLA method requires accurate calcu-
lation of the spectral absorbed energy for the TPs. In the
study, this is based on the TARTES and SBDART models,
but any other radiative model could be used (e.g. SNICAR
for snow, He et al., 2018; Bird and Riordan, 1986, for the at-
mosphere). The overall accuracy of the calculation depends
on the choice of the radiative transfer model for snow and
for the atmosphere. We believe that the VALHALLA method
is an especially efficient compromise between accuracy and
computational cost when only broadband (or large narrow-
bands) solar irradiance values are available from the atmo-
spheric model. This is the case, for example, for the detailed
snowpack model Crocus in the land surface model SURFEX
(Tuzet et al., 2017); this is also the case when surface simu-
lations are performed offline (not coupled), i.e. using atmo-
spheric reanalysis or measurements as inputs. In conclusion, the development of the method VAL-
HALLA presented here allows a considerable reduction in
calculation time while maintaining a good representation of
the spectral albedo. One of the perspectives would be to in-
tegrate this method into a radiative scheme of a global or
regional climate model in order to drastically reduce the cal-
culation time and to largely improve the albedo calculation
compared to more common broadband and/or narrowband
calculations. F. Veillon et al.: VALHALLA v1.0 The
use of reference profiles with an adequate value of SZA is
necessary for the good accuracy of the method. 4.3
Comparison to other existing methods Gardner and Sharp (2010) developed a snow broadband
albedo parameterization accounting for changes in the snow
and atmospheric properties. The computational cost of such
albedo parameterization is very small, and the accuracy is
around 0.01 for the broadband albedo (compared to refer-
ence calculations at 10 nm resolution). This accuracy is de-
picted in Fig. 6 by the grey dotted horizontal lines. The ac-
curacy of VALHALLA is roughly an order of magnitude
lower. However, the albedo parameterizations of Gardner
and Sharp (2010) and VALHALLA fulfil two different goals
since VALHALLA requires accurate snow and atmosphere
radiative transfer calculation for the TPs. The computational VALHALLA and SNOWBAL fulfil two different niches. SNOWBAL indeed required accurate snow radiative trans-
fer calculation and accurate atmospheric conditions (cloud https://doi.org/10.5194/gmd-14-7329-2021 Geosci. Model Dev., 14, 7329–7343, 2021 7341 F. Veillon et al.: VALHALLA v1.0 https://doi.org/10.5194/gmd-14-7329-2021 F. Veillon et al.: VALHALLA v1.0 land’s darkening since 2009, Nat. Geosci., 7, 509–512,
https://doi.org/10.1038/ngeo2180, 2014. land’s darkening since 2009, Nat. Geosci., 7, 509–512,
https://doi.org/10.1038/ngeo2180, 2014. Competing interests. The authors declare that they have no conflict
of interests. Competing interests. The authors declare that they have no conflict
of interests. Dumont, M., Arnaud, L., Picard, G., Libois, Q., Lejeune, Y., Nabat,
P., Voisin, D., and Morin, S.: In situ continuous visible and near-
infrared spectroscopy of an alpine snowpack, The Cryosphere,
11 1091–1110 https://doi org/10 5194/tc-11-1091-2017 2017 Disclaimer. Publisher’s note: Copernicus Publications remains
neutral with regard to jurisdictional claims in published maps and
institutional affiliations. 11, 1091–1110, https://doi.org/10.5194/tc-11-1091-2017, 2017. ECMWF: Part IV: Physical Processes, IFS Documentation, opera-
tional implementation 3 June 2008, ECMWF, Reading, England,
224 pp., 2009. Flanner, M. G. and Zender, C. S.: Linking snowpack micro-
physics and albedo evolution, J. Geophys. Res., 111, D12208,
https://doi.org/10.1029/2005JD006834, 2006. Acknowledgements. The authors are grateful to Ghislain Picard and
Quentin Libois for discussion on the VALHALLA method. The au-
thors are also grateful to the two referees, Joseph Cook and Chris-
tiaan Van Dalum, for very helpful and relevant comments on the
paper. Gardner, A. S. and Sharp, M. J.: A review of snow and ice
albedo and the development of a new physically based broad-
band albedo parameterization, J. Geophys. Res., 115, F01009,
https://doi.org/10.1029/2009JF001444, 2010. Grenfell, T. C., Warren, S. G., and Mullen, P. C.: Reflection of solar
radiation by the Antarctic snow surface at ultraviolet, visible, and
near-infrared wavelengths, J. Geophys. Res., 99, 18669–18684,
https://doi.org/10.1029/94JD01484, 1994. Financial support. CNRM/CEN is part of Labex OSUG@2020
(ANR-10-LABX-0056). This work was partly funded by CNES
APR MIOSOTIS and by ANR grant EBONI (ANR-16-CE01-
0006). Marie Dumont has received funding from the European Re-
search Council (ERC) under the European Union’s Horizon 2020
research and innovation programme (IVORI, grant agreement no. 949516). He, C., Flanner, M. G., Chen, F., Barlage, M., Liou, K.-N., Kang,
S., Ming, J., and Qian, Y.: Black carbon-induced snow albedo
reduction over the Tibetan Plateau: uncertainties from snow
grain shape and aerosol–snow mixing state based on an up-
dated SNICAR model, Atmos. Chem. Phys., 18, 11507–11527,
https://doi.org/10.5194/acp-18-11507-2018, 2018. Review statement. This paper was edited by Fabien Maussion and
reviewed by Christiaan van Dalum and Joseph Cook. Review statement. This paper was edited by Fabien Maussion and
reviewed by Christiaan van Dalum and Joseph Cook. Joseph, J. H., Wiscombe, W. J., and Weinman, J. References Bird, R. E. and Riordan, C.: Simple Solar Spectral Model
for Direct and Diffuse Irradiance on Horizontal and Tilted
Planes at the Earth’s Surface for Cloudless Atmospheres, J. Appl. Meteorol. Clim., 25, 87–97, https://doi.org/10.1175/1520-
0450(1986)025<0087:SSSMFD>2.0.CO;2, 1986. Kokhanovsky,
A. A. and
Zege,
E. P.:
Scattering
optics
of
snow,
Appl. Optics,
43,
1589–1602,
https://doi.org/10.1364/AO.43.001589, 2004. Kuipers Munneke, P., van den Broeke, M. R., Lenaerts, J. T. M., Flanner, M. G., Gardner, A. S., and van de Berg, W. J.: A new albedo parameterization for use in climate models
over the Antarctic ice sheet, J. Geophys. Res., 116, D05114,
https://doi.org/10.1029/2010JD015113, 2011. Cess, R. D., Potter, G. L., Zhang, M. H., Blanchet, J. P., Chalita,
S., Colman, R., Dazlich, D. A., Genio, A. D. D., Dymnikov, V.,
Galin, V., Jerrett, D., Keup, E., Lacis, A. A., Le Treut, H., Liang,
X. Z., Mahfouf, J. F., Mcavaney, B. J., Meleshko, V. P., Mitchell,
J. F. B., Morcrette, J. J., Norris, P. M., Randall, D. A., Rikus, L.,
Roeckner, E., Royer, J. F., Schlese, U., Sheinin, D. A., Slingo, J. M., Sokolov, A. S., Taylor, K. E., Washington, W. M., Wetherald,
R. T., and Yagai, I.: Interpretation of Snow-Climate Feedback as
Produced by 17 General Circulation Models, Science, 253, 888–
892, https://doi.org/10.1126/science.253.5022.888, 1991. Libois, Q., Picard, G., France, J. L., Arnaud, L., Dumont, M., Car-
magnola, C. M., and King, M. D.: Influence of grain shape
on light penetration in snow, The Cryosphere, 7, 1803–1818,
https://doi.org/10.5194/tc-7-1803-2013, 2013. eckner, E., Royer, J. F., Schlese, U., Sheinin, D. A., Slingo, J. Libois, Q., Picard, G., Dumont, M., Arnaud, L., Sergent, C.,
Pougatch, E., Sudul, M., and Vial, D.: Experimental determi-
nation of the absorption enhancement parameter of snow, J. Glaciol., 60, 714–724, https://doi.org/10.3189/2014JoG14J015,
2014. Clough,
S.,
Shephard,
M.,
Mlawer,
E.,
Delamere,
J.,
Ia-
cono, M., Cady-Pereira, K., Boukabara, S., and Brown,
P.: Atmospheric radiative transfer modeling: a summary of
the AER codes, J. Quant. Spectrosc. Ra., 91, 233–244,
https://doi.org/10.1016/j.jqsrt.2004.05.058, 2005. McKenzie Skiles, S. and Painter, T. H.: Assessment of Radiative
Forcing by Light-Absorbing Particles in Snow from In Situ Ob-
servations with Radiative Transfer Modeling, J. Hydrometeo-
rol., 19, 1397–1409, https://doi.org/10.1175/JHM-D-18-0072.1,
2018. Domine, F., Taillandier, A.-S., and Simpson, W. R.: A parameteri-
zation of the specific surface area of seasonal snow for field use
and for models of snowpack evolution, J. Geophys. Res., 112,
F02031, https://doi.org/10.1029/2006JF000512, 2007. Mlawer, E. J., Taubman, S. J., Brown, P. D., Iacono, M. 5
Conclusions Code and data availability. The VALHALLA v1.0 development
and data presented and described in this article are available for
download at https://doi.org/10.5281/zenodo.4570565 (Veillon et al.,
2021). In climate models, energy fluxes are most often given for nar-
row and large spectral bands. The low spectral resolution of
these fluxes therefore leads to uncertainties in the determi-
nation of radiative variables such as snow albedo that are
key for energy exchanges at the surface. This study presents
a new method, VALHALLA, for calculating the spectral
albedo of snow based on the determination of key atmo-
spheric and snow variables explaining variations in absorbed
energy using spectrally fixed variables. For this method, tie
points (TPs) and reference irradiance profiles are calculated
to incorporate the absorbed energy and the reference irra-
diance. The absorbed energy is then interpolated for each
wavelength present between two TPs with adequate kernel TARTES is available as a python module at https://pypi.org/
project/tartes/ (last access: 19 November 2021, Picard and Li-
bois, 2021). SBDART model is available at https://github.com/
paulricchiazzi/SBDART (last access: 19 November 2021, Ricchi-
azzi, 2018). Author contributions. FV, MD, and MF started this project and de-
veloped the method. FV ran the simulations and wrote the first draft
of the paper. FV, MD, and CA performed the analysis. All authors
discussed and revised the paper. https://doi.org/10.5194/gmd-14-7329-2021 Geosci. Model Dev., 14, 7329–7343, 2021 7342 F. Veillon et al.: VALHALLA v1.0 A.: The
Delta-Eddington Approximation for Radiative Flux Transfer,
J. Atmos. Sci., 33, 2452–2459, https://doi.org/10.1175/1520-
0469(1976)033<2452:TDEAFR>2.0.CO;2, 1976. Kokhanovsky, A., Lamare, M., Di Mauro, B., Picard, G., Arnaud,
L., Dumont, M., Tuzet, F., Brockmann, C., and Box, J. E.: On
the reflectance spectroscopy of snow, The Cryosphere, 12, 2371–
2382, https://doi.org/10.5194/tc-12-2371-2018, 2018. F. Veillon et al.: VALHALLA v1.0 for the longwave, J. Geophys. Res., 102, 16663–16682,
https://doi.org/10.1029/97JD00237, 1997. for the longwave, J. Geophys. Res., 102, 16663–16682,
https://doi.org/10.1029/97JD00237, 1997. Tuzet, F., Dumont, M., Lafaysse, M., Picard, G., Arnaud, L., Voisin,
D., Lejeune, Y., Charrois, L., Nabat, P., and Morin, S.: A mul-
tilayer physically based snowpack model simulating direct and
indirect radiative impacts of light-absorbing impurities in snow,
The Cryosphere, 11, 2633–2653, https://doi.org/10.5194/tc-11-
2633-2017, 2017. Morcrette, J.-J., Barker, H. W., Cole, J. N. S., Iacono, M. J., and
Pincus, R.: Impact of a New Radiation Package, McRad, in the
ECMWF Integrated Forecasting System, Mon. Weather Rev.,
136,
4773–4798,
https://doi.org/10.1175/2008MWR2363.1,
2008. Tuzet, F., Dumont, M., Picard, G., Lamare, M., Voisin, D., Nabat,
P., Lafaysse, M., Larue, F., Revuelto, J., and Arnaud, L.:
Quantification of the radiative impact of light-absorbing par-
ticles during two contrasted snow seasons at Col du Lautaret
(2058 m a.s.l., French Alps), The Cryosphere, 14, 4553–4579,
https://doi.org/10.5194/tc-14-4553-2020, 2020. Picard, G. and Libois, Q.: Two-stream radiative transfer in snow
model, Python Software Foundation [code], available at: https:
//pypi.org/project/tartes/, last access: 19 November 2021. Picard,
G.,
Arnaud,
L.,
Domine,
F.,
and
Fily,
M.:
De-
termining
snow
specific
surface
area
from
near-infrared
reflectance
measurements:
Numerical
study
of
the
influ-
ence of grain shape, Cold Reg. Sci. Technol., 56, 10–17,
https://doi.org/10.1016/j.coldregions.2008.10.001, 2009. van Dalum, C. T., van de Berg, W. J., Libois, Q., Picard, G., and van
den Broeke, M. R.: A module to convert spectral to narrowband
snow albedo for use in climate models: SNOWBAL v1.2, Geosci. Model Dev., 12, 5157–5175, https://doi.org/10.5194/gmd-12-
5157-2019, 2019. Picard, G., Domine, F., Krinner, G., Arnaud, L., and Lefebvre,
E.: Inhibition of the positive snow-albedo feedback by precip-
itation in interior Antarctica, Nat. Clim. Change, 2, 795–798,
https://doi.org/10.1038/nclimate1590, 2012. van Dalum, C. T., van de Berg, W. J., Lhermitte, S., and van
den Broeke, M. R.: Evaluation of a new snow albedo scheme
for the Greenland ice sheet in the Regional Atmospheric Cli-
mate Model (RACMO2), The Cryosphere, 14, 3645–3662,
https://doi.org/10.5194/tc-14-3645-2020, 2020. Ricchiazzi, P.: SBDART, GitHub [code], available at: https://github. com/paulricchiazzi/SBDART (last access: 19 November 2021),
2018. Ricchiazzi, P., Yang, S., Gautier, C., and Sowle, D.: SB-
DART: A Research and Teaching Software Tool for Plane-
Parallel Radiative Transfer in the Earth’s Atmosphere, B. Am. Meteorol. Soc., 79, 2101–2114, https://doi.org/10.1175/1520-
0477(1998)079<2101:SARATS>2.0.CO;2, 1998. van Dalum, C. T., van de Berg, W. J., and van den Broeke, M. References J., and Clough, S. A.: Radiative transfer for inhomoge-
neous atmospheres: RRTM, a validated correlated-k model Dumont, M., Brun, E., Picard, G., Michou, M., Libois, Q.,
Petit, J.-R., Geyer, M., Morin, S., and Josse, B.: Con-
tribution of light-absorbing impurities in snow to Green- https://doi.org/10.5194/gmd-14-7329-2021 Geosci. Model Dev., 14, 7329–7343, 2021 7343 F. Veillon et al.: VALHALLA v1.0 R.: Impact of updated radiative transfer scheme in snow and
ice in RACMO2.3p3 on the surface mass and energy budget
of the Greenland ice sheet, The Cryosphere, 15, 1823–1844,
https://doi.org/10.5194/tc-15-1823-2021, 2021. Schneider, T., Kaul, C. M., and Pressel, K. G.: Possible
climate transitions from breakup of stratocumulus decks
under
greenhouse
warming,
Nat. Geosci.,
12,
163–167,
https://doi.org/10.1038/s41561-019-0310-1, 2019. Veillon, F., Dumont M., Amory, C., and Fructus, M.: Veil-
lon
et
al. (2020),
Geoscientific
Model
Development:
data and development, version V1.0, Zenodo [data set],
https://doi.org/10.5281/zenodo.4570565, 2021. Warren, S. G.: Optical properties of snow, Rev. Geophys., 20, 67–
89, https://doi.org/10.1029/RG020i001p00067, 1982. Seity, Y., Brousseau, P., Malardel, S., Hello, G., Bénard, P., Bouttier,
F., Lac, C., and Masson, V.: The AROME-France convective-
scale operational model, Mon. Weather Rev., 139, 976–991,
2011. Warren, S. G. and Brandt, R. E.: Optical constants of ice from the
ultraviolet to the microwave: A revised compilation, J. Geophys. Res.,
113,
D14220,
https://doi.org/10.1029/2007JD009744,
2008. Stamnes, K., Tsay, S.-C., Wiscombe, W., and Jayaweera, K.: Nu-
merically stable algorithm for discrete-ordinate-method radiative
transfer in multiple scattering and emitting layered media, Appl. Optics, 27, 2502–2509, https://doi.org/10.1364/AO.27.002502,
1988. Wetherald,
R. T. and
Manabe,
S.:
Cloud
Feedback
Pro-
cesses
in
a
General
Circulation
Model,
J. Atmos. Sci.,
45,
1397–1416,
https://doi.org/10.1175/1520-
0469(1988)045<1397:CFPIAG>2.0.CO;2, 1988. Sterle, K. M., McConnell, J. R., Dozier, J., Edwards, R., and Flan-
ner, M. G.: Retention and radiative forcing of black carbon
in eastern Sierra Nevada snow, The Cryosphere, 7, 365–374,
https://doi.org/10.5194/tc-7-365-2013, 2013. Geosci. Model Dev., 14, 7329–7343, 2021 https://doi.org/10.5194/gmd-14-7329-2021
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Disclosure of non-recent (historic) childhood sexual abuse: What should researchers do?
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Journal of medical ethics
| 2,020
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cc-by
| 6,001
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ABSTRACT Researchers engage in the empirical study of
survivors’ experiences in order to understand the
ramifications for survivors’ social lives, their mental
and physical health, and their psychosexual well-
being. Findings often aim to increase public under-
standing of CSA,10 improve policies and services
for survivors, and inform clinical care for people
who have gone through similar experiences of
CSA (whether or not this is known). The need for
research is therefore clear. Non-recent (historic) childhood sexual abuse is an
important issue to research, though often regarded
as taboo and frequently met with caution, avoidance
or even opposition from research ethics committees. Sensitive research, such as that which asks victim-
survivors to recount experiences of abuse or harm,
has the propensity to be emotionally challenging for
both the participant and the researcher. However, most
research suggests that any distress experienced is usually
momentary and not of any clinical significance. Moreover,
this type of research offers a platform for voices which
have often been silenced, and many participants report
the cathartic effect of recounting their experiences
in a safe, non-judgemental space. With regard to the
course of such research, lines of inquiry which ask adult
participants to discuss their experiences of childhood
sexual abuse may result in a first-time disclosure of
that abuse by the victim-survivor to the researcher. Guidance about how researchers should respond to
first-time disclosure is lacking. In this article, we discuss
our response to one research ethics committee which
had suggested that for a qualitative study for which we
were seeking ethical approval (investigating experiences
of pregnancy and childbirth having previously survived
childhood sexual abuse), any disclosure of non-recent
(historic) childhood sexual abuse which had not been
previously reported would result in the researcher being
obliged to report it to relevant authorities. We assess
this to be inconsistent with both law and professional
guidance in the United Kingdom; and provide
information and recommendations for researchers and
research ethics committees to consider. 3Department of Midwifery,
King’s College London,
Waterloo, UK 4Maternity Services, University
College London Hospitals NHS
Foundation Trust, Fitzrovia, UK
5The Survivors Trust, Rugby, UK
6Centre for Midwifery, Child
and Family Health, University
of Technology Sydney, Sydney,
New South Wales, Australia
7Faculty of Laws, University
College London, Bloomsbury,
UK Research which asks participants to discuss uncom-
fortable or traumatic experiences or memories is
commonplace within health and psychological
sciences. Sergio A. Silverio
,1,2 Susan Bewley
,1 Elsa Montgomery
,3
Chelsey Roberts
,2 Yana Richens
,2,4 Fay Maxted,5 Jane Sandall
,1,6
Jonathan Montgomery
7 1Department of Women &
Children’s Health, King’s College
London, Westminster, UK
2Elizabeth Garrett Anderson
Institute for Women’s Health,
University College London,
Bloomsbury, UK
3Department of Midwifery,
King’s College London,
Waterloo, UK
4Maternity Services, University Clinical ethics Clinical ethics on November 15, 2020 by guest. Prote
http://jme.bmj.com/
J Med Ethics: first published as 10.1136/medethics-2020-106343 on 10 November 2020. Downloaded from ABSTRACT However, disclosure of previous trauma can
occur in research environments spontaneously, even
without the nature of the research being specifically
focused on trauma. These disclosures can also be diffi-
cult for researchers.11 It is widely accepted participants
may experience distress, even momentarily, when
recounting their experience for research.12 However,
evidence suggests for the large majority of partici-
pants, this distress is neither clinically significant nor
longstanding.13–17 It has been documented that these
research-based exchanges can even be cathartic due to
offering a non-judgmental ‘safe space’ in which partic-
ipants may achieve validation and find a voice.18 This
has led some to conclude ‘there is a low likelihood
of significant emotional harm from participating in
trauma-focused studies’ for participants involved in
research.13 on November 15, 2020 by guest. Pro
http://jme.bmj.com/
0 November 2020. Downloaded from Correspondence to
Mr. Sergio A. Silverio,
Department of Women &
Children’s Health, King’s College
London, London SE1 7EH, UK;
Sergio.Silverio@kcl.ac.uk Received 24 April 2020
Revised 1 October 2020
Accepted 6 October 2020 on November 15, 2020 by guest. Protected by copyright. http://jme.bmj.com/
om A particular ethical challenge facing researchers
who work with adult survivors of CSA may arise if
they receive disclosures of criminal activity perpe-
trated on the participant which have not previously
been recognised or reported to relevant authorities. This is sometimes described as non-recent (historic)
CSA,6 even though it may not be long ago for a young
adult survivor. This analysis piece was prompted by
comments received (by SAS, CR, YR) from a research
ethics committee (REC) on proposed work involving
qualitative interviews about the childbearing expe-
riences of women who had previously experienced
CSA. The REC asked what the researchers would do
if ‘specific information about criminal activities are
identified given that you would have the obligation
to report this’.i The source, nature and scope of this
supposed ‘obligation’ were not explained. This piece
considers how researchers should respond to such a
question. Correspondence to
Mr. Sergio A. Silverio,
Department of Women &
Children’s Health, King’s College
London, London SE1 7EH, UK;
Sergio.Silverio@kcl.ac.uk Received 24 April 2020
Revised 1 October 2020
Accepted 6 October 2020 i Our emphasis. Disclosure of non-recent (historic) childhood sexual
abuse: What should researchers do? Sergio A. Silverio
,1,2 Susan Bewley
,1 Elsa Montgomery
,3
Chelsey Roberts
,2 Yana Richens
,2,4 Fay Maxted,5 Jane Sandall
,1,6
Jonathan Montgomery
7 To cite: Silverio SA, Bewley S,
Montgomery E, et al.
J Med Ethics Epub ahead of
print: [please include Day
Month Year]. doi:10.1136/
medethics-2020-106343
© Author(s) (or their
employer(s)) 2020. Re-use
permitted under CC BY.
Published by BMJ. © Author(s) (or their
employer(s)) 2020. Re-use
permitted under CC BY.
Published by BMJ. BACKGROUND would apply. In the only clearly analogous case where reporting
is mandatory in England and Wales,28 concerning the female
genital mutilation (FGM) of minors, ‘historic’ cases are expressly
excluded.29 In Wales, there is a wider mandatory obligation on
organisations (but not individuals) to report current child protec-
tion concerns (where there is reasonable cause to suspect a child
is at risk).30 This applies to local authorities, police, providers of
probation services, local health boards, National Health Service
Trusts, and Youth Offending Teams. Some researchers will be
employees of such organisations, but this would be coincidental. These obligations are defined in terms of individuals being at
risk, not to the investigation of crime. The research setting of an unhurried, private interview might
be the first time and place in which the participant has ever felt
safe to tell their story. Most CSA is not revealed, and most adult
victim-survivors of CSA choose not to make a formal report to
the authorities.19 The reasons can be complex and numerous,
ranging from rational fears of disbelief or reprisals, to wishing
to protect other family members. This raises different challenges
from disclosure of current abusive behaviour. Studies about
experiences of non-recent (historic) CSA need an empathic
researcher; research itself will be hampered if the participant
fears anything they say will be reported to authorities not already
privy to the information. Removing choice from any victim-
survivor is deeply disempowering and re-enacts the dynamics of
abuse of power and loss of control which is often characteristic
of the original abuse.4 The process of reporting to authorities is
stressful; it may result in forced disclosure to family members or
partners, which the victim-survivor had not planned, which itself
can be (re)traumatising. Requiring researchers to report disclo-
sures of non-recent (historic) abuse might increase the risk of the
likelihood of significant harm being caused by participation in
research beyond ethically tolerable levels. on November 15, 2020 by guest. Protected by copyright. http://jme.bmj.com/
published as 10.1136/medethics-2020-106343 on 10 November 2020. BACKGROUND Downloaded from g
Similar considerations arise even in jurisdictions which have
general mandatory reporting laws, such as those that have existed
in parts of Australia since 1969 and which were considered in the
Royal Commission into Institutional Responses to Child Sexual
Abuse.31 In federal Australian law, and most states, the reporting
obligations are imposed only on certain authorities (as in Wales,
above) and researchers are unlikely to be covered unless they
work within the prescribed services. In the Northern Territo-
ries there is a more general rule that will apply to researchers
as members of the public, subject to a defence of ‘reasonable
excuse’ to the disclosure obligations.32 The arguments we make
in this paper support the reasonableness of declining to disclose
in order to protect the autonomy of adult research participants
to determine whether reports are made. This is specifically
accepted in New South Wales, also in Australia, where the law
states that the fact that an adult victim does not wish the matter
reported provides reasonable and sufficient justification for
non-disclosure.33 In therapeutic contexts, increasing awareness of CSA, and also
of the risk of ‘gaze aversion’ (where professionals do not want
to acknowledge it), has led to greater encouragement to share
information with safeguarding authorities. This is conducted
either via a ‘support to report’ pathway, or by breaching patient
confidentiality in order to report disclosures of situations which
suggest vulnerable persons are at risk.6 The latter route is some-
times undertaken, even where consent to report disclosures
has been refused by the person disclosing their experiences
of non-recent (historic) CSA.20 This raises significant ethical
challenges.21 It involves a deliberate breach of the element of
trust built between the patient/client (here, a victim-survivor of
CSA) and the authority figure in whom they confide and who is
meant to provide therapeutic counsel (whether that be a medical
professional, psychological or counselling practitioner, social
worker or other therapeutic practitioner). on November 15, 2020 by guest. Prote
http://jme.bmj.com/
vember 2020. Downloaded from These examples suggest that it is unlikely to be true that
researchers would be legally obliged to report disclosures of
historic abuse against the wishes of participants. The detailed
arguments that would need to be made to support this claim
will depend on the specific legal jurisdiction in which they work. INTRODUCTION The effects of childhood sexual abuse (CSA) are
often devastating and wide ranging; affecting phys-
ical,1 psychological2–4 and sexual health.5 The inci-
dence and prevalence of such abuses are difficult to
ascertain, not least because most incidents of CSA go
unrecognised or unreported by the victim.6 However,
incidence rates of CSA are likely to be significant. In
a representative sample from the United Kingdom
(UK), 18.6% of women aged 18–24 reported contact
sexual abuse (ranging from touching to rape) while
they were still children.7 This is consistent with inter-
national data. A meta-analysis of studies from 22
countries reported some form of sexual abuse prior
to the age of 18 in 19.2% of women and 7.4% of
men.8 Where reporting does occur, numerous factors
contribute to its delay or only partial disclosure.6 9 To cite: Silverio SA, Bewley S,
Montgomery E, et al. J Med Ethics Epub ahead of
print: [please include Day
Month Year]. doi:10.1136/
medethics-2020-106343
© Author(s) (or their
employer(s)) 2020. Re-use
permitted under CC BY. Published by BMJ. To cite: Silverio SA, Bewley S,
Montgomery E, et al. J Med Ethics Epub ahead of
print: [please include Day
Month Year]. doi:10.1136/
medethics-2020-106343
© Author(s) (or their
employer(s)) 2020. Re-use
permitted under CC BY. Published by BMJ. guest. Protected by copyright. To cite: Silverio SA, Bewley S,
Montgomery E, et al. J Med Ethics Epub ahead of
print: [please include Day
Month Year]. doi:10.1136/
medethics-2020-106343 i Our emphasis. Silverio SA, et al. J Med Ethics 2020;0:1–5. doi:10.1136/medethics-2020-106343 1 on November 15, 2020 by guest. Prote
http://jme.bmj.com/
J Med Ethics: first published as 10.1136/medethics-2020-106343 on 10 November 2020. Downloaded from Clinical ethics BACKGROUND We do not offer a comprehensive survey of the laws in different
jurisdictions, but the illustration suffices to show that even where
mandatory reporting exists, claims of an automatic or absolute
legal duty to disclose are unlikely to be well founded. on November 15, 2020 by guest. Protected by copyright. http://jme.bmj.com/
om For researchers, there is no specific guidance regarding previ-
ously unreported disclosures of non-recent (historic) CSA. Researchers must abide by their obligations to comply with
general legal, ethical, and data protection standards.6 22 When
these are ambiguous and fail to give guidance as to how choices
should be made between competing principles, or on strategies
to balance them fairly with each other, then researchers are ill-
equipped to address the concerns raised by the REC. FRAMEWORK FOR DISCLOSURE DECISIONS IN PUBLIC
INTEREST We therefore turn to consider the implications of the more
general protections of privacy in both law and ethics, and in
particular the way in which privacy rights are protected through
the obligation of confidentiality. The starting point for analysis is
that reporting of information disclosed to researchers would be
unlawful and unethical, because the researcher has only acquired
such information subject to legal and ethical obligations of
(research) confidentiality (which adds specific responsibilities to
those arising from general protections of privacy). LEGAL POSITIONS
Th
h The suggestion which prompted this article—that if non-recent
(historic) CSA which had not been previously reported was
disclosed to researchers then the researcher would be obliged
to report it to relevant authorities—is inconsistent with both
law and professional guidance in the UK. In the UK, there is no
general legal duty to report crimes, and mandatory reporting
obligations are tightly defined and rare.23 In 2016, the govern-
ment consulted on whether to make reporting of child abuse
mandatory and concluded it would not do so.24 The Indepen-
dent Inquiry into Child Sexual Abuse (IICSA) has taken evidence
on mandatory reporting, but has not yet reached a conclusion
on the issue.25–27 Any breach of said obligation would need clear justification. It is sometimes permissible, although not obligatory, to disclose
information in the ‘public interest’. Any jurisdiction which
recognises privacy rights will include an exception of this sort,
as in the New Zealand Privacy Act 2020 which states that other-
wise confidential information may be disclosed where necessary
to prevent or lessen a serious threat to public health or safety;
or to the life or health of individuals.34 In the UK there is also
a recognised justification for disclosure to ‘prevent, detect, or
prosecute, a serious crime’, which is generally thought to include
child abuse.35 guest. Protected by copyright. Existing reporting duties do not directly address the research
context, but the terms on which they are formulated make it
unlikely they would be interpreted such that mandatory reporting Silverio SA, et al. J Med Ethics 2020;0:1–5. doi:10.1136/medethics-2020-106343 2 on November 15, 2020 by guest. Protected by
http://jme.bmj.com/
J Med Ethics: first published as 10.1136/medethics-2020-106343 on 10 November 2020. Downloaded from Clinical ethics The implications of these powers, which are not duties such
as those considered in the previous section, to breach confiden-
tiality in the public interest can be considered against the frame-
work set out in the General Medical Council (GMC) Guidance
on Confidentiality. LEGAL POSITIONS
Th
h This was drawn up in order to ensure doctors
complied with UK law, including international human rights
requirements such as privacy.36 The GMC advises that when
considering breaching confidentiality, it is necessary to balance
various factors that point against disclosure, including the
‘potential harm or distress to the patient arising from the disclo-
sure’, harm to trust in professionals and the views expressed by
the patient.36 The GMC recognises breaching confidentiality
is exceptional and should only occur if ‘failure to disclose the
information would leave individuals or society exposed to a risk
so serious that it outweighs the patient’s and the public interest
in maintaining confidentiality’36 (i.e., both criteria are fulfilled). We have already seen that removing control from the partici-
pants itself causes harm and this raises the threshold required for
disclosure against a patient’s objection, which the GMC suggests
can only be justified in the public interest in cases where ‘failure
to do so would leave others at risk of death or serious harm’.36
It follows that the state interest in investigating crime is insuffi-
cient to justify overriding a participant who wishes to maintain
confidentiality. victim-survivors of CSA, but also be aware of the legal frame-
works which apply to the jurisdiction in which the research is
intended to be conducted. RECs should also draw researchers’
attention to the legal jurisdiction and its supposed ‘obligations’. We argue for a presumption that—in the UK at least—disclosure
would be unethical whatever the legal framework. Readers or
researchers working within other legal systems must carefully
review the issue before planning to start. The research itself might
become unethical (and re-traumatising) if the legal framework is
not understood in advance, does oblige mandatory reporting, or
is not made crystal clear to participants before engaging with the
research. Research ethics committees should ensure researchers
are able to offer all participants advice about, and appropriate
signposting to, a full range of current, independent agencies and
sources of support available. This can be coupled with ensuring
researchers know what to do and how best to facilitate should
participants wish to make contact with relevant agencies if they
are not already linked into them. What we strongly oppose,
however, is the enforcement of rigid protocols which encourage
researchers to follow ‘support to report’ pathways, or breach
participants’ confidentiality in order to report all disclosures. CONCLUSION
Childh
d Childhood sexual abuse has long existed, but been taboo, and
thus neglected, ignored or deemed too controversial to examine. Research must aim to understand better the experience of survi-
vors. Those who have experienced CSA belong to a group whose
voices are seldom heard, and research to better understand their
experiences should be undertaken in an appropriate and sensi-
tive manner. The analysis we present above, depicts a UK REC’s
suggestion that there was an ‘obligation’ or ‘duty to report’
which was in fact misconceived in law, ethics, and policy terms. We believe the proper response to the REC’s question is that
when a researcher receives a first-time disclosure of non-recent
(historic) CSA, they should generally respect the participants’
decision on whether a report should be made to the police or
safeguarding authorities. Only where there is clear evidence of
current risk should they consider breaching confidentiality, and
if they believe this to be the case, they should seek advice on an
anonymous basis. They should not proceed directly to disclosure. k
b
d
d on November 15, 2020 by guest. Protected by copyright. http://jme.bmj.com/
om LEGAL POSITIONS
Th
h Likewise, it is important for RECs to assess and ensure the
suitability of the researchers undertaking sensitive projects and
that of their own support networks or supervision.11 37 This is
important especially for times when conducting a sensitive inter-
view becomes emotionally intensive or exhaustive,38 challenging
to conduct,39 or indeed, has made the researcher question
whether or not there is a need to take action which would result
in the breach of the participant’s confidentiality.12 It is therefore
important for members of RECs to understand the complexity
of such sensitive research, and draw on both principles of ethics
and law when reaching decisions on whether or not to grant
ethical approval. The only plausible situation for a public interest disclosure
in the context of research is when the participant-survivor
discloses that the perpetrator of their own CSA poses a current
risk to vulnerable people (especially children aged under 18). However, researchers will rarely have sufficient information to
make a proper assessment of this risk and it would be unethical
to proceed from suspicion to breach of confidentiality without
seeking advice. This can usually be obtained on an anonymous
basis (without compromising the participant’s privacy) within
UK health providers via Caldicott Guardians, or through safe-
guarding processes. We recommend this step should precede any
non-consented reporting of disclosures in order to ensure the
balance is properly struck, and the researcher’s response is there-
fore appropriate and proportional. on November 15, 2020 by guest. P
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November 2020. Downloaded from Data availability statement There are no data associated with this work. Data availability statement There are no data associated with this work. i
q
17 Elmir R, Schmied V, Jackson D, et al. Interviewing people about potentially sensitive
topics. Nurse Res 2011;19(1):12–16. Author note SAS has expertise in qualitative research and has undertaken
significant study of women’s mental health. CR and EM have undertaken research
with women who have experienced childhood sexual abuse, and CR has worked
in a therapeutic setting with survivors. FM leads The Survivors Trust, the largest
umbrella agency for specialist rape and sexual abuse services in the UK. JS and
YR have extensive research and clinical experience in women’s health, maternity
service delivery and provision of midwifery care, with JS holding many internationally
reaching research grants. SB and JM are experts in the fields of health care ethics
and health care law, with SB having extensive clinical and research experience in
women’s health and sexual offences examination. p
;
( )
18 McClain N, Amar AF. Female survivors of child sexual abuse: finding voice through
research participation. Issues Ment Health Nurs 2013;34(7):482–7.i 19 Longfield A. Protecting children from harm: a critical assessment of child sexual abuse
in the family network in England and priorities for action. London, United Kingdom
Children’s Commissioner; 2015: 1–109. https://www.childrenscommissioner.gov.uk/
report/protecting-children-from-harm/i p
p
g
20 Harrington B. Disclosure of child sexual abuse. maintaining confidentiality is not in
best interest of woman or others. BMJ 1998;317(7152):208. 21 David TJ, Wynne G, Kessel AS, et al. Child sexual abuse: when a doctor’s duty
to report abuse conflicts with a duty of confidentiality to the victim. BMJ
1998;316(7124):55–7. Supplemental material This content has been supplied by the author(s). It
has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have
been peer-reviewed. Any opinions or recommendations discussed are solely those
of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and
responsibility arising from any reliance placed on the content. Where the content
includes any translated material, BMJ does not warrant the accuracy and reliability
of the translations (including but not limited to local regulations, clinical guidelines,
terminology, drug names and drug dosages), and is not responsible for any error
and/or omissions arising from translation and adaptation or otherwise. ;
(
)
22 Richards HM, Schwartz LJ. Ethics of qualitative research: are there special issues for
health services research? Fam Pract 2002;19(2):135–9.i 23 Independent Inquiry into Child Sexual Abuse. Data availability statement There are no data associated with this work. Seminar briefing note. mandatory
reporting: existing obligations to report child sexual abuse. London, United Kingdom
Independent Inquiry into Child Sexual Abuse; 2018: 1–6. https://www.iicsa.org.
uk/key-documents/7025/view/mandatory-reporting-seminar-existing-obligations-
reporting-child-sexual-abuse.-briefing-note.pdf p
gi
g
p
24 Home Office. Reporting and acting on child abuse and neglect: summary of
consultation responses and government action.. London, United Kingdom HM
Government; 2018: 1–35. https://assets.publishing.service.gov.uk/government/
uploads/system/uploads/attachment_data/file/685463/Reporting_and_acting_on_
child_abuse_and_neglect_-_response_print.pdf Open access This is an open access article distributed in accordance with the
Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits
others to copy, redistribute, remix, transform and build upon this work for any
purpose, provided the original work is properly cited, a link to the licence is given,
and indication of whether changes were made. See: https://creativecommons.org/
licenses/by/4.0/. 25 Independent Inquiry into Child Sexual Abuse. Mandatory reporting seminar 1 –
existing obligations to report child sexual abuse: a summary report. London, United
Kingdom Independent Inquiry into Child Sexual Abuse; 2018: 1–14. https://www.
iicsa.org.uk/key-documents/8725/view/mandatory-reporting-seminar-one-summary-
report.pdf on November 15, 2020 by guest
http://jme.bmj.com/
J Med Ethics: first published as 10.1136/medethics-2020-106343 on 10 November 2020. Downloaded from Elsa Montgomery http://orcid.org/0000-0002-4193-1261
Chelsey Roberts http://orcid.org/0000-0002-1960-1514
Yana Richens http://orcid.org/orcid.org/0000-0002-8280-4912
Jane Sandall http://orcid.org/0000-0003-2000-743X
Jonathan Montgomery http://orcid.org/0000-0002-4592-0930 to child protection authorities is an important ethical founda-
tion enabling them to trust researchers. In the case we describe
above, the UK REC to which we applied for ethical scrutiny and
approvals asserted a (mythological) blanket rule which would
have prevented the researchers responding sensitively to a
participant who chose to disclose the experience of abuse at an
‘ethically important moment’.40 Such a rule would undermine,
rather than promote good research ethics. Silverio SA, et al. J Med Ethics 2020;0:1–5. doi:10.1136/medethics-2020-106343 REFERENCES
h
b 1 Irish L, Kobayashi I, Delahanty DL. Long-term physical health consequences of
childhood sexual abuse: a meta-analytic review. J Pediatr Psychol 2010;35(5):450–61. 1 Irish L, Kobayashi I, Delahanty DL. Long-term physical health consequences of
childhood sexual abuse: a meta-analytic review. J Pediatr Psychol 2010;35(5):450–61. Twitter Sergio A. Silverio @Silverio_SA_, Susan Bewley @susan_bewley, Elsa
Montgomery @elsamwm, Yana Richens @Yanarichens, Jane Sandall @SandallJane
and Jonathan Montgomery @Prof_JonMont Twitter Sergio A. Silverio @Silverio_SA_, Susan Bewley @susan_bewley, Elsa
Montgomery @elsamwm, Yana Richens @Yanarichens, Jane Sandall @SandallJane
and Jonathan Montgomery @Prof_JonMont 2 Byrne J, Smart C, Watson G. "I felt like i was being abused all over again": how
survivors of child sexual abuse make sense of the perinatal period through their
narratives. J Child Sex Abus 2017;26(4):465–86. 2 Byrne J, Smart C, Watson G. "I felt like i was being abused all over again": how
survivors of child sexual abuse make sense of the perinatal period through their
narratives. J Child Sex Abus 2017;26(4):465–86. 3 Oliveira AGESde, Reichenheim ME, Moraes CL, et al. Childhood sexual abuse, intimate
partner violence during pregnancy, and posttraumatic stress symptoms following
childbirth: a path analysis. Arch Womens Ment Health 2017;20(2):297–309. Acknowledgements The authors would like to first thank all participant-survivors
who have already taken part and will, in the future, take part in studies addressing
this area of research. We would also like to extend a special thanks to Ms S i Acknowledgements The authors would like to first thank all participant-survivors
who have already taken part and will, in the future, take part in studies addressing
this area of research. We would also like to extend a special thanks to Ms S
Henley (Barnet, Enfield and Harringay Mental Health NHS Trust), Professor J Read
(University of East London) and Mr B Wright (The British Psychological Society), who
all provided invaluable information on the current published guidance for research
involving participant-survivors. Acknowledgements The authors would like to first thank all participant-survivors
who have already taken part and will, in the future, take part in studies addressing
this area of research. We would also like to extend a special thanks to Ms S
Henley (Barnet, Enfield and Harringay Mental Health NHS Trust), Professor J Read
(University of East London) and Mr B Wright (The British Psychological Society), who
all provided invaluable information on the current published guidance for research
involving participant-survivors. p
y
4 Montgomery E, Pope C, Rogers J. REFERENCES
h
b The prevalence of child sexual abuse in community
and student samples: a meta-analysis. Clin Psychol Rev 2009;29(4):328–38. 8 Pereda N, Guilera G, Forns M, et al. The prevalence of child sexual abuse in community
and student samples: a meta-analysis. Clin Psychol Rev 2009;29(4):328–38. 9 Leclerc B, Wortley R. Predictors of victim disclosure in child sexual abuse: additional
evidence from a sample of incarcerated adult sex offenders. Child Abuse Negl
2015;43:104–11. 9 Leclerc B, Wortley R. Predictors of victim disclosure in child sexual abuse: additional
evidence from a sample of incarcerated adult sex offenders. Child Abuse Negl
2015;43:104–11. Disclaimer The views expressed are those of the authors and not necessarily those
of the NIHR or the Department of Health and Social Care. 10 Mathews B, Collin-Vézina D. Child sexual abuse: raising awareness and empathy
is essential to promote new public health responses. J Public Health Policy
2016;37(3):304–14. Competing interests JM was Chair of the Health Research Authority from 2012-
19 and Chair of the Task and Finish Group that supported the development of the
GMC’s guidance on Confidentiality: Good practice in handling patient information
(2017). ;
( )
11 Dickson-Swift V, James EL, Kippen S, et al. Risk to researchers in qualitative research
on sensitive topics: issues and strategies. Qual Health Res 2008;18(1):133–44. 12 All
k P B
t J Ch
b
E
t l Ethi
l i
i th
f i d
th i t
i 12 Allmark P, Boote J, Chambers E, et al. Ethical issues in the use of in-depth interviews:
literature review and discussion. Res Ethics 2009;5(2):48–54. Patient and public involvement statement We are also grateful to The
Survivors Trust for their input to this paper. 13 Newman E, Risch E, Kassam-Adams N. Ethical issues in trauma-related research: a
review. J Empir Res Hum Res Ethics 2006;1(3):29–46. Patient consent for publication Not required. p
14 Hewitt J. Ethical components of researcher researched relationships in qualitative
interviewing. Qual Health Res 2007;17(8):1149–59. Ethics approval Not required. 15 Orb A, Eisenhauer L, Wynaden D. Ethics in qualitative research. J Nurs Scholarsh
2001;33(1):93–6.i Provenance and peer review Not commissioned; externally peer reviewed. 16 Opsal T, Wolgemuth J, Cross J, et al. "There are no known benefits ….": considering
the risk/benefit ratio of qualitative research. Qual Health Res 2016;26(8):1137–50. REFERENCES
h
b The re-enactment of childhood sexual abuse in
maternity care: a qualitative study. BMC Pregnancy Childbirth 2015;15(1):1–7. p
y
4 Montgomery E, Pope C, Rogers J. The re-enactment of childhood sexual abuse in
maternity care: a qualitative study. BMC Pregnancy Childbirth 2015;15(1):1–7. 4 Montgomery E, Pope C, Rogers J. The re-enactment of childhood sexual abuse in
maternity care: a qualitative study. BMC Pregnancy Childbirth 2015;15(1):1–7. 5 Beitchman JH, Zucker KJ, Hood JE, et al. A review of the long-term effects of child
sexual abuse. Child Abuse Negl 1992;16(1):101–18. p
Henley (Barnet, Enfield and Harringay Mental Health NHS Trust), Professor J Read
(University of East London) and Mr B Wright (The British Psychological Society), who
all provided invaluable information on the current published guidance for research
involving participant-survivors. maternity care: a qualitative study. BMC Pregnancy Childbirth 2015;15(1):1 7. 5 Beitchman JH, Zucker KJ, Hood JE, et al. A review of the long-term effects of child
sexual abuse. Child Abuse Negl 1992;16(1):101–18. y
q
y
g
y
5 Beitchman JH, Zucker KJ, Hood JE, et al. A review of the long-term effects of child
sexual abuse. Child Abuse Negl 1992;16(1):101–18. 6 Rouf K, Waites B, Weatherhead S, McKail R, Manser R. Guidance document on the
management of disclosures of non-recent (historic) child sexual abuse. Leicester,
United Kingdom The British Psychological Society; 2016: 1–32. https://www.bps.org.
uk/news-and-policy/guidance-management-disclosures-non-recent-historic-child-
sexual-abuse-2016 Contributors The paper was developed after receiving feedback on a research
ethics committee application for a study devised by CR, SAS and YR. The initial idea
for the paper was developed by SAS, JS, SB and EM. SAS prepared the first draft of
the paper, with SB and JM re-drafting sections, and EM and FM providing substantial
revisions. JS, YR and CR provided extensive feedback. Together the final version was
produced. 7 Radford L, Corral S, Bradley C, et al. The prevalence and impact of child maltreatment
and other types of victimization in the UK: findings from a population survey
of caregivers, children and young people and young adults. Child Abuse Negl
2013;37(10):801–13. Funding JS (King’s College London) is an NIHR Senior Investigator and with SAS
(King’s College London) is supported by the National Institute for Health Research
Applied Research Collaboration South London (NIHR ARC South London) at King’s
College Hospital NHS Foundation Trust. on November 15, 2020 by guest. Pro
http://jme.bmj.com/
10 November 2020. Downloaded from 8 Pereda N, Guilera G, Forns M, et al. Sergio A. Silverio http://orcid.org/0000-0001-7177-3471
Susan Bewley http://orcid.org/0000-0001-8064-652X RECOMMENDATIONS FOR RESEARCHERS Researchers in the UK have no legal or ethical duty to report
disclosures of previously unreported non-recent (historic) cases
of CSA by their participants. We have shown why we believe
that this is probably also the case for Australia where manda-
tory reporting laws exist. We recommend that reports should
not generally be made without the consent of research partici-
pants because to take away their control over disclosure is likely
to be particularly damaging in this context. Researchers should
only consider reporting these disclosures if they have reason to
believe the reported perpetrator of the abuse has access to, and
therefore may pose a current risk to, vulnerable persons (espe-
cially children under the age of 18). This would be an example
of the ‘public interest’ ground for breach of confidentiality. We
recommend if researchers believe there are exceptional circum-
stances which make the risk to vulnerable persons so immediate,
such that disclosure without consent may be necessary, the safety
risk should first be assessed with an independent agency (e.g.,
social services or police) on an anonymous basis. mber 15, 2020 by guest. Protected by copyright. Our key concerns about introducing mandatory reporting are
that it would undermine people’s control over the (re)telling of
their stories, make them even more reluctant to participate in
research, and further silence the voices of people who should
be heard, without prejudice and without fear of losing control
of their own story and survivorship. We have illustrated how
mandatory reporting systems do recognise reasonable excep-
tions to the obligation to share information and why it is usually
reasonable to preserve the confidentiality of disclosures by
adults, of abuse they have experienced in the past. Respect for
participants’ need to control whether or not data are reported guest. Protected by copyright. CONSIDERATIONS FOR RESEARCH ETHICS COMMITTEES
It is important for RECs to not only assess the ethical rigour
of any proposed research project, including those involving 3 Silverio SA, et al. J Med Ethics 2020;0:1–5. doi:10.1136/medethics-2020-106343 Clinical ethics ORCID iDs
l Sergio A. Silverio http://orcid.org/0000-0001-7177-3471
Susan Bewley http://orcid.org/0000-0001-8064-652X p
p
26 Independent Inquiry into Child Sexual Abuse. Mandatory reporting seminar 2: a
summary report. London, United Kingdom Independent Inquiry into Child Sexual p
p
26 Independent Inquiry into Child Sexual Abuse. Mandatory reporting seminar 2: a
summary report. London, United Kingdom Independent Inquiry into Child Sexual Silverio SA, et al. J Med Ethics 2020;0:1–5. doi:10.1136/medethics-2020-106343 4 Clinical ethics Clinical Abuse; 2019: 1–17. https://www.iicsa.org.uk/key-documents/13689/view/mandatory-
reporting-seminar-two-summary-report.pdf Abuse; 2019: 1–17. https://www.iicsa.org.uk/key-documents/13689/view/mandatory-
reporting-seminar-two-summary-report.pdf 33 Attorney General, Minister for the Prevention of Domestic Violence. Crimes Act 1900
No. 40, New South Wales, section 316A(f). New South Wales, Australia Parliamentary
Counsel’s Office; 2020: 1–309. https://www.legislation.nsw.gov.au/view/html/inforce/
current/act-1900-040 p
g
y
p
p
27 Matthews B. A model law for the mandatory reporting of child sexual abuse in
England and Wales. Brisbane, Australia Queensland University of Technology;
2020: 1–15. https://www.iicsa.org.uk/document/professor-ben-mathews-model-law-
mandatory-reporting-child-sexual-abuse-england-and-wales 34 Ministry of Justice. Privacy Act 2020, New Zealand, s 22 Principle 11(1)(f). Wellington,
New Zealand New Zealand Govenment; 2020: 1–192. http://www.legislation.govt.
nz/act/public/2020/0031/latest/LMS23223.html mandatory-reporting-child-sexual-abuse-england-and-wales y
p
g
g
28 HM Goverment. Female genital mutilation act 2003, s 5B, inserted by s 71 serious
crimes act 2015. London, United Kingdom Home Office; 2015: 1–129. https://www.
legislation.gov.uk/ukpga/2015/9/part/5/crossheading/female-genital-mutilation/
enacted 35 Department of Health Informatics. Confidentiality: NHS code of practice. Supplementary guidance: public interest disclosures. Leeds, United Kingdom
Department of Health; 2010: 1–15. https://www.gov.uk/government/publications/
confidentiality-nhs-code-of-practice-supplementary-guidance-public-interest-
disclosures 29 Home Office, Department for Education. Mandatory reporting of female genital
mutilation – procedural information (2016) para 2.1b. London, United Kingdom
Home Office; 2015: 1–20. https://www.gov.uk/government/publications/mandatory-
reporting-of-female-genital-mutilation-procedural-information 36 General Medical Council. Confidentiality: good practice in handling patient
information. Manchester, United Kingdom General Medical Council; 2017: 1–78. https://www.gmc-uk.org/ethical-guidance/ethical-guidance-for-doctors/
confidentiality p
g
g
p
30 National Assembly for Wales. Social services and well-being (Wales) act 2014, s 130. Cardiff, Wales, United Kingdom National Assembly for Wales; 2014: 1–208. https://
www.legislation.gov.uk/anaw/2014/4/contents 37 Limerick B, Burgess‐Limerick T, Grace M. The politics of interviewing: power relations
and accepting the gift. Int J Qual Stud Educ 1996;9(4):449–60. g
g
31 Royal Commission into Institutional Responses to Child Sexual Abuse. Final report. Barton, Australian Capital Territory, Australia Attorney-General’s Department; 2017. https://www.childabuseroyalcommission.gov.au/final-report 38 Kumar S, Cavallaro L. Researcher self-care in emotionally demanding research: a
proposed conceptual framework. Qual Health Res 2018;28(4):648–58. p
y
gi
p
32 Department of the Attorney-General and Justice, Department of Territory Families,
Housing and Communities. Care and Protection of Children Act 2007, Northern
Territories of Australia, s. 26(3). Silverio SA, et al. J Med Ethics 2020;0:1–5. doi:10.1136/medethics-2020-106343 ORCID iDs
l Northern Territory, Australia Northern Territory
Government; 2020: 1–166. https://legislation.nt.gov.au/en/Legislation/CARE-AND-
PROTECTION-OF-CHILDREN-ACT-2007 p p
p
39 Sands RG, Krumer-Nevo M. Interview shocks and Shockwaves. Qual Inquiry
2006;12(5):950–71. 40 Guillemin M, Gillam L. Ethics, reflexivity, and “ethically important moments” in
research. Qual Inquiry 2004;10(2):261–80. Silverio SA, et al. J Med Ethics 2020;0:1–5. doi:10.1136/medethics-2020-106343 5
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A post-decisional neural marker of confidence predicts information-seeking in decision-making
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bioRxiv (Cold Spring Harbor Laboratory)
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A Postdecisional Neural Marker of Confidence Predicts
Information-Seeking in Decision-Making XKobe Desender,1,2 XPeter Murphy,1 XAnnika Boldt,3 XTom Verguts,2 and XNick Yeung4
1Department of Neurophysiology and Pathophysiology, University Medical Center Hamburg-Eppendorf, Hamburg-Eppendorf 20251, Germany,
2Department of Experimental Psychology, Ghent University, Ghent 9000, Belgium, 3Institute of Cognitive Neuroscience, University College London, London
WC1N 3AZ, United Kingdom, and 4Department of Experimental Psychology, University of Oxford, Oxford OX2 6HG, United Kingdom XKobe Desender,1,2 XPeter Murphy,1 XAnnika Boldt,3 XTom Verguts,2 and XNick Yeung4
1Department of Neurophysiology and Pathophysiology, University Medical Center Hamburg-Eppendorf, Hamburg-Eppendorf 20251, Germany,
2Department of Experimental Psychology, Ghent University, Ghent 9000, Belgium, 3Institute of Cognitive Neuroscience, University College London, London
WC1N 3AZ, United Kingdom, and 4Department of Experimental Psychology, University of Oxford, Oxford OX2 6HG, United Kingdom Theoretical work predicts that decisions made with low confidence should lead to increased information-seeking. This is an adaptive
strategy because it can increase the quality of a decision, and previous behavioral work has shown that decision-makers engage in such
confidence-driven information-seeking. The present study aimed to characterize the neural markers that mediate the relationship
between confidence and information-seeking. A paradigm was used in which 17 human participants (9 male) made an initial perceptual
decision, and then decided whether or not they wanted to sample more evidence before committing to a final decision and confidence
judgment.Predecisionalandpostdecisionalevent-relatedpotentialcomponentsweresimilarlymodulatedbythelevelofconfidenceand
by information-seeking choices. Time-resolved multivariate decoding of scalp EEG signals first revealed that both information-seeking
choices and decision confidence could be decoded from the time of the initial decision to the time of the subsequent information-seeking
choice(within-conditiondecoding).Noabove-chancedecodingwasvisibleinthepreresponsetimewindow.Crucially,aclassifiertrained
to decode high versus low confidence predicted information-seeking choices after the initial perceptual decision (across-condition
decoding).Thistimewindowcorrespondstothatofapostdecisionalneuralmarkerofconfidence.Collectively,ourfindingsdemonstrate,
for the first time, that neural indices of confidence are functionally involved in information-seeking decisions. Key words: confidence; decision-making; error positivity; information sampling; information-seeking; metacognition Despite substantial current interest in neural signatures of our sense of confidence, it remains largely unknown how confidence is
used to regulate behavior. Here, we devised a task in which human participants could decide whether or not to sample additional
decision-relevant information at a small monetary cost. Using neural recordings, we could predict such information-seeking
choices based on a neural signature of decision confidence. Our study illuminates a neural link between decision confidence and
adaptive behavioral control. 2010) and medical (Butler et al., 2009) decisions, as well as in
more carefully controlled laboratory settings (Sniezek and Van
Swol, 2001). A Postdecisional Neural Marker of Confidence Predicts
Information-Seeking in Decision-Making Recent evidence suggests that information-seeking
depends crucially on explicit representation of decision confi-
dence (Desender et al., 2018). Theoretically, decision confidence
has been treated as an internal evaluation signal that can be used
to adapt behavior in the absence of external feedback (Yeung and
Summerfield, 2012; Meyniel et al., 2015). When confidence in a
decision is low, this implies that the probability of a decision being
correct is also low, and seeking additional information before com-
mitting to a decision might be particularly beneficial. In a previous
behavioralstudy,participantsengagedmoreininformation-seeking
in conditions associated with low compared with high confidence,
despite equal accuracy in both (Desender et al., 2018). The Journal of Neuroscience, April 24, 2019 • 39(17):3309–3319 • 3309 The Journal of Neuroscience, April 24, 2019 • 39(17):3309–3319 • 3309 Behavioral/Cognitive Received Oct. 10, 2018; revised Jan. 30, 2019; accepted Feb. 2, 2019.
Author contributions: K.D., A.B., and N.Y. designed research; K.D. performed research; K.D. analyzed data; K.D.
wrote the paper; P.M., T.V., and N.Y. contributed unpublished reagents/analytic tools; P.M., A.B., T.V., and N.Y.
edited the paper.
K.D. is an FWO (PEGASUS) Marie Sklodowska-Curie Fellow (Grant 12T9717N), and was supported by Research
Foundation Flanders, Belgium (FWO-Vlaanderen, Grants 11H3415N and V447115N). A.B. was supported by an
EconomicandSocialResearchCouncilUnitedKingdomPhDstudentship.WethankKerstinFro¨berforassistancewith
EEG prepping; and Mehdi Senoussi for advice on the decoding analyses.
The authors declare no competing financial interests.
Correspondence should be addressed to Kobe Desender at Kobe.Desender@gmail.com.
https://doi.org/10.1523/JNEUROSCI.2620-18.2019
Copyright © 2019 Desender et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution License
Creative Commons Attribution 4.0 International, which permits unrestricted use, distribution and reproduction in
anymediumprovidedthattheoriginalworkisproperlyattributed. Received Oct. 10, 2018; revised Jan. 30, 2019; accepted Feb. 2, 2019. Stimuli and apparatus
S i
li
d Stimuli and apparatus
Stimuli were presented on a gray background on a 20-inch CRT monitor
with a 75 Hz refresh rate, using the MATLAB toolbox Psychtoolbox3. Each stimulus consisted of eight colored shapes spaced regularly around
a fixation point (radius 2.8° visual arc). To manipulate task difficulty, the
mean and the variance of the eight elements varied across trials. The mean color of the eight shapes was determined by the variable C; the
variance across the eight shapes was determined by the variable V. The
mean color of the stimuli varied between red (1, 0, 0) and blue (0, 0, 1)
along a linear path in RGB space (C, 0, 1 C). At the start of the
experiment, C could take four different values: 0.450, 0.474, 0.526, and
0.550 (from blue to red, with 0.5 being the category boundary), and V
could take two different values: 0.0333 and 0.1000 (low and high vari-
ance, respectively). The current task was used because previous research
has shown it can dissociate subjective confidence from objective accu-
racy, by matching the difficulty of two conditions that either have a low
mean (i.e., average color close to the category boundary) or a high vari-
ance (i.e., a varied mix of colors across the eight shapes in the display),
with confidence found to be systematically lower in the latter case (Boldt
et al., 2017a; Desender et al., 2018). On every trial, the color of each
individual element was pseudo-randomly selected with the constraint
that the mean and variance of the eight elements closely matched the
mean of C and its variance V, respectively. Each combination of C and V
values occurred equally often. The individual elements did not vary in
shape. Responses were made using a USB mouse and a standard
QWERTY keyboard. The current study aimed to identify neural signatures of con-
fidence that are predictive of information-seeking behavior. In
our experimental paradigm, on each trial, participants made an
initial perceptual decision about the mean color of eight visual
elements, and then decided whether or not to sample additional
evidence (at a small cost) before committing to a final decision. Electrophysiological recordings allowed us to evaluate which
neural signatures of confidence were related to information-
seeking choices. To do so, we relied on time-resolved decoding of
EEG data to test for shared neural coding of confidence and de-
cisions to seek additional information. Introduction Humans seek information adaptively to improve the quality of
their decisions, for example, by requesting expert advice when
they lack the relevant domain knowledge or discriminating evi-
dence (for review, see Bonaccio and Dalal, 2006). This tendency
has been documented for everyday financial (Hung and Yoong, Author contributions: K.D., A.B., and N.Y. designed research; K.D. performed research; K.D. analyzed data; K.D. wrote the paper; P.M., T.V., and N.Y. contributed unpublished reagents/analytic tools; P.M., A.B., T.V., and N.Y. edited the paper. K.D. is an FWO (PEGASUS) Marie Sklodowska-Curie Fellow (Grant 12T9717N), and was supported by Research
Foundation Flanders, Belgium (FWO-Vlaanderen, Grants 11H3415N and V447115N). A.B. was supported by an
EconomicandSocialResearchCouncilUnitedKingdomPhDstudentship.WethankKerstinFro¨berforassistancewith
EEG prepping; and Mehdi Senoussi for advice on the decoding analyses. At present, however, it remains unclear how the neural coding
of confidence informs the decision to engage in additional
information-seeking. It has been argued that decision confidence
reflects the strength of the evidence in favor of a decision (Vick-
ers, 1979; Zylberberg et al., 2012), stressing the importance of Correspondence should be addressed to Kobe Desender at Kobe.Desender@gmail.com. Correspondence should be addressed to Kobe Desender at Kobe.Desender@gmail.com. https://doi.org/10.1523/JNEUROSCI.2620-18.2019 This is an open-access article distributed under the terms of the Creative Commons Attribution License
Creative Commons Attribution 4.0 International, which permits unrestricted use, distribution and reproduction in
anymediumprovidedthattheoriginalworkisproperlyattributed. Figure 1. Timeline of an experimental trial. A stimulus was presented for 200 ms, and participants made a speeded response with the mouse, deciding whether the average color of the eight
elementswasredorblue.Onfree-choicetrials(75%),participantssubsequentlyusedaverticalslidertochooseeithertoseethestimulusagaininaneasierversion(bymovingthegraycursortoward
S) or to give their response (by moving the gray cursor toward R). When the stimulus is shown again, the mean of the eight elements is more clearly red and the variance is smaller (note that the
displayedchangeisexaggeratedforillustrationpurposes).Onno-choicetrials(25%),participantscouldonlychoosetogivetheirresponse.Finally,onalltrials,participantsjointlyindicatedtheirfinal
responseandlevelofconfidenceonahorizontalcontinuousresponsescale.Beingaccuratewasrewarded(5points),errorswerepunished(5points),andtherewasasmallcostassociatedwith
sampling more information (1 point). 3310 • J. Neurosci., April 24, 2019 • 39(17):3309–3319
Desender et al. • Postdecisional Neural Marker of Confidence 3310 • J. Neurosci., April 24, 2019 • 39(17):3309–3319
Desender et al. • Postdecisional Neural Marker of Confidence 3310 • J. Neurosci., April 24, 2019 • 39(17):3309–3319 Desender et al. • Postdecisional Neural Marker of Confidence Figure 1. Timeline of an experimental trial. A stimulus was presented for 200 ms, and participants made a speeded response with the mouse, deciding whether the average color of the eight
elementswasredorblue.Onfree-choicetrials(75%),participantssubsequentlyusedaverticalslidertochooseeithertoseethestimulusagaininaneasierversion(bymovingthegraycursortoward
S) or to give their response (by moving the gray cursor toward R). Stimuli and apparatus
S i
li
d Specifically, we tested
whether a multivariate classifier trained to decode confidence
from EEG data would be predictive of information-seeking
choices. Such between-condition generalization isolates neural
processes integral to translating decision confidence into the
overt decision to sample additional information. Our main ques-
tion of interest was whether information-seeking choices could
be predicted based on neural markers of confidence observed
predecisional (P3), postdecisional (Pe), or both. Procedure
i
h Figure 1 shows an example trial during the main part of the experiment. After a 200 ms fixation interval, the stimulus was flashed for 200 ms. Participants were instructed to respond as quickly as possible, deciding
whether the average color of the eight elements was more blue or more
red, by clicking one of two mouse buttons. The mapping between color
and response was counterbalanced between participants. After a 200 ms
postresponse interval, there was a choice phase consisting of two condi- Introduction When the stimulus is shown again, the mean of the eight elements is more clearly red and the variance is smaller (note that the
displayedchangeisexaggeratedforillustrationpurposes).Onno-choicetrials(25%),participantscouldonlychoosetogivetheirresponse.Finally,onalltrials,participantsjointlyindicatedtheirfinal
responseandlevelofconfidenceonahorizontalcontinuousresponsescale.Beingaccuratewasrewarded(5points),errorswerepunished(5points),andtherewasasmallcostassociatedwith
sampling more information (1 point). (£20 plus up to £4.92 dependent on performance, range of the rounded
actual payments: £22-£24). The data of 2 participants were excluded
because accuracy of the primary responses was at chance level (48.9%
and 49.6% correct); thus, the final sample comprised 15 participants. All
provided written informed consent, reported normal or corrected-to-
normal vision, and were naive with respect to the hypothesis. All proce-
dures were approved by the local ethics committee. predecisional evidence in the computation of confidence (Kiani
and Shadlen, 2009). On such a view, neural signals involved in the
decision process itself should be predictive of information-
seeking (e.g., the P3 component of the scalp-recorded EEG)
(O’Connell et al., 2012; Twomey et al., 2015). Alternatively, de-
cision confidence has been quantified as a function of continued
evidence accumulation following a decision (Pleskac and Buse-
meyer, 2010; Moran et al., 2015), stressing the importance of
postdecisional signals in the computation of confidence. Re-
cently, a postdecisional centroparietal positivity was found in
scalp EEG recordings that reflects postdecisional neural evidence
accumulation, informing judgments about the accuracy of the
preceding decision (Murphy et al., 2015). This signal is com-
monly referred to as the error positivity (Pe) (Nieuwenhuis et al.,
2001) and has been shown to reflect fine-grained variations in
decision confidence (Boldt and Yeung, 2015). Statistical analyses Statistical analyses
Behavioral analysis. To test whether our different measures of perfor-
mance lawfully scaled with difficulty, indices of performance were calcu-
lated separately for the factors mean and variance. This was done for
median response times (RTs) on correct trials and mean accuracy of the
primary response (calculated based on all trials), mean accuracy of the
secondary response, mean confidence on the secondary response (calcu-
lated on no-choice data), and the proportion of see again choices (calcu-
lated on free-choice data). A repeated-measures ANOVA with the factors
mean (high or low) and variance (high or low) was then performed on all
these indices. Participants gained 5 points for correct answers and lost 5 points for
errors. They could win up to an additional £4.92 by scoring points (650
points £1). Choosing to see the stimulus again in an easier version cost
1 point, giving participants an incentive to sample more information
only when the benefit of doing so (in terms of increasing the probability
of making a correct choice) outweighed this cost. Participants were ex-
plicitly instructed that they could score more points by strategic use of the
see again option. Next, we fitted a mixed-regression model to the data to test whether
confidence predicts information-seeking over and above other factors. This analysis cannot be performed at the trial level because confidence
and see again choices are measured on separate parts of the data (no-
choice vs free-choice, respectively). Therefore, for all variables, we com-
puted eight data points (2 levels of mean 2 levels of variance 2
colors), separately for each participant. Specifically, we computed
(1) mean confidence based on the no-choice data, (2) the proportion of
see again choices based on the free-choice data, (3) mean accuracy of the
primary response based on all data, and (4) median RTs on the primary
response based on all data, separately for the factors evidence variance
(high or low), evidence mean (high or low), and color (red or blue). Values were calculated separately for each color to partition the data in a
more fine-grained manner, but this variable was not taken into account
in the analyses. For ease of interpretation, low variability and high mean
were dummy coded as reference categories so that a positive effect of each
factor corresponds to an increase in difficulty. EEG recording and preprocessing g
p p
g
Participants sat in a dimly lit, electrically shielded room. EEG data were
recorded using a fabric cap (QuickCap, Neuroscan) with 32 channels, all
referenced to the right mastoid online. Vertical and horizontal electro-
oculogram was measured from above and below the left eye and the outer
canthi of both eyes. Impedance was kept at 50 k. The data were
continuously recorded using SynAmps2 amplifiers (Neuroscan), sam-
pled at 1000 Hz. Stimulus-locked data were baselined 100 ms to 0 ms
before stimulus onset. In addition, these data were aligned to the time of
the primary response while keeping the same prestimulus baseline. Inde-
pendently from this, the raw data were also locked to the primary re-
sponse with a baseline 100 to 0 ms before response onset. For analyses
locked to the information-seeking decision, the response-locked data
(keeping the same preresponse baseline) were realigned to the onset of
the information-seeking decision (i.e., the space bar press confirming the
decision). In preprocessing, segments containing gross artifacts were first
identified by visual inspection and removed. Next, eye blinks were re-
moved using independent component analysis, and segments containing
values 200 V were excluded using extreme value rejection. Bad
(noisy) channels were replaced by an interpolated weighted average from
surrounding electrodes using the EEGLAB toolbox (Delorme and
Makeig, 2004) in MATLAB (The MathWorks). Finally, segments con-
taining further artifacts, identified by visual inspection, were removed
before averaging. For plotting purposes only, data were filtered using a 10
Hz low pass filter. When participants chose to see the stimulus again, the values of the
stimulus were slightly altered so that the distance to the boundary was
higher (C C 0.01) and the variability lower (V V 0.0167),
making the discrimination easier. They were presented with a fixation
point for 400 ms, this easier stimulus for 200 ms, and a fixation point for
400 ms. When participants opted (or were forced) to give their response
without seeing the stimulus again, they simply viewed the fixation point
for the same total amount of time (1000 ms). Afterward, a horizontal response scale appeared (0.4° high and 9.0°
wide) with a slider (0.4° high and 0.1° wide) in the center. The left-hand
side of the bar was labeled as “sure blue,” and the right-hand side was
labeled as “sure red” (depending on the counterbalancing of the response
mapping). Materials and Methods
Participants Participants Seventeen participants (9 males, mean SD age: 24.1 3.0 years; range:
21–32 years) took part at Oxford University for monetary compensation J. Neurosci., April 24, 2019 • 39(17):3309–3319 • 3311 Desender et al. • Postdecisional Neural Marker of Confidence tions: free-choice or no-choice. On free-choice trials (75% of trials), the
letters R and S appeared, indicating that participants could either choose
to request additional evidence by seeing the stimulus again in an easier
version (S) or to give their response (R). They indicated their choice by
moving a gray slider up or down with their mouse toward their choice,
and confirmed by pressing the space bar (locations of R and S were fixed
across trials and counterbalanced across participants). On no-choice tri-
als (25% of the trials), only an R appeared, and participants were forced
to select the option to give their response. tention of creating two conditions with equated performance but differ-
ent levels of confidence (Boldt et al., 2017a; Desender et al., 2018). However, because in the current data there were small but consistent
performance differences between conditions and no significant differ-
ence in confidence (see Results), our key analyses were performed re-
gardless of condition. Importantly, however, additional analyses were
performed to rule out that our findings are driven by task difficulty. EEG recording and preprocessing EEG recording and preprocessing The location of the slider on the scale was translated into a
numerical score, ranging from 50 (sure blue) to 50 (sure red), with
every three screen-pixel increments (0.09°) resulting in a difference of 1
confidence point. Participants moved the cursor with their mouse to
indicate jointly their response and their level of confidence, and con-
firmed by pressing the space bar. A response could not be given when the
cursor was exactly in the middle (0 on the scale), so participants were
always forced to make the categorical judgment between red or blue. They were instructed to make this judgment at their own pace. Accuracy
for the final response was scored as a binary variable (i.e., ignoring con-
fidence level). Confidence was scored as the absolute value on the re-
sponse scale. To account for between-participant variation in use of the
confidence scale and drift in confidence judgments over the course of the
experiment, ratings were z-scored separately for each participant and
each block. Behavioral results This approach iden-
tifies the spatial distribution of scalp EEG activity in a given time window
that maximally distinguishes two classes to deliver a scalar estimate of
component amplitude. Three sets of analyses were performed. First, it
was tested how well information-seeking choices and how well decision
confidence can be decoded from EEG data (within-condition decoding). These analyses were a first step in identifying the time window during
which information-seeking choices and decision confidence are decod-
able. Confidence judgments on no-choice trials were median split into
high and low values. In both analyses, both the training and testing sets
were restricted to trials with correct responses on the primary decision
only, to dissociate the coding of information-seeking and confidence
from the coding for errors. A 10-fold cross-validation approach was used
to avoid overfitting. To reduce noise due to the random assignment of
single trials in each fold, we repeated this procedure 100 times, resulting
in 1000 classifier predictions per time point. These scores were then
averaged, and below we report these averaged classification values. For
the third set of analyses, we tested how well see again choices can be
decoded from a classifier trained to discriminate EEG activity associated
with differing levels of confidence. The decoder was trained to discrimi-
nate high versus low confidence judgments (on correct trials only, from
no-choice trials) and tested on see again versus respond choices on cor-
rect trials from free-choice data; thus, there was no overlap between
training and testing data. This approach was repeated 1000 times. Vari-
ability across iterations arose due to the random selection of data neces-
sary to obtain an equal number of high and low confidence trials in the
training data and an equal number of see again and respond trials in the
free-choice data. (t(14) 2.47, p 0.027), even though, if anything, they made
numerically fewer errors in the former condition (p 0.189). A
one-way ANOVA on see again proportions across the two
medium-difficulty conditions, including the accuracy difference
as a covariate, confirmed the main effect of condition (F(1,13)
8.92, p 0.011), which was not modulated by differences in
accuracy (F(1,13) 2.75, p 0.139). RTs were slower in the high
mean/high variance compared with the low mean/low variance
condition (t(14) 2.819, p 0.014), and unexpectedly, there
was no difference in confidence between these conditions (p
0.570). Behavioral results Performance, confidence, and information-seeking
We first confirmed that all behavioral measures (performance,
confidence, and information-seeking) scaled with task difficulty
(Fig. 2). For the primary perceptual decision (speeded color dis-
crimination) data, a repeated-measures ANOVA showed that
both median RTs on correct trials and mean accuracy were sig-
nificantly affected by color mean (RTs: F(1,14) 14.96, p 0.002;
accuracy: F(1,14) 72.94, p 0.001) and by color variance (RTs:
F(1,14) 27.89, p 0.001; accuracy: F(1,14) 38.37, p 0.001),
but not by their interaction (RTs: F 1; accuracy: F 1). Mean
accuracy of the final response (regardless of the level of confi-
dence), calculated on the data of the no-choice condition in
which participants were forced to respond without viewing the
stimulus again, was likewise affected by both color mean
(F(1,14) 45.55, p 0.001) and color variance (F(1,14) 23.56,
p 0.001), but the interaction failed to reach significance
(F(1,14) 4.32, p 0.056). Mean confidence, again calculated on
the data of the no-choice condition (to avoid effects of seeing the
stimulus again), was affected by color mean (F(1,14) 32.18, p
0.001) and color variance (F(1,14) 21.73, p 0.001), but not by
their interaction (F 1). Finally, on free-choice trials, partici-
pants on average chose to see the stimulus again in an easier
version on 43.2% (range 2%–94%) of the trials. The proportion
of see again choices was affected by mean (F(1,14) 21.75, p
0.001) and variance (F(1,14) 20.02, p 0.001), with no signifi-
cant interaction between these factors (F(1,14) 3.22, p 0.09). The large variation in see again choices did not correlate with
individual differences in overall mean confidence on forced
choice trials (r(13) 0.22, p 0.428, Bayes Factor 0.26) or
accuracy on the primary response (r(13) 0.36, p 0.18, BF
0.47). Replicating previous work (Desender et al., 2018), partici-
pants chose more often to see the stimulus again in the high
mean/high variance compared with the low mean/low variance
condition Time-resolved decoding. To test for overlap in the neural coding of
confidence and see again choices, a classifier was trained separately for
each participant using single-trial logistic regression based on the linear
derivation method introduced by Parra et al. (2005). Statistical analyses We used mixed-regression
modeling (using the lme4 package in R) (Bates et al., 2015) to construct
models of increasing complexity. For each model, random slopes were
added for all variables for which this significantly increased the fit com-
pared with that model without random slopes. When required, degrees
of freedom were estimated using Satterthwaite’s approximation (using
the lmerTest package) (Kuznetsova et al., 2014). The main part of the experiment comprised 10 blocks of 64 trials, with
balanced numbers of trials for each combination of mean and variance
separately for each trial type (free-choice vs no-choice), in pseudo-
randomized order. Each block started with 8 additional practice trials in
which the free- versus no-choice phase was omitted and participants
received auditory feedback on the accuracy of their responses. This was
done to maintain a stable color criterion over the course of the experi-
ment. Before the main part of the experiment, several practice blocks
were administered. In the first block (64 trials), participants practiced the
color judgment task, and only were to give a speeded response with the
mouse, with auditory feedback to signal decision accuracy. In Blocks 2
and 3 (64 trials each), the second response (including the confidence
judgment) was added to the task. No feedback was delivered during these
blocks, which served to familiarize participants with the confidence rat-
ing scale. Finally, practice Block 4 was identical to the main part of the
experiment. At the end of each block (starting from Block 2), the C value in the
low-mean condition was adjusted depending on performance in that
block. Specifically, an inverse efficiency score (median RT/p(correct))
was calculated for the condition with low mean and low variance and the
condition with high mean and high variance (across all trials). When the
difference between the two was 100/50/10 in absolute value, the C value
of the low-mean condition was adjusted by 0.0025/0.0012/0.0005, re-
spectively, depending on the sign of the difference to match performance
in these two conditions. This manipulation was performed with the in- Event-related potentials (ERPs). In a first set of analyses, we wanted to
confirm that our data showed the usual modulation of predecisional and
postdecisional event-related components as a function of decision con- 3312 • J. Neurosci., April 24, 2019 • 39(17):3309–3319 Desender et al. • Postdecisional Neural Marker of Confidence fidence. Statistical analyses Building on previous work, ERP indices of confidence and see
again choices were tested at electrode CPz (Boldt and Yeung, 2015). Significant time windows during which ERPs for high and low confi-
dence trials (or for respond and see again trials) differed from each other
were identified using a standard two-tailed within-subjects cluster-based
permutation test using custom code in MATLAB. Elements that were
adjacent and significant (element-level p 0.05) were collected in a
cluster. Cluster-level test statistics consisted of the absolute sum of t
values within each cluster, and these were compared with a null distribu-
tion of test statistics created by drawing 1000 random permutations of
the observed data. A cluster was considered significant when its (cluster-
level) p value was 0.05. To examine whether the modulation of the
ERPs by confidence extended over and above our manipulation of task
difficulty, multivariate regression analyses were performed on each time
point (separately for each participant) predicting single-trial EEG ampli-
tude at electrode CPz by the factors mean (high or low), variance (high or
low), the interaction between mean and variance, and confidence (high
or low). We then performed cluster-based permutation tests on the t
values associated with each factor to examine whether confidence ex-
plains unique variance in EEG data after task difficulty is accounted for. Ti
l
d d
di
T
f
l
i
h
l
di
f old of 0.05. Elements were considered as neighbors when they were (car-
dinally or diagonally) adjacent. Cluster-level test statistics consisted of
the absolute sum of t values within each cluster, and these were compared
with a null distribution of test statistics created by drawing 1000 random
permutations of the observed data. A cluster was considered significant
when its (cluster-level) p value was 0.05. Behavioral results Because the two conditions of medium difficulty did not
differ in terms of confidence, we analyzed the EEG data combined
across conditions and then performed additional analyses to
demonstrate that the observed effects are not explained by task
difficulty. To increase the signal-to-noise ratio of data used for decoding, classi-
fiers were trained on time-averaged signals within discrete temporal win-
dows (window width of 106 ms, moving in 10 ms increments along entire
epochs aligned to stimulus onset, initial decisions, and see again choices). The ability to successfully classify individual trials was quantified by cal-
culating the Az score, which gives the area under the receiver operating
characteristic curve, derived from signal detection theory (Stanislaw and
Todorov, 1999). Classifiers were trained and tested on each time point of
the EEG data. Thus, this method produces a 2D (training time testing
time) decoding performance matrix. Specific dynamics of mental repre-
sentations can be unraveled by evaluating the shape of the decoding
matrix (King and Dehaene, 2014). The statistical reliability of this decod-
ing was determined via a bootstrap procedure, comparing actual classi-
fication with classifier performance on trials with randomized condition
labels (1000 iterations with different randomizations), to provide an es-
timate of the null classification. Clusters were formed via paired-samples
t tests for the entire 2D matrix, comparing true and null classifications. Neighboring elements that passed a threshold value corresponding to an
(element-level) p value of 0.01 (two-tailed) were collected into a separate
cluster. The same results were obtained when using a more liberal thresh- The relation between decision confidence and information-seeking
To further interrogate potential sources of variability in observed
information-seeking behavior, mixed-regression models of in-
creasing complexity were fit to the data predicting variation in the
proportion of see again choices across conditions of the experi- J. Neurosci., April 24, 2019 • 39(17):3309–3319 • 3313 Desender et al. • Postdecisional Neural Marker of Confidence Figure2. Behavioralperformanceismodulatedbymeanandvariance.A,Meanaccuracyoftheprimaryresponse(basedonalldata).B,Medianreactiontimesoftheprimaryresponse(basedon
alldata).C,Meanaccuracyofthefinalresponse(basedonno-choicedata).D,Meanstandardizedconfidence(basedonno-choicedata).E,Thenumberoftrialsonwhichparticipantswaivedthesee
again option (based on free-choice data). Figure2. Behavioralperformanceismodulatedbymeanandvariance.A,Meanaccuracyoftheprimaryresponse(basedonalldata).B,Medianreactiontimesoftheprimaryresponse(basedon
alldata).C,Meanaccuracyofthefinalresponse(basedonno-choicedata).D,Meanstandardizedconfidence(basedonno-choicedata).E,Thenumberoftrialsonwhichparticipantswaivedthesee
again option (based on free-choice data). Table 1. Models of different complexity predicting information seeking
Model
df
BIC
Test
2
p
0A. Variance
4
37
—
—
—
0B. Mean
4
23
—
—
—
1. Variance
Mean
5
43
1 vs 0A
10.86
0.001
1 vs 0B
25.11
0.001
2. Variance
Mean
RT
Accuracy
12
118
2 vs 1
108.2
0.001
3. Behavioral results Variance
Mean
RT
Accuracy
Confidencea
13
122
3 vs 2
9.13
0.002
aWinning model. Table 1. Models of different complexity predicting information seeking amount of variation in information-seeking in simpler models,
their statistical contributions were largely accounted for by
confidence. ERP markers of confidence f
f
To examine how ERP waveforms are modulated by confidence,
correct trials in the no-choice condition were split into high and
low confidence bins via median-split separately for each partici-
pant. As can be seen in Figure 3A, at electrode CPz, the stimulus-
locked ERP showed significant modulation by confidence from
414 to 581 ms (p 0.016, cluster level), corresponding to a
typical P300 component (Hillyard et al., 1971; Polich, 2007). Mean amplitude was more positive for trials on which participants
laterindicatedhigh(vslow)confidence.Bycontrast,theERPaligned
to and following the primary response showed the opposite pattern:
more negative amplitudes for high confidence trials were observed
from 403 ms after response until the end of the analyzed epoch (700
ms; p 0.008, cluster level), corresponding to a typical Pe compo-
nent (Ridderinkhof et al., 2009). The topographies associated with
these significant time windows showed that these effects had a sim-
ilar centroparietal scalp distribution. mental design (see Materials and Methods) by five predictors: the
factors color variance (high or low) and color mean (high or low),
and the variables median primary RTs (both on free-choice and
no-choice), mean accuracy of the primary response (both on
free-choice and no-choice), and mean confidence (on no-choice
trials). A model building strategy was used (Table 1). The exper-
imental variables mean and variance explained a significant part
of the variance in information-seeking (Model 1), and measures
of primary task performance (accuracy and RT) significantly in-
creased the fit (Model 2). Crucially, adding confidence to a model
that already contained these four variables provided the best fit to
the data. As predicted, in this final model (Model 3), there was a
clear negative effect of confidence on see again choices (
0.08, t(85.63) 3.09, p 0.003), whereas the effects of RT
( 0.15, p 0.065) and variance ( 0.03, p 0.069) were no
longer statistically significant. The effects of accuracy and mean
were not significant either (both p values 0.10). Thus, although
mean, variance, RTs, and accuracy explained a significant Postdecisional ERPs are modulated by confidence over and above
task condition Shadings represent SEM. Figure 4. Time-resolved multivariate regression of EEG data by confidence and task difficulty. Horizontal lines indicate clusters significant ression of EEG data by confidence and task difficulty. Horizontal lines indicate clusters significantly differing from 0. Shadings represent SEM. response (p 0.021). Finally, in the data locked to the
information-seeking choice, there was no effect of confidence (no
significant elements in the cluster-forming step), but a significant
main effect of mean between 196 ms before and 13 ms after the
information-seeking choice (p 0.007). In sum, in the stimulus-
locked P3 cluster, we found no reliable modulation by confidence
after controlling for task difficulty; whereas in the response-
locked Pe cluster, we did observe a modulation by confidence
beyond the effect of experimentally induced task difficulty. identifying the time window during which information-seeking
can be decoded. In this analysis, classifiers were both trained and
tested on data from free-choice trials, using 10-fold cross-
validation as described above. Classifiers were trained and tested
on each point in time, thus shedding light on the generalization of
the discriminate pattern over time. In the stimulus-locked matrix
(Fig. 5A), no robust decoding was possible in the prestimulus
period (all cluster p values 0.143). The decoder was only trained
on data up until 400 ms after stimulus (13th percentile of RTs) to
avoid contamination from postresponse data (from trials with
short RTs). To complement this, the same analysis was repeated
after the data were realigned to the time of the response (but
keeping the same prestimulus baseline). Again, no decoding was
visible before response (all p values 0.156), but there was a
significant cluster that largely fell after response (p 0.040; Fig. 5B). Next, we decoded EEG data measured in the postresponse
period, using a conventional preresponse baseline. This analysis
revealed that, in marked contrast to the preresponse epoch, it was
possible to classify see again choices reliably across the entire
epoch from the initial task response to the subsequent see again
choice: There was a highly significant cluster that began just be-
fore primary response execution (Fig. 5C; p 0.001, cluster level)
and peaked just before the decision of whether or not to sample
more information (Fig. 5D; p 0.001, cluster level). ERP markers of information-seeking choices ERP markers of information-seeking choices
Figure 3B shows the ERP waveforms at CPz, separately for see
again and respond directly choices, again averaged over correct
trials only. The results closely mirror the modulation of the ERPs
by confidence. The stimulus-locked ERPs showed more positive
amplitudes on respond compared with see again trials, from 357
until 534 ms (p 0.028, cluster level). The response-locked ERPs
showed more negative amplitudes on respond compared with see
again trials, from 250 ms until the end of the epoch (700 ms, p
0.001, cluster level). Finally, the latter difference remained signif-
icant up until 29 ms before the information-seeking decision
(p 0.008, cluster level). Similar to confidence, all significant
effects had a clear centroparietal scalp distribution, although the
stimulus-locked P3 component for information-seeking choices
has a slightly more anterior scalp distribution than that for
confidence. In sum, analysis of the ERPs provides preliminary evidence for
a link between confidence and information-seeking, given that
both processes have very similar neural markers. In the following,
we use multivariate single-trial decoding to provide a more rig-
orous appraisal of this link, testing the informational content of
these neural markers (i.e., whether information-seeking choices
can be decoded reliably) and whether neural markers of confi-
dence are predictive of information-seeking behavior. Next, we used the same approach to examine whether deci-
sion confidence could be decoded from EEG data. This analysis
used no-choice trials only, to avoid any contaminating effects of
the see again choice itself. In the stimulus-locked matrix (Fig. 5E),
no robust decoding was observed (all cluster p values 0.785). Complementing this, when these preresponse data were re- Postdecisional ERPs are modulated by confidence over and above
task condition Postdecisional ERPs are modulated by confidence over and above
task condition We next examined whether the modulation of the ERPs by con-
fidence extended over and above our manipulation of task diffi-
culty. To do so, we used multivariate regression analyses to
examine whether confidence explains unique variance in EEG
data after task difficulty is accounted for. Figure 4 shows the 3314 • J. Neurosci., April 24, 2019 • 39(17):3309–3319 Desender et al. • Postdecisional Neural Marker of Confidence age t value for each predictor from the multivariate regres-
h ti
p i t A
b
ft
t
lli
f
t k
significant (p values 0.079). In the response-locked da
b
d
d l ti
f th EEG d t b
fid
e 3. ERPs as a function of confidence (A) and information-seeking choices (B). Head plots represent the difference in scalp distribution during the significant time periods for
ence(A)andseeagain-respond(B).Grayhorizontallinesindicateclustersduringwhichbothconditionssignificantlydiffer.ShadingsrepresentSEM.Highandlowconfidenceiscalcul
ice data and information-seeking choices from free-choice trials. igure 3. ERPs as a function of confidence (A) and information-seeking choices (B). Head plots represent the difference in scalp distribution during the significant time periods for
onfidence(A)andseeagain-respond(B).Grayhorizontallinesindicateclustersduringwhichbothconditionssignificantlydiffer.ShadingsrepresentSEM.Highandlowconfidenceiscalcul
o-choice data and information-seeking choices from free-choice trials Figure 3. ERPs as a function of confidence (A) and information-seeking choices (B). Head plots represent the difference in scalp distribution during the significant time periods for low-high
confidence(A)andseeagain-respond(B).Grayhorizontallinesindicateclustersduringwhichbothconditionssignificantlydiffer.ShadingsrepresentSEM.Highandlowconfidenceiscalculatedfrom
no-choice data and information-seeking choices from free-choice trials. significant (p values 0.079). In the response-locked data, we
observed a modulation of the EEG data by confidence, even when
controlling for task difficulty, occurring between 481 and 615 ms
after response (p 0.042). Slightly earlier in time, there also was
a significant main effect of variance between 225 and 517 ms after average t value for each predictor from the multivariate regres-
sion on each time point. As can be seen, after controlling for task
difficulty, there is no consistent effect of confidence in the
stimulus-locked data (no significant elements in the cluster-
forming step). None of the factors capturing task difficulty was Desender et al. • Postdecisional Neural Marker of Confidence J. Neurosci., April 24, 2019 • 39(17):3309–3319 • 3315 Figure 4. Time-resolved multivariate regression of EEG data by confidence and task difficulty. Horizontal lines indicate clusters significantly differing from 0. Shadings represent SEM. Figure 4. Time-resolved multivariate regression of EEG data by confidence and task difficulty. Horizontal lines indicate clusters significantly differing from 0. Postdecisional ERPs are modulated by confidence over and above
task condition Decoding
performance was strongest along the diagonal, indicating best
decoding when the cross-validation test data came from the same
time period as the classifier training data. Nevertheless, both clusters
also displayed significant off-diagonal decoding, indicating that the
cross-validationtestdatacouldbepredictedabovechancelevel,even
when classifier training data were obtained from a different time
window. This strong temporal generalization suggests a neural ac-
tivity pattern that is consistent and sustained over time during the
formation of information-seeking choices. Within-condition decoding of information-seeking choices and
decision confidence Within-condition decoding of information-seeking choices and
decision confidence We first tested whether information-seeking choices can be de-
coded over time from the EEG data. This analysis is a first step in 3316 • J. Neurosci., April 24, 2019 • 39(17):3309–3319 Desender et al. • Postdecisional Neural Marker of Confidence Figure5. Within-conditiondecodingofinformation-seekingchoices(A–D)andconfidence(E–H).Classifiersweretrainedandtestedonalltimepoints(oncorrecttrialsonly;stepsof10msand
aslidingwindowof106ms).Topographiesrepresentthescalpprojectionsobtainedfromthelogisticregressionclassifieratthetrainingtimewhereclassificationismaximal.A,B,E,F,Prestimulus
baseline(100msuntil0ms).C,D,G,H,Preresponsebaseline(100msuntil0ms).Becauseofthisdifferenceinbaseline,theregionsofsignificantdecodinginB,CandF,Garenotidentical.Solid
blacklinesindicatesignificantclusters(p0.05).Thetrainingtimesofeachpanelcorrespondtothetestingtimeofthatpanel;forexample,t0correspondstostimulus,response,response,and
information-seeking decisions in panels A–D and E–H, respectively. 3316 • J. Neurosci., April 24, 2019 • 39(17):3309–3319
Desender et al. • Postdecisional Neural Marker of Confidence Figure5. Within-conditiondecodingofinformation-seekingchoices(A–D)andconfidence(E–H).Classifiersweretrainedandtestedonalltimepoints(oncorrecttrialsonly;stepsof10msand
aslidingwindowof106ms).Topographiesrepresentthescalpprojectionsobtainedfromthelogisticregressionclassifieratthetrainingtimewhereclassificationismaximal.A,B,E,F,Prestimulus
baseline(100msuntil0ms).C,D,G,H,Preresponsebaseline(100msuntil0ms).Becauseofthisdifferenceinbaseline,theregionsofsignificantdecodinginB,CandF,Garenotidentical.Solid
blacklinesindicatesignificantclusters(p0.05).Thetrainingtimesofeachpanelcorrespondtothetestingtimeofthatpanel;forexample,t0correspondstostimulus,response,response,and
information-seeking decisions in panels A–D and E–H, respectively. to-significance stimulus-locked cluster in Fig. 5E; 3 pixels, t(14)
3.69, p 0.002; and the closest-to-significance cluster in the
preresponse data with a prestimulus baseline in Fig. 5F; 23 pixels,
t(14) 5.52, p 0.001). To confirm that these results were not
specific to the particular cluster sizes used, we confirmed that
corresponding results were observed when taking clusters of 3
3, 5 5, 7 7, and 9 9 pixels around the peak pixel in each
matrix. aligned to the response (keeping the same prestimulus baseline),
no significant clusters were observed (all cluster p values 0.195;
Fig. 5F). In contrast, in the postresponse period, there was a
highly significant cluster (p 0.002) during which decoding of
confidence was possible (Fig. 5G). Although this cluster was lim-
ited to 400 ms after response, it extended up to 550 ms after
response when using a more liberal cluster threshold of 0.05. Finally, in the data locked to the information-seeking decision
(Fig. 5H), there were no significant clusters (p values 0.410). In sum, both information-seeking choices and decision con-
fidence could be decoded from EEG data, but only during the
time window following the primary task response. No above-
chance decoding was visible when classifiers were trained on EEG
data from the preresponse window. g
g
p
Collectively, the preceding analyses suggest more robust de-
coding of confidence and see again choices based on postresponse
EEG than preresponse EEG data. A final set of analyses aimed to
test these postresponse vs preresponse differences more directly. To this end, we first identified clusters in the preresponse matri-
ces that came closest to significance in the permutation analyses
(comprising neighboring pixels with a significant effect, uncor-
rected), as representing the strongest observed decoding in the
preresponse period. Within-condition decoding of information-seeking choices and
decision confidence We then selected a corresponding number of
pixels in the postresponse cluster, centered on its peak, and com-
pared decoding accuracies between clusters. When decoding see
again choices, decoding accuracy in the postresponse cluster (Fig. 5C) was significantly higher than the closest-to-significant cluster
in the stimulus-locked matrix (Fig. 5A; 5 pixels, t(14) 15.14, p
0.001) and the preresponse matrix with a prestimulus baseline
(Fig. 5B; 34 pixels, t(14) 26.83, p 0.001). The same was true
when decoding confidence (higher decoding than in the closest- Across-condition decoding of information-seeking choices
by confidence
h l
h
l
l
d h
d
h While the previous analyses already hinted at the importance of
postdecisional neural activity for information-seeking choices,
those analyses are uninformative about the role of confidence in
this process. Our final set of analyses provided a more direct
test of the hypothesis that confidence underpins adaptive
information-seeking. Specifically, we tested whether an EEG clas-
sifier trained to decode confidence on no-choice trials (i.e., in
which participants were not asked to decide whether or not to
seek further information before their final decision) would be
able to predict see again choices on free-choice trials (i.e., on the Desender et al. • Postdecisional Neural Marker of Confidence J. Neurosci., April 24, 2019 • 39(17):3309–3319 • 3317 Figure6. Across-conditiondecodingofinformation-seekingchoicesbyconfidence,lockedtothestimulus(A),theresponse(B,C)andtheinformation-seekingchoice(D).Classifiersaretrained
on high versus low confidence from no-choice data and tested on see again versus respond decisions from free-choice data (both on correct trials only). Above-chance decoding only occurs
postresponse. The same conventions as in Figure 5A–D apply. Figure6. Across-conditiondecodingofinformation-seekingchoicesbyconfidence,lockedtothestimulus(A),theresponse(B,C)andtheinformation-seekingchoice(D).Classifiersaretrained
on high versus low confidence from no-choice data and tested on see again versus respond decisions from free-choice data (both on correct trials only). Above-chance decoding only occurs
postresponse. The same conventions as in Figure 5A–D apply. there reliable decoding of task difficulty itself (p values 0.405;
within-condition decoding). Together, these findings provide fur-
ther evidence that confidence predicts information-seeking behav-
ior, even when controlling for possible confounding effects of
objective task difficulty. Significant decoding was not observed in
analysis of data time-locked to the information sampling decision
(Fig. 6D; all cluster p values 0.256). separate set of trials in which participants had the option of seek-
ing or declining additional information). Importantly, on no-
choice trials participants did not make an information sampling
choice themselves but were forced to select the option to give
their response. This rules out the possibility that our classifier is
decoding incidental processes related to this choice, such as
motor-related neural activity resulting from the information-
seeking choice. Moreover, to ensure that our classifier was decod-
ing decision confidence, and not achieving above-chance
classification by exploiting other correlated features of the data
(e.g., trials in which participants changed their mind), only trials
on which participants were correct in both their initial and their
final decision were used to train the classifier. Our final set of analyses directly compared decoding accuracy
in postresponse versus preresponse periods. These analyses re-
vealed that decoding accuracy in the postresponse cluster (Fig. Across-condition decoding of information-seeking choices
by confidence
h l
h
l
l
d h
d
h 6C) was significantly higher than the closest-to-significance clus-
ter in the stimulus-locked matrix (Fig. 6A; 1 pixel, t(14) 3.29,
p 0.005), and numerically higher compared with a cluster in
the preresponse data with a prestimulus baseline (Fig. 6B; 36
pixels, t(14) 1.56, p 0.140). To check that these results were
not specific to the particular cluster sizes used, we confirmed that
corresponding results were observed when taking clusters of 3
3, 5 5, 7 7, and 9 9 pixels around the peak pixel in each
matrix. In the resulting stimulus-locked matrix, there was no sign of
above-chance decoding (all p values 0.289; Fig. 6A). A comple-
mentary analysis in which these data were realigned to the time of
the response (keeping the same prestimulus baseline) also
showed no reliable preresponse decoding (all p values 0.218;
Fig. 6B). The response-locked data, by contrast, showed signifi-
cant decoding from 350 to 700 ms after the response (p
0.008, cluster level; Fig. 6C). Thus, a decoder trained to predict
confidence from EEG data from no-choice trials was able to pre-
dict information-seeking decisions from EEG data recorded on
free-choice trials, specifically in this postresponse window. As
becomes clear from the associated topography in Figure 6C, the
scalp projections show a very similar scalp distribution to the Pe
that was observed in the ERPs. Importantly, across-condition
decoding was not driven by a joint relation of confidence and
information-seeking to task difficulty (e.g., stimulus variance):
the same postresponse cluster was found (p 0.022) when the
factors mean (high or low), variance (high or low), and their
interaction were first regressed out from the EEG data (from both
the training and test datasets) on each time point at each elec-
trode, separately for each participant. Similar results were ob-
served in a further control analysis where these factors were
regressed out from participants’ confidence ratings before run-
ning the EEG decoding analysis. Conversely, after confidence
(high or low) was regressed out from the EEG data, a classifier
trainedtodecodetaskdifficultyitself(specifically,whetheratrialhad
an easy high mean/low variance stimulus vs a difficult low mean/
highvariancestimulus)didnotrobustlygeneralizeacrossconditions
to predict information-seeking choices (p values 0.101), nor was Discussion The current work significantly extends our previous behavioral
findings by characterizing the neural signatures integral to translat-
ing decision confidence into overt information-seeking choices. In
particular, we could predict information-seeking behavior based on
confidence; however, we could do so only in postdecisional EEG
activity,eventhoughaveragedERPwaveformsvariedsignificantlyas
a function of confidence and see again choices also in the predeci-
sional period. As such, our findings converge with theoretical work
arguing that postdecisional evidence accumulation plays a critical
roleinconfidencejudgments(PleskacandBusemeyer,2010;Moran
et al., 2015; Fleming and Daw, 2017) and subsequent actions. De-
pending on the strength of the postdecisional evidence (i.e., reflect-
ing the degree of confidence), participants will seek additional
information or not. Neither within-condition nor across-condition decoding
analyses yielded above-chance decoding of confidence or
information-seeking choices in the time window occurring be-
fore the response. This lack of decoding is striking given that
predecisional neural activity (corresponding to the well-studied
P3 component) was found to be sensitive to confidence and
information-seeking in univariate analyses. In this regard, it is
important to note that not all neural signals that are modulated
by confidence are also involved in the representation of explicit
(i.e., subjective) confidence (Pouget et al., 2016). Several studies
showed predecisional neural markers that are sensitive to the
level of confidence (Kiani and Shadlen, 2009; Gherman and Phili-
astides, 2015, 2018; Odegaard et al., 2018). For example,
Odegaard et al. (2018) demonstrated that opt-out decisions (pre-
sumably reflecting low confidence) are associated with weak
traces of neural evidence in monkey superior colliculus occurring
before the response. However, using a positive-evidence manip-
ulation that dissociates evidence quality from confidence, they
were able to show that the predecisional neural activity tracks
evidence quality, not subjective confidence (both of which are
typically closely associated). A similar observation was made in
the current work. Significant modulation of the predecisional
neural marker by confidence disappeared once stimulus variabil-
ity (i.e., a measure of evidence quality) was taken into account. Together, this might explain why predecisional neural markers
are modulated by confidence but do not predict information-
seeking choices. The postdecisional neural marker observed in the current
work closely resembles the classical Pe component of the ERP
(Ridderinkhof et al., 2009). This neural marker has been sug-
gested to reflect an evidence accumulation signal evaluating the
likelihood that the just-executed response was incorrect (Stein-
hauser and Yeung, 2010; Murphy et al., 2015). Discussion We
indeed observed large interindividual differences in the tendency to
prefer one of the information-seeking options, with some partici-
pants biased toward respond choices (N 10, on average 23% see
again choices, RTsee again 1245 ms vs RTrespond 800 ms, t(9)
4.18, p 0.002) and others toward see again choices (N 5, on
average 73% see again choices, RTsee again 797 ms vs RTrespond
953 ms, t(4) 2.02, p 0.11). For both subgroups, however, the
postdecisional neural marker was of higher amplitude when partic-
ipants chose to see more information (replicating Fig. 3B; both p
values 0.001). This observation is hard to reconcile with the idea
that postdecisional neural activity reflects processes related specifi-
cally to the information-sampling choice, but are compatible with
our interpretation that this activity indexes an evaluation of the ac-
curacy of the just-executed response, which then informs explicit
confidence reports and information-seeking choices. When confronted with difficult decisions, humans seek further
information to improve the quality of their decisions. Unsolicited
information does not affect decisions, whereas decisions are more
accuratewhenadditionalinformationisactivelysolicited(Hungand
Yoong, 2010). This suggests that the act of seeking further informa-
tionisdrivenbyaninternalevaluation.Astraightforwardhypothesis
is that humans internally compute the probability of making a cor-
rect decision; and when this probability is low, they seek additional
information. Indeed, in a recent behavioral study, we were able to
demonstratethatexplicitlyrepresenteddecisionconfidencepredicts
information-seeking, even across conditions matched for objective
difficulty (Desender et al., 2018). In contrast to that finding and
other previous work (Spence et al., 2016; Boldt et al., 2017a), in the
current study we did not observe a difference in confidence between
the two medium difficulty conditions (high mean/high variance vs
low mean/low variance) of the same task. This discrepancy might in
part reflect the failure of our psychophysical staircase to match be-
havioral performance across these conditions. Participants made
perceptualdecisionssignificantlymoreslowlyandnumericallymore
accurately with high variance stimuli, suggesting a more cautious
response strategy that would tend to inflate confidence (Vickers and
Packer, 1982). Moreover, in the current study, confidence was only
calculated from no-choice data, which constituted 25% of all data,
thus making our design less sensitive to find subtle differences be-
tween two specific conditions. Despite this, using mixed modeling,
we were able to demonstrate that decision confidence, not objective
accuracy or stimulus difficulty, was the main variable predicting
information-seeking. Discussion The current study examined whether neural markers of decision
confidence are predictive of information-seeking behavior. To
this end, we recorded scalp EEG while participants performed a
task in which they first made an initial decision about a stimulus,
then chose whether or not to sample more information, before
providing
their
final
response
and
level
of
confidence. Information-seeking choices and confidence similarly modu-
lated predecisional (P3) and postdecisional (Pe) ERP compo-
nents. Using multivariate classification, we then showed that
information-seeking choices could be decoded from EEG data
from the time of the initial decision to the time of the subsequent
information-seeking choice (within-condition decoding). How-
ever, no above-chance decoding was visible preceding the initial
decision. Crucially, a classifier trained to decode high versus low
confidence generalized to prediction of information-seeking
choices (across-condition decoding), and this too was restricted
to a post-RT window. The time period during which we observed
robust across-condition generalization (i.e., from no-choice trial
confidence ratings to free-choice trial information-seeking be-
havior) corresponds to that of a postdecisional neural marker of
decision confidence, suggesting that the latter reflects a neural 3318 • J. Neurosci., April 24, 2019 • 39(17):3309–3319 Desender et al. • Postdecisional Neural Marker of Confidence both confidence reports and information-seeking choices appear to
be reflected in the same underlying neural signal. process integral to translating one’s subjective sense of confi-
dence into overt decisions to sample more information. An alternative interpretation of our results could be that the
postdecisional neural marker we describe reflects the internal
decision to seek more information, rather than a common signal
that can be leveraged for making both information-seeking deci-
sions and graded confidence reports. In other words, perhaps
participants made internal information-seeking decisions on no-
choice trials despite there being no explicit requirement to do so,
and the neural signal that is central to our analyses reflects this. Such an explanation of our findings seems unlikely for two rea-
sons. First, our postdecisional neural signal highly resembles the
well-characterized Pe component, both in time and scalp distri-
bution (Ridderinkhof et al., 2009). The Pe is reliably observed in
paradigms that do not require any overt postdecisional response,
which makes it unlikely that it reflects a signal specifically related
to this additional response. Second, if this postdecisional neural sig-
nal directly reflects the information sampling choice, it should be
related to systematic biases that are observed in such choices. Discussion Accordingly, the
amplitude of this signal (reflecting the amount of evidence for an
error) has been shown to scale inversely with decision confidence
(Boldt and Yeung, 2015). Our observation of this signal after
participants made a primary decision in both the no-choice and
free-choice conditions suggests that, in both conditions, evidence
is accumulated about the likelihood of this decision being correct. This evaluation process could, in principle, be used to guide both
immediate, binary information-seeking choices (by imposing a sin-
gle threshold on the evolving tally of error evidence) (Murphy et al.,
2015) and later confidence reports (by translating the final continu-
ous evidence tally into a correspondingly graded expression of
confidence). The present finding of significant across-condition de-
coding provides important support for this idea by showing that Our findings are of relevance to research on the role of uncer-
tainty in action control (Daw et al., 2005; Yu and Dayan, 2005). J. Neurosci., April 24, 2019 • 39(17):3309–3319 • 3319 Desender et al. • Postdecisional Neural Marker of Confidence From a Bayesian perspective, uncertainty (i.e., the inverse of con-
fidence) can be used as a cue for behavioral control (Daw et al.,
2005). From this, it follows that decision confidence should pre-
dict strategic decisions, such as whether or not to sample more
evidence (Meyniel et al., 2015). Our findings are the first empir-
ical demonstration that decision confidence and the adaptive act
of information-seeking share the same neural signal. Another
relevant connection to our work is research on exploitation–
exploration dilemmas. When forced to decide from which of several
patches to harvest, participants are faced with the dilemma between
exploiting a known patch or exploring unknown but potentially
more rewarding patches. Empirical and modeling work has demon-
strated that participants’ uncertainty about these choices arbitrates
between the two strategies. Consistent with normative theories
(Sutton and Barto, 2018), participants actively explore unknown
patches to gain information and reduce their uncertainty (Badre
et al., 2012; Cavanagh et al., 2012). Interestingly, recent research
has indicated an important role for subjective confidence in this
process (Boldt et al., 2017b). Specifically, when confidence in
value representations was low, participants actively explored un-
known patches. Although the underlying construction of confi-
dence may differ (confidence in value representation vs
confidence in the accuracy of a decision), in both cases, low con-
fidence triggers the need for information-seeking. Discussion Gherman S, Philiastides MG (2015) Neural representations of confidence
emerge from the process of decision formation during perceptual choices. Neuroimage 106:134–143. Gherman S, Philiastides MG (2018) Human VMPFC encodes early signa-
tures of confidence in perceptual decisions. ELife 7:1–28. Hillyard SA, Squires KC, Bauer JW, Lindsay PH (1971) Evoked potential
correlates of auditory signal detection. Science 172:1357–1360. Hung AA, Yoong JK (2010) Asking for help survey and experimental evidence
change. RAND Working paper Series WR-714-1. Santa Monica, CA:
RAND Corporation. Kiani R, Shadlen MN (2009) Representation of confidence associated with a
decision by neurons in the parietal cortex. Science 324:759–764. King JR, Dehaene S (2014) Characterizing the dynamics of mental representa-
tions: the temporal generalization method. Trends Cogn Sci 18:203–210. Kuznetsova A, Brockhoff PB, Christensen RH (2014) lmerTest: test
in linear mixed effect models. R package version 2.0–20. Available at
http://CRAN.R-project.org/packagelmerTest. Meyniel F, Sigman M, Mainen ZF (2015) Confidence as Bayesian probabil-
ity: from neural origins to behavior. Neuron 88:78–92. Moran R, Teodorescu AR, Usher M (2015) Post choice information integra-
tion as a causal determinant of confidence: novel data and a computa-
tional account. Cogn Psychol 78:99–147. Murphy PR, Robertson IH, Harty S, O’Connell RG (2015) Neural evidence
accumulation persists after choice to inform metacognitive judgments. ELife 4:e11946. Nieuwenhuis S, Ridderinkhof KR, Blom J, Band GP, Kok A (2001) Error-
related brain potentials are differentially related to awareness of response
errors: evidence from an antisaccade task. Psychophysiology 38:752–760. In conclusion, it was shown that a classifier trained to decode
confidence based on EEG data was able to predict information-
seeking decisions, specifically in the time window following a
speeded decision about the stimulus. This suggests that postdeci-
sional neural processes are integral to translating decision confi-
dence into overt decisions to seek further information. O’Connell RG, Dockree PM, Kelly SP (2012) A supramodal accumulation-
to-bound signal that determines perceptual decisions in humans. Nat
Neurosci 15:1729–1735. Odegaard B, Grimaldi P, Cho SH, Peters MA, Lau H, Basso MA (2018)
Superior colliculus neuronal ensemble activity signals optimal rather than
subjective confidence. Proc Natl Acad Sci U S A 115:E1588–E1597. Parra LC, Spence CD, Gerson AD, Sajda P (2005) Recipes for the linear
analysis of EEG. NeuroImage 28:326–341. References Badre D, Doll BB, Long NM, Frank MJ (2012) Rostrolateral prefrontal cor-
tex and individual differences in uncertainty-driven exploration. Neuron
73:595–607. Pouget A, Drugowitsch J, Kepecs A (2016) Confidence and certainty: distinct
probabilistic quantities for different goals. Nat Neurosci 19:366–374. Ridderinkhof KR, Ramautar JR, Wijnen JG (2009) To P(E) or not to P(E): a
P3-like ERP component reflecting the processing of response errors. Psy-
chophysiology 46:531–538. Bates DM, Maechler M, Bolker B, Walker S (2015) Fitting linear mixed-
effects models using lme4. J Statist Software 67:1–48. Boldt A, Yeung N (2015) Shared neural markers of decision confidence and
error detection. J Neurosci 35:3478–3484. Sniezek JA, Van Swol LM (2001) Trust, confidence, and expertise in a judge-
advisor system. Organ Behav Hum Decis Process 84:288–307. Boldt A, de Gardelle V, Yeung N (2017a) The impact of evidence reliability
on sensitivity and bias in decision confidence. J Exp Psychol Hum Percept
Perform 43:1520–1531. Spence ML, Dux PE, Arnold DH, Spence ML, Dux PE, Arnold DH (2016)
Computations underlying confidence in visual perception computations
underlying confidence in visual perception. J Exp Psychol Hum Percept
Perform 42:671–682. Boldt A, Blundell C, De Martino B (2017b) Confidence modulates explora-
tion and exploitation in value-based learning. bioRxiv. Advance online
publication. Retrieved December 28, 2017. doi: 10.1101/236026. Stanislaw H, Todorov N (1999) Calculation of signal detection theory mea-
sures. Behav Res Methods Instrum Comput 31:137–149. Bonaccio S, Dalal RS (2006) Advice taking and decision-making: an integra-
tive literature review, and implications for the organizational sciences. Organ Behav Hum Decis Process 101:127–151. Steinhauser M, Yeung N (2010) Decision processes in human performance
monitoring. J Neurosci 30:15643–15653. Organ Behav Hum Decis Process 101:127–151. g
Sutton RS, Barto AG (2018) Reinforcement learning: an introduction. Cambridge MA: Massachusetts Institute of Technology g
Sutton RS, Barto AG (2018) Reinforcement learning: an introduction. Sutton RS, Barto AG (2018) Reinforcement learning: an intro
Cambridge, MA: Massachusetts Institute of Technology. Butler CW, Danby S, Emmison M, Thorpe K (2009) Managing medical
advice seeking in calls to child health line. Sociol Health Illn 31:817–834. Sutton RS, Barto AG (2018) Reinforcement learning: an
Cambridge, MA: Massachusetts Institute of Technology. Twomey DM, Murphy PR, Kelly SP, O’Connell RG (2015) The classic
P300 encodes a build-to-threshold decision variable. Eur J Neurosci
42:1636–1643. Cavanagh JF, Figueroa CM, Cohen MX, Frank MJ (2012) Frontal theta re-
flects uncertainty and unexpectedness during exploration and exploita-
tion. Cereb Cortex 22:2575–2586. All raw data can be freely accessed at https://osf.io/z7umt/. All raw data can be freely accessed at https://osf.io/z7umt/. Polich J (2007) Updating P300: an integrative theory of P3a and P3b. Clin
Neurophysiol 118:2128–2148. Notes Pleskac TJ, Busemeyer JR (2010) Two-stage dynamic signal detection: a the-
ory of choice, decision time, and confidence. Psychol Rev 117:864–901. References Vickers D (1979) Decision processes in visual perception. San Diego: Academic. Daw ND, Niv Y, Dayan P (2005) Uncertainty-based competition between
prefrontal and dorsolateral striatal systems for behavioral control. Nat
Neurosci 8:1704–1711. Vickers D, Packer J (1982) Effects of alternating set for speed or accuracy on
response time, accuracy and confidence in a unidimensional task. Acta
Psychol 50:179–197. Delorme A, Makeig S (2004) EEGLAB: an open source toolbox for analysis
of single-trial EEG dynamics including independent component analysis. J Neurosci Methods 134:9–21. Yeung N, Summerfield C (2012) Metacognition in human decision-mak-
ing: confidence and error monitoring. Philos Trans R Soc Lond B Biol Sci
367:1310–1321. Desender K, Boldt A, Yeung N (2018) Subjective confidence predicts
information-seeking in decision making. Psychol Sci 29:761–778. Yu AJ, Dayan P (2005) Uncertainty, neuromodulation, and attention. Neu-
ron 46:681–692. Fleming SM, Daw ND (2017) Self-evaluation of decision performance: a
general Bayesian framework for metacognitive computation. Psychol Rev
124:91–114. Zylberberg A, Barttfeld P, Sigman M (2012) The construction of confidence
in a perceptual decision. Front Integr Neurosci 6:79.
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Topical ivermectin in the treatment of pediculosis capitis
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ABSTRACT Background: Head lice infestation is considered a prominent issue because of its worldwide distribution, especially
among children. Millions of children are infested with pediculosis capitis every year. The increasing rate of lice infestation
has been reported even in the developed countries due to the resistance to known pediculicides. Besides, the louse is
a vector for serious diseases, such as epidemic typhus fever. Objective: The aim of this work was the search for a new
cheap and effective drug for pediculosis capitis. 2% ivermectin solution, a promising novel drug used for endoparasite
and ectoparasite infections, was used in this study. Materials and Methods: This was a clinical trial study. The in vitro
study was carried out in a period of five months. Samples of nits and lice were collected from female patients of the
Department of Dermatology and Venereology, Baghdad Teaching Hospital. The study was conducted during a period
from October 2002 through November 2003. One hundred fifty children from four primary schools in Hayy Al-A’amel,
Baghdad, participated in this part of the study. The preparation was applied to the children by the author. One hundred
children were tested with ivermectin solution and fifty children with rectified spirit. The first part of the study involved
a parasitological evaluation (in vitro study) of pediculicidal and ovicidal activity, in which 4% ivermectin solution was
discontinued because of contact dermatitis. Therefore, 2% ivermectin solution was used instead. Pediculicidal and
ovicidal activity was examined half an hour afterward. The second part of the study involved a clinical evaluation
(in situ study) of 2% ivermectin solution for half an hour. Results: Topical 2% ivermectin solution in rectified spirit
is a good pediculicidal (100%) and a good ovicidal (88%) drug with good therapeutic efficacy (82% after the first
application, 90% after the second application) against pediculosis capitis. Conclusion: The study described a novel
topical preparation for pediculosis capitis, which was proven effective and safe. Key words: Ivermectin; Pediculosis capitis; Iraq; Topical solution How to cite this article: Husni L, Al-Waiz M. Topical ivermectin in the treatment of pediculosis capitis. Our Dermatol Online. 2021;12(1):14-18.
Submission: 20.02.2020; Acceptance: 01.06.2020
DOI: 10.7241/ourd.20211.3 Our Dermatology Online
Original Article
Topical ivermectin in the treatment of pediculosis
Topical ivermectin in the treatment of pediculosis
capitis
capitis Original Article Luma Husni, Makram Al-Waiz Baghdad, Medical City, Baghdad Training Centre, Baghdad, Iraq Corresponding author: Dr. Luma Husni, E-mail: sizerluma@yahoo.com Topical ivermectin in the treatment of pediculosis
Topical ivermectin in the treatment of pediculosis
capitis
capitis Luma Husni, Makram Al-Waiz INTRODUCTION DDT, the pyrethrins, and the pyrethroids [6]. Numerous
treatment options have been used to control the spread of
pediculosis capitis with various drawbacks. For instance,
1% lindane exhibits central nervous system toxicity and
may cause severe seizures in children [7]. 5% benzyl
alcohol was the first non-neurotoxic pediculicide, and
pruritus, erythema, pyoderma, and ocular irritation
are its usual side effects. It is also non-ovicidal [8]. The percentage of patients who were louse-free after
treatment with a product containing tea tree oil and
lavender oil and a head lice suffocation product was
higher than with a product containing pyrethrins and
piperonyl butoxide [9]. Eucalyptus oils have been used
against permethrin-resistant pediculosis [10]. Population-based studies in European countries show
a highly divergent prevalence of pediculosis capitis,
ranging from 1% to 20% [1]. With an increasing rate
of treatment failure, it is worthwhile to consider the
issues of misdiagnosis, lack of adherence, inadequate
treatment, reinfestation, lack of ovicidal or residual killing
properties of the pediculicide, and/or resistance of lice to
the pediculicide [2]. Resistance to topical pediculicides
is an emerging concern in most parts of the world [3]. The problem of resistance is directly related to the
frequency of its use [4,5] in genotyping, demonstrating
that head lice exhibit resistance to compounds such as 14 © Our Dermatol Online 1.2021 www.odermatol.com Grapefruit juice [11], coconut, and anise spray have
been found to be more effective than permethrin in
pediculosis capitis, thereby demonstrating clinical
resistance [12]. mobile (active) lice were transferred to another clean
Petri dish. A hundred fully mobile lice were exposed to
test preparations by immersion at room temperature. The number of live and dead lice was recorded. After
three minutes, the lice were washed in tap water, then
microscopically examined. A louse was considered dead
if it lacked signs of internal and external movement. Fifty fully mobile lice were exposed to rectified spirit
half an hour after washing, then microscopically
examined. The percentage of dead lice represented the
pediculicidal activity of the test preparation. In head lice infestations that are difficult to treat, oral
ivermectin may prove more effective [13] or as effective
as topical 0.5% malathion lotion [14]. Oral ivermectin
demonstrates high efficacy and tolerability in the
treatment of pediculosis capitis. A significant number
of children required a second dose to ensure complete
eradication [15]. Ovicidal Test Hair shafts with attached viable ova (nits) were
removed with forceps and scissors, transferred onto a
sterile Petri dish, closed with self-adhesive tape, and
identified. An ovum was judged viable if it was plump,
had an intact operculum and ideal eyespot, and was
yellow or creamy white. Dark tan to brown or black
ova with shriveled or shrunken shells were judged
nonviable. An ovum that had the operculum popped
open and a translucent shell without a brown residue
was considered hatched. Nits were examined with a
binocular microscope. Empty (hatched) or possibly
dead (nonviable) nits were excluded from the ovicidal
test, whereas the plump (viable) nits were transferred
to another clean Petri dish. The viable ova, firmly
fixed to their support (hairs), were exposed to the test
preparation by immersion for half an hour at room
temperature. Afterward, the nits were washed three
times in tap water. Nits were allowed to dry at room
temperature and incubated again in similar conditions
at room temperature for two weeks for hatching. Next,
unhatched nits were counted under the microscope. The percentage of unhatched nits represented the
ovicidal activity of the test preparation. Then, the same
was done but with the control preparation after the
completion of the parasitological evaluation. INTRODUCTION A single oral dose of ivermectin 400 μg/
kg repeated throughout seven days has been shown to be
more effective than 0.5% malathion lotion [13]. It has also
been used successfully in school children [16]. In head
lice infestations that are difficult to treat, oral ivermectin
given twice every seven days had superior efficacy when
compared with topical 0.5% malathion lotion [17]. After a single dose, complete healing was achieved in
77.5% and 87.5% of ivermectin and malathion groups,
respectively. No major adverse effects were observed in
either group. Oral ivermectin is a promising and effective
approach for the treatment of head lice and might be
an ideal substitute for conventional pediculicides [14]. Community-based treatment with oral ivermectin in
susceptible, and poor sections of the society has given
us promising results [18]. MATERIALS AND METHODS In vitro parasitological evaluation: The activity of the
test preparation was evaluated on the Pediculus capitis
in ovicidal and pediculicidal tests. Samples of nits
and lice were collected from female patients of the
Department of Dermatology and Venereology, Baghdad
Teaching Hospital. The study was conducted during
a period from September 2002 through November
2003. 2% ivermectin solution l gm/50 cc rectified spirit
was used and tested by parasitological evaluation (in
vitro study). A control test was included as part of the
parasitological evaluation as well. Rectified spirit was
the control preparation. Ovicidal tests of each type of
test and control preparation were tested half an hour
after washing in tap water. RESULTS To be eligible for participation, each patient had
to have a diagnostically active head lice infestation
confirmed by direct visual identification of live adult
lice or nymphs. The presence of nits on the hair shafts
was insufficient to qualify a child for inclusion in the
study. A child was excluded if there were any other
dermatological conditions present or if a pediculicide
was used within two weeks of the initial evaluation. A total of 150 lice were included in the study. A hundred
were tested with ivermectin and fifty with rectified
spirit. A total of 150 lice were included in the study. A hundred
were tested with ivermectin and fifty with rectified
spirit. The following pediculicidal activity was observed: The following pediculicidal activity was observed: 1. 2% ivermectin solution The parents agreed not to wash the hair of their
children for half an hour of solution application and
not to use pediculicides, medicated shampoos, or
lotions, other than ordinary shampoo, during the study. During the first visit, a visual estimation of live adult
louse and nymph populations in the children’s hair
was conducted and the test preparation was applied
to the dry scalp in amounts sufficient to thoroughly
wet the hair and skin of the infested areas. An amount
of 25–50 mL was sufficient to wet the hair and scalp,
but extremely long and thick hair sometimes required
larger amounts. During the application of the test
preparation, the children were instructed to protect
their eyes from the preparation by holding a folded
towel against their forehead. The hair was covered with
a cup for half an hour, then washed. After exposure for three minutes, all of the 100 ova
were dead. The pediculicidal activity of the preparation
was 100%. 2. Rectified spirit
After half an hour, 7 ova out of the 50 were dead. The
pediculicidal activity was 14%. In Situ Study Results Hundred fifty primary school children infested with
active pediculosis capitis were included in the study. One hundred of them were treated with 2% ivermectin
solution at the first and second visit and fifty with a control
preparation (rectified spirit). All children were female, and
their age ranged from 6 to 12 years with a mean of 9 years. Hundred fifty primary school children infested with
active pediculosis capitis were included in the study. One hundred of them were treated with 2% ivermectin
solution at the first and second visit and fifty with a control
preparation (rectified spirit). All children were female, and
their age ranged from 6 to 12 years with a mean of 9 years. A clinical evaluation was performed 30–60 minutes
following day fourteen. The children were examined
for three dermal signs (edema, erythema, and rash)
and five dermal symptoms (pruritus, burning, stinging,
numbness, and tingling). Eighty-two children had no live lice at the first
examination (one week after the first application of
the test preparation). The therapeutic efficacy of the
test preparation after one application was 82%, and
90% after two applications. In Situ Study Ivermectin solution was tested for its therapeutic
efficacy on primary school children infested with
pediculosis capitis. One hundred fifty children from
four primary schools in Hayy Al-A’amel, Baghdad,
participated in this part of the study. Pediculicidal test: lice and nymphs were collected
from the patients’ heads with a fine-tooth comb,
gently transferred with forceps onto a clean Petri dish,
closed with self-adhesive tape, and identified. Lice
were examined microscopically. Lice that were dead,
damaged, or less than fully mobile were discarded. Fully The children were in grades one to six. The preparation
was applied to the children by the author. One hundred
children were tested with ivermectin solution and fifty
children with rectified spirit. © Our Dermatol Online 1.2021 15 www.odermatol.com A total of 150 eggs were included in the study. 1. Out of the 100 eggs, 88 eggs were dead. The ovicidal
activity of the ivermectin solution was 88% after
half an hour. 2. Out of the 50 eggs, 6 eggs were dead. The ovicidal
activity of the rectified spirit was 12% after half an
hour. The therapeutic index (i.e., eradication of live lice and
nymphs) was evaluated on days seven and fourteen
following the treatment. The children were examined
for a minimum of five minutes for the presence of one
or more live adult lice or nymphs on either evaluation. The first application of the preparation was given at the
first visit, and the second application was given at the
second visit (after one week) with the same procedure
as on the first application. The parents of the children
were instructed to boil or steam sheets, pillowcases, and
other formats for about 15–20 minutes. The lice were found to be able to survive for 2–4 days
at 30°C without food (away from the human host). The lice were found to be able to survive for 2–4 days
at 30°C without food (away from the human host). Ovicidal Test Results A total of 150 eggs were included in the study. CONCLUSION This is the first Iraqi study on the use of topical
ivermectin for pediculosis capitis. After the first application of 2% ivermectin solution
for half an hour, the therapeutic efficacy (percentage
of dead lice and ova) was very good (82%) and, after
the second application, it was still very good (90%). Topical 2% ivermectin solution in rectified spirit
is a good pediculicidal (100%) and a good ovicidal
(88%) drug for the in vitro treatment of pediculosis
capitis. However, a second application seems to
be necessary because of its incomplete ovicidal
activity. A burning sensation was reported in twelve children
30 minutes after the application. These results
support that topical ivermectin solutions produce a
low frequency of side effects, whereas, with the use of
lindane, toxicity has been reported [7]. It is deemed safe and has no or few side effects. The result of the study indicates that a 2% ivermectin
solution is a good pediculicidal and ovicidal drug
that appears to be a suitable alternative, especially
considering the reported worldwide spread of resistance
to both pyrethroids and malathion. It is a good pediculicide drug, with 82% therapeutic
efficacy on the first application and 90% therapeutic
efficacy on the second application, and a good choice
for a primary treatment of pediculosis capitis and
resistant cases. Therefore, 2% ivermectin solution is a good and cheap
pediculicide with a low frequency of side effects. Its
main advantages are low adverse reactions and rapid
insecticide action, which requires a short contact time
and makes it suitable for home treatment. At the time of the study, there had been few similar
studies, which shows that there is a need for more
clarification on Iraqi patients because of the geographical
diversity of resistant cases. Ethics Statement While out of the 50 children infested with pediculosis
capitis treated with rectified spirit applied for half an
hour before washing, 47 children had live lice (adults
or nymphs) at the first examination (one week after the
first application), and 43 children had live lice (adults or
nymphs) at the second examination (one week after the
second application). Therefore, the therapeutic efficacy All authors hereby declare that the study has been
approved by the scientific committee of the Scientific
Council of Dermatology and have, therefore, been
performed in accordance with the ethical standards
defined by the Iraqi Board of Dermatology and
Venereology. 16 © Our Dermatol Online 1.2021 www.odermatol.com of the control preparation (rectified spirit) was 6% after
the first application and 14% after the second application. 2% ivermectin solution was found to have good ovicidal
activity (88%) and, if the time of exposure is increased,
the ovicidal activity may increase; therefore, a second
application is of extreme importance. A burning sensation was reported in twelve children
(12%) 15–30 minutes after the application of the test
solution (2% ivermectin solution). No other dermal
symptoms or signs were observed after the application
of either preparation. In the study, the lice were able to survive for 2–4 days
without food (away from the human host). Causes of treatment failure: Active lice, adults and
nymphs, in parasitological evaluation, exposed to
the test preparation under optimal conditions with
complete exposure of all lice to the preparation in a
Petri dish, while the application of the preparation
on the hair of the children may not be in close
contact with all the lice. Some nits may need more
than one week to hatch. Therefore, the nits will
hatch on the second application, and this may lead
to failure in treatment and reinfestation since all
failed cases in our study had live lice. Most of them
were small (nymphs) but several were of a mature
size (adults), suggesting that these children had
been reinfested. DISCUSSION Ivermectin was chosen because of its role in the
treatment of ectoparasitic infestations and its relative
lack of toxicity as compared with other modalities by
recent studies and, because of the increasing number
of treatment failures, possibly due to the development
of resistance, a change in treatment is to be considered. Ivermectin is a new drug for the treatment of
ectoparasites. To determine its effect on pediculosis
capitis, in this study, we applied it as a topical 2%
solution in rectified spirit and found that the in vitro
pediculicidal activity of the ivermectin solution after
three minutes was exceptionally good (100%) and that
its ovicidal activity was good as well (88%). Recommendations The study suggests that topical ivermectin may be a
promising treatment for head lice and that a second
dose on day seven might be appropriate. 1. Further studies regarding ivermectin solutions for
longer periods of exposure. © Our Dermatol Online 1.2021 17 www.odermatol.com J Cutan Med Surg. 2010;14:115-8. 2. Further comparative studies of ivermectin solutions
with other pediculicides. 4. Bialek R, Zelck UE, Fölster-Holst R. Permethrin treatment of
head lice with knockdown resistance-like gene. N Engl J Med. 2011;364:386-7. 3. There is an urgent need for monitoring the
development of resistance through official control
of sales and prescriptions. 5. Lee SH, Gao J-R, Yoon KS, Mumcuoglu KY, Taplin D, Edman JD,
et al. Sodium channel mutations associated with knockdown
resistance in the human head louse, Pediculus capitis (De Geer). Pestic Biochem Physiol. 2003;75:79-91. 4. New products such as topical ivermectin are
required and, once introduced, careful control of
their use would be of benefit. 6. Hodgdon HE, Yoon KS, Previte DJ, Kim HJ, Aboelghar GE, Lee SH,
et al. Determination of knockdown resistance allele frequencies in
global human head louse populations using the serial invasive signal
amplifi cation reaction. Pest Manag Sci. 2010;66:1031-40. 5. Control of head lice can be attempted by head
shaving and wet combing. 7. Nolan K, Kamrath J, Levitt J. Lindane Toxicity: A comprehensive
review of the medical literature. Pediatr Dermatol. 2012;29:141-6. g
g
6. We recommend the use of a fine-toothed louse
comb in addition to treatment for its role in:
a. The prevention of lice infestation;
b. Anti-louse treatment methods as an
accessory tool;
Th
l f
i 6. We recommend the use of a fine-toothed louse
comb in addition to treatment for its role in: 8. Meinking TL, Villar ME, Vicaria M, Eyerdam DH, Paquet D, Mertz-
Rivera K, et al. The clinical trials supporting benzyl alcohol lotion
5% [Ulesfi a]: A safe and effective topical treatment for head lice
[pediculosis humanus capitis]. Pediatr Dermatol. 2010;27:19–24. a. The prevention of lice infestation; b. Anti-louse treatment methods as an
accessory tool; 9. Barker SC, Altman PM. A randomized, assessor blind, parallel-group
comparative effi cacy trial of three products for the treatment of
head lice in children-melaleuca oil and lavender oil, pyrethrins and
piperonyl butoxide, and a “suffocation” product. BMC Dermatol. 2010;10:6. c. The removal of nits. l Mass family treatment is necessary. 10. Toloza AC, Lucía A, Zerba E, Masuh H, Picollo MI. Recommendations Eucalyptus
essential oil toxicity against permethrin-resistant Pediculus humanus
capitis [Phthiraptera: Pediculidae]. Parasitol Res. 2010;106:409–14. Statement of Human and Animal Rights 13. Chosidow O, Giraudeau B, Cottrell J, Izri A, Hofmann R, Mann SG,
et al. Oral ivermectin versus malathion lotion for diffi cult-to-treat
head lice. N Engl J Med. 2010;362:896–905. All the procedures followed were in accordance with the ethical
standards of the responsible committee on human experimentation
(institutional and national) and with the 2008 revision of the
Declaration of Helsinki of 1975. 14. Nofal A. Oral ivermectin for head lice: A comparison with 0.5%
topical malathion lotion. J Dtsch Dermatol Ges. 2010;8:985–8. 15. Ameen M, Arenas R, Villanueva-Reyes J, Ruiz-Esmenjaud J,
Millar D, Domínguez-Dueñas F, et al. Oral ivermectin for treatment
of pediculosis capitis. Pediatr Infect Dis J. 2010;29:991–3. Statement of Informed Consent 16. Currie MJ, Reynolds GJ, Glasgow NJ, Bowden FJ. A pilot study
of the use of oral ivermectin to treat head lice in primary school
students in Australia. Pediatr Dermatol. 2010;27:595–9. As per the national standard, written consent has been collected
from the patient or a parent of the patient and preserved by the
author(s). 17. Chosidow O, Giraudeau B, Cottrell J, Izri A, Hofmann R, Mann SG,
Burgess I. Ivermectin versus malathion lotion for diffi cult-to-treat
head lice. N Engl J Med. 2010;362:896-905. ACKNOWLEDGMENTS 11. Abdel-Ghaffar F, Semmler M, Al-Rasheid K, Klimpel S,
Mehlhorn H. Effi cacy of a grapefruit extract on head lice: A clinical
trial. Parasitol Res. 2010;106:445–9. We are grateful to Dr. Abdul-Rahman Al-Tae and Dr. Raad Al-Sady for their encouragement and help in
providing us with necessary observations. 12. Burgess IF, Brunton ER, Burgess NA. Clinical trial showing
superiority of a coconut and anise spray over permethrin 0.43%
lotion for head louse infestation, ISRCTN96469780. Eur J Pediatr. 2010;169:55–62. Copyright by Luma Husni, et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited. © Our Dermatol Online 1.2021 Copyright by Luma Husni, et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited. REFERENCES 18. Pilger D, Heukelbach J, Khakban A, Oliveira FA, Fengler G,
Feldmeier H. Household-wide ivermectin treatment for head lice
in an impoverished community: Randomized observer-blinded
controlled trial. Bull World Health Organ. 2010;88:90-6. 1. Feldmeier H. Pediculosis capitis: New insights into epidemiology,
diagnosis and treatment. Eur J Clin Microbiol Infect Dis. 2012;31:2105–10. 1. Feldmeier H. Pediculosis capitis: New insights into epidemiology,
diagnosis and treatment. Eur J Clin Microbiol Infect Dis. 2012;31:2105–10. 2. Frankowski BL, Bocchini JA, Jr. Council on School Health
and Committee on Infectious Diseases. Head lice. Pediatrics. 2010;126:392–403. 2. Frankowski BL, Bocchini JA, Jr. Council on School Health
and Committee on Infectious Diseases. Head lice. Pediatrics. 2010;126:392–403. Copyright by Luma Husni, et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited. Source of Support: Nil, Confl ict of Interest: None declared. Copyright by Luma Husni, et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited. 3. Marcoux D, Palma KG, Kaul N, Hodgdon H, Van Geest A,
Previte DJ, et al. Pyrethroid pediculicide resistance of head lice in
Canada evaluated by serial invasive signal amplifi cation reaction. 3. Marcoux D, Palma KG, Kaul N, Hodgdon H, Van Geest A,
Previte DJ, et al. Pyrethroid pediculicide resistance of head lice in
Canada evaluated by serial invasive signal amplifi cation reaction. 18 © Our Dermatol Online 1.2021
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Dietary cadmium and risk of breast cancer subtypes defined by hormone receptor status: A prospective cohort study
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International journal of cancer
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cc-by
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Dietary cadmium and risk of breast cancer subtypes defined by
hormone receptor status: A prospective cohort study Sara Grioni1, Claudia Agnoli1, Vittorio Krogh
1, Valeria Pala1, Sabina Rinaldi
2, Marco Vinceti3,4, Paolo Contiero5,
Luciano Vescovi3, Marcella Malavolti3 and Sabina Sieri
1 Sara Grioni1, Claudia Agnoli1, Vittorio Krogh
1, Valeria Pala1, Sabina Rinaldi
2, Marco Vinceti3,4, Pa
Luciano Vescovi3, Marcella Malavolti3 and Sabina Sieri
1 1Epidemiology and Prevention Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy
2International Agency for Research on Cancer, Lyon, France
3CREAGEN, Environmental, Genetic and Nutritional Epidemiology Research Center, Section of Public Health, Department of Biomedical, Metabolic and
Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy
4Department of Epidemiology, Boston University School of Public Health, Boston, MA, USA
5Environmental Epidemiology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 1Epidemiology and Prevention Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy
2International Agency for Research on Cancer, Lyon, France
3CREAGEN, Environmental, Genetic and Nutritional Epidemiology Research Center, Section of Public Health, Department of Biomedical, Metabolic and
Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy
4Department of Epidemiology, Boston University School of Public Health, Boston, MA, USA
5Environmental Epidemiology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy Cancer Epidemiology Cancer Epidemiology Diet is the primary source of cadmium—a proven Group 1 human carcinogen—for non-smokers. Observational studies
investigating the effect of cadmium from food sources on breast cancer risk have produced inconsistent results. We examined
the association between dietary cadmium and risk of breast cancer defined by estrogen receptor (ER), progesterone receptor
(PR) and HER2 status, in 8924 women recruited to a prospective study between 1987 and 1992. Dietary cadmium intake was
estimated using a semi-quantitative food frequency questionnaire at baseline. During a median of 22 years of follow-up,
451 incident cases of breast cancer were identified through the Varese Cancer Registry. Multivariable-adjusted hazard ratios
(HRs) with 95% confidence intervals (CIs) for breast cancer and receptor-defined breast cancer subtypes were estimated for
quintiles of dietary cadmium intake, adjusting for confounding factors. Mean dietary cadmium intake was 7.8 (standard
deviation 1.4) μg/day. Women with highest quintile of cadmium intake had a greater risk of breast cancer (HR 1.54; 95% CI,
1.06–2.22; p trend = 0.028) than those with lowest quintile of intake. Women premenopausal at recruitment had HR = 1.73
(95% CI, 1.10–2.71, highest vs. lowest quintile); postmenopausal women had HR = 1.32 (95% CI, 1.05–1.66 for each standard
deviation increase in cadmium). Introduction synthesize cadmium-inducible proteins called metallothioneins
that protect cells by binding toxic Cd2+. In addition circulating
Cd-metallothionein is taken up by proximal tubular cells of
kidney resulting in cadmium accumulation in kidney cortex.2 Cadmium (Cd) is a proven human carcinogen (group I of
International Agency for Research on Cancer classification).1
Cadmium is absorbed into the body from dietary sources, ciga-
rette smoke, and by inhalation in industrial or polluted envi-
ronments. Cadmium
levels
increase
with
age
since
the
elimination half-life is long (10–30 years).2–4 The metal accu-
mulates mainly in liver and kidney because these tissues Cadmium (Cd) is a proven human carcinogen (group I of
International Agency for Research on Cancer classification).1
Cadmium is absorbed into the body from dietary sources, ciga-
rette smoke, and by inhalation in industrial or polluted envi-
ronments. Cadmium
levels
increase
with
age
since
the
elimination half-life is long (10–30 years).2–4 The metal accu-
mulates mainly in liver and kidney because these tissues In non-smokers, diet is the main source of cadmium.5,6 In
smokers, inhaled tobacco smoke is usually the main source of
cadmium. One cigarette contains about 1–2 μg cadmium, and
about 10% of inhaled cadmium in tobacco smoke is absorbed.2 The Joint FAO/WHO Expert Committee on Food Additives7
recommends 25 μg/kg body weight as the maximum tolerable
monthly intake of cadmium. Concentrations of cadmium in
most foods are usually less than 0.15 mg/kg. Notable exceptions
are shellfish and kidneys, which contain 1–2 mg/kg and
0.5 mg/kg, respectively.8 Cadmium in vegetables and cereals
mainly derives from the soil. Cadmium in soil is usually derived
from cadmium-rich industrial or urban wastes, but may be pre-
sent naturally in geological terrains characterized by the presence
of zinc- or lead-rich minerals.8 Phosphate fertilizer and sewage
sludge, used agriculturally, are important contributors to cad-
mium in the environment and human foods.9 Key words: cadmium, breast cancer, estrogen receptors, progesterone
receptors, HER2
Abbreviations: ER: estrogen receptor; PR: progesterone receptor;
HRs: hazard ratios; CIs: confidence intervals; Cd: Cadmium; FFQ:
food frequency questionnaire; BMI: body mass index; SD: standard
deviation
The authors declare no potential conflicts of interest. Dietary cadmium and risk of breast cancer subtypes defined by
hormone receptor status: A prospective cohort study Cadmium-related risk of breast cancer did not vary with ER, PR or HER2 status (p-heterogeneity
not significant). These findings support the hypothesis that dietary cadmium is a risk factor for breast cancer. International Journal of Cancer
IJC 1Epidemiology and Prevention Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy
2International Agency for Research on Cancer, Lyon, France
3CREAGEN, Environmental, Genetic and Nutritional Epidemiology Research Center, Section of Public Health, Department of Biomedical, Metabolic and
Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy
4Department of Epidemiology, Boston University School of Public Health, Boston, MA, USA
5Environmental Epidemiology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy Introduction The lack of
significant heterogeneity in risk estimates between different ER and PR status neither supports nor refutes the hypothesis that
cadmium acts as a metalloestrogen. status of zinc, iron or calcium is high, and enhanced if the
nutritional status of these important micronutrients is low.11 hormone therapy, smoking, and education) and a self-reporting
semi-quantitative food frequency questionnaire (FFQ) designed
to capture the dietary habits of northern Italians.24 Anthropo-
metric measurements were taken using a standardized protocol. Body mass index (BMI) was calculated as weight (kg) divided
by height squared (m2). Women were considered postmeno-
pausal at recruitment if they reported no menses over the
preceding 12 months. All other women were considered pre-
menopausal. The main mechanisms of cadmium carcinogenesis are
thought to be promotion of oxidative stress and inflammation,
induction of DNA damage, alteration of DNA repair mechanisms,
and interferencewith apoptosis.12 Cadmiumalso hasestrogen-like
effects that include stimulation of human breast cancer cell
proliferation,12,13 increased expression of estrogen-regulated
genes,14activationofestrogenreceptor(ER)alpha,15andincreased
progesteronereceptor(PR)levelsinbreastcancercells.14 Cancer Epidemiology Cancer Epidemiology Cancer Epidemiology The FFQ investigated the quantities and types of foods and
drinks consumed in the year up to reporting, from which
average daily diet—food items and portion sizes—was esti-
mated for each person. Nutrient values of food items were
obtained from Italian food composition tables.26 Chemical
analyses of foods eaten in northern Italy were performed at
the University of Modena and Reggio Emilia. Cadmium levels
in foods were determined by inductively coupled plasma mass
spectrometry after wet-ashing with 50% aqueous nitric acid in
a microwave digestion system. Quantification limit was
0.02 μg/kg, and detection limit was 0.007 μg/kg.27 The cad-
mium levels thus obtained were added to the Italian food
composition tables to thereby afford estimates of dietary cad-
mium intake (μg/day) for each participant. Few
epidemiological
studies
have
assessed
associations
between dietary cadmium and breast cancer risk with conflicting
results. A meta-analysis of cohort and case–control studies
reported a significant positive association between dietary cad-
mium intake and breast cancer,16 while a subsequent update
which included two additional cohort studies, found that the posi-
tive association was no longer statistically significant.17 A recent
meta-analysis of studies on postmenopausal women found that
dietary cadmium was unrelated to breast cancer risk.18 Few stud-
ies have investigated associations between dietary cadmium and
the ER/PR status of breast cancers that develop19–23 again with
discordant results. Introduction DOI: 10.1002/ijc.32039
History: Received 17 Jul 2018; Accepted 21 Nov 2018;
Online 4 Dec 2018
Correspondence to: Sabina Sieri, PhD, Fondazione IRCCS Istituto
Nazionale dei Tumori, Via Venezian 1, 20133 Milano, Italy,
E-mail: sabina.sieri@istitutotumori.mi.it; Tel.: +39 02 239 03506; Fax:
+39 02 2390 3516 Key words: cadmium, breast cancer, estrogen receptors, progesterone
receptors, HER2 Abbreviations: ER: estrogen receptor; PR: progesterone receptor;
HRs: hazard ratios; CIs: confidence intervals; Cd: Cadmium; FFQ:
food frequency questionnaire; BMI: body mass index; SD: standard
deviation The authors declare no potential conflicts of interest. The authors declare no potential conflicts of interest. The authors declare no potential conflicts of interest. DOI: 10.1002/ijc.32039
History: Received 17 Jul 2018; Accepted 21 Nov 2018;
Online 4 Dec 2018
Correspondence to: Sabina Sieri, PhD, Fondazione IRCCS Istituto
Nazionale dei Tumori, Via Venezian 1, 20133 Milano, Italy,
E-mail: sabina.sieri@istitutotumori.mi.it; Tel.: +39 02 239 03506; Fax:
+39 02 2390 3516 About 5% of the cadmium ingested by adults is absorbed,
with
marked
between-individual
variation.10
Cadmium
absorption from dietary sources is reduced if the nutritional E-mail: sabina.sieri@istitutotumori.mi.it; Tel.: +39 02 239 03506; Fax:
+39 02 2390 3516 Int. J. Cancer: 144, 2153–2160 (2019) © 2018 UICC What’s new? Diet is the primary source of cadmium – a proven Group 1 human carcinogen – for non-smokers. Observational studies
investigating the effect of cadmium from food sources on breast cancer risk have produced inconsistent results, however. This
first cohort study to investigate the effects of dietary cadmium on the risk of breast cancer and breast cancer subtypes defined
by the expression of ER, PR, and HER2 provides further evidence that dietary cadmium increases breast cancer risk. The lack of
significant heterogeneity in risk estimates between different ER and PR status neither supports nor refutes the hypothesis that
cadmium acts as a metalloestrogen. 2154
Dietary cadmium and breast cancer risk 2154 Dietary cadmium and breast cancer risk Dietary cadmium and breast cancer risk Dietary cadmium and breast cancer risk Diet is the primary source of cadmium – a proven Group 1 human carcinogen – for non-smokers. Observational studies
investigating the effect of cadmium from food sources on breast cancer risk have produced inconsistent results, however. This
first cohort study to investigate the effects of dietary cadmium on the risk of breast cancer and breast cancer subtypes defined
by the expression of ER, PR, and HER2 provides further evidence that dietary cadmium increases breast cancer risk. Introduction As far as we are aware no studies have investi-
gated dietary cadmium exposure and risk of breast cancer accord-
ing to HER2 status. In the present study, we investigated a cohort
of Italian women to ascertain whether dietary cadmium intake
was associated with risk of breast cancer, and also with the risk of
breast cancer subtypes defined by ER, PR, and HER2 status. Cancer incidence, available from the Varese Cancer Regis-
try, was linked to the ORDET database to identify breast can-
cer cases incident up to the end of December 2012. The
ORDET database was also linked to the regional file of Varese
residents to check vital status at December 31st 2012. Int. J. Cancer: 144, 2153–2160 (2019) © 2018 UICC Statistical Methods Dietary cadmium was adjusted for energy intake using the
regression-residual method.31 Cadmium intake was categorized
into quintiles based on the distribution of intake in the whole
cohort, with lowest quartile as reference. Hazard ratios (HRs)
with 95% confidence intervals (CIs) for developing breast can-
cer in relation to quintiles of dietary cadmium were estimated
from multivariable Cox proportional hazard models, with age
as primary time variable. HRs were also calculated for 1 stan-
dard deviation increases in cadmium intake as a continuous
variable. We ran a minimally adjusted model, with age and
energy intake (continuous, kcal/day) as covariates. We also ran
models additionally adjusted for menopausal status (pre- or
postmenopausal), age at menarche (> or ≤13 years), height
(continuous), BMI (continuous), age at first birth (nulliparous,
≤30, >30 years), years of education (≤5, 8–12, >12 years),
smoking status (never, current, former), alcohol intake (contin-
uous, g/day), vegetable intake (excluding potatoes and pulses,
g/day). We also ran fully adjusted models, additionally adjusted
for dietary zinc, iron and calcium (continuous, mg/day) (these
micronutrients may interfere with the intestinal absorption of
cadmium). Linear trends were tested using median cadmium
values within quintiles as a continuous variable. We also
assessed whether the effect of cadmium exposure was influ-
enced by menopausal status, smoking status, and BMI (all at
recruitment) using a likelihood ratio test that compared a
model that included the cross-product term with one that did
not include it. We also performed analyses stratified by meno-
pausal status at recruitment (pre or postmenopausal). Compet-
ing risks Cox regression models were used to analyze the risk
of developing one breast cancer subtype with other subtypes
considered as competing risks. Women who developed a com-
peting breast cancer subtype were censored at the time of diag-
nosis. The heterogeneity of associations of cadmium intake
with different subtypes was assessed after applying data aug-
mentation, as described by Lunn and McNeil.32 All analyses
were performed with STATA software (version 14.0; Stata
Corp, College Station, TX). Cancer Epidemiology Cancer Epidemiology Table 2 shows HRs (with 95% CIs) for breast cancer by
quintiles of energy-adjusted dietary cadmium. By the addi-
tionally adjusted model women in the highest intake quintile
had a significantly greater risk of breast cancer (HR = 1.43;
95% CI, 1.02–2.01; p trend = 0.047) than those in the lowest
quintile (reference). Materials and Methods Fifty-one women who moved out of the area, or who were
lost to follow-up immediately after recruitment were excluded. An additional 1552 women were excluded because the FFQ was
not available. We also decided to exclude women diagnosed with
breast cancer in the first 90 days of follow-up. The cohort was
further reduced by excluding women with missing values for
covariates included in the fully adjusted risk models, or for
whom the ratio of total energy intake (determined from the
FFQ) to basal metabolic rate, determined by Herris-Benedict
equation,28 was at either extreme (first and last half-percentiles)
of the distribution. The analyses were conducted on 8924
women. Participants were women recruited to the study on hormones,
diet, and the etiology of breast cancer (ORDET), which was
designed to prospectively investigate associations of hormones
and diet with breast cancer risk. Study methods are available
elsewhere.24,25 Briefly, between June 1987 and June 1992,
10,786 healthy women aged 34–70 years, resident in Varese
province, northern Italy, were recruited from the general pop-
ulation. Women taking hormone therapy in the 3 months
before recruitment, with a history of cancer or liver disease, or
who had undergone bilateral ovariectomy, were excluded. The
study protocol was approved by the ethics committee of the
National Cancer Institute of Milan. The study complied with
the
Helsinki
Declaration,
and
participants
gave
written
informed consent to use their clinical data for research. ER, PR and HER2 status was assessed from pathology
records or determined (where possible) from ad hoc analyses
of slides or tissue blocks, as described elsewhere.29 Specimens
examined immunohistochemically were considered to be hor-
mone receptor-positive when ≥10% of tumor cell nuclei were At baseline participants completed a lifestyle questionnaire
(that included questions on reproductive history, menstruation, Int. J. Cancer: 144, 2153–2160 (2019) © 2018 UICC 2155 Grioni et al. was available for 456 cases (361 ER+ and 95 ER−); PR status
was available for 435 cases (293 PR+ and 142 PR−); HER2 sta-
tus was available for 421 cases (351 HER2−and 70 HER2+). was available for 456 cases (361 ER+ and 95 ER−); PR status
was available for 435 cases (293 PR+ and 142 PR−); HER2 sta-
tus was available for 421 cases (351 HER2−and 70 HER2+). stained, and negative when staining was <10%. HER2 was
considered overexpressed (positive) when >30% of cancer
Cells exhibited complete intense membrane staining: i.e. Materials and Methods score
3+ according to ASCO 2007 guidelines.30 The determinations
were performed at the Molecular Biology Unit Laboratories of
the National Cancer Institute, Milan. Estimated dietary energy-adjusted cadmium intake ranged
from 0.5 to 16.1 μg/day, mean 7.8 (standard deviation, SD, 1.4)
μg/day, or 0.89 (SD 0.31) μg/week/kg body weight. Women in
the highest quintile of dietary cadmium consumed more vegeta-
bles on average (Table 1), less alcohol, were less educated, more
likely to be never-smokers, older at menarche, less likely to be
premenopausal, and reported older age at first birth, than
women in the lowest quintile. Women in the highest quintile
also had lower mean dietary calcium intake, and higher mean
dietary iron intake, than women in the lowest quintile. In each
quintile,
mean
estimated
dietary
iron
intake
was
under
18 mg/day—below the level recommended for premenopausal
women by the Italian Human Nutrition Society.33 The main
contributors to dietary cadmium (data not shown in tables) were
grain-based foods (46.7%), vegetables (22.8%), wine (11.8%), and
potatoes (8.4%). Meat, fish and offal products contributed least
to cadmium intake (2.3%, 1.4% and 0.7% respectively). The study period was from date of recruitment (variable)
to closure on December 31, 2012, with participants censored
at date of cancer diagnosis, death, or loss to follow-up, which-
ever came first. Statistical Methods In the fully adjusted model, the risk was
slightly higher (HR = 1.54, 95% CI, 1.06–2.22; p trend = 0.028). In the fully adjusted model with cadmium as a continuous
variable (1.40 μg/day increments), increasing cadmium was
associated with an HR of 1.20 (95% CI, 1.05–1.37) for breast
cancer. This association persisted after excluding current and
former smokers (data not shown). For women premenopausal
at recruitment risks were somewhat higher: by the additionally
adjusted model those in the highest quintile of intake had a
significantly greater risk of breast cancer than those in the
lowest quintile (HR = 1.71; 95% CI, 1.12–2.59, p trend = 0.046);
the corresponding HR for the fully adjusted model was 1.73
(95% CI, 1.10–2.71, p trend = 0.063). For women postmenopausal at recruitment the increased
risk of breast cancer was significant only in the model in
which cadmium was a continuous variable (HR = 1.32; 95%
CI, 1.05–1.66, for 1.40 μg/day increments in cadmium intake). Interactions of risk estimates with menopausal status and
other potential effect modifiers (data not shown for BMI and
smoking status) were not significant. We performed competing risks analyses of risk of breast
cancer defined by ER, PR and HER2 expression (Table 3). For
ER+ disease, fully adjusted model, the HR was 1.64 (95% CI,
1.06–2.54; highest vs. lowest quintile). Associations were
weaker and not significant for ER−disease (HR = 1.30; 95%
CI, 0.60–2.83). There was no significant heterogeneity in risk
estimates between ER+ and ER−disease. Results During a median 22.1 years of follow-up (179,540 person-
years), 481 incident breast cancer cases (451 invasive and
30 in situ) were identified among the 8924 women. ER status While for PR−disease there was no evidence that cadmium
intake influenced risk, for PR+ disease, risks were significantly Int. J. Cancer: 144, 2153–2160 (2019) © 2018 UICC Dietary cadmium and breast cancer risk 2156 tics of 8924 ORDET women by quintiles of estimated dietary cadmium intake Table 1. Summary baseline characteristics of 8924 ORDET women by quintiles of estimated dietary cadmium intake
Quintiles of cadmium intake (energy-adjusted, residual method)
Q1
Q2
Q3
Q4
Q5
Cadmium (μg/day)
0.45–6.72
6.73–7.40
7.41–8.02
8.03–8.81
8.82–16.10
N women
1785
1785
1785
1785
1784
Age (years); mean (SD)
47.8 (8.3)
48.3 (8.6)
48.7 (8.6)
48.9 (8.8)
49.0 (8.6)
Body mass index (kg/m2); mean (SD)
25.0 (4.0)
25.3 (4.3)
25.3 (4.3)
25.4 (4.2)
25.6 (4.4)
Height (m); mean (SD)
1.58 (0.1)
1.58 (0.1)
1.58 (0.1)
1.58 (0.1)
1.58 (0.1)
>30 years at first birth %
11.9
12.9
12.7
12
13.3
>13 years at menarche %
31.2
30.7
34.9
34.2
35.7
Premenopausal at baseline %
70.5
68.8
67.1
65.4
64.1
Never used oral contraceptive %
64.5
66.4
65.2
66.8
69.1
≤5 years of schooling %
42.5
47.5
47.6
51.4
52.4
Never smokers %
60.9
65.0
64.2
67.7
66.3
Energy intake (kcal/day); mean (SD)
1873 (510.7)
1707 (468.2)
1687 (467.2)
1734 (468.2)
1847 (484.5)
Vegetable intake1 (g/day); mean (SD)
157.0 (76.0)
166.4 (78.6)
189.3 (79.0)
216.6 (83.0)
303.0 (118.9)
>12 g/day alcohol %
52.3
42.5
35.9
31.3
25.3
Zinc (mg/day); mean (SD)
12.0 (3.8)
10.9 (3.0)
10.8 (3.3)
11.2 (3.3)
11.9 (3.6)
Iron (mg/day); mean (SD)
11.8 (3.3)
11.1 (3.0)
11.2 (3.1)
11.8 (3.2)
13.5 (3.5)
Calcium (mg/day); mean (SD)
877.5 (412.7)
704.0 (306.4)
656 (286.5)
652.8 (272.6)
710.4 (287.0)
1Leafy vegetables, tomatoes, carrots, fruit and fruiting vegetables. Cancer Epidemiology Cancer Epidemiology risk,19–22,34 while the cohort study of Julin et al.23 found a sig-
nificant positive association, with relative risk (RR) of 1.21
(95% CI, 1.06–1.37) for highest vs. lowest tertile of cadmium
intake. These six studies (321,315 women, 11,978 breast can-
cer cases) formed the data for a recent meta-analysis to evalu-
ate the combined RR using a random-effects model. our study
did not find a significant association between dietary cad-
mium and overall breast cancer risk with a combined RR of
1.01 (95% CI, 0.88–1.14) for highest vs. Results lowest category of die-
tary cadmium.17 Similarly, a recent meta-analysis of studies
on postmenopausal women found no relation between dietary
cadmium and breast cancer risk.18 Thus literature findings are
conflicting and suggest the need for further studies on the role
of cadmium in breast cancer. higher in the 2nd to 5th quintiles of intake compared to the
1st, in both the additionally adjusted and fully adjusted
models. For the fully adjusted model HRs were 1.67 (95% CI,
1.12–2.49),
1.83
(95%
CI,
1.21–2.76),
2.01
(95%
CI,
1.30–3.09), and 1.90 (95% CI, 1.15–3.13). Nevertheless there
was no significant heterogeneity in risk estimates between the
PR+ and PR−subtypes. No significant association of cadmium intake with risk of
HER2+ or HER2−disease was found, with the single exception
that, with cadmium as a continuous variable, the HR for
HER2−disease was 1.18 (95% CI, 1.01–1.37). Again there was
no significant heterogeneity of risk estimates between the two
HER2 subtypes. Discussion Cadmium has long half-life (10–30 years) and accumulates
in tissues, including breast tissue.35 In vivo and in vitro
studies show that cadmium stimulates the proliferation of
breast cancer cells,36,37 activates and increases the expression
of estrogen-regulated genes,14 activates ER alpha,14,15,37 and
upregulates PR expression in breast cancer cells.14 These find-
ings suggest that cadmium is an estrogen mimetic and hence
may promote the development of estrogen-dependent breast
cancer.23,38,39 We found that ER+ disease was significantly
associated high dietary cadmium while ER−disease was not,
however there was no significant heterogeneity in risk esti-
mates between ER+ and ER−subtypes. Furthermore PR+ dis-
ease was significantly associated with high cadmium, while
PR−disease was not, but once again there was no significant
heterogeneity of risk between the PR subtypes. A Danish pro-
spective cohort, which found that dietary cadmium was not
associated with breast cancer, also found no evidence of an In our study we found that increasing dietary cadmium intake
was significantly associated with increasing risk of breast can-
cer. Analyses stratified by menopausal status showed that this
association persisted in premenopausal women but almost dis-
appeared in postmenopausal women, nevertheless interactions
of risk estimates with menopausal status were not significant. Analysis by disease subtype showed that cadmium intake
was associated with increased risk of both ER+ and PR+ breast
cancer. The associations were stronger for the fully adjusted
model (which also adjusted for dietary iron, calcium and
zinc). To our knowledge this is the first prospective study to
investigate associations between dietary cadmium and breast
cancer defined by hormone receptor expression in an Italian
population. Several cohort and case–control studies found no signifi-
cant association between dietary cadmium and breast cancer Int. J. Cancer: 144, 2153–2160 (2019) © 2018 UICC 2157 Grioni et al. 1Adjusted for age and energy intake. 2Additionally adjusted for menopausal status (pre-, post-menopausal in model including all women), age at menarche (≤13, >13 years), height, BMI, age at first childbirth (nulliparous, ≤30, >30 years),
smoking status (current, former, never smoker), years of education (≤5, 8-12, >12 years), alcohol (g/day ethanol), vegetable intake (g/day leafy vegetables, tomatoes, carrots, fruits, fruiting
vegetables). 3Additionally adjusted for dietary iron (mg/day), dietary calcium (mg/day) and dietary zinc (mg/day). 4Test for linear trend performed using median intake of each quintile. ntake. Int. J. Cancer: 144, 2153–2160 (2019) © 2018 UICC Discussion Hazard ratios (HRs) with 95% confidence intervals (CIs) for receptor-defined breast cancer in ORDET women by quintiles of dietary cadmium
Quintiles of dietary cadmium (energy-adjusted, residual method)
Q1
Q2
Q3
Q4
Q5
p trend7
Continuous
p heterogeneity8
Cadmium intake (μg/day)
0.45–6.72
6.73–7.40
7.41–8.02
8.03–8.81
8.82–16.10
ER–disease4
N cases
21
18
20
15
21
Minimally adjusted HR (95% CI)1
1.00
0.88 (0.47–1.65)
0.97 (0.52–1.80)
0.74 (0.38–1.44)
1.03 (0.56–1.88)
0.952
1.04 (0.85–1.27)
Additionally adjusted HR (95% CI)2
1.00
0.93 (0.49–1.76)
1.08 (0.57–2.04)
0.88 (0.44–1.78)
1.44 (0.71–2.96)
0.408
1.22 (0.94–1.57)
Fully adjusted HR (95% CI)3
1.00
0.91 (0.47 -1.73)
1.03 (0.54–2.00)
0.83 (0.40–1.73)
1.30 (0.60–2.83)
0.600
1.19 (0.90- 1.59)
ER+disease4
N cases
63
78
81
74
65
Minimally adjusted HR (95% CI)1
1.00
1.22 (0.87–1.71)
1.24 (0.89–1.74)
1.16 (0.83–1.63)
1.03 (0.73–1.46)
0.988
0.99 (0.89–1.10)
Additionally adjusted HR (95% CI)2
1.00
1.29 (0.92–1.81)
1.40 (1.00–1.97)
1.41 (0.99–2.01)
1.44 (0.96–2.16)
0.067
1.14 (1.00–1.31)
Fully adjusted HR (95% CI)3
1.00
1.35 (0.96–1.90)
1.49 (1.04–2.12)
1.53 (1.05–2.23)
1.64 (1.06–2.54)
0.025
1.22 (1.04–1.42)
ER+ vs ER–
0 9218 Table 3. Hazard ratios (HRs) with 95% confidence intervals (CIs) for receptor-defined breast cancer in ORDET women by quintiles of dietary cadmium
Quintiles of dietary cadmium (energy-adjusted, residual method)
Q1
Q2
Q3
Q4
Q5
p trend7
Continuous
p heterogeneity8
Cadmium intake (μg/day)
0.45–6.72
6.73–7.40
7.41–8.02
8.03–8.81
8.82–16.10
ER–disease4
N cases
21
18
20
15
21
Minimally adjusted HR (95% CI)1
1.00
0.88 (0.47–1.65)
0.97 (0.52–1.80)
0.74 (0.38–1.44)
1.03 (0.56–1.88)
0.952
1.04 (0.85–1.27)
Additionally adjusted HR (95% CI)2
1.00
0.93 (0.49–1.76)
1.08 (0.57–2.04)
0.88 (0.44–1.78)
1.44 (0.71–2.96)
0.408
1.22 (0.94–1.57)
Fully adjusted HR (95% CI)3
1.00
0.91 (0.47 -1.73)
1.03 (0.54–2.00)
0.83 (0.40–1.73)
1.30 (0.60–2.83)
0.600
1.19 (0.90- 1.59)
ER+disease4
N cases
63
78
81
74
65
Minimally adjusted HR (95% CI)1
1.00
1.22 (0.87–1.71)
1.24 (0.89–1.74)
1.16 (0.83–1.63)
1.03 (0.73–1.46)
0.988
0.99 (0.89–1.10)
Additionally adjusted HR (95% CI)2
1.00
1.29 (0.92–1.81)
1.40 (1.00–1.97)
1.41 (0.99–2.01)
1.44 (0.96–2.16)
0.067
1.14 (1.00–1.31)
Fully adjusted HR (95% CI)3
1.00
1.35 (0.96–1.90)
1.49 (1.04–2.12)
1.53 (1.05–2.23)
1.64 (1.06–2.54)
0.025
1.22 (1.04–1.42)
ER+ vs. Discussion ER–
0.9218
PR–disease5
N cases
35
28
31
19
29
Minimally adjusted HR (95% CI)1
1.00
0.77 (0.46–1.27)
0.83 (0.51–1.35)
0.53 (0.30–0.92)
0.82 (0.50–1.35)
0.269
0.95 (0.81–1.13)
Additionally adjusted HR (95% CI)2
1.00
0.85 (0.51–1.39)
0.98 (0.59–1.61)
0.68 (0.38–1.22)
1.29 (0.71–2.33)
0.656
1.16 (0.93–1.45)
Fully adjusted HR (95% CI)3
1.00
0.83 (0.50–1.39)
0.96 (0.57–1.62)
0.66 (0.36–1.23)
1.22 (0.64–2.32)
0.793
1.17 (0.91–1.48)
PR+disease5
N cases
43
65
67
66
52
Minimally adjusted HR (95% CI)1
1.00
1.53 (1.04–2.25)
1.55 (1.06–2.28)
1.56 (1.06–2.29)
1.22 (0.81–1.82)
0.462
1.03 (0.92–1.15)
Additionally adjusted HR (95% CI)2
1.00
1.59 (1.07–2.34)
1.70 (1.15–2.52)
1.82 (1.21–2.72)
1.63 (1.02–2.59)
0.031
1.16 (1.01 -1.35)
Fully adjusted HR (95% CI)3
1.00
1.67 (1.12–2.49)
1.83 (1.21–2.76)
2.01 (1.30–3.09)
1.90 (1.15–3.13)
0.011
1.24 (1.05–1.47)
PR+ vs. PR–
0.6551 HER2– disease6
N cases
62
79
74
67
69
Minimally adjusted HR (95% CI)1
1.00
1.27 (0.91–1.78)
1.16 (0.83–1.63)
1.08 (0.76–1.52)
1.11 (0.79–1.57)
0.860
1.02 (0.91–1.13)
Additionally adjusted HR (95% CI)2
1.00
1.32 (0.95–1.86)
1.25 (0.89–1.78)
1.23 (0.85–1.77)
1.40 (0.94–2.10)
0.172
1.12 (0.98–1.29)
Fully adjusted HR (95% CI)3
1.00
1.36 (0.97–1.92)
1.31 (0.91–1.88)
1.30 (0.88–1.91)
1.53 (0.99–2.37)
0.102
1.18 (1.01–1.37)
HER2+disease6
N cases
17
11
15
13
14
Minimally adjusted HR (95% CI)1
1.00
0.67 (0.31–1.44)
0.92 (0.46–1.85)
0.81 (0.39–1.67)
0.86 (0.42–1.75)
0.811
0.95 (0.75–1.20)
Additionally adjusted HR (95% CI)2
1.00
0.80 (0.37–1.73)
1.29 (0.62–2.67)
1.33 (0.61 -2.89)
2.05 (0.88–4.77)
0.073
1.32 (0.97–1.79)
Fully adjusted HR (95% CI)3
1.00
0.79 (0.36–1.73)
1.25 (0.58–2.67)
1.31 (0.58–2.99)
2.02 (0.81–5.05)
0.097
1.34 (0.95–1.89)
HER2+ vs. HER2−
0.5075
1Competing risks Cox model adjusted for age and energy intake. 2Competing risks Cox model additionally adjusted for menopausal status, age at menarche, height, BMI, age at first child, smoking status, years of education, alcohol, and vegetables. 3Competing risks Cox model additionally adjusted for dietary iron (mg/day), dietary calcium (mg/day), and dietary zinc (mg/day). 4Excluding 25 cases with no information on ER status. 5Excluding 46 cases with no information on PR status. 6Excluding 60 cases with no information on HER2 status. 7Test for linear trend performed using median intake of each quintile. 8Test for heterogeneity performed using data augmentation technique, as described by Lunn and McNeil. Table 3. Discussion Hazard ratios (HRs) with 95% confidence intervals (CIs) for receptor-defined breast cancer in ORDET women by quintiles of dietary cadmium
Quintiles of dietary cadmium (energy-adjusted, residual method)
Q1
Q2
Q3
Q4
Q5
p trend7
Continuous
p heterogeneity
Cadmium intake (μg/day)
0.45–6.72
6.73–7.40
7.41–8.02
8.03–8.81
8.82–16.10
ER–disease4
N cases
21
18
20
15
21
Minimally adjusted HR (95% CI)1
1.00
0.88 (0.47–1.65)
0.97 (0.52–1.80)
0.74 (0.38–1.44)
1.03 (0.56–1.88)
0.952
1.04 (0.85–1.27)
Additionally adjusted HR (95% CI)2
1.00
0.93 (0.49–1.76)
1.08 (0.57–2.04)
0.88 (0.44–1.78)
1.44 (0.71–2.96)
0.408
1.22 (0.94–1.57)
Fully adjusted HR (95% CI)3
1.00
0.91 (0.47 -1.73)
1.03 (0.54–2.00)
0.83 (0.40–1.73)
1.30 (0.60–2.83)
0.600
1.19 (0.90- 1.59)
ER+disease4
N cases
63
78
81
74
65
Minimally adjusted HR (95% CI)1
1.00
1.22 (0.87–1.71)
1.24 (0.89–1.74)
1.16 (0.83–1.63)
1.03 (0.73–1.46)
0.988
0.99 (0.89–1.10)
Additionally adjusted HR (95% CI)2
1.00
1.29 (0.92–1.81)
1.40 (1.00–1.97)
1.41 (0.99–2.01)
1.44 (0.96–2.16)
0.067
1.14 (1.00–1.31)
Fully adjusted HR (95% CI)3
1.00
1.35 (0.96–1.90)
1.49 (1.04–2.12)
1.53 (1.05–2.23)
1.64 (1.06–2.54)
0.025
1.22 (1.04–1.42)
ER+ vs. ER–
0.9218
PR–disease5
N cases
35
28
31
19
29
Minimally adjusted HR (95% CI)1
1.00
0.77 (0.46–1.27)
0.83 (0.51–1.35)
0.53 (0.30–0.92)
0.82 (0.50–1.35)
0.269
0.95 (0.81–1.13)
Additionally adjusted HR (95% CI)2
1.00
0.85 (0.51–1.39)
0.98 (0.59–1.61)
0.68 (0.38–1.22)
1.29 (0.71–2.33)
0.656
1.16 (0.93–1.45)
Fully adjusted HR (95% CI)3
1.00
0.83 (0.50–1.39)
0.96 (0.57–1.62)
0.66 (0.36–1.23)
1.22 (0.64–2.32)
0.793
1.17 (0.91–1.48)
PR+disease5
N cases
43
65
67
66
52
Minimally adjusted HR (95% CI)1
1.00
1.53 (1.04–2.25)
1.55 (1.06–2.28)
1.56 (1.06–2.29)
1.22 (0.81–1.82)
0.462
1.03 (0.92–1.15)
Additionally adjusted HR (95% CI)2
1.00
1.59 (1.07–2.34)
1.70 (1.15–2.52)
1.82 (1.21–2.72)
1.63 (1.02–2.59)
0.031
1.16 (1.01 -1.35)
Fully adjusted HR (95% CI)3
1.00
1.67 (1.12–2.49)
1.83 (1.21–2.76)
2.01 (1.30–3.09)
1.90 (1.15–3.13)
0.011
1.24 (1.05–1.47)
PR+ vs. Discussion opausal status (pre-, post-menopausal in model including all women), age at menarche (≤13, >13 years), height, BMI, age at first childbirth (nulliparous, ≤30, >30 years),
er, never smoker), years of education (≤5, 8-12, >12 years), alcohol (g/day ethanol), vegetable intake (g/day leafy vegetables, tomatoes, carrots, fruits, fruiting Table 2. Hazard ratios (HR) with 95% confidence intervals (CIs) for breast cancer in all ORDET women, and according to menopausal status at recruitment, by quintiles of dietary cadmium
Quintiles of dietary cadmium (energy-adjusted, residual method)
Q1
Q2
Q3
Q4
Q5
p trend4
Continuous
Cadmium intake (μg/day)
0.45–6.72
6.73–7.40
7.41–8.02
8.03–8.81
8.82–16.10
All women
N cases
92
100
105
91
93
Minimally adjusted HR (95% CI)1
1.00
1.08 (0.81–1.43)
1.12 (0.84–1.48)
0.99 (0.74–1.32)
1.01 (0.76–1.35)
0.869
0.99 (0.91–1.09)
Additionally adjusted HR (95% CI)2
1.00
1.15 (0.86–1.53)
1.26 (0.94–1.68)
1.20 (0.88–1.63)
1.43 (1.02–2.01)
0.047
1.15 (1.02–1.29)
Fully adjusted HR (95% CI)3
1.00
1.17 (0.88–1.57)
1.30 (0.96–1.76)
1.25 (0.90–1.74)
1.54 (1.06–2.22)
0.028
1.20 (1.05–1.37)
Premenopausal women
N cases
60
74
70
51
62
Minimally adjusted HR (95% CI)1
1
1.25 (0.89–1.76)
1.21 (0.85–1.71)
0.91 (0.63–1.33)
1.15 (0.81–1.64)
0.938
0.99 (0.88–1.10)
Additionally adjusted HR (95% CI)2
1
1.36 (0.96–1.92)
1.40 (0.98–2.00)
1.13 (0.77–1.69)
1.71 (1.12–2.59)
0.046
1.14 (0.99–1.32)
Fully adjusted HR (95% CI)3
1
1.36 (0.96–1.94)
1.40 (0.97–2.04)
1.15 (0.76–1.74)
1.73 (1.10–2.71)
0.063
1.14 (0.97–1.34)
Postmenopausal women
N cases
32
26
35
40
31
Minimally adjusted HR (95% CI)1
1
0.77 (0.45–1.29)
0.95 (0.59–1.53)
1.08 (0.68–1.72)
0.80 (0.49–1.31)
0.718
1.01 (0.86–1.19)
Additionally adjusted HR (95% CI)2
1
0.80 (0.47–1.35)
1.03 (0.63–1.69)
1.25 (0.76–2.05)
1.03 (0.58–1.85)
0.485
1.17 (0.95–1.43)
Fully adjusted HR (95% CI)3
1
0.85 (0.50–1.46)
1.14 (0.68–1.92)
1.43 (0.84–2.44)
1.29 (0.68–2.44)
0.190
1.32 (1.05–1.66)
p Interaction5 Premenopausal vs. Postmenopausal = 0.769
1Adjusted for age and energy intake. 2Additionally adjusted for menopausal status (pre-, post-menopausal in model including all women), age at menarche (≤13, >13 years), height, BMI, age at first childbirth (nulliparous, ≤30, >30 years)
smoking status (current, former, never smoker), years of education (≤5, 8-12, >12 years), alcohol (g/day ethanol), vegetable intake (g/day leafy vegetables, tomatoes, carrots, fruits, fruiting
vegetables). 3Additionally adjusted for dietary iron (mg/day), dietary calcium (mg/day) and dietary zinc (mg/day). 4Test for linear trend performed using median intake of each quintile. 5Test for interaction was performed using the likelihood ratio test. Cancer Epidemiology Int. J. Cancer: 144, 2153–2160 (2019) © 2018 UICC 2158 Dietary cadmium and breast cancer risk Table 3. Discussion association between cadmium and specific PR or ER sub-
types.21 A case–control study on Japanese women, which
found no overall association between dietary cadmium and
breast cancer risk, and weak or absent associations with PR+
and PR−disease, found that high cadmium intake was associ-
ated with significantly increased risk of ER+ disease risk
(p trend = 0.032) among postmenopausal women.22 A large
Swedish prospective study on postmenopausal women, found
that dietary cadmium was significantly associated with risk of
invasive breast cancer overall and also ER+ disease, but not
ER−disease.23 Strengths of the present study are its prospective design,
availability of information on diet, availability of ER, PR and
HER2 status of the cancers in most cases, and availability of
abundant additional information making it possible to adjust
for potential confounders including smoking. Furthermore
breast cancer cases were identified by the local cancer registry,
and it is unlikely that any cases were lost.44 Other strengths
are the large number of food samples used for the analytical
determinations of cadmium, collected from local groceries
and supermarkets in order to mimic the real sources of food-
stuffs in the population.27 Thus, literature findings on the risk of breast cancer
defined by ER and PR status are contrasting, as they are for
risk of breast cancer as a whole. Our data suggest that cad-
mium does have a breast-cancer-promoting effect, but the evi-
dence is insufficient to suggest whether this effect occurs
occur via estrogen-mimetic mechanisms. Furthermore a poten-
tial estrogenic effect of cadmium might be partly masked by
the effect of endogenous estrogens in premenopausal women in
our cohort. Cancer Epidemiology Cancer Epidemiology The fact that diet was only investigated on one occasion
(at recruitment) is a limitation, since dietary habits may have
changed over time. The FFQ assessed diet over the year up to
recruitment, permitting assessment of long-term dietary cad-
mium intake, even though the FFQ was not originally
designed to assess cadmium. Although cadmium levels in
foods were determined accurately, their levels vary with soil
type, water source, fertilizer used, and levels of anthropogenic
contamination.41,45 In addition we had no information on
environmental/occupational exposure to cadmium, although
data indicate that in most non-smokers diet is the main
source of cadmium.46 Also, and as noted above, cadmium
absorption is affected by nutritional status,11 so dietary intake
may not accurately reflect the cadmium absorbed by individ-
uals. Discussion All these factors will have resulted in some unquantifi-
able misclassification of exposure. It is noteworthy however
that our estimates of daily cadmium intake were closely simi-
lar to those estimated by other Italian studies47,48 while the
ranking of cadmium sources (cereals > vegetables > sweets >
fish/seafood) was the same as that found in other Italian,27,48
and European studies.45 As regards the HER2 status of breast cancer, although we
found a significant positive association between dietary cad-
mium as a continuous variable and HER2−disease (Table 3),
in general associations of cadmium with breast cancer defined
by HER2 status were not significant. More importantly, there
was no significant heterogeneity of risk between HER2 sub-
types. To our knowledge no other study has examined the asso-
ciation between dietary cadmium and breast cancer defined by
HER2 status. However Strumylaite et al. examined urinary cad-
mium (marker of long-term cadmium exposure) and the HER2
status of breast cancer in a case–control study,40 finding a sig-
nificant association (p trend <0.001) between risk of HER2−
breast cancer and urinary cadmium.38,39 Mean intake of cadmium from the diet in our cohort was
0.89 (SD 0.31) μg/week/kg body weight, which is well below
the intake limit of 7 μg/week/kg considered safe by the
WHO.41 The daily mean intake of dietary cadmium was
7.8 μg (standard deviation, SD, 1.4), which is similar to the
intake reported in a recent study in a Northern Italy commu-
nity (median 5.0 μg/day, interquartile range 3.17–7.65).27 Finally, the limited number of breast cancer cases likely
resulted in insufficient power to assess whether the cancer-
promoting effect of cadmium was due to estrogen-mimetic
mechanisms. Similarly we were unable to analyze subtypes
such as ER++PR+ which might have provided insight in the
role of cadmium in cancer subtypes known to differ in etiol-
ogy and prognosis. Cadmium is absorbed from the intestine and moved into
the blood by the same transporters used for essential metals
like zinc, iron and calcium,42 and since these transporters are
up-regulated when iron status is low,43 cadmium absorption is
increased when iron status is low. Discussion PR–
0.6551
HER2– disease6
N cases
62
79
74
67
69
Minimally adjusted HR (95% CI)1
1.00
1.27 (0.91–1.78)
1.16 (0.83–1.63)
1.08 (0.76–1.52)
1.11 (0.79–1.57)
0.860
1.02 (0.91–1.13)
Additionally adjusted HR (95% CI)2
1.00
1.32 (0.95–1.86)
1.25 (0.89–1.78)
1.23 (0.85–1.77)
1.40 (0.94–2.10)
0.172
1.12 (0.98–1.29)
Fully adjusted HR (95% CI)3
1.00
1.36 (0.97–1.92)
1.31 (0.91–1.88)
1.30 (0.88–1.91)
1.53 (0.99–2.37)
0.102
1.18 (1.01–1.37)
HER2+disease6
N cases
17
11
15
13
14
Minimally adjusted HR (95% CI)1
1.00
0.67 (0.31–1.44)
0.92 (0.46–1.85)
0.81 (0.39–1.67)
0.86 (0.42–1.75)
0.811
0.95 (0.75–1.20)
Additionally adjusted HR (95% CI)2
1.00
0.80 (0.37–1.73)
1.29 (0.62–2.67)
1.33 (0.61 -2.89)
2.05 (0.88–4.77)
0.073
1.32 (0.97–1.79)
Fully adjusted HR (95% CI)3
1.00
0.79 (0.36–1.73)
1.25 (0.58–2.67)
1.31 (0.58–2.99)
2.02 (0.81–5.05)
0.097
1.34 (0.95–1.89)
HER2+ vs. HER2−
0.5075
1Competing risks Cox model adjusted for age and energy intake. 2Competing risks Cox model additionally adjusted for menopausal status, age at menarche, height, BMI, age at first child, smoking status, years of education, alcohol, and vegetables. 3Competing risks Cox model additionally adjusted for dietary iron (mg/day), dietary calcium (mg/day), and dietary zinc (mg/day). 4Excluding 25 cases with no information on ER status. 5Excluding 46 cases with no information on PR status. 6Excluding 60 cases with no information on HER2 status. 7Test for linear trend performed using median intake of each quintile. 8Test for heterogeneity performed using data augmentation technique, as described by Lunn and McNeil. Int. J. Cancer: 144, 2153–2160 (2019) © 2018 UICC 2159 Grioni et al. calcium intake would be deficient in 33% of the cohort. Based
on these considerations, we suggest that the increased breast
cancer risk, particularly in premenopausal women, is in part
be due to increased cadmium absorption due to mild chronic
iron deficiency10 and perhaps also deficient zinc and calcium
intake. Discussion In fact cadmium absorption
is increased when calcium, iron or zinc are deficient11; and
blood levels of cadmium are significantly increased in preme-
nopausal women with low iron status (serum ferritin <20 μg/l).10
For 26% of our cohort dietary zinc was under the recom-
mended level 9 mg/day; and for 95% of the cohort women who
were premenopausal at recruitment, dietary iron intake was
below the 18 mg/day recommended.33 As regards calcium
intake, this was estimated from food sources only, but if we
assume that women were drinking about 1.5 l of water/day, References 1. Arsenic, metals, fibres, and dusts. IARC Monogr
Eval Carcinog Risks Hum 2012;100(Pt C):11–465. 1. Arsenic, metals, fibres, and dusts. IARC Monogr
Eval Carcinog Risks Hum 2012;100(Pt C):11–465. postmenopausal women: a systematic review and
meta-analysis. Environ Int 2016;86:1–13. 34. Sawada N, Iwasaki M, Inoue M, et al. Long-term
dietary cadmium intake and cancer incidence. Epidemiology 2012;23:368–76. postmenopausal women: a systematic review and
meta-analysis. Environ Int 2016;86:1–13. 2. Bernard A. Cadmium & its adverse effects on
human health. Indian J Med Res 2008;128:557–64. 2. Bernard A. Cadmium & its adverse effects on
human health. Indian J Med Res 2008;128:557–64. 19. Adams SV, Newcomb PA, White E. Dietary cad-
mium and risk of invasive postmenopausal breast
cancer in the VITAL cohort. Cancer Causes Con-
trol 2012;23:845–54. 35. Byrne C, Divekar SD, Storchan GB, et al. Metals
and breast cancer. J Mammary Gland Biol Neopla-
sia 2013;18:63–73. 3. Nawrot T, Plusquin M, Hogervorst J,
et al. Environmental exposure to cadmium and
risk of cancer: a prospective population-based
study. Lancet Oncol 2006;7:119–26. 20. Adams SV, Quraishi SM, Shafer MM,
et al. Dietary cadmium exposure and risk of
breast, endometrial, and ovarian cancer in the
Women’s Health Initiative. Environ Health Per-
spect. 2014;122:594–600. 36. Brama M, Gnessi L, Basciani S, et al. Cadmium
induces mitogenic signaling in breast cancer cell
by an ERalpha-dependent mechanism. Mol Cell
Endocrinol 2007;264:102–8. 4. Amzal B, Julin B, Vahter M, et al. Population tox-
icokinetic modeling of cadmium for health risk
assessment. Environ Health Perspect. 2009;117:
1293–301. Cancer Epidemiology 37. Martinez-Campa C, Alonso-Gonzalez C,
Mediavilla MD, et al. Melatonin inhibits both ER
alpha activation and breast cancer cell prolifera-
tion induced by a metalloestrogen, cadmium. J Pineal Res 2006;40:291–6. 21. Eriksen KT, Halkjaer J, Sorensen M, et al. Dietary
cadmium intake and risk of breast, endometrial
and ovarian cancer in Danish postmenopausal
women: a prospective cohort study. PLoS One
2014;9:e100815. 5. WHO. Environmental health criteria 134: cad-
mium. Geneva: World Health Organization, 1992. 6. Vahter M, Berglund M, Lind B, et al. Personal
monitoring of lead and cadmium exposure--a
Swedish study with special reference to methodo-
logical aspects. Scand J Work Environ Health
1991;17:65–74. 38. Aquino NB, Sevigny MB, Sabangan J, et al. The
role of cadmium and nickel in estrogen receptor
signaling and breast cancer: metalloestrogens or
not? J Environ Sci Health C Environ Carcinog Eco-
toxicol Rev 2012;30:189–224. 22. Itoh H, Iwasaki M, Sawada N, et al. References Dietary cad-
mium intake and breast cancer risk in Japanese
women: a case-control study. Int J Hyg Environ
Health 2014;217:70–7. 7. World Health Organization. Safety Evaluation of
Certain Food Additives and Contaminants:
Eighty-second Meeting of the Joint Fao/Who
Expert Committee on Food Additives Jecfa. 2017. 23. Julin B, Wolk A, Bergkvist L, et al. Dietary cad-
mium exposure and risk of postmenopausal breast
cancer: a population-based prospective cohort
study. Cancer Res 2012;72:1459–66. 39. Silva N, Peiris-John R, Wickremasinghe R,
et al. Cadmium a metalloestrogen: are we con-
vinced? J Appl Toxicol 2012;32:318–32. Expert Committee on Food Additives Jecfa. 2017. 8. World Health Organization, International Atomic
Energy Agency & Food and Agriculture Organiza-
tion of the United Nations. (1996). Trace elements
in human nutrition and health. Geneva: World
Health Organization. 40. Strumylaite L, Kregzdyte R, Bogusevicius A,
et al. Association between cadmium and breast
cancer risk according to estrogen receptor and
human epidermal growth factor receptor 2: epide-
miological evidence. Breast Cancer ResTreat 2014;
145:225–32. 24. Sieri S, Krogh V, Muti P, et al. Fat and protein
intake and subsequent breast cancer risk in post-
menopausal women. Nutr Cancer 2002;42:10–7. 25. Sieri S, Pala V, Brighenti F, et al. Dietary glycemic
index, glycemic load, and the risk of breast cancer
in an Italian prospective cohort study. Am J Clin
Nutr 2007;86:1160–6. 9. Satarug S, Baker JR, Urbenjapol S, et al. A global
perspective on cadmium pollution and toxicity in
non-occupationally exposed population. Toxicol
Lett 2003;137:65–83. 41. World Health Organization. Cadmium. Food
additives series. Geneva: Joint FAO/WHO Expert
Committee on Food Additives (JECFA), 2011. 26. Salvini S, Parpinel M, Gnagnarella P, et al. Banca
dati di composizione degli alimenti per studi epi-
demiologici in Italia. Milano: European Institute
of. Oncology 1998;958. 10. Berglund M, Akesson A, Nermell B,
et al. Intestinal absorption of dietary cadmium in
women depends on body iron stores and fiber
intake. Environ Health Perspect 1994;102:1058–66. 42. Satarug S, Haswell-Elkins MR, Moore MR. Safe
levels of cadmium intake to prevent renal
toxicity in human subjects. Br J Nutr 2000;84:
791–802. intake. Environ Health Perspect 1994;102:1058–66. 27. Filippini T, Cilloni S, Malavolti M, et al. Dietary
intake of cadmium, chromium, copper, manga-
nese, selenium and zinc in a northern Italy com-
munity. J Trace Elem Med Biol 2018;50:508–517. 43. Kim DW, Kim KY, Choi BS, et al. References Regulation of
metal transporters by dietary iron, and the rela-
tionship between body iron levels and cadmium
uptake. Arch Toxicol 2007;81:327–34. 11. Reeves PG, Chaney RL. Bioavailability as an
issue in risk assessment and management of food
cadmium: a review. Sci Total Environ 2008;
398:13–9. 28. Harris JA, Benedict FG. A biometric study of
human basal metabolism. Proc Natl Acad Sci USA
1918;4:370–3. 12. Joseph P. Mechanisms of cadmium carcinogene-
sis. Toxicol Appl Pharmacol 2009;238:272–9. 44. Tagliabue G, Maghini A, Fabiano S,
et al. Consistency and accuracy of diagnostic can-
cer codes generated by automated registration:
comparison with manual registration. Popul
Health Metr 2006;4:10. 29. Sieri S, Krogh V, Bolelli G, et al. Sex hormone
levels, breast cancer risk, and cancer receptor sta-
tus in postmenopausal women: the ORDET
cohort. Cancer Epidemiol Biomarkers Prev 2009;
18:169–76. 13. Waalkes MP. Cadmium carcinogenesis. Mutat Res
2003;533:107–20. 14. Garcia-Morales P, Saceda M, Kenney N,
et al. Effect of cadmium on estrogen receptor
levels and estrogen-induced responses in human
breast cancer cells. J Biol Chem 1994;269:
16896–901. 45. European Food Safety Authority (EFSA). Cad-
mium in food - scientific opinion of the panel on
contaminants in the food chain. EFSA J 2009;7:
980 n/a. 30. Wolff AC, Hammond ME, Schwartz JN,
et al. American Society of Clinical Oncology/Col-
lege of American Pathologists guideline recom-
mendations for human epidermal growth factor
receptor 2 testing in breast cancer. Arch Pathol
Lab Med 2007;131:18–43. 15. Stoica A, Katzenellenbogen BS, Martin MB. Acti-
vation of estrogen receptor-alpha by the heavy
metal cadmium. Mol Endocrinol 2000;14:545–53. 46. Jarup L, Akesson A. Current status of cadmium as
an environmental health problem. Toxicol Appl
Pharmacol 2009;238:201–8. 16. Cho YA, Kim J, Woo HD, et al. Dietary cadmium
intake and the risk of cancer: a meta-analysis. PLoS.One. 2013;8:e75087. 47. Filippini T, Michalke B, Malagoli C,
et al. Determinants of serum cadmium levels in a
northern Italy community: a cross-sectional study. Environ Res 2016;150:219–26. 31. Willett W, Stampfer MJ. Total energy intake:
implications for epidemiologic analyses. Am J Epidemiol 1986;124:17–27. 17. Wu X, Zhu X, Xie M. Association between dietary
cadmium exposure and breast cancer risk: an
updated meta-analysis of observational studies. Med Sci Monit 2015;21:769–75. 48. Turconi G, Minoia C, Ronchi A, et al. Dietary
exposure estimates of twenty-one trace
elements from a Total diet study carried out
in Pavia, northern Italy. Br J Nutr 2009;101:
1200–8. 32. Lunn M, McNeil D. Acknowledgements estrogens. This may be because relatively few breast cancer cases
were analyzed, resulting in insufficient power. Thus, further
studies on the effects of prolonged exposure to low levels of cad-
mium on breast cancer development are required. We
are
indebted
to
the
10786
ORDET
participants. We
thank
A. Evangelista and D. Del Sette for technical support, and Don Ward for
help with the English. Conclusions We have found that the highest levels of dietary cadmium are
associated with increased risk of breast cancer in this large
population-based prospective cohort. The lack of significant het-
erogeneity in risk estimates between ER+ and ER−disease and
between PR+ and PR−disease, mean that our data neither sup-
port nor refute the hypothesis that cadmium acts as a metalloes-
trogen, mimicking or interfering with the action of physiological Int. J. Cancer: 144, 2153–2160 (2019) © 2018 UICC 2160 References Applying cox regression to
competing risks. Biometrics 1995;51:524–32. 33. Società Italiana di Nutrizione Umana (SINU). LARN: livelli di assunzione di riferimento di
nutrienti ed energia, 4th edn. Milan: SICS, 2014. 18. Maele-Fabry G, Lombaert N, Lison D. Dietary
exposure to cadmium and risk of breast cancer in Int. J. Cancer: 144, 2153–2160 (2019) © 2018 UICC
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Chromosome Malorientations after Meiosis II Arrest Cause Nondisjunction
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Molecular biology of the cell
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INTRODUCTION moved, stuck together, to the same pole. Actually, nondis-
junction often occurs because partners disconnect from one
another prematurely (Lamb et al., 1996; Bickel et al., 1998;
Rebollo and Arana, 1998). Normally, the back-to-back asso-
ciation of partner chromosomes helps them orient (attach
via kinetochore fibers) to opposite poles, and this ensures
that they segregate to opposite poles. In the absence of such
pairing, orientation and hence the segregation it specifies,
may be anomalous (Khodjakov et al., 1997; Nicklas, 1997; Yu
and Dawe, 2000), leading to nondisjunction (Angell, 1997;
Bickel et al., 1998; Hassold and Hunt, 2001; Tanaka, 2002;
Kops et al., 2005). Nondisjunction is the outcome of anomalous mitosis or mei-
osis in which one daughter nucleus gains an extra chromo-
some and the other daughter loses a chromosome. When
nondisjunction occurs in meiosis, a resulting gamete having
an extra chromosome can participate in fertilization to create
an embryo with an extra chromosome. Such embryos suffer
from serious disorders, Down’s syndrome (trisomy 21) be-
ing a common example (Hassold and Hunt, 2001; Lamb and
Hassold, 2004). Gametes with missing chromosomes pro-
duce embryos that are nonviable, except for sex chromo-
somes. Nondisjunction is one of two routes to aneuploidy
(any change, be it a gain or a loss, in chromosome number
other than complete multiples of the normal set). The other
route to aneuploidy is chromosome loss from both daughter
cells, generally attributed to chromosome lagging near the
equator at anaphase (Ford et al., 1988). Somatic cell aneu-
ploidy is associated with tumorigenesis and other disorders
(Cimini and Degrassi, 2005; Kops et al., 2005). Marie A. Janicke,* Loren Lasko,* Rudolf Oldenbourg,†
and James R. LaFountain, Jr.* Marie A. Janicke,* Loren Lasko,* Rudolf Oldenbourg,†
and James R. LaFountain, Jr.* *Department of Biological Sciences, State University of New York at Buffalo, Amherst, NY 14260; and †Marine
Biological Laboratory, Woods Hole, MA 02543 *Department of Biological Sciences, State University of New York at Buffalo, Amherst
Biological Laboratory, Woods Hole, MA 02543 Submitted October 27, 2006; Revised January 16, 2007; Accepted February 9, 2007
Monitoring Editor: Kerry Bloom This study investigated the basis of meiosis II nondisjunction. Cold arrest induced a fraction of meiosis II crane fly
spermatocytes to form (n 1) and (n 1) daughters during recovery. Live-cell liquid crystal polarized light microscope
imaging showed nondisjunction was caused by chromosome malorientation. Whereas amphitely (sister kinetochore fibers
to opposite poles) is normal, cold recovery induced anaphase syntely (sister fibers to the same pole) and merotely (fibers
to both poles from 1 kinetochore). Maloriented chromosomes had stable metaphase positions near the equator or between
the equator and a pole. Syntelics were at the spindle periphery at metaphase; their sisters disconnected at anaphase and
moved all the way to a centrosome, as their strongly birefringent kinetochore fibers shortened. The kinetochore fibers of
merotelics shortened little if any during anaphase, making anaphase lag common. If one fiber of a merotelic was more
birefringent than the other, the less birefringent fiber lengthened with anaphase spindle elongation, often permitting
inclusion of merotelics in a daughter nucleus. Meroamphitely (near amphitely but with some merotely) caused sisters to
move in opposite directions. In contrast, syntely and merosyntely (near syntely but with some merotely) resulted in
nondisjunction. Anaphase malorientations were more frequent after longer arrests, with particularly long arrests required
to induce syntely and merosyntely. This article was published online ahead of print in MBC in Press
(http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06–10–0963)
on February 21, 2007. Molecular Biology of the Cell
Vol. 18, 1645–1656, May 2007 Molecular Biology of the Cell
Vol. 18, 1645–1656, May 2007 This article was published online ahead of print in MBC in Press
(http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06–10–0963)
on February 21, 2007.
□
V The online version of this article contains supplemental material
at MBC Online (http://www.molbiolcell.org).
Address correspondence to: Marie A. Janicke (mjanicke@buffalo.edu).
Abbreviations used: LC-PolScope, liquid crystal polarized light mi-
croscope. □
V The online version of this article contains supplemental material
at MBC Online (http://www.molbiolcell.org). © 2007 by The American Society for Cell Biology INTRODUCTION Comparison of incidence of anaphase lag at anaphase II 60 min into recovery with the incidence of nondisjunction at telophase
II/cytokinesis 90 min into recovery
Duration of arrest
Incidence of anaphase lag (%)a
Incidence of nondisjunction (%)b
23–24 h
43 (of 475 anaphase II cells from 4 testes)
1 (of 595 telophase II/cytokinesis cells from 6 testes)
47–48 h
64 (of 2134 anaphase II cells from 8 testes)
3 (of 999 telophase II/cytokinesis cells from 13 testes)
72 h
72 (of 1692 anaphase II cells from 8 testes)
8 (of 485 telophase II/cytokinesis cells from 6 testes)
Data are based on analysis of fixed cell smears after cold arrests at 2°C for the indicated durations. a Anaphase laggards defined in Materials and Methods. b Data on cells that conform in all respects to that presented in Figure 1, G and I. p
gg
b Data on cells that conform in all respects to that presented in Figure 1, G and I. sis. Live-cell imaging with an LC-PolScope (Cambridge Re-
search and Instrumentation, Woburn, MA) made it possible
to directly observe kinetochore fibers of chromosomes
whose subsequent behavior could then be followed. When the screen was for incidence of nondisjunction, the contents of a testis
were scanned in a similar manner, but cells in which cytokinesis II was
virtually complete were scored according to whether, based on the size
difference of the two nuclei, the two daughter nuclei resulting from meiosis II
seemed to have unequal numbers of chromosomes. Cells were not scored if
cytokinesis was abortive or if they had chromatin either separate from the two
daughter nuclei or extending equatorially from a daughter nucleus. Cells in
which the two daughter nuclei seemed “balanced” were scored as normal
(Figure 1F), and those in which the two nuclei seemed significantly different
were scored as nondisjunction (Figure 1G). Results of this screen for nondis-
junction are presented in Table 1 as percentage of otherwise normal cytoki-
nesis II cells that show evidence for nondisjunction. To check the accuracy of
this methodology, the locations of nondisjunction pairs were noted using the
x-y coordinates of the stage vernier, so that they could be relocated for
segmentation analysis. Nondisjunction was confirmed by quantitative fluo-
rescence as described below. Crane Fly Spermatocytes Crane Fly Spermatocytes Fixed cells were analyzed using a Zeiss Photomicroscope II with transmitted
phase contrast and epifluorescence optics. With both, a 40/0.75 numerical
aperture (NA) Neofluar objective was used to locate cells for screening and
for segmentation analysis of selected cells undergoing cytokinesis to confirm
nondisjunction that was detected by eye during the initial screening regimen. When screening necessitated just phase contrast optics, a Wild research stand
with a 40/0.75 NA Fluotar objective was used. A colony of crane flies (Nephrotoma suturalis) that is maintained in the labo-
ratory year-round was the source of spermatocytes for this study. Secondary
(meiosis II) spermatocytes were obtained from selected fourth instars and
prepared for microscopy. p
p
py
For live-cell specimens, testes were isolated from larvae in tricine insect
buffer (Begg and Ellis, 1979), and they were ruptured under oil to make a
spermatocyte culture that was viable for several hours. Two types of live-cell
specimens were made. 1) Well preparations, typically used on inverted mi-
croscopes, were made by rupturing testes at the oil–glass interface of a well
slide that was constructed as described by Janicke and LaFountain (1986). 2)
“Sandwich” preparations, used with the high numerical aperture oil immer-
sion lenses on the LC-PolScope (LaFountain and Oldenbourg, 2004), were
made by rupturing testes under oil on a coverglass, which was subsequently
mounted onto a glass microscope slide for observation. Data on chromosome malorientations and kinetochore fiber birefringence
were obtained with the LC-PolScope, equipped with polarized light and DIC
optics (60/1.4 NA plan apochromat objective) used previously (LaFountain
and Oldenbourg, 2004). A kinetochore fiber (Scarcello et al., 1986) is the
bundle of microtubules that extends from the kinetochore to the pole; it
contains kinetochore microtubules, which end at the kinetochore, and may
contain a few nonkinetochore microtubules interspersed among the kineto-
chore microtubules. With the LC-PolScope, birefringence is revealed regard-
less of specimen orientation. Z-focus series were made at steps of 0.3 m to
maintain high spatial resolution. g
p
For fixed cell specimens, testes were isolated as described above, but then
their entire contents were smeared over the dry surface of a cover glass and
fixed by air drying. Fixed cells were stained with 0.1 g/ml Hoechst 33528 in
4% formaldehyde and then mounted on a microscope slide. Excess stain was
blotted away, and the coverglass was sealed to the slide with nail polish. INTRODUCTION p
Some of the few clear facts about nondisjunction are that
1) it is induced in mitotic or meiotic cells recovering from
experimental spindle arrest (Hildreth and Ulrichs, 1969;
Kato and Yosida, 1970; Tokunaga, 1970; Karp and Smith,
1975; Cimini et al., 1999); 2) it can occur naturally in mitosis
(Cimini et al., 1999) or in the first meiotic division or the
second meiotic division of either the male or female germ-
line (Nicolaidis and Petersen, 1998), where it sometimes but
not always correlates with premature disconnection of part-
ner chromosomes (Hawley et al., 1994; Hassold and Hunt,
2001); and 3) it is more frequent with age in human oocytes,
correlating with longer periods of meiotic arrest that have
been suggested to lead to defects in some unknown aspect of
a spindle-related mechanism (Hawley et al., 1994; Koehler et
al., 1996; Lamb et al., 1996; Orr-Weaver, 1996; Hassold and
Hunt, 2001). The reason so little is known about mechanisms
of nondisjunction is that in many cell types, the process
leading to it is difficult to study cytologically. The goal of the
present study was to investigate the basis of nondisjunction
during experimental arrest recovery in meiosis II crane fly
spermatocytes, a cell type well suited for cytological analy- The term nondisjunction may seem to imply a process in
which partner chromosomes that should have segregated at
anaphase failed to “disjoin” (Cimini et al., 1997) and thus This article was published online ahead of print in MBC in Press
(http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06–10–0963)
on February 21, 2007. □
V The online version of this article contains supplemental material
at MBC Online (http://www.molbiolcell.org). □
V The online version of this article contains supplemental material
at MBC Online (http://www.molbiolcell.org). Address correspondence to: Marie A. Janicke (mjanicke@buffalo.edu). Address correspondence to: Marie A. Janicke (mjanicke@buffalo.edu). Abbreviations used: LC-PolScope, liquid crystal polarized light mi-
croscope. Abbreviations used: LC-PolScope, liquid crystal polarized light mi-
croscope. 1645 1645 © 2007 by The American Society for Cell Biology M. A. Janicke et al. Table 1. INTRODUCTION q
We present in this report a newly observed mechanism for
nondisjunction that involves neither failure of partners to
lose cohesion nor premature loss of cohesion, but, rather, a
spindle-related defect. The defects that we have discovered
are monopolar and nearly monopolar malorientations of
cohered sister chromosomes that are located near the equa-
tor at metaphase, disconnect at anaphase onset, and then are
included during anaphase in the same nucleus as a result of
malorientation, producing (n 1) and (n 1) daughter
cells. Cytokinesis in fixed cells was scored as being successful if the cleavage
furrow had constricted the cell in that area to 75% of the width of the cell at
its widest point. Crane Fly Spermatocytes g
p
Other live-cell observations were made with a Leica DMIRE2 with differ-
ential interference optics (60/1.4 NA planachromatic objective). For these
observations, anaphase II cells were imaged in time-lapse using a script that
made a Z-focus series (0.5 m steps) at each time point. y
g
p
Cold treatments for the induction of chromosome malorientation were
performed as in previous studies (Janicke and LaFountain, 1982; LaFountain
and Oldenbourg, 2004). For 2°C treatments, larvae were placed on refriger-
ated moist tissue paper in a Petri dish in the meat keeper of a refrigerator. For
0.2°C treatments, the Petri dish was put on an ice bath in the refrigerator. Recovery was in moist tissue paper at 22°C. Image Acquisition and Analysis Images of fixed cells were acquired in both phase contrast and fluorescence
modes with a Princeton Instruments Micromax CCD camera and stored as
TIFF files for subsequent analysis. In fluorescence mode, images of Hoechst-
stained cells were used to determine differences in the DNA content of
daughter nuclei. With both daughter nuclei in the same field of view, the
exposure time was kept constant for the two daughter nuclei being compared
by maximizing the 12-bit dynamic range for the brightest nucleus in the
sample. Using IP lab software (Scanalytics, Fairfax, VA), the analysis of DNA
content (based on Hoechst binding) of daughter nuclei was performed as
follows. Boundaries of in-focus daughter nuclei were established by interac-
tively segmenting the image until the segment precisely matched the area of
the Hoechst-labeling. Densitometry was performed on the segmented images. Pixel values within each nucleus were summed and total pixel value was
calculated for the Hoechst-labeled DNA within each nucleus. Analysis of Anaphase Lag and Nondisjunction in Fixed
Spermatocytes Nondisjunction during Meiosis II Figure 1. (A) Diagram of the karyotype of N. suturalis. Spermato-
cytes entering meiosis II have three metacentric autosomes and a
small X or Y chromosome. (B and D) Normally, chromosome seg-
regation during meiosis II produces two haploid daughter nuclei,
each containing 50% of the chromatin. (C and E) One nondisjunction
event produces one nucleus with just 35% and one nucleus with
65% of the chromatin. (F and H) Phase and Hoechst 33528 fluores-
cence images of an untreated control spermatocyte that has essen-
tially completed cytokinesis after meiosis II. Daughter nuclei have
similar sizes. (G and I) Images of a cold-recovering spermatocyte
that has essentially completely cytokinesis after meiosis II. Its
daughter nuclei display nondisjunction. The nucleus on the left
seems smaller than the one nucleus on the right. The cell containing
the small nucleus also is smaller. Bar, 5 m. (J and K) Plots of
fluorescence emitted along the lines drawn across the nuclei in H
and I demonstrate similarities in size and fluorescence emitted from
control nuclei and differences between the two cold-recovering
daughters and between both cold-recovering daughters and control
nuclei. Segmentation analysis (see Materials and Methods for details)
of each of those nuclei demonstrated that the total pixel brightness
of the two control nuclei differed by only 1%, whereas the difference
between the two nuclei in Figure 3I differed by 40%. lytics, Fairfax, VA) and then imported into Microsoft Excel (Microsoft, Red-
mond, WA). With the LC-PolScope, the duration of intervals between Z-slices
was irregular, due to the variation in the time spent calibrating the system
between scans. Birefringence retardation, also called retardance, in images made with the
LC-PolScope was quantified by using a computer algorithm that computed
retardance area within a domain of a selected kinetochore fiber as described
previously (LaFountain and Oldenbourg, 2004). The program is built on the
direct proportionality between the gray scale level (brightness) of a kineto-
chore fiber image in polarized light and the measured retardance within the
fiber image. The retardance area is the integrated retardance across the fiber
width and is directly proportional to the number of microtubules, with each
microtubule contributing 7.5 nm2 to the retardance area of the fiber
(Oldenbourg et al., 1998). Corrections are made for the angle of inclination of
the kinetochore fiber with respect to the pole-to-pole axis of the spindle. RESULTS Crane fly spermatocytes entering meiosis II contain three
morphologically indistinguishable metacentric autosomes
and a small telocentric X or Y (Figure 1A). Each of the three
autosomal dyads contains 30% of the total chromatin, and
the sex dyad contains 10%. Thus, nondisjunction of just
one pair of autosomal sisters increases the chromatin content
of one daughter nucleus by 15% and decreases that of the
other by 15% (Figure 1, B and C). That means an (n 1)
daughter would contain roughly twice the amount of chro-
matin (65% of the total) of that in an (n 1) daughter (35%
of the total) (Figure 1, D and E). Corresponding disparities in
DNA content after staining with the DNA-specific fluoro-
phore Hoechst 33528 (Figure 1, H–K) were readily detected
by eye and quantified by fluorescence emission. Based on
measurements made from 48 such pairs fixed during cold
recovery (discussed below), the average nuclear diameter
was 1.9 m for the smaller nucleus of a pair and 2.6 m for
the larger (Figure 1, F and G). Screening for nondisjunction
in fixed smears of control spermatocytes revealed no evi-
dence for disparate daughter nuclei among 1470 cytokinesis
II cells analyzed, indicating that baseline levels of nondis-
junction are very low in this system, as they are in many
other cells, including human fibroblasts (Cimini et al., 1999). At metaphase II, each dyad is bioriented, having a bire-
fringent bundle of 30–40 microtubules—a kinetochore fi-
ber—extending from the kinetochores of sisters to opposite
poles (Figure 2A). This arrangement is called amphitelic
orientation (Figure 3A). Dyads are stably positioned on the
spindle equator and do not exhibit the oscillatory move-
ments (directional instability) that are characteristic of chro-
mosomes in mitotic cells (Skibbens et al., 1993). At anaphase
onset, sister chromatids disconnect and kinetochore fibers
shorten (anaphase A) over the course of 10–15 min until
sisters reach the edge of the polar centrosomes and pole-
ward movement stops. Usually, all kinetochore fibers
shorten at roughly the same rate (0.5 m/min), so segre-
gating chromosomes reach the edge of the centrosomes at
about the same time (Figure 2, B and C). As the chromo-
somes move poleward, the spindle elongates by 20–25%
(Figures 2C and 3C and Supplemental Video 1). Spindle Crane fly spermatocytes entering meiosis II contain three
morphologically indistinguishable metacentric autosomes
and a small telocentric X or Y (Figure 1A). Time-lapse differential interference contrast (DIC) movies made with the
Leica DMIRE2 (Leica Microsystems, Deerfield, IL) provided data on chromo-
some velocities, K-fiber lengths, and spindle lengths, all of which were
obtained with the linear measurement tool included in IPLab software (Scana- Nondisjunction during Meiosis II p
p
p
p
We estimate that the minimum number of bundled microtubules that is still
detectable with the LC-PolScope in this study is three. This estimate is based
on 1) the average fluctuations or noise of background retardance in LC-
PolScope images of spermatocyte spindles and 2) the uniform fiber retardance
that extends for at least several microns in the image. The morphologically
distinct feature of an elevated retardance in an in-focus fiber section improves
the detectability of the fiber above the noise background. y
g
To depict the positions of different chromosomes that were imaged in
different planes of a Z-focus series, an outline of the best in-focus image of
each chromosome was traced using the pencil tool in Image J. The positions
of the basal bodies were also marked. Tracings were stored as a separate
image stack, which was then projected into a single plane. Afterward, the
outlines of the maloriented chromosomes of interest in each two-dimensional
(2-D) projection were highlighted by painting within the boundary of its
tracing. Analysis of Anaphase Lag and Nondisjunction in Fixed
Spermatocytes Fixed cell specimens were used for assessing both the incidence of anaphase
lagging and the incidence of nondisjunction in all of the spermatocytes
contained within the population released from each of the testes studied. A
systematic screening regimen was used by which the smeared contents of a
testis were scanned manually back and forth from top to bottom while
observing the contents in a phase-contrast microscope. When the screen was for incidence of anaphase lagging, each late anaphase
cell (in which the segregating chromosomes had not yet fused together upon
reaching the poles) was scored according to whether any of the chromosomes
were laggards. A laggard was defined as a chromosome that did not overlap
along the long axis of the spindle with any of the properly segregating
chromosomes. The results of such a screen are presented in Table 1 as
percentage of all late anaphase II cells that contain laggards. For live-cell imaging, Z-series of well specimens were made with a Q-
imaging RETIGA EXi camera on the Leica DMIRE2; the LC-PolScope was
equipped with the same type of camera. All images were stored as TIFF files
that were imported into either IP Lab or ImageJ (public domain software
available from NIH: http://rsb.info.nih.gov/ij/index.html) for analysis. 1646 Molecular Biology of the Cell Nondisjunction during Meiosis II RESULTS Segmentation analysis (see Materials and Methods for details)
of each of those nuclei demonstrated that the total pixel brightness
of the two control nuclei differed by only 1%, whereas the difference
between the two nuclei in Figure 3I differed by 40%. Figure 1. (A) Diagram of the karyotype of N. suturalis. Spermato-
cytes entering meiosis II have three metacentric autosomes and a
small X or Y chromosome. (B and D) Normally, chromosome seg-
regation during meiosis II produces two haploid daughter nuclei,
each containing 50% of the chromatin. (C and E) One nondisjunction
event produces one nucleus with just 35% and one nucleus with
65% of the chromatin. (F and H) Phase and Hoechst 33528 fluores-
cence images of an untreated control spermatocyte that has essen-
tially completed cytokinesis after meiosis II. Daughter nuclei have
similar sizes. (G and I) Images of a cold-recovering spermatocyte
that has essentially completely cytokinesis after meiosis II. Its
daughter nuclei display nondisjunction. The nucleus on the left
seems smaller than the one nucleus on the right. The cell containing
the small nucleus also is smaller. Bar, 5 m. (J and K) Plots of
fluorescence emitted along the lines drawn across the nuclei in H
and I demonstrate similarities in size and fluorescence emitted from
control nuclei and differences between the two cold-recovering
daughters and between both cold-recovering daughters and control
nuclei. Segmentation analysis (see Materials and Methods for details)
of each of those nuclei demonstrated that the total pixel brightness
of the two control nuclei differed by only 1%, whereas the difference
between the two nuclei in Figure 3I differed by 40%. ,
g
(
,
)
At metaphase II, each dyad is bioriented, having a bire-
fringent bundle of 30–40 microtubules—a kinetochore fi-
ber—extending from the kinetochores of sisters to opposite
poles (Figure 2A). This arrangement is called amphitelic
orientation (Figure 3A). Dyads are stably positioned on the
spindle equator and do not exhibit the oscillatory move-
ments (directional instability) that are characteristic of chro-
mosomes in mitotic cells (Skibbens et al., 1993). At anaphase
onset, sister chromatids disconnect and kinetochore fibers
shorten (anaphase A) over the course of 10–15 min until
sisters reach the edge of the polar centrosomes and pole-
ward movement stops. Vol. 18, May 2007 RESULTS Each of the three
autosomal dyads contains 30% of the total chromatin, and
the sex dyad contains 10%. Thus, nondisjunction of just
one pair of autosomal sisters increases the chromatin content
of one daughter nucleus by 15% and decreases that of the
other by 15% (Figure 1, B and C). That means an (n 1)
daughter would contain roughly twice the amount of chro-
matin (65% of the total) of that in an (n 1) daughter (35%
of the total) (Figure 1, D and E). Corresponding disparities in
DNA content after staining with the DNA-specific fluoro-
phore Hoechst 33528 (Figure 1, H–K) were readily detected
by eye and quantified by fluorescence emission. Based on
measurements made from 48 such pairs fixed during cold
recovery (discussed below), the average nuclear diameter
was 1.9 m for the smaller nucleus of a pair and 2.6 m for
the larger (Figure 1, F and G). Screening for nondisjunction
in fixed smears of control spermatocytes revealed no evi-
dence for disparate daughter nuclei among 1470 cytokinesis
II cells analyzed, indicating that baseline levels of nondis-
junction are very low in this system, as they are in many
other cells, including human fibroblasts (Cimini et al., 1999). Figure 1. (A) Diagram of the karyotype of N. suturalis. Spermato-
cytes entering meiosis II have three metacentric autosomes and a
small X or Y chromosome. (B and D) Normally, chromosome seg-
regation during meiosis II produces two haploid daughter nuclei,
each containing 50% of the chromatin. (C and E) One nondisjunction
event produces one nucleus with just 35% and one nucleus with
65% of the chromatin. (F and H) Phase and Hoechst 33528 fluores-
cence images of an untreated control spermatocyte that has essen-
tially completed cytokinesis after meiosis II. Daughter nuclei have
similar sizes. (G and I) Images of a cold-recovering spermatocyte
that has essentially completely cytokinesis after meiosis II. Its
daughter nuclei display nondisjunction. The nucleus on the left
seems smaller than the one nucleus on the right. The cell containing
the small nucleus also is smaller. Bar, 5 m. (J and K) Plots of
fluorescence emitted along the lines drawn across the nuclei in H
and I demonstrate similarities in size and fluorescence emitted from
control nuclei and differences between the two cold-recovering
daughters and between both cold-recovering daughters and control
nuclei. Chromosome Segregation during Meiosis II in
Cold-recovering Spermatocytes elongation (anaphase B) may continue somewhat even after
anaphase A has been completed. A chromosome was scored as a laggard if it was located at
late anaphase far enough behind the properly segregating
chromosomes that it did not overlap with them. It is impor-
tant to note that there is no one standardized definition of
anaphase lag. Some consider a chromosome to be a laggard
only if it remains immobilized in the interzone, not included
in either daughter nucleus, becoming a micronucleus. Oth-
ers, including us, define a laggard by its anaphase position
relative to nonlaggard chromosomes, irrespective of its ul-
timate fate. In our material, many anaphase laggards shifted
poleward to become included within daughter nuclei (also
see Falck et al., 2002). For example, after 72 h at 2°C, 72% of
anaphase II cells had laggards (Table 1). At 90 min of recov-
ery after the same cold treatment, only 24% had distinct
laggards in the interzone (i.e., micronuclei), whereas 38%
had chromatin that trailed from a daughter nucleus equato-
rially, deriving from former laggards that had fused with a
daughter nucleus but had not been compactly included. Hence, the frequency of micronuclei was substantially lower
than the frequency of lagging using our scoring criteria. Overview of Meiosis II in Cold-recovering Spermatocytes
Previous work on meiosis I spermatocytes (Janicke and
LaFountain, 1982) showed that exposure of crane fly larvae
to cold (0.2–2°C) has two effects: 1) it causes existing spindles
to depolymerize rapidly, and 2) it prevents assembly of
spindles in spermatocytes entering meiosis after having un-
dergone nuclear envelope breakdown in the cold. Both out-
comes generate a condition we call cold-prometaphase, de-
fined by absence of spindle microtubules and of random
positioning of chromosomes in the cytoplasm. When cold-
prometaphase I spermatocytes are returned to room temper-
ature, they assemble prometaphase I spindles rapidly, and
bivalents become oriented and congress to a metaphase I
plate; anaphase I begins at 60 min of recovery, and at
mid-to-late anaphase (90 min of recovery), there is a high
incidence of anaphase lag. With longer cold arrests, more
cells accumulated in cold-prometaphase I due to nuclear
membrane breakdown in the cold, and frequencies of an-
aphase lag were high in those cells when they reached
anaphase (Janicke and LaFountain, 1982, 1984). Any chromosome and any number of chromosomes could
lag in cold-recovering meiosis II. Chromosome Segregation during Meiosis II in
Cold-recovering Spermatocytes The incidence of laggard
induction correlated with the duration of cold arrest, data
for 2°C exposures being given in Table 1. Data for 0.2°C
were similar. At 60 min of recovery after 1-d exposures to
2°C, 43% of anaphase cells had one or more laggards, but at
60 min of recovery after 2–3 d at 2°C, the majority of an-
aphase II cells (64–72%) had laggards. In untreated cells,
laggards are found in only 5% of anaphase cells. p
(J
,
,
)
Here, we found the same is true for meiosis II. When cells
arrested in cold-prometaphase II were returned to room
temperature, they assembled spindles rapidly, allowing dy-
ads to become attached to microtubules rapidly, they began
anaphase II at 35 min of recovery, and, when they reached
late anaphase II at 60 min of recovery, they had a high
incidence of anaphase lag. With longer cold arrests, larger
numbers of cold-prometaphase cells accumulated. These
were likely made up of a small fraction of cells that had
regressed from prometaphase II (with a spindle) to cold-
prometaphase II upon exposure to cold as well as a larger
fraction of cold-prometaphase cells that had been in interki-
nesis in the cold and had undergone nuclear envelope
breakdown in the absence of spindle formation. Anaphase II
was completed by 60 min of recovery and cytokinesis at
90 min of recovery. The time course of meiosis II during
cold recovery closely paralleled that in untreated control
cells, except that a slightly longer period (45 min compared
with 35 min for cold recovery) elapses in untreated cells
between the onset of prometaphase II (at nuclear membrane
breakdown) and the beginning of anaphase II. We applied the term “nondisjunction” only to examples
that conformed in all respects to Figure 1, F–I: daughter
nuclei were compact and disparately sized, there was no
evidence of a micronucleus, and the cell had essentially
completed cytokinesis to form two daughter cells that were
still contiguous. Of such cells fixed at 90 min of recovery, the
percentage that had daughter cells with at least a 2:1 dispar-
ity in nuclear size was 1% after 1-d exposures to 2°C, 3%
after 2 d, and 8% after 3 d (Table 1). Thus, the incidence of
nondisjunction was low in comparison with that of an-
aphase lagging, but it increased with increased duration of
cold treatment. RESULTS Usually, all kinetochore fibers
shorten at roughly the same rate (0.5 m/min), so segre-
gating chromosomes reach the edge of the centrosomes at
about the same time (Figure 2, B and C). As the chromo-
somes move poleward, the spindle elongates by 20–25%
(Figures 2C and 3C and Supplemental Video 1). Spindle Vol. 18, May 2007 1647 1647 M. A. Janicke et al. Figure 2. LC-PolScope images of anaphase of meiosis II in two crane fly secondary spermatocytes that originated from the same primary
spermatocyte. Taken from the time-lapse movie in Supplemental Video 1. In LC-PolScope images and movies, image brightness expresses
measured retardance independent of the orientation of the birefringence axis. White corresponds to 2.5 nm or larger retardance (retardance
ceiling 2.5 nm). (A) Metaphase: dyad chromosomes are bioriented, exhibiting amphitelic orientation (Figure 3A) with birefringent kinetochore
fibers extending from sisters to opposite poles (frame 2/60 of movie). (B) Midanaphase A: kinetochore fibers shorten and disjoined sisters
move to opposite poles (frame 16/60 of movie). (C) Completion of anaphase A: poleward movement of sisters ceases upon their making
contact with the polar centrosomes (frame 30/60 of movie). The spindle elongates during the course of anaphase A (concurrent anaphase B)
as well as somewhat further subsequent to the completion of anaphase A. Bar, 5 m. Time interval between frames of the movie is 30 s. Figure 2. LC-PolScope images of anaphase of meiosis II in two crane fly secondary spermatocytes that originated from the same primary
spermatocyte. Taken from the time-lapse movie in Supplemental Video 1. In LC-PolScope images and movies, image brightness expresses
measured retardance independent of the orientation of the birefringence axis. White corresponds to 2.5 nm or larger retardance (retardance
ceiling 2.5 nm). (A) Metaphase: dyad chromosomes are bioriented, exhibiting amphitelic orientation (Figure 3A) with birefringent kinetochore
fibers extending from sisters to opposite poles (frame 2/60 of movie). (B) Midanaphase A: kinetochore fibers shorten and disjoined sisters
move to opposite poles (frame 16/60 of movie). (C) Completion of anaphase A: poleward movement of sisters ceases upon their making
contact with the polar centrosomes (frame 30/60 of movie). The spindle elongates during the course of anaphase A (concurrent anaphase B)
as well as somewhat further subsequent to the completion of anaphase A. Bar, 5 m. Time interval between frames of the movie is 30 s. Chromosome Segregation during Meiosis II in
Cold-recovering Spermatocytes Molecular Biology of the Cell Chromosome Segregation during Meiosis II in
Cold-recovering Spermatocytes Of 72-
h–arrested 90-min–recovered telophase II displaying only
nondisjunction, 80% were scored as successfully cleaving
(Figure 1G) (contrast Shi and King, 2005). Thus, the success-
ful completion of cytokinesis seems to be more affected by
the presence of chromatin at the equator than by imbalance
in chromosome numbers at the two poles (also see Weaver et
al., 2006). About half the cells with chromatin trailing equa- To compare the contribution of lagging versus nondis-
junction to aneuploidy, we considered only cells that were
cleaving successfully. After 90 min of recovery from 72 h of
cold, only 1% of cleaving otherwise normal telophase cells
had micronuclei (still discrete laggards), compared with the
8% that exhibited nondisjunction. We have no evidence that
those micronuclei either disintegrated or were lost, but it is
possible that they resulted in some (n 1) spermatids. Thus,
nondisjunction seems to have contributed more to aneu-
ploidy than did chromosome loss (also see Cimini et al.,
1999). Chromosome Segregation during Meiosis II in
Cold-recovering Spermatocytes Both fibers of the merotelic elongated as it remained near its starting point, slightly closer to the pole to which its nonmerotelic
sister moved. (G) Syntely with merotely in both kinetochores: (3 examples recorded in 2 cells; all 3 cells first observed at metaphase). The dyad
began anaphase nearer to one pole. Neither chromatid moved all the way to the pole during anaphase. (H) Merosyntely with merotely in
one kinetochore: (recorded in 2 cells, both cells first observed at metaphase, including cell 125 [Figure 5]). (I) Syntely without merotely:
(recorded in 6 cells, all cells first observed at metaphase, including cell 132 [Figure 4]). behavior their chromosomes were displaying. (A) Amphitely: the normal orientation for meiosis II found in all untreated spermatocytes that were studied (an example being illustrated
in Figure 2) and in normally segregating chromosomes of all cold-recovering spermatocytes studied. (B) Meroamphitely with merotely in
both kinetochores: (recorded in 3 cells; 1 cell first observed at early anaphase, and 2 cells first observed at midanaphase). The less birefringent
fiber of each laggard elongated via anaphase B. As the initial positions of both laggards was at the equator, both shifted away from the
equator into opposite half-spindles (with limited kinetochore fiber shortening), so that laggards were included within opposite daughter
nuclei. (C) Meroamphitely with merotely in one kinetochore: (recorded in 5 cells; 1 cell first observed at anaphase onset [cell 99; Figure 6],
1 cell first observed at early anaphase, and 3 cells first observed at midanaphase). (D) Balanced merotely in both kinetochores: (recorded in
1 cell first observed at midanaphase). Both kinetochore fibers of each sister elongated, and the laggards remained at the equator. (E) Balanced
merotely in one kinetochore and unbalanced merotely in the other: (recorded in 5 cells; 1 cell first observed at metaphase, 3 cells first observed
at anaphase onset, and 1 cell first observed at midanaphase). The unbalanced merotelic moved away from its less birefringent fiber and hence
away from the equator, whereas the balanced merotelic made no progress from its starting position. (F) Balanced merotely in one kinetochore
and nonmerotely in the other: (5 examples recorded in 4 cells; 1 cell first observed at anaphase onset and 4 cells first observed at
midanaphase). Both fibers of the merotelic elongated as it remained near its starting point, slightly closer to the pole to which its nonmerotelic
sister moved. Chromosome Segregation during Meiosis II in
Cold-recovering Spermatocytes Neither chromatid moved all the way to the pole during anaphase. (H) Merosyntely with merotely in
one kinetochore: (recorded in 2 cells, both cells first observed at metaphase, including cell 125 [Figure 5]). (I) Syntely without merotely:
(recorded in 6 cells, all cells first observed at metaphase, including cell 132 [Figure 4]). torially from a daughter nucleus were scored as aborting
cytokinesis. arations, cytokinesis commonly fails even without treat-
ment. Cytokinesis was scored as successful in fixed cells if
the cleavage furrow had constricted the equator at least 75%
(as in Figure 1F). We found success rates of 90% in cytoki-
nesis II cells from untreated larvae and 86% in normal
cold-recovering telophase II cells (with balanced, compact
daughter nuclei and no micronuclei) fixed at 90 min of
recovery after 72 h at 2°C. At the same recovery time from
the same cold exposure, cytokinesis was scored as successful
in only 3% of the cells that had micronuclei and no other
anomalies. These results show that, of the laggards that
became micronuclei, the majority inhibited cytokinesis and
hence likely led to production of cells with diploid chromo-
some complements rather than aneuploid cells. Of 72-
h–arrested 90-min–recovered telophase II displaying only
nondisjunction, 80% were scored as successfully cleaving
(Figure 1G) (contrast Shi and King, 2005). Thus, the success-
ful completion of cytokinesis seems to be more affected by
the presence of chromatin at the equator than by imbalance
in chromosome numbers at the two poles (also see Weaver et
al., 2006). About half the cells with chromatin trailing equa- arations, cytokinesis commonly fails even without treat-
ment. Cytokinesis was scored as successful in fixed cells if
the cleavage furrow had constricted the equator at least 75%
(as in Figure 1F). We found success rates of 90% in cytoki-
nesis II cells from untreated larvae and 86% in normal
cold-recovering telophase II cells (with balanced, compact
daughter nuclei and no micronuclei) fixed at 90 min of
recovery after 72 h at 2°C. At the same recovery time from
the same cold exposure, cytokinesis was scored as successful
in only 3% of the cells that had micronuclei and no other
anomalies. These results show that, of the laggards that
became micronuclei, the majority inhibited cytokinesis and
hence likely led to production of cells with diploid chromo-
some complements rather than aneuploid cells. Chromosome Segregation during Meiosis II in
Cold-recovering Spermatocytes To assess the impact of the induced anomalies on cytoki-
nesis, we analyzed fixed cells, because in live-cell oil prep- 1648 Molecular Biology of the Cell Nondisjunction during Meiosis II osis II found in all untreated spermatocytes that were studied (an example being illustrated
mosomes of all cold-recovering spermatocytes studied. (B) Meroamphitely with merotely in
st observed at early anaphase, and 2 cells first observed at midanaphase). The less birefringent
B. As the initial positions of both laggards was at the equator, both shifted away from the
ted kinetochore fiber shortening), so that laggards were included within opposite daughter
ne kinetochore: (recorded in 5 cells; 1 cell first observed at anaphase onset [cell 99; Figure 6],
ells first observed at midanaphase). (D) Balanced merotely in both kinetochores: (recorded in
tochore fibers of each sister elongated, and the laggards remained at the equator. (E) Balanced
erotely in the other: (recorded in 5 cells; 1 cell first observed at metaphase, 3 cells first observed
idanaphase). The unbalanced merotelic moved away from its less birefringent fiber and hence
merotelic made no progress from its starting position. (F) Balanced merotely in one kinetochore
recorded in 4 cells; 1 cell first observed at anaphase onset and 4 cells first observed at
gated as it remained near its starting point, slightly closer to the pole to which its nonmerotelic
kinetochores: (3 examples recorded in 2 cells; all 3 cells first observed at metaphase). The dyad
hromatid moved all the way to the pole during anaphase. (H) Merosyntely with merotely in
ls first observed at metaphase, including cell 125 [Figure 5]). (I) Syntely without merotely:
metaphase, including cell 132 [Figure 4]). Figure 3. Diagrams of various types of mei-
osis II dyad orientations observed in untreated
(A) and in cold-recovering (A–I) spermato-
cytes and fates of chromosomes derived from
them. Normal chromosome distribution re-
sults from the orientations in (A–C), whereas
in (D–F) anaphase laggards remain near the
plane of the cleavage furrow, and in G–I the
outcome is nondisjunction. Horizontal lines
indicate the plane of the spindle equator. Dashed circles represent polar centrosomes,
open circles represent kinetochores (chromo-
somes arms are not shown), and vertical lines
are kinetochore fibers. Thickness of the fiber
signifies its level of brirefringence in compar-
ison with the other fibers. Chromosome Segregation during Meiosis II in
Cold-recovering Spermatocytes Kinetochore fibers
are drawn only for the central dyad; the other
kinetochores are shown as they would behave
if they exhibited normal amphitelic orienta-
tion (A). Depictions of kinetochore fiber short-
ening, lengthening, and spindle elongation do
not attempt to quantify exactly how these pa-
rameters changed but only give qualitative
general illustrations of how positions of kinet-
ochores and spindle poles changed relative to
one another. The number of cells recorded in
each category is indicated, but it does not re-
flect the actual frequency of different orienta-
tions in the population, because cells were not
recorded at random, but rather they were se-
lected because of our interest in the particular
behavior their chromosomes were displaying. (A) Amphitely: the normal orientation for meiosis II found in all untreated spermatocytes that were studied (an example being illustrated
in Figure 2) and in normally segregating chromosomes of all cold-recovering spermatocytes studied. (B) Meroamphitely with merotely in
both kinetochores: (recorded in 3 cells; 1 cell first observed at early anaphase, and 2 cells first observed at midanaphase). The less birefringent
fiber of each laggard elongated via anaphase B. As the initial positions of both laggards was at the equator, both shifted away from the
equator into opposite half-spindles (with limited kinetochore fiber shortening), so that laggards were included within opposite daughter
nuclei. (C) Meroamphitely with merotely in one kinetochore: (recorded in 5 cells; 1 cell first observed at anaphase onset [cell 99; Figure 6],
1 cell first observed at early anaphase, and 3 cells first observed at midanaphase). (D) Balanced merotely in both kinetochores: (recorded in
1 cell first observed at midanaphase). Both kinetochore fibers of each sister elongated, and the laggards remained at the equator. (E) Balanced
merotely in one kinetochore and unbalanced merotely in the other: (recorded in 5 cells; 1 cell first observed at metaphase, 3 cells first observed
at anaphase onset, and 1 cell first observed at midanaphase). The unbalanced merotelic moved away from its less birefringent fiber and hence
away from the equator, whereas the balanced merotelic made no progress from its starting position. (F) Balanced merotely in one kinetochore
and nonmerotely in the other: (5 examples recorded in 4 cells; 1 cell first observed at anaphase onset and 4 cells first observed at
midanaphase). Chromosome Segregation during Meiosis II in
Cold-recovering Spermatocytes (G) Syntely with merotely in both kinetochores: (3 examples recorded in 2 cells; all 3 cells first observed at metaphase). The dyad
began anaphase nearer to one pole. Neither chromatid moved all the way to the pole during anaphase. (H) Merosyntely with merotely in
one kinetochore: (recorded in 2 cells, both cells first observed at metaphase, including cell 125 [Figure 5]). (I) Syntely without merotely:
(recorded in 6 cells, all cells first observed at metaphase, including cell 132 [Figure 4]). p y
g
(A) Amphitely: the normal orientation for meiosis II found in all untreated spermatocytes that were studied (an example being illustrated
in Figure 2) and in normally segregating chromosomes of all cold-recovering spermatocytes studied. (B) Meroamphitely with merotely in
both kinetochores: (recorded in 3 cells; 1 cell first observed at early anaphase, and 2 cells first observed at midanaphase). The less birefringent
fiber of each laggard elongated via anaphase B. As the initial positions of both laggards was at the equator, both shifted away from the
equator into opposite half-spindles (with limited kinetochore fiber shortening), so that laggards were included within opposite daughter
nuclei. (C) Meroamphitely with merotely in one kinetochore: (recorded in 5 cells; 1 cell first observed at anaphase onset [cell 99; Figure 6],
1 cell first observed at early anaphase, and 3 cells first observed at midanaphase). (D) Balanced merotely in both kinetochores: (recorded in
1 cell first observed at midanaphase). Both kinetochore fibers of each sister elongated, and the laggards remained at the equator. (E) Balanced
merotely in one kinetochore and unbalanced merotely in the other: (recorded in 5 cells; 1 cell first observed at metaphase, 3 cells first observed
at anaphase onset, and 1 cell first observed at midanaphase). The unbalanced merotelic moved away from its less birefringent fiber and hence
away from the equator, whereas the balanced merotelic made no progress from its starting position. (F) Balanced merotely in one kinetochore
and nonmerotely in the other: (5 examples recorded in 4 cells; 1 cell first observed at anaphase onset and 4 cells first observed at
midanaphase). Both fibers of the merotelic elongated as it remained near its starting point, slightly closer to the pole to which its nonmerotelic
sister moved. (G) Syntely with merotely in both kinetochores: (3 examples recorded in 2 cells; all 3 cells first observed at metaphase). The dyad
began anaphase nearer to one pole. Chromosome Malorientation as a Cause of Lagging
and Nondisjunction This is best visualized by viewing the Z-focus series made with this cell, provided in Supplemental Video 2. Also note in the
video the absence of birefringence on the equatorial side of the syntelic dyad, offering no evidence for merotely. (B) Twenty-eight minutes
into recovery: anaphase begins. The separation of the two chromatids of the syntelic dyad is resolved as they move poleward, and the
left-most kinetochore of the syntelic dyad advances poleward slightly ahead of the other kinetochore. (C and D) Thirty-four minutes and 41
min into recovery: continuation of anaphase A and the shortening of syntelic kinetochore fibers. (E) Forty-nine minutes into recovery:
cosegregating sisters (arrowhead) reach the spindle pole. (F) Sixty-one minutes into recovery: DIC image of four autosomes that segregated
to the upper pole and just two autosomes segregated to the lower pole. Bar, 5 m. (AA–FF) 2-D projections (see Materials and Methods) of the
Z-focus series containing the images presented in A–F show metaphase and anaphase positions of all of the chromosomes in cell 132. Dots
locate basal bodies within the polar centrosomes. (AA) Twenty-one minutes into recovery: at metaphase, the syntelic dyad (painted gray) is
positioned slightly off the equator. (BB) Twenty-eight minutes into recovery, anaphase begins. (CC and DD) Thirty-four and 41 min into
recovery: cosegregation of syntelic sisters to the same pole. (EE as in E and FF as in F) The outcome is nondisjunction. Quite significant is that we observed syntelic dyads that
were positioned near the spindle equator at metaphase (Fig-
ure 4, A and B) and that did not exhibit either oscillatory
behavior (Skibbens et al., 1993) or orientation instability
(Henderson et al., 1970). Although shorter at metaphase than
those of amphitelic dyads in the same cells, syntelic kineto-
chore fibers were robust (Figure 7A and Table 2), containing
similar numbers of microtubules as amphitelic fibers in the
same cell, and upon disjunction of sisters at anaphase onset,
they exhibited anaphase A shortening (Figure 4, C–F, and
AA–FF) just like that of amphitelic fibers. Because syntelic
dyads were somewhat closer at metaphase to the pole to
which they were oriented, their chromatids in many cases
preceded the other chromosomes in making contact with the
centrosomes. and nondisjunction. Chromosome Malorientation as a Cause of Lagging
and Nondisjunction Cold treatment: 72 h at 2°C
(retardance ceiling 2.5 nm). (A) Twenty-one minutes into recovery: at metaphase, the syntelic dyad (arrowhead) is at the left periphery of the
spindle. Both kinetochores have robust kinetochore fibers (97 kinetochore microtubules for the pair; see Figure 7A, compare with Table 2)
oriented to the upper pole. The kinetochore fibers of this dyad are tilted away from the spindle axis more than the fibers of the bioriented
chromosomes. This is best visualized by viewing the Z-focus series made with this cell, provided in Supplemental Video 2. Also note in the
video the absence of birefringence on the equatorial side of the syntelic dyad, offering no evidence for merotely. (B) Twenty-eight minutes
into recovery: anaphase begins. The separation of the two chromatids of the syntelic dyad is resolved as they move poleward, and the
left-most kinetochore of the syntelic dyad advances poleward slightly ahead of the other kinetochore. (C and D) Thirty-four minutes and 41
min into recovery: continuation of anaphase A and the shortening of syntelic kinetochore fibers. (E) Forty-nine minutes into recovery:
cosegregating sisters (arrowhead) reach the spindle pole. (F) Sixty-one minutes into recovery: DIC image of four autosomes that segregated
to the upper pole and just two autosomes segregated to the lower pole. Bar, 5 m. (AA–FF) 2-D projections (see Materials and Methods) of the
Z-focus series containing the images presented in A–F show metaphase and anaphase positions of all of the chromosomes in cell 132. Dots
locate basal bodies within the polar centrosomes. (AA) Twenty-one minutes into recovery: at metaphase, the syntelic dyad (painted gray) is
positioned slightly off the equator. (BB) Twenty-eight minutes into recovery, anaphase begins. (CC and DD) Thirty-four and 41 min into
recovery: cosegregation of syntelic sisters to the same pole. (EE as in E and FF as in F) The outcome is nondisjunction. Figure 4. Syntelic orientation of sisters that cosegregate to the same pole during anaphase A (cell 132). Cold treatment: 72 h at 2°C
(retardance ceiling 2.5 nm). (A) Twenty-one minutes into recovery: at metaphase, the syntelic dyad (arrowhead) is at the left periphery of the
spindle. Both kinetochores have robust kinetochore fibers (97 kinetochore microtubules for the pair; see Figure 7A, compare with Table 2)
oriented to the upper pole. The kinetochore fibers of this dyad are tilted away from the spindle axis more than the fibers of the bioriented
chromosomes. Chromosome Malorientation as a Cause of Lagging
and Nondisjunction All anomalous segregation observed by live-cell imaging
with the LC-PolScope was attributable to chromosome mal-
orientation. That is, recovering dyads had a high probability
for making improper connections with the two spindle
poles, and those malorientations resulted in anaphase lag Vol. 18, May 2007 1649 1649 M. A. Janicke et al. Figure 4. Syntelic orientation of sisters that cosegregate to the same pole during anaphase A (cell 132). Cold treatment: 72 h at 2°C
(retardance ceiling 2.5 nm). (A) Twenty-one minutes into recovery: at metaphase, the syntelic dyad (arrowhead) is at the left periphery of the
spindle. Both kinetochores have robust kinetochore fibers (97 kinetochore microtubules for the pair; see Figure 7A, compare with Table 2)
oriented to the upper pole. The kinetochore fibers of this dyad are tilted away from the spindle axis more than the fibers of the bioriented
chromosomes. This is best visualized by viewing the Z-focus series made with this cell, provided in Supplemental Video 2. Also note in the
video the absence of birefringence on the equatorial side of the syntelic dyad, offering no evidence for merotely. (B) Twenty-eight minutes
into recovery: anaphase begins. The separation of the two chromatids of the syntelic dyad is resolved as they move poleward, and the
left-most kinetochore of the syntelic dyad advances poleward slightly ahead of the other kinetochore. (C and D) Thirty-four minutes and 41
min into recovery: continuation of anaphase A and the shortening of syntelic kinetochore fibers. (E) Forty-nine minutes into recovery:
cosegregating sisters (arrowhead) reach the spindle pole. (F) Sixty-one minutes into recovery: DIC image of four autosomes that segregated
to the upper pole and just two autosomes segregated to the lower pole. Bar, 5 m. (AA–FF) 2-D projections (see Materials and Methods) of the
Z-focus series containing the images presented in A–F show metaphase and anaphase positions of all of the chromosomes in cell 132. Dots
locate basal bodies within the polar centrosomes. (AA) Twenty-one minutes into recovery: at metaphase, the syntelic dyad (painted gray) is
positioned slightly off the equator. (BB) Twenty-eight minutes into recovery, anaphase begins. (CC and DD) Thirty-four and 41 min into
recovery: cosegregation of syntelic sisters to the same pole. (EE as in E and FF as in F) The outcome is nondisjunction. Figure 4. Syntelic orientation of sisters that cosegregate to the same pole during anaphase A (cell 132). Molecular Biology of the Cell Chromosome Malorientation as a Cause of Lagging
and Nondisjunction In the merotelic chromosome (right-pointing arrowhead), both its more birefringent fiber
(right-pointing arrow), to the closer pole, and the long less birefringent fiber (left-pointing arrow), to the distal pole, have elongated in the
time interval after A and B above, the former by less than a micrometer (0.8 m), whereas the less birefringent fiber elongated 4–5 times
that (3.8 m). Importantly, the merotelic sister is still located in the same half-spindle as its properly segregating sister. (E) Fifty-four
minutes into recovery: DIC image upon the initiation of cytokinesis. The merotelic sister (arrowhead) is near the group of chromosomes that
reached the spindle pole, but the merotelic sister does not make contact with the polar centrosome as normally segregating chromosomes do. (F) Ninety-six minutes into recovery: cytokinesis is well underway. The merotelic sister is included in the same (n 1) nucleus as its sister. Bar, 5 m. (AA–EE) 2-D projections of Z-focus series of cell 125 illustrate merosyntely causing nondisjunction. (AA) From Z-focus series at
anaphase onset at 27 min into recovery. Chromosomes derived from the merosyntelic dyad are painted gray. (BB) Thirty-two minutes into
recovery as in A–C. (CC) Thirty-five minutes into recovery. (DD) Forty-four minutes into recovery as in D (sex chromosome at lower pole
was not included in the Z-focus series.) (EE) (n 1) chromosomes are at the upper pole, and (n 1) chromosomes are at the lower pole as
in E (polar centrosomes could not be located). Nondisjunction during Meiosis II Figure 5
Merosyntely in cell 125 causes nondisjunction Cold treatment: 72 h at 2°C (retardance ceiling 2 5 nm) (A C) Thirty two minutes Figure 5. Merosyntely in cell 125 causes nondisjunction. Cold treatment: 72 h at 2°C (retardance ceiling 2.5 nm). (A–C) Thirty-two minutes
into recovery: anaphase onset in three slices taken from the Z-focus series that is supplied as Supplemental Video 3 to illustrate the behavior
of chromosomes from a merosyntelic dyad (in A and B) and two other chromosomes that come from properly bioriented dyads and are
segregating normally (in C). (A) Slice 7/27. (B) Slice 8/27. (C) Slice 15/27. (A and B) The right-side daughter (left-pointing arrowhead) is not
merotelic and is closer (7.2 m) to the upper pole than the merotelic kinetochore of the left-side sister (right-pointing arrowhead), which
is 7.8 m from that pole. Chromosome Malorientation as a Cause of Lagging
and Nondisjunction All laggards that were studied exhib-
ited merotelic orientation, i.e., the single kinetochore of the
laggard had kinetochore fibers connecting it to both spindle
poles (Figure 3, B–H), a malorientation first documented at
anaphase in crane fly meiosis I spermatocytes recovering
from cold (Janicke and LaFountain, 1984) or from microtu-
bule inhibitors (Ladrach and LaFountain, 1986). Thus, a
laggard’s failure to move properly to one of the poles was
caused by its having another kinetochore fiber to the oppo-
site pole (see also Cimini et al., 2001). p
The only chromosomes observed to undergo nondisjunc-
tion were those that were oriented either syntelically, mean-
ing sisters had their kinetochore fibers directed toward the
same pole (Figures 3I and 4), or merosyntelically, meaning
near-syntelic orientation but with one or both kinetochores
having a second, less robust fiber directed toward the op-
posite pole (Figures 3, G and H, and 5). Direct observation of
these malorientations causing nondisjunction (Figures 4,
AA–FF, and 5, AA–EE) is a new finding with several re-
markable features. The pathway to nondisjunction by sisters from merosyn-
telic dyads (Figures 3, G and H, and 5, A–E and AA–EE) was
different from that of sisters from syntelic dyads. In mer-
osyntely, shortening of the leading kinetochore fiber of the
merotelic kinetochore(s), and hence, poleward movement of 1650 Nondisjunction during Meiosis II Figure 5. Merosyntely in cell 125 causes nondisjunction. Cold treatment: 72 h at 2°C (retardance ceiling 2.5 nm). (A–C) Thirty-two minutes
into recovery: anaphase onset in three slices taken from the Z-focus series that is supplied as Supplemental Video 3 to illustrate the behavior
of chromosomes from a merosyntelic dyad (in A and B) and two other chromosomes that come from properly bioriented dyads and are
segregating normally (in C). (A) Slice 7/27. (B) Slice 8/27. (C) Slice 15/27. (A and B) The right-side daughter (left-pointing arrowhead) is not
merotelic and is closer (7.2 m) to the upper pole than the merotelic kinetochore of the left-side sister (right-pointing arrowhead), which
is 7.8 m from that pole. The merotelic kinetochore has 33 microtubules (right-pointing arrow) extending to the upper pole (Figure 7B) and
15 microtubules (left-pointing arrow) extending to the lower pole (Figure 7C). (D) Forty-four minutes into recovery: anaphase progresses with
nonmerotelic chromosomes approaching the pole. Chromosome Malorientation as a Cause of Lagging
and Nondisjunction The merotelic kinetochore has 33 microtubules (right-pointing arrow) extending to the upper pole (Figure 7B) and
15 microtubules (left-pointing arrow) extending to the lower pole (Figure 7C). (D) Forty-four minutes into recovery: anaphase progresses with
nonmerotelic chromosomes approaching the pole. In the merotelic chromosome (right-pointing arrowhead), both its more birefringent fiber
(right-pointing arrow), to the closer pole, and the long less birefringent fiber (left-pointing arrow), to the distal pole, have elongated in the
time interval after A and B above, the former by less than a micrometer (0.8 m), whereas the less birefringent fiber elongated 4–5 times
that (3.8 m). Importantly, the merotelic sister is still located in the same half-spindle as its properly segregating sister. (E) Fifty-four
minutes into recovery: DIC image upon the initiation of cytokinesis. The merotelic sister (arrowhead) is near the group of chromosomes that
reached the spindle pole, but the merotelic sister does not make contact with the polar centrosome as normally segregating chromosomes do. (F) Ninety-six minutes into recovery: cytokinesis is well underway. The merotelic sister is included in the same (n 1) nucleus as its sister. Bar, 5 m. (AA–EE) 2-D projections of Z-focus series of cell 125 illustrate merosyntely causing nondisjunction. (AA) From Z-focus series at
anaphase onset at 27 min into recovery. Chromosomes derived from the merosyntelic dyad are painted gray. (BB) Thirty-two minutes into
recovery as in A–C. (CC) Thirty-five minutes into recovery. (DD) Forty-four minutes into recovery as in D (sex chromosome at lower pole
was not included in the Z-focus series.) (EE) (n 1) chromosomes are at the upper pole, and (n 1) chromosomes are at the lower pole as
in E (polar centrosomes could not be located). Figure 5. Merosyntely in cell 125 causes nondisjunction. Cold treatment: 72 h at 2°C (retardance ceiling 2.5 nm). (A–C) Thirty-two minutes
into recovery: anaphase onset in three slices taken from the Z-focus series that is supplied as Supplemental Video 3 to illustrate the behavior
of chromosomes from a merosyntelic dyad (in A and B) and two other chromosomes that come from properly bioriented dyads and are
segregating normally (in C). (A) Slice 7/27. (B) Slice 8/27. (C) Slice 15/27. (A and B) The right-side daughter (left-pointing arrowhead) is not
merotelic and is closer (7.2 m) to the upper pole than the merotelic kinetochore of the left-side sister (right-pointing arrowhead), which
is 7.8 m from that pole. Vol. 18, May 2007 Chromosome Malorientation as a Cause of Lagging
and Nondisjunction The merotelic kinetochore has 33 microtubules (right-pointing arrow) extending to the upper pole (Figure 7B) and
15 microtubules (left-pointing arrow) extending to the lower pole (Figure 7C). (D) Forty-four minutes into recovery: anaphase progresses with
nonmerotelic chromosomes approaching the pole. In the merotelic chromosome (right-pointing arrowhead), both its more birefringent fiber
(right-pointing arrow), to the closer pole, and the long less birefringent fiber (left-pointing arrow), to the distal pole, have elongated in the
time interval after A and B above, the former by less than a micrometer (0.8 m), whereas the less birefringent fiber elongated 4–5 times
that (3.8 m). Importantly, the merotelic sister is still located in the same half-spindle as its properly segregating sister. (E) Fifty-four
minutes into recovery: DIC image upon the initiation of cytokinesis. The merotelic sister (arrowhead) is near the group of chromosomes that
reached the spindle pole, but the merotelic sister does not make contact with the polar centrosome as normally segregating chromosomes do. (F) Ninety-six minutes into recovery: cytokinesis is well underway. The merotelic sister is included in the same (n 1) nucleus as its sister. Bar, 5 m. (AA–EE) 2-D projections of Z-focus series of cell 125 illustrate merosyntely causing nondisjunction. (AA) From Z-focus series at
anaphase onset at 27 min into recovery. Chromosomes derived from the merosyntelic dyad are painted gray. (BB) Thirty-two minutes into
recovery as in A–C. (CC) Thirty-five minutes into recovery. (DD) Forty-four minutes into recovery as in D (sex chromosome at lower pole
was not included in the Z-focus series.) (EE) (n 1) chromosomes are at the upper pole, and (n 1) chromosomes are at the lower pole as
in E (polar centrosomes could not be located). by our criteria (see Materials and Methods), although it never
fully reached the centrosome (Figure 5E and EE). the kinetochore, was inhibited compared with that of non-
merotelic chromatids segregating to the same pole (Figure 5,
A–C and AA–EE). During the late stages of anaphase,
lengthening of the trailing fiber, always the less birefringent
of the two fibers of the merotelic kinetochore (Figures 5, A
and B, and 7, B and C), allowed the merotelic to move far
enough away from the equator to fuse with the chromo-
somes of the nucleus that contained its sister (Figure 5, D–F). The merotelic initially extended from the nucleus in the
direction of the equator before becoming more compactly
included. Chromosome Malorientation as a Cause of Lagging
and Nondisjunction Because a merosyntelic dyad was closer to its pole
at metaphase than an amphitelic dyad (Figure 5AA), the
nonmerotelic chromatids started out behind the merotelic
chromatids but then passed them (Figure 5, CC–EE). Thus, a
merotelic chromatid from a merosyntelic dyad never was far
enough behind the nonmerotelics to be scored as a laggard the kinetochore, was inhibited compared with that of non-
merotelic chromatids segregating to the same pole (Figure 5,
A–C and AA–EE). During the late stages of anaphase,
lengthening of the trailing fiber, always the less birefringent
of the two fibers of the merotelic kinetochore (Figures 5, A
and B, and 7, B and C), allowed the merotelic to move far
enough away from the equator to fuse with the chromo-
somes of the nucleus that contained its sister (Figure 5, D–F). The merotelic initially extended from the nucleus in the
direction of the equator before becoming more compactly
included. Because a merosyntelic dyad was closer to its pole
at metaphase than an amphitelic dyad (Figure 5AA), the
nonmerotelic chromatids started out behind the merotelic
chromatids but then passed them (Figure 5, CC–EE). Thus, a
merotelic chromatid from a merosyntelic dyad never was far
enough behind the nonmerotelics to be scored as a laggard We reported previously that a bundle of 10 microtubules
might not be detected with the LC-PolScope (LaFountain and
Oldenbourg, 2004), but we have revised that lower limit to
be less than three microtubules in images recorded for this
study (see Materials and Methods). We therefore questioned
whether the observed syntelic dyads, despite having only
one birefringent fiber extending from each kinetochore,
might have one or two undetected microtubules extending
from them toward the opposite pole. The behavior of the
chromosomes we are calling syntelics argues in three ways
against their having undetected, oppositely directed micro-
tubules. 1) Anaphase A movement is impaired in merotelic
chromatids. We know from previous electron microscopy
analysis of meiosis I laggards that just one merotelic kinet- 1651 M. A. Janicke et al. Table 2. Chromosome Malorientation as a Cause of Lagging
and Nondisjunction Comparison of data regarding kinetochore fibers in control versus cold-recovering meiosis II spermatocytes
Control
spermatocytes
Only amphitelic
orientation
Cold-recovering spermatocytes
Cells having only
amphitelic orientations
Amphitelic orientations in
cells with a syntelic dyad
Syntelic dyads
Avg length (m)a of metaphase
kinetochore fibers
8.9 0.9 (n 86)
7.3 0.7 (n 13)
7.8 0.6 (n 34)
6.1 0.8 (n 12)
Tilt angle (degrees) of kinetochore fiber
relative to pole-to-pole axis
8.6 4.3 (n 14)
7.0 3.9 (n 19)
26.8 10.8 (n 6)
Microtubules/kinetochoreb
37.5 8.1 (n 17)
53.2 13.1 (n 9)
50.3 10.0 (n 3)
a Distance from kinetochore center to basal body at the core of the centrosome. b From retardance area analysis (see Figure 7). Comparison of data regarding kinetochore fibers in control versus cold-recovering meiosis II spermatocytes a Distance from kinetochore center to basal body at the core of the centrosome. b From retardance area analysis (see Figure 7). greatly impaired during anaphase, but the less birefringent
kinetochore fiber elongated along with the separation of the
two poles When both sisters were merotelic (Figure 3B) and
the dyad was meroamphitelic (almost amphitelic but with
some merotely), dyads were near the equator at metaphase. Their sister chromatids moved away from the equator to-
ward opposite poles during anaphase spindle elongation,
but they were far enough behind the nonmerotelics to be
scored as laggards. One meroamphitelic dyad was observed
that had merotely in just one kinetochore (Figure 3C). The
dyad was located at metaphase in the half-spindle of the less
birefringent fiber of the merotelic (Figure 6, A, B, and AA). Lengthening of the less birefringent fiber of the merotelic
therefore caused the initial direction of the anaphase move-
ment of the merotelic to be toward, rather than away from,
the equator. The merotelic then moved slightly beyond the
equator and into the opposite half-spindle (Figures 6, C and
DD, and 7, D and E), where it was still lagging when
cytokinesis began. Movement toward the equator at an-
aphase is an unusual finding that may relate to results
reported by Pidoux et al. (2000) in yeast. It confirms that the
direction of anaphase B shifting does indeed depend on
relative robustness of kinetochore fibers (Cimini et al., 2004)
and not on the half-spindle in which a merotelic begins
anaphase. Ruling Out Other Potential Causes of Nondisjunction Ruling Out Other Potential Causes of Nondisjunction g
f
j
To evaluate whether factors other than malorientation may
have contributed to the incidence of nondisjunction in fixed
cell smears, we investigated four known alternative sources
of aneuploidy, and all were ruled out, as follows. 1) Multi-
polar spindles. Had cold exposure of secondary spermato-
cytes caused them to form tripolar or tetrapolar spindles
during recovery, (n 1) and (n 1) daughter nuclei may
have resulted. To test this, meiosis II cells were analyzed
from testicular contents fixed at recovery times (60 min) too
short for them to have been in meiosis I during exposure. Frequencies of multipolar anaphase II spindles were similar
to those in untreated smears. 2) Sticky sisters. We found no
evidence for intact dyads at any postmetaphase II stage. 3)
Precocious disjunction of sisters. Smears of cells were fixed
when the “wave” of meiosis II cells peaked at metaphase II
(45 min of recovery), and other smears were made of cells
fixed in the cold without recovery. Of 1553 metaphase II
cells, only three cells had what seemed to be a pair of
disjoined sisters. None of the dyads in cold-prometaphase II
seemed to have precociously disjoined. 4) Chromosomes Molecular Biology of the Cell Chromosome Malorientation as a Cause of Lagging
and Nondisjunction Importantly, whether the merotely was in one or
both kinetochores, meroamphitely (Figure 3, B and C) al-
ways resulted in sisters moving away from one another. ochore microtubule can cause a chromosome to trail behind
the others at anaphase (LaFountain, 1985). In contrast, chro-
matids from syntelic dyads moved poleward at the same
time and same approximate velocity as properly oriented
chromosomes, sometimes reaching the pole before properly
oriented chromosomes. 2) Because their leading fibers do
not shorten substantially during anaphase, merotelic chro-
mosomes do not make actual contact with the centrosome
even after anaphase spindle elongation. In contrast, the
length of kinetochore fibers of syntelics shortened to zero as
the chromatids of syntelics moved all the way to the centro-
some. 3) At metaphase, we have observed only central lo-
cations and shallow tilt angles of 15° or less for bioriented
dyads (Figure 3, A–H, and Table 2), including merosyntelics
(Figure 3, G and H), the merosyntelic dyad in Figure 5 being
tilted only 7° relative to the pole-to-pole axis. In contrast,
syntelic dyads were positioned at the spindle periphery with
kinetochore fiber tilt angles ranging between 18 and 47°
(Figure 4A and Table 2), consistent with their not being
subject to forces focused toward the opposite pole. Even a
single kinetochore microtubule extending toward the oppo-
site pole has been reported to be capable of exerting force
toward that pole (McEwen et al., 1997). The difference be-
tween the tilt angle of syntelic kinetochore fibers and the
fibers of other chromosomes in the cell also suggests that the
kinetochores of syntelics were not interacting laterally with
other kinetochore fibers to achieve congression (Kapoor et
al., 2006). The Full Spectrum of Malorientations and the Ultimate
Fate of Laggards Bar, 5 m. (AA–DD) 2-D projections of chromosome positions in cell 99. (AA) Thirty-one minutes into recovery: at anaphase onset, sisters derived from
the meroamphitelic sex dyad (painted gray) are positioned in the upper half-spindle. (BB) Thirty-four minutes into recovery: one of the sex
sisters exhibits anaphase lag as its sister moves to the upper pole. (The upper centrosome was not included in the Z-focus series.) (CC)
Thirty-nine minutes into recovery: the laggard is positioned near the equator. (DD) Forty-eight minutes into recovery: the laggard is
positioned in the lower half-spindle. anaphase to wait for achievement of bipolar orientations and
that, if disrupted, cause monopolar chromosomes to be
“stuck at the poles” (Kline-Smith et al., 2004). Tension, usu-
ally achieved through bipolar orientation, is thought to be
necessary for chromosomes to maintain their orientation,
achieve full microtubule complements, keep from moving to
a pole before anaphase, and inactivate the anaphase check-
point (Nicklas, 1997). Although cold-recovering syntelics are
monopolar, they had metaphase positions away from the
poles, had robust kinetochore fibers of sizable length, and
did not delay anaphase. We suggest these syntelics did
experience tension, not from oppositely directed kinetochore
fibers but from polar ejection forces that counteracted the
poleward forces of their kinetochore fibers. In crane fly
spermatocytes, transverse equilibrium forces move acentric
fragments to the spindle periphery, where they are driven
equatorially by polar ejection forces hypothetically involv-
ing a yet-to-be-characterized kinesin-10 orthologue (i.e., anaphase to wait for achievement of bipolar orientations and
that, if disrupted, cause monopolar chromosomes to be
“stuck at the poles” (Kline-Smith et al., 2004). Tension, usu-
ally achieved through bipolar orientation, is thought to be
necessary for chromosomes to maintain their orientation,
achieve full microtubule complements, keep from moving to
a pole before anaphase, and inactivate the anaphase check-
point (Nicklas, 1997). Although cold-recovering syntelics are
monopolar, they had metaphase positions away from the
poles, had robust kinetochore fibers of sizable length, and
did not delay anaphase. We suggest these syntelics did
experience tension, not from oppositely directed kinetochore
fibers but from polar ejection forces that counteracted the
poleward forces of their kinetochore fibers. In crane fly
spermatocytes, transverse equilibrium forces move acentric
fragments to the spindle periphery, where they are driven
equatorially by polar ejection forces hypothetically involv-
ing a yet-to-be-characterized kinesin-10 orthologue (i.e., “stuck” at the poles. The Full Spectrum of Malorientations and the Ultimate
Fate of Laggards The less birefringent fiber (right-pointing arrow) is now 3.4 m
longer than at anaphase onset, whereas the length of the more birefringent fiber (left-pointing arrow) has increased only 0.4 m during the
same interval. (D) Sixty-seven minutes into recovery: cytokinesis begins. (E) Ninety-four minutes into recovery: cytokinesis fails. Bar, 5 m. (AA–DD) 2-D projections of chromosome positions in cell 99. (AA) Thirty-one minutes into recovery: at anaphase onset, sisters derived from
the meroamphitelic sex dyad (painted gray) are positioned in the upper half-spindle. (BB) Thirty-four minutes into recovery: one of the sex
sisters exhibits anaphase lag as its sister moves to the upper pole. (The upper centrosome was not included in the Z-focus series.) (CC)
Thirty-nine minutes into recovery: the laggard is positioned near the equator. (DD) Forty-eight minutes into recovery: the laggard is
positioned in the lower half-spindle. Nondisjunction during Meiosis II Nondisjunction during Meiosis II 6
A meroamphitelic sex dyad that exhibited merotely in one of its two kinetochores in cell 99 The merotelic chromosome moves Figure 6. A meroamphitelic sex dyad that exhibited merotely in one of its two kinetochores in cell 99. The merotelic chromosome moves
toward the equator as the spindle elongates and its less birefringent fiber lengthens. Neither of its kinetochore fibers shortens. Cold treatment:
75 h at 2°C. (retardance ceiling 2.0 nm). Anaphase begins 27 min into recovery, and first measurements of kinetochore fibers at 31 min into
recovery (see AA) indicate the merotelic kinetochore is 7.9 m from the upper pole and 9.4 m from the lower pole. (A and B) Forty
minutes into recovery: two slices from a Z-focus series of cell 99 illustrate kinetochore fibers of the merotelic sex chromosome (arrowhead). (A) Slice 20/38. (B) Slice 22/38. The fiber to the lower pole (left-pointing arrow) contains 37 microtubules (Figure 7D), whereas the fiber to
the upper pole (right-pointing arrow) contains 24 microtubules (Figure 7C). With the progression of anaphase, the merotelic chromosome
moved toward the equator as the less birefringent fiber elongated. (C) Forty-eight minutes into recovery: as anaphase progresses, the
merotelic laggard shifts past the equator and into the lower half-spindle. The less birefringent fiber (right-pointing arrow) is now 3.4 m
longer than at anaphase onset, whereas the length of the more birefringent fiber (left-pointing arrow) has increased only 0.4 m during the
same interval. (D) Sixty-seven minutes into recovery: cytokinesis begins. (E) Ninety-four minutes into recovery: cytokinesis fails. The Full Spectrum of Malorientations and the Ultimate
Fate of Laggards Very few metaphase II cells (2/985)
were found with an off-equator dyad after 44-h exposures,
whereas more (64/565 cells) were found during recovery
from 72 h. Off-equator dyads were not observed adjacent to
the pole as would have been expected if forces that act away
from the pole were inoperative (Weaver et al., 2006). Instead,
they were in positions similar to those of maloriented dyads
in our live-cell preparations. Vol. 18, May 2007 The Full Spectrum of Malorientations and the Ultimate
Fate of Laggards The Full Spectrum of Malorientations and the Ultimate
Fate of Laggards Like merotelic chromatids from merosyntelic dyads (Figure
3, G and H), other merotelic chromatids (Figure 3, B–F) also
failed to reach the polar centrosomes, and when they were
included in a daughter nucleus, it was only because they
were close enough to nonmerotelics to fuse with them at
telophase. Unlike merotelic chromatids from merosyntelic
dyads, the merotelic chromatids in Figure 3, B–F were, at
some point in anaphase, far enough behind the nonmerotel-
ics to be scored as laggards by our criteria. gg
y
If the two fibers of a merotelic had approximately equal
birefringence (Figure 3, D–F), then both fibers elongated. This resulted in little or no net anaphase movement of the
laggard away from the equator at anaphase, as also reported
by Cimini et al. (2004). y
Laggards that had one fiber that was more birefringent
than the other (unbalanced merotely; Figure 3, B, C, and E)
behaved as described for merotelics from merosyntelic dy-
ads: shortening of the kinetochore fibers of merotelics was Molecular Biology of the Cell Molecular Biology of the Cell 1652 Figure 6. A meroamphitelic sex dyad that exhibited merotely in one of its two kinetochores in cell 99. The merotelic chromosome moves
toward the equator as the spindle elongates and its less birefringent fiber lengthens. Neither of its kinetochore fibers shortens. Cold treatment:
75 h at 2°C. (retardance ceiling 2.0 nm). Anaphase begins 27 min into recovery, and first measurements of kinetochore fibers at 31 min into
recovery (see AA) indicate the merotelic kinetochore is 7.9 m from the upper pole and 9.4 m from the lower pole. (A and B) Forty
minutes into recovery: two slices from a Z-focus series of cell 99 illustrate kinetochore fibers of the merotelic sex chromosome (arrowhead). (A) Slice 20/38. (B) Slice 22/38. The fiber to the lower pole (left-pointing arrow) contains 37 microtubules (Figure 7D), whereas the fiber to
the upper pole (right-pointing arrow) contains 24 microtubules (Figure 7C). With the progression of anaphase, the merotelic chromosome
moved toward the equator as the less birefringent fiber elongated. (C) Forty-eight minutes into recovery: as anaphase progresses, the
merotelic laggard shifts past the equator and into the lower half-spindle. DISCUSSION (D and E) Slice 20/38 (presented
as Figure 6A) and slice 22/38 (Figure 6B) from a Z-focus series of the merotelic sex chromosome in cell 99 were similarly analyzed, revealing
a less birefringent fiber containing 24 microtubules to the upper pole and a more birefringent fiber containing 37 microtubules to the lower
pole. Figure 7. Duplicate images of maloriented chromosomes depicted in Figures 4–6 to illustrate how the retardance area data were obtained
(retardance ceiling 2.5 nm) (A) The selected portion of slice 14/28 from cell 132 (presented as Figure 4A) was analyzed using an algorithm
(described in detail by LaFountain and Oldenbourg, 2004) to generate a plot of retardance (in units of nanometers) as a function of distance
(micrometers). A line scan having the shape of an elongated rectangle 5 m long by 4 pixels wide was made perpendicular to the kinetochore
fiber(s) of interest. The portion of the scan corresponding to the kinetochore fiber(s) was identified (the shaded area in the plot). The area
under that portion of the plot is the retardance area (in units of square nanometers) of the selected kinetochore fiber(s). The conversion factor
from retardance area to number of microtubules is 7.5 nm2/microtubule (MT), yielding an apparent value of 96 microtubules for the two
kinetochore fibers from this syntelic dyad. Because these fibers were inclined somewhat (by 4°) in relation to the x-y plane of the image,
computed retardance area was adjusted to take that into account (see LaFountain and Oldenbourg, 2004), yielding an actual value of 97
microtubules in these two fibers. (B and C) Likewise, retardance area analysis was performed on slice 7/27 (presented as Figure 5A) and on
slice 8/27 (Figure 5B) of the merotelic sister that was derived from the merosyntelic dyad in cell 125, revealing 33 microtubules in the fiber
extending from the merotelic to the upper pole and 15 microtubules in the fiber extending to the lower pole. (D and E) Slice 20/38 (presented
as Figure 6A) and slice 22/38 (Figure 6B) from a Z-focus series of the merotelic sex chromosome in cell 99 were similarly analyzed, revealing
a less birefringent fiber containing 24 microtubules to the upper pole and a more birefringent fiber containing 37 microtubules to the lower
pole. rules” (Cimini et al., 2004; Salmon et al., 2005): 1) their
kinetochore fibers shorten little, but 2) their less robust fiber
lengthens with spindle elongation. DISCUSSION Cold-recovering meiosis II spermatocytes displayed a spec-
trum of chromosome orientations, from fully monopolar to
fully bipolar. Only syntelic (fully monopolar) and merosyn-
telic (nearly monopolar) orientation caused nondisjunction. Cold-recovering meiosis II spermatocytes displayed a spec-
trum of chromosome orientations, from fully monopolar to
fully bipolar. Only syntelic (fully monopolar) and merosyn-
telic (nearly monopolar) orientation caused nondisjunction. Monopolar orientations normally are detected and cor-
rected by mechanisms (Shannon and Salmon, 2002; Biggins,
2004; Gassmann et al., 2004; Maiato et al., 2004) that cause y
p
j
Monopolar orientations normally are detected and cor-
rected by mechanisms (Shannon and Salmon, 2002; Biggins,
2004; Gassmann et al., 2004; Maiato et al., 2004) that cause 1653 M. A. Janicke et al. Figure 7. Duplicate images of maloriented chromosomes depicted in Figures 4–6 to illustrate how the retardance area data were obtained
(retardance ceiling 2.5 nm) (A) The selected portion of slice 14/28 from cell 132 (presented as Figure 4A) was analyzed using an algorithm
(described in detail by LaFountain and Oldenbourg, 2004) to generate a plot of retardance (in units of nanometers) as a function of distance
(micrometers). A line scan having the shape of an elongated rectangle 5 m long by 4 pixels wide was made perpendicular to the kinetochore
fiber(s) of interest. The portion of the scan corresponding to the kinetochore fiber(s) was identified (the shaded area in the plot). The area
under that portion of the plot is the retardance area (in units of square nanometers) of the selected kinetochore fiber(s). The conversion factor
from retardance area to number of microtubules is 7.5 nm2/microtubule (MT), yielding an apparent value of 96 microtubules for the two
kinetochore fibers from this syntelic dyad. Because these fibers were inclined somewhat (by 4°) in relation to the x-y plane of the image,
computed retardance area was adjusted to take that into account (see LaFountain and Oldenbourg, 2004), yielding an actual value of 97
microtubules in these two fibers. (B and C) Likewise, retardance area analysis was performed on slice 7/27 (presented as Figure 5A) and on
slice 8/27 (Figure 5B) of the merotelic sister that was derived from the merosyntelic dyad in cell 125, revealing 33 microtubules in the fiber
extending from the merotelic to the upper pole and 15 microtubules in the fiber extending to the lower pole. DISCUSSION For meroamphitelic dy-
ads, the less robust kinetochore fiber of the merotelic ex-
tends toward the pole opposite that to which its sister ex-
tends, so the merotelic shifts away from its sister during
anaphase B. As predicted by Cimini’s rules, if a merotelic
starts anaphase in the half-spindle of its less birefringent
fiber (because its nonmerotelic sister is oriented toward the
pole of the half-spindle), the merotelic shifts equatorially at
anaphase (also see Pidoux et al., 2000) into the opposite
half-spindle, still away from its sister. So far, no evidence,
including ours, exists for merotelics disobeying Cimini’s
rules. chromokinesin) on chromosome arms (LaFountain et al.,
2002). Regardless of where cold-recovering dyads were at
recovery onset (undetermined due to time lost in specimen
preparation), failure to biorient should locate them at the
spindle periphery and then ejection forces would drive them
toward the equator at an angle parallel to peripheral micro-
tubules lying at steep angles to the pole-to-pole axis. Ac-
cordingly, cold-recovering syntelics were at the periphery
and had tilted kinetochore fibers. Failure of syntely to elicit a wait-anaphase response may
be a peculiarity of this cell type (LeMaire-Adkins et al., 1997;
Forer and Pickett-Heaps, 1998) or of cold recovery (but see
Cimini et al., 2002). Alternatively, the anaphase checkpoint
may be satisfied by the full kinetochore microtubule occu-
pancy of these syntelics and/or by the tension exerted by
polar ejection forces on them (Cimini and Degrassi, 2005;
Pinsky and Biggins, 2005). At anaphase, kinetochore fibers
of syntelics shortened in concert with those of normally
oriented chromosomes, presumably because proteolysis of
chromokinesins (Antonio et al., 2000; Funabiki and Murray,
2000) allowed their chromatids to move poleward after loss
of cohesion. Merosyntely (Figure 3, G and H) has been postulated
previously, although not observed (Salmon et al., 2005), to
explain nondisjunction in nocodazole-recovering mitotic
cells (Cimini et al., 1999). Our demonstration that merosyn-
tely is indeed induced by arrest recovery is the first for either
mitosis or meiosis II and establishes it as a direct cause of
nondisjunction. In contrast to chromatids of syntelics, which
move all the way to the centrosome, a merotelic from a
merosyntelic dyad is included in a nucleus because it starts
anaphase close enough to it to fuse with nonmerotelic chro-
mosomes that move there during anaphase. Meroamphitely (Figure 3, B and C) does not cause non-
disjunction. Molecular Biology of the Cell Nondisjunction during Meiosis II matocytes, equatorial laggards usually cause cleavage fur-
row regression. Abortive cytokinesis II produces a meiotic
product with twice its normal chromosome complement. In
mitosis, cytokinesis failure can cause subsequent divisions
to be multipolar, leading to aneuploidy (Shi and King, 2005). Similarly, laggard-induced meiosis I cytokinesis failure was
shown previously to lead to a tripolar meiosis II spindle and
to a spermatid containing chromosomes from one of those
poles (Janicke and LaFountain, 1982). In mitotic cells where
cytokinesis succeeds despite equatorial laggards, indirect
nondisjunction could occur if a balanced merotelic from a
dyad oriented as in Figure 3, E or F, is included in the same
daughter cell as its sister, forms a micronucleus, then, during
the next division, incorporates into a daughter nucleus, mak-
ing it (n 1) (Rizzoni et al., 1989). Alternatively, balanced
merotely might lead to aneuploidy through laggard disinte-
gration or micronucleus loss (Sugawara and Mikamo, 1980;
Ford et al., 1988), neither of which was observed here. Antonio, C., Ferby, I., Wilhelm, H., Jones, M. Karsenti, E. Nebreda, A. R., and
Vernos, I. (2000). Xkid, a chromokinesin required for chromosome alignment
on the metaphase plate. Cell 102, 425–435. Battaglia, D. G., Goodwin, P., and Klein, N. A. (1996). Influence of maternal
age on meiotic spindles in oocytes from naturally cycling women. Hum. Reprod. 11, 2217–2222. Begg, D. A., and Ellis, G. W. (1979). Micromanipulation studies of chromo-
some movement. 2. Birefringent chromosomal fibers and the mechanical
attachment of chromosomes to the spindle. J. Cell Biol. 82, 542–554. Bickel, S. E., Moore, D. P., Lai, C., and Orr-Weaver, T. L. (1998). Genetic
interactions between meiS332 and ord in the control of sister-chromatid
cohesion. Genetics 150, 1467–1476. Biggins, S. (2004). Correcting SYNful attachments. Nat. Cell Biol. 6, 181–183. Cimini, D., Antoccia, A., Tanzarella, C., and Degrassi, F. (1997). Topoisomer-
ase II inhibition I mitosis produces numerical and structural chromosomal
aberrations I human fibroblasts. Cytogenet. Cell Genet. 76, 61–67. Cimini, D., Cameron, L. A., and Salmon, E. D. (2004). Anaphase spindle
mechanics prevent mis-segregation of merotelically oriented chromosomes. Curr. Biol. 14, 2149–2155. )
Coinduction of bipolar and monopolar malorientations
may be common. If so, that might help explain why coin-
duction of anaphase lag and nondisjunction has been re-
ported in diverse systems (Sugawara and Mikamo, 1980;
Elhajouji et al., 1997; Parry et al., 2002; Salmon et al., 2005; Sun
et al., 2005). Nondisjunction during Meiosis II Using our scoring criteria, after 24-h arrests, the
ratio of percentage of cells with anaphase lag (of all an-
aphase cells) at 60 min of recovery to percentage of cells with
nondisjunction (of successfully cleaving cells) at 90 min of
recovery was 40:1; it was 20:1 after 48-h arrests and 9:1 after
72-h arrests (Table 1). Thus, syntely and merosyntely were
induced less efficiently than other malorientations, but
lengthening cold exposure increased the proportion of mal-
orientations that were syntelic or merosyntelic. Cimini, D., and Degrassi, F. (2005). Aneuploidy: a matter of bad connections. Trends Cell Biol. 15, 442–451. Cimini, D., Fioravanti, D., Salmon, E. D., and Degrassi, F. (2002). Merotelic
kinetochore orientation versus chromosome mono-orientation in the origin of
lagging chromsomes in human primary cells. J. Cell Sci. 115, 507–515. Cimini, D., Howell, B., Maddox, P., Khodjakov, A. Degrassi, F., and Salmon,
E. D. (2001). Merotelic kinetochore orientation is a major mechanism of
aneuploidy in mitotic ammalian tissue cells. J. Cell Biol. 153, 517–527. Cimini, D., Moree, B., Canman, J. C., and Salmon, E. D. (2003). Merotelic
kinetochore orientation occurs frequently during early mitosis in mammalian
tissue cells and error correction is achieved by two different mechanisms. J. Cell Sci. 116, 4213–4225. Cimini, D., Tanzarella, C., and Degrassi, F. (1999). Differences in mal-segre-
gation rate obtained by scoring ana-telophase or binucleate cells. Mutagenesis
14, 563–568. y
y
With longer arrests, metaphase II dyads in cold arrest-recov-
ering spermatocytes more frequently displayed off-equator
metaphase dyad positions, which resulted from bipolar or
monopolar malorientations. Human oocytes naturally arrest in
meiosis I until ovulation and in meiosis II until fertilization and
have unusually high frequencies of nondisjunction. Studies on
human oocytes have shown that metaphase II dyad mis-
alignment correlates with age-dependent increased nondis-
junction (Battaglia et al., 1996, Volarcik et al., 1998; Hodges et
al., 2002; Page and Hawley, 2003). Perhaps, as in cold recov-
ery, off-equator metaphase II dyads in oocytes resuming
meiosis after natural arrests have bipolar or monopolar mal-
orientations. If so, those that are monopolar or nearly mo-
nopolar could cause nondisjunction, as has been demon-
strated in spermatocytes by the present study. Elhajouji, A., Tibaldi, F., and Kirsch-Volders, M. (1997). Indication for thresh-
olds of chromosome non-disjunction versus chromosome lagging induced by
spindle inhibitors in vitro in human lymphocytes. Mutagenesis 12, 133–140. Falck, G. C., Catala´n, J., and Norppa, H. (2002). Nondisjunction during Meiosis II Nature of anaphase laggards
and micronuclei in female cytokinesis-blocked lymphocytes. Mutagenesis 17,
111–117. Ford, J. H., Schultz, C. J., and Correll, A. T. (1988). Chromosome elimination
in micronuclei: a common cause of hypoploidy. Am. J. Hum. Genet. 43,
733–740. Forer, A., and Pickett-Heaps, J. D. (1998). Checkpoint control in crane-fly
spermatocytes: unattached chromosomes induced by cytochalasin D or la-
trunculin do not prevent or delay the start of anaphase. Protoplasma 203,
100–111. Funabiki, H., and Murray, A. W. (2000). The Xenopus chromokinesinXkid is
essential for metaphase chromosome alignment and must be degraded to
allow anaphase chromosome movement. Cell 102, 411–424. How malorientation is induced by arrest recovery remains
to be resolved, as does the relationship between induction of
bipolar versus monopolar malorientations. Further investi-
gation should elucidate not only errors leading to nondis-
junction but also mechanisms (Lampson et al., 2004; Biggins,
2004) by which chromosomes achieve the proper orientation
that ensures equidistribution. Gassmann, R., Carvalho, A., Henzing, A. J., Ruchaud, S., Hudson, D. F.,
Honda, R., Nigg, E. A., Gerloff, D. L., and Earnshaw, W. C. (2004). Borealin:
a novel chromosomal passenger required for stability of the bipolar meiotic
spindle. J. Cell Biol. 166, 179–191. Hassold, T., and Hunt, P. (2001). To err (meiotically) is human: the genesis of
human aneuploidy. Nat. Rev. Genet. 2, 280–291. Hawley, R. S., Frazier, J., and Rasooly, R. (1994). Separation anxiety: the
biology of non-disjunction in flies and people. Hum. Mol. Genet. 3, 1521–1528. ACKNOWLEDGMENTS Henderson, S. A., Nicklas, R. B., and Koch, C. A. (1970). Temperature-induced
orientation instability during meiosis: an experimental analysis. J. Cell Sci. 6,
323–350. We thank Alan Siegel and Grant Harris for technical assistance and Jim
Stamos for assisting with illustrations. This study was supported by National
Science Foundation grant MCB-0235934 (to J.L.) and grants GM 49210 from
the National Institute of General Medical Sciences and EB002045 from the
National Institutes of Biomedical Imaging and Bioengineering (to R.O.). Hildreth, P. D., and Ulrichs, P. C. (1969). A temperature effect on nondisjunc-
tion of the X chromosomes among eggs from aged Drosophila females. Ge-
netica 40, 191–197. Hodges, C. A., Ilagen, A., Jennings, D., Keri, R., Nilson, J., and Hunt, P. A. (2002). Experimental evidence that changes in oocyte growth influence mei-
otic chromosome segregation. Hum. Reprod. 17, 1171–1180. Angell, R. (1997). First-meiotic division nondisjunction in human oocytes.
Am. J. Hum. Genet. 61, 23–32. DISCUSSION This is despite the fact that, probably because it
is bipolar, merotely does not trigger a wait-anaphase re-
sponse (Cimini et al., 2002). If merotely escapes the prean-
aphase orientation correction mechanism, it then encounters
a second mechanism (Cimini et al., 2003) to help prevent it
from causing aneuploidy. That second mechanism is an-
aphase B. During anaphase B, merotelics follow “Cimini’s g
p
While meroamphitely and amphitely lead to proper dis-
tribution (Figure 3, A–C), and merosyntely and syntely lead
to nondisjunction (Figure 3, G–I), balanced merotelics,
which remain near the equator (Figure 3, D–F), may elicit
different outcomes depending on cell type. In crane fly sper- 1654 Molecular Biology of the Cell REFERENCES Rebollo, E., and Arana, P. (1998). Chromosomal factors affecting the trans-
mission of univalents. Chromosome Res. 6, 67–69. Koehler, K. E., Boulton, C. L., Collings, H. E., French, R. L., Herman, K. C.,
Lacefield, S. M., Madden, L. D., Schuetz, C. D., and Hawley, R. S. (1996). Spontaneous X chromosome MI and MII nondisjunction events in Drosophila
melanogaster oocytes have different recombinational histories. Nat. Genet. 14,
406–414. Rizzoni, M., Tanzarella, C., Gustavino, B., Degrassi, F., Guarino A, and
Vitagliano, E. (1989). Indirect mitotic nondisjunction in Vicia faba and Chinese
hamster cells. Chromosoma 97, 339–346. Salmon, E. D., Cimini, D., Cameron, L. A., and DeLuca, J. G. (2005). Merotelic
kinetochores in mammalian tissue cells. Phil. Trans. R. Soc. B 360, 553–568. Kops, G.J.P.L., Weaver, A. A., and Cleveland, D. W. (2005). On the road to
cancer: aneuploidy and the mitotic checkpoint. Nat. Rev. Cancer 5, 773–785. Scarcello, L. A., Janicke, M. A., and LaFountain, J. R. (1986). Kinetochore
microtubules in crane-fly spermatocytes: untreated, 2°C treated, and 6°C
grown spindles. Cell Motil. Cytoskeleton 6, 428–438. Ladrach, K. S., and LaFountain, J. R., Jr. (1986). Malorientation and abnormal
segregation of chromosomes during recovery from colcemid and nocodazole. Cell Motil. Cyotskeleton 6, 419–427. Shannon, K. B., and Salmon, E. D. (2002). Chromosome dynamics: new light
on aurora B kinase function. Curr. Biol. 12, R458–R460. LaFountain, J. R., Jr. (1985). Chromosome segregation and spindle structure in
crane fly spermatocytes following Colcemid treatment. Chromosoma 91, 329–
336. Shi, Q., and King, R. W. (2005). Chromosome nondisjunction yields tetraploid
rather than aneuploid cells in human cell lines Nature 437, 1038–1042. Skibbens, R. V., Skeen, V. P., and Salmon, E. D. (1993). Directional instability
of kinetochore motility during chromosome congression and segregation in
mitotic newt lung cells: a push-pull mechanism. J. Cell Biol. 122, 859–875. LaFountain, J. R., Jr., Cole, R. W., and Rieder, C. L. (2002). Polar ejection forces
are operative in crane-fly spermatocytes, but their action is limited to the
spindle periphery. Cell Motil. Cytoskeleton 51, 16–26. Sugawara, S., and Mikamo, K. (1980). An experimental approach to the
analysis of mechanisms of meiotic nondisjunction and anaphase lagging in
primary oocytes. Cytogenet. Cell Genet. 28, 251–264. LaFountain, J. R., Jr., and Oldenbourg, R. (2004). Maloriented bivalents have
metaphase positions at the spindle equator with more kinetochore microtu-
bules to one pole than to the other. Mol. Biol. Cell 15, 5346–5355. REFERENCES Janicke, M. and LaFountain, J. R., Jr. (1982). Chromosome segregation in
crane-fly spermatocyts: cold treatment and cold recovery induce anaphase
lag. Chromosoma 85, 619–631. Vol. 18, May 2007 1655 M. A. Janicke et al. mechanisms of chromosome congression and anaphase onset. J. Cell Biol. 137,
1567–1580. Janicke, M. A., and LaFountain, R., Jr. (1984). Malorientation in half-bivalents
at anaphase: analysis of autosomal laggards in untreated, cold-treated, and
cold recovering crane-fly spermatocytes. J. Cell Biol. 98, 859–869. Nicklas, R. B. (1997). How cells get the right chromosomes. Science 275,
632–637. Janicke, M. A., and LaFountain, R., Jr. (1986). Bivalent orientation and behav-
ior in crane-fly spermatocytes recovering from cold exposure. Cell Motil. Cytoskeleton 6, 492–501. Nicolaidis, P., and Petersen, M. B. (1998). Origin and mechanisms of nondis-
junction in human autosomal trisomies. Hum. Reprod. 13, 313–319. Kapoor, T. M., Lampson, M. A., Hergert, P., Cameron, L., Cimini, D., Salmon,
E. D., McEwen, B., and Khodjakov, A. (2006). Chromosomes can congress to
the metaphase plate before biorientation. Science 311, 388–391. Oldenbourg, R., Salmon, E. D., and Tran, P. T. (1998). Birefringence of single
and bundled microtubules. Biophys. J. 74, 645–654. Orr-Weaver, T. (1996). Meiotic nondisjunction does the two-step. Nat. Genet. 14, 374–376. Karp, L. E., and Smith, W. D. (1975). Experimental production of aneuploidy
in mouse oocytes. Gynecol. Investig. 6, 337–341. Page, S. L., and Hawley, R. S. (2003). Chromosome choreography: the meiotic
ballet. Science 301, 785–789. Kato, H., and Yosida, T. H. (1970). Nondisjunction of chromosomes in a
synchronized cell population initiated by reversal of colcemid inhibition. Exp. Cell Res. 60, 459–464. Parry, E. M. et al. (2002). Detection and characterization of action of aneugenic
chemicals. Mutagenesis 17, 509–521. Khodjakov, A., Cole, R. W., McEwen, B. R., Buttle, K. F., and Rieder, C. L. (1997). Chromosome fragments possessing only one kinetochore can congress
to the spindle equator. J. Cell Biol. 136, 229–240. Pidoux, A. L., Uzawa, S., Perry, P. E., Cande, W. Z., and Allshire, R. C. (2000). Live analysis of lagging chromosomes during anaphase and their effect on
spindle elongation rate in fission yeast. J. Cell Sci. 113, 4177–4191. Kline-Smith, A., Khoakov, A., Herget, P., and Walczak, C. E. (2004). Depletion
of centromeric MCAK leads to chromosome congression and segregation
defects due to improper kinetochore attachments. Mol. Biol. Cell 15, 1146–
1159. Pinsky, B. A., and Biggins, S. (2005). The spindle checkpoint: tension versus
attachment. Trends Cell Biol. 15, 486–493. REFERENCES Sun, F., Betzendahl, I., Pacchierotti, F., Randaldi, R., Smitz, J., Cortvrindt, R.,
and Eichenlaub-Ritter, U. (2005). Aneuploidy in mouse metaphase II oocytes
exposed in vivo and in vitro in preantral follicle culture to nocodazole. Mutagenesis 10, 65–75. Lamb, N. E., and Hassold, T. J. (2004). Nondisjunction—a view from ringside. N. Engl. J. Med. 351, 1931–1934. Lamb, N. E. et al. (1996). Susceptible chiasmate configurations of chromosome
21 predispose to nondisjunction in both maternal meiosis I and meiosis II. Nat. Genet. 14, 400–405. Tanaka, T. (2002). Bi-orienting chromosomes on the mitotic spindle. Curr. Opin. Cell Biol. 14, 365–371. Lampson, M. A., Renduchitala, K., Khodjakov, A., and Kapoor, T. M. (2004). Correcting improper chromosome-spindle attachments during cell division. Nat. Cell Biol. 6, 232–237. Tokunaga, C. (1970). Aspects of low-temperature-induced meiotic nondis-
junction in Drosophila females. Genetics 66, 653–661. Volarcik, K., Sheean, L., Goldfarb, J., Woods, L., Abdul-Karim, F. W., and
Hunt, P. (1998). The meiotic competence of in-vitro matured human oocytes
is influenced by donor age: evidence that folliculogenesis is compromised in
the reproductively aged ovary. Hum. Reprod. 13, 154–160. LeMaire-Adkins, R., Radke, K., and Hunt, P. A. (1997). Lack of checkpoint
control at the metaphase/anaphase transition: a mechanism of meiotic non-
disjunction in mammalian females. J. Cell Biol., 139, 1611–1619. Maiato, H., DeLuca, J., Salmon, E. D., and Earnshaw, W. C. (2004). The
dynamic kinetochore-microtubule interface. J. Cell Sci. 117, 5461–5477. Weaver, B.A.A., Silk, A., and Cleveland, D. (2006). Nondisjunction, aneu-
ploidy and tetraploidy. Nature 442, E9–E10. Yu, H.-G., and Dawe, R. K. (2000). Functional redundancy in the maize
meiotic kinetochore. J. Cell Biol. 151, 131–141. McEwen, B. F., Heagle, A. B., Cassels, G. O., Buttle, K. F., and Rieder, C. L. (1997). Kinetochore fiber maturation in PtK1 cells and its implications for the 1656 Molecular Biology of the Cell
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Vertical characterization of highly oxygenated molecules (HOMs) below and above a boreal forest canopy
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To cite this version: To cite this version:
Qiaozhi Zha, Chao Yan, Heikki Junninen, Matthieu Riva, Nina Sarnela, et al.. Vertical characteriza-
tion of highly oxygenated molecules (HOMs) below and above a boreal forest canopy. Atmospheric
Chemistry and Physics, 2018, 18 (23), pp.17437-17450. 10.5194/acp-18-17437-2018. hal-01975352 Qiaozhi Zha, Chao Yan, Heikki Junninen, Matthieu Riva, Nina Sarnela, et al.. Vertical characteriza-
tion of highly oxygenated molecules (HOMs) below and above a boreal forest canopy. Atmospheric
Chemistry and Physics, 2018, 18 (23), pp.17437-17450. 10.5194/acp-18-17437-2018. hal-01975352 Qiaozhi Zha, Chao Yan, Heikki Junninen, Matthieu Riva, Nina Sarnela, Juho
Aalto, Lauriane Quéléver, Simon Schallhart, Lubna Dada, Liine Heikkinen, et
al. Vertical characterization of highly oxygenated molecules (HOMs)
below and above a boreal forest canopy Qiaozhi Zha1, Chao Yan1, Heikki Junninen1, Matthieu Riva1, Nina Sarnela1, Juho Aalto1, Lauriane Quéléver1,
Simon Schallhart1, Lubna Dada1, Liine Heikkinen1, Otso Peräkylä1, Jun Zou2, Clémence Rose1, Yonghong Wang1,
Ivan Mammarella3, Gabriel Katul4,5, Timo Vesala1, Douglas R. Worsnop1,6, Markku Kulmala1, Tuukka Petäjä1,
Federico Bianchi1, and Mikael Ehn1 1Institute for Atmospheric and Earth System Research/Physics, Faculty of Science, University of Helsinki,
P.O. Box 64, 00014 Helsinki, Finland nstitute for Atmospheric and Earth System Research/Physics, Faculty of Science, University of Hel
O. Box 64, 00014 Helsinki, Finland 2CMA-NJU Joint Laboratory for Climate Prediction Studies, Institute for Climate and Global Cha
S h
l f A
h i S i
ji
i
i
ji
Chi 2CMA-NJU Joint Laboratory for Climate Prediction Studies, Institute for Climate and Global Change Research,
School of Atmospheric Sciences, Nanjing University, Nanjing, China School of Atmospheric Sciences, Nanjing University, Nanjing, China 3Institute for Atmospheric and Earth System Research/Physics, Faculty of Science, University of Helsinki,
P.O. Box 68, 00014 Helsinki, Finland 3Institute for Atmospheric and Earth System Research/Physics, Faculty of Science, University of Helsinki,
P.O. Box 68, 00014 Helsinki, Finland 4Nicholas School of the Environment, Duke University, Durham, North Carolina, USA
5D
f Ci il
d E
i
l E
i
i
D k U i
i
D
h
N
h C
li 4Nicholas School of the Environment, Duke University, Durham, North Carolina, USA
5Department of Civil and Environmental Engineering, Duke University, Durham, North Carolina, USA
6Aerodyne Research, Inc., Billerica, MA 01821, USA 4Nicholas School of the Environment, Duke University, Durham, North Carolina, USA
5Department of Civil and Environmental Engineering, Duke University, Durham, North Carolina, USA
6Aerodyne Research, Inc., Billerica, MA 01821, USA Correspondence: Qiaozhi Zha (qiaozhi.zha@helsinki.fi) and Chao Yan (chao.yan@helsinki.fi) Correspondence: Qiaozhi Zha (qiaozhi.zha@helsinki.fi) and Chao Yan (chao.yan@helsinki.fi) Received: 24 November 2017 – Discussion started: 8 December 2017
Revised: 28 August 2018 – Accepted: 30 October 2018 – Published: 10 December 2018 tire nocturnal boundary layer. This could, in turn, influence
the growth of newly formed particles and SOA formation be-
low the canopy where the large majority of measurements are
typically conducted. Abstract. While the role of highly oxygenated molecules
(HOMs) in new particle formation (NPF) and secondary or-
ganic aerosol (SOA) formation is not in dispute, the interplay
between HOM chemistry and atmospheric conditions contin-
ues to draw significant research attention. Vertical characterization of highly oxygenated molecules (HOMs)
below and above a boreal forest canopy During the Influ-
ence of Biosphere-Atmosphere Interactions on the Reactive
Nitrogen budget (IBAIRN) campaign in September 2016,
profile measurements of neutral HOMs below and above the
forest canopy were performed for the first time at the boreal
forest SMEAR II station. The HOM concentrations and com-
position distributions below and above the canopy were sim-
ilar during daytime, supporting a well-mixed boundary layer
approximation. However, much lower nighttime HOM con-
centrations were frequently observed at ground level, which
was likely due to the formation of a shallow decoupled layer
below the canopy. Near the ground HOMs were influenced
by the changes in the precursors and oxidants and enhance-
ment of the loss on surfaces in this layer, while the HOMs
above the canopy top were not significantly affected. Our
findings clearly illustrate that near-ground HOM measure-
ments conducted under stably stratified conditions at this site
might only be representative of a small fraction of the en- tire nocturnal boundary layer. This could, in turn, influence
the growth of newly formed particles and SOA formation be-
low the canopy where the large majority of measurements are
typically conducted. HAL Id: hal-01975352
https://uca.hal.science/hal-01975352v1
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lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers. Atmos. Chem. Phys., 18, 17437–17450, 2018
https://doi.org/10.5194/acp-18-17437-2018
© Author(s) 2018. This work is distributed under
the Creative Commons Attribution 4.0 License. 1
Introduction Highly oxygenated molecules (HOMs), a subgroup of the ox-
idation products of volatile organic compounds (VOCs) iden-
tified by their high oxidation states, have been recognized as
important precursors for organic aerosol in the atmosphere
(Ehn et al., 2014). They have also been found to enhance
new particle formation (NPF) and growth (Kulmala et al.,
2013; Zhao et al., 2013; Ehn et al., 2014; Bianchi et al., 2016;
Kirkby et al., 2016; Tröstl et al., 2016). The importance of
HOMs has been confirmed in ambient environments, espe-
cially in monoterpene-dominated regions such as the boreal
forest (Kulmala et al., 2013; Ehn et al., 2014), but also in
high-altitude mountain regions (Bianchi et al., 2016) and in
rural areas (Jokinen et al., 2014; Kürten et al., 2016). In labo-
ratory studies, HOM formation has been observed from var- Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. Q. Zha et al.: Vertical characterization of HOMs below and above a boreal forest canopy 17438 ious precursor molecules (Ehn et al., 2017), including both
biogenic and anthropogenic emissions (Molteni et al., 2018). boundary layer (Chen et al., 2018). In contrast, the nocturnal
boundary layer was shallow with stability regimes that de-
pended on radiative cooling within the canopy and turbulent
shear stresses at the canopy top. In Hyytiälä, the depletion
of O3 below the canopy has been frequently observed during
nighttime, while the O3 above the canopy was less affected
(Chen et al., 2018). The MT concentration at ground level in-
creased when O3 was depleted (Eerdekens et al., 2009). The
inhomogeneous distribution of the precursors and oxidants
below and above the canopy might further impact nocturnal
HOM distributions, which frames the scope of this study. Un-
til now, all CI-APi-TOF deployments have been at ground
level, and the main subject of inquiry here is the vertical in-
formation on HOMs and the role of meteorological condition
in shaping them. A characterization of the HOMs at different
heights provides a decisive advantage in disentangling the
role of nonuniform mixing within the atmospheric layers im-
pacted by strong thermal stratification, especially inside the
canopy volume. The direct observation of HOMs has only recently become
possible, following the developments of the atmospheric-
pressure-interface time-of-flight (APi-TOF, measures the
naturally charged HOMs) (Junninen et al., 2010) and
chemical-ionization atmospheric-pressure-interface time-of-
flight (CI-APi-TOF, measures the neutral HOMs) (Jokinen et
al., 2012) mass spectrometers. Ehn et al. (2010) and Bianchi
et al. (2017) found that the naturally charged HOM clusters
could be observed every night in the boreal forest during
spring. Out of the observed ambient mass spectra, a signif-
icant part could be reproduced in a chamber by introducing
the monoterpene α-pinene (C10H16, the major biogenic VOC
in the boreal forest) and ozone (O3) (Ehn et al., 2012). )
(
) (
)
Further investigations of HOM formation chemistry have
been carried out in both laboratory and field studies. Published by Copernicus Publications on behalf of the European Geosciences Union. Based
on current understanding from laboratory experiments, the
formation of HOMs involves three main steps: (1) initial
formation of peroxy radicals (RO2) from VOC oxidation;
(2) RO2 autoxidation, that is, the isomerization of the RO2
via intramolecular H shifts and subsequent oxygen (O2) ad-
ditions; and (3) radical termination, forming closed-shell
molecules (Crounse et al., 2013; Ehn et al., 2014; Joki-
nen et al., 2014, 2016; Rissanen et al., 2014; Mentel et al.,
2015). In the atmosphere, HOM formation studies are com-
plicated by the plethora of different compounds and pro-
cesses taking place. However, recent ambient measurements
together with factor analysis were able to shed light on the
HOM formation pathways in the boreal forest (Yan et al.,
2016). They showed that the majority of the daytime pro-
duction of HOMs was from reactions initiated by the oxi-
dation of monoterpenes (MTs) with hydroxyl radicals (OH)
or O3. The RO2 after autoxidation were often terminated
by hydroperoxyl radicals (HO2) or self-termination (Orlando
and Tyndall, 2012) to form a non-nitrate HOM monomer
(CHOmonomer, mainly C9 and C10 compounds, with masses
between 290 and 450 Th after clustering with the charg-
ing ion (NO−
3 ) of the instrument), or reacting with nitro-
gen oxides (NOx = NO + NO2) to form organonitrate HOM
monomers (CHONmonomer). During nighttime, MTs were
mainly oxidized by O3 and NO3 radicals. Furthermore, due
to the lower nocturnal HO2 and NOx concentrations, in addi-
tion to the production of CHONmonomer, the RO2 products
readily reacted with other RO2 to form either non-nitrate
HOM dimers (CHOdimer, mainly C16–20 compounds with
masses between 450 and 600 Th after clustering with NO−
3 )
or organonitrate HOM dimers (CHONdimer), depending on
the oxidants forming the RO2 (Ehn et al., 2014; Jokinen et
al., 2014; Yan et al., 2016; Berndt et al., 2018). The first measurements of the HOM concentrations at two
different heights (36 m and 1.5 m a.g.l.) are presented and
discussed. The influence of boundary layer dynamics on the
HOMs at these different heights at SMEAR II station is ana-
lyzed and characterized in conjunction with auxiliary turbu-
lence and micrometeorological measurements. 2.2
Instrumentation This value is much smaller than the observed devia-
tion of HOM concentrations during inversion nights, as will
be discussed later. Additionally, an intercomparison between
the two instruments with a permeation tube containing trini-
trotriazinane (C3H6N6O6) was conducted in the field right
after the campaign. The results showed good agreement with
the relative TE, lending confidence to the method used here. Finally, it should be noted that the difference in TE between
the two instruments was larger than one would normally ex-
pect since the tower CI-APi-TOF had been tuned for higher
sensitivity at the largest masses (at the expense of transmis-
sion at the lower masses). In comparison to the direct determination of TE (Hein-
ritzi et al., 2016), this method increases the uncertainty in Concentration of HOMs was measured with two nitrate-ion-
based CI-APi-TOF mass spectrometers. The CI-APi-TOF
measuring at higher altitude was deployed at the top of a 35 m
tower located ∼20 m horizontally from the ground measure-
ment location. Both instruments were working in rooms with
air-conditioning and room temperatures controlled at 25 ◦C. The inlets of the two instruments were pointed to the south-
east direction and fixed at ∼36 and ∼1.5 m above ground. The tower measurement is at about twice the canopy height,
which is still within the roughness sublayer of the forest
(Raupach and Thom, 1981). The instrument setup of the
two CI-APi-TOF mass spectrometers was similar. In brief,
the CI-APi-TOF was the combination of a chemical ioniza-
tion (CI) inlet and an APi-TOF mass spectrometer (Aero-
dyne Research Inc., USA, and Tofwerk AG, Switzerland). The ambient air was first drawn into the inlet with a sam-
ple flow of 7 L min−1 (liter per minute) and then centered
to an ion reaction tube surrounded by sheath flow (filtered
air, 35 L min−1). Meanwhile, the nitrate ions carried by the
sheath gas, which were generated by exposing the nitric acid
(HNO3) to soft X-ray radiation, were guided into the sample
gas by an electrical field at ambient pressure (∼100 ms re-
action time). Neutral molecules (M) in the sample air were
ionized by either clustering with charged nitrate / nitric acid
((HNO3)n=0−2·NO−
3 ) to form (M)·NO−
3 cluster ions or los-
ing a proton to the charging ions to form deprotonated ions
(e.g., H2SO4 + NO−
3 →HSO−
4 + HNO3). 2.2
Instrumentation The ions then en-
tered the APi part, which was a three-stage vacuum cham-
ber, through a pinhole. In the APi, two quadrupoles and a
stack of ion lenses guided the ions into the TOF mass ana-
lyzer, in which ions were separated based on their mass-to-
charge (m/z) ratios. A more detailed description of this in-
strument has been given by Junninen et al. (2010) and Joki-
nen et al. (2012), and discussion on selectivity of this nitrate
ion charging was provided by Hyttinen et al. (2015). Mass
spectra obtained from the instrument were analyzed using the
“tofTools” program described in Junninen et al. (2010). De-
termination of the concentration of a measured molecule M
was based on the following equation: [M] =
PM
Preagent ion count rates × C,
(1) (1) where the sum of ion count rates (PM) in the numerator
includes all detected ions relating to compound M, whether
deprotonated or in clusters with reagent ions, and the sum of
reagent ion count rates in the denominator is the total sig-
nal of the nitrate ions. C is the calibration coefficient, which
was assigned the same value for all detected compounds. This assignment is only valid for compounds that cluster with
the reagent ions at the collision limit, such as H2SO4 (Vig-
giano et al., 1997), and have equal collision rates. The col-
lision rates of nitrate ions with H2SO4 and with HOMs are
expected to be very close (Ehn et al., 2014). Here, a cali- where the sum of ion count rates (PM) in the numerator
includes all detected ions relating to compound M, whether
deprotonated or in clusters with reagent ions, and the sum of
reagent ion count rates in the denominator is the total sig-
nal of the nitrate ions. C is the calibration coefficient, which
was assigned the same value for all detected compounds. This assignment is only valid for compounds that cluster with
the reagent ions at the collision limit, such as H2SO4 (Vig-
giano et al., 1997), and have equal collision rates. The col-
lision rates of nitrate ions with H2SO4 and with HOMs are
expected to be very close (Ehn et al., 2014). Here, a cali- In comparison to the direct determination of TE (Hein-
ritzi et al., 2016), this method increases the uncertainty in
the quantification of HOM concentrations. 2.2
Instrumentation bration coefficient of 1 × 1010 molec cm−3, estimated from
previous calibrations with similar settings using sulfuric acid
and theoretical constraints (Ehn et al., 2014), with an uncer-
tainty of at least −50%/ + 100%, was used in calculating
the HOM concentrations for both instruments. Ultimately,
the absolute HOM concentrations in this work are of sec-
ondary importance, as we focus on the relative comparison
of HOM concentrations measured at different heights. How-
ever, the comparability of the two CI-APi-TOF instruments
is of great importance, and results cannot be allowed to vary
as a result of inevitable differences in the mass-dependent
transmission efficiency (TE), for example. For a detailed dis-
cussion on factors affecting the TE of a CI-APi-TOF, we re-
fer to Heinritzi et al. (2016). To this end, instead of directly
evaluating the TE of each instrument, a “relative” TE of the
two CI-APi-TOFs was used for data correction: we selected
a time period at noontime on 9 September with a well-mixed
boundary layer, identified by the clear and sunny weather
and homogeneous vertical distribution of monoterpene and
other trace gases, and we assumed the HOM concentrations
at the two heights to be the same. Thus, the relative TE was
obtained from the concentration ratio between the two CI-
APi-TOFs at each m/z (Fig. 1). A fitted relative TE curve
(R2 = 0.97), which represents how the TE of the tower CI-
APi-TOF was changed at each m/z over the TE of the ground
one, was obtained using power-law regression. Weaker cor-
relation was obtained in the 200–250 and 500–600 Th mass
ranges, but in the mass range in which most of the HOMs
were located (290–500 Th) there is very little scatter around
the fitted curve, clearly suggesting that observed differences
in the two instruments’ responses were mainly due to differ-
ences in TE. To test our assumption of negligible vertical gra-
dients of HOMs during daytime, we analyzed the behavior of
sulfuric acid. We found that the uncertainty related to this as-
sumption corresponds to a value of 26 % (see Fig. S1 in the
Supplement). An upper limit of uncertainty relating to our
TE correction (Fig. 1) was also estimated, yielding a value
of 10 %, giving a total uncertainty from these two sources of
28 %. 2.1
Measurement site description The measurements were performed at the SMEAR II station
(Station for Measuring Ecosystem–Atmosphere Relations)
in the boreal forest in Hyytiälä, southern Finland (61◦51′ N,
24◦17′ E, 181 m a.s.l.; Hari and Kulmala, 2005; Hari et al.,
2013) during September 2016. There is no large anthro-
pogenic emission source at or near the site. The closest
sources are the two sawmills ∼5 km southeast of the site
and the city area of Tampere (∼60 km away). The forest
surrounding the station is primarily Scots pine with a mean
canopy height of ∼17.5 m, a total leaf area index (LAI) of ∼
6.5 m2m−2, a stand density of ∼1400 trees ha−1, and an av-
erage diameter at breast height (DBH) of ∼0.16 m (Bäck et
al., 2012; Launiainen et al., 2013). The forest floor is majorly
covered with a shallow dwarf shrub (a LAI of ∼0.5 m2m−2)
and moss layer (a LAI of ∼1 m2m−2) (Kulmala et al., 2008;
Launiainen et al., 2013). The planetary boundary layer height
at the SMEAR II station has been determined from previous
studies using radiosondes (Lauros et al., 2007; Ouwersloot
et al., 2012) and balloon soundings (Eerdekens et al., 2009). Roughly, these heights span some 400 m (March) to 1700 m
(August) at noontime and 100 m (March) to < 160 m (April)
at midnight. Beyond those chemical pathways, varied meteorological
conditions are also factors influencing the MT and oxidants
at different heights above the forest floor. Unsurprisingly, the
oxidants producing HOMs (e.g., O3) have been found al-
most uniformly distributed within the well-mixed daytime www.atmos-chem-phys.net/18/17437/2018/ Atmos. Chem. Phys., 18, 17437–17450, 2018 17439 2.2
Instrumentation However, as men-
tioned, a more accurate knowledge of the exact HOM con- Atmos. Chem. Phys., 18, 17437–17450, 2018 3.1
Data overview The Influence of Biosphere-Atmosphere Interactions on the
Reactive Nitrogen budget (IBAIRN) campaign was con-
ducted from 1 to 25 September 2016. After data quality
checks, only the measurements collected after 5 September
were used. Figure 2 shows the overall time series of the mete-
orological parameters measured at ground and tower levels,
including the temperature, RH, global radiation, concentra-
tions of trace gases, MT, and total HOMs (Zha, 2018). The
weather was generally sunny and clear during the campaign
except for a few cloudy (10, 15, and 22–23 September) and
drizzling (24 and 25 September) days. The mean air tempera-
ture and RH observed at ground level were 10.8±3.3 ◦C and
87±13 % (1σ standard deviation) and at the tower level were
10.5±3.0 ◦C and 88±14 %, respectively. The O3 concentra-
tions measured at ground and tower levels were 21 ± 8 and
25 ± 6 ppbv, respectively. The air temperature, RH, and O3
measured at the two heights were close to each other during
daytime. The NOx concentrations were quite low through-
out the campaign; the mean NOx concentrations were mostly
around the reported detection limit at 0.4±0.4 ppbv (ground)
and 0.4±0.5 ppbv (tower), yet showed an overall good agree-
ment between the measurements at the different heights. The
MT concentrations at ground level (0.38±0.34 ppbv on aver-
age) were generally higher than those above the canopy level
(0.20 ± 0.16 ppbv). Figure 1. The relative transmission curve between the two CI-APi-
TOF mass spectrometers, determined during a period of strong tur-
bulent mixing. Intercomparison results using a permeation tube con-
taining trinitrotriazinane (C3H6N6O6) are shown in red circles. The
fitted green line was used to scale the measured signals between the
two instruments to match, in order to compare relative changes dur-
ing times of limited vertical mixing. centrations would not influence the main findings of this
study. y
The MT, trace gases, and meteorological parameters were
continuously monitored at the different heights (4.2, 8.4,
16.8, 33.6, 50.4, 67.2, 101, and 125 m) on a 126 m mast
∼100 m away from the location of the CI-APi-TOFs. The
data at 4.2 and 33.6 m were used in this study to represent the
concentrations at near ground and tower levels, respectively. 3.1
Data overview MT concentrations were measured every third hour using a
proton transfer reaction mass spectrometer with a lower de-
tection limit of 1 pptv (PTR-MS, Ionicon Analytik GmbH;
Taipale et al., 2008). The O3 concentration was measured
with a UV light absorption analyzer that had a lower detec-
tion limit of 1 ppbv (TEI model 49C, Thermo Fisher Scien-
tific, USA). The NOx measurement was conducted using a
chemiluminescence analyzer (TEI model 42C TL, Thermo
Fisher Scientific, USA). The lower detection limit of the NOx
analyzer is 100 pptv. The CO2 measurement was performed
using an infrared detection system (LI-840, LI-COR Bio-
sciences, Lincoln, NE, USA). The aerosol number concen-
tration size distributions were obtained with a twin differen-
tial mobility particle sizer (twin DMPS) for the size range
from 3 to 1000 nm (Aalto et al., 2001) at 8 m in height above
ground, and was used to calculate condensation sink (CS)
based on the method from Kulmala et al. (2001). Air tem-
perature was measured with PT-100 resistance thermome-
ters. Air relative humidity (RH) was measured with RH sen-
sors (Rotronic HygroMet model MP102H with HygroClip
HC2S3, Rotronic AG, Switzerland). Global radiation (solar
radiation in the wavelength range of 0.3–4.8 µm) was ob-
tained with a Pyranometer (Reemann TP3, Astrodata, Esto-
nia) above the canopy top at 18 m. All the data presented The estimated total HOM concentration is representative
for the overall concentration level of HOMs and is here de-
fined as the sum of the detected signals among ions from
m/z 200 to 600 after removing identified background peaks. The gaps in the estimated total HOM at ground level were
due to automatic zero checks. During the campaign, a sig-
nificant difference was found in the estimated total HOM
concentrations below and above the canopy (mean and me-
dian concentrations of 1.1±1.7×108 and 0.8×108 cm−3 at
ground level and 1.7±1.3×108 and 1.3×108 cm−3 at tower
level). The causes of these differences (∼55 % in mean and
∼71 % in median) frame the upcoming discussion. Q. Zha et al.: Vertical characterization of HOMs below and above a boreal forest canopy Q. Zha et al.: Vertical characterization of HOMs below and above a boreal forest canop 17440 Figure 1. The relative transmission curve between the two CI-APi-
TOF mass spectrometers, determined during a period of strong tur-
bulent mixing. Intercomparison results using a permeation tube con-
taining trinitrotriazinane (C3H6N6O6) are shown in red circles. The
fitted green line was used to scale the measured signals between the
two instruments to match, in order to compare relative changes dur-
ing times of limited vertical mixing. are at 10 min averaging intervals, except for the MT (at a 1 h
averaging interval). A schematic figure showing sampling lo-
cations of all the measured parameters is provided in Fig. S2. are at 10 min averaging intervals, except for the MT (at a 1 h
averaging interval). A schematic figure showing sampling lo-
cations of all the measured parameters is provided in Fig. S2. www.atmos-chem-phys.net/18/17437/2018/ www.atmos-chem-phys.net/18/17437/2018/ Q. Zha et al.: Vertical characterization of HOMs below and above a boreal forest canopy 3.2
Intercomparison of estimated total HOM
concentrations The estimated total HOM concentrations at the two heights
were not different during the day (mean ±1σ standard de-
viation and median concentrations of 4.1 ± 2.3 × 108 and
3.6 × 108 cm−3 at ground level, 4.3 ± 2.6 × 108 and 4.0 ×
108 cm−3 at tower level), which validates the use of only 1
day of data for scaling the TE of the ground CI-APi-TOF to Atmos. Chem. Phys., 18, 17437–17450, 2018 www.atmos-chem-phys.net/18/17437/2018/ Q. Zha et al.: Vertical characterization of HOMs below and above a boreal forest canopy Q. Zha et al.: Vertical characterization of HOMs below and above a boreal forest canopy 17441 Q. Zha et al.: Vertical characterization of HOMs below and above a boreal forest canopy
17441
Figure 2. The overall time series of the measured trace gases, mete-
orological parameters, and estimated total HOM concentrations at
the ground (blue) and tower (red) levels. Figure 3. Comparison between ground (x axis) and tower (y axis)
measurements of the estimated total HOM concentrations. The
black line denotes the 1 : 1 ratio. Color code indicates the sampling
time of HOMs. Figure 2. The overall time series of the measured trace gases, mete-
orological parameters, and estimated total HOM concentrations at
the ground (blue) and tower (red) levels. Figure 3. Comparison between ground (x axis) and tower (y axis)
measurements of the estimated total HOM concentrations. The
black line denotes the 1 : 1 ratio. Color code indicates the sampling
time of HOMs. Figure 4. Mean mass spectra with the averaging periods of daytime
(09:00–15:00) and nighttime (21:00–03:00) at the ground and tower
levels. Figure 2. The overall time series of the measured trace gases, mete-
orological parameters, and estimated total HOM concentrations at
the ground (blue) and tower (red) levels. match the HOM signals of the two instruments. The good
daytime agreement throughout the campaign period also ver-
ifies that the response of each instrument stayed stable. Con-
trary to the daytime results, the estimated total HOM con-
centration at ground level usually diverged from the tower
measurement in the nocturnal boundary layer. The concen-
tration below the canopy became even lower when temper-
ature inversions were observed, accompanied by a decreas-
ing ground-level O3 and increasing MT concentrations. Fig-
ure 3 shows a comparison between the estimated total HOM
concentrations observed at two heights. 3.2
Intercomparison of estimated total HOM
concentrations Herein, good agree-
ment could be found for the group of points representing the
concentrations around noontime (R2 = 0.89). The points in-
dicating the nighttime estimated total HOM concentrations
were scattered (R2 = 0.28), and the ground concentrations
were found to be much lower than the tower ones. Figure 4. Mean mass spectra with the averaging periods of daytime
(09:00–15:00) and nighttime (21:00–03:00) at the ground and tower
levels. set periods. During daytime, a good agreement (R2 = 0.87)
was obtained from the mass-by-mass comparison using the
UMR concentrations extracted from daytime mean spectra,
suggesting a uniform composition distribution in the day-
time boundary layer condition. During nighttime, the mean
concentrations of all HOMs in the ground mean spectra were
much lower than the tower spectra. The HOM concentrations
shown in the ground and tower mean spectra were also less
correlated. Therefore, a logical outcome is that the condi-
tions below and above the canopy experience different turbu- Figure 4 shows the mean mass spectra (in unit mass resolu-
tion, UMR, for m/z 200–600) obtained from the ground and
tower. It is worth mentioning that there might be some sig-
nals not attributable to HOMs in the plotted spectra, but only
as a small proportion. Only selected periods (09:00–15:00
for daytime and 21:00–03:00 for nighttime; all the times are
given in Finnish winter time, UTC + 2) are included in the
averaging period to eliminate the effect of sunrise and sun- 3.3.1
Statistics of the non-inversion night and inversion
night types Table 1 shows the overall statistics including the mean and
median values of the temperatures, O3, NOx, MT, and esti-
mated total HOM concentrations for the non-inversion night
and inversion night types. On the non-inversion nights, the air
below and above the canopy was relatively well mixed. The
mean and median concentrations of the ground O3 (21 ± 8
and 22 ppbv) were close to the tower values (25 ± 6 and
24 ppbv). The slight difference might be attributed to the
higher VOC emissions (Rantala et al., 2014) and larger sink
near ground level. In contrast, during the inversion nights,
the mean estimated total HOM concentration and O3 at
ground level were generally much lower, only ∼33 % and
∼69 % of the tower concentrations, respectively. Instead,
the mean and median ground MT concentration (0.70±0.28
and 0.70 ppbv) were ∼3 times higher than the tower ones
(0.24 ± 0.04 and 0.23 ppbv). The measured NOx levels were
similar in both categories and at both heights, though the
ambient concentrations were close to the detection limit and
therefore small differences might not be observable. All the HOM groups in Fig. 5a show stable patterns, and
good agreement is observed between the ground and tower
measurements in the first half of the night. Variations were
observed when air mass change occurred at around 01:00,
as indicated by the drop of NOx concentration and horizon-
tal wind shift (not shown here). A rapid decrease was found
in CS, which represents the rate of condensation of low-
volatility vapors onto the existing aerosol particles (Dada et
al., 2017), implying that the aerosol population also changed. However, the HOM groups were still well correlated with
each other, suggesting well-mixed conditions in the non-
inversion night. Figure 5b shows the time series of the trace gases, MT,
and HOM groups of both ground and tower measurements
during an inversion night case (8–9 September, from 21:00
to 03:00). Ri was generally higher during this night, and in-
creased from ∼0.03 (indicating a weakly stable condition;
Mammarella et al., 2007), at around midnight, to a maxi-
mum of ∼1.13 (indicating a very stable condition) for the
rest of the night. Roughly, Ri values in excess of unity indi-
cate that stably stratified conditions appreciably diminish the
inverse turbulent Prandtl number (Pr) and the efficiency of
turbulence to mix heat when compared to momentum (Katul
et al., 2014). 3.3
Influence of nocturnal boundary layer dynamics
and micrometeorological processes The nighttime HOMs at ground level are likely influenced
by transport processes below the canopy since the estimated
total HOM concentrations were found much lower on the
nights when temperature inversions were observed. To fur-
ther investigate the potential impact of such micrometeoro-
logical phenomena on ground-level HOMs, the nights dur-
ing the campaign without precipitation or instrument fail-
ure were selected (14 nights in total) and categorized into
two types based on the occurrence of temperature inversions:
(1) the “non-inversion night” type included seven nights
when no temperature inversion was recorded; (2) the “inver-
sion night” type category consisted of seven nights that had
encountered temperature inversions, and the ground temper-
atures were generally ∼1 ◦C lower than tower temperatures
during these nights. Ri = g1θ1z
θ(u)2 ,
(2) Ri = g1θ1z
θ(u)2 , (2) where g is the gravitational acceleration, 1θ and 1z are
the mean potential temperature (10 min averaging interval,
same as measurement data) and height difference between
the ground and tower levels, respectively, and θ and u are the
mean potential temperature and mean wind velocity at tower
level, respectively. During the selected non-inversion night,
Ri was generally positive but close to 0 (shown in Fig. 5a),
indicating a weakly stable and relatively well-mixed (i.e.,
1θ →0) condition (Mahrt, 1998; Mammarella et al., 2007). This was also confirmed using the well-correlated ground
and tower MT and trace gas concentrations. Selected HOMs representing the major HOM types (and
formation pathways) were summed up and categorized into
four groups, as shown in Table 2. Each pathway might
be influenced differently by boundary layer dynamics and
micrometeorological processes. In this study, OH-initiated
HOMs were assumed negligible due to the very low OH level
in the nocturnal boundary layer. Q. Zha et al.: Vertical characterization of HOMs below and above a boreal forest canopy lent mixing strength and/or source–sink regimes during the
night. mospheric stability parameter or a Richardson number (flux
based, gradient based, or bulk). Because of its simplicity and
the availability of high-resolution mean air temperature pro-
files, the bulk Richardson number (Ri) was used here (Mahrt
et al., 2001; Mammarella et al., 2007; Vickers et al., 2012;
Alekseychik et al., 2013). It is calculated using 3.3.1
Statistics of the non-inversion night and inversion
night types The parameters measured at tower level were
not significantly affected by strong Ri fluctuations through-
out the night; in contrast, significant variations were observed
at ground level. www.atmos-chem-phys.net/18/17437/2018/ www.atmos-chem-phys.net/18/17437/2018/ Atmos. Chem. Phys., 18, 17437–17450, 2018 17442 3.3.2
Case study Two individual nights representing the non-inversion night
and inversion night types were selected and further com-
pared. Figure 5a shows the time series of the meteorological
parameters, trace gases, and HOMs measured at ground and
tower levels of one selected night for the non-inversion night
type (11–12 September, from 21:00 to 03:00). A number of
measures can be used to assess the local atmospheric stabil-
ity conditions at a given layer. These measures are commonly
based on either the Obukhov length and its associated at- Atmos. Chem. Phys., 18, 17437–17450, 2018 www.atmos-chem-phys.net/18/17437/2018/ www.atmos-chem-phys.net/18/17437/2018/ Q. Zha et al.: Vertical characterization of HOMs below and above a boreal forest canopy
17443
Table 1. Summary of the “Non-inversion night” and “Inversion night” types. Type
Non-inversion night
Inversion night
Date
6, 7, 9, 11, 15, 16, 21 Septembera
5, 8, 10, 12, 13, 14, 19 Septemberb
Parameters
Temperature
O3
NOx
MT
Estimated
Temperature
O3
NOx
MT
Estimated
(◦C)
(ppbv)
(ppbv)
(ppbv)
total HOM
(◦C)
(ppbv)
(ppbv)
(ppbv)
total HOM
(108 cm−3)
(108 cm−3)
Tower
Mean ±1σ standard
10.2 ± 2.6
25 ± 6
0.5 ± 0.5
0.31 ± 0.31
2.9 ± 1.9
9.5 ± 1.7
24 ± 2
0.4 ± 0.3
0.24 ± 0.04
2.4 ± 0.8
deviation
Median
10.9
24
0.4
0.17
2.8
9.2
23
0.3
0.23
2.3
Ground
Mean ±1σ standard
10.6 ± 2.7
21 ± 8
0.4 ± 0.4
0.52 ± 0.74
1.6 ± 0.6
8.3 ± 2.2
16 ± 6
0.3 ± 0.2
0.70 ± 0.28
0.8 ± 0.4
deviation
Median
11.5
22
0.3
0.22
1.7
8.5
17
0.3
0.70
0.7
a MT data not available on 5 and 19 September. b MT data not available on 15 and 16 September. Q. Zha et al.: Vertical characterization of HOMs below and above a boreal forest canopy 17443 Q. Zha et al.: Vertical characterization of HOMs below and above a boreal forest canopy Table 2. Compositions of selected HOMs and their main oxidants (Yan et al., 2016). Table 2. Compositions of selected HOMs and their main oxidants (Yan et al., 2016). Molecule compositions
Main oxidants
Main terminators
CHOmonomer
C10H14O7, C10H14O9
O3
Self-termination or RO2
CHONmonomer
C10H15O9N, C10H15O11N
O3 or NO3
NO or self-termination–RO2
CHOdimer
C19H28O11, C20H30O14
O3
RO2
CHONdimer
C20H32O12N2, C20H31O13N
NO3
RO2 The ground O3 concentration experienced a rapid decrease
at midnight. 3.3.2
Case study In about an hour (from 23:30 to 00:30), ground
O3 concentration dropped by more than half (from 20 to
9 ppbv), and CO2 concentration increased as well (from 404
to 423 ppbv). Conversely, the MT concentration at ground
level almost doubled (from 0.49 to 0.80 ppbv) during the
same period. Theoretically, the enhancement of HOM pre-
cursor VOC and the decrease in oxidant would compen-
sate for each other if the sink remained the same, and the
ground HOM concentrations should also remain constant. However, all the HOM groups showed a significant decrease
after midnight, despite the CS (generally the main sink for
HOM in the atmosphere) staying practically constant. In par-
ticular, the concentration of the CHOmonomer group dropped
∼80 %, from 8.6 × 106 to 1.7 × 106 cm−3, and the concen-
tration of the CHOdimer group decreased from 1.5×106 to ∼
0.1×106 cm−3. The concentrations of the CHONmonomer and
CHONdimer groups also experienced large declines (∼34 %
and ∼50 %, respectively) in the latter half of the night. At
03:00, the CHONdimer concentration was already below the
detection limit (1 × 104 cm−3). Therefore, the much lower
ground HOM concentrations might not be totally explained
by the change of HOM production but rather due to some
other processes such as additional losses. 2009, 2012; Alekseychik et al., 2013; Chen et al., 2018) and
could also explain the occurrence of the strong temperature
inversion during the inversion nights. To explore the possible
mechanism resulting in significantly different O3, MT, and
HOM concentrations below the canopy, the mean continuity
equation for high-Reynolds-number flows within the canopy
is formulated as (e.g., Katul et al., 2006) ∂C
∂t + U ∂C
∂x + W ∂C
∂z = −S −∂w′c′
∂z
−∂u′c′
∂x ,
(3)
N1 + N2 + N3 = N4 + N5 + N6,
(4) (3) (3) (4) where t is time, x and z are the longitudinal and vertical di-
rections, respectively, C is the scalar concentration, U and W
are the longitudinal and vertical velocity components, w′c′
and u′c′ are the turbulent scalar fluxes in the vertical and
horizontal, respectively, S represents the net sources or sinks
(physical, chemical, and biological) of C, and the overline
represents time averaging over turbulent scales. Atmos. Chem. Phys., 18, 17437–17450, 2018 3.3.2
Case study The six terms
in this equation represent the following (left to right): local
rate of change (N1), horizontal advection by the mean ve-
locity (N2), vertical advection by the mean velocity (N3),
net sources or sinks (N4), net vertical transport by the ver-
tical turbulent flux gradient (N5), and net horizontal trans-
port by the horizontal turbulent flux gradient (N6). Generally,
|N6| ≪|N5| and is hereafter ignored in the discussion. A previous study by Alekseychik et al. (2013) at the
SMEAR II station showed that nocturnal decoupled air lay-
ers were frequently (with a fraction of 18.6 % based on a
long-term dataset) observed under high-Ri conditions in the
boreal forest. The decoupled layer could strongly influence
the ground O3, MT, and CO2 concentrations (Rannik et al., During the non-inversion night, the ground O3 could be re-
plenished by vertical turbulent transport (N5), mean vertical
advection from the upper boundary layer (N3), or horizon-
tal advection below the canopy (N2) (as shown in Fig. 6). However, for highly stratified flows, N5 becomes small, as www.atmos-chem-phys.net/18/17437/2018/ Atmos. Chem. Phys., 18, 17437–17450, 2018 17444 Q. Zha et al.: Vertical characterization of HOMs below and above a boreal forest canopy Q
py
Figure 5. (a) Time series of ground and tower concentrations of CO2, NOx, O3, MT, and selected HOM groups in the selected non-inversion
night (11 September) and (b) inversion night (8 September) types. Ri is calculated with the meteorology data of the ground and tower levels. CS is determined based on the aerosol data measured at 8 m above ground level. Figure 5. (a) Time series of ground and tower concentrations of CO2, NOx, O3, MT, and selected HOM groups in the selected non-inversion
i h (11 S
b )
d (b) i
i
i h (8 S
b )
Ri i
l
l
d
i h h
l
d
f h
d
d
l
l Figure 5. (a) Time series of ground and tower concentrations of CO2, NOx, O3, MT, and selected HOM groups in the selected non-inversion
night (11 September) and (b) inversion night (8 September) types. Ri is calculated with the meteorology data of the ground and tower levels. CS is determined based on the aerosol data measured at 8 m above ground level. forming and eventually reached a much lower concentration. Q. Zha et al.: Vertical characterization of HOMs below and above a boreal forest canopy Q. Zha et al.: Vertical characterization of HOMs below and above a boreal forest canopy Q. a et a .: Ve t ca c a acte
at o
o
O
s be ow a d above a bo ea
o est ca opy
Figure 6. Schematic figure showing how vertical mixing, vertical advection, and horizontal advection influence ground-level O3 concentra-
tions differently on non-inversion nights and inversion nights at the SMEAR II station. Figure 6. Schematic figure showing how vertical mixing, vertical advection, and horizontal advection influence ground-level O3 concentra-
tions differently on non-inversion nights and inversion nights at the SMEAR II station. wing how vertical mixing, vertical advection, and horizontal advection influence ground-level O3 concentra-
on nights and inversion nights at the SMEAR II station. between the two measurement heights. Moreover, a signifi-
cant fraction of the ground HOMs disappeared on the inver-
sion night, and the concentrations of the remaining HOMs
were also lower, confirming the aforementioned results ob-
tained with the selected HOM groups. no longer balanced by a large flux of O3 from upper layers. However, the stabilization of ground-level O3 concentrations
at nonzero values after the initial fast decrease suggests that
a small amount of inflow, either via N3 or N5, is still taking
place. Therefore, the differences between the ground and tower
measurements were due to the joint effects of (i) decou-
pling between the stably stratified near-ground layer and the
canopy top and the consequent formation of a shallow layer,
(ii) weakening of advective and turbulent flux transport terms
thereby inhibiting mass exchange between the ground decou-
pled layer and the remaining nocturnal boundary layer, and
(iii) an increased surface area-to-volume ratio within the de-
coupled layer thereby enhancing N4. 3.3.2
Case study The decoupled layer also affected MT and CO2 below the
canopy in the inversion night but resulted in concentration
increases as opposed to O3. The weakened vertical turbu-
lence (N5) tended to retain the emissions from ground and
understory vegetation within the layer, though N4 also in-
creased. In general, the increased CO2 (primary source from
the ground) and MT (primary source from the canopy) at
ground level are good indicators for the extent of the mix-
ing in the shallow decoupled layer. At the same time, the
strong decrease in O3 shows how the sinks in this layer are the efficiency of turbulence to transport O3 to layers near the
ground becomes weak (Katul et al., 2014). Vertical and hor-
izontal advection were also small within such a stable layer,
and the reduced mean velocity would result in smaller con-
tributions from N2 and N3. Note that these advective terms
tend to be opposite in sign by the virtue of the mean fluid
continuity equation (Katul et al., 2006). Instead, the sink of
O3 (N4) was stronger because of the increasing loss due to
a higher surface area-to-volume density (S/V ) in this shal-
low decoupled layer. Under this circumstance, the ground O3
concentration dramatically decreased when the air layer was www.atmos-chem-phys.net/18/17437/2018/ Atmos. Chem. Phys., 18, 17437–17450, 2018 17445 Q. Zha et al.: Vertical characterization of HOMs below and above a boreal forest canopy Figure 7. Mass defect (MD) plots of the selected (a) non-inversion night case (11 September) and (b) inversion night case (8 September). Color code indicates the ratios between tower and ground HOM concentrations. The grey shaded area denotes the dimer range (m/z 450–
600 Th). Figure 8. Hourly changes of the ratios between estimated tower and ground HOM concentrations from 12:00 on 11 September to 11:00 on
12 September (non-inversion night). Markers are sized by ground HOM concentrations and colored by the O3 difference between the tower
and ground (O3tower −O3ground). Hourly ambient temperatures at ground (Tg) and tower (Tt) levels and RH at ground (RHg) and tower (RHt)
levels are shown in each subplot. Figure 8. Hourly changes of the ratios between estimated tower and ground HOM concentrations from 12:00 on 11 September to 11:00 on
12 September (non-inversion night). Markers are sized by ground HOM concentrations and colored by the O3 difference between the tower
and ground (O3tower −O3ground). Hourly ambient temperatures at ground (Tg) and tower (Tt) levels and RH at ground (RHg) and tower (RHt)
levels are shown in each subplot. two instruments throughout the night, despite large variabil-
ity in temperatures and RHs. in HOMs were observed only when the ground temperature
was lower than the tower temperature and when the ozone
concentration at ground level was several parts per billion
lower than at the tower (shown as a color scale in Figs. 8 and
9). Absolute temperatures or RHs at the two heights were
not able to explain the changes. As a concrete example, good
agreement was observed at 07:00 on 12 September, while
ambient temperatures were low (ground and tower temper-
atures were 9.3 and 8.6 ◦C, respectively) and RHs were
high (ground and tower RHs were 92 % and 96 %, respec-
tively), but large deviations were found at 20:00, 8 Septem-
ber, when higher temperatures (ground and tower tempera-
tures were 10.2 and 12.1 ◦C, respectively) and lower RHs
(ground and tower RHs were 88 % and 76 %, respectively)
were observed. In other words, neither low temperatures nor
high RHs caused large changes to our instruments. Instead, In contrast, during a 24h period with nighttime temper-
ature inversion (8 September, shown in Fig. 9), the ratios
agreed well only during daytime (from 12:00 to 17:00, and
09:00 to 11:00 on the next day). 3.4
Study limitations Several limitations still exist in this study. From the mea-
surement side, one major concern was the comparability be-
tween our two CI-APi-TOF mass spectrometers. In the worst
case, our conclusion might be biased if instrument responses
changed due to some parameter that correlated with the ob-
served inversions. The main parameters in this case would
be ambient temperature and RH. As both instruments were
located in temperature-controlled containers and the sample
flow was mixed 1 : 2 with dry sheath air in the CI-APi-TOF
drift tube, neither of these were expected to yield such large
changes. However, for confirmation, we compared the de-
tailed spectral evolution during days and nights of the study. Figure 8 shows an example of hourly changes of the ratios
between tower and ground HOMs, over a 24 h period without
nighttime temperature inversion (11 September). During this
period, ambient temperatures changed from 19.1 ◦C (12:00)
to 8.8 ◦C (07:00) at ground level, and from 17.9 to 8.1 ◦C at
tower level. Ambient RHs also increased from 72 % to 96 %
at ground level, and from 74 % to 98 % at tower level. While
some scatter is visible in the 200–300 Th range during some
parts of the night, good agreement was observed between the Examination of the selected HOMs was useful and effi-
cient to assess the changes in HOMs; however, such an analy-
sis might only indicate the major formation pathways. Hence,
it was also worthwhile to have a holistic view of the entire
mass spectra and all the detected HOMs. The mass defect
(MD) plot (Fig. 7) separates all identified compounds accord-
ing to their exact masses on the x axis and the deviation from
the integer mass on the y axis. Each circle represents a com-
pound, with the areas scaled by concentrations and colored
by the ratios between tower and ground concentrations. Fig-
ure 7a and b are MD plots showing the mean spectra of the
selected non-inversion night (11 September) and inversion
night (8 September), respectively. Without the formation of a
decoupled layer, nearly the same concentration distributions
of HOMs were observed. In contrast, during the inversion
night (8 September, Fig. 7b), large differences could be found www.atmos-chem-phys.net/18/17437/2018/ Atmos. Chem. Phys., 18, 17437–17450, 2018 17446
Q. Zha et al.: Vertical characterization of HOMs below and above a boreal forest canopy
Figure 7. 3.4
Study limitations Mass defect (MD) plots of the selected (a) non-inversion night case (11 September) and (b) inversion night case (8 September). Color code indicates the ratios between tower and ground HOM concentrations. The grey shaded area denotes the dimer range (m/z 450–
600 Th). 17446 Q. Zha et al.: Vertical characterization of HOMs below and above a boreal forest canopy Hourly changes of the ratios between estimated tower and ground HOM concentrations from 12:00 on 8 September to 11:00 on
9 September (inversion night). Markers are sized by ground HOM concentrations and colored by the O3 difference between tower and ground
(O3tower −O3ground). Hourly ambient temperatures at ground (Tg) and tower (Tt) levels and RH at ground (RHg) and tower (RHt) levels are
shown in each subplot. the large discrepancies between the two CI-APi-TOFs were
only observed when other key parameters (like ozone) were
found to deviate considerably between the two heights. out strong inversion), HOM concentrations and other mea-
sured species were overall similar between the ground and
tower measurements. In contrast, much lower ground-level
HOM concentrations were observed when nighttime temper-
ature inversion and formation of a decoupled layer occurred
below the canopy. On the one hand, the production of the
ground-level HOMs could be affected by the decreasing O3
concentrations and the increasing MT concentration in the
shallow layer. On the other hand, the surface area-to-volume
ratio dramatically increased in the shallow layer compared
to the nocturnal boundary layer. The possibility of losses on
surfaces for ground-level HOMs became much larger than
usual during inversion nights. The enhanced interaction of
air in the decoupled layer with the forest floor was supported
by increased concentrations of CO2, emitted mainly from the
ground, in this layer. From the micrometeorology side, the contribution from
the potential micrometeorological processes in the layer be-
tween 1.5 and 4.2 m (between the sampling heights of the
ground HOMs and other parameters) could not be estimated
with the current experiment design (i.e., only two measure-
ment heights). Similarly, the influence from horizontal ad-
vection could not be entirely ruled out as a reason for the
reduced ground-level HOM concentrations (and other signif-
icantly changed species) because of the possible horizontal
inhomogeneity of HOM precursors and oxidants below the
canopy. However, our conclusion was confirmed by the in-
compatibility between the increasing ground-level MT and
CO2 concentrations and the advection hypothesis (i.e., all
species would show similar tendencies if advection played
a major role), indicating the influence of horizontal and ver-
tical advection is probably minor when compared to the in-
creasing sink. However, conclusive evidence is still needed,
which highlights the need for joint vertical–planar HOM
studies, measuring both vertical and horizontal distribution
of HOM concentrations. Q. Zha et al.: Vertical characterization of HOMs below and above a boreal forest canopy Between these two peri-
ods, temperature and RH were most of the time in the same
range as on 11 September (when no strong deviations were
observed), but now the HOM behavior changed dramatically
between the two heights. The ratios increased from ∼1 (dur-
ing daytime) up to ∼20 at 07:00 for some of the measured
molecules. Figures 8 and 9 clearly imply that the large differences be-
tween ground and tower HOM concentrations were driven by
temperature inversions and consequent changes in the com-
position of the air in the two detached layers. Large changes Atmos. Chem. Phys., 18, 17437–17450, 2018 www.atmos-chem-phys.net/18/17437/2018/ Q. Zha et al.: Vertical characterization of HOMs below and above a boreal forest canopy
17447
20
15
10
5
0
20
15
10
5
0
20
15
10
5
0
20
15
10
5
0 200
300
400
500
600 200
300
400
500 600 200
300
400
500 600 200
300
400
500
600 200
300
400
500 600 200
300
400
500 600
13:00
Tg=15.7°C, Tt=14.6°C
RHg=59 %, RHt=61%
14:00
Tg=15.9 °C, Tt=14.9°C
RHg=58%, RHt=61%
15:00
Tg=15.7°C, Tt=14.9°C
RHg=60%, RHt=60%
16:00
Tg=15.3°C, Tt=14.8°C
RHg=63%, RHt=61%
17:00
Tg=15.3°C, Tt=14.8°C
RHg=62%, RHt=62%
12:00
Tg=15.6°C, Tt=14.5°C
RHg=61%, RHt=63%
19:00
Tg=11.3°C, Tt=13.0°C
RHg=83%, RHt=70%
20:00
Tg=10.2°C, Tt=12.1°C
RHg=88%, RHt=76%
21:00
Tg=10.1°C, Tt=11.3°C
RHg=89%, RHt=81%
22:00
Tg=10.1°C, Tt=10.5°C
RHg=88%, RHt=85%
23:00
Tg=9.2°C, Tt=9.6°C
RHg=93%, RHt=90%
18:00
Tg=13.8°C, Tt=13.9°C
RHg=69%, RHt=66%
01:00
Tg=6.2°C, Tt=8.9 °C
RHg=99%, RHt=89%
02:00
Tg=5.5°C, Tt=8.2°C
RHg=99%, RHt=92%
03:00
Tg=5.1°C, Tt=7.6°C
RHg=100%, RHt=95%
04:00
Tg=5.1°C, Tt=7.0°C
RHg=99 %, RHt=99%
05:00
Tg=4.6°C, Tt=7.1°C
RHg=100 %, RHt=97%
00:00
Tg=7.0°C, Tt=9.1°C
RHg=97%, RHt=86%
07:00
Tg=6.3°C, Tt=6.5°C
RHg=99%, RHt=100%
08:00
Tg=8.5°C, Tt=7.9 °C
RHg=92%, RHt=95 %
09:00
Tg=10.4°C, Tt=9.3°C
RHg=85%, RHt=90 %
10:00
Tg=12.0°C, Tt=11.0 °C
RHg=76%, RHt=79%
11:00
Tg=13.3°C, Tt=12.1°C
RHg=65%, RHt=69%
06:00
Tg=4.7°C, Tt=6.1°C
RHg=99%, RHt=100%
Figure 9. Hourly changes of the ratios between estimated tower and ground HOM concentrations from 12:00 on 8 September to 11:00 on
9 September (inversion night). Markers are sized by ground HOM concentrations and colored by the O3 difference between tower and ground
(O3tower −O3ground). Hourly ambient temperatures at ground (Tg) and tower (Tt) levels and RH at ground (RHg) and tower (RHt) levels are
shown in each subplot. . Zha et al.: Vertical characterization of HOMs below and above a boreal forest canopy Q. Zha et al.: Vertical characterization of HOMs below and above a boreal forest canopy 17447 Figure 9. Q. Zha et al.: Vertical characterization of HOMs below and above a boreal forest canopy We have presented the first detailed measurements of
HOMs below and above the canopy across a wide range of at-
mospheric stability conditions. The results highlight the sig-
nificance of near-ground boundary layer dynamics and mi-
crometeorological processes on the ambient HOMs, show-
ing that ground-based HOM measurement at this site might
not be representative for the entire nocturnal boundary layer. Conventionally, field measurements of HOMs and other pa-
rameters are mostly performed close to the ground, and the
possible effect of boundary layer dynamics and micromete-
orological processes to the HOM concentrations has rarely
been considered. Aerosol particle growth and SOA forma-
tion rates at ground level are likely to be influenced by the
reduced HOM concentrations on the inversion nights. How-
ever, there are still limitations due to current experiment de-
sign, such as horizontal separation in instrument setup, or the
uncertainties from using point measurements at two heights
to infer larger-scale exchange. Clearly, more vertical and
planar measurements of HOMs are needed to confirm the 4
Conclusion Highly oxygenated molecules (HOMs) were measured above
the canopy and at ground level (below the canopy) in a bo-
real forest environment during the IBAIRN campaign that
took place in September 2016. Boundary layer dynamics and
micrometeorology were found to be important factors that
influence the abundance of and trends in HOMs at ground
level, by perturbing both their sources and sinks. In the well-
mixed boundary layer (e.g., during daytime or nights with- www.atmos-chem-phys.net/18/17437/2018/ Q. Zha et al.: Vertical characterization of HOMs below and above a boreal forest canopy Q. Zha et al.: Vertical characterization of HOMs below and above a boreal forest canopy 17448 emerging picture presented here. Influence of boundary layer
dynamics should be better characterized and evaluated in fu-
ture field campaigns. in a pine forest canopy, Agr. Forest. Meteorol., 174, 15–27,
https://doi.org/10.1016/j.agrformet.2013.01.011, 2013. in a pine forest canopy, Agr. Forest. Meteorol., 174, 15–27,
https://doi.org/10.1016/j.agrformet.2013.01.011, 2013. Bäck, J., Aalto, J., Henriksson, M., Hakola, H., He, Q., and
Boy, M.: Chemodiversity of a Scots pine stand and implica-
tions for terpene air concentrations, Biogeosciences, 9, 689–702,
https://doi.org/10.5194/bg-9-689-2012, 2012. Data availability. The time series of the measured trace gases,
meteorological parameters, and estimated total HOM concentra-
tions at the ground and tower levels will be available at the end
of December 2018 (https://doi.org/10.5281/zenodo.1489079, Zha,
2018). For other data please contact the first author via email
(qiaozhi.zha@helsinki.fi). Berndt, T., Scholz, W., Mentler, B., Fischer, L., Herrmann,
H., Kulmala, M., and Hansel, A.: Accretion Product For-
mation from Self- and Cross-Reactions of RO2 Radicals in
the Atmosphere, Angew. Chem. Int. Edit., 57, 3820–3824,
https://doi.org/10.1002/anie.201710989, 2018. p
g
Bianchi, F., Tröstl, J., Junninen, H., Frege, C., Henne, S., Hoyle,
C. R., Molteni, U., Herrmann, E., Adamov, A., Bukowiecki,
N., Chen, X., Duplissy, J., Gysel, M., Hutterli, M., Kangaslu-
oma, J., Kontkanen, J., Kürten, A., Manninen, H. E., Münch, S.,
Peräkylä, O., Petäjä, T., Rondo, L., Williamson, C., Weingart-
ner, E., Curtius, J., Worsnop, D. R., Kulmala, M., Dommen, J.,
and Baltensperger, U.: New particle formation in the free tropo-
sphere: A question of chemistry and timing, Science, 352, 1109–
12, https://doi.org/10.1126/science.aad5456, 2016. Supplement. The supplement related to this article is available
online at: https://doi.org/10.5194/acp-18-17437-2018-supplement. Author contributions. QZ, MR, and ME designed the study. QZ,
CY, HJ, MR, NS, JA, and SS collected the data. QZ and CY an-
alyzed the data. QZ wrote the paper. All coauthors contributed to
data interpretation and commented on the paper. Bianchi, F., Garmash, O., He, X., Yan, C., Iyer, S., Rosendahl, I.,
Xu, Z., Rissanen, M. P., Riva, M., Taipale, R., Sarnela, N., Petäjä,
T., Worsnop, D. R., Kulmala, M., Ehn, M., and Junninen, H.: The
role of highly oxygenated molecules (HOMs) in determining the
composition of ambient ions in the boreal forest, Atmos. Chem. Phys., 17, 13819–13831, https://doi.org/10.5194/acp-17-13819-
2017, 2017. Competing interests. The authors declare that they have no con-
flict of interest. Douglas R. Q. Zha et al.: Vertical characterization of HOMs below and above a boreal forest canopy Worsnop is affiliated with Aerodyne
Research, who produce the CI-APi-TOF instruments used in this
study. Chen, X., Quéléver, L. L. J., Fung, P. L., Kesti, J., Rissanen, M. P., Bäck, J., Keronen, P., Junninen, H., Petäjä, T., Kerminen, V.-
M., and Kulmala, M.: Observations of ozone depletion events
in a Finnish boreal forest, Atmos. Chem. Phys., 18, 49–63,
https://doi.org/10.5194/acp-18-49-2018, 2018. Acknowledgements. This work was supported by the IBAIRN
project, the Academy of Finland Center of Excellence in Atmo-
spheric Science, European commission Actris2 and Actris PPP, the
European Research Council (grant 638703-COALA), transnational
access from ENVRI plus, and the SMEAR II technical team. Qiaozhi Zha thanks ATM-DP (Doctoral Program in Atmospheric
Sciences) graduate programs, John Crowley and the Max Plank
Institute in association with the IBAIRN proposal, and the
tofTools team for providing tools for mass spectrometry analysis. Gabriel Katul acknowledges the support from the US National
Science Foundation (NSF-EAR-1344703, NSF-AGS-1644382), the
U.S. Department of Energy (DE-SC0011461), and the University of
Helsinki for supporting a 3-month sabbatical leave at the Division
of Atmospheric Sciences. Otso Peräkylä thanks the Vilho,
Yrjö
and Kalle Väisälä Foundation. Crounse, J. D., Nielsen, L. B., Jørgensen, S., Kjaergaard, H. G., and Wennberg, P. O.: Autoxidation of Organic Com-
pounds in the Atmosphere, J. Phys. Chem. Lett., 4, 3513–3520,
https://doi.org/10.1021/jz4019207, 2013. Dada, L., Paasonen, P., Nieminen, T., Buenrostro Mazon, S., Kon-
tkanen, J., Peräkylä, O., Lehtipalo, K., Hussein, T., Petäjä,
T., Kerminen, V.-M., Bäck, J., and Kulmala, M.: Long-term
analysis of clear-sky new particle formation events and non-
events in Hyytiälä, Atmos. Chem. Phys., 17, 6227–6241,
https://doi.org/10.5194/acp-17-6227-2017, 2017. Eerdekens, G., Yassaa, N., Sinha, V., Aalto, P. P., Aufmhoff, H.,
Arnold, F., Fiedler, V., Kulmala, M., and Williams, J.: VOC mea-
surements within a boreal forest during spring 2005: on the oc-
currence of elevated monoterpene concentrations during night
time intense particle concentration events, Atmos. Chem. Phys.,
9, 8331–8350, https://doi.org/10.5194/acp-9-8331-2009, 2009. Edited by: Kyung-Eun Min
Reviewed by: two anonymous referees Edited by: Kyung-Eun Min
Reviewed by: two anonymous referees Ehn, M., Junninen, H., Petäjä, T., Kurtén, T., Kerminen, V.-M.,
Schobesberger, S., Manninen, H. E., Ortega, I. K., Vehkamäki,
H., Kulmala, M., and Worsnop, D. R.: Composition and tem-
poral behavior of ambient ions in the boreal forest, Atmos. Chem. Phys., 10, 8513–8530, https://doi.org/10.5194/acp-10-
8513-2010, 2010. www.atmos-chem-phys.net/18/17437/2018/ Atmos. Chem. Phys., 18, 17437–17450, 2018 Q. Zha et al.: Vertical characterization of HOMs below and above a boreal forest canopy Q. Zha et al.: Vertical characterization of HOMs below and above a boreal forest canopy E., Seinfeld, J. H., Sipilä, M., Steiner, G., Stozhkov, Y., Stratmann, F., Tomé, A., Virtanen, A., Vogel, A. L., Wag-
ner, A. C., Wagner, P. E., Weingartner, E., Wimmer, D., Winkler, Hari, P. and Kulmala, M.: Station for Measuring Ecosystem-
Atmosphere Relations (SMEAR II), Boreal Environ. Res., 10,
315–322, 2005. P. M., Ye, P., Zhang, X., Hansel, A., Dommen, J., Donahue, N. M., Worsnop, D. R., Baltensperger, U., Kulmala, M., Carslaw, K. S., and Curtius, J.: Ion-induced nucleation of pure biogenic parti-
cles, Nature, 533, 521–526, https://doi.org/10.1038/nature17953,
2016. Hari, P., Nikinmaa, E., Pohja, T., Siivola, E., Bäck, J., Vesala, T.,
and Kulmala, M.: Station for measuring ecosystem-atmosphere
relations: SMEAR, Phys. Physiol. For. Ecol., 9789400756, 471–
487, https://doi.org/10.1007/978-94-007-5603-8_9, 2013. Kulmala, L., Launiainen, S., Pumpanen, J., Lankreijer, H., Lin-
droth, A., Hari, P., and Vesala, T.: H2O and CO2 fluxes at
the floor of a boreal pine forest, Tellus B, 60B, 167–178,
https://doi.org/10.1111/j.1600-0889.2007.00327.x, 2008. Heinritzi, M., Simon, M., Steiner, G., Wagner, A. C., Kürten,
A., Hansel, A., and Curtius, J.: Characterization of the mass-
dependent transmission efficiency of a CIMS, Atmos. Meas. Tech., 9, 1449–1460, https://doi.org/10.5194/amt-9-1449-2016,
2016. Kulmala,
M.,
Dal
Maso,
M.,
Mäkelä,
J. M.,
Pirjola, L.,
Väkevä, M., Aalto, P., Miikkulainen, P., Hämeri, K., and
O’Dowd, C. D.: On the formation, growth and composi-
tion of nucleation mode particles, Tellus B, 53, 479–490,
https://doi.org/10.3402/tellusb.v53i4.16622, 2001. Hyttinen, N., Kupiainen-Määttä, O., Rissanen, M. P., Muuro-
nen, M., Ehn, M., and Kurtén, T.: Modeling the Charging
of Highly Oxidized Cyclohexene Ozonolysis Products Using
Nitrate-Based Chemical Ionization, J. Phys. Chem. A, 119,
6339–6345, https://doi.org/10.1021/acs.jpca.5b01818, 2015. p
g
Kulmala, M., Kontkanen, J., Junninen, H., Lehtipalo, K., Manni-
nen, H. E., Nieminen, T., Petäjä, T., Sipilä, M., Schobesberger,
S., Rantala, P., Franchin, A., Jokinen, T., Järvinen, E., Äijälä, M.,
Kangasluoma, J., Hakala, J., Aalto, P. P., Paasonen, P., Mikkilä,
J., Vanhanen, J., Aalto, J., Hakola, H., Makkonen, U., Ruuska-
nen, T., Mauldin, R. L., Duplissy, J., Vehkamäki, H., Bäck,
J., Kortelainen, A., Riipinen, I., Kurtén, T., Johnston, M. V,
Smith, J. N., Ehn, M., Mentel, T. F., Lehtinen, K. E. J., Laakso-
nen, A., Kerminen, V.-M., and Worsnop, D. R.: Direct observa-
tions of atmospheric aerosol nucleation., Science, 339, 943–946,
https://doi.org/10.1126/science.1227385, 2013. Jokinen, T., Sipilä, M., Junninen, H., Ehn, M., Lönn, G., Hakala,
J., Petäjä, T., Mauldin III, R. L., Kulmala, M., and Worsnop,
D. R.: Atmospheric sulphuric acid and neutral cluster measure-
ments using CI-APi-TOF, Atmos. References Aalto, P., Hämeri, K., Becker, E., Weber, R., Salm, J., Mäkelä, J. M.,
Hoell, C., O’dowd, C. D., Hansson, H.-C., Väkevä, M., Koponen,
I. K., Buzorius, G., and Kulmala, M.: Physical characterization
of aerosol particles during nucleation events, Tellus B, 53, 344–
358, https://doi.org/10.3402/tellusb.v53i4.17127, 2001. Aalto, P., Hämeri, K., Becker, E., Weber, R., Salm, J., Mäkelä, J. M.,
Hoell, C., O’dowd, C. D., Hansson, H.-C., Väkevä, M., Koponen,
I. K., Buzorius, G., and Kulmala, M.: Physical characterization
of aerosol particles during nucleation events, Tellus B, 53, 344–
358, https://doi.org/10.3402/tellusb.v53i4.17127, 2001. Ehn, M., Kleist, E., Junninen, H., Petäjä, T., Lönn, G., Schobes-
berger, S., Dal Maso, M., Trimborn, A., Kulmala, M., Worsnop,
D. R., Wahner, A., Wildt, J., and Mentel, Th. F.: Gas phase
formation of extremely oxidized pinene reaction products in Alekseychik, P., Mammarella, I., Launiainen, S., Rannik, Ü.,
and Vesala, T.: Evolution of the nocturnal decoupled layer www.atmos-chem-phys.net/18/17437/2018/ Atmos. Chem. Phys., 18, 17437–17450, 2018 Q. Zha et al.: Vertical characterization of HOMs below and above a boreal forest canopy 17449 chamber and ambient air, Atmos. Chem. Phys., 12, 5113–5127,
https://doi.org/10.5194/acp-12-5113-2012, 2012. chamber and ambient air, Atmos. Chem. Phys., 12, 5113–5127,
https://doi.org/10.5194/acp-12-5113-2012, 2012. Katul, G. G., Porporato, A., Shah, S., and Bou-Zeid, E.:
Two phenomenological constants explain similarity laws in
stably
stratified
turbulence,
Phys. Rev. E,
89,
023007,
https://doi.org/10.1103/PhysRevE.89.023007, 2014. Ehn, M., Thornton, J. A., Kleist, E., Sipilä, M., Junninen, H.,
Pullinen, I., Springer, M., Rubach, F., Tillmann, R., Lee, B.,
Lopez-Hilfiker, F., Andres, S., Acir, I.-H., Rissanen, M., Joki-
nen, T., Schobesberger, S., Kangasluoma, J., Kontkanen, J.,
Nieminen, T., Kurtén, T., Nielsen, L. B., Jørgensen, S., Kjaer-
gaard, H. G., Canagaratna, M., Maso, M. D., Berndt, T.,
Petäjä, T., Wahner, A., Kerminen, V.-M., Kulmala, M., Worsnop,
D. R., Wildt, J., and Mentel, T. F.: A large source of low-
volatility secondary organic aerosol, Nature, 506, 476–479,
https://doi.org/10.1038/nature13032, 2014. Kirkby, J., Duplissy, J., Sengupta, K., Frege, C., Gordon, H.,
Williamson, C., Heinritzi, M., Simon, M., Yan, C., Almeida, J.,
Tröstl, J., Nieminen, T., Ortega, I. K., Wagner, R., Adamov, A.,
Amorim, A., Bernhammer, A.-K., Bianchi, F., Breitenlechner,
M., Brilke, S., Chen, X., Craven, J., Dias, A., Ehrhart, S., Flagan,
R. C., Franchin, A., Fuchs, C., Guida, R., Hakala, J., Hoyle, C. R., Jokinen, T., Junninen, H., Kangasluoma, J., Kim, J., Krapf,
M., Kürten, A., Laaksonen, A., Lehtipalo, K., Makhmutov, V.,
Mathot, S., Molteni, U., Onnela, A., Peräkylä, O., Piel, F., Petäjä,
T., Praplan, A. P., Pringle, K., Rap, A., Richards, N. A. D., Riip-
inen, I., Rissanen, M. P., Rondo, L., Sarnela, N., Schobesberger,
S., Scott, C. E., Seinfeld, J. H., Sipilä, M., Steiner, G., Stozhkov,
Y., Stratmann, F., Tomé, A., Virtanen, A., Vogel, A. L., Wag-
ner, A. C., Wagner, P. E., Weingartner, E., Wimmer, D., Winkler,
P. M., Ye, P., Zhang, X., Hansel, A., Dommen, J., Donahue, N. M., Worsnop, D. R., Baltensperger, U., Kulmala, M., Carslaw, K. S., and Curtius, J.: Ion-induced nucleation of pure biogenic parti-
cles, Nature, 533, 521–526, https://doi.org/10.1038/nature17953,
2016. R. C., Franchin, A., Fuchs, C., Guida, R., Hakala, J., Hoyle, C. Ehn, M., Berndt, T., Wildt, J., and Mentel, T.: Highly Oxygenated
Molecules from Atmospheric Autoxidation of Hydrocarbons:
A Prominent Challenge for Chemical Kinetics Studies, Int. J. Chem. Kinet., 49, 821–831, https://doi.org/10.1002/kin.21130,
2017. y
j
T., Praplan, A. P., Pringle, K., Rap, A., Richards, N. A. D., Riip-
inen, I., Rissanen, M. P., Rondo, L., Sarnela, N., Schobesberger,
S., Scott, C. Atmos. Chem. Phys., 18, 17437–17450, 2018 Q. Zha et al.: Vertical characterization of HOMs below and above a boreal forest canopy Chem. Phys., 12, 4117–4125,
https://doi.org/10.5194/acp-12-4117-2012, 2012. Jokinen, T., Sipilä, M., Richters, S., Kerminen, V.-M., Paasonen, P.,
Stratmann, F., Worsnop, D., Kulmala, M., Ehn, M., Herrmann,
H., and Berndt, T.: Rapid Autoxidation Forms Highly Oxidized
RO2 Radicals in the Atmosphere, Angew. Chem. Int. Edit., 53,
14596–14600, https://doi.org/10.1002/anie.201408566, 2014. Kürten, A., Bergen, A., Heinritzi, M., Leiminger, M., Lorenz, V.,
Piel, F., Simon, M., Sitals, R., Wagner, A. C., and Curtius, J.: Ob-
servation of new particle formation and measurement of sulfuric
acid, ammonia, amines and highly oxidized organic molecules at
a rural site in central Germany, Atmos. Chem. Phys., 16, 12793–
12813, https://doi.org/10.5194/acp-16-12793-2016, 2016. Jokinen, T., Kausiala, O., Garmash, O., Peräkylä, O., Junninen, H.,
Schobesberger, S., Yan, C., Sipilä, M., and Rissanen, M. P.: Pro-
duction of highly oxidized organic compounds from ozonolysis
of β-caryophyllene: laboratory and field measurements, Boreal
Environ. Res., 21, 262–273, 2016. Junninen, H., Ehn, M., Petäjä, T., Luosujärvi, L., Kotiaho, T., Kos-
tiainen, R., Rohner, U., Gonin, M., Fuhrer, K., Kulmala, M., and
Worsnop, D. R.: A high-resolution mass spectrometer to mea-
sure atmospheric ion composition, Atmos. Meas. Tech., 3, 1039–
1053, https://doi.org/10.5194/amt-3-1039-2010, 2010. Launiainen, S., Katul, G. G., Grönholm, T., and Vesala, T.: Parti-
tioning ozone fluxes between canopy and forest floor by mea-
surements and a multi-layer model, Agr. Forest. Meteorol., 173,
85–99, https://doi.org/10.1016/j.agrformet.2012.12.009, 2013. Lauros, J., Nilsson, E. D., Dal Maso, M., and Kulmala, M.:
Contribution of mixing in the ABL to new particle formation
based on observations, Atmos. Chem. Phys., 7, 4781–4792,
https://doi.org/10.5194/acp-7-4781-2007, 2007. Katul, G. G., Finnigan, J. J., Poggi, D., Leuning, R., and Belcher, S. E.: The Influence of Hilly Terrain on Canopy-Atmosphere Car-
bon Dioxide Exchange, Bound.-Lay. Meteorol., 118, 189–216,
https://doi.org/10.1007/s10546-005-6436-2, 2006. Atmos. Chem. Phys., 18, 17437–17450, 2018 www.atmos-chem-phys.net/18/17437/2018/ Q. Zha et al.: Vertical characterization of HOMs below and above a boreal forest canopy 17450 Mahrt, L.: Nocturnal Boundary-Layer Regimes, Bound.-Lay. Me-
teorol., 88, 255–278, https://doi.org/10.1023/A:1001171313493,
1998. Taipale, R., Ruuskanen, T. M., Rinne, J., Kajos, M. K., Hakola,
H., Pohja, T., and Kulmala, M.: Technical Note: Quanti-
tative long-term measurements of VOC concentrations by
PTR-MS – measurement, calibration, and volume mixing ra-
tio calculation methods, Atmos. Chem. Phys., 8, 6681–6698,
https://doi.org/10.5194/acp-8-6681-2008, 2008. Mahrt,
L.,
Vickers,
D.,
Nakamura,
R.,
Soler,
M. R.,
Sun,
J.,
Burns,
S.,
and
Lenschow,
D. H.:
Shallow
Drainage
Flows,
Bound.-Lay. Meteorol.,
101,
243–260,
https://doi.org/10.1023/A:1019273314378, 2001. p
g
p
Tröstl, J., Chuang, W. K., Gordon, H., Heinritzi, M., Yan, C.,
Molteni, U., Ahlm, L., Frege, C., Bianchi, F., Wagner, R., Si-
mon, M., Lehtipalo, K., Williamson, C., Craven, J. S., Du-
plissy, J., Adamov, A., Almeida, J., Bernhammer, A.-K., Bre-
itenlechner, M., Brilke, S., Dias, A., Ehrhart, S., Flagan, R. C., Franchin, A., Fuchs, C., Guida, R., Gysel, M., Hansel, A.,
Hoyle, C. R., Jokinen, T., Junninen, H., Kangasluoma, J., Kesk-
inen, H., Kim, J., Krapf, M., Kürten, A., Laaksonen, A., Lawler,
M., Leiminger, M., Mathot, S., Möhler, O., Nieminen, T., On-
nela, A., Petäjä, T., Piel, F. M., Miettinen, P., Rissanen, M. P.,
Rondo, L., Sarnela, N., Schobesberger, S., Sengupta, K., Sip-
ilä, M., Smith, J. N., Steiner, G., Tomè, A., Virtanen, A., Wag-
ner, A. C., Weingartner, E., Wimmer, D., Winkler, P. M., Ye, P.,
Carslaw, K. S., Curtius, J., Dommen, J., Kirkby, J., Kulmala,
M., Riipinen, I., Worsnop, D. R., Donahue, N. M., and Bal-
tensperger, U.: The role of low-volatility organic compounds in
initial particle growth in the atmosphere, Nature, 533, 527–531,
https://doi.org/10.1038/nature18271, 2016. Mammarella, I., Kolari, P., Rinne, J., Keronen, P., Pumpanen, J., and
Vesala, T.: Determining the contribution of vertical advection to
the net ecosystem exchange at Hyytiälä forest, Finland, Tellus B,
59, 900–909, https://doi.org/10.1111/j.1600-0889.2007.00306.x,
2007. Mentel, T. F., Springer, M., Ehn, M., Kleist, E., Pullinen, I., Kurtén,
T., Rissanen, M., Wahner, A., and Wildt, J.: Formation of highly
oxidized multifunctional compounds: autoxidation of peroxy
radicals formed in the ozonolysis of alkenes – deduced from
structure–product relationships, Atmos. Chem. Phys., 15, 6745–
6765, https://doi.org/10.5194/acp-15-6745-2015, 2015. Molteni, U., Bianchi, F., Klein, F., El Haddad, I., Frege, C., Rossi,
M. J., Dommen, J., and Baltensperger, U.: Formation of highly
oxygenated organic molecules from aromatic compounds, At-
mos. Chem. Phys., 18, 1909–1921, https://doi.org/10.5194/acp-
18-1909-2018, 2018. Orlando, J. J. and Tyndall, G. Q. Zha et al.: Vertical characterization of HOMs below and above a boreal forest canopy S.: Laboratory studies of organic
peroxy radical chemistry: an overview with emphasis on recent
issues of atmospheric significance, Chem. Soc. Rev., 41, 6294,
https://doi.org/10.1039/c2cs35166h, 2012. Vickers,
D.,
Irvine,
J.,
Martin,
J. G.,
and
Law,
B. E.:
Nocturnal
subcanopy
flow
regimes
and
missing
car-
bon
dioxide,
Agr. Forest. Meteorol.,
152,
101–108,
https://doi.org/10.1016/j.agrformet.2011.09.004, 2012. Ouwersloot, H. G., Vilà-Guerau de Arellano, J., Nölscher, A. C.,
Krol, M. C., Ganzeveld, L. N., Breitenberger, C., Mammarella,
I., Williams, J., and Lelieveld, J.: Characterization of a boreal
convective boundary layer and its impact on atmospheric chem-
istry during HUMPPA-COPEC-2010, Atmos. Chem. Phys., 12,
9335–9353, https://doi.org/10.5194/acp-12-9335-2012, 2012. Viggiano, A. A., Seeley, J. V., Mundis, P. L., Williamson, J. S., and
Morris, R. A.: Rate Constants for the Reactions of XO3−(H2O)n
(X = C, HC, and N) and NO3-(HNO3)n with H2SO4: Implica-
tions for Atmospheric Detection of H2SO4, J. Phys. Chem. A,
101, 8275–8278, https://doi.org/10.1021/jp971768h, 1997. ,
,
p
g
jp
,
Yan, C., Nie, W., Äijälä, M., Rissanen, M. P., Canagaratna, M. R., Massoli, P., Junninen, H., Jokinen, T., Sarnela, N., Häme,
S. A. K., Schobesberger, S., Canonaco, F., Yao, L., Prévôt, A. S. H., Petäjä, T., Kulmala, M., Sipilä, M., Worsnop, D. R., and
Ehn, M.: Source characterization of highly oxidized multifunc-
tional compounds in a boreal forest environment using posi-
tive matrix factorization, Atmos. Chem. Phys., 16, 12715–12731,
https://doi.org/10.5194/acp-16-12715-2016, 2016. Rannik, Ü., Mammarella, I., Keronen, P., and Vesala, T.: Ver-
tical advection and nocturnal deposition of ozone over a
boreal pine forest, Atmos. Chem. Phys., 9, 2089–2095,
https://doi.org/10.5194/acp-9-2089-2009, 2009. Rannik, Ü., Altimir, N., Mammarella, I., Bäck, J., Rinne, J., Ru-
uskanen, T. M., Hari, P., Vesala, T., and Kulmala, M.: Ozone
deposition into a boreal forest over a decade of observations:
evaluating deposition partitioning and driving variables, Atmos. Chem. Phys., 12, 12165–12182, https://doi.org/10.5194/acp-12-
12165-2012, 2012. Zha, Q.: Data for “Vertical characterization of highly oxygenated
molecules (HOMs) below and above a boreal forest canopy”,
https://doi.org/10.5281/zenodo.1489079, 2018. Rantala, P., Taipale, R., Kajos, M. K., Patokoski, J., Ruuska-
nen, T. M., Rinne, J., and Aalto, J.: Continuous flux measure-
ments of VOCs using PTR-MS – Reliability and feasibility
of disjunct-eddy-covariance, surface-layer-gradient, and surface-
layer-profile methods, Boreal Environ. Res., 19, 87–107, 2014. Zhao, J., Ortega, J., Chen, M., McMurry, P. H., and Smith, J. N.: De-
pendence of particle nucleation and growth on high-molecular-
weight gas-phase products during ozonolysis of α-pinene, At-
mos. Chem. Q. Zha et al.: Vertical characterization of HOMs below and above a boreal forest canopy Phys., 13, 7631–7644, https://doi.org/10.5194/acp-
13-7631-2013, 2013. Raupach, M. R. and Thom, A. S.: Turbulence in and above
Plant
Canopies,
Annu. Rev. Fluid
Mech.,
13,
97–129,
https://doi.org/10.1146/annurev.fl.13.010181.000525, 1981. Rissanen, M. P., Kurtén, T., Sipilä, M., Thornton, J. A., Kangaslu-
oma, J., Sarnela, N., Junninen, H., Jørgensen, S., Schallhart, S.,
Kajos, M. K., Taipale, R., Springer, M., Mentel, T. F., Ruuska-
nen, T., Petäjä, T., Worsnop, D. R., Kjaergaard, H. G., and Ehn,
M.: The Formation of Highly Oxidized Multifunctional Prod-
ucts in the Ozonolysis of Cyclohexene, J. Am. Chem. Soc., 136,
15596–15606, https://doi.org/10.1021/ja507146s, 2014. Atmos. Chem. Phys., 18, 17437–17450, 2018 www.atmos-chem-phys.net/18/17437/2018/
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In vitro methods in autophagy research: Applications in neurodegenerative diseases and mood disorders
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Frontiers in molecular neuroscience
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TYPE Review
PUBLISHED 12 April 2023
DOI 10.3389/fnmol.2023.1168948 TYPE Review
PUBLISHED 12 April 2023
DOI 10.3389/fnmol.2023.1168948 OPEN ACCESS EDITED BY
Roy Chun-laam NG,
The University of Hong Kong,
Hong Kong SAR, China
REVIEWED BY
Ashok Iyaswamy,
Hong Kong Baptist University,
Hong Kong SAR, China
Marius Baeken,
Okinawa Institute of Science and Technology
Graduate University,
Japan
*CORRESPONDENCE
Dalinda Isabel Sánchez-Vidaña
dalinda-isabel.sanchezvidana@polyu.edu.hk;
dalinda.sanchezvidana@connect.polyu.hk
SPECIALTY SECTION
This article was submitted to
Methods and Model Organisms,
a section of the journal
Frontiers in Molecular Neuroscience
RECEIVED 18 February 2023
ACCEPTED 14 March 2023
PUBLISHED 12 April 2023 EDITED BY
Roy Chun-laam NG,
The University of Hong Kong,
Hong Kong SAR, China Dalinda Isabel Sánchez-Vidaña 1,2*, Jingjing Li 1, Samuel Abokyi 3,
Jackie Ngai-Man Chan 1, Shirley Pui-Ching Ngai 1 and Benson
Wui-Man Lau 1,2 1 Department of Rehabilitation Sciences, Faculty of Health and Social Sciences, The Hong Kong
Polytechnic University, Kowloon, Hong Kong SAR, China, 2 Mental Health Research Centre, The Hong
Kong Polytechnic University, Kowloon, Hong Kong SAR, China, 3 School of Optometry, Faculty of Health
and Social Sciences, The Hong Kong Polytechnic University, Kowloon, Hong Kong SAR, China Background: Autophagy is a conserved physiological intracellular mechanism
responsible for the degradation and recycling of cytoplasmic constituents (e.g.,
damaged organelles, and protein aggregates) to maintain cell homeostasis. Aberrant autophagy has been observed in neurodegenerative diseases, including
Alzheimer’s Disease (AD), Parkinson’s Disease (PD), Amyotrophic Lateral Sclerosis
(ALS), and Huntington’s Disease (HD), and recently aberrant autophagy has been
associated with mood disorders, such as depression. Several in vitro methods
have been developed to study the complex and tightly regulated mechanisms
of autophagy. In vitro methods applied to autophagy research are used to
identify molecular key players involved in dysfunctional autophagy and to screen
autophagy regulators with therapeutic applications in neurological diseases and
mood disorders. Therefore, the aims of this narrative review are (1) to compile
information on the cell-based methods used in autophagy research, (2) to discuss
their application, and (3) to create a catalog of traditional and novel in vitro
methods applied in neurodegenerative diseases and depression. Sánchez-Vidaña DI, Li J, Abokyi S, Chan JN-M,
Ngai SP-C and Lau BW-M (2023) In vitro
methods in autophagy research: Applications in
neurodegenerative diseases and mood
disorders. Front. Mol. Neurosci. 16:1168948. doi: 10.3389/fnmol.2023.1168948 COPYRIGHT
© 2023 Sánchez-Vidaña, Li, Abokyi, Chan, Ngai
and Lau. This is an open-access article
distributed under the terms of the Creative
Commons Attribution License (CC BY). COPYRIGHT
© 2023 Sánchez-Vidaña, Li, Abokyi, Chan, Ngai
and Lau. This is an open-access article
distributed under the terms of the Creative
Commons Attribution License (CC BY). The
use, distribution or reproduction in other
forums is permitted, provided the original
author(s) and the copyright owner(s) are
credited and that the original publication in this
journal is cited, in accordance with accepted
academic practice. No use, distribution or
reproduction is permitted which does not
comply with these terms. Frontiers in Molecular Neuroscience KEYWORDS 1. Introduction Extracellular stress induced by nutrient deprivation, ischemia,
and hypoxia leads to autophagy activation (Nie et al., 2021). Intracellularly, metabolic stress, and the presence of damaged
organelles, unfolded proteins, or protein aggregates also activate
the autophagy machinery (Ni et al., 2013; Saha et al., 2018). The
mechanisms of autophagy are classified according to the mode of
cargo
delivery
to
the
lysosome
into
macroautophagy,
microautophagy, and chaperone-mediated autophagy (CMA)
(Mizushima and Levine, 2020). Autophagy can also be categorized
as selective autophagy in which specific cargo to be degraded is
recognized, and non-selective autophagy in which random
transport of organelles and other components to the lysosome
occurs (Nie et al., 2021). Selective autophagy recognizes specific
cargo (e.g., mitochondria, lipids, protein aggregates) for its
degradation and can be categorized based on the type of cargo to
be degraded (e.g., mitophagy, lipophagy, and aggregophagy)
(Mizushima and Levine, 2020; Nie et al., 2021). CMA does not require the formation of membranous structures
(Nie et al., 2021). In CMA, the cytosolic proteins to be degraded get
translocated across the lysosomal membrane through a molecular
chaperone complex in the cytosol that is associated with the
lysosomal membrane (Dice, 2007; Mizushima and Levine, 2020). The translocation process starts with the recognition of a KFERQ-
like pentapeptide sequence in the substrate protein by the heat
shock protein of 70 kDa (Hsc70) and cochaperones (Dice, 2007;
Mizushima and Komatsu, 2011). The substrate protein and
chaperone complex interact with the lysosomal-associated
membrane protein type 2A (LAMP2A) receptor located on the
lysosomal membrane to be transported into the lysosome (Dice,
2007; Lynch-Day et al., 2012). Macroautophagy has been extensively studied and is
considered the major type of autophagy mechanism (Mizushima
and Komatsu, 2011). A cytosolic double-membrane structure
known as autophagosome is the intermediate organelle in
macroautophagy and is formed from an isolation membrane
known as phagophore (Mizushima and Komatsu, 2011; Bar-Yosef
et al., 2019). The phagophore engulfs cytosolic components such
as protein aggregates and damaged organelles for degradation and
seals to form an autophagosome (Singh and Cuervo, 2011). The
autophagosome travels along microtubules to reach the lysosome
to fuse with it, becoming an autolysosome and starting the
degradation and recycling of the cargo components through acidic
lysosomal hydrolases (Mizushima and Komatsu, 2011; Bar-Yosef
et al., 2019). An important parameter to measure in
macroautophagy research is autophagy flux, defined as the dynamic
process of autophagy. 1. Introduction biogenesis (Tong et al., 2022). Lysosomal enzymes and lysosomal
functions are pivotal in functional autophagy (Ding et al., 2022). The induction of lysosomal biogenesis has been suggested as a
potentially effective therapeutic strategy (Ding et al., 2022). For
instance, lysosomal acidity is normally reduced with aging which
generates a suboptimal environment for the enzymatic activity in
the catalytic process mediated by autophagy which has been seen
to reduce the clearance of amyloid-β (AB) and tau aggregates in
AD models (Tong et al., 2022). Autophagy is a vital intracellular machinery responsible for the
clearance and recycling of cellular components (e.g., damaged
organelles, protein aggregates, lipids, nucleic acids) (Bar-Yosef et al.,
2019). Through this mechanism, the cytoplasmic cargo containing
cellular waste is transported to the lysosome for degradation (Levine
and Klionsky, 2017). Autophagy functions include maintaining cell
homeostasis, supplying the cells with building blocks (e.g., amino
acids, fatty acids) for the synthesis of new cellular components,
supporting the generation of energy during cell renovation processes,
and assisting cell growth and development (Mizushima et al., 2008;
Mizushima and Komatsu, 2011; Park and Cuervo, 2013). Autophagy
is a response mechanism to extracellular and intracellular stressors
to drive the cell toward cell survival (Bar-Yosef et al., 2019; Nie et al.,
2021). Autophagy is also part of the repertoire of programmed cell
death mechanisms, the type II cell death mechanism, that is different
from apoptosis (type I programmed cell death) (Nie et al., 2021). In microautophagy, the lysosome itself engulfs cytoplasmic
components by inward invagination of the lysosomal membrane that
traps the cytosolic cargo and internalizes it for degradation (Singh
and Cuervo, 2011; Mizushima and Levine, 2020). The invaginations
of the lysosomal membrane form multivesicular bodies (MVB) (Sahu
et al., 2011). After the cargo is wrapped around by the lysosomal
membrane, the membrane is rapidly degraded allowing the cargo to
get released into the lysosome where the degradation process starts
(Nie et al., 2021). Hydrolases in the lysosome catabolize the cargo and
the recycled components are released to ensure the reuse of the
nutrients by the cell (Nie et al., 2021). In microautophagy, the
selective targeting of cytosolic proteins is done through the Hsc70 to
late endosomes using the same KFERQ motif as in the mechanism of
CMA (Scrivo et al., 2018). This process is known as endosomal
microautophagy (Scrivo et al., 2018). OPEN ACCESS The
use, distribution or reproduction in other
forums is permitted, provided the original
author(s) and the copyright owner(s) are
credited and that the original publication in this
journal is cited, in accordance with accepted
academic practice. No use, distribution or
reproduction is permitted which does not
comply with these terms. Methods: Pubmed and Google Scholar were used to retrieve relevant in vitro
studies on autophagy mechanisms in neurological diseases and depression using a
combination of search terms per mechanism and disease (e.g., “macroautophagy”
and “Alzheimer’s disease”). A total of 37 studies were included (14 in PD, 8 in AD,
5 in ALS, 5 in %, and 5 in depression). Results: A repertoire of traditional and novel approaches and techniques was
compiled and discussed. The methods used in autophagy research focused on
the mechanisms of macroautophagy, microautophagy, and chaperone-mediated
autophagy. The in vitro tools presented in this review can be applied to explore
pathophysiological mechanisms at a molecular level and to screen for potential
therapeutic agents and their mechanism of action, which can be of great
importance to understanding disease biology and potential therapeutic options
in the context of neurodegenerative disorders and depression. Conclusion: This is the first review to compile, discuss, and provide a catalog of
traditional and novel in vitro models applied to neurodegenerative disorders and
depression. autophagy, cell-based assays, depression, neurodegenerative disorders, mood disorders autophagy, cell-based assays, depression, neurodegenerative disorders, mood disorders 01 Frontiers in Molecular Neuroscience frontiersin.org frontiersin.org Sánchez-Vidaña et al. Sánchez-Vidaña et al. 10.3389/fnmol.2023.1168948 Sánchez-Vidaña et al. Frontiers in Molecular Neuroscience 2. Methodological considerations of
this review Therefore, the aims
of this narrative review are (1) to compile information on the cell-
based methods used in autophagy research, (2) to discuss their
application, and (3) to create a catalog of traditional and novel in
vitro methods applied in neurodegenerative diseases and
depression. Our hypothesis was that traditional in vitro methods are
still being used as they are designed to explore relevant and well-
known molecular key players in autophagy research of
neurodegenerative disorders and depression. Also, we hypothesized
that new methods applied to autophagy research have emerged
thanks to technological advances. Therefore, the analysis and
compilation of both traditional and novel in vitro methods can
guide autophagy researchers in the field of neurological disorders
and depression to adapt their experimental designs for future
autophagy research. The catalog of in vitro models applied to
autophagy research of neurodegenerative disorders and depression
include a repertoire of traditional and novel approaches and
techniques. These in vitro tools can be applied to explore
pathophysiological mechanisms at a molecular level and to screen A narrative review was conducted to qualitatively summarize the
current in vitro methods used in autophagy research in
neurodegenerative diseases and mood disorders. Pubmed and Google
Scholar were used to retrieve relevant in vitro studies on autophagy
mechanisms in neurological diseases and depression using a
combination of search terms per mechanism and disease (Figure 1). The terms “macroautophagy” and “Alzheimer’s disease” were used to
retrieve studies on macroautophagy in AD. Since this review is a
narrative review rather than a systematic review, no record of the total
number of articles retrieved or excluded was kept. Instead, a qualitative
screening of articles took place considering the following: (1) studies
published in the past 10 years (2) that used in vitro platforms, and (3)
allowed the compilation of a varied repertoire of approaches and
techniques used in autophagy research in neurodegenerative disorders
and depression. Studies in English were included and abstracts and
conference proceedings were excluded. Studies published more than
10 years ago were only considered when the in vitro methods are still
used and add-up to the variety of the list of methods presented. Furthermore, the reference list of relevant studies was revised to
identify articles that could be included. This is the first review article
compiling relevant information on the application of in vitro methods
in autophagy research focused on neurodegenerative diseases and
mood disorders. 2. Methodological considerations of
this review In this context, cell-
based methods to study autophagy in neurodegenerative diseases
and mood disorders as well as to screen for autophagy modulators
are research tools that play a pivotal role in understanding
autophagy-mediated neuroprotection and therapeutics. Cell-based
models are used to resemble relevant disease hallmarks and are
powerful tools in drug discovery research to shed light on the
identification of drug targets (Cetin et al., 2022). Therefore, the aims
of this narrative review are (1) to compile information on the cell-
based methods used in autophagy research, (2) to discuss their
application, and (3) to create a catalog of traditional and novel in
vitro methods applied in neurodegenerative diseases and
depression. Our hypothesis was that traditional in vitro methods are
still being used as they are designed to explore relevant and well-
known molecular key players in autophagy research of
neurodegenerative disorders and depression. Also, we hypothesized
that new methods applied to autophagy research have emerged
thanks to technological advances. Therefore, the analysis and
compilation of both traditional and novel in vitro methods can
guide autophagy researchers in the field of neurological disorders
and depression to adapt their experimental designs for future
autophagy research. The catalog of in vitro models applied to
autophagy research of neurodegenerative disorders and depression
include a repertoire of traditional and novel approaches and
techniques. These in vitro tools can be applied to explore
pathophysiological mechanisms at a molecular level and to screen
for potential therapeutic agents and their mechanism of action. 1. Introduction The evaluation of autophagy flux is essential
in autophagy research as this process considers all the autophagy
steps from autophagosome formation, maturation, fusion with
lysosomes, and the release of catabolized components back to the
cytosol (Zhang et al., 2013). Therefore, the evaluation of autophagy
flux in in vitro models gives a complete picture of autophagy
function as a dynamic and multistep process (Zhang et al., 2013). Another important concept in macroautophagy is lysosomal Autophagy is constitutively active and particularly important
in post-mitotic cells such as neurons because they cannot use cell
division to dilute protein aggregates or other cytosolic waste to
prevent their accumulation (Nah et al., 2015). The special
morphology of the neuron, large dendritic and axonal cytoplasm,
makes it difficult for them to eliminate waste products in time to
prevent cytotoxicity (Guo et al., 2018). In neuronal autophagy, key
autophagy components are located at different places in the neuron
(Guo et al., 2018). Autophagosomes formed in the axons travel
long distances to reach the cell body where the lysosomes are
located and fuse with the lysosomes to initiate the degradation of
the autophagosome cargo (Guo et al., 2018). Consequently,
autophagy alterations and impaired axonal transport make neurons
highly susceptible to cell damage and death (Nah et al., 2015;
Bar-Yosef et al., 2019). Neural autophagy is crucial as this
mechanism plays an important role in axonal homeostasis,
dendrite spine, and synapse formation (Wang et al., 2019; Valencia
et al., 2021). Neuronal autophagy is also crucial to meet the energy
demands and support synaptic plasticity because synapses require
high energy and protein turnover (Bar-Yosef et al., 2019; Valencia
et al., 2021). 02 frontiersin.org Sánchez-Vidaña et al. Sánchez-Vidaña et al. 10.3389/fnmol.2023.1168948 2. Methodological considerations of
this review Defective
autophagy
is
closely
associated
with
the
pathophysiology of neurodegenerative disorders such as Parkinson’s
disease (PD), Alzheimer’s disease (AD), amyotrophic lateral
sclerosis (ALS), and Huntington’s disease (HD) and mood disorders
such as depression (Jia and Le, 2015; Nah et al., 2015; Wang et al.,
2019). Understanding the underlying molecular pathophysiology
of neurological disorders and depression is crucial due to the
prevalence of these disorders. The number of neurodegenerative
disease cases is alarmingly increasing, and these disorders are
becoming a major global cause of mortality and morbidity
(Erkkinen et al., 2018; Santomauro et al., 2021). Estimations
indicate that about 6.5 million people around the world are affected
by AD (Alzheimer’s Association, 2022), about 1% of the global
population is affected by PD, the prevalence of ALS accounts for 7.7
per 100,000 only in the USA (Mehta et al., 2023), the prevalence of
HD is 10.6–13.7 cases per 100,000 in Western populations and 1 to
7 cases per million in Eastern populations (McColgan and Tabrizi,
2018) whereas depression affects about 10% of the global population
(Alcocer-Gómez et al., 2017). Evidence suggests that the activation
of autophagy could slow down or stop neurodegenerative changes
(Jia and Le, 2015; Nah et al., 2015). For instance, recent studies have
shown the potential therapeutic properties of autophagy inducers
such as bromo-protopine, a protopine derivative that stimulates
CMA and reduces the presence of pathological tau in AD models
(Sreenivasmurthy et al., 2022) and klotho, a single-pass
transmembrane protein in the brain, that induces autophagy
leading to increased autophagy mediated clearance of amyloid-β in
AD models (Fung et al., 2022). Moreover, research on the
mechanism of autophagy and autophagy inducers contributes to the
identification of autophagy targets with therapeutic relevance in
neurodegenerative diseases such as KIF5B/kinesin-1, an autophagy-
related target in AD (Selvarasu et al., 2022). In this context, cell-
based methods to study autophagy in neurodegenerative diseases
and mood disorders as well as to screen for autophagy modulators
are research tools that play a pivotal role in understanding
autophagy-mediated neuroprotection and therapeutics. Cell-based
models are used to resemble relevant disease hallmarks and are
powerful tools in drug discovery research to shed light on the
identification of drug targets (Cetin et al., 2022). Frontiers in Molecular Neuroscience 2. Methodological considerations of
this review The information in the review is presented starting
with a discussion of the role of autophagy in neurodegenerative
disorders, including PD, AD, ALS, and HD, and mood disorders such
as depression to establish the key autophagy molecular players in each
disorder. Next, the cell-based methods used to investigate relevant
autophagy-related mechanisms and to screen for autophagy mediators
are discussed. The effect of therapeutic strategies on autophagy was
not considered in the present review. Instead, emphasis was put on
cell-based platforms and their application in the study of autophagy. A total of 37 studies were included (14 in PD, 8 in AD, 5 in ALS, 5 in
HD, and 5 in depression). sclerosis (ALS), and Huntingtons disease (HD) and mood disorders
such as depression (Jia and Le, 2015; Nah et al., 2015; Wang et al.,
2019). Understanding the underlying molecular pathophysiology
of neurological disorders and depression is crucial due to the
prevalence of these disorders. The number of neurodegenerative
disease cases is alarmingly increasing, and these disorders are
becoming a major global cause of mortality and morbidity
(Erkkinen et al., 2018; Santomauro et al., 2021). Estimations
indicate that about 6.5 million people around the world are affected
by AD (Alzheimer’s Association, 2022), about 1% of the global
population is affected by PD, the prevalence of ALS accounts for 7.7
per 100,000 only in the USA (Mehta et al., 2023), the prevalence of
HD is 10.6–13.7 cases per 100,000 in Western populations and 1 to
7 cases per million in Eastern populations (McColgan and Tabrizi,
2018) whereas depression affects about 10% of the global population
(Alcocer-Gómez et al., 2017). Evidence suggests that the activation
of autophagy could slow down or stop neurodegenerative changes
(Jia and Le, 2015; Nah et al., 2015). For instance, recent studies have
shown the potential therapeutic properties of autophagy inducers
such as bromo-protopine, a protopine derivative that stimulates
CMA and reduces the presence of pathological tau in AD models
(Sreenivasmurthy et al., 2022) and klotho, a single-pass
transmembrane protein in the brain, that induces autophagy
leading to increased autophagy mediated clearance of amyloid-β in
AD models (Fung et al., 2022). Moreover, research on the
mechanism of autophagy and autophagy inducers contributes to the
identification of autophagy targets with therapeutic relevance in
neurodegenerative diseases such as KIF5B/kinesin-1, an autophagy-
related target in AD (Selvarasu et al., 2022). Sánchez-Vidaña et al. 3. Parkinson’s disease (PD) First reported by the British doctor James Parkinson in 1817,
PD is nowadays the second most common neurodegenerative
disorder after AD that affects the motor system (Guo et al., 2018;
Hou et al., 2020). This chronic movement disorder gradually
develops over time and is prevalent in 1% of the elderly population
over 60 years of age and in 5–6% of the population over 85 years old
(Hou et al., 2020). PD is characterized by resting tremors, muscle
rigidity, and bradykinesia as a result of selective loss of dopamine
neurons in the substantia nigra pars compacta and the accumulation
of mutant proteins in inclusions of Lewy bodies (Winslow et al.,
2010; Guo et al., 2018; Bar-Yosef et al., 2019). The majority of PD
cases are sporadic in which the etiology is unknown while 5% of the
cases are hereditary and have been associated with mutations (Nah
et al., 2015). Mutations in the genes encoding the proteins
α-synuclein, leucine-rich repeat kinase 2 (LRRK2), vacuolar protein
sorting 35 (VSPS35), the chaperone protein REM-8, and the gene
CHCHD2 encoding a mitochondrial protein have been found in
cases of autosomal dominant PD (Moloudizargari et al., 2017). 03 frontiersin.org 10.3389/fnmol.2023.1168948 Sánchez-Vidaña et al. FIGURE 1
Flowchart of study screening and selection. FIGURE 1
Flowchart of study screening and selection. FIGURE 1
Flowchart of study screening and selection. Flowchart of study screening and selection. Mutations in genes encoding proteins such as the ubiquitin-ligase
parkin, PTEN-induced kinase 1 (PINK1), and the mitochondrial
protein DJ-1 involved in the regulation of oxidative stress have been
reported in autosomal recessive cases of PD (Moloudizargari et al.,
2017; Bar-Yosef et al., 2019; Meng et al., 2019). Other rare mutations
include the regulators of autophagy ATP13A2, the lysosomal
hydrolase glucocerebrosidase β acid (GBA), and the lysosomal
integral membrane protein-2 (LIMP-2) (Moloudizargari et al.,
2017). In PD, mutant proteins act at different stages of autophagy
through different mechanisms. Misfolded and aggregated mutant
α-synuclein and polyubiquitinated proteins are the main component
of Lewy bodies, one of the key pathological hallmarks in PD
suggesting that insufficient protein clearance leads to aberrant
protein accumulation (Guo et al., 2018; Meng et al., 2019). Post-
mortem samples from PD patients show abnormal accumulation of
autophagosomes in neurons which indicates that dysfunctional
autophagy may play a role in PD (Guo et al., 2018; Bar-Yosef et al.,
2019). 3. Parkinson’s disease (PD) Mitochondrial dysfunction, and oxidative stress, all of which
are closely associated with autophagy, also contribute to the
pathogenesis of PD (Lynch-Day et al., 2012). through the macroautophagy pathway (Guo et al., 2018). At the
elongation phase of macroautophagy, α-synuclein, a pre-synaptic
regulator of dopamine neurotransmission, inhibits autophagy as its
accumulation causes mislocalization of the autophagy protein Atg9,
which affects autophagosome formation (Kim and Guan, 2015;
Bar-Yosef et al., 2019). Although the presence of mutant α-synuclein
suppresses autophagy contributing to its accumulation and
cytotoxicity (Meng et al., 2019), the accumulation of α-synuclein
can also be triggered by other mechanisms. For instance, changes
in post-translational modifications, such as higher lysine acetylation
levels in histones (e.g., SIRT2 that deacetylates α-synuclein) also
contribute to the accumulation of α-synuclein in dopaminergic
neurons of PD patients (Wang R. et al., 2020). Therefore, mutations
should be regarded as part of the array of triggers associated with
the accumulation of α-synuclein. Other mutations associated with
PD are VPS35, a protein involved in the trafficking of proteins in
the cytoplasm, and LRRK2 proteins are observed in autosomal
dominant PD cases (Moloudizargari et al., 2017; Meng et al., 2019). Mutant VPS35 affects autophagosome formation through
mislocalization of the autophagy protein Atg9 (Guo et al., 2018;
Meng et al., 2019). Autophagosome aggregation and recruitment of
PI3KIII complex and Rubicon, a negative regulator of autophagy,
have been observed in the presence of mutant LRRK2 causing
deficits in autophagy, shortening of dendrites, and inhibition of
phagosome maturation leading to autophagosome aggregation
(Guo et al., 2018; Meng et al., 2019; Nakamura et al., 2019). At the
lysosome level, alterations in the lysosomal hydrolase GBA affect
the autophagosome-lysosome mechanism leading to the
accumulation of α-synuclein (Meng et al., 2019). Lysosomal
ATPases play an essential role in maintaining the optimal conditions
in the lysosome (e.g., lysosomal pH) for protease activity (Meng
et al., 2019). Mutant ATP13A2 causes down regulation of the
degradation processes taking place inside the lysosomes leading to Frontiers in Molecular Neuroscience 3.3. Chaperone-mediated autophagy in PD the accumulation of α-synuclein in dopaminergic neurons and
mutant LIMP-2 is involved in dysfunctional autophagosome and
lysosome function (Meng et al., 2019). Parkin and PINK1 are
proteins involved in the selective degradation of damaged
mitochondria by mitophagy (Guo et al., 2018). Mutations in both
parkin and PINK1 affect the recruitment of autophagy components
leading to reduced mitophagy (Bar-Yosef et al., 2019; Broadway
et al., 2022). Impaired mitophagy caused by mutant parkin and
PINK1 enhances sensitivity to oxidative stress due to the
accumulation of dysfunctional mitochondria and excessive
production of reactive oxygen species which has been associated
with PD (Lynch-Day et al., 2012; Meng et al., 2019). TMEM175, a
component of the lysosome proteome responsible for the regulation
of lysosomal pH and function, plays a role in mitophagy by
influencing respiration and regulation of energy homeostasis in the
mitochondria (Meng et al., 2019). the accumulation of α-synuclein in dopaminergic neurons and
mutant LIMP-2 is involved in dysfunctional autophagosome and
lysosome function (Meng et al., 2019). Parkin and PINK1 are
proteins involved in the selective degradation of damaged
mitochondria by mitophagy (Guo et al., 2018). Mutations in both
parkin and PINK1 affect the recruitment of autophagy components
leading to reduced mitophagy (Bar-Yosef et al., 2019; Broadway
et al., 2022). Impaired mitophagy caused by mutant parkin and
PINK1 enhances sensitivity to oxidative stress due to the
accumulation of dysfunctional mitochondria and excessive
production of reactive oxygen species which has been associated
with PD (Lynch-Day et al., 2012; Meng et al., 2019). TMEM175, a
component of the lysosome proteome responsible for the regulation
of lysosomal pH and function, plays a role in mitophagy by
influencing respiration and regulation of energy homeostasis in the
mitochondria (Meng et al., 2019). Alterations in the CMA mechanism have also been implicated
in the pathophysiology of PD (Nah et al., 2015). The presence of
mutant α-synuclein can affect both macroautophagy and CMA
(Bar-Yosef et al., 2019). In normal conditions, α-synuclein is
translocated into the lysosomes to undergo degradation by the CMA
machinery (Bar-Yosef et al., 2019). However, the mutant form of
α-synuclein interrupts the CMA process by binding to LAMP2A and
thus blocking it and affecting the protein translocation into the
lysosome for degradation (Nah et al., 2015; Bar-Yosef et al., 2019). 3.2. Microautophagy in PD Microautophagy has also been involved in the pathophysiology
of PD, and this autophagy process involves intricate coordination
of multiple mediators (Perrett et al., 2015) Structures such as the
late endosome and multivesicular bodies are responsible for the
selective uptake of proteins in microautophagy (Krause and Cuervo,
2021). The Hsc70 protein binds to the KFERQ-like motif present in
cytosolic proteins, and the Hsc70-labeled proteins get engulfed into
the multivesicular body compartment for lysosomal degradation
(Krause and Cuervo, 2021). Other key players participate in
microautophagy, including proteins such as Rab5 and Rab7 that
regulate endosomal maturation, VPS35 and REM-8 that participate
in actin polymerization, and SNARE proteins that contribute to the
fusion of the multivesicular bodies with the lysosome (Perrett et al.,
2015). Genetic mutations found in α-synuclein as well as mutations
in microautophagy regulators (e.g., VPS3) suggest aberrant
microautophagy in PD (Perrett et al., 2015; Krause and Cuervo,
2021). Other molecular players and signaling pathways also
interfere with the microautophagy degradation process. For
instance, the neutral sphingomyelinase 2 (nSMase2)- and ESCRT-
mediated signaling pathways are possibly involved in regulating the
uptake of α-synuclein by endosomal multivesicular bodies
(Sackmann et al., 2019). The relationship between microautophagy
and the above-mentioned pathways was evident when the inhibition
of nSMase2 led to a significant reduction in the cell-to-cell transfer
of oligomeric α-synuclein between neuron-like cells and decreased
the aggregation of α-synuclein (Sahu et al., 2011; Sackmann et al.,
2019). Moreover, α-synuclein with Lys-63 polyubiquitin chains
could be taken up by late endosomes in an ESCRT-dependent
manner and subsequently degraded by the lysosomes (Sackmann
et al., 2019). Another kay player that participates in the
microautophagy-mediated degradation of α-synuclein is a neural
precursor cell expressed developmentally down-regulated protein
4, known as Nedd4 (Sackmann et al., 2019). Nedd4 regulates the
ubiquitination of Lys-63 residues present in α-synuclein and
promotes α-synuclein internalization in SH-SY5Y cells (Sugeno
et al., 2014; Sackmann et al., 2019). The Needd4-mediated
ubiquitination mechanism is in turn regulated by CHMP2B
(Sugeno et al., 2014; Sackmann et al., 2019). 3.3. Chaperone-mediated autophagy in PD A53T mutant α-synuclein and A30P mutant α-synuclein can
strongly bind to the LAMP2A receptor preventing binding the
degradation of other targets through the CMA pathway (Lynch-Day
et al., 2012; Lu et al., 2020). Furthermore, the mutant form of the
LAMP2A receptor disrupts the degradation pathway mediated by
CMA leading to the accumulation of toxic aggregates in the
cytoplasm as observed in PD post-mortem tissues (Lynch-Day et al.,
2012). Blockade of the LAMP2A receptor leads to the upregulation
of autophagy as a compensatory response that may trigger cell death
and the accumulation of toxic components in the cell (Das et al.,
2012; Bar-Yosef et al., 2019). For instance, LRRK2 is degraded by
CMA but not the mutant form, thus blockade of the CMA
degradation pathway will impair the normal process of degradation
of LRRK2 leading to its accumulation in the cell (Nah et al., 2015). DJ1, which is a mitochondrial protein involved in the regulation of
oxidative stress, has also been associated with PD (Meng et al.,
2019). Overexpression of DJ1 in astrocytes can have a positive effect
on CMA suggesting that the presence of the mutant DJ1 could affect
CMA (Meng et al., 2019). Inhibition of the CMA pathway promotes
the buildup of toxic compounds in the neuron through the formation
of aggregates and prevents the protective activity of transcription
factors crucial in neuronal survival such as the protein myocyte
enhancer factor 2D (MEF2D) (Lynch-Day et al., 2012; Lu et al.,
2020). MEF2D activity is regulated by CMA-dependent degradation. When the CMA pathway is inhibited, there is an accumulation of
inactive MEF2D which cannot longer bind to the DNA to assist in
the transcription process (Lynch-Day et al., 2012). 3.1. Macroautophagy in PD Dysfunctional macroautophagy is implicated in PD (Nah et al.,
2015). α-synuclein has been considered one of the crucial key
players as it is the main component of Lewy bodies observed in PD
brains (Lu et al., 2020). This protein is prone to form highly toxic
aggregates, especially the mutant forms, and the formation of Lewy
bodies (Lu et al., 2020). Mutant forms of α-synuclein can affect both
the autophagosome-mediated and CMA degradation pathways
leading to different levels of toxicity in the cell (Lynch-Day et al.,
2012). Degradation of all forms of α-synuclein can be conducted 04 frontiersin.org 10.3389/fnmol.2023.1168948 Sánchez-Vidaña et al. frontiersin.org Frontiers in Molecular Neuroscience 3.4. Autophagy in vitro methods in PD
research Several in vitro platforms and analytical techniques have been
used to understand the contribution of autophagy-mediated
degradation to the pathogenesis of PD. Table 1 summarizes studies
that used PD models on different mechanisms of autophagy and
molecular targets, and a brief description of the application and
readout of the cell-based methods used. Macroautophagy and CMA are most commonly studied in PD
with only a few studies focusing on microautophagy. Studies on
macroautophagy are mainly concerned with autophagy induction,
expansion, autophagosome maturation, and autophagic flux, while
CMA studies focus on the cargo recognition, translocation, and
substrate degradation steps, and studies on microautophagy are
limited to the formation of endosomal multivesicular bodies (Table 1). Monitoring the accumulation and degradation of α-synuclein as well
as the use of autophagy inhibitors and activators, cell engineering 05 frontiersin.org Application (A), description (D), and readout (R)
References
s
A: Evaluation of a mutant form of α-synuclein and identification of biomarkers at different stages of MA
using the autophagy activator rapamycin and AMPK inhibitor compound C. D: The generation of mutant α-synuclein was induced by treatment with doxycycline. Phosphorylated
and non-phosphorylated mutant α-synuclein were measured to assess its autophagy-mediated clearance. Autophagy induction (Ulk1, AMPK, mTOR), TFBE activation, autophagic flux (LC3I and LC3II), and
lysosomal biogenesis (LAMP1) were measured. R: (1) Protein expression of the phosphorylated and non-phosphorylated form of α-synuclein by Western
blot. (2) Nuclear translocation of TFEB by immunostaining and Western blot of flag-TFEB. (3) Protein
expression of LC3I and L3CII by Western blot. (4) Protein activation by measuring Ulk1 and p-Ulk1,
AMPK, p-AMPK, mTOR, and p-mTOR using Western blot. Xu et al. (2022)
ons
rom human
stem cells
A: Monitoring MA markers specific to different stages of the autophagy process using cells treated with
an autophagy activator (AZD8055) and an inhibitor of autophagosome-lysosome fusion (bafilomycin A1,
BafA1). D: Upon autophagy activation by AZD8055, MA markers were measured. An autolysosome-fusion
inhibitor was used to assess autolysosome formation. R: (1) Gene expression of MA markers (beclin 1, LC3, p62, Optn, Ambra1) by qRT-PCR. (2) Protein
expression by immunostaining targeting LC3 and WIPI2, Western blot (LC3 and p62), and detection of
green fluorescent protein-tagged LC3 and Atg5 by time-lapse wide-field microscopy. (3) Quantitation of
autophagic vacuoles and autophagic flux using the CYTO-ID autophagy kit that labels accumulated
autophagic vacuoles. (4) Analysis of autolysosomal vacuoles by ultrastructural analysis using electron
microscopy. Stathakos et al. 3.4. Autophagy in vitro methods in PD
research (2021)
cephalic
A: Degradation of several sirtuin proteins in dopaminergic neuronal cells using different PD models. D: LUHMES cells were subjected to 1-methyl-4-phenylpyridinium, rotenone, and paraquat, which inhibit
autophagy, and the autophagy-mediated degradation of sirtuin proteins in the presence or absence of the
t
h
l
f
i
i hibit
B fA1
it
d
Baeken et al. (2021) autophagosome-lysosome fusion inhibitor BafA1 was monitored. R: (1) Protein expression of sirtuin proteins and the MA mediator LC3B by Western blot and
immunostaining. A: Assessment of the expression of the autophagy marker Atg5 by treating the cells with an autophagy
inhibitor (3-ethyladenine, 3-MA) and oligomerized α-synuclein. D: Cells were treated with different concentrations of oligomeric α-synuclein and the levels of Atg5 were
monitored. R: Protein expression by Western blot analysis of Atg5. Wang X. et al. (2020)
(Continued) Application (A), description (D), and readout (R)
References
A: Evaluation of a mutant form of α-synuclein and identification of biomarkers at different stages of MA
using the autophagy activator rapamycin and AMPK inhibitor compound C. D: The generation of mutant α-synuclein was induced by treatment with doxycycline. Phosphorylated
and non-phosphorylated mutant α-synuclein were measured to assess its autophagy-mediated clearance. Autophagy induction (Ulk1, AMPK, mTOR), TFBE activation, autophagic flux (LC3I and LC3II), and
lysosomal biogenesis (LAMP1) were measured. R: (1) Protein expression of the phosphorylated and non-phosphorylated form of α-synuclein by Western
blot. (2) Nuclear translocation of TFEB by immunostaining and Western blot of flag-TFEB. (3) Protein
expression of LC3I and L3CII by Western blot. (4) Protein activation by measuring Ulk1 and p-Ulk1,
AMPK, p-AMPK, mTOR, and p-mTOR using Western blot. Xu et al. (2022)
ns
om human
stem cells
A: Monitoring MA markers specific to different stages of the autophagy process using cells treated with
an autophagy activator (AZD8055) and an inhibitor of autophagosome-lysosome fusion (bafilomycin A1,
BafA1). D: Upon autophagy activation by AZD8055, MA markers were measured. An autolysosome-fusion
inhibitor was used to assess autolysosome formation. R: (1) Gene expression of MA markers (beclin 1, LC3, p62, Optn, Ambra1) by qRT-PCR. (2) Protein
expression by immunostaining targeting LC3 and WIPI2, Western blot (LC3 and p62), and detection of
green fluorescent protein-tagged LC3 and Atg5 by time-lapse wide-field microscopy. (3) Quantitation of
autophagic vacuoles and autophagic flux using the CYTO-ID autophagy kit that labels accumulated
autophagic vacuoles. (4) Analysis of autolysosomal vacuoles by ultrastructural analysis using electron
microscopy. Stathakos et al. 3.4. Autophagy in vitro methods in PD
research (2021)
ephalic
A: Degradation of several sirtuin proteins in dopaminergic neuronal cells using different PD models. D: LUHMES cells were subjected to 1-methyl-4-phenylpyridinium, rotenone, and paraquat, which inhibit
autophagy, and the autophagy-mediated degradation of sirtuin proteins in the presence or absence of the
autophagosome-lysosome fusion inhibitor BafA1 was monitored. R: (1) Protein expression of sirtuin proteins and the MA mediator LC3B by Western blot and
immunostaining. Baeken et al. (2021)
A: Assessment of the expression of the autophagy marker Atg5 by treating the cells with an autophagy
inhibitor (3-ethyladenine, 3-MA) and oligomerized α-synuclein. D: Cells were treated with different concentrations of oligomeric α-synuclein and the levels of Atg5 were
monitored. R: Protein expression by Western blot analysis of Atg5. Wang X. et al. (2020)
(Continued) ed) Application (A), description (D), and readout (R)
References
A: Identification of induction and autophagosome formation using the markers beclin 1, p62, mTOR, and
phospho-mTOR, and LC3I/LC3II after treatment with an autophagy inhibitor (rotenone) and a
neuroprotective agent (icariin) to assess autophagy induction and autophagic flux. D: Cells were pretreated with a neuroprotective agent and then challenged with an autophagy inhibitor. R: (1) Protein expression by Western blot analysis targeting α-synuclein and autophagy markers. (2) Cell
viability by lactate dehydrogenase assay. Zeng et al. (2019)
A: Detection of MA induction and autophagic flux upon treatment with a neurotoxin was used as a PD
model. An autophagy activator (rapamycin) and an autophagy inhibitor (3-MA) were also used. D: Cells were treated with a neurotoxin as a PD model. Autophagy markers were monitored upon
treatment with an autophagy activator and inhibitor. R: (1) Protein expression by Western blot analysis of beclin 1, LC3, and Atg12. (2) Gene expression by
mRNA analysis of beclin 1, LC3, and Atg12 by qRT-PCR. (3) Autophagosome formation by identification
of double-membrane enclosed autophagosome by transmission electron microscopy. Liu et al. (2019)
A: Study of the role of Park9 and Atg7 in autophagic flux and autophagosome maturation in SH-SY5Y
cells and primary neurons. D: Small interfering RNA was used to knock down Park9 and Atg7 in both types of cells. A gene-reported
approach was used to tag LC3 with a green and red fluorescent protein. Dual green and red positive cells
indicated early autophagosomes and red fluorescence indicated autophagosome maturation. R: (1) Autophagic flux was assessed by Western blot targeting LC3II and LC3I. 3.4. Autophagy in vitro methods in PD
research (2) Autophagy maturation
was assessed in green fluorescent and red fluorescent protein-tagged LC3 cells. Gusdon et al. (2012)
A: Monitoring the degradation of monomeric and oligomeric forms of α-synuclein and a CMA substrate
using autophagy inhibitors (rotenone and ammonium chloride). D: Cells were treated with autophagy inhibitors to assess the degradation of different forms of
α-synuclein. MA induction and autophagosome formation were monitored to evaluate α-synuclein
degradation. Cells were treated with the CMA substrate MEF2D to assess its degradation. R: (1) Gene and (2) protein expression of α-synuclein and autophagy markers of induction and
autophagosome formation (beclin1, LC3I, LC3II, p62) and CMA markers (LAMP2A, Hsc70) were
assessed using qRT-PCR and Western blot, respectively. (3) Substrate degradation by measuring the
extracellular concentration of α-synuclein was assessed by Dot blot. Sala et al. (2021)
(Continued) Application (A), description (D), and readout (R)
References
A: Monitoring CMA-mediated degradation of α-synuclein and MA biomarkers in cells derived from PD
patients with a mutation in the LRRK2 gene and healthy volunteers. Also, the role of mutant LRRK2 and
the downregulation of LAMP2A was assessed by cell engineering. D: α-synuclein was detected in both cells derived from patients and healthy subjects and engineered cells
to assess CMA-mediated degradation of α-synuclein and flagged α-synuclein. Also, LAMP2A was
knocked down in astrocytes. MA biomarkers to assess induction and autophagosome formation were
measured. R: (1) CMA-mediated degradation of α-synuclein by measuring the accumulation of α-synuclein and
transfer of α-synuclein using flagged α-synuclein assessed by immunostaining. (2) Protein expression by
immunostaining and Western blot targeting LAMP2A, α-synuclein, LC3II, LC3I, and p62. di Domenico et al. (2019)
A: Monitoring the MA and CMA degradation of α-synuclein in cells derived from PD patients with
GBA1 mutation to study α-synuclein degradation mediated by MA and CMA. D: An autophagy inducer (ambroxol) was used to assess autophagic flux and CMA. OR: (1) Protein expression of autophagy and autophagic flux markers (LC3II, LC3I, and p62) and CMA
autophagy (LAMP2A and LAMP1) by Western blot. (2) Substrate degradation (α-synuclein) by
immunostaining. Yang et al. (2017)
A1: Identification of the CMA pathway as a major route of degradation for WT α-synuclein. WT and
mutant α-synuclein turnover by activating autophagy with doxycycline in cells overexpressing of
α-synuclein and mutant α-synuclein and well as downregulated LAMP2A receptor to decrease CMA-
mediated degradation. A2: Identification of the contribution of MA-dependent degradation of wild type α-synuclein. 3.4. Autophagy in vitro methods in PD
research Therefore, BafA1
disrupts the autophagy pathway by preventing the acidification of the
endosomes and lysosomes, altering autophagosome maturation
(Redmann et al., 2016). Acidification is crucial for the proper fusion
of the autophagosome and lysosome which will ultimately lead to the
degradation of the sequestered cargo in the autolysosome (Yamamoto
et al., 1998). Ammonium chloride is used as an autophagy inhibitor
that acts at the lysosome level similarly to BafA1 (Sala et al., 2021;
Mavroeidi et al., 2022). Ammonium chloride inhibits the fusion of the
autophagosome with the lysosome and therefore interrupts cargo
degradation (Ghosh and Pattison, 2018). Another approach applied to study autophagy is cell engineering methods, and the use of cells from PD patients are common
approaches in autophagy research of PD using in vitro systems. pp
p
gy
g
y
Measuring the accumulation of different forms of α-synuclein is
crucial in in vitro autophagy research of PD (Guzzo et al., 2022). In
this line, either monitoring of α-synuclein accumulation (Xu et al.,
2022) or exposing the cells to α-synuclein (e.g., oligomeric
α-synuclein) (Wang X. et al., 2020) to induce a pathological state are
approaches used in PD research. Protein expression analysis by
Western blot is commonly used to measure different forms of
α-synuclein such as monomeric (Alvarez-Erviti et al., 2013; di
Domenico et al., 2019; Sackmann et al., 2019; Zeng et al., 2019; Sala
et al., 2021; Mavroeidi et al., 2022), oligomeric (Sala et al., 2021), and
phosphorylated α-synuclein (Xu et al., 2022). About 90% of the
α-synuclein aggregates found in Lewy Bodies of brain tissue from PD
patients are phosphorylated at serine 129 (Ser129-phosphorylated
α-synuclein) (Sasaki et al., 2015). The analysis of Ser129-
phosphorylated α-synuclein is crucial as high concentrations of
phosphorylated α-synuclein is an early event that promotes
α-synuclein aggregation (Sasaki et al., 2015). Therefore, measurement
of monomeric α-synuclein that is available for phosphorylation and
subsequent degradation, measurement of the phosphorylated form,
which accelerates the aggregation process, or measurement of the
oligomeric form which represents a late state of α-synuclein
aggregation should be considered a priori in the design of an in vitro
experiment to assess α-synuclein degradation. Other methods to
monitor α-synuclein accumulation and degradation include gene
expression analysis by qRT-PCR (Alvarez-Erviti et al., 2013),
immunostaining (Yang et al., 2017; Mavroeidi et al., 2022),
immunohistochemistry (Vogiatzi et al., 2008), radiolabeling and
subsequent immunoprecipitation (Vogiatzi et al., 2008), and dot blot
(Sala et al., 2021). Frontiers in Molecular Neuroscience 3.4. Autophagy in vitro methods in PD
research Proteasome (epoxomicin) and MA (3-MA) inhibitors were used. D: Overexpression of α-synuclein and expression of a mutant form of α-synuclein was induced in PC12
and SH-SY5Y cells. LAMP2A expression in PC12 was downregulated in PC12 cells to block the CMA
Vogiatzi et al. (2008) and SH SY5Y cells. LAMP2A expression in PC12 was downregulated in PC12 cells to block the CMA
pathway. Primary cultures were used as reference. R: (1) Half-life of WT (endogenous and overexpressed) and mutant α-synuclein in all cell types using
radiolabeling and subsequent immunoprecipitation against α-synuclein. (2) Protein expression by
Western blot analysis targeting α-synuclein, LAMP2A, and ubiquitin and by immunocytochemistry
targeting α-synuclein. (3) Gene expression of α-synuclein by RT-PCR. (Continued) d) escription (D), and readout (R)
References
CMA degradation of α-synuclein in engineered cells overexpressing human
PPP as well as cells with downregulation of LAMP2A and Atg5. -synuclein and TPPP were treated with the inhibitor 3-MA and CMA lysosomal
oride) and α-synuclein degradation was monitored. Downregulation of MA and
cted while monitoring α-synuclein degradation. of MA markers (LC3II, LC3I, p62), (2) Gene expression of MA (Atg5) and
umulation of α-synuclein by immunostaining and Western blot. Mavroeidi et al. (2022)
gradation of α-synuclein in normal cells and cells overexpressing wild-type
ere transfected with eight miRNAs identified in PD brains that regulate the
nd Hsc70. with stressors (rotenone and paraquat). The expression of LAMP2A, Hsc70, and
Regulation of LAMP2A and Hsc70 was assessed using a luciferase reporter
RNAs derived from PD brains. e expression of LAMP2A, Hsc70, and α-synuclein by Western blot and qRT-
of LAMP2A and Hsc70 by luciferase reporter assay. Alvarez-Erviti et al. (2013)
tion of neutral sphingomyelinase 2 (nSMase2) expression, a hydrolyze that acts
gomyelin to produce ceramide that forms extracellular vesicles. Increased levels
und in PD patients. prevents oligomeric α-synuclein aggregation in SH-SY5Y cells. Mase2 using siRNA transfection and CRISPR/Cas9 technique. (2) Protein
ot analysis targeting nSMase2 and α-synuclein. (3) Assessment of nSMase2
y assay. Sackmann et al. (2019) Sánchez-Vidaña et al. 10.3389/fnmol.2023.1168948 (Ghosh and Pattison, 2018). Rapamycin also acts on other autophagy
regulators. For instance, it activates Atg1 and improves the binding
affinity of Atg1 to Atg13 and Atg17 (Ghosh and Pattison, 2018). Another activator that acts at the induction stage of macroautophagy
is the compound AZD8055, a potent mTOR inhibitor (Hu et al., 2014;
Stathakos et al., 2021). Autophagy activation can also be induced at
other stages. 3.4. Autophagy in vitro methods in PD
research For instance, ambroxol acts at the lysosomal level (Yang
et al., 2017; Magalhaes et al., 2018). This compound is an expectorant
drug that promotes the translocation of glucocerebrosidase, an
enzyme that breaks down glucocerebroside into glucose and ceramide
in the lysosome, to the lysosome improving lysosomal enzymatic
activity and reducing oxidative stress (McNeill et al., 2014; Moors
et al., 2017). Concerning autophagy inhibitors, the neurotoxic agent
rotenone is commonly used in PD cell models (Alvarez-Erviti et al.,
2013; Zeng et al., 2019; Baeken et al., 2021; Sala et al., 2021). Rotenone,
a herbicide, and pesticide is a neurotoxin found in tropical plants that
can cross the blood–brain barrier (Salari and Bagheri, 2019). It
damages dopaminergic neurons and causes aggregation of α-synuclein
and Lewy body inclusions (Salari and Bagheri, 2019). Rotenone
promotes cell death through the induction of mitochondrial
dysfunction, increases oxidative stress, aggregation of α-synuclein,
post-translational modifications, and accumulation of autophagy
vacuoles leading to neuronal death (Mader et al., 2012; Ioghen et al.,
2023). Paraquat (Alvarez-Erviti et al., 2013; Baeken et al., 2021) is a
herbicide that affects the mitochondria causing increased oxidative
stress that leads to cellular damage and eventually death of
dopaminergic neurons (Salari and Bagheri, 2019). Similar to ratonone,
paraquat causes the accumulation of autophagy vacuoles (Lynch-Day
et al., 2012). 3-methyladenine (3-MA) is another compound used to
inhibit autophagy (Vogiatzi et al., 2008; Liu et al., 2019; Wang X. et al.,
2020; Mavroeidi et al., 2022). 3-MA, a PI3K inhibitor, acts at the
induction phase of macroautophagy by blocking the mTOR-mediated
activation pathway (Albanese et al., 2019). At the autophagosome
level, 3-MA inhibits the formation of autophagosomes (Ghosh and
Pattison, 2018). The compound 1-methyl-4-phenylpyridinium
(MPP+),
a
metabolite
of
1-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine (MPTP), a gold standard inducer of PD in research
settings, that selectively damages dopaminergic neurons, exhibits high
affinity to the dopamine transporter leading to its accumulation inside
the cells (Salari and Bagheri, 2019; Ioghen et al., 2023). MPP+ disrupts
the mitochondrial electron transport chain increasing oxidative stress
and causing cell damage and neuronal death (Ioghen et al., 2023). The
antibiotic bafilomycin A1 (BafA1) is used as an autophagy inhibitor
(Baeken et al., 2021; Stathakos et al., 2021). BafA1 acts at the lysosome
level through the inhibition of the vacuolar H + ATPase that regulates
the pH inside the lysosome (Redmann et al., 2016). frontiersin.org 4.1. Macroautophagy in AD Dysfunctional macroautophagy in AD has been observed at the
autophagy induction level, autophagosome maturation, and trafficking
of the autophagosome to the neuronal cell body as well as at the
autophagosome-lysosome function level (Nah et al., 2015; Bar-Yosef
et al., 2019). The first piece of evidence that autophagy may be involved
in the pathophysiology of AD was the striking accumulation of
immature autophagosome vesicles observed in dystrophic neurites in
AD brain tissue suggesting impaired autophagy (Nah et al., 2015; Guo
et al., 2018). Downregulation of beclin 1, a key component of the
macroautophagy machinery at the autophagy induction phase, has
been reported in AD brains (Nah et al., 2015; Guo et al., 2018). Another abnormality in macroautophagy in AD includes the cleavage
of beclin 1 by caspase 3, a component of the apoptosis pathway
disrupting autophagy (Guo et al., 2018). Alterations in autophagosome
trafficking in AD contribute to the accumulation of autophagic
vacuoles, also known as immature autophagic vacuoles, in neurons
and prevents the autophagosome from reaching the lysosome for
fusion and subsequent cargo degradation (Nah et al., 2015). Furthermore, the accumulation of immature autophagic vacuoles
stimulates autophagy through AMPK activation, a well-known
autophagy
activation
pathway,
suggesting
that
autophagy
overactivation is the result of autophagosome accumulation caused by
reduced trafficking (Nah et al., 2015). Autophagosome trafficking is
also affected by the decreased levels of beclin 1 (Nah et al., 2015). Disturbances at the autophagosome-lysosome fusion level have been
observed in AD (Guo et al., 2018). For instance, PS1 participates in
the process of autophagosome and lysosome fusion, and lack of
phosphorylated PS1 interrupts this process resulting in decreased
autophagosome-mediated clearance (Guo et al., 2018; Bar-Yosef et al.,
2019). Aberrant PS1 can degrease the degradation of cargo causing the
accumulation of autophagosomes in neurons (Guo et al., 2018;
Bar-Yosef et al., 2019). Additionally, PS1 regulates the internal
environment in the lysosome by promoting acidification and
activation of cathepsins which are proteases responsible for proteolytic
degradation inside the lysosome (Bar-Yosef et al., 2019). Recent
evidence showed the connection between plaque formation and
autolysosome acidification. Before extracellular AB deposition, a
decreased in autolysosome acidification was observed in neurons
which led to the build-up of AB and the pathogenic APP metabolite
APP-βCTF that, in normal autophagy functioning, are delivered to
lysosomes for degradation (Lee et al., 2022). 3.4. Autophagy in vitro methods in PD
research However, those methods are mainly used to detect
monomeric and non-phosphorylated α-synuclein. In terms of the
evaluation of specific stages or molecular players of the autophagy
process, in addition to the traditional methods mentioned above,
more sophisticated methods include the CYTO-ID autophagy kit that
labels accumulated autophagic vacuoles for the quantitation of
autophagic vacuoles and autophagic flux (Stathakos et al., 2021), time-
lapse wide-field microscopy for the detection of green fluorescent
protein-tagged LC3 and Atg5 (Stathakos et al., 2021), ultrastructural
analysis using electron microscopy for the analysis of autolysosomal
vacuoles (Stathakos et al., 2021), transmission electron microscopy to
assess autophagosome formation by identification of double-
membrane enclosed autophagosomes (Liu et al., 2019), and cell
engineering to generate green fluorescent and red fluorescent protein-
tagged LC3 cells to evaluate autophagy maturation (Gusdon
et al., 2012). The activation and inhibition of autophagy mechanisms are used
to understand the regulation and contribution of autophagy and to
identify potential drug targets (Yang et al., 2013). Activators and
inhibitors act at different stages of the autophagy pathway. Activators
of the macroautophagy pathway include rapamycin (Liu et al., 2019;
Xu et al., 2022), AZD8055 (Stathakos et al., 2021), and ambroxol (Yang
et al., 2017). Rapamycin is a drug that inhibits the mTOR signaling
pathway, thus leading to the activation of the macroautophagy leading
to a reduced accumulation of α-synuclein (Moors et al., 2017). The
rapamycin-mediated activation of macroautophagy starts with the
formation of a complex with the FK506-binding protein that
subsequently binds to and inhibits the activity of mTOR kinase Another approach applied to study autophagy is cell engineering
to generate cell profiles relevant to PD. Cells of rat origin can 10 frontiersin.org 10.3389/fnmol.2023.1168948 Sánchez-Vidaña et al. be modified to overexpress human α-synuclein and human tubulin
polymerization promoting protein, TPPP/p25α (Mavroeidi et al.,
2022). More recently, cell reprogramming to generate induced
pluripotent stem cells (iPSC) from PD patients can be used to obtain
disease-specific iPSC to mimic cellular and molecular mechanisms
involved in the pathophysiology of PD (Torrent et al., 2015; Yang et al.,
2017; di Domenico et al., 2019). Astrocytes and ventral midbrain
dopaminergic neurons can be obtained using patient-specific iPSC (di
Domenico et al., 2019). In this model, cells from PD patients with
LRRK2 mutation can be engineered using CRISPR/Cas9 technology
(di Domenico et al., 2019). 4.1. Macroautophagy in AD In compromised neurons,
autophagy vacuoles containing AB pack into large structures forming
flower-like perikaryal rosettes known as PANTHOS (poisonous
Anthos or flower) which occur in the brains of AD patients (Lee et al.,
2022). Another key player in autolysosome acidification is SIRT5, a
sirtuin protein found in the mitochondria (Baeken, 2023). SIRT5
mediates the deacetylation and activation of the enzyme lactate 3.4. Autophagy in vitro methods in PD
research Subsequently, the CMA-mediated
degradation of α-synuclein and macroautophagy markers can
be monitored to assess autophagy-mediated degradation and the
contribution of different autophagy-mediated mechanisms (di
Domenico et al., 2019). Another example of a cell reprograming
method in a PD model is the use of human adipose neural crest stem
cells (haNCSCs) from subjects with PD and GBA1 mutation which is
a gene linked to increased α-synuclein production (Yang et al., 2017). Cell reprograming offers the opportunity to address questions specific
to relevant molecular key players, genetic contributors, and patient-
specific profiles in PD (Torrent et al., 2015). Furthermore, the presence of mutant PS1 has also been associated
with decreased autophagosome-lysosome fusion, lysosomal
dysfunction, and neuronal loss (Nah et al., 2015; Bar-Yosef et al.,
2019). Autophagy is also responsible for the clearance of tau forms
including the phosphorylated form (Nah et al., 2015). Alterations in
the autophagy pathway delay tau clearance leading to its accumulation
and formation of neurofibrillary tangles (Nah et al., 2015). Frontiers in Molecular Neuroscience frontiersin.org 4. Alzheimer’s disease AD is the most prevalent neurodegenerative disease (Li et al.,
2017). Only in the USA, about 6.5 million people aged 65 years or
above are currently living with AD and estimates indicate that the
number of cases could increase to 13.8 million by 2060 (Alzheimer’s
Association, 2022). AD is the most common type of dementia
characterized by progressive loss of memory and cognitive function
(Li et al., 2017). As the disease progresses, other symptoms such as
confusion, irritability, aggression, mood swings, and reading and
writing difficulties manifest (Nah et al., 2015). The hallmarks of AD
include the presence of two abnormal structures, namely senile
plaques consisting of extracellular amyloid-β (AB) deposits distributed
along axons and dendrites and intra-neural neurofibrillary tangles
aggregated into filaments inside the cells (Li et al., 2017; Guo et al.,
2018; Bar-Yosef et al., 2019). AB plaques form as a result of aggregation
of cleaved products of the amyloid precursor protein (APP) while
neurofibrillary tangles are composed of hyperphosphorylated tau
protein which is prone to form aggregates (Guo et al., 2018). Both
aberrant structures lead to the progressive loss of neurons followed by
the disruption of synapses leading to the clinical manifestations
observed in AD (Li et al., 2017). Although the etiology of AD is now
clear, recent evidence points out dysfunctional autophagy that fails to
clear misfolded, hyperphosphorylated proteins, or protein aggregates
preceding the formation of AB plaques or neurofibrillary tangles (Li
et al., 2017). Some components involved in the APP catalytic pathway
are key contributors to the formation of AB plaques and are associated
with dysfunctional autophagy. An important molecular player in AD
is presenilin 1 (PS1), a transmembrane protein ubiquitously expressed,
which participates in the cleavage of APP as part of the APP-cleaving
γ-secretase complex (Guo et al., 2018; Bar-Yosef et al., 2019). In
normal conditions, APP is cleaved by β-secretase to generate the
β-C-terminal fragment that is later cleaved by PS1 to produce Aβ
(Guo et al., 2018). In AD, a mutant form of PS1 has been suggested to
interfere with the APP catalytic process (Guo et al., 2018). 11 frontiersin.org 10.3389/fnmol.2023.1168948 Sánchez-Vidaña et al. surface interfering with the lysosomal membrane integrity and
affecting CMA’s normal functioning (Cuervo and Wong, 2014). Lysosome rupture releases mutant tau oligomers that serve as seeds
for subsequent tau aggregation in the cytosol aggravating the
formation of neurofibrillary tangles (Cuervo and Wong, 2014). 4.2. Microautophagy in AD In microautophagy, cell membrane components are captured
through the formation of the multivesicular body (MVB), or
intracellular components are engulfed at the lysosomal membrane
(Lata et al., 2009). A variety of complexes or proteins are involved
in the process of MVB-mediated microautophagy, including
endosomal sorting complex required for transport (ESCRT),
glyceraldehyde-3-phosphate
dehydrogenase,
aldolase,
and
cyclophilin (Shpilka and Elazar, 2011). Lysosomes and endosomes
can directly trap cytosolic cargo through the boundary membrane,
and MVBs are directly involved in the regulation of intracellular
lysosome and endosome formation. Therefore, multivesicular
lysosomes and MVBs are always considered the major biomarkers
of microautophagy (Li et al., 2012). Mutations of several
microautophagy-related genes in the promotion of different
neurodegenerative diseases have been reported. For example,
charged MVB protein 2B (CHMP2B) is a subunit of ESCRT and
mutation of CHMP2B could lead to the pathogenesis of
amyotrophic lateral sclerosis (Parkinson et al., 2006) and
frontotemporal dementia (Skibinski et al., 2005). Mutations of
VPS4A, a dominant negative form of the CHMP2B-interacting
protein in primary neurons, lead to accumulation and aggregation
of Aβ and phosphorylation of tau, suggesting that dysfunction of
microautophagy could be considered as a risk factor for the
pathogenesis of AD (Willen et al., 2017). Additionally,
microautophagy deficit may be implicated in the regulation of
tau-induced AD. For instance, IST1 is a positive regulator for the
formation of the ESCRT complex. Feng et al. (2020) reported that
overexpression of IST1 reduces the accumulation and aggregation
of tau, and ameliorates synaptic plasticity and cognitive functions
in human tau transgenic mice (Feng et al., 2020). 4.4. Autophagy in vitro methods in AD
research Table 2 shows a list of cell-based approaches used in autophagy
research in AD. The methods used to study the mechanism of
selective (e.g., mitophagy) and non-selective macroautophagy focus
on
cargo
recognition,
induction,
autophagosome
flux,
autophagosome maturation, and autolysosome formation stages as
well as lysosomal function. In vitro platforms to study CMA in AD
models mostly focus on the induction phase and the mechanism of
cargo recognition and translocation while the formation of late
endosomes and MVBs is assessed in microautophagy in vitro
studies. Several AD in vitro models have been generated to
manipulate the gene expression of relevant key molecular players,
to assess the activity of key enzymes involved in the pathophysiology
of AD, and to monitor substrate degradation in the presence and
absence of autophagy inhibitors and activators. 4. Alzheimer’s disease Another protein implicated in dysfunctional CMA in AD is the
regulator of calcineurin 1 (RCAN1). RCAN1 undergoes
CMA-mediated degradation and is highly expressed in AD brains
(Cuervo and Wong, 2014). It has been suggested that pathogenic
tau clocking the CMA pathway leads to elevated levels of RCAN1 in
the neuron (Cuervo and Wong, 2014). However, the contribution
of RCAN1 to AD phenotype is still not well understood (Cuervo
and Wong, 2014). More interestingly, a novel CMA-activating drug,
metformin, recently identified during a high-throughput screening,
has been found to reduce Aβ protein levels and improve cognitive
impairment in a mouse model of AD, via Hsc70 signaling (Xu et al.,
2021). The Aβ protein precursor contains three putative KFERQ-
like CMA motifs that are strongly conserved across species. Metformin-induced CMA activity and Aβ plaque degradation
involved these motifs, as the results revealed that mutation of the
three KFERQ-like motifs blocked Aβ degradation as well as
inhibiting the interaction of Aβ precursor protein with endogenous
Hsc70 or LAMP2A (Xu et al., 2021). dehydrogenase B found on the lysosomal membrane supplying the
lysosomes with protons for lysosomal acidification (Baeken, 2023). Due to both its role in lysosomal acidification and its decreased
expression in AD, SIRT5 has been regarded as an attractive autophagy-
related target in AD therapeutics (Baeken, 2023). Frontiers in Molecular Neuroscience frontiersin.org 4.3. Chaperone-mediated autophagy in
AD (2022)
Murine blastocysts
expressing different
PS1 genotypes
A: Identification of loss of MA through deletion of PS1 by engineering cells and using MA
activators and inhibitors. D: Cells with different PS1 genotypes including wild-type cells, PS1 knockout, mutant PS2, and
wild-type human PS1 were treated to identify the role of PS1 in lysosome acidification and
function. Autophagic/lysosomal degradation was induced through serum withdrawal, proteolysis
was blocked using ammonium chloride, and MA was induced using rapamycin and inhibited using
3-methyladenine. R: (1) Classification and quantitation of autophagy vacuoles by ultrastructural and morphometric
analysis using electron microscopy. (2) Protein degradation measured by pulse-chase tracking
[3H]-leucine incorporation into proteins. (3) Protein phosphorylation by Western blot targeting
p70S6 kinase and its phosphor-epitope to identify mTOR-mediated autophagy induction. (4)
Protein expression by immunostaining targeting LC3, Western blot of LC3I and L3CII, and
immunofluorescence targeting LC3 and LAMP2A. Lee et al. (2010)
Ba/F3 cells
A: Identification of beclin 1 as a substrate of caspases as a cause of lower levels of beclin 1 in AD. D: Use of a site-directed caspase-cleavage antibody targeting beclin 1 to assess whether beclin 1
proteolytic cleavage by caspases is responsible for the lower levels of beclin 1 in AD. Detection of a
35 kDa C-terminal fragment of beclin 1 by a novel antibody is followed by incubation with
caspase-3 to detect caspase-mediated cleavage of beclin 1. Cells were engineered to express two
mutant beclin 1 forms expressing potential caspase cleavage sites
Rohn et al. (2011) Cell line
Application (A), description (D), and readout (R)
References
SH-SY5Y cells
Primary neurons
A: Identification of mitophagy triggered by amyloid-β (Aβ) oligomers. D: Cells were treated with Aβ oligomers in the presence and absence of the inhibitor of autophagic
vacuole maturation bafilomycin. The co-localization of the autophagy markers p62 and LC3 with a
mitochondria marker was identified as an indication of induced mitophagy. R: (1) Protein expression by Western blot and detection of p62 and LC3 by immunofluorescence. (3) Calculation of the amount of colocalized mitochondria by Velocity software. de la Cueva et al. (2022)
Murine blastocysts
expressing different
PS1 genotypes
A: Identification of loss of MA through deletion of PS1 by engineering cells and using MA
activators and inhibitors. D: Cells with different PS1 genotypes including wild-type cells, PS1 knockout, mutant PS2, and
wild-type human PS1 were treated to identify the role of PS1 in lysosome acidification and
function. 4.3. Chaperone-mediated autophagy in
AD Cell-based methods that mimic AD hallmarks (e.g., over
production of Aβ oligomers) are particularly useful in AD research. A mutation known as 7PA2, an APP isoform containing Val → Phe
mutation at residue 717, can be introduced to Chinese hamster
ovary cells to generate 7PA2 cells that overproduce Aβ oligomers
(Portelius et al., 2012). The characteristics of the 7PA2 cell model
make it a first-choice model to study Aβ-induced toxicity, a
physiological feature relevant to the pathogenesis of AD (Portelius
et al., 2012). Macroautophagy induction can be triggered by the
presence of high levels of Aβ in 7PA2 cells (Caccamo et al., 2010). In this cell model, the role of autophagy key signaling such as the
mTOR pathway could be monitored in function of Aβ levels when
treating the cells with autophagy activators and inhibitors (Caccamo
et al., 2010). At the autophagosome level, primary cortical neurons
can be engineered to express LC3 tagged with a green fluorescent
protein (GFP) to monitor autophagic flux by confocal microscopy The presence of mutant tau proteins contributes to the
formation of neurofibrillary tangles arising from mutant tau
aggregation in AD (Cuervo and Wong, 2014). Tau contains
CMA-target motifs in the C-terminal region that make it prone to
interact with the chaperone protein Hsc70, which is responsible for
the translocation of CMA substrates to the lysosome for degradation
(Wang et al., 2009). In AD brain tissue, Hsc70 associates with
neurofibrillary tangles indicating that the CMA system is also
involved in the degradation of neurofibrillary tangles (Wang et al.,
2009). Mutant tau variants are processed by the CMA degradation
system in which mutant tau is bound to the LAMP2A receptor and
only partially internalized resulting in the formation of smaller
amyloidogenic tau fragments in the lysosome (Cuervo and Wong,
2014). The tau fragments generated oligomerize at the lysosomal 12 frontiersin.org Cell line
Application (A), description (D), and readout (R)
References
SH-SY5Y cells
Primary neurons
A: Identification of mitophagy triggered by amyloid-β (Aβ) oligomers. D: Cells were treated with Aβ oligomers in the presence and absence of the inhibitor of autophagic
vacuole maturation bafilomycin. The co-localization of the autophagy markers p62 and LC3 with a
mitochondria marker was identified as an indication of induced mitophagy. R: (1) Protein expression by Western blot and detection of p62 and LC3 by immunofluorescence. (3) Calculation of the amount of colocalized mitochondria by Velocity software. de la Cueva et al. 4.3. Chaperone-mediated autophagy in
AD Autophagic/lysosomal degradation was induced through serum withdrawal, proteolysis
was blocked using ammonium chloride, and MA was induced using rapamycin and inhibited using
3-methyladenine. R: (1) Classification and quantitation of autophagy vacuoles by ultrastructural and morphometric
analysis using electron microscopy. (2) Protein degradation measured by pulse-chase tracking
[3H]-leucine incorporation into proteins. (3) Protein phosphorylation by Western blot targeting
p70S6 kinase and its phosphor-epitope to identify mTOR-mediated autophagy induction. (4)
Protein expression by immunostaining targeting LC3, Western blot of LC3I and L3CII, and
immunofluorescence targeting LC3 and LAMP2A. Lee et al. (2010)
Ba/F3 cells
A: Identification of beclin 1 as a substrate of caspases as a cause of lower levels of beclin 1 in AD. D: Use of a site-directed caspase-cleavage antibody targeting beclin 1 to assess whether beclin 1
proteolytic cleavage by caspases is responsible for the lower levels of beclin 1 in AD. Detection of a
35 kDa C-terminal fragment of beclin 1 by a novel antibody is followed by incubation with
caspase-3 to detect caspase-mediated cleavage of beclin 1. Cells were engineered to express two
mutant beclin 1 forms expressing potential caspase-cleavage sites. R: (1) Protein expression analysis by Western blot targeting a caspase-resistant form of beclin 1. Rohn et al. (2011)
7PA2 cells and
Chinese hamster
ovary cells were
used as control
A: Assessment of autophagy induction using cells engineered to express an AD mutation that leads
to the production of high levels of Aβ oligomers. D: mTOR phosphorylation was monitored to assess the induction of autophagy in both cell types. Autophagy activation mediated by Aβ and amyloid β-precursor protein was evaluated using the
γ-secretase inhibitor compound E. mTOR inhibition using rapamycin was done to assess the effects
of mTOR function on the levels of Aβ. R: (1) Protein expression by Western blot targeting mTOR, p-mTOR to assess autophagy induction. (2) Levels of intracellular Aβ measured by ELISA. Caccamo et al. (2010)
(Continued) Application (A), description (D), and readout (R)
References
A: Evaluation of autophagosome and autolysosome formation using cells engineered to label LC3. Also, evaluation of protease activity and vesicle acidification using a fluorescent probe (BODIPY FL
pepstatin A) to inhibit cathepsin D in lysosomes. D: Cells were treated with rapamycin to induce autophagy and measure mTOR-mediated
autophagy activation. Labeled LC3 and BODIPY-pepstatin-FL labeling were used to assess
autophagosome formation and maturation. 5. Amyotrophic lateral sclerosis (ALS) ALS is an age-dependent neurodegenerative disease characterized
by the selective degeneration of motor neurons in the brain and spinal
cord (Guo et al., 2018; Amin et al., 2020). The clinical manifestation
of ALS includes progressive muscle weakness, muscle wasting,
atrophy, and paralysis starting in the distal muscles of limbs moving
toward proximal muscles as the disease progresses (Masrori and Van
Damme, 2020). Most ALS cases manifest between 50 and 60 years of
age and culminate in death caused by respiratory failure (Amin et al.,
2020). Accumulation of protein aggregates and mitochondrial damage
are responsible for motor neuron degeneration and death in ALS
(Chen et al., 2012). It is estimated that about 90% of ALS cases are
sporadic and 10% are associated with genetic mutations (Nguyen
et al., 2019). The hallmark of ALS is the cytoplasmic aggregation of
TDP-43 protein, which is observed in more than 95% of ALS cases
(Amin et al., 2020). Furthermore, about 20% of the classic familiar
ALS cases are attributed to mutations in SOD1 (Chen et al., 2012). Other genes associated with ALS include fused in sarcoma (FUS),
sequestosome1 (SQSTM1 also known as p62), optineurin (OPTN),
TANK binding kinase 1 (TBK1), VAMP-associated protein B (VAPB),
valosin-containing protein (VCP), ubiquilin 2 (UBQLN2), alsin,
charged multivesicular body protein 2B (CHMP2B), dynactin
(DCTN), profilin 1 (PFN1), factor-induced gene 4, and a
hexanucleotide repeat expansion in the gene C9orf72 (Guo et al.,
2018; Nguyen et al., 2019). The accumulation of mutant SOD1
aggregates and autophagic vacuoles in motor neurons of the spinal
cords points out at aberrant autophagic flux or specific dysfunctional
autophagy regulatory processes that may lead to motor neuron
degeneration and death (Chen et al., 2012). Mutant SOD1 can
be recognized by ubiquitin-interacting proteins such as p62 for
selective autophagy clearance, but this mutant protein can inhibit the
autophagy pathway leading to decreased clearance (Chen et al., 2012;
Nguyen et al., 2019). Moreover, mutant SOD1 alters the microtubule-
localized dynein-dynactin complex affecting autophagosome
trafficking (Nguyen et al., 2019). Alterations in autophagosome
maturation and fusion with the lysosome have also been attributed to
the presence of mutant SOD1 (Chen et al., 2012). For instance, mutant
SOD1 leads to the accumulation of mitochondria and membrane-
bound organelles by disruption of the axonal transport (Chen et al.,
2012). Frontiers in Molecular Neuroscience 4.3. Chaperone-mediated autophagy in
AD In
this model, cells are challenged with AB oligomers in the presence
and absence of the inhibitor of autophagic vacuole maturation
BafA1 (de la Cueva et al., 2022). The colocalization of the autophagy
markers p62 and LC3 with the mitochondrial marker CxVβ allows
monitoring of the mitophagy process using immunofluorescence
and Western blot analysis as well as the quantitation of colocalized
mitochondria by immunofluorescence (de la Cueva et al., 2022). 4.3. Chaperone-mediated autophagy in
AD R: (1) Protein expression analysis by Western blot targeting mTOR, p-mTOR, LC3I, and LC3II and
immunohistochemistry to detect LC3. (2) Evaluation of autophagosomes and autolysosomes by
morphometric analysis using confocal microscopy. Boland et al. (2008)
H4
A: Evaluation of Aβ protein degradation mediated by Hsc70 and LAMP2A using engineered cells. D: HEK293T cells were engineered to express HK2-GFP in a doxycycline (DOX)-inducible manner
(293THK). Cells were treated with the autophagy inhibitors Spautin-1 and Fms-like tyrosine
kinase-3 (Flt3) inhibitor AC220 to induce CMA-mediated degradation of HK2. Hsc70 and
LAMP2A were knockdown to assess CMA activation and monitor HK2 degradation. R: (1) Protein degradation by immunofluorescence to assess CMA-mediated degradation of HK2. (2) Gene and (3) protein expression of Hsc70, p-Hsc70, HK2 by qRT-PCR and Western blot,
respectively. Xu et al. (2021)
A: Monitoring the degradation of APP in a Hsc70 and LAMP2A-dependent manner by cell
engineering to flag Hsc70 and LAMP2A. D: Cells were treated with a CMA inducer and lysosomal inhibitors such as E-64D, bafilomycin A1
(BafA1), leupeptin combined with ammonium chloride, and the protease inhibitor MG132 to
assess lysosome-dependent degradation, Hsc70 phosphorylation, and APP degradation. R: (1) Protein expression by Western blot targeting Hsc70, pHsc70, LAMP2A, and APP. Xu et al. (2021)
al
A: Identification of the overexpression of VPS4 can lead to Aβ accumulation in enlarged endocytic
compartments. D: Overexpression of VPS4 increased the intracellular pool of Aβ and decreased the amounts of Aβ
secreted into the medium. R: (1) Protein expression by Western blot analysis targeting Aβ and by immunocytochemistry
targeting Aβ. Willen et al. (2017) 10.3389/fnmol.2023.1168948 Sánchez-Vidaña et al. system, the intra and extracellular accumulation of AB is monitored
to assess late endosome and MVB formation (Willen et al., 2017). (Boland et al., 2008). To assess autolysosome formation and
function in the same type of modified cells, the fluorescent probe
BODIPY FL pepstatin A is used to monitor protease activity and
vesicle acidification through the inhibition of cathepsin D, a
protease found in the lysosome that participates in the APP
degradation (Chen et al., 2000; Boland et al., 2008; Uddin et al.,
2018). A key molecular player in AD is PS1, a protein involved in
the cleavage of APP, as the presence of mutant forms of PS1 not only
disrupts APP catalysis but also interrupts the macroautophagy
process at different stages (e.g., autophagosome-lysosome function)
(Guo et al., 2018; Bar-Yosef et al., 2019). 4.3. Chaperone-mediated autophagy in
AD The genetic manipulation
of PS1 in in vitro models is useful to identify the effect of PS1 in
macroautophagy. For
instance,
murine
blastocysts
can
be engineered to express different PS1 genotypes, including PS1
knockout, mutant PS1, and wild-type human PS1, to assess the PS1
effect at the lysosome level using ultrastructural and morphometric
analysis by electron microscopy to analyze autophagy vacuoles (Lee
et al., 2010). An in vitro method to study mitophagy, a selective
macroautophagy mechanism, focuses on the assessment of cargo
recognition, autophagic flux, and autophagosome maturation in
SH-SY5Y cells and primary neurons (de la Cueva et al., 2022). In
this model, cells are challenged with AB oligomers in the presence
and absence of the inhibitor of autophagic vacuole maturation
BafA1 (de la Cueva et al., 2022). The colocalization of the autophagy
markers p62 and LC3 with the mitochondrial marker CxVβ allows
monitoring of the mitophagy process using immunofluorescence
and Western blot analysis as well as the quantitation of colocalized
mitochondria by immunofluorescence (de la Cueva et al., 2022). (Boland et al., 2008). To assess autolysosome formation and
function in the same type of modified cells, the fluorescent probe
BODIPY FL pepstatin A is used to monitor protease activity and
vesicle acidification through the inhibition of cathepsin D, a
protease found in the lysosome that participates in the APP
degradation (Chen et al., 2000; Boland et al., 2008; Uddin et al.,
2018). A key molecular player in AD is PS1, a protein involved in
the cleavage of APP, as the presence of mutant forms of PS1 not only
disrupts APP catalysis but also interrupts the macroautophagy
process at different stages (e.g., autophagosome-lysosome function)
(Guo et al., 2018; Bar-Yosef et al., 2019). The genetic manipulation
of PS1 in in vitro models is useful to identify the effect of PS1 in
macroautophagy. For
instance,
murine
blastocysts
can
be engineered to express different PS1 genotypes, including PS1
knockout, mutant PS1, and wild-type human PS1, to assess the PS1
effect at the lysosome level using ultrastructural and morphometric
analysis by electron microscopy to analyze autophagy vacuoles (Lee
et al., 2010). An in vitro method to study mitophagy, a selective
macroautophagy mechanism, focuses on the assessment of cargo
recognition, autophagic flux, and autophagosome maturation in
SH-SY5Y cells and primary neurons (de la Cueva et al., 2022). 5.4. Autophagy in vitro methods in ALS
research A summary of cell-based platforms used in ALS research is
presented in Table 3. Most studies target substrate degradation,
autophagy induction, autophagic flux, and autophagosome-
autolysosome fusion in macroautophagy and substrate recognition
and degradation in CMA (Table 3). Crucial molecular features of ALS
pathophysiology include the cytoplasmatic aggregation of TDP-43
and mutant SOD1 that are responsible for the disruption of the
autophagy machinery in motor neurons of the spinal cord (Chen
et al., 2012; Amin et al., 2020). Most cell-based methods in ALS
research are therefore concerned with the role of TDP-43 and SOD1
at different stages of the mechanism of autophagy. f
g
p
gy
In several in vitro models to study selective and non-selective
macroautophagy as well as CMA, cells are engineered to express
mutant SOD1 and TDP-43. For instance, the spinal cord
neuroblastoma hybrid cell line NSC-34, a cell line of choice in ALS
research, has been modified to express mutant form SOD1 and
TDP-43 (Perera et al., 2017; Amin et al., 2020). NSC-34 cells
expressing those mutant proteins are then challenged with
autophagy activators and inhibitors (e.g., rilmenidine, BafA1, or
rotenone) to assess macroautophagy, mitophagy, and CMA at
different stages of the autophagy-mediated degradation process
(Perera et al., 2017). By applying traditional (e.g., Western blot)
and more sophisticated analytical techniques such as gene reporter
assays (e.g., DsRed-T3 or mCherry-GFP-LC3B reporter assays),
key autophagy players (e.g., LC3I, LC3II, P62, LAMP2A, and
Hsc70) can be monitored to assess aberrant autophagy and the
effect of the presence of mutant proteins relevant to ALS (Perera
et al., 2017). For instance, the mCherry-GFP-LC3B reporter system
is a tandem system to identify mature autolysosomes and measure
autophagic flux by confocal microscopy (Perera et al., 2017). Furthermore, the fusion of mitochondria with the lysosome can
be monitored using a ratiometric pHluorin DsRed-T3 system
(Perera et al., 2017). In this system, the mitochondria are labeled,
and the presence of labeled mitochondria in lysosomes can be used
as an indication of the successful fusion of the mitochondria-
containing autophagosome with the lysosome (Perera et al., 2017). A variation of the cell-based approach mentioned-above in NSC-34
cells involves monitoring the degradation of SOD1 and TDP-43
aggregates in cells overexpressing mutant SOD1 (Gomes et al.,
2010). Upon autophagy induction, the degradation of SOD1,
TDP-43 aggregates, and ubiquitinated TDP-43 can be analyzed by
Western blot and immunofluorescence (Gomes et al., 2010). 5.1. Macroautophagy in ALS toxic protein in cells with macroautophagy inhibition (Huang et al.,
2014). Besides the TDP-43 protein, the regulatory role of CMA in
other ALS-associated toxic mutant proteins, such as SOD1 found in
familial forms of ALS, has not yet been explicitly shown (Crippa
et al., 2010). Dysregulation at different macroautophagy stages, particularly at
induction, trafficking, and autophagosome-lysosome fusion, has been
implicated in ALS (Guo et al., 2018; Amin et al., 2020). For instance,
increased levels of LC3, beclin 1, p62, and the Atg5-Atg12 complex,
which are mediators involved in the early stages of the macroautophagy
process, have been detected in spinal motor neurons of both sporadic
and familial ALS that later extend to other cells such as astrocytes and
microglia (Amin et al., 2020). Aberrant accumulation of immature
autophagosomes observed in spinal motor neurons suggest alterations
of the macroautophagy machinery at autophagosome maturation and
subsequent lysosomal function level in which mutant forms of VAPB,
UBQLN2, VCP, PC2, OPTN, and TBK1 proteins have been implicated
(Guo et al., 2018; Amin et al., 2020). The dynein-dynactin complex is
responsible for the retrograde transport of autophagosomes (Nguyen
et al., 2019). Autophagosomes mature during transportation as a result
of acidification that occurs before lysosomal fusion when the
autophagosome reaches the cell soma (Nguyen et al., 2019). However,
misfolded mutant SOD1 is prone to increase its interaction with
dynein leading to an overload of dynein-mediated retrograde axonal
transport which in turn affects autophagosome trafficking (Nguyen
et al., 2019). At autophagosome and lysosome fusion levels, aberrant
autophagy regulators such as C9orf72, VCP, CHMP2B, ALS2, and
(Table 3) affect the formation of the autolysosome leading to reduced
cargo degradation and build-up of autophagosomes and aberrant
proteins in motor neurons and glial cells (Amin et al., 2020). 5.2. Microautophagy in ALS The relationship between microautophagy and ALS is rarely
reported. However, a study reported that mutation of CHMP2B can
lead to ALS pathogenesis (Parkinson et al., 2006), suggesting the
potential role of microautophagy. Additionally, in Drosophila models
of ALS and human neuronal cells, scientists found that overexpression
of TAR DNA-binding protein 43 (TDP-43) can inhibit HSPA8
transcription, leading to the dysfunction of synaptic vesicle cycling all
of which is associated with microautophagy dysfunction (Coyne
et al., 2017). 5. Amyotrophic lateral sclerosis (ALS) Ubiquitinated aggregates in ASL are mainly formed by the
accumulation of abnormally modified and cleavage forms of TDP-43
that cannot be efficiently cleared by either autophagy or the
proteasome system (Chen et al., 2012). Aberrant TDP-43 impairs its
ubiquitination leading to dysfunctional protein clearance and
destabilizing Atg7 mRNA impairing autophagosome formation
(Amin et al., 2020). Mutant forms of other proteins also alter the
autophagy machinery at different stages. For example, mutant FUS,
VAPB, C9orf72, and CHMP2B affect autophagosome formation,
mutant OPTN, TBK1, CHMP2B, PFN1, and DCTN impair retrograde
transport for autolysosome formation, mutant C9orf72, OPTN, and
CHMP2B disrupt autolysosome formation, and mutant p62, OPTN,
and UBQLN2 affect cargo degradation (Amin et al., 2020). l
A useful approach to studying CMA combines the
downregulation of pivotal key players in CMA such as Hsc70 and
LAMP2A with overexpression of CMA substrates such as
hexokinase 2 (HK2), an enzyme that participates in glucose
metabolism, which is targeted by the CMA machinery for
degradation (Xu et al., 2021). This reporter system is CMA-specific
and suitable for high-throughput screening and is characterized by
HEK293 cells modified to express the HK2-GFP complex in
doxycycline (DOX), a second-generation tetracycline that activates
autophagy,-inducible manner to obtain 293THK cells (Xing et al.,
2017; Xu et al., 2021). The system allows the identification of CMA
activation and substrate degradation using immunofluorescence,
Western blot, qRT-PCR, and flow cytometry (Xu et al., 2021). APP
degradation as well as CMA induction, cargo recognition and
translocation can be assessed in SH-SY5Y cells expressing Hsc70-
and LAMP2A-Flag treated with CMA inducers and lysosomal
inhibitors such as E-64D, BafA1, leupeptin, ammonium chloride,
and the protease inhibitor MG132 in combination with the
detection of the accumulation of AB using immunocytochemistry
and Western blot (Xu et al., 2021). A cell-engineering approach to generate an AD model to study
the mechanism of microautophagy involves primary neuronal cells
that overexpress VPS4A (Willen et al., 2017). VPS4A is an enzyme
that regulates endosomal sorting complexes (ESCRTs) responsible
for membrane modeling and topology processes such as the
formation of late endosomal MVBs (Willen et al., 2017; Rodger
et al., 2020). Overexpression of VPS4A mimics AD hallmarks such
as the accumulation and aggregation of AB in MVBs and enlarged
late endocytic compartments (Willen et al., 2017). In this in vitro 15 frontiersin.org Sánchez-Vidaña et al. 10.3389/fnmol.2023.1168948 Frontiers in Molecular Neuroscience 5.4. Autophagy in vitro methods in ALS
research TDP-43 C-terminal fragments such as TDP-25 and TDP-35 exhibit
pathogenic properties as observed in diseased brains (Wang et al.,
2010). These TDP-43-derived fragments of lower molecular weight
are also prone to aggregation and worth attention in cell-based
platforms (Wang et al., 2010). A cell model to monitor the
degradation of TDP-43 fragments through macroautophagy frontiersin.org 5.3. Chaperone-mediated autophagy in
ALS The involvement of CMA in ALS is quite poorly understood
despite the insight into the contribution of dysfunctional autophagy
at large in the pathogenesis of the disease (Lee et al., 2015). Of the
many ALS-associated protein aggregates, TDP-43 is the most studied
in terms of its correlation with CMA activity (Liao et al., 2021). It has
been found that the TDP-43 protein contained the CMA-recognition
motif sequence Q134VKKD138, a KFERQ-like motif, suggesting that
TDP-43 could be a substrate for CMA (Huang et al., 2014). Mutation
of the KFERQ-like motif in the TDP-43 coding gene inhibited the
CMA degradation of TDP-43 and led to the aggregation of TDP-43. By immunoprecipitation, it was demonstrated that TDP-43 proteins
with the KFERQ-like motif in transfected N2a cells interacted with the
molecular chaperone Hsc70, which facilitated the degradation of the 16 frontiersin.org osis research. line
Application (A), description (D), and readout (R)
References
4 cells
neurons
ntiated from
man
onic stem cells
s)
A: Monitoring autophagy at different stages using cells expressing mutant SOD1 and mutant TDP-43 and validation in hESC-
derived spinal motor neurons. D: NSC-34 cells expressing mutant SOD1 and TDP-43 were treated with the autophagy activator rilmenidine and autophagy
inhibitors bafilomycin A1 (BafA1) and rotenone. MA and CMA markers, autophagic flux, were evaluated to assess autophagy
induction. NSC-34 cells were engineered to obtain a mCherry-GFP-LC3B reporter system to assess autophagic flux. The fusion
of the mitochondria with lysosomes was analyzed to assess mitophagy in cells engineered with the ratiometric pHluorin
DsRed-T3 to evaluate the percentage of DsRed-T3-positive mitochondria fused with lysosomes. hESC-derived spinal motor
neurons were engineered to express wild-type SOD1 and treated with an autophagy inducer. R: (1) Protein expression by Western blot targeting LC3I and LC3II, p62, LAMP2A, and Hsc70. (2) Autophagosome and
autolysosome fusion by confocal microscopy analysis of mature autolysosomes. (3) Fusion of the mitochondria with lysosomes
by confocal microscopy to identify DsRed.T3-positive mitochondria. (4) Autophagic flux using a tandem mCherry-GFP-LC3B
reporter construct to identify mature autolysosomes. Perera et al. (2017)
4 cells
A: Identification of SOD1 and TDP-43 aggregates in cells overexpressing mutant SOD1. D: Cells were engineered to overexpress SOD1 and treated with the autophagy inductor trehalose. The presence of SOD1 and
TDP-43 aggregates was assessed. R: (1) Protein expression by Western blot targeting SOD1 and immunofluorescence targeting TDP-43 and ubiquitin. Gomes et al. lin
34 ce
r neu
entia
uman
yonic
Cs) 6.1. Macroautophagy in HD Alterations in the macroautophagy pathway and decreased
macroautophagy-mediated clearance have been associated with the
pathogenesis of HD (Bar-Yosef et al., 2019). Macroautophagy
contributes to the elimination of mutant huntingtin aggregates, a
process also known as aggregophagy (Croce and Yamamoto, 2019). Although the mechanisms for macroautophagy-mediated clearance
of aggregates remain elusive, key mediators such as beclin 1, p62,
Nbr1, Optn, and Alfy have been implicated in the mechanism of
mutant huntingtin aggregate degradation (Croce and Yamamoto,
2019). At the cargo recognition level, mutant huntingtin interacts
with p62 in HD cells causing problems in efficiently recognizing
and engulfing the cytosolic cargo for degradation leading to the
accumulation of toxic cellular components (Nah et al., 2015). In
the normal cargo recognition process, the cargo gets sequestered
by p62 binding to Atg8 cytosolic orthologues to bring the emerging
autophagy membrane to the aggregate structure (Croce and
Yamamoto, 2019). Other proteins such as LC3B and Alfy are also
needed at this stage. Alfy binds to p62-tagged inclusions and the
complex formed by the autophagy proteins Atg5, Atg12, and Atg16
to facilitate the conjugation of Atg8 proteins to the autophagosome
membrane (Croce and Yamamoto, 2019). Furthermore, the beclin
1-complex involved in the autophagy initiation phase is affected
because beclin 1 gets recruited by mutant huntingtin aggregates
(Nah et al., 2015). In turn, the lack of autophagy initiation causes
further accumulation of mutant huntingtin leading to neuronal
death (Nah et al., 2015). The continuous accumulation of
aggregates as a result of decreased macroautophagy-mediated
clearance activates cell death pathways through apoptosis or
autophagy leading to a dramatic loss of cells (Bar-Yosef et al., 2019). 6. Huntington’s disease (HD) First described by George Huntington in 1872, HD is an
autosomal dominant progressive neurodegenerative disorder
characterized by loss of motor control, cognitive impairment,
bradykinesia, rigidity, muscle wasting, weight loss, and eventually
death (Nah et al., 2015; Bar-Yosef et al., 2019; Stoker et al., 2022). Other symptoms in HD include ticks and impaired gait and postural
stability that manifest as a result of dystonia, rigidity, chorea, and
ataxia (Stoker et al., 2022). The onset of symptoms occurs between
35 and 50 years (Bar-Yosef et al., 2019). The prevalence of HD is
10.6–13.7 cases per 100,000 in Western populations while a lower
prevalence, 1–7 cases per million, has been reported in Eastern
populations such as Japan, Taiwan, and Hong Kong (McColgan and
Tabrizi, 2018). HD is caused by the production of a mutant form of
huntingtin protein (Nah et al., 2015). Huntingtin is a large
scaffolding protein that has many interaction sites (Ghosh and
Tabrizi, 2018). In HD, a mutation in huntingtin makes it interact
with other proteins in an aberrant way leading to the disruption of
cellular functions (Ghosh and Tabrizi, 2018). The presence of
mutant huntingtin causes changes in its conformation that make it
prone to form aggregates which affect several neuronal mechanisms
such as cellular proteostasis, axonal transport, transcription,
translation, mitochondrial and synaptic function and ultimately
lead to neuronal death (Nah et al., 2015; Ghosh and Tabrizi, 2018; 5.3. Chaperone-mediated autophagy in
ALS Mutant huntingtin is particularly
damaging to medium spiny neurons of the striatum, which play a
critical role in the regulation of signals from the cortex to output
pathways, leading to the clinical manifestations observed in HD
(Pan and Feigin, 2021). Although autophagy is the mechanism
responsible for the clearance of mutant huntingtin, the presence of
this aberrant protein affects the autophagy process (Nah et al.,
2015). Therefore, the accumulation of aberrant huntingtin leads to
inefficient autophagy contributing to HD pathogenesis (Nah
et al., 2015). In vitro platforms focusing on the CMA degradation process
monitor TDP-43 recognition using cell systems that modify the
TDP-43 site in a fast-growing mouse neuroblastoma cell line, N2a
(Huang et al., 2014). In this cell model, N2a cells are engineered to
modify the KFERQ-like motif recognition site present in TDP-43
leading to assessing the interaction of this aberrant protein with
Hsc70 for substrate recognition in CMA (Huang et al., 2014). The
use of lysosome inhibitors (e.g., 3-MA and ammonium chloride)
and cells with downregulated expression of LAMP2A in parallel
with the analysis of TDP-43-derived fractions and the CMA key
players LAMP2A and Hsc70 are also approaches to assess the CMA
degradation of TDP-43 (Huang et al., 2014). Other systems
targeting TDP-43 degradation by CMA use patient-derived cells
and cells in combination with other cell lines commonly used in
neurodegeneration research such as SH-SY5Y cells (Arosio et al.,
2020). For instance, the accumulation of TDP-43 as well as gene and
protein expression of CMA markers can be evaluated in
immortalized lymphoblastoid cells obtained from peripheral blood
mononuclear cells isolated from sporadic ALS patients (Arosio
et al., 2020). A filter retardation assay using a Bio-Dot SF
microfiltration system to conduct Dot Blot analysis can be employed
to measure the presence of insoluble TDP-43 (Arosio et al., 2020). Downregulation of Hsc70 in SH-SY5Y cells is a useful approach to
evaluate the role of Hsc70 in TDP-43 degradation (Arosio
et al., 2020). Frontiers in Molecular Neuroscience 5.3. Chaperone-mediated autophagy in
ALS (2010)
93 cells
A: Monitoring the degradation of TDP-43 in TDP-43 and TDP-25 overexpressing cells. D: Cells were engineered to overexpress TDP-43 and TDP-25 in a green fluorescence protein system. Then, cells were treated
with the autophagy inducer trehalose and autophagy inhibitor 3-methyladenine. Cells were further modified to tag LC3 with a
red fluorescent protein and treated with the same autophagy inducer and inhibitor to assess MA-mediated substrate
degradation. R: (1) Protein expression by Western blot, immunofluorescence, and immunoprecipitation targeting TDP-43, TDP-25, and
ubiquitin. Wang et al. (2010)
ells
A: Evaluation of TDP-43 degradation through the CMA system by modifying the CMA recognition motif in TDP-43. D: Cells were modified to express a mutation in the KFERQ-like motif of TDP-43. The interaction of the mutant TPD-43 and
Hsc70 was assessed. Cells were treated with the lysosome inhibitor ammonium chloride and 3-methyladenine (3-MA). Also,
LAMP2A was knocked down to assess CMA-mediated degradation. R: (1) Protein expression by Western blot and immunofluorescence targeting TDP-43, TDP-35, TDP-25, LAMP2A, and Hsc70. Huang et al. (2014)
ry
omonocytes of
dic ALS
ts,
oblastoid cell
erived from
dic ALS
ts, and SH-
cells
A: Monitoring the expression of TDP-43 and Hsc70 activity in an ALS model consisting of cells engineered to silence Hsc70 to
assess CMA-mediated degradation of TDP-43. D: Peripheral blood mononuclear cells from sporadic ALS patients were used to obtain immortalized lymphoblastoid cells. The
expression of Hsc70, LAMP2A, Bag1, Bag3, and LPD-43 was measured in both cell lines to assess CMA and LPD-43
accumulation. The expression of Hsc70 was downregulated in SH-SY5Y cells to assess the participation of Hsc70 in TDP-43
degradation. R: (1) Gene and (2) protein expression assessed by qRT-PCR and Western blot, respectively, targeting Hsc70, LAMP2A, and
co-chaperones Bag1 and Bag3. Substrate degradation was evaluated by immunofluorescence analysis targeting TDP-43. (3)
Identification of insoluble TDP-43 by filter retardation assay using a Bio-Dot SF microfiltration apparatus and Dot Blot analysis. Arosio et al. (2020) Sánchez-Vidaña et al. 10.3389/fnmol.2023.1168948 Sánchez-Vidaña et al. involves HEK293 cells engineered to overexpress TDP-43 and
TDP-25 using a green fluorescent protein system (Wang et al.,
2010). Using autophagy inhibitors and activators, autophagic flux
and substrate degradation are evaluated by Western blot,
immunofluorescence,
and
immunoprecipitation
(Wang
et al., 2010). McColgan and Tabrizi, 2018). 6.3. Chaperone-mediated autophagy in HD Another cell system includes
medium sized-spiny neurons and cortico-striatal slice cultures
treated with the autophagy inhibitors AZD8055 and BafA1 followed
by the evaluation of mutant huntingtin degradation by
immunohistochemistry and Western blot (Proenca et al., 2013). Another approach to identifying the role of key autophagy players
in mutant huntingtin degradation is to engineer PC12 cells to
downregulate the expression of CMA markers Hsc70 and LAMP2A
and measure the accumulation of mutant huntingtin by
immunofluorescence, immunoprecipitation, and Western blot (Qi
et al., 2012). Modifying cells to express mutant huntingtin and
manipulating the expression of key autophagy mediators contribute
to the mechanistic understanding of mutant huntingtin degradation. Cell engineering can be even extended to molecular players outside
the autophagy machinery to identify potential therapeutic targets. For instance, mouse striatal cell lines expressing mutant human
huntingtin and STHdh cells can be modified to overexpress the
miRNA lethal-7b (let7b), a member of the let7 family responsible
for the regulation of cell differentiation processes in neural stem
cells (Choi and Cho, 2021; Chan et al., 2022). In this cell system, the
role of let7 on CMA can be evaluated by monitoring CMA markers
and mutant huntingtin accumulation in cells overexpressing let7b
(Choi and Cho, 2021). The chaperone Hsc70 is highly selective in recognizing the
KFERQ-like motif in a protein substrate destined for degradation
by CMA (Kirchner et al., 2019). Hence, the direct identification of
KFERQ-like motifs in protein aggregates indicates the compromise
of the CMA. The normal huntingtin protein has two KFERQ-like
motifs: one at amino acid 99–103 (KDRVN), and the other at
248–252 (NEIKV). The 99-KDRVN-103 motif in the huntingtin
protein functionally interacts with Hsc70, resulting in the delivery
of the huntingtin protein to lysosomes for degradation by CMA
(Fred Dice, 1990). In HD, the mutant huntingtin protein contains
abnormally extended polyglutamine (polyQ) repeats, which are
prone to fragmentation, misfolding, and aggregation in the basal
ganglia and cortex (Cortes and La Spada, 2014). The polyQ-
expanded mutant huntingtin fragments are also degraded by CMA,
except that its degradation is relatively much slower leading to
protein aggregation (Qi et al., 2012). The mutant huntingtin protein
contained KFERQ-like motifs, and by manipulating the Hsc70 and
LAMP2A levels, it was possible to promote mutant huntingtin
degradation through CMA (Qi et al., 2012). The huntingtin-552
(Htt-552) overexpression model of HD was generated by adenoviral
transfection of PC12 cells. 6.3. Chaperone-mediated autophagy in HD The role of CMA in the degradation of
Htt-552 was determined by overexpression or silencing of Hsc70
and LAMP2A. While the above evidence may suggest a decline in
CMA activity in HD, rather studies have reported the constitutive
upregulation of CMA in HD (Koga et al., 2011; Choi and Cho,
2021). CMA activity increases as a compensatory response to
impaired macroautophagy in the early stages of HD. In cellular and
mouse models of HD, LAMP2A, and Hsc70 were markedly
increased (Koga et al., 2011). In another HD model with extended
polyQ in striatal neurons, LAMP2A levels became elevated,
inducing neuroinflammation. These results suggest that LAMP2A
related to CMA capacity might play an important role in HD onset
and progression (Choi and Cho, 2021). Thus, these pieces of
evidence that (1) levels of mutant huntingtin fragments could
be reduced by the upregulation of the CMA biomarkers LAMP2A
and Hsc70; and (2) the observation of spontaneous changes in these
biomarkers as the disease progresses give a strong justification to
link CMA with HD. A critical step in mutant huntingtin degradation is substrate
recognition by the autophagy machinery. Modifications can
be added to mutant huntingtin to express a series of truncation
mutants in primary and COS-7 cells (Ehrnhoefer et al., 2018). In
this macroautophagy cell-based approach, the interaction of
mutant huntingtin forms with p62 can be monitored by
co-immunoprecipitation (Ehrnhoefer et al., 2018). In HD studies
focusing on macroautophagy, autophagic flux is monitored using
different techniques such as protein expression by Western blot
targeting LC3II and LC3I, immunostaining of LC3, or using the
novel tandem fluorescent-tagged mCherry-GFP-LC3 system
(Proenca et al., 2013; Ehrnhoefer et al., 2018). In studies focusing
on CMA autophagy in HD, traditional methods to monitor CMA
markers such as Western blot or qRT-PCR are commonly applied
(Koga et al., 2011; Qi et al., 2012; Choi and Cho, 2021). However,
novel methods have been developed to assess CMA at the
lysosomal level. For example, lysosomal integrity, purity, and
analysis of the lysosomal content can be conducted using
subcellular fractionation with a self-Percoll gradient followed by
the analysis of the enzymatic activity of the lysosomal enzyme
ß-hexosaminidase and detection of LAMP2A, huntingtin, and
Hsc70 by Western blot (Qi et al., 2012). A recently developed
method allows the evaluation of cargo delivery to the lysosome
using a photoswitchable reporter KFERQ-PS-CFP2 system that can
be tracked by analyzing intracellular immunofluorescence (Choi
and Cho, 2021). Frontiers in Molecular Neuroscience 6.2. Microautophagy in HD One particular form of microautophagy, RN/DNautophagy, a
degradation mechanism responsible for the direct uptake of RNA and
DNA into lysosomes, has been reported as closely implicated in HD
(Martin-Aparicio et al., 2001; Wang et al., 2023). Specifically, SID1
transmembrane family member 2 (SIDT2) can bind to exon 1 of the
polyglutamine-expanded huntingtin, one of the hallmarks of HD
pathology (Martin-Aparicio et al., 2001). Overexpression of SIDT2
can lead to the degradation of huntingtin mRNA and further reduce
the level of the huntingtin complex (Hase et al., 2020). Currently, the
biological functions of microautophagy and its role in the pathogenesis
of HD are rarely reported. 18 frontiersin.org 10.3389/fnmol.2023.1168948 Sánchez-Vidaña et al. 6.3. Chaperone-mediated autophagy in HD Proenca et al., 2013). To identify specific autophagy key players and
autophagy stages that are crucial for mutant huntingtin degradation,
cell-based methods are designed to measure the accumulation of
mutant huntingtin after treatment with different autophagy
inhibitors. For instance, accumulation of mutant huntingtin in cells
treated with autophagy inhibitors such as the lysosomal proteolysis
disruptors ammonium chloride and leupeptin and the autophagy
induction
inhibitor
3-MA
can
be
monitored
using
immunofluorescence in both cells isolated from HD patients and
mouse cells (Koga et al., 2011). Another cell system includes
medium sized-spiny neurons and cortico-striatal slice cultures
treated with the autophagy inhibitors AZD8055 and BafA1 followed
by the evaluation of mutant huntingtin degradation by
immunohistochemistry and Western blot (Proenca et al., 2013). Another approach to identifying the role of key autophagy players
in mutant huntingtin degradation is to engineer PC12 cells to
downregulate the expression of CMA markers Hsc70 and LAMP2A
and measure the accumulation of mutant huntingtin by
immunofluorescence, immunoprecipitation, and Western blot (Qi
et al., 2012). Modifying cells to express mutant huntingtin and
manipulating the expression of key autophagy mediators contribute
to the mechanistic understanding of mutant huntingtin degradation. Cell engineering can be even extended to molecular players outside
the autophagy machinery to identify potential therapeutic targets. For instance, mouse striatal cell lines expressing mutant human
huntingtin and STHdh cells can be modified to overexpress the
miRNA lethal-7b (let7b), a member of the let7 family responsible
for the regulation of cell differentiation processes in neural stem
cells (Choi and Cho, 2021; Chan et al., 2022). In this cell system, the
role of let7 on CMA can be evaluated by monitoring CMA markers
and mutant huntingtin accumulation in cells overexpressing let7b
(Choi and Cho, 2021). Proenca et al., 2013). To identify specific autophagy key players and
autophagy stages that are crucial for mutant huntingtin degradation,
cell-based methods are designed to measure the accumulation of
mutant huntingtin after treatment with different autophagy
inhibitors. For instance, accumulation of mutant huntingtin in cells
treated with autophagy inhibitors such as the lysosomal proteolysis
disruptors ammonium chloride and leupeptin and the autophagy
induction
inhibitor
3-MA
can
be
monitored
using
immunofluorescence in both cells isolated from HD patients and
mouse cells (Koga et al., 2011). 6.4. Autophagy in vitro methods in HD
research The hallmark in HD is the presence of mutant huntingtin that
is prone to aggregation disrupting crucial cellular processes that are
relevant for neuronal survival (Ghosh and Tabrizi, 2018). Because
of the damaging nature of mutant huntingtin aggregates, HD cell-
based methods primarily focus on monitoring the autophagy-
mediated molecular mechanisms of mutant huntingtin degradation. A selection of cell-based methods used to investigate autophagy in
HD is shown in Table 4. In vitro HD models mainly target the
mechanism of macroautophagy and CMA.h The most commonly used model is the overexpressing human
mutant huntingtin in a wide variety of cells including embryonic
fibroblasts, COS-7, medium sized-spiny neurons, cortico-striatal
slice cultures, and PC12 cells (Koga et al., 2011; Qi et al., 2012; 19 frontiersin.org Application (A), description (D), and readout (R)
References
A: Monitoring the interaction of mutant huntingtin with autophagy markers in primary cells engineered
to overexpress mutant huntingtin and assessment of autophagic flux. D: Primary cells derived from mouse cell models were generated to determine the effect on autophagy
of cleavable or C6R mutant huntingtin. Cells were treated with bafilomycin A1 (BafA1) to inhibit
autophagic flux. Cells were modified to express a series of truncation mutants and treated with BafA1 to
assess the interaction between mutant huntingtin and p62. Cos-7 cells were modified to express
cleavable or a C6R mutant huntingtin form and p62 to assess huntingtin degradation. R: (1) Protein expression by Western blot (targeting LC3II, LC3I, and p62) and immunostaining (LC3
and p62) to measure the expression of autophagy markers. (2) Gene expression of p62 was evaluated by
qRT-PCR. (3) Protein–protein interaction (mutant huntingtin forms and p62) by co-
immunoprecipitation. Ehrnhoefer et al. (2018)
A: Monitoring of autophagy induction and identification of Atg4b-dependent autophagic flux in cells
prone to aggregation of mutant huntingtin and engineered to overexpress Atg4B. D: Cells were treated with an mTOR inhibitor (AZD8055) and a lysosomal inhibitor (BafA1) to evaluate
the accumulation of mutant huntingtin. A tandem fluorescent-tagged mCherry-GFP-LC3 system was
used to monitor autophagic flux in the presence of BafA1. R: (1) Protein expression by Western blot targeting, LC3II, LC3I, Atg4B, and p62. (2) Substrate
accumulation by immunohistochemistry and Western blot analyses targeting mutant huntingtin. (3)
Autophagic flux by immunofluorescence (LC3). Proenca et al. (2013)
A: Monitoring of mutant huntingtin degradation through CMA and MA using engineered and HD
cells. 6.4. Autophagy in vitro methods in HD
research D: Fibroblasts engineered to express mutant human huntingtin, as well as human lymphoblasts, and
primary striatal cultures were treated with the autophagy inhibitors ammonium chloride and leupeptin
to assess inhibition of lysosomal proteolysis relevant to the CMA pathway and 3-methyladenine to
Koga et al. (2011) y
p
y
p
y
y
assess MA-mediated autophagy. CMA activity was assessed in control and HC cells using a
photoswitchable reporter (KFERQ-PS-CFP2) to track cargo delivery to the lysosome. R: (1) Protein expression by immunofluorescence targeting mutant huntingtin. (2) Gene expression by
qRT-PCR targeting LAMP2A and Hsc70. (3) Cargo delivery to the lysosome using the photoswitchable
reporter KFERQ-PS-CFP2 to analyze intracellular immunofluorescence. (Continued) Application (A), description (D), and readout (R)
References
: Monitoring the interaction of mutant huntingtin with autophagy markers in primary cells engineered
o overexpress mutant huntingtin and assessment of autophagic flux. D: Primary cells derived from mouse cell models were generated to determine the effect on autophagy
f cleavable or C6R mutant huntingtin. Cells were treated with bafilomycin A1 (BafA1) to inhibit
utophagic flux. Cells were modified to express a series of truncation mutants and treated with BafA1 to
ssess the interaction between mutant huntingtin and p62. Cos-7 cells were modified to express
eavable or a C6R mutant huntingtin form and p62 to assess huntingtin degradation. : (1) Protein expression by Western blot (targeting LC3II, LC3I, and p62) and immunostaining (LC3
nd p62) to measure the expression of autophagy markers. (2) Gene expression of p62 was evaluated by
RT-PCR. (3) Protein–protein interaction (mutant huntingtin forms and p62) by co-
mmunoprecipitation. Ehrnhoefer et al. (2018)
: Monitoring of autophagy induction and identification of Atg4b-dependent autophagic flux in cells
rone to aggregation of mutant huntingtin and engineered to overexpress Atg4B. D: Cells were treated with an mTOR inhibitor (AZD8055) and a lysosomal inhibitor (BafA1) to evaluate
he accumulation of mutant huntingtin. A tandem fluorescent-tagged mCherry-GFP-LC3 system was
sed to monitor autophagic flux in the presence of BafA1. : (1) Protein expression by Western blot targeting, LC3II, LC3I, Atg4B, and p62. (2) Substrate
ccumulation by immunohistochemistry and Western blot analyses targeting mutant huntingtin. (3)
utophagic flux by immunofluorescence (LC3). Proenca et al. (2013)
: Monitoring of mutant huntingtin degradation through CMA and MA using engineered and HD
ells. 7. Depression these molecules stimulate the macroautophagy machinery at the
initiation phase through the activation of protein phosphatase 2A,
Ulk1, beclin 1, and LC3B (Rein, 2019). Other studies have
demonstrated a reduction in autophagy markers after induction of
depressive-like behavior such as beclin 1, LC3II, and LC3I suggesting
dysfunctional mechanisms in the induction and autophagosome
formation stages (Pierone et al., 2020). Furthermore, the Hsp90
cochaperone FKBP5, a regulator of the NR3C1/glucocorticoid
receptor, has been associated with antidepressant treatment response
through the inhibition of Akt, which controls the activity of beclin 1
leading to increased levels of beclin 1, LC3II, LC3I, and Atg12 (Gassen
et al., 2015). A positive correlation between the levels of FKBP5 and
beclin 1, LC3II, LC3I, and Atg12 and a negative correlation with the
phosphorylation of Akt1 suggesting that FKBP5 acts as an autophagy
inducer (Gassen et al., 2015). To date, evidence on autophagy
regulation in depression shows conflicting results (Zhang et al., 2020)
suggesting that the mechanism to maintain the autophagy balance in
depression is a dynamic process. For instance, depression-like
behavior induced by chronic unpredictable mild stress could activate
or inhibit the macroautophagy pathway through beclin 1 and LC3
(Zhang et al., 2020). In other animal models of depression, these
macroautophagy markers may behave differently. Upregulation of
beclin 1 and LC3 have been observed in depression-like behavior
induced by electroconvulsive shot and downregulation occurs in
pain-, bacterial endotoxin lipopolysaccharide-, and maternal
separation-induced depression-like behavior (Pierone et al., 2020;
Zhang et al., 2020). The conflicting results suggest that the autophagic
response varies depending on the different stimuli and treatments in
depression (Zhang et al., 2020). At the autolysosome level, the number
and size of autolysosomes were higher after induction of depression-
like behavior by chronic unpredictable mild stress compared to the
treatment with serotonin selective reuptake inhibitors (Zhang
et al., 2020). Major depressive disorder is a severe psychiatric illness that limits
psychosocial functioning and diminishes the quality of life (Malhi and
Mann, 2018). This multifactorial disorder significantly affects up to
10% of the global population and represents a tremendous economic
burden to society (Jia and Le, 2015; Alcocer-Gómez et al., 2017). The
lifetime risk of depression is high with estimations indicating that one
in every five people experiences an episode of depression at some
point in their lives (Malhi and Mann, 2018). 6.4. Autophagy in vitro methods in HD
research D: Fibroblasts engineered to express mutant human huntingtin, as well as human lymphoblasts, and
rimary striatal cultures were treated with the autophagy inhibitors ammonium chloride and leupeptin
o assess inhibition of lysosomal proteolysis relevant to the CMA pathway and 3-methyladenine to
ssess MA-mediated autophagy. CMA activity was assessed in control and HC cells using a
hotoswitchable reporter (KFERQ-PS-CFP2) to track cargo delivery to the lysosome. : (1) Protein expression by immunofluorescence targeting mutant huntingtin. (2) Gene expression by
RT-PCR targeting LAMP2A and Hsc70. (3) Cargo delivery to the lysosome using the photoswitchable
eporter KFERQ-PS-CFP2 to analyze intracellular immunofluorescence. Koga et al. (2011)
(Continued) d readout (R)
References
sess the effect of increased let7b on CMA by
in expression by Western blot and
d mutant huntingtin. Choi and Cho (2021)
Hsc70 and LAMP2A levels in a cell model of HD
The contribution of MA degradation of mutant
untingtin and its degradation was monitored in cells
e CMA markers Hsc70 and LAMP2A. Starvation was
immunoprecipitation, and Western blot analysis
d beclin 1. (2) Lysosomal integrity, purity, and content
on with a self-Percoll gradient followed by the
enzyme ß-hexosaminidase, and the detection of
rn blot. Qi et al. (2012) Sánchez-Vidaña et al. 10.3389/fnmol.2023.1168948 7.1. Macroautophagy in depression Although the exact autophagy-modulating mechanisms of
antidepressants have not been fully elucidated, recent evidence shows
that macroautophagy induction occurs through antidepressant
treatment (Rein, 2019). For instance, fluoxetine and amitriptyline
cause the accumulation of sphingomyelin in the lysosomes and
ceramide in the endoplasmic reticulum (Rein, 2019). Subsequently, Frontiers in Molecular Neuroscience 7. Depression The prevalence of
depression is twice as common in women than in men with a peak in
prevalence observed around the second and third decade of life (Malhi
and Mann, 2018). Depression is characterized by depressed mood,
anhedonia, feelings of worthlessness, low self-esteem, fatigue, changes
in eating and sleeping patterns, and altered cognition (Alcocer-Gómez
et al., 2017; Bar-Yosef et al., 2019). The pathogenesis of depression is
still not well understood due to the complex multifactorial nature that
involves both genetic and environmental factors (Jia and Le, 2015). Consequently, several hypotheses including the monoamine,
hypothalamic–pituitary–adrenal (HPA) axis, neuroplasticity and
neurogenesis, and more recently the autophagy hypothesis have been
developed to understand the molecular mechanisms of the
pathogenesis of depression (Malhi and Mann, 2018; Bar-Yosef et al.,
2019). The first observations indicating that autophagy could
be implicated in depression were the upregulation of autophagy
markers (e.g., beclin 1 and LC3II) and activation of the autophagy
signaling pathway upon treatment with antidepressants (Jia and Le,
2015; Gassen and Rein, 2019). In patients with depression, high
circulating levels of beclin 1, phosphorylated Akt, and LC3II/LC3I
predicted improved clinical antidepressant effect suggesting that
antidepressants
stimulate
macroautophagy
induction
and
autophagosome formation (Rein, 2019). Also, gene expression studies
revealed higher expression of autophagy-related genes in blood
samples from patients with depression compared with healthy controls
(Zhang et al., 2020). Other studies have shown a positive correlation
between the expression of autophagy initiators such as beclin 1 and
clinical treatment effectiveness (Zhang et al., 2020). Further evidence
that suggested the participation of autophagy in depression was the
effect of antidepressants on the co-chaperone FKBP51, a regulator of
the NR3C1-glucocorticoid receptor involved in the stress response
and inhibitor of Akt1 that in turn regulates the activity of beclin 1 in
the autophagy signaling pathway (Gassen et al., 2015; Jia and Le,
2015). FKBP51 acts synergically with antidepressants by binding to
beclin 1 leading to its phosphorylation which activates the autophagy
machinery (Jia and Le, 2015). In animal models of depression, a
decrease in autophagy regulators such as Ulk1, AMPK, LC3II/LC3I
and an increase in p62 and phosphorylated mTOR suggest reduced
autophagy activation (Pierone et al., 2020). 7.2. Microautophagy in depression The potential role of microautophagy in the pathogenesis of
depression remains elusive as there are not many studies focused on
microautophagy and depression. Glucocorticoids are a class of steroid
hormones that are synthesized and secreted in response to stress
(Chan et al., 2017). Notably, glucocorticoids are reported to
be involved in the pathogenesis of depression by inhibiting
neurogenesis in the hippocampus (Chan et al., 2017). Recently, a study
showed that glucocorticoids impair both microautophagy and CMA
in primary cultured cortical neurons (Sato et al., 2020). Moreover,
another recent study reported that glucocorticoids inhibit the
degradation of synaptic vesicular proteins in an ESCRT-dependent
manner (Sheehan et al., 2016). These findings indicate that
glucocorticoid-induced impairment of microautophagy plays an
indispensable role in the pathogenesis of depression. 7.3. Chaperone-mediated autophagy in
depression In in vitro models of depression,
antidepressants are used to assess their effect on autophagy
mechanisms. SH-SY5Y and HEK293T cells engineered with mCherry-
GFP-LC3 were subjected to treatment with the antidepressant
amitriptyline, the autophagy activator rapamycin, and the autophagy
inhibitors MG132 and ammonium chloride followed by the analysis
of macroautophagy and CMA markers to monitor the autophagy
process at different stages (Kwon et al., 2020). depression is an abnormal brain response to normal hormone changes
(Nogrady, 2022). One of the hormones implicated is glucocorticoids,
which are released by the adrenal cortex for the regulation of various
physiological functions. Chronic stress upregulates glucocorticoid
levels, causing degeneration of hippocampal neurons in the central
nervous system and mental disorders such as depression (Conrad,
2008). In vitro, it was observed that glucocorticoids impaired CMA
and microautophagy (Sato et al., 2020). Following chronic treatment
of human-derived embryonic kidney cell line (AD293 cells) and
primary cultured rat cortical neurons with dexamethasone, a potent
glucocorticoid receptor agonist, it was found that CMA activity
declined due to a decrease in lysosomal Hsc70 (Sato et al., 2020). Since
glucocorticoids are involved in depression, understanding the impact
of these agents on CMA activity in neurons can provide an indirect
clue to the CMA mechanisms. Also, an endoplasmic reticulum
(ER)-stress model of depression was generated recently by
hippocampal injection of the antibiotic tunicamycin, which blocks
protein glycosylation to induce ER stress (Yuan et al., 2023). Behavioral
tests result from the open field test, and forced swimming test in the
tunicamycin-injected rats revealed depressive and anxiety-like
phenotypes within 8 days. It was determined that synaptic and
CMA-protein levels were significantly reduced in the hippocampus of
the rats, indicating that these pathways played an important role in the
ER-stress depressive rats (Yuan et al., 2023). The disruption of ER
homeostasis induces the accumulation of unfolded/misfolded
proteins, which in turn causes ER stress (Sicari et al., 2020). Hence, the
activation of CMA is a possible defense mechanism for restoring ER
homeostasis (Sicari et al., 2020). The evidence backs altered neuronal
proteostasis (i.e., protein homeostasis) as a core mechanism
underlying the effects of physiological stress on synaptic plasticity
(Yang et al., 2014; Martinelli et al., 2018). A terrified-sound stress
induced proteomic changes in adult male rat hippocampus (Yang
et al., 2014). 7.3. Chaperone-mediated autophagy in
depression The influence of hormonal changes as an important risk factor in
depressive-like behavior is starting to be understood. Data suggest that 22 frontiersin.org 10.3389/fnmol.2023.1168948 Sánchez-Vidaña et al. depression chemically induced conditions are immunolabeled against
LC3 (Kallergi et al., 2022). After treatment of the cells with autophagy
inhibitors such as wortmannin, a PI3K and Vasp34 inhibitor, and
SBI-0206965, a Ulk1 selective inhibitor, autophagy induction,
autophagosome formation, and flux can be monitored by measuring
autophagy mediators such as LC3, Atg13, Atg101, Atg5, Atg16L1E-10,
Atg9α, Ulk1, p62, and WIPI2 (Kallergi et al., 2022). The chemically
induced long-term depression in vitro system can be modified further
by engineering the cells to knock down autophagy regulators such as
Atg7 and express EGFP-LC3 for the assessment of autophagic flux
(Shehata et al., 2012). After treatment with the autophagy inhibitor
BafA1, the phosphorylated forms of Akt and mTOR can be detected
to monitor autophagy induction and quantitation of autophagosomes
and autolysosomes can be monitored by morphological analysis using
confocal microscopy targeting LC3 (Shehata et al., 2012). Another
platform used to study autophagy in depression-like conditions in
vitro involves the evaluation of KFBP51, a regulator of the
glucocorticoid receptor using HEK293 cells, mouse embryonic cells,
primary neurons, and astrocytes, and peripheral blood mononuclear
cells from healthy subjects and patients with depression under
antidepressant treatment (Gassen et al., 2014). In this depression
model, cells were engineered to knockout FKBP51 and Akt1/2 and
treated with different antidepressants, including paroxetine,
amitriptyline, and fluoxetine, and autophagy induction was assessed
by identifying autophagy markers and autophagic flux by monitoring
the LC3 fluorescence signal (Gassen et al., 2014). Also, FKBP51-
mediated induction of autophagy can be monitored by
co-immunoprecipitation to analyze the interaction between beclin 1
and FKBP51 and Akt (Gassen et al., 2014). A novel depression method
to explore the effect of glucocorticoids on autophagy uses the marker
of CMA and microautophagy activity known as glyceraldehyde
3-phosphate dehydrogenase fused with a HaloTag (GAPDH-HT) in a
human-derived cell line (AD293 cells) and cerebral cortex primary
neurons (Sato et al., 2020). This system relies on the use of
GAPDH-HT as a substrate of the CMA and microautophagy systems,
measurement of substrate accumulation by confocal microscopy, and
monitoring of CMA and microautophagy markers (Sato et al., 2020). In this model, lysosomes were isolated using a lysosome enrichment
kit and the lysosomal content was evaluated by a bicinchoninic acid
protein assay (Sato et al., 2020). 7.3. Chaperone-mediated autophagy in
depression Similarly, mimicking acute physiological stress in
SH-SY5Y cells with a synthetic glucocorticoid receptor agonist was
found to alter CMA activity and the levels of synaptic proteins
(Martinelli et al., 2018). Expectedly, the knockdown of LAMP2A or
the Hsp90 co-chaperone FKBP51 increased the stability of CMA
target proteins relevant to synaptic plasticity (Martinelli et al., 2018). These data highlight the significance of different forms of stress on
synaptic plasticity and neural activity and reveal the pivotal role of
CMA in regulating the neuronal protein changes underlying
depressive-like behaviors. Thus, the changes in the expression of
chaperones (including Hsc70) and LAMP2A under physiological
stress modulate the CMA activity in the central nervous system. Frontiers in Molecular Neuroscience 8. Conclusion To study the role of autophagy in depression, several in vitro
models have been developed and a list of approaches is shown in
Table 5. Cell-based platforms use glucocorticoid or chemically-
induced long-term depression settings as well as antidepressant
treatment to create an environment relevant to depression research. Autophagy is an important cellular process associated with the
pathophysiology of neurodegenerative diseases and mood
disorders. In this narrative review, cell-based models designed to
study the role of different mechanisms of autophagy in
neurodegenerative disorders and depression were presented and
discussed. It is now clear that the advances in in vitro systems offer
the possibility to manipulate different types of cells to exhibit A commonly used cell-based platform involved the chemical
induction of long-term depression in primary neurons (Shehata et al.,
2012; Kallergi et al., 2022). To assess the interplay and interdependence
of long-term depression and macroautophagy, cells under long-term 23 frontiersin.org on (A), description (D), and readout (R)
References
n of the interplay and interdependence between long-term depression and autophagy. m depression was chemically induced in neurons. Neurons were immunolabeled
Cells were treated with the autophagy inhibitor wortmannin which inhibits PI3K and
0206965 that selectively inhibits Ulk1. n expression by immunofluorescence targeting and Western blot targeting LC3, Atg13,
, Atg16L1E-10, Atg9α, Ulk1, p62, and WIPI2. Kallergi et al. (2022)
ion of the role of a regulator of the glucocorticoid receptor (FKBP51) on autophagy
ressant treatment using cells engineered to knockout KFBP51 and Akt1/2. s were engineered to express GFP-LC3 and treated with the autophagy inhibitor BafA1. s and mouse embryonic fibroblasts were engineered to knockout FKBP51 and Akt1/2
nteraction of FKBP51 with beclin 1. Mouse embryonic cells, HEK293, and astrocytes
with different antidepressants (e.g., paroxetine, amitriptyline, and fluoxetine). Cells
patients with depression were treated with fluoxetine. n expression by Western blot targeting beclin 1, p-beclin1, Atg12, LC3II, LC3I, P3K,
) Protein interaction was evaluated by co-immunoprecipitation (interaction between
beclin 1, and Akt and beclin 1). (3) Autophagic flux was evaluated by confocal
argeting LC3. Gassen et al. (2014)
ion of macroautophagy regulation by neuronal activity after neuronal stimulation by
g-term depression. treated with the autophagy inhibitor BafA1 in chemical long-term depression
combination with chemical long-term depression inhibitors. Cells were engineered to
the expression of Atg7 and to express EGFP-LC3B. n expression by Western blot targeting LC3, Akt, p-Akt, mTOR, p-mTOR, and Atg7. 8. Conclusion ion of autophagosomes and lysosomes and (4) cell morphology were conducted by
roscopy targeting LC3. Shehata et al. (2012) on (A), description (D), and readout (R)
References
of the interplay and interdependence between long-term depression and autophagy. m depression was chemically induced in neurons. Neurons were immunolabeled
Cells were treated with the autophagy inhibitor wortmannin which inhibits PI3K and
0206965 that selectively inhibits Ulk1. n expression by immunofluorescence targeting and Western blot targeting LC3, Atg13,
Atg16L1E-10, Atg9α, Ulk1, p62, and WIPI2. Kallergi et al. (2022)
ion of the role of a regulator of the glucocorticoid receptor (FKBP51) on autophagy
ressant treatment using cells engineered to knockout KFBP51 and Akt1/2. were engineered to express GFP-LC3 and treated with the autophagy inhibitor BafA1. and mouse embryonic fibroblasts were engineered to knockout FKBP51 and Akt1/2
nteraction of FKBP51 with beclin 1. Mouse embryonic cells, HEK293, and astrocytes
with different antidepressants (e.g., paroxetine, amitriptyline, and fluoxetine). Cells
patients with depression were treated with fluoxetine. n expression by Western blot targeting beclin 1, p-beclin1, Atg12, LC3II, LC3I, P3K,
) Protein interaction was evaluated by co-immunoprecipitation (interaction between
beclin 1, and Akt and beclin 1). (3) Autophagic flux was evaluated by confocal
argeting LC3. Gassen et al. (2014)
ion of macroautophagy regulation by neuronal activity after neuronal stimulation by
g-term depression. treated with the autophagy inhibitor BafA1 in chemical long-term depression
combination with chemical long-term depression inhibitors. Cells were engineered to
the expression of Atg7 and to express EGFP-LC3B. n expression by Western blot targeting LC3, Akt, p-Akt, mTOR, p-mTOR, and Atg7. ion of autophagosomes and lysosomes and (4) cell morphology were conducted by
roscopy targeting LC3. Shehata et al. (2012)
(Continued) description (D), and readout (R)
References
of an antidepressant on macroautophagy by monitoring protein aggregates
acroautophagy and CMA markers. th the antidepressant amitriptyline in combination with the autophagy
inhibitors MG132 and ammonium chloride. Autophagy markers and the
gregates were evaluated. Cells were engineered using the mCherry-GFP-
ophagic flux. HEK293T cells were engineered to express GFP-Arl8 and
ocation of the lysosome. Cells were treated with a PI3K inhibitor
MA induction. n by Western blot and immunostaining to identify p62, ubiquitin, LC3II,
Akt, p-mTOR, and beclin 1. (2) Lysosome translocation was evaluated by
d Western blot targeting KLC1, SKIP, and GFP. Kwon et al. (2020)f
ct of glucocorticoids on CMA and MiA in a human-derived cell line and
neurons. 8. Conclusion gineered to express glyceraldehyde 3-phosphate dehydrogenase fused with a
a marker for CMA and MiA activity, and to knockdown the expression of
as used to activate microautophagy. Cells were treated with the
agonist dexamethasone to decrease CMA and MiA. GAPDH-HT was assessed by confocal microscopy. (2) Lysosomal isolation
sosome enrichment kit. (3) Protein concentration of lysosomal lysates was
honinic acid protein assay. (4) Protein expression by Western blot targeting
70, MEF2D, and the HaLo tag. Sato et al. (2020) description (D), and readout (R)
Referencesf
t of an antidepressant on macroautophagy by monitoring protein aggregates
acroautophagy and CMA markers. ith the antidepressant amitriptyline in combination with the autophagy
d inhibitors MG132 and ammonium chloride. Autophagy markers and the
gregates were evaluated. Cells were engineered using the mCherry-GFP-
utophagic flux. HEK293T cells were engineered to express GFP-Arl8 and
location of the lysosome. Cells were treated with a PI3K inhibitor
MA induction. n by Western blot and immunostaining to identify p62, ubiquitin, LC3II,
-Akt, p-mTOR, and beclin 1. (2) Lysosome translocation was evaluated by
nd Western blot targeting KLC1, SKIP, and GFP. Kwon et al. (2020)f
ect of glucocorticoids on CMA and MiA in a human-derived cell line and
y neurons. gineered to express glyceraldehyde 3-phosphate dehydrogenase fused with a
), a marker for CMA and MiA activity, and to knockdown the expression of
was used to activate microautophagy. Cells were treated with the
agonist dexamethasone to decrease CMA and MiA. GAPDH-HT was assessed by confocal microscopy. (2) Lysosomal isolation
ysosome enrichment kit. (3) Protein concentration of lysosomal lysates was
choninic acid protein assay. (4) Protein expression by Western blot targeting
c70, MEF2D, and the HaLo tag. Sato et al. (2020) Sánchez-Vidaña et al. 10.3389/fnmol.2023.1168948 organization of the information in the manuscript and scientifically,
revised, and approved the submission of the manuscript. organization of the information in the manuscript and scientifically,
revised, and approved the submission of the manuscript. relevant disease hallmarks and modify the expression of key
autophagy players to bring to light the mechanistic relationship
between disease biology and autophagy. Furthermore, cell-based
models that combine the use of autophagy activators and
inhibitors are available to provide helpful insights into the role of
aberrant autophagy. The catalog of in vitro models applied to
autophagy research of neurodegenerative disorders and depression
include a repertoire of traditional and novel approaches and
techniques. References Cetin, S., Knez, D., Gobec, S., Kos, J., and Pišlar, A. (2022). Cell models for Alzheimer’s
and Parkinson’s disease: At the interface of biology and drug discovery. Biomed. Pharmacother. 149:112924. doi: 10.1016/j.biopha.2022.112924 Albanese, F., Novello, S., and Morari, M. (2019). Autophagy and LRRK2 in the aging
brain. Front. Neurosci. 13:1352. doi: 10.3389/fnins.2019.01352 Albanese, F., Novello, S., and Morari, M. (2019). Autophagy and LRRK2 in the aging
brain. Front. Neurosci. 13:1352. doi: 10.3389/fnins.2019.01352 Alcocer-Gómez, E., Casas-Barquero, N., Williams, M. R., Romero-Guillena, S. L.,
Cañadas-Lozano, D., Bullón, P., et al. (2017). Antidepressants induce autophagy
dependent-NLRP3-inflammasome inhibition in major depressive disorder. Pharmacol. Res. 121, 114–121. doi: 10.1016/j.phrs.2017.04.028 Chan, J. N.-M., Lee, J. C.-D., Lee, S. S. P., Hui, K. K. Y., Chan, A. H. L., Fung, T. K.-H.,
et al. (2017). Interaction effect of social isolation and high dose corticosteroid on
neurogenesis and emotional behavior. Front. Behav. Neurosci. 11:18. doi: 10.3389/
fnbeh.2017.00018 Alvarez-Erviti, L., Seow, Y., Schapira, A. H., Rodriguez-Oroz, M. C., Obeso, J. A., and
Cooper, J. M. (2013). Influence of microRNA deregulation on chaperone-mediated
autophagy and α-synuclein pathology in Parkinson’s disease. Cell Death Dis. 4:e545. doi:
10.1038/cddis.2013.73 Alvarez-Erviti, L., Seow, Y., Schapira, A. H., Rodriguez-Oroz, M. C., Obeso, J. A., and
Cooper, J. M. (2013). Influence of microRNA deregulation on chaperone-mediated
autophagy and α-synuclein pathology in Parkinson’s disease. Cell Death Dis. 4:e545. doi:
10.1038/cddis.2013.73 Chan, J. N.-M., Sánchez-Vidaña, D. I., Anoopkumar-Dukie, S., Li, Y., and Benson
Wui-Man, L. (2022). RNA-binding protein signaling in adult neurogenesis. Fron. Cell
Dev. Biol. 10:982549. doi: 10.3389/fcell.2022.982549 Chen, C. S., Chen, W. N., Zhou, M., Arttamangkul, S., and Haugland, R. P. (2000). Probing the cathepsin D using a BODIPY FL-pepstatin A: Applications in fluorescence
polarization and microscopy. J. Biochem. Biophys. Methods 42, 137–151. doi: 10.1016/
s0165-022x(00)00048-8 Alzheimer’s Association (2022). 2022 Alzheimer’s disease facts and figures. Alzheimer’s
Dement. 18, 700–789. doi: 10.1002/alz.12638 Amin, A., Perera, N. D., Beart, P. M., Turner, B. J., and Shabanpoor, F. (2020). Amyotrophic lateral sclerosis and autophagy: Dysfunction and therapeutic targeting. Cells 9:E2413. doi: 10.3390/cells9112413 Chen, S., Zhang, X., Song, L., and Le, W. (2012). Autophagy dysregulation in
amyotrophic
lateral
sclerosis. Brain
Pathol. 22,
110–116. doi:
10.1111/j.1750-3639.2011.00546.x Arosio, A., Cristofani, R., Pansarasa, O., Crippa, V., Riva, C., Sirtori, R., et al. (2020). HSC70 expression is reduced in lymphomonocytes of sporadic ALS patients and
contributes to TDP-43 accumulation. Amyotrophic. Lateral Sclerosis. Frontotemporal
Degener. 21, 51–62. doi: 10.1080/21678421.2019.1672749 Choi, S. H., and Cho, K. Author contributions All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated organizations,
or those of the publisher, the editors and the reviewers. Any product
that may be evaluated in this article, or claim that may be made by its
manufacturer, is not guaranteed or endorsed by the publisher. DS-V designed the framework and conceptualization of the
review. DS-V, JL, and SA conducted the literature search. All authors
contributed to the manuscript writing, revision of the structure and Funding This work was supported by the Start-up Fund for research
assistant professors under the Strategic Hiring Scheme (Grant no. P0041471) from The Hong Kong Polytechnic University, Hong Kong
SAR awarded to DS-V. Conflict of interest The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could
be construed as a potential conflict of interest. 8. Conclusion These in vitro tools can be applied to explore
pathophysiological mechanisms at a molecular level and to screen
for potential therapeutic agents and their mechanisms of action. Altogether, the use of in vitro methods significantly assists in the
identification of promising targets to restore autophagy function
which can be of great importance to understanding disease
biology and potential therapeutic options in the context of
neurodegenerative disorders and depression. This is the first
review to compile, discuss, and provide a catalog of traditional
and novel in vitro models applied to neurodegenerative disorders
and depression. Frontiers in Molecular Neuroscience References (2021). Dysfunction of
chaperone-mediated autophagy in human diseases. Mol. Cell. Biochem. 476, 1439–1454. doi: 10.1007/s11010-020-04006-z Ghosh, R., and Pattison, J. S. (2018). Macroautophagy and chaperone-mediated
autophagy in heart failure: The known and the unknown. Oxidative Med. Cell. Longev. 2018:e8602041. doi: 10.1155/2018/8602041 Liu, C., Huang, X., Qiu, S., Chen, W., Li, W., Zhang, H., et al. (2019). Chinese herbal
complex ‘Bu Shen Jie Du fang’ (BSJDF) modulated autophagy in an MPP+-induced cell
model of Parkinson’s disease. Evid. Based Complement. Alternat. Med. 2019, 1–10. doi:
10.1155/2019/8920813 Ghosh, R., and Tabrizi, S. J. (2018). “Chapter 17 - Huntington disease” in Handbook
of clinical neurology Neurogenetics, Part I. eds. D. H. Geschwind, H. L. Paulson and C. Klein (Amsterdam, The Netherlands: Elsevier), 255–278. Gomes, C., Escrevente, C., and Costa, J. (2010). Mutant superoxide dismutase 1
overexpression in NSC-34 cells: Effect of trehalose on aggregation, TDP-43 localization
and levels of co-expressed glycoproteins. Neurosci. Lett. 475, 145–149. doi: 10.1016/j. neulet.2010.03.065 Lu, J., Wu, M., and Yue, Z. (2020). “Autophagy and Parkinson’s disease” in Autophagy:
Biology and diseases: Clinical science advances in experimental medicine and biology. ed. W. Le (Singapore: Springer), 21–51. Lynch-Day, M. A., Mao, K., Wang, K., Zhao, M., and Klionsky, D. J. (2012). The role
of autophagy in Parkinson’s disease. Cold Spring Harb. Perspect. Med. 2:a009357. doi:
10.1101/cshperspect.a009357 Guo, F., Liu, X., Cai, H., and Le, W. (2018). Autophagy in neurodegenerative diseases:
Pathogenesis and therapy. Brain Pathol. 28, 3–13. doi: 10.1111/bpa.12545 Gusdon, A. M., Zhu, J., Van Houten, B., and Chu, C. T. (2012). ATP13A2 regulates
mitochondrial bioenergetics through macroautophagy. Neurobiol. Dis. 45, 962–972. doi:
10.1016/j.nbd.2011.12.015 Mader, B. J., Pivtoraiko, V. N., Flippo, H. M., Klocke, B. J., Roth, K. A., Mangieri, L. R.,
et al. (2012). Rotenone inhibits autophagic flux prior to inducing cell death. ACS Chem. Neurosci. 3, 1063–1072. doi: 10.1021/cn300145z Guzzo, A., Delarue, P., Rojas, A., Nicolaï, A., Maisuradze, G. G., and Senet, P. (2022). Wild-type α-Synuclein and variants occur in different disordered dimers and pre-
Fibrillar conformations in early stage of aggregation. Front. Mol. Biosci. 9:910104. doi:
10.3389/fmolb.2022.910104 Magalhaes, J., Gegg, M. E., Migdalska-Richards, A., and Schapira, A. H. (2018). Effects
of ambroxol on the autophagy-lysosome pathway and mitochondria in primary cortical
neurons. Sci. Rep. 8:1385. doi: 10.1038/s41598-018-19479-8 Malhi, G. S., and Mann, J. J. (2018). Depression. Lancet 392, 2299–2312. doi: 10.1016/
S0140-6736(18)31948-2 Hase, K., Contu, V. R., Kabuta, C., Sakai, R., Takahashi, M., Kataoka, N., et al. (2020). References Structure and function of ESCRT-III. Biochem. Soc. Trans. 37, 156–160. doi:
10.1042/BST0370156 Feng, Q., Luo, Y., Zhang, X. N., Yang, X. F., Hong, X. Y., Sun, D. S., et al. (2020). MAPT/Tau accumulation represses autophagy flux by disrupting IST1-regulated
ESCRT-III complex formation: A vicious cycle in Alzheimer neurodegeneration. Autophagy 16, 641–658. doi: 10.1080/15548627.2019.1633862 Lee, J. K., Shin, J. H., Lee, J. E., and Choi, E. J. (2015). Role of autophagy in the
pathogenesis of amyotrophic lateral sclerosis. Biochim. Biophys. Acta Mol. basis Dis. 1852, 2517–2524. doi: 10.1016/j.bbadis.2015.08.005 Fred Dice, J. (1990). Peptide sequences that target cytosolic proteins for lysosomal
proteolysis. Trends Biochem. Sci. 15, 305–309. doi: 10.1016/0968-0004(90)90019-8 Lee, J.-H., Yang, D.-S., Goulbourne, C. N., Im, E., Stavrides, P., Pensalfini, A., et al. (2022). Faulty autolysosome acidification in Alzheimer’s disease mouse models induces
autophagic build-up of Aβ in neurons, yielding senile plaques. Nat. Neurosci. 25,
688–701. doi: 10.1038/s41593-022-01084-8 Fung, T. Y., Iyaswamy, A., Sreenivasmurthy, S. G., Krishnamoorthi, S., Guan, X.-J.,
Zhu, Z., et al. (2022). Klotho an autophagy stimulator as a potential therapeutic target
for
Alzheimer’s
disease:
A
review. Biomedicine
10:705. doi:
10.3390/
biomedicines10030705 Lee, J.-H., Yu, W. H., Kumar, A., Lee, S., Mohan, P. S., Peterhoff, C. M., et al. (2010). Lysosomal proteolysis and autophagy require presenilin 1 and are disrupted by
Alzheimer-related PS1 mutations. Cells 141, 1146–1158. doi: 10.1016/j.cell.2010.05.008 Gassen, N. C., Hartmann, J., Schmidt, M. V., and Rein, T. (2015). FKBP5/FKBP51
enhances autophagy to synergize with antidepressant action. Autophagy 11, 578–580. doi: 10.1080/15548627.2015.1017224 Levine, B., and Klionsky, D. J. (2017). Autophagy wins the 2016 nobel prize in
physiology or medicine: Breakthroughs in baker’s yeast fuel advances in biomedical
research. Proc. Natl. Acad. Sci. U. S. A. 114, 201–205. doi: 10.1073/PNAS.1619876114 Gassen, N. C., Hartmann, J., Zschocke, J., Stepan, J., Hafner, K., Zellner, A., et al. (2014). Association of FKBP51 with priming of autophagy pathways and mediation of
antidepressant treatment response: Evidence in cells, mice, and humans. PLoS Med. 11:e1001755. doi: 10.1371/journal.pmed.1001755 Li, W. W., Li, J., and Bao, J. K. (2012). Microautophagy: Lesser-known self-eating. Cell. Mol. Life Sci. 69, 1125–1136. doi: 10.1007/s00018-011-0865-5 Li, Q., Liu, Y., and Sun, M. (2017). Autophagy and Alzheimer’s disease. Cell. Mol. Neurobiol. 37, 377–388. doi: 10.1007/s10571-016-0386-8 Gassen, N. C., and Rein, T. (2019). Is there a role of autophagy in depression and
antidepressant action? Front. Psych. 10:337. doi: 10.3389/fpsyt.2019.00337 Liao, Z., Wang, B., Liu, W., Xu, Q., Hou, L., Song, J., et al. References 31, 427–434. doi: 10.1007/
s12264-015-1548-2 Das, G., Shravage, B. V., and Baehrecke, E. H. (2012). Regulation and function of
autophagy during cell survival and cell death. Cold Spring Harb. Perspect. Biol. 4:a008813. doi: 10.1101/cshperspect.a008813 Kallergi, E., Daskalaki, A.-D., Kolaxi, A., Camus, C., Ioannou, E., Mercaldo, V., et al. (2022). Dendritic autophagy degrades postsynaptic proteins and is required for long-
term synaptic depression in mice. Nat. Commun. 13:680. doi: 10.1038/
s41467-022-28301-z de la Cueva, M., Antequera, D., Ordoñez-Gutierrez, L., Wandosell, F., Camins, A.,
Carro, E., et al. (2022). Amyloid-β impairs mitochondrial dynamics and autophagy in
Alzheimer’s disease experimental models. Sci. Rep. 12:10092. doi: 10.1038/
s41598-022-13683-3 Kim, Y. C., and Guan, K. L. (2015). mTOR: A pharmacologic target for autophagy
regulation. J. Clin. Invest. 125, 25–32. doi: 10.1172/JCI73939 di Domenico, A., Carola, G., Calatayud, C., Pons-Espinal, M., Muñoz, J. P.,
Richaud-Patin, Y., et al. (2019). Patient-specific iPSC-derived astrocytes contribute to
non-cell-autonomous neurodegeneration in Parkinson’s disease. Stem Cell Rep. 12,
213–229. doi: 10.1016/j.stemcr.2018.12.011 Kirchner, P., Bourdenx, M., Madrigal-Matute, J., Tiano, S., Diaz, A., Bartholdy, B. A.,
et al. (2019). Proteome-wide analysis of chaperone-mediated autophagy targeting
motifs. PLoS Biol. 17:e3000301. doi: 10.1371/JOURNAL.PBIO.3000301 Dice, J. F. (2007). Chaperone-mediated autophagy. Autophagy 3, 295–299. doi:
10.4161/auto.4144 Koga, H., Martinez-Vicente, M., Arias, E., Kaushik, S., Sulzer, D., and Cuervo, A. M. (2011). Constitutive upregulation of chaperone-mediated autophagy in Huntington’s
disease. J. Neurosci. 31, 18492–18505. doi: 10.1523/JNEUROSCI.3219-11.2011 Ding, X., Yang, X., Zhao, Y., Wang, Y., Fei, J., Niu, Z., et al. (2022). Identification of
active natural products that induce lysosomal biogenesis by lysosome-based screening
and biological evaluation. Heliyon 8:e11179. doi: 10.1016/j.heliyon.2022.e11179 Krause, G. J., and Cuervo, A. M. (2021). Assessment of mammalian endosomal
microautophagy. Methods Cell Biol. 164, 167–185. doi: 10.1016/bs.mcb.2020.10.009 Ehrnhoefer, D. E., Martin, D. D. O., Schmidt, M. E., Qiu, X., Ladha, S., Caron, N. S.,
et al. (2018). Preventing mutant huntingtin proteolysis and intermittent fasting promote
autophagy in models of Huntington disease. Acta Neuropathol. Commun. 6:16. doi:
10.1186/s40478-018-0518-0 Kwon, Y., Bang, Y., Moon, S.-H., Kim, A., and Choi, H. J. (2020). Amitriptyline
interferes with autophagy-mediated clearance of protein aggregates via inhibiting
autophagosome maturation in neuronal cells. Cell Death Dis. 11, 874–817. doi: 10.1038/
s41419-020-03085-6 Erkkinen, M. G., Kim, M.-O., and Geschwind, M. D. (2018). Clinical neurology and
epidemiology of the major neurodegenerative diseases. Cold Spring Harb. Perspect. Biol. 10:a033118. doi: 10.1101/cshperspect.a033118 Lata, S., Schoehn, G., Solomons, J., Pires, R., Gottlinger, H. G., and Weissenhorn, W. (2009). References (2021). LAMP2A-mediated autophagy involved in
Huntington’s disease progression. Biochem. Biophys. Res. Commun. 534, 561–567. doi:
10.1016/j.bbrc.2020.11.042 Baeken, M. W. (2023). Sirtuins and their influence on autophagy. J. Cell. Biochem,
1–13. doi: 10.1002/jcb.30377 Conrad, C. D. (2008). Chronic stress-induced hippocampal vulnerability: The
glucocorticoid vulnerability hypothesis. Rev. Neurosci. 19, 395–411. doi: 10.1515/
REVNEURO.2008.19.6.395 Baeken, M. W., Schwarz, M., Kern, A., Moosmann, B., Hajieva, P., and Behl, C. (2021). The selective degradation of sirtuins via macroautophagy in the MPP+ model of
Parkinson’s disease is promoted by conserved oxidation sites. Cell Death Discov 7:286. doi: 10.1038/s41420-021-00683-x Cortes, C. J., and La Spada, A. R. (2014). The many faces of autophagy dysfunction in
Huntington’s disease: From mechanism to therapy. Drug Discov. Today 19, 963–971. doi:
10.1016/j.drudis.2014.02.014 Bar-Yosef, T., Damri, O., and Agam, G. (2019). Dual role of autophagy in diseases
of the central nervous system. Front. Cell. Neurosci. 13:196. doi: 10.3389/
FNCEL.2019.00196 Coyne, A. N., Lorenzini, I., Chou, C. C., Torvund, M., Rogers, R. S., Starr, A., et al. (2017). Post-transcriptional inhibition of Hsc70-4/HSPA8 expression leads to synaptic
vesicle cycling defects in multiple models of ALS. Cell Rep. 21, 110–125. doi: 10.1016/j. celrep.2017.09.028 Boland, B., Kumar, A., Lee, S., Platt, F. M., Wegiel, J., Yu, W. H., et al. (2008). Autophagy induction and autophagosome clearance in neurons: Relationship to
autophagic pathology in Alzheimer’s disease. J. Neurosci. 28, 6926–6937. doi: 10.1523/
JNEUROSCI.0800-08.2008 Crippa, V., Sau, D., Rusmini, P., Boncoraglio, A., Onesto, E., Bolzoni, E., et al. (2010). The small heat shock protein B8 (HspB8) promotes autophagic removal of misfolded
proteins involved in amyotrophic lateral sclerosis (ALS). Hum. Mol. Genet. 19,
3440–3456. doi: 10.1093/hmg/ddq257 Broadway, B. J., Boneski, P. K., Bredenberg, J. M., Kolicheski, A., Hou, X.,
Soto-Beasley, A. I., et al. (2022). Systematic functional analysis of PINK1 and PRKN
coding variants. Cells 11:2426. doi: 10.3390/cells11152426 Croce, K. R., and Yamamoto, A. (2019). A role for autophagy in Huntington’s disease. Neurobiol. Dis. 122, 16–22. doi: 10.1016/j.nbd.2018.08.010 Caccamo, A., Majumder, S., Richardson, A., Strong, R., and Oddo, S. (2010). Molecular interplay between mammalian target of rapamycin (mTOR), amyloid-β, and
tau. J. Biol. Chem. 285, 13107–13120. doi: 10.1074/jbc.M110.100420 Cuervo, A. M., and Wong, E. (2014). Chaperone-mediated autophagy: Roles in disease
and aging. Cell Res. 24, 92–104. doi: 10.1038/cr.2013.153 Frontiers in Molecular Neuroscience 26 frontiersin.org frontiersin.org Sánchez-Vidaña et al. 10.3389/fnmol.2023.1168948 Jia, J., and Le, W. (2015). Molecular network of neuronal autophagy in the
pathophysiology and treatment of depression. Neurosci. Bull. References Selective autophagy: Talking with the UPS. Cell
Biochem. Biophys. 67, 3–13. doi: 10.1007/s12013-013-9623-7 Shpilka, T., and Elazar, Z. (2011). Shedding light on mammalian microautophagy. Dev. Cell 20, 1–2. doi: 10.1016/j.devcel.2010.12.010 Parkinson, N., Ince, P. G., Smith, M. O., Highley, R., Skibinski, G., Andersen, P. M.,
et al. (2006). ALS phenotypes with mutations in CHMP2B (charged multivesicular body
protein 2B). Neurology 67, 1074–1077. doi: 10.1212/01.wnl.0000231510.89311.8b Sicari, D., Delaunay-Moisan, A., Combettes, L., Chevet, E., and Igbaria, A. (2020). A
guide to assessing endoplasmic reticulum homeostasis and stress in mammalian
systems. FEBS J. 287, 27–42. doi: 10.1111/febs.15107 Perera, N. D., Sheean, R. K., Lau, C. L., Shin, Y. S., Beart, P. M., Horne, M. K., et al. (2017). Rilmenidine promotes MTOR-independent autophagy in the mutant SOD1
mouse model of amyotrophic lateral sclerosis without slowing disease progression. Autophagy 14, 534–551. doi: 10.1080/15548627.2017.1385674 Singh, R., and Cuervo, A. M. (2011). Autophagy in the cellular energetic balance. Cell
Metab. 13, 495–504. doi: 10.1016/J.CMET.2011.04.004 Skibinski, G., Parkinson, N. J., Brown, J. M., Chakrabarti, L., Lloyd, S. L.,
Hummerich, H., et al. (2005). Mutations in the endosomal ESCRTIII-complex subunit
CHMP2B in frontotemporal dementia. Nat Genet 37, 806–808. doi: 10.1038/ng1609 Perrett, R. M., Alexopoulou, Z., and Tofaris, G. K. (2015). The endosomal pathway in
Parkinson’s disease. Mol. Cell. Neurosci. 66, 21–28. doi: 10.1016/j.mcn.2015.02.009 Sreenivasmurthy, S. G., Iyaswamy, A., Krishnamoorthi, S., Reddi, R. N.,
Kammala, A. K., Vasudevan, K., et al. (2022). Bromo-protopine, a novel protopine
derivative, alleviates tau pathology by activating chaperone-mediated autophagy for
Alzheimer’s disease therapy. Front. Mol. Biosci. 9:1030534. doi: 10.3389/
fmolb.2022.1030534 Pierone, B. C., Pereira, C. A., Garcez, M. L., and Kaster, M. P. (2020). Stress and
signaling pathways regulating autophagy: From behavioral models to psychiatric
disorders. Exp. Neurol. 334:113485. doi: 10.1016/j.expneurol.2020.113485 Portelius, E., Olsson, M., Brinkmalm, G., Rüetschi, U., Mattsson, N., Andreasson, U.,
et al. (2012). Mass spectrometric characterization of amyloid-β species in the 7PA2 cell
model of Alzheimer’s disease. J. Alzheimers Dis. 33, 85–93. doi: 10.3233/
JAD-2012-120994 Stathakos, P., Jiménez-Moreno, N., Crompton, L. A., Nistor, P. A., Badger, J. L.,
Barbuti, P. A., et al. (2021). A monolayer hiPSC culture system for autophagy/mitophagy
studies in human dopaminergic neurons. Autophagy 17, 855–871. doi:
10.1080/15548627.2020.1739441 Proenca, C. C., Stoehr, N., Bernhard, M., Seger, S., Genoud, C., Roscic, A., et al. (2013). Atg4b-dependent autophagic flux alleviates Huntington’s disease progression. PLoS One 8:e68357. doi: 10.1371/journal.pone.0068357 Stoker, T. B., Mason, S. L., Greenland, J. C., Holden, S. References Depletion of Beclin-1 due to proteolytic cleavage by caspases in the Alzheimer's
disease brain. Neurobiol Dis, 43:68–78. doi: 10.1016/j.nbd.2010.11.003 Mehta, P., Raymond, J., Punjani, R., Han, M., Larson, T., Kaye, W., et al. (2023). Prevalence of amyotrophic lateral sclerosis in the United States using established and
novel methodologies, 2017. Amyotrophic. Lateral Sclerosis. Frontotemporal Degener. 24,
108–116. doi: 10.1080/21678421.2022.2059380 Sackmann, V., Sinha, M. S., Sackmann, C., Civitelli, L., Bergström, J., Ansell-Schultz, A.,
et al. (2019). Inhibition of nSMase2 reduces the transfer of oligomeric α-synuclein
irrespective of hypoxia. Front. Mol. Neurosci. 12:200. doi: 10.3389/fnmol.2019.00200 Saha, S., Panigrahi, D. P., Patil, S., and Bhutia, S. K. (2018). Autophagy in health and
disease: A comprehensive review. Biomed. Pharmacother. 104, 485–495. doi: 10.1016/j. biopha.2018.05.007 Meng, T., Lin, S., Zhuang, H., Huang, H., He, Z., Hu, Y., et al. (2019). Recent progress
in the role of autophagy in neurological diseases. Cell Stress 3, 141–161. doi: 10.15698/
cst2019.05.186 Sahu, R., Kaushik, S., Clement, C. C., Cannizzo, E. S., Scharf, B., Follenzi, A., et al. (2011). Microautophagy of cytosolic proteins by late endosomes. Dev. Cell 20, 131–139. doi: 10.1016/j.devcel.2010.12.003 Mizushima, N., and Komatsu, M. (2011). Autophagy: Renovation of cells and tissues. Cells 147, 728–741. doi: 10.1016/j.cell.2011.10.026 Mizushima, N., and Levine, B. (2020). Autophagy in human diseases. N. Engl. J. Med. 383, 1564–1576. doi: 10.1056/NEJMra2022774 Sala, G., Bocci, T., Borzì, V., Parazzini, M., Priori, A., and Ferrarese, C. (2021). Direct
current stimulation enhances neuronal alpha-synuclein degradation in vitro. Sci. Rep. 11:2197. doi: 10.1038/s41598-021-81693-8 Mizushima, N., Levine, B., Cuervo, A. M., and Klionsky, D. J. (2008). Autophagy fights
disease through cellular self-digestion. Nature 451, 1069–1075. doi: 10.1038/nature06639 Salari, S., and Bagheri, M. (2019). In vivo, in vitro and pharmacologic models of
Parkinson’s disease. Physiol. Res. 68, 17–24. doi: 10.33549/physiolres.933895 Moloudizargari, M., Asghari, M. H., Ghobadi, E., Fallah, M., Rasouli, S., and
Abdollahi, M. (2017). Autophagy, its mechanisms and regulation: Implications in
neurodegenerative diseases. Ageing Res. Rev. 40, 64–74. doi: 10.1016/j.arr.2017.09.005 Santomauro, D. F., Mantilla Herrera, A. M., Shadid, J., Zheng, P., Ashbaugh, C.,
Pigott, D. M., et al. (2021). Global prevalence and burden of depressive and anxiety
disorders in 204 countries and territories in 2020 due to the COVID-19 pandemic. Lancet 398, 1700–1712. doi: 10.1016/S0140-6736(21)02143-7 Moors, T. E., Hoozemans, J. J. M., Ingrassia, A., Beccari, T., Parnetti, L.,
Chartier-Harlin, M.-C., et al. (2017). Therapeutic potential of autophagy-enhancing
agents in Parkinson’s disease. Mol. Neurodegener. 12:11. References Cytosolic domain of SIDT2 carries an arginine-rich motif that binds to RNA/DNA and
is important for the direct transport of nucleic acids into lysosomes. Autophagy 16,
1974–1988. doi: 10.1080/15548627.2020.1712109 Martin-Aparicio, E., Yamamoto, A., Hernandez, F., Hen, R., Avila, J. S., and Lucas, J. J. (2001). Proteasomal-dependent aggregate reversal and absence of cell death in a
conditional mouse model of Huntington’s disease. J. Neurosci. 21, 8772–8781. doi:
10.1523/Jneurosci.21-22-08772.2001 Hou, X., Watzlawik, J. O., Fiesel, F. C., and Springer, W. (2020). Autophagy in
Parkinson’s disease. J. Mol. Biol. 432, 2651–2672. doi: 10.1016/j.jmb.2020.01.037 Martinelli, S., Park, D., Hafner, H., Nagaraj, N., Binder, E., and Gassen, N. C. (2018). T180. Unraveling the effect of physiological stress on chaperone mediated autophagy-
dependent synaptic Proteostasis. Biol. Psychiat. 83, S197–S198. doi: 10.1016/j. biopsych.2018.02.517 HU, M., HUANG, H., ZHAO, R., LI, P., LI, M., MIAO, H., et al. (2014). AZD8055
induces cell death associated with autophagy and activation of AMPK in hepatocellular
carcinoma. Oncol. Rep. 31, 649–656. doi: 10.3892/or.2013.2890 Masrori, P., and Van Damme, P. (2020). Amyotrophic lateral sclerosis: A clinical
review. Eur. J. Neurol. 27, 1918–1929. doi: 10.1111/ene.14393 Huang, C. C., Bose, J. K., Majumder, P., Lee, K. H., Huang, J. T. J., Huang, J. K., et al. (2014). Metabolism and mis-metabolism of the neuropathological signature protein
TDP-43. J. Cell Sci. 127, 3024–3038. doi: 10.1242/jcs.136150 Mavroeidi, P., Arvanitaki, F., Vetsi, M., Becker, S., Vlachakis, D., Jensen, P. H., et al. (2022). Autophagy mediates the clearance of oligodendroglial SNCA/alpha-synuclein
and TPPP/p25A in multiple system atrophy models. Autophagy 18, 2104–2133. doi:
10.1080/15548627.2021.2016256 Ioghen, O. C., Ceafalan, L. C., and Popescu, B. O. (2023). SH-SY5Y cell line in vitro
models for Parkinson disease research—old practice for new trends. J. Integr. Neurosci. 22:20. doi: 10.31083/j.jin2201020 Frontiers in Molecular Neuroscience 27 frontiersin.org Sánchez-Vidaña et al. 10.3389/fnmol.2023.1168948 McColgan, P., and Tabrizi, S. J. (2018). Huntington’s disease: A clinical review. Eur. J. Neurol. 25, 24–34. doi: 10.1111/ene.13413 Rodger, C., Flex, E., Allison, R. J., Sanchis-Juan, A., Hasenahuer, M. A., Cecchetti, S.,
et al. (2020). De novo VPS4A mutations cause multisystem disease with abnormal
neurodevelopment. Am. J. Hum. Genet. 107, 1129–1148. doi: 10.1016/j.ajhg.2020.10.012 McNeill, A., Magalhaes, J., Shen, C., Chau, K.-Y., Hughes, D., Mehta, A., et al. (2014). Ambroxol improves lysosomal biochemistry in glucocerebrosidase mutation-linked
Parkinson disease cells. Brain 137, 1481–1495. doi: 10.1093/brain/awu020 Rohn, T. T., Wirawan, E., Brown, R. J., Harris, J. R., Masliah, E., and Vandenabeele, P.. (2011). References doi: 10.1186/s13024-017-0154-3 Sasaki, A., Arawaka, S., Sato, H., and Kato, T. (2015). Sensitive western blotting for
detection of endogenous Ser129-phosphorylated α-synuclein in intracellular and
extracellular spaces. Sci. Rep. 5:14211. doi: 10.1038/srep14211 Nah, J., Yuan, J., and Jung, Y.-K. K. (2015). Autophagy in neurodegenerative diseases:
From mechanism to therapeutic approach. Mol Cells, 38, 381–389. doi: 10.14348/
molcells.2015.0034 Sato, M., Ueda, E., Konno, A., Hirai, H., Kurauchi, Y., Hisatsune, A., et al. (2020). Glucocorticoids
negatively
regulates
chaperone
mediated
autophagy
and
microautophagy. Biochem. Biophys. Res. Commun. 528, 199–205. doi: 10.1016/j. bbrc.2020.04.132 Nakamura, S., Oba, M., Suzuki, M., Takahashi, A., Yamamuro, T., Fujiwara, M., et al. (2019). Suppression of autophagic activity by Rubicon is a signature of aging. Nat. Commun. 10:847. doi: 10.1038/s41467-019-08729-6 Nguyen, D. K. H., Thombre, R., and Wang, J. (2019). Autophagy as a common pathway
in amyotrophic lateral sclerosis. Neurosci. Lett. 697, 34–48. doi: 10.1016/j. neulet.2018.04.006 Scrivo, A., Bourdenx, M., Pampliega, O., and Cuervo, A. M. (2018). Selective
autophagy as a potential therapeutic target for neurodegenerative disorders. Lancet
Neurol, 17, 802–815. doi: 10.1016/S1474-4422(18)30238-2 Ni, C.-H., Hou, W.-H., Kao, C.-C., Chang, M.-L., Yu, L.-F., Wu, C.-C., et al. (2013). The anxiolytic effect of aromatherapy on patients awaiting ambulatory surgery: A
randomized controlled trial. Evid. Based Complement. Alternat. Med. 2013:927419, 1–5. doi: 10.1155/2013/927419 Selvarasu, K., Singh, A. K., Iyaswamy, A., Gopalkrishnashetty Sreenivasmurthy, S.,
Krishnamoorthi, S., Bera, A. K., et al. (2022). Reduction of kinesin I heavy chain
decreases tau hyperphosphorylation, aggregation, and memory impairment in
Alzheimer’s disease and tauopathy models. Front. Mol. Biosci. 9:1050768. doi: 10.3389/
fmolb.2022.1050768 Nie, T., Zhu, L., and Yang, Q. (2021). “The classification and basic processes of
autophagy” in Autophagy: Biology and diseases: Technology and methodology advances
in experimental medicine and biology. ed. Z. Xie (Singapore: Springer), 3–16. Sheehan, P., Zhu, M., Beskow, A., Vollmer, C., and Waites, C. L. (2016). Activity-
dependent degradation of synaptic vesicle proteins requires Rab35 and the ESCRT
pathway. J. Neurosci. 36, 8668–8686. doi: 10.1523/JNEUROSCI.0725-16.2016 Nogrady, B. (2022). The hormonal keys to depression. Nature 608, S44–S45. doi:
10.1038/d41586-022-02208-7 Shehata, M., Matsumura, H., Okubo-Suzuki, R., Ohkawa, N., and Inokuchi, K. (2012). Neuronal stimulation induces autophagy in hippocampal neurons that is involved in
AMPA receptor degradation after chemical long-term depression. J. Neurosci. 32,
10413–10422. doi: 10.1523/JNEUROSCI.4533-11.2012 Pan, L., and Feigin, A. (2021). Huntington’s disease: New Frontiers in therapeutics. Curr. Neurol. Neurosci. Rep. 21:10. doi: 10.1007/s11910-021-01093-3 Park, C., and Cuervo, A. M. (2013). References T., Santini, H., and Barker, R. A. (2022). Huntington’s disease: Diagnosis and management. Pract. Neurol. 22, 32–41. doi:
10.1136/practneurol-2021-003074 Qi, L., Zhang, X. D., Wu, J. C., Lin, F., Wang, J., DiFiglia, M., et al. (2012). The role of
chaperone-mediated autophagy in huntingtin degradation. PLoS One 7:e46834-16. doi:
10.1371/journal.pone.0046834 Sugeno, N., Hasegawa, T., Tanaka, N., Fukuda, M., Wakabayashi, K., Oshima, R., et al. (2014). Lys-63-linked ubiquitination by E3 ubiquitin ligase Nedd4-1 facilitates
endosomal sequestration of internalized alpha-synuclein. J. Biol. Chem. 289,
18137–18151. doi: 10.1074/jbc.M113.529461 Redmann, M., Benavides, G. A., Berryhill, T. F., Wani, W. Y., Ouyang, X.,
Johnson, M. S., et al. (2016). Inhibition of autophagy with bafilomycin and chloroquine
decreases mitochondrial quality and bioenergetic function in primary neurons. Redox
Biol. 11, 73–81. doi: 10.1016/j.redox.2016.11.004 Tong, B. C.-K., Huang, A. S., Wu, A. J., Iyaswamy, A., Ho, O. K.-Y., Kong, A. H.-Y.,
et al. (2022). Tetrandrine ameliorates cognitive deficits and mitigates tau aggregation in
cell and animal models of tauopathies. J. Biomed. Sci. 29:85. doi: 10.1186/
s12929-022-00871-6 Rein, T. (2019). Is autophagy involved in the diverse effects of antidepressants? Cells
8:44. doi: 10.3390/cells8010044 28 Frontiers in Molecular Neuroscience frontiersin.org Sánchez-Vidaña et al. 10.3389/fnmol.2023.1168948 Torrent, R., De Angelis Rigotti, F., Dell’Era, P., Memo, M., Raya, A., and Consiglio, A. (2015). Using iPS cells toward the understanding of Parkinson’s disease. J. Clin. Med. 4,
548–566. doi: 10.3390/jcm4040548 Implications for Parkinson’s disease. J. Cell Biol. 190, 1023–1037. doi: 10.1083/
jcb.201003122 Xing, Y., Liqi, Z., Jian, L., Qinghua, Y., and Qian, Y. (2017). Doxycycline induces
mitophagy and suppresses production of interferon-β in IPEC-J2 cells. Front. Cell. Infect. Microbiol. 7:21. doi: 10.3389/fcimb.2017.00021 Uddin, M. S., Stachowiak, A., Mamun, A. A., Tzvetkov, N. T., Takeda, S.,
Atanasov, A. G., et al. (2018). Autophagy and Alzheimer’s disease: From molecular
mechanisms to therapeutic implications. Front. Aging Neurosci. 10:4. doi: 10.3389/
fnagi.2018.00004 Xu, J., Ao, Y.-L., Huang, C., Song, X., Zhang, G., Cui, W., et al. (2022). Harmol
promotes α-synuclein degradation and improves motor impairment in Parkinson’s
models via regulating autophagy-lysosome pathway. NPJ Parkinsons Dis. 8:100. doi:
10.1038/s41531-022-00361-4 Valencia, M., Kim, S. R., Jang, Y., and Lee, S. H. (2021). Neuronal autophagy:
Characteristic features and roles in neuronal pathophysiology. Biomol Ther 29, 605–614. doi: 10.4062/biomolther.2021.012 Xu, X., Sun, Y., Cen, X., Shan, B., Zhao, Q., Xie, T., et al. (2021). Metformin activates
chaperone-mediated autophagy and improves disease pathologies in an Alzheimer
disease mouse model. Protein Cell 12, 769–787. References doi: 10.1007/s13238-021-00858-3 Vogiatzi, T., Xilouri, M., Vekrellis, K., and Stefanis, L. (2008). Wild type α-synuclein
is degraded by chaperone-mediated autophagy and macroautophagy in neuronal cells. J. Biol. Chem. 283, 23542–23556. doi: 10.1074/jbc.M801992200 Yamamoto, A., Tagawa, Y., Yoshimori, T., Moriyama, Y., Masaki, R., and Tashiro, Y. (1998). Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion
between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E cells. Cell
Struct. Funct. 23, 33–42. doi: 10.1247/csf.23.33 Wang, X., Chi, J., Huang, D., Ding, L., Zhao, X., Jiang, L., et al. (2020). α-Synuclein
promotes progression of Parkinson’s disease by upregulating autophagy signaling
pathway to activate NLRP3 inflammasome. Exp. Ther. Med. 19, 931–938. doi: 10.3892/
etm.2019.8297 Yang, S.-Y., Beavan, M., Chau, K.-Y., Taanman, J.-W., and Schapira, A. H. V. (2017). A
human neural crest stem cell-derived dopaminergic neuronal model recapitulates biochemical
abnormalities in GBA1 mutation carriers. Stem Cell Rep. 8, 728–742. doi: 10.1016/j. stemcr.2017.01.011 Wang, X., Fan, H., Ying, Z., Li, B., Wang, H., and Wang, G. (2010). Degradation of
TDP-43 and its pathogenic form by autophagy and the ubiquitin-proteasome system. Neurosci. Lett. 469, 112–116. doi: 10.1016/j.neulet.2009.11.055 Yang, J., Hu, L., Wu, Q., Liu, L., Zhao, L., Zhao, X., et al. (2014). A terrified-sound
stress induced proteomic changes in adult male rat hippocampus. Physiol. Behav. 128,
32–38. doi: 10.1016/j.physbeh.2014.01.038 Wang, M.-M., Feng, Y.-S., Yang, S.-D., Xing, Y., Zhang, J., Dong, F., et al. (2019). The
relationship between autophagy and brain plasticity in neurological diseases. Front. Cell. Neurosci. 13:228. doi: 10.3389/fncel.2019.00228 Wang, L., Klionsky, D. J., and Shen, H.-M. (2023). The emerging mechanisms and
functions of microautophagy. Nat. Rev. Mol. Cell Biol. 24, 186–203. doi: 10.1038/
s41580-022-00529-z Yang, Y., Hu, L., Zheng, H., Mao, C., Hu, W., Xiong, K., et al. (2013). Application and
interpretation of current autophagy inhibitors and activators. Acta Pharmacol. Sin. 34,
625–635. doi: 10.1038/aps.2013.5 Wang, Y., Martinez-Vicente, M., Krüger, U., Kaushik, S., Wong, E.,
Mandelkow, E.-M., et al. (2009). Tau fragmentation, aggregation and clearance: The
dual role of lysosomal processing. Hum. Mol. Genet. 18, 4153–4170. doi: 10.1093/
hmg/ddp367 Yuan, G., Xiao, L., Xie, Y., Si, L., Xu, H., Xu, W., et al. (2023). Tunicamycin induces
depression-like behaviors in male rats, accompanied by initiated chaperon-mediated
autophagy and decreased synaptic protein expression in the hippocampus. Neurosci. Lett. 798:137058. doi: 10.1016/j.neulet.2023.137058 Wang, R., Sun, H., Wang, G., and Ren, H. (2020). Imbalance of lysine acetylation
contributes to the pathogenesis of Parkinson’s disease. Int. J. References Mol. Sci. 21:7182. doi:
10.3390/ijms21197182 Zeng, R., Zhou, Q., Zhang, W., Fu, X., Wu, Q., Lu, Y., et al. (2019). Icariin-mediated
activation of autophagy confers protective effect on rotenone induced neurotoxicity
in vivo and in vitro. Toxicol. Rep. 6, 637–644. doi: 10.1016/j.toxrep.2019.06.014 Willen, K., Edgar, J. R., Hasegawa, T., Tanaka, N., Futter, C. E., and Gouras, G. K. (2017). Abeta accumulation causes MVB enlargement and is modelled by dominant
negative VPS4A. Mol. Neurodegener. 12:61. doi: 10.1186/s13024-017-0203-y Zhang, Z., Cai, X., Yao, Z., Wen, F., Fu, Z., Zhang, J., et al. (2020). EA ameliorated
depressive behaviors in CUMS rats and was related to its suppressing autophagy in the
hippocampus. Neural Plast. 2020, 1–9. doi: 10.1155/2020/8860968 Zhang, X., Chen, S., Huang, K., and Le, W. (2013). Why should autophagic flux
be assessed? Acta Pharmacol. Sin. 34, 595–599. doi: 10.1038/aps.2012.184 Winslow, A. R., Chen, C.-W., Corrochano, S., Acevedo-Arozena, A.,
Gordon, D. E., Peden, A. A., et al. (2010). α-Synuclein impairs macroautophagy: 29 frontiersin.org Frontiers in Molecular Neuroscience
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13–16 metų paauglių, gyvenančių modernėjančioje Lietuvoje, tautinės vertybės
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13–16 metų paauglių, gyvenančių modernėjančioje
Lietuvoje, tautinės vertybės Valdonė Šaulienė
Magistrė socialinių mokslų (edukologijos) doktorantė
Klaipėdos universiteto
Vaikystės pedagogikos katedra
S. Nėries g. 5, LT-92227, Klaipėda
Tel. +370 677 74754
El. paštas: valdone.saule@gmail.com Edukologijos mokslas pripažįsta, kad paauglystėje ugdymas gali daryti didžiausią įtaką asmeny-
bės savivertės formavimuisi. Straipsnyje analizuojamas 13–16 metų paauglių tautinių vertybių
suvokimas ir raiška moderniosios lietuvybės kontekste interpretuojant respondentų savireflek-
sijas. Tyrimo instrumento – esė – pagalba nustatomos jaunesniojo amžiaus paauglių tautinės
vertybės, analizuojama vertybinė jaunų žmonių patirtis, jausmai ir nuostatos, kultūrą moder-
niosios lietuvybės kontekste suprantant kaip visos tautos sukurtą, iš kartos į kartą per ugdymą
perduodamą ir nuolat atnaujinamą kultūros vertybių visumą – bendra kalba, bendra praeitis ir
likimas, tautinis mentalitetas, tradicijos, kraštovaizdis – patriotiniu, meilės Tėvynei, jos saugoji-
mo ir išlikimo požiūriu. Pagrindiniai žodžiai: paauglystė, tautinės vertybės, moderni lietuvybė, savivertė. Uždaviniai: Uždaviniai: Išanalizuoti mokslinę literatūrą tautinių vertybių būklės modernioje visuomenėje požiū-
1. riu. Nustatyti nepriklausomus ir ugdomuosius veiksnius, formuojančius jaunų žmonių požiūrį
2. į tautines vertybes;ii Atskleisti jaunų žmonių ,,buvimo lietuviu“ filosofiją. 3. Įvadas Gyvename postmoderniame pasaulyje, modernėjančioje Lietuvoje, kai vis labiau tolstame nuo
tautinių vertybių, kurių simbolinė prasmė tėra deklaratyvi, nes tautinis savitumas nebėra savi-
monės pamatas, kurį reikėtų, kaip liudija tautos istorijos faktai, ginti nuo išorės priešų, saugoti ir
puoselėti slapta. XXI globalizacijos amžiuje siekiama formuoti universalų, kosmopolitišką žmo-
gų. Komercializuojamas kultūros paveldas, net ugdymas, nes verslumas modernioje visuomenėje
užima išskirtinę vertę. Kyla klausimas, ką reiškia būti moderniu lietuviu? Ar tai būti savimi –
brandžia, susipratusia, savo tautines šaknis turinčia asmenybe, gebančia priimti kultūrinę įvai-
rovę, kitoniškumą, juo domėtis ir vertinti? O gal tai reiškia atsisakyti savo istorinės atminties,
kultūros ir strimgalviais vytis senosios Europos patirtį arba globalaus pasaulio technokratinį he-
donizmo vaiduoklį? Europos Sąjungos ir Lietuvos švietimo dokumentuose akcentuojama tautinių vertybių ug-
dymo problema (Mokymosi visą gyvenimą memorandume, 2001; Pradinio ir pagrindinio ugdy-
mo programose, 2008; Valstybinės švietimo strategijos 2003–2012 nuostatose, Etninės kultūros
plėtros švietimo įstaigose strategijos 2003–2012 nuostatose). Tautinių vertybių studijos teoriniu
ir praktiniu lygmeniu yra pedagogikos mokslininkų akiratyje. Lietuvių tautinių vertybių integra-
vimo į ugdymo turinį svarbą asmenybės branduolio formavimui (si) edukologiniu aspektu yra
analizavę daugelis autorių: M. Lukšienė (2000) išskyrė tris galimus kultūros modelio perteikimo
jaunimui scenarijus, O. Tijūnėlienė (2002) nagrinėjo tautinio ir kultūrinio tapatumo ugdymą mo- 53 kykloje, I. Trinkūnienė (2005) akcentavo etnokultūrinio savitumo išlaikymo nuostatas, I. Čepie-
nė (2008) įvertino etninės kultūros reikšmę tautiniam ir patriotiniam ugdymui, I. Stonkuvienė
(2011) analizavo etninės kultūros perdavimo iš kartos į kartą mechanizmus, R. Grigas (2001),
B. Kuzmickas (2007) aktualizavo tautinės savivokos ir savimonės reikšmę. Tačiau tautiškumas
kaip vertybė ugdymo procese yra nepakankamai suprastas, tebėra plūduriuojantis tarp individu-
alumo ir bendruomeniškumo, etnocentrizmo ir etnoreliatyvizmo, idealizmo ir pragmatizmo, tad
nuolat keičiantis šalies politinėms, ekonominėms, socialinėms ir kultūrinėms sąlygoms, moder-
nėjant visuomenei, ugdytojams kyla vis nauji tautinių vertybių ugdymo iššūkiai, ypač ugdant
paauglius – šiuo požiūriu jautriausią žmonių grupę. Pasak E. Gellner’io (1996, p. 21), žmogus be tautos yra tarsi ,,žmogus be šešėlio“, vadinasi,
jis yra netikras, nerealus. Modernybės kontekste asmens tapatumas suvokiamas kaip projektas,
kuris A. Giddens’o (2000) stratifikacijos modelyje išplečiamas iki ontologinio nesaugumo. Dėl
šiuolaikiniame pasaulyje sparčiai vykstančios kaitos paaugliai dažnai patiria įtampą, beprasmiš-
kumą, net agresiją, kurios neigiamai veikia besiformuojančią jaunų žmonių tautinio tapatumo
jauseną, santykius, požiūrį į tautines vertybes. Todėl šiame darbe tikslinga nagrinėti šiandienos
Lietuvos paauglių nuostatas savivertės, tautiškumo požiūriu. Tyrimo mokslinė problema formuluojama klausimais: kokios yra Lietuvoje gyvenančių
13–16 metų jaunų žmonių vertybinės nuostatos ir kokie veiksniai lemia tautinės savivertės for-
mavimąsi modernėjančioje Lietuvoje? Įvadas mo objektas – 13–16 metų paauglių tautinės vertybės modernios lietuvybės kontekste. Tyrimo objektas – 13–16 metų paauglių tautinės vertybės modernios lietuvybės kontekste. Tyrimo tikslas – atskleisti jauno žmogaus buvimo tautos atstovu vertybes remiantis jo paties
tautinės kultūros samprata. Tyrimo tikslas – atskleisti jauno žmogaus buvimo tautos atstovu vertybes remiantis jo paties
tautinės kultūros samprata. Tyrimo teoriniai pagrindai edukologiniame kontekste Moderni lietuvybė – dvasios slėpinių turtinga, atvira pasauliui, organiška ir kūrybiška būklė,
turinti neginčijamų demokratijos pranašumų, dialogo, diskusijos su pasauliu galimybę. Tačiau
modernėjančioje Lietuvoje vyksta sparti tradicinių, dvasinių vertybių erozija nuo pat Nepriklau-
somybės atkūrimo. Atlikti tyrimai (Kuzmickas, Astra, 1996) rodo, kad nemažai žmonių įgijo var-
tojamojo, sumaterialėjusio elgesio formas. Paauglių dvasines vertybes tyrusi E. Martišauskienė
(2004) susidariusią padėtį aiškina tuo, jog Nepriklausomybės laikais atsivėrė sovietmečio sufor-
muotos žmogiškosios ydos, kurias dar pagilina vakarietiškasis pragmatizmas. Postmoderniems
tarpžemyniniams tinklams ir globaliems valstybių dariniams tautiškumas šiandien yra atgyvena,
provincialumo žymė, net pavojus visuomenės stabilumui. Vadinasi, išlaisvėjus globalioms min-
ties sistemoms, nebelieka pagrindo, palaikančio kolektyvinį idealizmą. Postmodernus žinojimas
sustiprina nebendramatiškumo toleravimą (Barth, 1969; Anderson, 1999). Patriotizmas, kaip tau-
tos ir jos narių bendro likimo jausmas, „neegoistinis nacionalizmas“ (Gellner, 1996) modernioje
valstybėje keičiamas pilietiškumo sąvoka, kuri savo turiniu reiškia mažiau dvasingumo, daugiau 54 formalių įsipareigojimų valstybei. Formalizuoti, biurokratiniai, vartojamieji valstybės ir jos pilie-
čių santykiai gilina bedvasiškumo prarają. Tenka ginti pačią modernią lietuvybę nuo jos piliečių
savanaudiškumo, abejingumo, tamsumo. Žmogaus dvasingumo turinys – internalizuotų vertybių visuma. Vertybės – tai objektai, reikš-
mingai tenkinantys asmens ar visuomenės poreikius, taip pat asmenybės veiklos ir elgesio moty-
vai, susiję su objektais, labiausiai atitinkančiais materialinius, kultūrinius ar dvasinius asmeny-
bės poreikius (Jovaiša, 2007). Vertybės yra tam tikri kultūros principai, standartai, padedantys
žmogui teigiamai arba neigiamai įvertinti objektus ir įvykius (Zaleskienė, 2004). Tautinės ver-
tybės – tautos mentalitetas ir emocijos, jos dvasinis nusiteikimas, tautinė savimonė ir savijauta,
tai – gimtoji kalba, kurioje dera dorovinis ir estetinis auklėjimas, tai – kultūra, tradicijos, papročiai,
t. y. tautos dvasinių klodų turinys, forma ir veikimo laukas, tai – pagarba savo tautiniam tapa-
tumui ir kitoms tautoms, tai ir charakterio vertybės (Bajoriūnas, 1997, p. 103–128). Pagaliau –
tautos kultūra, kaip simbolių formų ir praktikų samprata, sukuria atvirumo perspektyvą ir leidžia
priimti naujas idėjas, neprarandant savo tapatybės (Kavolis, 1996). Tai rodo būtinybę pažinti ir
vertinti savąją, lietuvių etininę kultūrą. Tautinė savivertė atsiranda tik kryptingai auklėjant jauną žmogų (Maceina (1991), Šalkauskis
(1991), Bitinas (1995), Grigas (2001)), puoselėjant meilę tautos paveldui per teigiamų emocijų
sukeltus ir taip ilgiau išliekančius vaizdinius, prisiminimus, jausmus. Raidos psichologai (Na-
vickas, Vaičiulienė, 2010) mano, kad jausminis išgyvenimas ir prasmės kūrimas yra daug reikš-
mingesnis, nes žmogaus savitumą sukuria ne nuogi faktai, bet išgyventos patirtys. Kognityviniu
lygmeniu požiūris į vertybes formuoja paauglių pažintinį santykį. Tyrimo teoriniai pagrindai edukologiniame kontekste Emociniu lygmeniu vertybės
motyvacijos struktūroje užima skirtingą vietą, atsižvelgiant į tai, kiek jos yra žmogui reikšmin-
gos, kokius tenkina poreikius. Todėl patiriamos emocijos įgyja skirtingą traukos galią – tampa
malonios ir patrauklios arba neprasmingos, sukeliančios neigiamus vaizdinius (Aramavičiūtė,
2006). Asmens savivertė yra svarbus psichosocialinis reiškinys, kurį paauglystėje lengva pažeisti. Savivertė pirmiausia susijusi su žinojimu: ,,kas aš esu“ ir ,,kaip aš jaučiuosi“. Paaugliai pasaulį,
gyvenamąją realybę ir santykius išgyvena sensityviai – tai yra jų nuostatų, vėliau ir charakterio
formavimosi pamatas. Pagaliau tai yra sąmoningo pasirinkimo kuo būti preliudija, nes paaug
lystėje perkainojamos žmogaus vertybinės nuostatos, formuojasi kritinio mąstymo užuomazgos. Šiuo amžiaus tarpsniu ugdymas asmeniui gali daryti didžiausią įtaką, nes paaugliai ieško idėjų ir
žmonių, kuriais galėtų tikėti ir pasitikėti, o savo baimę būti apgautiems išreiškia cinišku netikėji-
mu, atžariu elgesiu (Erikson, 1968). Metodologija ir metodika:
Empirinis tyrimas grindžiamas požiūriu į žmogų ka Postmodernistinė filosofija laikosi nuostatos, kad vertybės ir vertės egzistuoja žmogaus ir pasaulio
santykiuose, pasirinkimo teisė suteikiama kiekvienam individui gyvenamosios realybės rėmuose –
vyrauja nuomonių pliuralizmas, viskas leista ir įmanoma, tačiau laisvė nuo netampa laisve kam
savaime. Ji yra socialiai kuriama, veikiama, reklamuojama, peršama vartojamojo pragmatizmo. Kai kultūros pasiekia postmodernizmo amžių, tada keičiasi pažinimo būklė ir įgyti žinių nebėra
asmenybės ugdymo tikslas, bet tampa preke, gal net kovos objektu pasaulinėse varžybose dėl val-
džios (Lyotard, 2010). Postmodernus pasaulis – atsitiktinumų pasaulis, kuriame atsitinka kažkas,
bet neatsitinka niekam konkrečiai ir niekas nėra atsakingas (Bauman, 2002). 55 Fenomenologijos požiūriu ugdymo procese svarbi refleksija, kuri labiausiai yra siejama su
patirtimi. Pažinimui vienodai svarbus subjektyvus patyrimas ir objektai. Idėjos, objektai, daiktai
žmonėms įgauna reikšmę tik per jų sąmonę, t. y. pasaulis yra toks, kokią reikšmę daiktams sutei-
kia žmonės (Husserl, 2005). Taip nereikšmingi objektai tampa reikšmingi, gali veikti žmogaus
sąmonę ir elgesį, kurti prasmes. Priimtąją prasmę kiekvienas individas gali patvirtinti tik savo
esybės nepakartojamumu, Aš ir Tu santykio spontaniškumu (Buber, 1998, 2001). Hodegetiniu požiūriu egzistencinė žmogaus pajauta, jo išgyvenimai yra unikalūs ir todėl ver-
tingi (Duoblienė, 2006). Auklėjimo paskirtis – ugdyti brandžią ir harmoningą asmenybę, adek
vačia informacija artinant žmogų prie universalios realybės, jos reikšmės ir prasmės supratimo
(Aramavičiūtė, 2005; Jovaiša, 2009), kartu nepuoselėjant bešakniškumo ideologijos, kuri paranki
su globalizacija susijusiems procesams. Vadinasi, svarbiausia auklėjimo paskirtis – padėti indivi-
dui įgyti dvasinių vertybių, formuoti pasaulėžiūrą. Remiantis šiomis nuostatomis pasirinktas tyrimo instrumentas – esė. Tyrimui pritaikyta
V. Čiubrinsko (2008, p. 23–24) pasiūlyta eminė prieiga, kai paaugliai patys atsako į klausimus:
Ką man reiškia būti lietuviu? Kuo pasižymi ši raiška? Eminio metodo esmė ta, kad tiriamieji pa-
tys sukuria lietuvio tapatumo sampratą ir aprašymą, jie yra ekspertai, o tyrėjas fiksuoja – aprašo
vietinę kultūrą, identifikuoja respondentų tautines vertybes. i
j
p
y
Tyrime dalyvavo 203 septintų–devintų klasių mokiniai, kurie anonimiškai rašė struktūruotą
esė. 13–16 metų paaugliai – tai karta, kuri gimė ir gyvena nepriklausomoje Lietuvoje. Tiriamųjų
imtis orientuota į amžių (13–16 metų) ir gyvenamąją vietą. Geografiniu požiūriu tyrimas apėmė
Lietuvos regionų didžiųjų miestų (Kauno, Klaipėdos, Šiaulių), rajonų centrų (Anykščių, Vilka-
viškio, Jurbarko, Radviliškio, Telšių, Plungės, Šilutės), miestelių (Palangos) bei kaimo vietovių
(Platelių) moksleivius. Darant prielaidą, kad šeima turi didelę įtaką paauglių nuostatų formavi-
muisi, pateikiami šeimos statistiniai duomenys: 37,1 proc. respondentų šeimas sudaro 4 asmenys,
19,9 proc. – 5 nariai ir daugiau, 13,6 proc. yra vienturčiai, 24,4 proc. gyvena su vienu iš tėvų, iš jų
7 proc. Metodologija ir metodika:
Empirinis tyrimas grindžiamas požiūriu į žmogų ka turi patėvį, 1 proc. sudaro mišrios šeimos. Santykius šeimoje respondentai apibūdino kaip
gerus, normalius. Bendravimo problemų šeimoje turi 5,1 proc. paauglių. 76,7 proc. apklaustųjų
artimųjų tyrimo metu buvo emigravę arba dirbo užsienyje Tiriamųjų pasiskirstymą pagal amžių
ir lytį iliustruoja 1 lentelė: 1 lentelė. Tiriamųjų pasiskirstymas pagal amžių ir lytį
13 metų (n = 52)
14 metų (n = 50)
15 metų (n = 59)
16 metų (n = 42)
mergaitės
berniukai
mergaitės
berniukai
mergaitės
berniukai
mergaitės
berniukai
46,2 proc. 53,8 proc. 43,6 proc. 57,4 proc. 62,1 proc. 37,9 proc. 65,0 proc. 35,0 proc. 1 lentelė. Tiriamųjų pasiskirstymas pagal amžių ir lytį Tyrimo duomenų analizė, siekiant atskleisti respondentų subjektyviai išgyvenamų vertinimų
įvairovę, atlikta taikant fenomenografinę turinio analizę (Žydžiūnaitė, Jonušaitė, 2006). Analizės
principu pasirinkta pozicijos / opozicijos sklaida, kuri visapusiškiau parodo tiriamo reiškinio su-
dėtingumą. Artikuliuojant opozicijas išvengiama logocentrizmo (Derrida, 2006). Tyrimas vykdytas 2012 m vasario – balandžio mėn Tyrimas vykdytas 2012 m. vasario – balandžio mėn. Empirinio tyrimo duomenų analizė: Analizuojant empirinius duomenis išskirti keturi teminiai laukai, kurie reprezentuoja paauglių
buvimo lietuviu sampratą (žr. 2 lentelę). 56 2 lentelė. Paauglių „buvimo lietuviu“ teminiai laukai
Tautinių vertybių suvokimas
Patriotiškumo sklaida
Pasireiškia supratimu, kad kitoniškumas yra
kiekvienos tautos unikali savastis
Patriotiškumas yra visokeriopai ugdoma savybė;
Patriotiškumas, kaip santykis su tauta, išreiškiamas
įvardžiais aš, mes, jie ir veiksmažodžiais reikia, turėtų
būti;
Nepatriotiškai nuteikia išoriniai veiksniai
Meilės Tėvynei gramatika
Pasaulėžiūra
Meilė – tai prisirišimas prie meilės objekto;
Meilė – tai ištikimybė, noras būti kartu,
neemigruoti;
Meilė – tai saugojimas senųjų ir kūrimas naujų
bendrų vertybių savo gimtinėje;
Kai stokojama meilės Tėvynei – tarpsta meilė
sau
Diskusijų laukas 2 lentelė. Paauglių „buvimo lietuviu“ teminiai laukai Tautinių vertybių suvokimas Tautines vertybes paaugliai supranta kaip kitoniškumą, kaip kiekvienos tautos išskirtinius,
unikalius bruožus. Tačiau kad lietuvių tauta vis dar yra unikali, sutinka ne visi respondentai
(42,6 proc.). Opozicinės nuomonės verčia suklusti, permąstyti ugdymo turinio ir gyvenamos re-
alybės sąsajas. Visiems paaugliams suprantama ir dažniausiai esė tekstuose reflektuojama sąvoka – patrio-
tiškumas. Paaugliai mano, kad patriotiškumas yra išugdoma savybė, pateikia pavyzdžių, kada jie
yra pasijutę tikrais patriotais, įvardija ugdytojus. Tai dažniausiai pasireiškia valstybinių švenčių
metu, dalyvaujant renginiuose, visuomeninėse akcijose, šeimos susibūrimuose, istorijos pamo-
kose ir kt. Kita kategorija – meilės Tėvynei, jos objektams istorijos. Apie šį patyrimą paaugliai pasakoja
emocionaliai, dažniausiai susiedami su kritinėmis situacijomis, kada reikia rinktis, likti Lietuvoje
ar emigruoti, ir kt. Skirtingos paauglių nuostatos, jausmai ir patirtys susikryžiuoja diskusijų lauke – būti lietu-
viu garbė ar gėda, svarbiau tautos išlikimas ar asmeninių interesų tenkinimas, tautinės vertybės
yra gerbtinos ir puoselėtinos ar yra tik praeities balastas, trukdantis tobulėti, įsilieti į pasaulio
šurmulį? Tautinių vertybių suvokimas Tautines vertybes paaugliai suvokia įvairiai. Yra manančių, kad tautos ir žmonės nesiski-
ria. Kiti mano, kad skiriasi „tik odos spalva“, „kalba“, „tradicijos“, kad „su kitomis tautomis
mus vienija Dievas“. Kai kuriems svarbiausia yra „žmogiškos savybės, nesvarbu, kokiai tautai
priklausai“. Universalios vertybės, be abejonės, rodo asmenybės brandą doriniu aspektu. Bet
dauguma paauglių (83,4 proc.) įsitikinę, kad tautos ir žmonės yra skirtingi – „kaip snaigės“. Lietuvių kitoniškumas pasireiškia tautinių vertybių akumuliacija, tačiau paauglių nuostatos tauti-
nių vertybių atžvilgiu taip pat skiriasi. Kiekvienai pozicijai apie paveldo savitumą jaunų žmonių
mąstymuose galima atrasti opoziciją (žr. 3 lentelę): 57 3 lentelė. Paauglių nuostatos tautinių vertybių atžvilgiu 3 lentelė. Tautinių vertybių suvokimas Paauglių nuostatos tautinių vertybių atžvilgiu
Kategorija
Subkategorija
Pozicija
Opozicija
Unikalumas
Teritorija
„...kiek žinau, nėra panašios valstybės
į Lietuvą...“
„...Lietuvoje neliko išskirtinu-
mų, kuriais žavėtųsi kitos tau-
tos...“
Tautinis
tapatumas
„...būti lietuviu – tai mūsų prigimimas,
mūsų tapatybė ir tai privalu puoselė-
ti...“
„...kad esu lietuvė, nėra duo-
tybė ir galima susikonstruoti
savąją tapatybę taip, kaip tu
nori...“
„...daug lietuvių yra patriotai, ne visos
tautos tokių turi...“
„...būti tikru lietuviu, tikru pa-
triotu yra sunku ir šiais laikais
tokiais būti nori nedaugelis...“
Istorija
„...tautos
kitoniškumas
pasireiškia
unikalia praeitimi, istorija...“
„...na ir kas iš to, gi eidami gat
ve
nepasakosime
užsienie-
čiams to, kad buvome stiprūs
ir galingi, ne veltui sakoma, kas
buvo – pražuvo...“
„...lietuvių tauta išsiskiria iš kitų, nes
jau nuo pat senovės buvo garbinga ir
kilni tauta...“
Kalba
„...lietuviai nuo kitų tautybių skiriasi
kalba, kurios daugiau niekur, išskyrus
Lietuvoje, nėra ir turime keturias tar-
mes...“
„...nors myliu lietuvių kalbą,
prie širdies man arčiau anglų,
mėgstu šnekėti ja, man labai
lengva ir labai patinka...“
Tradicijos
„...lietuviai yra kitokie, labiau gerbia
senas lietuviškas tradicijas...“
„...tautos skiriasi kalba ir viskas,
tradicijos visur panašios, dau-
guma vienodų...“
„...unikali šalis, nes turi įdomius ir
išlaikytus savo papročius...“
„...buvome šventėje, kur pagal
papročius vyko įvairiausi daly-
kai, bet kad viskas buvo lietu-
viška, nemanau...“
„...lietuviai turi tikrai įdomių šven-
čių...“
„...daugelis švenčių yra skirtos
pasipelnyti
prekybos
cent
rams...“
Tautos kultūra
„...turime gražias tautines dainas bei
linksmus ir daug energijos reikalau-
jančius šokius...“
„...man nelabai patinka liaudiš-
kos dainos ar šokiai, nesijaučiu
taip, jog pagerbdama savo
tautą turėčiau jų mokintis...“
Paveldo
objektai
„...turime daug lankytinų vietų, sena-
miesčių...“
„...Lietuvoje nieko nepamatysi
įdomaus, visur viskas bran-
gu...“
„...turime savitą kraštovaizdį...“
„...gyvename prie jūros ir turime gin-
tarą...“
„...turime savos veislės žirgus – žemai-
tukus...“
„...nuo senų laikų likęs pagrindinis
maistas – cepelinai, balandėliai, ku-
gelis...“ 58 Unikali tautos savastis, neturinti opozicijos, respondentams yra Lietuvos gamta. Gyvenan-
tiems pajūryje jūra ir gintaras yra tautos vertybės. Paaugliams, kurie domisi jodinėjimo sportu,
nacionalinė vertybė yra žemaitukų veislės žirgai. Tai patvirtina nuostatą, kad vertybė yra tai, ką
žmogus iš tikrųjų žino ir pažįsta. Paauglių išvardytos tautinės vertybės – Lietuvos žemė, tauti-
nis tapatumas, istorija, kalba, tradicijos, tautos kultūra, paveldo objektai – skleidžiasi žinojimo
/ išmokimo perspektyvoje, šia savastimi didžiuojamasi. Žinojimui trūksta dvasingumo pajautos. Opoziciškos nuomonės, paremtos realybės pastebėjimais, tikroviškiau atspindi tautinių vertybių
būklę – išskirtinumų mažėja, tautos panašėja dėl masinės kultūros invazijos. Būti tikru lietuviu,
tikru patriotu darosi sunku. Patriotiškumo sklaida Patriotiškumas yra išugdoma vertybė – jausmas, atsirandantis kaip auklėjimo pasekmė, – teigia
respondentė. Tai reiškia mylėti savo žemę, kalbą, šalį žinant už ką, kodėl, vardan ko: „Manyčiau,
jog patriotiškumo ką tik gimęs kūdikis neturi. Tai išugdoma vertybė, ir ar ją turėsi ar ne, priklau-
sys tik nuo tavo auklėjimo. Nuo mažens buvau ugdoma, kad reikia mylėti savo žemę, kalbą, šalį. Taigi, jei tave išugdė lietuviu, tai bus lengva, tačiau, jei ne – bus sunku mylėti savo šalį, nežinant
už ką, kodėl, kam...“ Sektinas patriotinio auklėjimo pavyzdys yra kai kurių bendruomenių puoselėjamos valstybi-
nių švenčių minėjimo tradicijos, kai jaunam žmogui įskiepijamas pareigos jausmas kaip palikimas
ateinančiai kartai: „Mūsų kaimas turi tradiciją švęsti Kovo 11-ąją, Vasario 16-ąją. Pirmiausia
iškyla trispalvės, tada susirenkame buvusiųjų tremtinių sodyboje, giedame giesmes, dainuojame
dainas, vyresnieji dalinasi prisiminimais, keliame klausimus: „Ką man reiškia būti lietuviu.“ Ta
nuotaika, kai esame visi drauge, man leidžia pasijusti tikra patriote.“ Patriotiškumo jausmą žadina edukaciniai renginiai, ekskursijos, nes pagarba ir meilė Tėvynei
prasideda nuo gimtųjų namų, tėviškės, krašto pažinimo. Tai skatina mąstyti apie lietuviškumą,
pasijausti Lietuvos dalimi: „Nuvykęs į Kernavę, atsistojęs ant piliakalnio ar pažvelgęs į Nemuną,
įsivaizdavau, kaip kalavijuočiai puolė mus – lietuvius. O šalia augantis miškas padėjo triuškinti
priešus. Toje aplinkoje ir pasijutau, kad esu lietuvis“; „Prisimenu kalnus, miškus, ežerus, vaka-
ronę, dainas ir senovines apeigas. Tą dieną ilgiausiai mąsčiau apie lietuvybę.“ Ugdant patriotizmą svarbiomis tampa istorijos žinios. Jos sukelia pasididžiavimo, net meilės
jausmus: „Jei atvirai, didžiuojuosi, kad esu lietuvis ir žinau, kad kažkada mano protėviai vaikš-
čiojo LDK žemėmis, ir tai man kiekvienąkart primena istorijos vadovėlis“; „Tikra lietuve pasiju-
tau, kaip nebūtų keista, per istorijos pamoką kalbant apie mūsų šalies praeitį, apie tai, kaip mes
kovojome dėl savo nepriklausomybės. Tuomet mane pirmąkart užplūdo meilė Lietuvai.“ Tačiau gimtoji kalba nebėra ištikimybės tėvynei objektas, nors beveik visi paaugliai
(95,6 proc.) teigia, jog mokėti gimtąją kalbą yra svarbu, patriotiška, nes tai – „išskirtinė kalba“,
„senovinė kalba“, „sunki ir nepaprastinta per tiek metų“. Tačiau daugelis jų (87,2 proc.) pabrė-
žia, kad anglų kalba daug lengvesnė ir įdomesnė. Anglų kalbos populiarumą lemia tai, kad „šia
kalba galima susikalbėti beveik visame pasaulyje, o lietuviškai tik Lietuvoje“; „tėvai mano, kad
anglų kalba ateityje bus reikalingesnė“; „lietuvių kalbos mokausi visą gyvenimą ir niekaip nega-
liu išmokti“ ir pan. Tai gali būti priežastys, kodėl daugelis šiuolaikinių paauglių rašo su klaidomis
ir ne visi moka nuosekliai reikšti savo mintis. Kolektyvinio tapatumo – vienybės – jausmas dažnai painiojamas su patriotiškumu. Patriotiškumo sklaida Respon-
dentai patiria patriotinius jausmus per krepšinio varžybas: „Tačiau didžiausias patriotiškumo 59 antplūdis mane ištinka stebint krepšinio varžybas. Tas jausmas man asocijuojasi su aistra, įtam-
pa. Tik tada suprantu, kad Lietuva man labai brangi.“ Bet tai tik laikina būsena: „Lietuviai būna
patriotai tik tuomet, kai vyksta krepšinio varžybos. Jie serga už savo šalį, o kai varžybos baigiasi,
taip pat ir patriotiškumas baigiasi.“ Patriotiškumas, kaip santykis su tauta, respondentų darbuose išreiškiamas įvardžiais aš, mes –
tu, jie. Sakydamas aš, žmogus prisiima atsakomybę už savo žodžius: „aš čia gimiau ir patyriau
savo geriausius laikus ir šventes“, „kada nors aš tikrai paaukosiu savo gyvybę dėl savo tautos“. Žodis mes turi kolektyvinio tapatumo, susitapatinimo su savo tauta konotaciją: „tai mūsų gimtoji
tauta, tik čia mes galime geriausiai pritapti“, „net širdį suspaudžia, kaip tada kovojom už laisvę,
nepasidavėm ir vieni kitiems, buvome šilti.“ Sakant tu, jie, tarsi atsiribojama: „turi dėl jos auko-
tis“, „žmonės parodo, kaip myli ir tiki Lietuvos valstybe.“ Patriotiškumas kognityviniu lygmeniu išreiškiamas veiksmažodžiais reikia, turėtų būti: „rei-
kia švęsti lietuviškas šventes“, „reikia neužmiršti savo kalbos“, „reikia neišsilakstyti į užsienį“,
„reikia būti savimi ir nebijoti parodyti, kad esi lietuvis“, „lietuvis turėtų garsinti Lietuvą“. Galima teigti, kad daugelis paauglių žino, ką reikia daryti, „kad išliktume savita šalimi“. Tačiau žinojimas neparodo, ar vertybė yra internalizuota: laikui bėgant žinios ir vertybės gali
pasikeisti. Tai patvirtina paauglių išvardyti nepatriotiškai nuteikiantys išoriniai veiksniai – ša-
lies ekonominės sąlygos, nedarbas, šeimų skurdas: „Mane, gyvenantį krizės ištiktoje Lietuvoje,
matantį suaugusiųjų sutrikimą, irgi veikia daug dalykų“; „kiekvieną dieną pagalvoju, kad gal
geriau būtų išvykti į užsienį ir ten gyventi, nes Lietuvoje vyrauja nedarbas, visi liūdni, nusimi-
nę“; „čia mažas pragyvenimo lygis ir ten, kur aš buvau išvykęs, tikrai geriau“ ir pan. Kartais tai
skamba kaip pagalbos šauksmas: „Tai yra mano šalis, kurioje gimiau, augau ir dabar gyvenu! Tai – mano šalis, kurioje norėčiau gyventi visada, bet nežinau, ar tai bus galima įgyvendinti, nes
mūsų finansinė būklė tikrai labai prasta!!!“ Nepatriotiškai nuteikia kai kurie tautiečių būdo bruožai: „Visi gyvena sau, sudaro kliūčių,
kad kitiems nepasisektų taip, kaip pasisekė jam“; „žmonės yra labai pikti: jei turi pinigų, tave
priims visi, bet jei esi neturtingas, negali sklaidytis pinigais, tavęs niekas nepriims.“ Negarbingas
bendratautiečių elgesys tampa blogu pavyzdžiu ir pasiteisinimu: „Kas mane suformavo? Baisūs
dalykai, baisi aplinka. Ir kaip gali būti geras, nepiktas, kai visi aplinkui tokie. Juk negali būti
kitoks – tave tuoj pat suvalgys“ ir pan. Patriotiškumo sklaida Išanalizavus paauglių patriotines nuostatas galima teigti, kad patriotiškumas pasireiškia tam
tikromis aplinkybėmis ir yra labiau susijęs su žinojimu, negu su buvimu. Šv. Rašte nėra pasakyta,
kad reikia mylėti savo artimą, yra pasakyta „mylėk...“ Gali būti, kad meilės Tėvynei jausmas yra
ta trūkstama grandis tarp žinojimo ir veikimo. Meilės Tėvynei gramatika Gramatika reiškia stiliaus, sakinio, žodžio ir formų mokslą. Daugelio respondentų esė vyrauja
poetinė sintaksė. A. J. Greimas (1991) yra teigęs, kad poezija yra kalba, vartojama ne tik susiži-
noti su kitu asmeniu, bet ji turi būti suprasta globaliai, iš karto, išjausta. S. Matulaitienė (1997)
„Poezijos gramatikoje“ laikosi nuostatos, kad kiekvieną teksto komponentą reikia nagrinėti ats-
kirai, tačiau būtina turėti prieš akis visumą. Siekdami atskleisti respondentų meilės Tėvynei,
prisirišimo prie jos nuostatas, nagrinėjome esė gramatiniu požiūriu: kaip stilistiškai atskleistas
jausmas, kokios veiksmažodžių gramatinės formos vartojamos, kaip, kokiais žodžiais įvardytas
supratimas, kokie veiksniai rašančiajam svarbūs. 60 Respondentė teigia: „Kad esu lietuvė, žinojau ir jutau visą laiką. Tai ypač stipriai pajutau,
kai vieną kartą tėvai kalbėjo, kad Lietuvoje nėra nieko gero ir visiems lietuviams reikia „mauti“
lauk iš Lietuvos. Tada aš susinervinusi tėvams pasakiau, kad jie nors ir dabar gali važiuoti, bet aš
pasiliksiu, nors būčiau ir vienintelė Lietuvoje. Nė už ką nepalikčiau Lietuvos. Čia yra visas mano
gyvenimas: draugai, žirgai, miškai ir pievos, kuriose aš pažįstu kiekvieną akmenį ar medį.“ Meilės
ir prieraišumo jausmai išreikšti veiksmažodžiais „žinojau“, „jutau“. Jausmas toks stiprus, kad
nebaisu pasipriešinti tėvų ketinimams. Sakinys, „nors būčiau ir vienintelė Lietuvoje“, „nė už ką
nepalikčiau“, reiškia stiprų apsisprendimą, asmenybės tvirtumą. Toliau logiškai išdėstoma, kas yra
lietuviškumo konkrečiu atveju pagrindas, vertybinė nuostata, meilės pamatas: „Čia yra visas mano
gyvenimas: draugai, žirgai, miškai ir pievos, kuriose aš pažįstu kiekvieną akmenį ar medį.“ Kita respondentė rašo: „Prisimenu save su tėvais sėdinčius kylančiame lėktuve ir keistą jaus-
mą, maudžiantį širdį, paliekant savo šalį trumpoms atostogoms. Tada supratau, kad niekur kitur
nesijausčiau taip gerai, kaip po savo tėvynės dangumi. Tikra lietuve pasijutau sėdėdama jau at-
galiniam skrydžiui pasibaigus, besileidžiant lėktuvui ant žalios lietuviškos žolės, aidint džiugiems
lietuvių keleivių plojimams. Nuotaikos nesugadino net tą kartą pradėjęs kapsėti lietus. Net jis
buvo mielas širdžiai ir atrodė kažkoks kitoks...lietuviškas!“ Tai būta „keisto jausmo, maudžiančio
širdį“: „žalia lietuviška žolė“, „mielas širdžiai lietuviškas lietus“. Yra įvardytas ir jausmo paska-
tintas supratimas, kad „niekur kitur nesijausčiau taip gerai, kaip po savo tėvynės dangumi“. Paauglė prisimena: „Pamenu, vieną rytą pabudau pas močiutę kaime. Paukščiai čiulba, šunys
loja, upelis čiurlena, saulės spinduliai man glosto veidą. Kas gali būti širdžiai maloniau? Išėjus į
lauką, pamačius platų žydintį sodą, mano širdis džiaugėsi, jog tame nepaprastai gražiame krašte
gyvenu. Atrodo, viskas sava! Ir senoji obelis, ir gėlės, ir žolė, vėjo linguojama. Nuostabu! Meilės Tėvynei gramatika Visuo-
met noriu ten sugrįžti ir pajusti tą Lietuvos, tikrosios Lietuvos gamtą ir grožį.“ Meilės jausmas
pasireiškia per „širdžiai malonius“, „džiuginančius širdį“ dalykus, kurie priklauso „tikrajai Lie-
tuvai“, kur „viskas sava“, nes „tikrosios Lietuvos gamta“, jos grožis, respondentės pajautimu,
yra likęs tik kaimo aplinkoje. Kitas respondentas reiškia mintis protesto forma: „Žmonės važiuoja į užsienį gyventi, laimės
ieškoti. Ne!!! Jokiu būdu nevažiuosiu iš Lietuvos, nes myliu savo šalį. Bet, kaip sakant, niekada
nesakyk niekada, bet nevažiuosiu iš Lietuvos. Gyvensiu, dirbsiu, kursiu šeimą tik čia! Savo vaikus
ugdysiu tikrais Lietuvos piliečiais. Ir nesuprantu tų žmonių, kurie bėga iš Lietuvos, kurie ją smer-
kia. Nereikia to daryti!!!“ Paauglystei būdingi šiek tiek egzaltuoti jausmai. Respondentas savo
norą būti gimtinėje, neemigruoti išreiškia aistringa priesaika: „Gyvensiu, dirbsiu, kursiu šeimą tik
čia! Savo vaikus ugdysiu tikrais Lietuvos piliečiais“, o emigraciją, laimės ieškojimą užsienyje,
Lietuvos smerkimą palydi šūksniais: „Ne!!!“, „Nereikia to daryti!!!“ Šie pavyzdžiai rodo, kad meilė Tėvynei turi ryškų romantizmo atspalvį, su visais romantizmui
priskiriamais atributais – gamta, prieraišumo objektais, asmenybės jausmingumu. Brandų tautiškumą rodo šis rašinys: „Manau, lietuviu būti, tai galvoti apie jos ateitį, jos gero-
vę, manau, kad mes daug labiau padėtume Lietuvai jos nesmerkdami, galvodami visi vieningai
apie ateitį ir prisimindami „senąją“ Lietuvą. Kai žmonės kovojo už Lietuvą, tai argi jie kovojo
šiaip? Lietuva buvo svarbi kiekvienam piliečiui, jiems ji rūpėjo. Taigi, reikia stengtis gyventi
taip, kad galėtume kiekvienas pasakyti, jog didžiuojamės būdami lietuviais. Galbūt tai nėra taip
lengva, betgi mes esame lietuviai, mus daug kartų puolė, grasino, buvo atėmę brangiausią daly-
ką – kalbą. Bet jeigu mes, mūsų tėvai, proseneliai iškentė visa tai, tai argi mes negalime pakovoti
dėl Lietuvos, galvodami apie jos gražų, nerūpestingą rytojų?“ Šis pavyzdys rodo internalizuotą 61 tautinę savivoką, nors respondentė nevartoja žodžių patriotiškumas, meilė, vertybės. Tačiau iš-
reiškia aiškią poziciją – būti lietuviu, tai vieningai galvoti apie ateitį ir, prisimenant praeitį, kie-
kvienam „pakovoti“ dėl gražaus Lietuvos rytojaus. Galima teigti, kad meilės Tėvynei fenomenas pasižymi neprognozuojamomis prasmėmis, in-
dividualia aplinkybių, patirčių samplaika ir santykio su aplinka unikalumu. Tai nepriklausomų
veiksnių zona – jausmai, nuotaikos, prisiminimai, suvokimai ir žinojimai, tapę savastimi. Visai
priešingus (opozicinius) jausmus Lietuvai reprezentuoja šie respondentų pasisakymai: Lietuviu būti nereiškia nieko gero: „Pasaulyje būti lietuviu, manau, nėra čia nieko gero. Nei
lietuvių kalba kur nors daug susikalbėsi, nei tau jos kur nors prireiks. Man Lietuvoje svarbiausia
gauti išsilavinimą bei baigti mokslus, o ateityje – važiuoti dirbti į užsienį. Man būti lietuviu nėra
vertybė. Meilės Tėvynei gramatika Šiais laikais Lietuva yra nusiritusi ir visas jaunimas tai žino. Jaunimas gerai daro, kad
išskrenda iš Lietuvos, kitoje šalyje bus laimingesnis ir neskurs, ir nebus išnaudojamas kaip čia.“
Nemylėti savo gimtinės, reiškia niekinti viską, kas su ja susiję. Apmaudas – „nėra čia nieko
gero“ parodo, kad respondentas jaučiasi nelaimingas, skurstantis ir išnaudojamas. Vienintelis
kelias – „gauti išsilavinimą“ ir „važiuoti dirbti į užsienį“. Lietuviu būti reiškia jaustis antrarūšiam: „Šiaip mano gyvenimo kelyje buvo daug kelionių:
trumpų ir ilgų, smagių ir varginančių. Kelionės po Lietuvą su tėvais ir mokytojais. Kaunas, Vil-
nius, Anykščiai, Nida, Palanga, Druskininkai. Vėliau – Lenkija, Latvija, Olandija, Graikija, Por-
tugalija. Smagu buvo šiose šalyse. Bet ar jaučiausi tikru lietuviu? Ar didžiavausi tuo, jog esu
lietuvis? Greičiau ne negu taip, nes Graikijoje perplaukęs baseiną greičiau už vokietuką, vis
tiek prizo negavau – buvau TIK lietuvis. Antrarūšis kažkoks. Portugalijoje – laaabai smagu, bet
lietuviškumo ten dar mažiaiu. Tokioje šalyje pabuvęs ilgiau, greičiau visai pamirši savo tautybę. Štai toks mano kelias. Kiek tas kelias geltonai žaliai raudonas? Nežinau...“ Šiuo atveju „smagu
buvo“, „bet ar jaučiausi“, „ar didžiavausi“ reiškia abejojimą, neapsisprendimą, kuris linksta
ne meilės gimtinei pusėn – „buvau TIK lietuvis“, „antrarūšis kažkoks“. Argumentais tampa du
veiksniai: „perplaukęs baseiną greičiau už vokietuką, vis tiek prizo negavau“ – kai užgaunamos
paauglio asmeninės ambicijos, „laaabai smagu, bet lietuviškumo ten dar mažiau“, „pabuvęs
ilgiau, greičiau visai pamirši savo tautybę“ – tautinės vertybės gali būti užgožtos ryškesnių as-
meninių įspūdžių. Būti amerikiete – didesnis džiaugsmas: „Nors aš nesigailiu, kad esu lietuvė, norėčiau, kad ši
šalis būtų didesnė, jos miestai – judresni. Bent vienas. Man Lietuvoje trūksta gyvenimo, triukš-
mo... Džiaugiuosi būdama lietuvė, bet būdama amerikiete džiaugčiausi labiau.“ Šiame pavyzdy-
je išsakytas tikros miestietės noras, kad „šalis būtų didesnė“, „miestai judresni“, „bent vienas“,
nes „Lietuvoje trūksta gyvenimo, triukšmo.“ Miestietiška aplinka formuoja kitokį supratimą apie
pasaulį – galima suprasti, kad trokštama įspūdžiais užpildyto gyvenimo. Būti lietuviu – nesuvokiamas dalykas: „Dirbu internete. Ką sugebu: galiu sukurti folderį,
greit rašau, galiu pakeisti nuotraukos formatą. Noriu uždirbti 100 000 lt/mėn. LIETUVIU? Kas
čia do itemas?“ Šis atvejis demonstruoja pasididžiavimą pragmatiniu gebėjimu: „dirbu“, „suge-
bu“, „noriu uždirbti“. Apie lietuviškumą visiškai nemąstoma – tai nereikalingas daiktas (angl. item – vienas iš pažymėtų dalykų, daiktas, elementas, pranešimas, prekė). Tai gali reikšti prisiri-
šimą prie virtualaus pasaulio dalykų, kuriuose nėra romantinių jausmų, vertybių – yra vertės. Taigi, meilė respondentų rašiniuose atsiskleidžia kaip stabilesnė būsena, kuri neišnyksta kei-
čiantis išorinėms aplinkybėms. Buvimo lietuviu filosofija – diskusijų laukas Apibendrinant paauglių tapatinimosi su tauta ir jos vertybėmis nuostatas / jausmus sąlygiškai
galima išskirti po du pozityvių / negtyvių pozicijų kategorinius branduolius, išreiškiančius buvi-
mo lietuviu filosofiją (žr. 4 lentelę). Tai nėra konstanta, nes daugeliu atvejų, paauglių nuomone,
„lietuviu būti gera, tik reikia kai kuriuos dalykus pakeisti“, „reikia perauklėti Lietuvos žmones“,
kartais abejojama – tai reiškia, kad pasaulėžiūros pamatai nėra iki galo susiformavę. Pozityvus
mąstymas ir jausmai priklauso idealistinei paradigmai, demonstruoja idealistinio realizmo raišką,
kuri buvo S. Šalkauskio pedagoginės sitemos ašis – idealas, kurio galima siekti, bet neįmanoma
tobulai įgyvendinti ugdant (1991). Negatyvus mąstymas remiasi gyvenamosios realybės kritika,
atsižvelgiant į aplinkybes, atliepia realistinei paradigmai arba išreiškia hedonistinę, vartojamąją
pasaulėžiūrą: 4 lentelė. Paauglių „buvimo lietuviu“ filosofija
Mąstymas pozityvus
Diskusijų laukas
Mąstymas negatyvus
Lietuva išsiskiria savo paveldu, unikalia istorija ir kalba,
grynu oru, gražia gamta ir aistra krepšiniui. Lietuva yra eilinė šalis ir gyventi joje
yra sunku. Būti lietuviu yra garbė ir vertybė. Tai reiškia: „mylėti Tė-
vynę kokia ji bebūtų“, „saugoti Lietuvos žemę“, „vertinti
save kaip svarbią gimtinės dalelę“, „gerbti tautos istoriją ir
kalbą“, „laikytis įstatymų“, „puoselėti tautiškumą“, „bran-
ginti šeimos vertybes“, „saugoti gamtą“, „norėti, kad sek-
tųsi ne tik tau, bet ir Lietuvai, nes jeigu blogai Lietuvai, visa
tai atsilieps ir tau“, „kartu gintis nuo krizės“, „susivienyti ir
kurti gražią ateitį“, „būti savo šalyje ir padėti jai“, „būti kar-
tu ir padėti vienas kitam“, „būti tuo, kuo esi“, „kalba, gimti-
nė, papročiai, šventės, giminės – viskas, kas svarbiausia ir
artima“, „kai nuvažiuosiu į kitą šalį, turėsiu pasakyti, kad
aš – lietuvis ir kaip aš elgsiuosi, kalbėsiu, tokį žmonės susi-
darys apie mane įspūdį, pasitikės manimi arba ne“, „įgijęs
gerą išsilavinimą Lietuvoje gali gyventi geriau, negu tar-
naudamas užsieniečiams“, „gal mes būsime ta karta, kuri
išgarsins ir sustiprins mūsų valstybę“
Būti lietuviu nėra vertybė, yra gėda,
nes: „Lietuva yra apkerpėjusi ir atsi-
likusi“, „mes eilinė tauta“, „Lietuvoje
labai mažai galimybių, bjaurūs žmo-
nės ir apskritai, Lietuva atsilieka nuo
pasaulio meno, kultūros“, „čia niekada
nieko nepasieksi“, „Lietuva nori pilkos
masės“, „netolerantiška tauta, nedrau-
giška, niekina vienas kitą“, „tokioje ne-
vieningoje valstybėje sunku prisitaiky-
ti“, „atlyginimai mažesni negu kainos ir
mokesčiai“, „didžiausi nusikaltėliai pa-
saulyje yra lietuviai“, „užsienyje geres-
nės perspektyvos“, „gyventi Lietuvoje
yra laiko gaišinimas“ 4 lentelė. Paauglių „buvimo lietuviu“ filosofija Diskusijų laukas rodo būtinybę ne tik kalbėtis su jaunais žmonėmis apie tautiškumą, tauti-
nes vertybes ir savigarbą – kas svarbiau, būti ar turėti, dirbti ar kritikuoti, kurti ar vartoti? Meilės Tėvynei gramatika Galima daryti prielaidą, kad meilė Tėvynei padeda išsaugoti tau-
tines vertybes ir modernėjant pasauliui neprarasti dvasingumo. 62 Išvados Modernioje valstybėje tautinių vertybių adoracija užleidžia vietą pilietiniams, dažnai formaliems
įsipareigojimams tautai. Tačiau galima daryti teorinę prielaidą, kad pilietiškumas, kurio politinį
pagrindą sudaro žmogaus teisių ir laisvių pirmumas, internalizavus dvasinį pradmenį, pajėgus
sukurti tokias sąlygas, kad patriotizmas taptų tylia ir savaime suprantama žmogaus ir piliečio
būsena. Tyrimas patvirtino, kad patriotiškumas yra išugdomas kolektyvinis jausmas, pasireiškiantis
žinojimu, kodėl reikia mylėti ir ginti tėvynę, saugoti tautos vertybes. Tačiau dažniausiai tai iš-
provokuota būsena, kuri „išgaruoja“ pasibaigus renginiui arba kai reikia prisiimti atsakomybę už
savo troškimus ir poelgius. Nepasitikėjimas valdžios sprendimais, ekonominiai sutrikimai, emi-
gracijos problemos, bendrabūvio ar atotolio nuotaikos šeimose veikia jaunų žmonių savimonę ir
kuria emocinio nepasitenkinimo, nesaugumo, nestabilumo, nepasitikėjimo artimiausia aplinka,
savo tauta ir valstybės ateitimi būsenas. Galima daryti prielaidą, kad šalia vartojamojo godumo
turėti daugiau materialių galimybių, šeimos skurdėjimo patirtis gali būti svarbus išorinis veiks-
nys, darantis įtaką vertybių formavimuisi. Taip pat išoriniu veiksniu, negatyviai veikiančiu tau-
tinių vertybių priėmimą, tampa savanaudiškas bendratautiečių elgesys – paaugliai ypač pastabūs
ir imlūs, kai kurie tai išreiškia prisipažinimu ir aš taip darau. Todėl apie atsakomybę ir pareigą
Tėvynei dažniau kalbama vartojant veiksmažodžius reikia, turėtų būti, kurie rodo neinternalizuo-
tą vertybių formą. Kita vertus, jauni žmonės išreiškė labai brandžių minčių apie Tėvynės reikšmę
žmogaus gyvenime. Tėvynės meilės fenomenui būdingas emocionalumas, individuali patirtis,
turinti romantizmo bruožų. Gali būti, kad meilės jausmas – trūkstama jungtis tarp žinojimo ir
veikimo Tėvynės labui. Tyrimas atskleidė, kad pragmatizmas jau suleidęs šaknis paauglių vertybinių nuostatų lau-
ke. Dažniau pamąstoma, ar verta prisirišti prie nykstančių tautinių vertybių, teigiama, kad dau-
giau naudos asmeniui suteikia turėjimas, negu buvimas. Pasaulėžiūra yra asmenybės formavimo
pagrindas, tai parodo ir paauglių buvimo lietuviu filosofijos gairės. Idealistai labiau negu kriti-
kuojantys realistai, pragmatikai, individualistai, vartotojai atsakingi už kitą, už tautinių vertybių
branginimą ir bendrabūvio gimtoje žemėje kūrimą ir kitoniškumo toleravimą. Kai menksta tautos
dvasingumo pradai, nyksta ir kolektyvinis idealizmas. Buvimo lietuviu filosofija – diskusijų laukas – bet
auklėjant sudaryti sąlygas, skatinti veikti iš meilės gimtajam kraštui, jo žmonių savitumui. Nesa-
vanaudiška veikla atskleidžia dorovinį jos pobūdį, aukšta tautos dora demonstruoja tikrąjį patrio-
tiškumą – patriotiška kovoti su negerovėmis, tačiau šiai kovai reikia dvasios stiprybės ir auklėtojų
kompetencijos. Teigiamas kritikos bruožas – pažinti negeroves, bet dar svarbiau išmokyti jauną
žmogų ieškoti būdų pakeisti neigiamus veiksnius, kaip kiekvienam prisidėti prie modernios vals-
tybės gerovės kūrimo. Skirtingos nuomonės visada buvo pažangos variklis – Lietuvos paaugliai
yra subrendę diskusijai apie tautines vertybes. 63 Literatūra Nacionalinis sąmoningumas šiandien: jaunuome ir pilietiškumas. Atskirtis ar dermė? Vilnius: VPU leidykla, 37–46. Huserl, E. (2005). Karteziškosios meditacijos. Vilnius: Aidai. Jovaiša, L. (2007). Enciklopedinis edukologijos žodynas. Vilnius: Gimtasis žodis. Jovaiša, L. (2009). Gyvenimo sėkmės ugdymas. Vilnius: AGORA. Kavolis, V. (1996). Kultūros dirbtuvės. Vilnius: Pradai. Kavolis, V. (1996). Kultūros dirbtuvės. Vilnius: Pradai. Kuzmickas, B.; Astra, L. (1996). Šiuolaikinė lietuvių tautinė savimonė.Vilnius: Rasma. mickas, B.; Astra, L. (1996). Šiuolaikinė lietuvių Kuzmickas, B. (2007). Tautos tapatumo savimonė. Vilnius: Mykolo Romerio universitetas. Kuzmickas, B. (2007). Tautos tapatumo savimonė. Vilnius: Mykolo Romerio universit Lukšienė, M. (2000). Jungtys. Vilnius: Alma littera. Lukšienė, M. (2000). Jungtys. Vilnius: Alma littera. Lukšienė, M. (2000). Jungtys. Vilnius: Alma littera. Lyotard, J. F. (2010). Postmodernus būvis. Vilnius: Baltos lankos. Š Lyotard, J. F. (2010). Postmodernus būvis. Vilnius: Baltos lankos. Š Lyotard, J. F. (2010). Postmodernus būvis. Vilnius: Baltos lankos. Maceina A. (1991). Tautinis auklėjimas. Kaunas: Šviesa. Maceina A. (1991). Tautinis auklėjimas. Kaunas: Šviesa. Martišauskienė, E. (2004). Paauglių dvasingumas kaip pedagoginis reiškinys. Vilnius: VPU leidykla. Martišauskienė, E. (2004). Paauglių dvasingumas kaip pedagoginis reiškinys. Vilnius: VPU leidykla. Matulaitienė, S. (1997). Poezijos gramatika.Vilnius: Gimtasis žodis. Matulaitienė, S. (1997). Poezijos gramatika.Vilnius: Gimtasis žodis. Mokymosi visą gyvenimą memorandumas. [žiūrėta 2013-02-11]. Prieiga per internetą: www.lssa.smm. lt/docs/Memorandumas_2001. doc. Mokymosi visą gyvenimą memorandumas. [žiūrėta 2013-02-11]. Prieiga per internetą: www.lssa.smm. lt/docs/Memorandumas_2001. doc. Pradinio ir pagrindinio ugdymo bendrosios programos. Lietuvos Respublikos švietimo ir mokslo mi-
nistro 2008 m. rugpjūčio 26 d. įsakymas Nr. ISAK-2433. Vilnius. Pradinio ir pagrindinio ugdymo bendrosios programos. Lietuvos Respublikos švietimo ir mokslo mi-
nistro 2008 m. rugpjūčio 26 d. įsakymas Nr. ISAK-2433. Vilnius. Stonkuvienė I. (2011). Etninės kultūros perdavimas: teorinis ir praktinis aspektas, Acta paedagogica
Vilnensia. Mokslo darbai, 26 : 9–21. Stonkuvienė I. (2011). Etninės kultūros perdavimas: teorinis ir praktinis aspektas, Acta paedagogica
Vilnensia. Mokslo darbai, 26 : 9–21. Šalkauskis, St. (1991). Pedagoginiai raštai. Kaunas: Šviesa. Šalkauskis, St. (1991). Pedagoginiai raštai. Kaunas: Šviesa. Tijūnėlienė, O. (2002). Tautinio kultūrinio tapatumo ugdymas mokykloje – mokytojų įsisąmonintas
uždavinys. Pedagogika. Mokslo darbai, 60 : 94–100. Tijūnėlienė, O. (2002). Tautinio kultūrinio tapatumo ugdymas mokykloje mokytojų įsisąmonintas
uždavinys. Pedagogika. Mokslo darbai, 60 : 94–100. T i kū i
ė I (2005) Et
k ltū i i
it
išl ik
t t
Li t
E
S j
j
i uždavinys. Pedagogika. Mokslo darbai, 60 : 94–100. Trinkūnienė, I. (2005). Etnokultūrinio savitumo išlaikymo nuostatos, Lietuva Europos Sąjungoje: pir- Trinkūnienė, I. (2005). Etnokultūrinio savitumo išlaikymo nuostatos, Lietuva Europos Sąjungoje: pir-
mieji metai. Vilnius: Fridas, 217–233. Literatūra Anderson, B. (1999). Įsivaizduojamos bendruomenės. Apmąstymai apie nacionalizmo kilmę ir plitimą. Anderson, B. (1999). Įsivaizduojamos bendruomenės. Apmąstymai apie nacionalizmo kilmę ir plitimą. Vilnius: Baltos lankos Vilnius: Baltos lankos. Aramavičiūtė, V. (2005). Auklėjimas ir dvasinė asmenybės branda. Vilnius: Gimtasis žodis. Bajoriūnas, Z. (1997). Šeimos edukologija. Vilnius: Jošara. Barth, F. (1969). Ethnic Groups and Boundaries: the Social Organizacion of Culture Difference. Boston: Aramavičiūtė, V. (2005). Auklėjimas ir dvasinė asmenybės branda. Vilnius: Gimtasis žodis. Barth, F. (1969). Ethnic Groups and Boundaries: the Social Organizacion of Culture Difference. Boston:
Little, Brown and Company. Bauman, Z. (2002). Globalizacija: pasekmės žmogui. Vilnius: Strofa. Little, Brown and Company. Bauman, Z. (2002). Globalizacija: pasekmės žmogui. Vilnius: Strofa. Little, Brown and Company. Bauman, Z. (2002). Globalizacija: pasekmės žmogui. Vilnius: Strofa. Bitinas, B. (1995). Auklėjimo procesas. Šiauliai. Buber, M. (1998). Dialogo principas I. Vilnius: Katalikų pasaulis. Buber, M. (2001). Dialogo principas II. Vilnius: Katalikų pasaulis. Čepienė, I. (2008). Lietuvių etninė kultūra. Raidos įžvalgos. Vilnius: Alma littera. Čiubrinskas, V. (2008). Tautinio identiteto antropologinio tyrinėjimo klausimai, Lietuviškojo identiteto
trajektorijos, sud. V. Čiubrinskas, J. Kuznecovienė. Kaunas: VDU, 13–25. Čiubrinskas, V. (2008). Tautinio identiteto antropologinio tyrinėjimo klausimai, Lietuviškojo identiteto
trajektorijos, sud. V. Čiubrinskas, J. Kuznecovienė. Kaunas: VDU, 13–25. 64 Derrida, J. (2006). Apie gramatologiją. Vilnius: Baltos lankos.iil Duoblienė, L. (2006). Šiuolaikinė ugdymo filosofija: refleksijos ir dialo Duoblienė, L. (2006). Šiuolaikinė ugdymo filosofija: refleksijos ir dialogo link. Vilnius: Tyto Erikson, E. H. (1968). Identity : Youth and Crisis. New York: Norton. Etninės kultūros plėtros švietimo įstaigose strategija 2003–2012. Lietuvos Respublikos švietimo ir
mokslo ministro 2009 m. lapkričio 19 d. įsakymas Nr. ISAK-2365. Vilnius. Gellner, E. (1996). Tautos ir nacionalizmas. Vilnius: Pradai. Gellner, E. (1996). Tautos ir nacionalizmas. Vilnius: Pradai. Giddens, A. (2000). Modernybė ir asmens tapatumas. Vilnius: Pradai. Girnius, K. K. (2011). Tauta, išbandymai, dorovė. Tarp komunizmo ir demokratijos. Naujasis židinys-
Aidai. Gellner, E. (1996). Tautos ir nacionalizmas. Vilnius: Pradai. Giddens, A. (2000). Modernybė ir asmens tapatumas. Vilnius: Pradai. Giddens, A. (2000). Modernybė ir asmens tapatumas. Vilnius: Pradai. Girnius, K. K. (2011). Tauta, išbandymai, dorovė. Tarp komunizmo ir demokratijos. Naujasis židinys-
Aidai. Greimas, A. J. (1991). Iš arti ir iš toli: literatūra, kultūra, grožis. Vilnius: Gimtasis žodis. Grigas, R. (2001). Tautinė savivoka. Vilnius: Rosma. Grigas, R. (2001). Tautinė savivoka. Vilnius: Rosma. Grigas, R. (2007). Nacionalinis sąmoningumas šiandien: jaunuomenės pasirinkimo drama, Tautiškumas
ir pilietiškumas Atskirtis ar dermė? Vilnius: VPU leidykla 37–46 Grigas, R. (2007). Nacionalinis sąmoningumas šiandien: jaunuomenės pasirinkimo drama, Ta Grigas, R. (2007). Summary As stated by the science of educology, at the tage of adolescence, education can make the greatest impact
on the formation of a personality and the contextualization of one’s own value. The article deals with the
13-to 16-year-old adolescents’ perception of national values and its expression in the context of modern
Lithuanianness. The research instrument – an essay – helps to determine younger teens’ national values,
analysis of young people’s experience of values, feelings, and attitudes. The context of modern Lithuanianness
is understood as a totality of cultural values created by the nation, handed on from generation to generation
via education and the regularly updated cultural whole – the common language, common past and destiny,
national mentality, traditions, landscape – by the patriotic love of homeland, its preservation and survival
point of view. The scientific problem of research is formulating by questions: what are the moral values
of Lithuanians aged 13 to 16 years and what factors affect the formation of national self-value in modern
Lithuania? The aim of the research was to reveal the values of a young man as the representative of the
nation, on the basis his its own national concept of culture.i The analysis of the scientific literature found that the adoration of national values in modern state
is giving way to civic, often formal, commitment to the nation. However, it is a theoretical assumption
that the citizenship, its political base being composed of human rights and freedoms, is able to create an
environment that patriotism would become a quiet and of course human and civil status by internalising the
spiritual primer. An empirical analysis of the data has revealed that external factors militate young people
unpatrioticly: distrust of government decisions, problems of economics and emigration, countrymens’ selfish
behaviour. On the other hand, young people express very mature thoughts about the value of human life in
the homeland. Patriotic love is characterized by emotions, personal experience with romantic elements. It
may be that the feeling of love is the missing conection between knowing and acting in the interests of the
homeland. But pragmatism is already rooted in adolescents. young people are often pondering whether it
is worth to be attached to the endangered national values; they understand that more benefits are affered
to a person by owning than by being. When the spirituality of a nation goes down the collective idealism
disappears. Literatūra mieji metai. Vilnius: Fridas, 217–233. Valstybinės švietimo strateginės 2003–2012 m. nuostatos. Lietuvos Respublikos Seimo nutarimas Valstybinės švietimo strateginės 2003–2012 m. nuostatos. Lietuvos Respublikos Seimo nutarimas
2003 07 04 Nr. IX-1700. Vilnius. Valstybinės švietimo strateginės 2003 2012 m. nuostatos. Lietuvos Respublikos Seimo nutarimas
2003 07 04 Nr. IX-1700. Vilnius. Z l
ki
ė I 2004 Pili i i
d
i li ė di
ij
Vil i
R
i
i i
ili ti ė
d k
ij 2003 07 04 Nr. IX-1700. Vilnius. Zaleskienė, I. 2004. Pilietinio ugdymo socialinė dimensija. Vilnius: Regioninis pilietinės edukacijos
centras.i Žydžiūnaitė, V.; Jonušaitė, S. 2006. Fenomenografija – kokybinės diagnostikos metodas: metodologinis
pagrindimas. [žiūrėta 2013-03-08]. Prieiga per internetą: www.biblioteka.vpu.lt/pedagogika/PDF/2007/85/
zj76-80.pdf. 13–16 metų paauglių apklausos raštu – esė – duomenys. 2012. Asmeninis autorių archyvas. Klaipėda. 13–16 metų paauglių apklausos raštu – esė – duomenys. 2012. Asmeninis autorių archyvas. Klaipėda. 65 Summary Key words: adolescence, national values, modern Lithuanianness, self-value. 66
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Expertise in Evaluating Choreographic Creativity: An Online Variation of the Consensual Assessment Technique
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Frontiers in psychology
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Expertise in Evaluating
Choreographic Creativity: An Online
Variation of the Consensual
Assessment Technique
Lucie Clements1,2*, Emma Redding2, Naomi Lefebvre Sell2 and Jon May3
1 Department of Psychology and Counselling, University of Chichester, Chichester, United Kingdom, 2 Dance Science, Trinity
Laban Conservatoire of Music and Dance, London, United Kingdom, 3 School of Psychology, Cognition Institute, University
of Plymouth, Plymouth, United Kingdom Lucie Clements1,2*, Emma Redding2, Naomi Lefebvre Sell2 and Jon May3 1 Department of Psychology and Counselling, University of Chichester, Chichester, United Kingdom, 2 Dance Science, Trinity
Laban Conservatoire of Music and Dance, London, United Kingdom, 3 School of Psychology, Cognition Institute, University
of Plymouth, Plymouth, United Kingdom In contemporary dance, experts evaluate creativity in competitions, auditions, and
performances, typically through ratings of choreography or improvisation. Audiences
also implicitly evaluate choreographic creativity, so dancers’ livelihoods also hinge upon
the opinions of non-expert observers. However, some argue that the abstract and often
pedestrian nature of contemporary dance confuses non-expert audiences. Therefore,
agreement regarding creativity and appreciation amongst experts and non-experts
may be low. Finding appropriate methodologies for reliable and real-world creativity
evaluation remains the subject of considerable debate within the psychology creativity
research field. Although considerably variant in methodological operationalisation, the
Consensual Assessment Technique (CAT) asks individuals to use an implicit definition to
assess creativity in others’ work. This study aimed to investigate the role of experience
and expertise in the evaluation of choreographic creativity, with a secondary aim of
testing the feasibility of an online snowballing methodology for large-scale dance-
specific research, informed by the methodology of the CAT. We filmed 23 Contemporary
Dance students each performing a 3-min peer-choreographed solo and then recruited
850 online evaluators with varying degrees of expertise and experience in dance and
creativity. Evaluators viewed at least one randomly selected video and rated creativity,
technical ability, appreciation and understanding of the work, each using a seven-point
Likert scale. A one-way ANOVA showed a significant difference in creativity ratings
across the 23 videos, and creativity correlated significantly with the other variables. We
then categorized evaluators on nine aspects of their dance and creative experience and
entered the data into a repeated-measures linear mixed model. Two of the fixed effects
yielded differences in creativity evaluations: (i) contemporary choreographic experience
and (ii) self-reported creative expertise, as did the random effect of the video. The
results indicate that personal experience of the choreographic process impacts creativity
assessment, above and beyond experience in dance class participation. Implications for
creativity assessment within creativity research and practice are discussed. Keywords: creativity, choreography, contemporary dance, expertise, audience, assessment ORIGINAL RESEARCH
published: 24 August 2018
doi: 10.3389/fpsyg.2018.01448 Edited by: Edited by:
Kathryn Friedlander,
University of Buckingham,
United Kingdom Reviewed by:
Glenna Batson,
Wake Forest University, United States
Luke Stephen Hopper,
Edith Cowan University, Australia Reviewed by:
Glenna Batson,
Wake Forest University, United States
Luke Stephen Hopper,
Edith Cowan University, Australia *Correspondence:
Lucie Clements
l.clements@chi.ac.uk Specialty section:
This article was submitted to
Performance Science,
a section of the journal
Frontiers in Psychology
Received: 29 April 2018
Accepted: 23 July 2018
Published: 24 August 2018
Citation:
Clements L, Redding E, Lefebvre
Sell N and May J (2018) Expertise
in Evaluating Choreographic
Creativity: An Online Variation of the
Consensual Assessment Technique. Front. Psychol. 9:1448. Specialty section:
This article was submitted to
Performance Science,
a section of the journal
Frontiers in Psychology Received: 29 April 2018
Accepted: 23 July 2018
Published: 24 August 2018 INTRODUCTION Audiences of varying
levels of expertise, levels or types of training, may, therefore,
assess creativity differently. Research in dance indicates that non-
expert dance audiences may fail to understand the meaning
behind contemporary dance, perhaps because contemporary
dance is detached from the ‘magic’ seen in dance which makes
use of popular music, costumes and staging (Stevens et al.,
2009). Contemporary dance has not become rooted in modern
westernized culture in the same way other art forms or classical
ballet have. For example, a dance director reports that his
audiences mainly consist of friends, family or supporters of
those directly involved in the performance rather than members
of the public (Van Dyke, 2010). Contemporary dancers are
often dressed in plain, everyday clothes or speaking directly
to audiences; the movement is often pedestrian and effortless,
or, hugely effortful. Often, dancers create movement without
music, and the music is added later in the choreographic process. Thus contemporary dance may be a particularly unique and ripe
area for novel research into creativity, and given this previous
research we were interested in the broad role of expertise and
understanding of contemporary dance in assessing creativity. The lack of creativity research within the performing arts
more broadly may be due to scientists’ misinformed beliefs
that performing artists are replicators who express work
generated by others, rather than creators, and are therefore
not a population of interest (Kogan, 2002; Sawyer, 2014;
Thomson and Jaque, 2017). Butterworth (2004) notes that this
traditional hierarchy of ‘choreographer-as-creative’ and ‘dancer-
as-reproducer’ is no longer the sole means by which creativity
occurs, citing numerous ways in which the dancer expresses
their creativity in choreography. The boundary between dancer
and choreographer is blurred, and dance students now learn
both performance and creative skills. Professional contemporary
dancers often contribute to the development of movement
material, through ‘exploring, selecting, and developing dance
material’ (Stevens and McKechnie, 2005, p. 40). The process
is often guided by ‘tasking’, the use of a problem set by the
choreographer, and solved by the dancers (May et al., 2011). Typically, each dancer’s material will contribute in some way,
through refinement of the movement and changes to timing,
resulting in a creative product (Stevens et al., 2001). Farrer (2014)
notes that whether improvising, choreographing, transforming
a phrase of movement, or completing a task, dancers embody
numerous creative roles, yet even dancers themselves do not
recognize their creativity. INTRODUCTION called ‘creativity’ is revealed in the products and that judges
and raters can recognize it” (p. 314). Csikszentmihalyi (2014)
argues that the interaction between three elements of a
system constitute creativity. A culture contains symbolic
rules
for
creativity,
the
individual
brings
that
creativity
into the domain, but creativity is only brought to fruition
when experts from that domain recognize the creativity. Recognition
of
creativity
occurs
in
contemporary
dance
education (for example, the ability to demonstrate creative
engagement in improvisation is a typical entry requirement
to
higher
education
dance
training),
subsequent
student
assessments, and in reviewing professional work. Although
experts are imperative to real-world creativity assessment,
non-experts also play a role in the day-to-day sustenance
of
creative
careers,
and
varying
levels
of
expertise
or
knowledge may predict differences in assessment of creativity
(Hong and Lee, 2015). ‘Contemporary dance’ loosely refers to a range of dance styles
that use the body to explore and express conceptual ideas or
images (Strauss and Nadel, 2012). In contemporary dance, there
are no set movement sequences to draw from so there is an
expectation of finding new and inventive movement. The focus is,
therefore, less on the formulaic construction of movement than in
classical forms such as ballet, with an often-deliberate rebellion
against codified technique. It is this freedom that supports
the argument that contemporary dance is creative by nature
(H’Doubler, 1998). Researchers commonly cite Guilford’s (1950)
presidential address to the American Psychological Association
as the defining moment in persuading psychology researchers
of the value and importance of scientific research into creativity
(Kaufman and Sternberg, 2010; Runco, 2014). Psychological
research has facilitated depth of understanding of the predictors,
correlates and consequences of creativity, but typically focuses on
general population research, with less research within specialist
domains (Kaufman and Sternberg, 2010; Runco and Acar, 2012;
Long et al., 2014; Runco, 2014; Simonton, 2015). Little has been
published drawing on scientific methods within the domain of
contemporary dance (Thomson and Jaque, 2017). Since participation in contemporary dance is an increasingly
popular recreational, educational and professional pursuit, one
could argue that the audiences who engage with and see
this creativity should also be increasing too. Burrows (2010)
highlights that contemporary dance audiences seek novelty, but
alternative research has also shown that some less experienced
contemporary dance audiences report confusion, failure to
understand the choreographic intention, and lack of enjoyment
(Stevens et al., 2007, 2009; Van Dyke, 2010). Citation: Clements L, Redding E, Lefebvre
Sell N and May J (2018) Expertise
in Evaluating Choreographic
Creativity: An Online Variation of the
Consensual Assessment Technique. Front. Psychol. 9:1448. doi: 10.3389/fpsyg.2018.01448 August 2018 | Volume 9 | Article 1448 1 Frontiers in Psychology | www.frontiersin.org Expertise in Evaluating Choreographic Creativity Clements et al. Frontiers in Psychology | www.frontiersin.org INTRODUCTION This
generalist perspective arguably lacks sensitivity to the individual
nuances of creative specialization that manifest in different ways
across different fields (Baer, 1998; Feist, 1998; Hu and Adey, 2002;
Julmi and Scherm, 2015). Divergent thinking tests may assess
only narrow ranges of ability and may not be conclusive about
measuring ‘creativity’ itself. Instead, they indicate abilities related
to creativity, which may not be as relevant in specialized domains
(Amabile, 1982; Baer and McKool, 2009). Thus it is important
also to develop methodologies that are sensitive to the individual
nuances of creativity in each domain. Although a large body of research has investigated audience
responses to classical dance as a performance (See Calvo-Merino
et al., 2005; Reason and Reynolds, 2010), there is a paucity
of research into contemporary dance audiences which focuses
on perceptions of creativity. Research has been undertaken to
explore the associative and affective results of performance (e.g.,
Stevens and McKechnie, 2005), but no research has considered
audience evaluations of creativity using the psychology of
creativity methods such as the CAT. Research using the CAT
supports that expert and non-expert creativity assessments of
poems differed significantly different, with expert raters giving a
higher rating than non-experts, thus is a suitable methodology
for investigating choreographic creativity (Kaufman et al., 2008). Kokotsaki and Newton (2015) suggest a continuum of insider-
outsider status that potential creativity assessors have, depending
on their expertise and experience. Therefore, using a simple
dichotomy of expert or non-expert may be too restrictive,
particularly in dance where individuals gain experience through
doing, making and watching. Choreographic creativity, for example, implicates embodied
cognition: cognitive processes are rooted in physical interaction
with the world (Wilson, 2002; Stevens and McKechnie, 2005). Embodiment
emphasizes
both
physical
exploration
and
knowledge (Kogan, 2002). Dancers understand the intention
and action of others moving in the same space and use the
body for problem-solving, demonstrating creativity by thinking
with the body (Kirsh, 2011). Choreographic creativity uses both
awareness of kinesthetic knowledge and experience in/through
the body and explicit knowledge of the external world; cognition
is situated (Risner, 2000; Kirsh, 2010, 2011). Thus creativity in
dance is a process of using the body in novel ways in response
to a task and the ability to successfully and fluidly link body
positions into a developed sequence (Stevens et al., 2001;
Stevens and McKechnie, 2005; Kirsh, 2011). INTRODUCTION A small number of research studies have used problem-
solving approaches to dancers’ creativity, using measures of
divergent thinking (the ability to produce multiple responses
to a problem) which is considered the ‘backbone of creativity
assessment’ (Runco, 2014, p. 14). Stinson (1993) found students
in Chinese dance education were significantly less creative (in
divergent thinking) than a non-dancing control group. Fink and
Woschnjak (2011) found differences in divergent thinking across
contemporary, ballet and jazz dance, suggesting that creativity
differs within dance genres. These studies suggest that differences
in creativity occur at the microdomain level of dance, yet their
generalized approach to assessing creativity may limit their
usefulness. are critical, which differs starkly from the pen and paper medium
emphasized in time-limited psychology measurement traditions;
thus studying creativity in dance would be wise to use dance in
its natural movement based form. One of the most widely advocated domain specific means
of assessing situated creativity is the Consensual Assessment
Technique (CAT; Amabile, 1982). The CAT is popular in
psychology since it is unrelated to any specific creativity theory,
meaning that its use is broad and relevant to any domain of
creativity (Baer and McKool, 2009). In the CAT methodology,
experts assess creativity using an implicit understanding within
their specific domain (Amabile, 1982; Amabile and Pillemer,
2012). Similarly, assessors in dance use an implicit creativity
definition to assess. For example, it is common to obtain mean
scores from panels assessments during improvisation at an
audition. However, the CAT has some challenges. Namely, there are
no clear guidelines for implementation, and many variations
have been used to investigate specific domains. It is a process
of obtaining evaluations from raters without using a formal tool
or needing to provide explicit criteria against which creativity
must be assessed. Conventionally, it is expected that raters should
share some common understanding of the domain to support a
consensus. There are reasons why traditional divergent thinking measures
may be of limited use for choreographic creativity. Most
importantly, some criticize the problem-solving approach to
creativity assessment for constituting just one type of creativity,
which assumes domain generality of the cognitive processes
(i.e., attention, perception, memory, language, and intelligence)
underpinning creativity (Kaufman and Baer, 2004; Runco, 2014). At this level, creativity is a nomothetic process shared by, and
accessible to, all humans (Simonton, 1999; Gl˘aveanu, 2010). INTRODUCTION These multiple perspectives highlight
a broad lack of awareness of dancers’ choreographic creativity,
calling for greater scientific attention to this unique domain of
creativity. Williams et al. (2016) note that despite the growth of
the
psychology
of
creativity
over
the
last
25
years,
in
particular, many fundamental complexities remain. One such
challenge is finding appropriate methodologies for investigating
previously underresearched domains of creativity. Problem
solving approaches are perhaps the most common methodology
seen in psychology research, where ‘creativity’ lies in the process
or means by which an individual arrives at a solution (Lubart,
2001). Problem-solving measures predominantly investigate
insight, also known as the ‘aha moment’ (e.g., the Remote
Associates Tests, Wallas, 1926; Mednick and Mednick, 1971; The purpose of our work was to investigate how experience
in contemporary dance impacts assessment of choreographic
creativity, because contemporary dance requires communication
of creative ideas to an audience (Humphrey, 1959; Burrows,
2010; Risner, 2000). Thus, creativity in dance is a social
phenomenon (Łucznik, 2015). As Csikszentmihalyi (1999)
notes, “The underlying assumption is that an objective quality August 2018 | Volume 9 | Article 1448 Frontiers in Psychology | www.frontiersin.org 2 Expertise in Evaluating Choreographic Creativity Clements et al. Runco and Jaeger, 2012). In these tests, problem solving
involves a two-stage process of divergent and convergent
thinking; restructuring the problem by reframing one’s mental
approach, to find the one appropriate answer (Guilford, 1956). A small number of research studies have used problem-
solving approaches to dancers’ creativity, using measures of
divergent thinking (the ability to produce multiple responses
to a problem) which is considered the ‘backbone of creativity
assessment’ (Runco, 2014, p. 14). Stinson (1993) found students
in Chinese dance education were significantly less creative (in
divergent thinking) than a non-dancing control group. Fink and
Woschnjak (2011) found differences in divergent thinking across
contemporary, ballet and jazz dance, suggesting that creativity
differs within dance genres. These studies suggest that differences
in creativity occur at the microdomain level of dance, yet their
generalized approach to assessing creativity may limit their
usefulness. Runco and Jaeger, 2012). In these tests, problem solving
involves a two-stage process of divergent and convergent
thinking; restructuring the problem by reframing one’s mental
approach, to find the one appropriate answer (Guilford, 1956). Frontiers in Psychology | www.frontiersin.org August 2018 | Volume 9 | Article 1448 INTRODUCTION These processes use
memory, language and perception as well as space, time, motion
and physical expression, with decreased emphasis on verbal
and greater emphasis on nonverbal communication (Bläsing
et al., 2010; Thomson and Jaque, 2017). Hagood (2001) writes
that dance, in general, is “an extremely complex experience to
attempt to measure” (p. 27). However, embodiment and process The role of creativity has been the subject of considerable
interest in psychology research but is yet to be explored in depth
in dance within a scientific framework. Therefore, the purpose of
this research was to establish an understanding of expertise on the
attribution of creativity in contemporary dance choreography. We aimed to recruit a large sample of assessors to judge
choreographic creativity of contemporary dance. Informed by the
method of the CAT, we used a quantitative methodology to assess
the impact of expertise in assessing creativity in contemporary
dance to rate video clips of student choreographies (Amabile,
1996). Additionally, we collected measures of perceptions of
technical ability, liking and ability to find meaning, as previous
research has indicated that non-experts use these variables to
assess creativity (e.g., Kozbelt, 2004; Glass and Stevens, 2005). August 2018 | Volume 9 | Article 1448 Frontiers in Psychology | www.frontiersin.org 3 Expertise in Evaluating Choreographic Creativity Clements et al. Participants
Choreographers Students (n = 24; male n = 6, female n = 18, mean age = 20.2 years;
SD = 1.6 years) studying in the 1st year of a BA Contemporary
Dance at Trinity Laban, a leading UK Dance Conservatoire,
consented to participate in the research. Students entered
onto the degree having been assessed for both technical and
creative skill at audition (evaluated through a panel marked
improvisation), thus had been selected onto the program
for their creative potential. Their dance training consists of
technique classes in Contemporary Dance (such as Graham
and Cunningham) and Ballet, as well as Choreography classes
focused on developing processes of exploratory non-stylistic
ways of moving from within the body. Students take additional
modules in performance and contextual studies. Students were
all members of the same choreography class, taught by the same
teacher, and had been randomly allocated to this teacher’s class at
the start of the academic year (from four possibilities). Procedure We
obtained
institutional
ethical
approval. Following
this, a choreography teacher provided initial consent to
approach her first-year choreography students to provide
choreographic material for creativity assessment in the research. The contemporary dance students consented at the end
of a timetabled choreography class, 2 weeks before their
choreography assessment. Each student’s assessed work was a
three-minute solo performed by a peer in the same class, so each
student consented once for the inclusion of their choreography
and a second time as a performer in a peer’s work. Creativity Ratings Creativity was assessed using a seven-point Likert scale (How
creative did you think the piece was?; 1. Not at all creative – 7. Very
creative) informed by the method of the CAT (Amabile, 1983). In addition to the target question, participants answered three
additional questions; How much did you like the piece? (1. Not
at all – 7. Very Much); How technically skilled did you think the
dancer was? (1. Not at all technically skilled – 7. Very technically
skilled); How able were you to find meaning in the piece? (1. Not
at all able to find meaning – 7. Very able to find meaning). Creativity Raters We recruited creativity raters (n = 1084) from a variety of levels
of expertise to the research. After data screening and cleaning, the
final sample size was 850 raters (female n = 682, male n = 158,
other n = 10). Participants ranged in age from 18 to 77 years
(M = 31.6, SD = 12.9). We created dummy variables using the
nine categories of experience and expertise seen in Table 1,
whereby an individual who’s answer was ‘No’ is coded as the
reference category of ‘0’, and an individual who’s answer was ‘Yes’
to any degree of experience is coded as ‘1’. The employment
categories were answered qualitatively and coded by the first
author as ‘No’ or ‘Yes’. An overview of rater experience and
expertise in dance and creativity are shown in Table 1. On the day of the assessment and filming for the research,
each participant provided secondary verbal consent to confirm
his or her inclusion. One participant was injured so did not
undertake her performance, resulting in 23 videos. We embedded
the clips into an online survey via a video hosting site. Snowball
sampling was used to recruit creativity online raters through
online platforms, social media and email groups. A variety
of groupings were targeted, including those with experience
in dance, those with experience in creative fields, and those
who had no experience in dance and/or creativity. Participants
completed comprehensive demographic questions to provide
information about their background and training in dance,
creativity and the arts. They then watched a randomly selected
video, before completing the four assessment scales (creativity,
liking, technique and meaning), which appeared in a random
order. Each participant had the option to watch as many clips as
they wished to, before completing the four scales at the end of
each piece. MATERIALS AND METHODS studio in natural lighting to standardize the videos and remove
confounding variables relating to production. We used a wide
shot of the dance studio which replicated a head-on audience
view. All dancers dressed in plain, dark colored practice clothes. An audio-visual expert removed the music and added a fade in
and out at the start and end of each piece. Video Stimuli We obtained videos of a short solo choreography (n = 23;
duration 172–194 s), which were created for the students’
choreography module assessment. The choreography was danced
by a classmate of the student, rather than the choreographer
themselves. We filmed the choreographies in a mirrorless dance TABLE 1 | Participant experience and expertise in dance and creativity. Experience/expertise
No (N)
Yes (N)
Experience in child/adult dance classes
n = 166
n = 684
Experience in child/adult contemporary dance classes
n = 444
n = 406
Current/previous attendance at the dance institution
n = 763
n = 87
Experience in watching live contemporary dance
n = 504
n = 346
Experience in choreographing dance
n = 262
n = 588
Experience choreographing contemporary dance
n = 605
n = 245
Employed in any creative domain
n = 383
n = 467
Employed in an artistic, creative domain
n = 483
n = 367
Are you an expert in creativity? n = 714
n = 136 TABLE 1 | Participant experience and expertise in dance and creativity. After 6 weeks, we had obtained sufficient data. Data were
downloaded to Microsoft Excel and cleaned and screened, where
participants with missing data or insufficient information were
removed. We then transferred data into the Statistical Package
for Social Sciences Version 23 (IBM Corp, 2016), and undertook
preliminary analyses of variance and correlation. We conducted
main analyses using the LAVAAN package (Rosseel, 2012) within
R version 3.2 (R Core Team, 2015). A repeated measures linear
mixed model was used to predict creativity score and determine
the impact of experience and expertise at the nine levels. We used August 2018 | Volume 9 | Article 1448 4 Expertise in Evaluating Choreographic Creativity Clements et al. According to Cohen’s Kappa statistic, an IRR of greater than 0.8
indicates a very good level of reliability between raters (McHugh,
2012). a repeated measures mixed model as it is suitable for missing
data, therefore allowing for the variation in the number of videos
observed. We entered each of the experience or expertise categories in
to the repeated measures linear mixed model as a fixed effect. Contemporary choreographic experience significantly predicted
creativity, F(1,2052.33) = 6.61, p < 0.001, as did self-attributed
creative expertise F(1,2032.13) = 17.82, p < 0.001, but none of
other categories were significant. Repeated Measures Linear Mixed Model A colleague of the authors’ who was blind to the purpose of the
research coded a random sample of 50 participants’ qualitative
employment responses ‘Employed in any creative domain’ and
‘Employed in an artistic creative domain’ to assess the reliability
of the expertise and experience coding seen in Table 1. A positive
inter-rater reliability (IRR) correlation = 0.83 was achieved. The results show that when an individual has experience in
choreography, they rate creativity higher. That is, one needs
experience in the choreographic process to judge a piece to be
more creative. This supports the idea by Corazza (2016) that
creativity is related to an ability to see the potential expression
of a process. This is in line with the emphasis on the creative
process in dance pedagogy (Butterworth, 2004; Farrer, 2014), yet
suggests that this emphasis may be preventing those who do
not have experience of choreography from identifying creativity. Our findings suggest that this level of expertise is essential in
evaluator selection; experience in physically dancing or watching
contemporary dance does not lead an individual to rate creativity
higher. Instead, experience in knowing the process of making
dance allows an individual to judge a piece as more creative. TABLE 2 | Descriptive statistics of creativity, likeability, meaning and technique
ratings. Min. Max. Mean
SD
Skewness
Kurtosis
Creativity
1.0
7.0
4.62
1.44
−0.45
−0.17
Likeability
1.0
7.0
4.13
1.61
−0.21
−0.67
Meaning
1.0
7.0
3.86
1.64
−0.17
−0.78
Technique
1.0
7.0
4.97
1.36
−0.55
−0.05
TABLE 3 | Pearson’s correlation coefficients for creativity, likeability, meaning and
technique ratings. Creativity
Likeability
Meaning
Creativity
Likeability
0.71∗
Meaning
0.60∗
0.67∗
Technique
0.62∗
0.57∗
0.45∗
∗Correlation is significant at the 0.05 level (2-tailed). Frontiers in Psychology | www.frontiersin.org
5 TABLE 2 | Descriptive statistics of creativity, likeability, meaning and technique
ratings. These findings have implications with regards to accessibility
of contemporary dance, in suggesting that training in dance per se
does not necessarily facilitate an understanding of choreographic
creativity, but that only those who learnt to make dance
understand and rate higher. The level of expertise suggested
by our findings regarding creativity is more specific than that
which has been reported in the literature on dance performance,
even beyond those studies involving fMRI recordings of audience
responses (e.g., Calvo-Merino et al., 2005). Here, physical
participation has led to significant differences in brain activity
when watching dance. Descriptive Statistics The numbers of videos viewed by each of the 850 creativity
raters ranged from one to 21 videos (M = 2.53, SD = 2.63). In
total, we obtained 2153 individual ratings with between 81 and
102 creativity ratings on each video (M = 91.61, SD = 6.37). Descriptive statistics of overall ratings from the 23 videos are
shown in Table 2. Video Stimuli In those with contemporary
choreographic experience, creativity was rated higher compared
to the reference group [b = 0.24, t(2067.48) = 2.71, p < 0.05
(95% CI = −0.044 to 0.39)]. In those with self-attributed creative
expertise, creativity was rated lower compared to the reference
group [b = −0.13, t(2032.13) = −4.44, p < 0.001 (95% CI = −0.52
to −0.19)]. Next, video was entered as a random effect. Both the
intercept (b = −0.19, Wald Z = 32.56, p < 0.001) and video were
significant (b = −0.12, Wald Z = 2.81, p < 0.05), indicating that
slopes were significantly different across the 23 videos. DISCUSSION Next, Pearson’s correlation analyses were conducted to
obtain an understanding of the relationships between creativity,
likeability, technique and meaning. Table 3 shows significant
moderate to strong positive correlations between all four
variables, suggesting that people rate contemporary dance highly
on creativity when it is also perceived as liked, well understood
and well executed. We explored the role of experience and expertise in assessing
choreographic creativity, using a novel online methodology
that facilitated dance specific research. 850 assessors assessed
creativity in 23 individual contemporary dance choreographies. Assessor experience and expertise were sampled from a
continuum of expertise from those who had never taken a dance
class to professional choreographers. The results demonstrate the
impact of both dance specific experience and broader creative
expertise in the assessment of choreographic creativity. Preliminary Analyses We undertook a series of one-way ANOVAS to determine
a difference in the mean ratings of the videos. Creativity
[F(22,2130) = 6.85, p < 0.001], likeability [F(22,2130) = 5.90,
p < 0.001], meaning [F(22,2130) = 4.77, p = < 0.001] and
technique [F(22,2130) = 11.44, p < 0.001] all showed significant
variation in scores between videos. Repeated Measures Linear Mixed Model However, our findings indicate that
experience of making or choreographing, beyond physical August 2018 | Volume 9 | Article 1448 5 Expertise in Evaluating Choreographic Creativity Clements et al. participation in dancing, impacts creativity assessment (e.g.,
Calvo-Merino et al., 2005). present research was a considerable variation on the original. The use of this variation was beneficial since it is arguably the
only available research methodology for creativity which is not
inherently tied to a theory of creativity but facilitated a means of
assessing dance specific creativity (Baer and McKool, 2009). The
methodology assessed the manifestation of creativity through
the body (Kirsh, 2010 and without pen and paper tests, while
focusing on product also increased validity. The results of the correlational analyses showed that creativity
score is related to choreography that the evaluator likes,
can find meaning in, and is performed by a dancer whom
the evaluator perceives as technically skilled. Collectively,
these correlational results indicate that an audience evaluates
creativity in line with subjective elements which go beyond
the criteria which underpin problem-solving tests such as the
RAT (Mednick and Mednick, 1971) and TTCT (Torrance, 1974). Standard creativity tests previously used in dance, operationalise
creativity by the ability to rapidly produce a large number of
infrequent responses (e.g., Fink and Woschnjak, 2011). Two
critical elements of creativity underpin most theoretical and
research-based definitions) originality or novelty and b) usefulness
or appropriateness (Stein, 1953; Barron, 1955; Amabile, 1983; ). This dualistic criterion remains the most commonly accepted
definition of creativity (Runco and Jaeger, 2012). Since creativity
correlated highly with making meaning of the piece, one
could argue that those who rated higher in the contemporary
dance choreography subgroup had a clearer insight into the
meaning of the work, because they had experience of the
process and understood intention. Creativity in the arts may be
assessed concerning intention at the moment of creation, with a
proposition that it is intentionality rather than novelty which is
vital (Kharkhurin, 2014; Weisberg, 2015). In turn, this supports
previous authors who have discussed the lack of outsider dance
audiences and the failure to understand contemporary dance
(Van Dyke, 2010). It is of note to consider the relationship between the
choreographer and the dancer who is performing the work. Repeated Measures Linear Mixed Model Whilst our intention was to assess the choreographer’s creativity,
one could argue that the audience perception may also be related
to the performer’s creative interpretation of the movement, in
the same way that it is related to their technical skill. Thus
an additional facet in dance may be the dancer’s ability to
communicate and interpret the choreographic interpretation
which is as important as the choreographer’s creative skill at
constructing the work (Smith-Autard, 2014). The study is strengthened by the inclusion of 23 videos
and a large sample of respondents, allowing a more substantial
variation of scores to be given and to facilitate a broad
audience, which is more reminiscent of real-life choreographic
settings. Future research should endeavor to establish reliability
amongst experts in dance specific creativity which is solely
reliant on expert opinions, such as auditions. The present
research was not intended to undertake IRR correlation
analysis; however, IRR between experts has been highlighted as
methodologically important (Kaufman et al., 2008; Haller et al.,
2011). Furthermore, there is debate regarding the width of the
Likert scale, with no consistent recommendations, aside from to
include a neutral point. Thus, findings are not comparable across
studies. However, in sum, although the method underpinning
the CAT may be perceived to lack methodological stability, the
breadth of application and validity has been demonstrated. A second finding was that scores by those who self-assigned
themselves as creative experts were lower than those who did
not. This supports the value of the chosen method, and that
asking judges to self-select whether they are an expert may be
valuable when seeking to recruit judges. Experts will have had
considerably greater exposure to creativity and therefore do not
consider the work to be as creative; there is some interaction of
expertise at this level, yet cause and effect cannot be established. pp
y
We had 87 (of 850) participants who currently/previously
attended
the
institution,
so
we
added
‘current/previous
attendance at the institution’ as a predictor. This was not
significant, thus did not impact on creativity ratings. Therefore
the possibility of this as a confound was deemed to be minor,
since only a small number of participants were potential
classmates and this did not have a significant impact. Frontiers in Psychology | www.frontiersin.org August 2018 | Volume 9 | Article 1448 Repeated Measures Linear Mixed Model In addition,
although we did not ask whether the viewer knew the performer
in the video, the video appeared randomly, so if they knew any
performer, there was a 1 in 23 chance of them knowing the
performer on video 1, 1 in 22 chance for video 2 and so forth. Since the average views were 2.5, the chances of knowing the
performer were again relatively small. The implications for these findings are numerous when
discussing the need for widening audience engagement in
contemporary dance. These findings may imply a need for
educating audiences about creative processes underpinning the
dance product. Glass and Stevens (2005) note that ‘Priming
audience members about a particular work should assist them to
engage with the work at a greater level of understanding’ (p.17). Educating an audience about the creative process might bridge
the gap between the audience’s understanding of creativity in
dance and subsequent enjoyment of the work. This may be
particularly true in an art form where the emphasis is on the
process and the dancer’s experience of making or creating a dance
for the dancer’s enjoyment (Lavender, 2009). The online methodology and use of snowballing enabled
meaningful participant diversity, which was also sensitive to
differences both in expertise and in evaluations of the videos. We recognize that snowballing can result in the loss of crucial
information over participants, however, for the present research
it facilitated a meaningful audience-like participant set. The use
of such an online evaluation might facilitate repeated testing
over time. Previous efforts to research dancers’ creativity focused
on domain-general measures and tended to be cross-sectional
in nature; longitudinal research looking at the impact of the Importantly, the results of the analyses showed variation in
the mean ratings of the videos, demonstrating that the snowball
sampling method does not neutralize differences; that is, a varied
audience collectively distinguish varying levels of creativity. Using a simple Likert scale for the CAT is therefore advocated
as a simple yet effective measure of creativity. We recognize
that there are numerous ways of implementing the CAT and the August 2018 | Volume 9 | Article 1448 6 Expertise in Evaluating Choreographic Creativity Clements et al. was to use a large scale online methodology which went beyond
the pen and paper problem-solving approaches which have
predominated the literature. REFERENCES Csikszentmihalyi, M. (1999). “Implications of a systems perspective for the study
of creativity,” in Handbook of Creativity, ed. R. J. Sternberg (Cambridge:
Cambridge University Press), 313–335. Amabile, T. M. (1982). Social psychology of creativity: a consensual assessment
technique. J. Pers. Soc. Psychol. 43, 997–1013. doi: 10.1037/0022-3514.43.5.997 g
y
Csikszentmihalyi, M. (2014). Society, Culture, and Person: A Systems View of
Creativity. Dordrecht: Springer. Amabile, T. M. (1983). The social psychology of creativity: a componential
conceptualisation. J. Pers. Soc. Psychol. 45, 357–376. doi: 10.1037/0022-3514. 45.2.357 Farrer, R. (2014). The creative dancer. Res. Dance Educ. 15, 95–104. doi: 10.1080/
14647893.2013.786035 Amabile,
T. M. (1996). Creativity
in
Context:
Update
to”
the
Social
Psychology
of
Creativity.”
Boulder,
NV:
Westview
Press. Feist, G. J. (1998). A meta-analysis of personality in scientific and artistic
creativity. Pers. Soc. Psychol. Rev. 2, 290–309. doi: 10.1207/s15327957pspr
0204_5 Amabile, T. M., and Pillemer, J. (2012). Perspectives on the social psychology of
creativity. J. Creat Behav. 46, 3–15. doi: 10.1002/jocb.001 Fink, A., and Woschnjak, S. (2011). Creativity and personality in professional
dancers. Pers. Individ. Diff. 51,
754–758. doi:
10.1016/j.paid.2011. 06.024 Baer, J. (1998). The case for domain specificity of creativity. Creat. Res. J. 11,
173–177. doi: 10.1207/s15326934crj1102_7 Glass, R., and Stevens, C. (2005). Making Sense of Contemporary Dance: An
Australian Investigation into Audience Interpretation and Enjoyment Levels. Sydney, NSW: University of Sydney. Baer, J., and McKool, S. S. (2009). “Assessing creativity using the consensual
assessment technique,” in Handbook of Research on Assessment Technologies,
Methods, and Applications in Higher Education, ed. C. Schreiner (Lawrenceville,
NJ: Rider University), 65–77. Gl˘aveanu, V. P. (2010). Paradigms in the study of creativity: introducing the
perspective of cultural psychology. New Ideas Psychol. 28, 79–93. doi: 10.1016/j. newideapsych.2009.07.007 Barron, F. (1955). The disposition towards originality. J. Abnorm. Soc. Psychol. 51,
478–485. doi: 10.1037/h0048073 Guilford, J. P. (1956). The structure of intellect. Psychol. Bull. 53, 267. doi: 10.1037/
h0040755 Bläsing, B., Puttke, M., and Schack, T. (Eds). (2010). The Neurocognition of Dance:
Mind, Movement and Motor Skills. New York, NY: Psychology Press. Guilford, J. P. (1950). Creativity. Am. Psychol. 5, 444–454. ows, J. (2010). A Choreographer’s Handbook. New York, NY: Routl Hagood, T. K. (2001). Dance to read or dance to dance? Arts Educ. Policy Rev. 102,
27–29. doi: 10.1080/10632910109600014 Butterworth,
J. (2004). Teaching
choreography
in
higher
education:
a
process continuum model. Res. Dance Educ. 5, 45–67. doi: 10.1080/
1464789042000190870 Haller, C. S., Courvoisier, D. S., and Cropley, D. H. ETHICS STATEMENT This
study
was
carried
out
in
accordance
with
the
recommendations of Trinity Laban Ethics Guidelines and the
BPS Code of Ethics. The protocol was approved by the Trinity
Laban Conservatoire of Music and Dance Ethics Committee. All
subjects gave written informed consent in accordance with the
Declaration of Helsinki. AUTHOR CONTRIBUTIONS LC led the work carried out on the paper, including the writing,
research design, data collection, and analysis. JM supported
the development and undertaking of statistical analyses. NLS
assisted in developing the methodology. ER and JM were Ph.D. supervisors of the work. CONCLUSION This research aimed to understand the role of expertise in
assessing creativity in choreographic creativity. A secondary aim Repeated Measures Linear Mixed Model The use of choreographic videos
allowed the expression of embodied creativity and recruitment of
a large audience with varying degrees of expertise and experience
in dance. The results showed that personal experience of the
creative process increased ratings of creativity, while creative
experts rated creativity lower. The use of online methodologies
for assessing creativity is advocated across multiple domains of
creativity. environment or training on dancers’ creativity has not yet
been possible (e.g., Kalliopuska, 1989; Stinson, 1993; Fink and
Woschnjak, 2011). Although we note that there are limitations of
online methodologies, such as being unable to establish reliability
between evaluators (as is common in the original version), the
results of the study support the viability of an online snowball
sampling method to recruit both experts and non-experts. In
particular, the effectiveness of adapting the CAT for research
purpose is advocated. p
p
The present online adaption has strength in its flexibility for
use across many unique domains of creativity. Thus, by assuming
neither domain generality nor specificity, it is a method which
could be replicated using any creative performances or artifact
across many arts such as music, or visual art, allow recruitment of
both large samples of creative works and raters. In this variation,
a methodological strength was that unknown to the raters, the
individual performing the work was not the creator. Future
research within the domain of dance should continue to use
the CAT in its most original form, aiming to establish reliability
between assessors in real life creative performance scenarios
such as an audition, to understand selection methods, as well
as evaluation of students in choreography and improvisation
courses. REFERENCES (2011). Perhaps there is
accounting for taste: evaluating the creativity of products. Creat. Res. J. 23,
99–109. doi: 10.1080/10400419.2011.571182 Calvo-Merino, B., Glaser, D. E., Grèzes, J., Passingham, R. E., and Haggard, P. (2005). Action observation and acquired motor skills: an fMRI study with expert
dancers. Cereb. Cortex 15, 1243–1249. doi: 10.1093/cercor/bhi007 H’Doubler, M. N. (1998). Dance: A Creative Art Experience. Madison, WI:
University of Wisconsin press. Hong, S. W., and Lee, J. S. (2015). Nonexpert evaluations on architectural design
creativity across cultures. Creat. Res. J 27, 314–321. doi: 10.1080/10400419.2015. 1087245 Corazza, G. E. (2016). Potential originality and effectiveness: the dynamic
definition of creativity. Creat. Res. J. 28, 258–267. doi: 10.1080/10400419.2016. 1195627 August 2018 | Volume 9 | Article 1448 7 Frontiers in Psychology | www.frontiersin.org Expertise in Evaluating Choreographic Creativity Clements et al. Hu, W., and Adey, P. (2002). A scientific creativity test for secondary school
students. Int. J. Sci. Educ. 24, 389–403. doi: 10.1080/09500690110098912 Runco, M. A., and Acar, S. (2012). Divergent thinking as an indicator of creative
potential. Creat. Res. J. 24, 66–75. doi: 10.1080/10400419.2012.652929 Humphrey, D. (1959). The Art of Making Dances. New York, NY: Grove Press. IBM Corp (2016). IBM SPSS Statistics: Version 23. Runco, M. A., and Jaeger, G. J. (2012). The standard definition of creativity. Creat. Res. J. 24, 92–96. doi: 10.1080/10400419.2012.650092 Sawyer, R. K. (2014). Group creativity: Music, Theatre, Collaboration. New York,
NY: Psychology Press. Julmi, C., and Scherm, E. (2015). The domain-specificity of creativity: insights from
new phenomenology. Creat. Res. J. 27, 151–159. doi: 10.1080/10400419.2015. 1030310 Simonton, D. K. (1999). Origins of Genius: Darwinian Perspectives on Creativity. Oxford: Oxford University Press. Kalliopuska, M. (1989). Empathy, self-esteem and creativity among junior ballet
dancers. Percept. Mot. Skills 69, 1227–1234. doi: 10.2466/pms.1989.69.3f.1227 Simonton, D. K. (2015). On praising convergent thinking: creativity as blind
variation and selective retention. Creat. Res J. 27, 262–270. doi: 10.1080/
10400419.2015.1063877 Kaufman, J. C., and Baer, J. (2004). Sure, I am creative—but not in mathematics!:
Self-reported creativity in diverse domains. Emp. Stud. Arts 22, 143–155. doi: 10.2190/26HQ-VHE8-GTLN-BJJM Smith-Autard, J. M. (2014). Dance Composition: A Practical Guide to Creative
Success
in
Dance
Making. New
York,
NY:
Routledge. doi:
10.4324/
9781315060033 Kaufman, J. C., Baer, J., Cole, J. C., and Sexton, J. D. (2008). A comparison of expert
and non-expert raters using the consensual assessment technique. Creat. Res. J. 20, 171–178. doi: 10.1080/10400410802059929 Stein, M. I. (1953). Creativity and culture. J. Psychol. REFERENCES 36, 311–322. doi: 10.1080/
00223980.1953.9712897 Kaufman, J. C., and Sternberg, R. J. (2010). The Cambridge Handbook of Creativity. New York, NY: Cambridge University Press. doi: 10.1017/CBO9780511763205 Stevens, C., Glass, R., Schubert, E., Chen, J., and Winskel, H. (2007). “Methods
for measuring audience reactions,” in Proceedings of the inaugural International
Conference on Music Communication Science, Sydney, NSW, 155. Kharkhurin, A. V. (2014). Creativity. 4in1: four-criterion construct of creativity. Creat. Res. J. 26, 338–352. doi: 10.1080/10400419.2014.929424 Kirsh, D. (2010). Thinking with external representations. AI Soc. 25, 441–454. doi: 10.1007/s00146-010-0272-8 Stevens, C., Malloch, S., and McKechnie, S. (2001). Moving mind: the cognitive
psychology of contemporary dance. Brolga 12, 7–14. Kirsh, D. (2011). “Creative cognition in choreography,” in Proceedings of the 2nd
International Conference on Computational Creativity, Mexico, 1–6. Stevens, C., and McKechnie, S. (2005). Thinking in action: thought made
visible in contemporary dance. Cogn. Process. 6, 243–252. doi: 10.1007/
s10339-005-0014-x Kogan, N. (2002). Careers in the performing arts: a psychological perspective. Commun. Res. J. 14, 1–16. doi: 10.1207/S15326934CRJ1401_1 Stevens, C. J., Schubert, E., Morris, R. H., Frear, M., Chen, J., Healey, S., et al. (2009). Cognition and the temporal arts: investigating audience response to
dance using PDAs that record continuous data during live performance. Int. Kokotsaki, D., and Newton, D. P. (2015). Recognising creativity in the music
classroom. Int. J. Music Educ. 3, 491–508. doi: 10.1177/0255761415607081 Kozbelt, A. (2004). Originality and technical skill as components of artistic
quality. Empirical studies of the arts 22, 157–170. doi: 10.2190/NDR5-G09N-X7
RE-34H7 J. Hum. Comput. Stud. 67, 800–813. doi: 10.1016/j.ijhcs.2009.06.001 Stinson, S. W. (1993). Testing creativity of dance students in the People’s Republic
of China. Dance Res. J. 25, 65–68. doi: 10.1017/S0149767700008056 Lavender, L. (2009). Dialogical practices in teaching choreography. Dance
Chronicle 32, 377–411. doi: 10.1080/01472520903276735 Strauss, M. R., and Nadel, M. (2012). Looking at Contemporary Dance: A Guide for
the Internet Age. Princeton, NJ: Princeton Book Company. Thomson, P., and Jaque, S. V. (2017). Creativity and the Performing Artist. San
Diego, CA: Elsevier. Long, H., Plucker, J. A., Yuc, Q., Ying, D., and Kaufman, J. C. (2014). Research productivity and performance of journals in the creativity sciences:
a bibliometric analysis. Creat. Res. J. 26, 353–360. doi: 10.1080/10400419.2014. 929425 Torrance, E. P. (1974). Norms-technical Manual: Torrance Tests of Creative
Thinking. Lexington, MA. Ginn & Company. Lubart, T. I. (2001). Models of the creative process: past, present and future. Creat. Res. J. 13, 295–308. REFERENCES doi: 10.1207/S15326934CRJ1334_07 Van
Dyke,
J. (2010). Vanishing:
dance
audiences
in
the
postmodern
age. Dance
Chronicle
33,
208–230. doi:
10.1080/01472526.2010.48
5902 Łucznik, K. (2015). Between minds and bodies: some insights about creativity from
dance improvisation. Technoetic Arts 13, 301–308. doi: 10.1386/tear.13.3.301_1 Wallas, G. (1926). The Art of Thought. London: CAPE. May, J., Calvo-Merino, B., Delahunta, S., McGregor, W., Cusack, R., Owen, A. M.,
et al. (2011). Points in mental space: an interdisciplinary study of imagery
in movement creation. Dance Res. 29(Suppl.), 404–432. doi: 10.3366/drs.2011. 0026 Weisberg, R. W. (2015). On the usefulness of “value” in the definition of creativity. Creat. Res. J. 27, 111–124. doi: 10.1080/10400419.2015.1030320 Williams, R., Runco, M. A., and Berlow, E. (2016). Mapping the themes, impact,
and cohesion of creativity research over the last 25 years. Creat. Res. J. 28,
385–394. doi: 10.1080/10400419.2016.1230358 McHugh, M. L. (2012). Interrater reliability: the kappa statistic. Biochem. Med. 22,
276–282. doi: 10.11613/BM.2012.031 Wilson, M. (2002). Six views of embodied cognition. Psychon. Bull. Rev. 9, 625–636. doi: 10.3758/BF03196322 Mednick, S. A., and Mednick, M. (1971). Remote Associates Test: Examiner’s
Manual. Boston, MA: Houghton Mifflin. Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest. R Core Team (2015). R: A Language and Environment for Statistical Computing. Vienna: R Foundation for Statistical Computing. Vienna: R Foundation for Statistical Computing. Reason, M., and Reynolds, D. (2010). Kinesthesia, empathy, and related pleasures:
an inquiry into audience experiences of watching dance. Dance Res. J. 42, 49–75. doi: 10.1017/S0149767700001030 Copyright © 2018 Clements, Redding, Lefebvre Sell and May. This is an open-access
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the original author(s) and the copyright owner(s) are credited and that the original
publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these
terms. Risner, D. (2000). Making dance, making sense: epistemology and Choreography. Res. Dance Educ. 1, 155–172. doi: 10.1080/713694259 Res. Dance Educ. 1, 155–172. doi: 10.1080/713694259 Rosseel, Y. (2012). Lavaan: an R package for structural equation modelling. J. Stat. Softw. 48, 1–36. doi: 10.18637/jss.v048.i02 Rosseel, Y. (2012). Lavaan: an R package for structural equation modelling. J. Stat. Softw. 48, 1–36. doi: 10.18637/jss.v048.i02 Runco, M. REFERENCES A. (2014). Creativity: Theories and themes: Research, development, and
practice. San Diego, CA: Elsevier. Runco, M. A. (2014). Creativity: Theories and themes: Research, development, and
practice. San Diego, CA: Elsevier. August 2018 | Volume 9 | Article 1448 Frontiers in Psychology | www.frontiersin.org 8
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Fairness of Academic Performance Prediction for the Distribution of Support Measures for Students: Differences in Perceived Fairness of Distributive Justice Norms
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Technology, Knowledge and Learning (2024) 29:1079–1107
https://doi.org/10.1007/s10758-023-09698-y Technology, Knowledge and Learning (2024) 29:1079–1107
https://doi.org/10.1007/s10758-023-09698-y ORIGINAL RESEARCH Extended author information available on the last page of the article Fairness of Academic Performance Prediction
for the Distribution of Support Measures for Students:
Differences in Perceived Fairness of Distributive Justice
Norms Marco Lünich1 · Birte Keller1 · Frank Marcinkowski1 Accepted: 15 October 2023 / Published online: 11 November 2023
© The Author(s) 2023 Accepted: 15 October 2023 / Published online: 11 November 2023
© The Author(s) 2023 1 Introduction Artificial Intelligence (AI) applications in the higher education sector have been acknowl-
edged to have great and diverse potential (Attaran et al., 2018; Daniel, 2015). For example,
there are already AI applications that assign grades (Kotsiantis, 2012), predict dropouts
or academic performance (Alyahyan & Düştegör, 2020; Alturki et al., 2022; Berens et al.,
2019; Kemper et al., 2020; Armatas et al., 2022), or chatbots that can answer various ques-
tions from students (Nguyen et al., 2021; Pelletier et al., 2021; Vieira et al., 2022). Devel-
opers and users hope that Artificial Intelligence in Education (AIEd) will lead to a higher
number of graduates and improved student performance, for example, by offering students
better individual feedback and support to reduce dropout rates (Attaran et al., 2018; Daniel,
2015; Zawacki-Richter et al., 2019). However, debates about the social justice of corre-
sponding systems repeatedly come into focus (Baker & Hawn, 2022; Fazelpour & Danks,
2021; Hsu et al., 2021; Keller et al., 2022; Marcinkowski et al., 2020; Muñoz et al., 2016;
Slade & Prinsloo, 2013). An illustrative example is provided by an automated study place
allocation system from France called ParcourSup (Wenzelburger & Hartmann, 2022). The
system was quickly criticized because, in addition to a lack of transparency, it was feared
that individual student groups would be discriminated against in the allocation of study
places, thus threatening to exacerbate existing social inequalities (Orwat, 2020). Further
concerns have been voiced in instances where algorithms predict the likelihood of gradu-
ation even before students start studying. It is feared that such a system could lead to dis-
crimination against students from lower-income backgrounds (Muñoz et al., 2016). Similar
applications that predict academic performance based on past performance, among other
factors, also point to the danger of different forms of bias, for example, regarding the input
data (Fazelpour & Danks, 2021). p
Accordingly, questions of algorithmic fairness are becoming virulent not only in the
higher education sector but also in numerous other areas, such as criminal justice or hiring
processes (Angwin et al., 2016; Kaibel et al., 2019). Consequently, the state of research
in fair machine learning (FAIR ML) offers numerous mathematical fairness notions that
enable equitable distribution (Verma & Rubin, 2018; Makhlouf et al., 2021; Friedler et al.,
2021). However, there has yet to be a consensus on which of these notions seems most
appropriate in preventing systematic discrimination. Abstracti Artificial intelligence in higher education is becoming more prevalent as it promises
improvements and acceleration of administrative processes concerning student support,
aiming for increasing student success and graduation rates. For instance, Academic Perfor-
mance Prediction (APP) provides individual feedback and serves as the foundation for dis-
tributing student support measures. However, the use of APP with all its challenges (e.g.,
inherent biases) significantly impacts the future prospects of young adults. Therefore, it
is important to weigh the opportunities and risks of such systems carefully and involve
affected students in the development phase. This study addresses students’ fairness percep-
tions of the distribution of support measures based on an APP system. First, we examine
how students evaluate three different distributive justice norms, namely, equality, equity,
and need. Second, we investigate whether fairness perceptions differ between APP based
on human or algorithmic decision-making, and third, we address whether evaluations differ
between students studying science, technology, engineering, and math (STEM) or social
sciences, humanities, and the arts for people and the economy (SHAPE), respectively. To
this end, we conducted a cross-sectional survey with a 2 × 3 factorial design among n =
1378 German students, in which we utilized the distinct distribution norms and decision-
making agents as design factors. Our findings suggest that students prefer an equality-
based distribution of support measures, and this preference is not influenced by whether
APP is based on human or algorithmic decision-making. Moreover, the field of study does
not influence the fairness perception, except that students of STEM subjects evaluate a dis-
tribution based on the need norm as more fair than students of SHAPE subjects. Based
on these findings, higher education institutions should prioritize student-centric decisions
when considering APP, weigh the actual need against potential risks, and establish con-
tinuous feedback through ongoing consultation with all stakeholders. Keywords Algorithmic decision-making · Higher education · Algorithmic fairness ·
Distributive fairness · Student support measures Keywords Algorithmic decision-making · Higher education · Algorithmic fairness ·
Distributive fairness · Student support measures 56789)
3 56789)
3 23451 M. Lünich et al. 1080 1 3 2 A Question of Justice: Perils and Pitfalls of Academic Performance
Prediction Concerning AIEd, a systematic review by Zawacki-Richter et al. (2019) shows four appli-
cation areas: “profiling and prediction, intelligent tutoring systems, assessment and evalu-
ation, and adaptive systems and personalisation” (p. 20). A so-called APP system is an
example that falls within the first and third category. As the name suggests, the system’s
function is to use predictive analytics to forecast students’ performance or, in some cases,
to prognosticate whether they will complete their studies (so-called dropout detection). APP, like other systems of AIEd, is based on historical student performance data, some-
times supplemented in part by non-academic data (e.g., sociodemographics) (Abu Saa
et al., 2019; Olaya et al., 2020). Universities, as the systems’ users, hope to increase reten-
tion and graduation rates based on the predictions (Attaran et al., 2018; Mah, 2016). APP
is already being used at various higher education institutions (HEI) worldwide, for exam-
ple, in the USA (Arnold & Pistilli, 2012; Ornelas & Ordonez, 2017), Australia (Adams
et al., 2017; Niall & Mullan, 2017), Germany (Berens et al., 2019; Kemper et al., 2020), or
Bangladesh (Ahmed & Khan, 2019). Based on the algorithmic predictions, HEIs may offer
individual feedback and support measures that can be tailored explicitly to the needs of
the students and automatically assigned to them with the help of APP (Ekowo et al., 2016;
Muñoz et al., 2016; Pistilli & Arnold, 2010). Universities use APP to allocate limited student support resources faster and more equi-
tably and, as a result, increase student success and receive higher graduation rates. Such
support resources could be academic and non-academic interventions (Keller et al., 2022),
as Tinto (1975) distinguishes two types of integration that are important goals to achieve
student retention: namely, academic integration and social integration. Interventions
for academic integration are used to improve the student’s academic performance. Such
improvement can be achieved, for example, through measures such as tutorials, advising
sessions for study planning, or student coaching (Bettinger & Baker, 2014; Chiteng Kot,
2014) but also through individually tailored feedback (Brade et al., 2018; Tinto, 1975),
which could be greatly facilitated and improved by an APP system. While these support
interventions come from above, others come from below (Morosanu et al., 2010), such as
those for social integration. These latter interventions build a social support network and
can be fostered especially through contact with experienced teachers and other students,
for instance, through mentoring programs. 1 Introduction Instead, the choice of an adequate
notion of fairness is highly context-dependent, and as a result, the perceptions of fairness
may vary (Lee et al., 2019; Starke et al., 2022; Wong, 2020). While the various fairness
notions tend to be developed at the theoretical level, only scattered evidence on stake-
holders’ perceptions of the same is available (Saxena et al., 2020; Srivastava et al., 2019). Thereby, it is shown that the fairness perception of an algorithm has a significant impact
on the trust in and acceptance of the AI system in question as well as the legitimacy of the
ensuing decision-making (Lünich & Kieslich, 2022; Shin, 2020, 2021; Shin et al., 2020;
Sun & Tang, 2021). Against this background, it is necessary to include the fairness percep-
tions of important stakeholders in the development of an AI system (Cheng et al., 2021;
Keller et al., 2022; Webb et al., 2018) in order to achieve trustworthy AI (European Com-
mission, 2019; Mäntymäki et al., 2022; Shneiderman, 2020).f To answer this call for research on fairness perceptions of those most affected by AIEd,
in this paper, we investigate students’ preferences for different understandings of justice
in the context of the distribution of support measures based on an academic performance
prediction (APP) system. However, we do not focus on specific mathematical fairness
notions but on students’ underlying justice principles concerning the AI-based distribution 1 3 Fairness of Academic Performance Prediction for the Distribution… 1081 of support measures. We draw on the four-dimensional concept of organizational justice
(Cropanzano & Ambrose, 2015; Greenberg, 1993) and focus primarily on the dimension
of distributive fairness. Based on a factorial survey of German students, we provide an
explanatory analysis for the three following questions: First, we investigate how students
perceive different norms for just distribution as fair when evaluating the APP system. Fur-
thermore, we investigate whether the fairness perceptions of AI-based APP systems (i.e.,
so-called algorithmic decision-making, ADM) differ from those of human decision-making
(HDM). Third, we look at the different fields of study and investigate whether the choice of
field of study impacts the fairness perception of the APP system. In answering these ques-
tions, this paper adds to the literature on AIEd and points to relevant concerns of students,
as the arguably most important stakeholders in this field, concerning distributive justice,
connecting to the literature on FAIR ML. 2 A Question of Justice: Perils and Pitfalls of Academic Performance
Prediction In addition, however, the sense of belonging 1 3 3 M. Lünich et al. 1082 to the institution–that is, the university–also plays a decisive role (Hausmann et al., 2009;
Neugebauer et al., 2019; Tinto, 1975). Assuming limited organizational resources (finan-
cial and human) that a university can muster to support students, these interventions often
cannot be made available to all students. Instead, they can only be deployed efficiently, for
instance, if they are utilized (only) by distressed students in need or–should a university
pursue a strategy of promoting only the best students–by promising and already successful
students. According to the 2021 EDUCAUSE Horizon Report, the COVID-19 pandemic has fur-
ther accelerated the development and use of hybrid learning models and AIEd (Pelletier
et al., 2021). However, the authors further state that “the arrival of AI in higher education
has opened a Pandora’s box. Going forward, higher education will need to be a careful
and ethical user of AI” (Pelletier et al., 2021, 15). In this sense, APP’s implementation
must be thoroughly reflected and systematically evaluated. Almost all commonly discussed
issues that come along with AI, in general, can also be applied to AIEd and the APP con-
text, respectively. These concerns range from technological over methodological to soci-
etal challenges (Hagendorff & Wezel, 2020; Wirtz et al., 2019) and include, for example,
questions of transparency (de Fine Licht, 2020; Ananny & Crawford, 2018), accountabil-
ity (Busuioc, 2021; Diakopoulos, 2016), and fairness (Veale & Binns, 2017; Shin, 2019). Because the use of AIEd can significantly impact the prospects of young adults, it is cru-
cial to weigh the opportunities and risks of any proposed system carefully.f For instance, an automated assessment system in the UK offers a negative example of
problematic AI systems in the educational sector. Since no A-level examinations could be
conducted during the first lockdown of the COVID-19 pandemic in 2020, an algorithm was
implemented to predict the A-level grades of students in the UK. However, this led to mas-
sive protests because students claimed the prediction fell far from their expectations and
actual performance as they were downgraded (Edwards, 2021). The procedure was particu-
larly problematic from a fairness perspective, as the algorithm disadvantaged students from
more deprived families compared to those from private schools, and the grades awarded
impact further education and working life (Adams & McIntyre, 2020; Smith, 2020). 2 A Question of Justice: Perils and Pitfalls of Academic Performance
Prediction While
the British algorithmic grading system was abandoned after the protests, a similar algo-
rithm in France, which also predicted baccalaureate grades, was accepted. However, it
resulted in higher pass rates than in prior years, leading to higher demand for university
places (Smith, 2020).l These examples show that without reflection and adjustment, APP systems may per-
petuate social injustices and biases inherent to human social structures (Attaran et al.,
2018; Fazelpour & Danks, 2021). Thus, when it comes to the use of APP, it is essential to
ensure not only that the system provides an appropriate level of transparency and explain-
ability (of both process and outcome) but that it is clear who can be held accountable in the
event of poor decisions, or that privacy concerns are addressed, but also that APP is free
of bias and that no one is systematically disadvantaged (Keller et al., 2022). Faced with
the threat of discrimination by AIEd, as evidenced by the UK grading algorithm, much of
the academic literature addresses the issue of fair distribution, the translation of fairness
into mathematical notions, and the reduction of algorithmic bias in AIEd (Jiang & Pardos,
2021; Martinez Neda et al., 2021). Beyond this, however, special attention must be paid to
the perceptions of those affected. Therefore, many authors stress the importance of stake-
holder involvement in developing and introducing AI systems (Cheng et al., 2021; Howell
et al., 2018; Keller et al., 2022; Knox et al., 2022; Lee et al., 2019; Webb et al., 2018). Otherwise, an APP system introduced with initially good intentions and presumably bias-
mitigating execution may still fail in the face of reality. 1 3 1 Fairness of Academic Performance Prediction for the Distribution… 1083 Consequently, in addition to the properties of the system itself, such as the use of sensi-
tive attributes (e.g., sociodemographics) (Nyarko et al., 2021), it can be assumed that the
consequences that follow from APP also influence people’s perceptions of fairness. Such
individual perceptions, however, are often neglected in the literature (Starke et al., 2022). So, what does the cognitive process entail in concrete terms for the students affected? In addition to their self-reflective fairness assessment of the APP assigned to them, stu-
dents’ perceptions may also relate to the resulting distribution of support measures based
on the forecast. 2 A Question of Justice: Perils and Pitfalls of Academic Performance
Prediction For example, students may perceive it as unfair if those measures are not
open to everyone in principle. The prediction could then become problematic if it leads to
unintended adverse perception effects, which affect students’ attitudes and behavior; for
instance, should non-prioritized students also demand access to support measures. Con-
sequently, a positive and a negative performance prediction could hurt study motivation
(Fazelpour & Danks, 2021; Legault et al., 2006; Mah, 2016) and potentially put students
under additional pressure (Yilmaz et al., 2020). While high-performing students might
rest on the positive feedback, lower-performing students might become frustrated given
their previous study efforts. Furthermore, students might see the prediction as an incentive
to adapt to the algorithm’s logic and change their behavior accordingly (Dai et al., 2021;
Fazelpour & Danks, 2021), which may also lead to positive and negative effects. Eventu-
ally, negative perceptions of prediction outcomes of APP and decisions based on such pre-
dictions may have detrimental effects on students’ attitudes towards HEIs deploying AIEd
and the perceived legitimacy of AI systems (Lünich & Kieslich, 2022; Marcinkowski et al.,
2020). Therefore, the question of a fair distribution of student support measures based on
the APP system arises. 3.1 Distributive Justice Norms In light of the question of how different norms of just distribution are perceived by students
when evaluating the APP system, we focus on the dimension of distributive fairness that
asks for the validity of a decision, respectively an outcome (Cropanzano et al., 2001). This
dimension is based on equity theory (Adams, 1965). According to equity theory, employ-
ees compare the ratio of their individually received outcome and the individually perceived
input with the corresponding ratio of other employees. If this comparison of the two ratios
does not match, the employee with the greater share is considered to be unjustly overpaid. In comparison, the employee with a lower share is considered to be unjustly underpaid
(Greenberg, 1990). According to this, equity is oriented toward merit and is based on com-
paring the input produced by oneself with the output received (Cropanzano & Ambrose,
2015). However, such a meritocratic distribution is only one of three different distributive
justice norms besides equality and needDeutsch (1975). In the case of APP, a distribu-
tion according to the equity principle could mean that only students who have already per-
formed well so far are entitled to the support measures presented. The need principle offers
an alternative option. This norm stipulates that only those students in particular need are
provided with support interventions. Accordingly, only students with poor APP outcomes
could benefit from support measures. Furthermore, a distribution according to the equality
principle is also conceivable. In this case, no distinction is made, and all students receive
access to support measures, which could reach its limits in everyday life because of the
institution’s limited resources. Of course, when it comes to applied distribution rules, these
ideal-typical distribution norms may come in any combination and with different grada-
tions and overlaps. 3 Justice, Fairness and the Distribution of Support Measures Rawls (1999) lays the foundation of the question of fair distribution by describing fairness
as the basis of justice. In his understanding, negotiating principles of justice requires a fair
societal system that allows cooperation between equal and free individuals (Rawls, 1999;
Sen, 2009). Murphy (2011) also highlights the link between both constructs by describ-
ing: “If proposed principles are deemed unfair, they are unjust” (S. 337). Accordingly, fair-
ness judgments arise from a perceived injustice, whereby a person must be held account-
able for that injustice (Folger & Cropanzano, 2001). A perceived (in-)justice usually refers
to the distribution of goods but can also include the distribution of rights and freedoms
(Rawls, 1999). While the distribution of support measures, in our case, refers to goods,
denied access could also be perceived as a restriction on student freedom. As HEIs may
see APP as a basis for decision-making on how to support and enable students to improve
their academic performance, the ensuing distribution of said support measures poses a dis-
tribution issue, if only because it must be partly about the distribution of limited goods and
resources. For example, as mentioned before, due to limited resources, offering tutoring
and additional personal counseling may not be available to all students. The perception of an equitable distribution of goods is also addressed by the organi-
zational justice literature, which examines how intra-organizational decision processes
should be designed to achieve a maximum degree of satisfaction and commitment of
organizational members (Greenberg, 1987), which in turn is necessary to prevent students
from protesting or, at worst, leaving the university as a result of using or being dissatisfied
with APP (Marcinkowski et al., 2020). Concerning the concept of organizational justice 1 3 3 M. Lünich et al. 1084 four dimensions of fairness have emerged: In addition to distributive fairness, there is pro-
cedural, interactional, and informational fairness (Greenberg, 1993). In this paper, how-
ever, we focus exclusively on distributive fairness to capture the essence of the distributive
justice norms of a fair distribution of support measures. four dimensions of fairness have emerged: In addition to distributive fairness, there is pro-
cedural, interactional, and informational fairness (Greenberg, 1993). In this paper, how-
ever, we focus exclusively on distributive fairness to capture the essence of the distributive
justice norms of a fair distribution of support measures. 3.2 Fairness Perceptions of Distributive Justice Norms Empirical research gives evidence that the choice of the right (i.e., publicly approved) dis-
tributive justice norm is highly context-dependent (Lee et al., 2017; Starke et al., 2022;
Wong, 2020) and can also vary among different stakeholders (Cheng et al., 2021; Lee et al.,
2019). To date, however, the literature on FAIR ML focuses mainly on the formulation of
mathematical fairness notions (Dwork et al., 2012; Kusner et al., 2017; Verma & Rubin,
2018), their evaluation by those affected (Cheng et al., 2021; Debjani et al., 2020; Sriv-
astava et al., 2019; Zhou et al., 2021) or takes distributive fairness in general into consid-
eration (Hannan et al., 2021; Marcinkowski et al., 2020; Wang et al., 2020). However, the
underlying norms of just distribution and respective preferences for distinct norms should
be addressed. Nevertheless, some authors conclude that an egalitarian distribution, as suggested by the
equality principle, is preferred in many contexts (Cropanzano & Ambrose, 2015; van Hoot-
egem et al., 2020). Such a preference seems to be especially the case when stakeholders see
themselves as part of a (small) group, and resources must be shared collectively (Allison
et al., 1992), as empathy is also a predictor of a preference for equality as a distributive
justice norm (Huppert et al., 2019). This preference is explained with the help of decision 1 3 Fairness of Academic Performance Prediction for the Distribution… 1085 heuristics, which are based on the assumption that all parties concerned unspokenly accept
the most simple solution of an equal distribution as the correct decision since they assume
that this represents a satisfactory outcome for all (Allison & Messick, 1990; Allison et al.,
1992). Therefore, this type of heuristic is also called equality heuristic or inequality aver-
sion and is shown to come into play in different contexts (Asaria et al., 2023; Morand et al.,
2020). Concerning ADM systems, Lee and colleagues (Lee & Baykal, 2017), for example,
show within qualitative interviews that some stakeholders prefer altruistic distributions
and are even willing to compromise and forgo unequally distributed allocations. Some of
them reasoned for this preference by prioritizing the group’s happiness. While the authors
substantiated this result in a second study, an international comparison between India and
America also showed cultural differences in evaluating distributive justice norms. In India,
need was preferred, while for Americans, the equality or equity norm was more in favor
(Lee et al., 2017). 3.2 Fairness Perceptions of Distributive Justice Norms In the case of a healthcare scheduling system, employees primarily pre-
ferred an equal distribution norm, although consideration of individual needs received
equal attention (Uhde et al., 2020). In a similar use case of a denied vacation request,
however, Schlicker et al. (2021) do not find any evidence for the assumption that equal-
ity explanations lead to a higher distributive fairness perception than equity explanations. Saxena et al. (2020), on the other hand, come to a different conclusion when asking for a
preferred loan decision. Their results indicate that respondents favor a proportional ‘ratio’
distribution compared to meritocratic or equality norms. Kasinidou et al. (2021) confirm
this insight by surveying computer science students who prefer a proportional distribution
to an equality norm. However, due to the contextual nature of fairness perceptions and
the inconsistent empirical insights, the potential perceptions of students toward a distribu-
tion of support interventions based on an APP system can only be inferred with qualifica-
tion based on these findings. Therefore, evaluating different fairness norms in the specific
context of APP and the distribution of student support measures is crucial. As a result, in
RQ1, we ask: How fair are different norms for fair distribution perceived by students when
evaluating an APP system? 3.3 Algorithmic vs. Human—Based Distribution of Support Measures The main driver of the implementation of AI technologies, especially when it comes to
AI in public administration, are expectations for AI to lead to cheaper, faster, more objec-
tive, and reliable results in decision-making compared to humans (König & Wenzelburger,
2022; Wirtz & Müller, 2019; Gomes de Sousa et al., 2019). While the superiority of tem-
porary AI applications may often be dubious, even if AI proves to make better decisions,
this must not necessarily lead to positive evaluations by the persons affected. In analogy
to the potential difference between notions of factual and perceived fairness, preferences
regarding the nature of the decision-makers may not solely be based on the quality or
goodness of the outcome. Two contrasting strands of literature address whether people pre-
fer AI systems over humans regarding decision-making. On the one hand, there is algo-
rithm aversion (Dietvorst et al. 2015; Dietvorst & Bharti 2020; for an overview, see Burton
et al.2020), and on the other hand, there is algorithmic appreciation (Araujo et al., 2020;
Logg et al., 2019; Thurman et al., 2019). Here, the former suggests a preference for HDM
over ADM. Such preference is especially the case when errors are observed, as people lose
confidence in algorithms more quickly than in other humans (Dietvorst et al., 2015). This
rejection is particularly evident in the case of unpredictable and risky decisions (Dietvorst 1 3 3 M. Lünich et al. 1086 & Bharti, 2020). The latter, algorithmic appreciation, however, sees ADM as superior and
therefore assumes that people are more likely to follow the recommendation of algorithms
than that of humans (Logg et al., 2019), based on the assumption that algorithms are less
biased and decide more objectively (Araujo et al., 2020). j
y (
j
)
Closely connected to aversion or appreciation of ADM is whether AIEd is wanted by
students, administrators, and other educational sector stakeholders at all. Again, several
studies indicate that the answer to this question can vary in different contexts and regard-
ing the stakeholders involved (Starke et al., 2022; Wong, 2020; Marcinkowski et al., 2020). Empirical research, on the one hand, gives evidence that ADM systems are perceived as
fairer than HDM, for instance, in logistics, even if those results do not explicitly focus on
the dimension of distributive fairness (Bai et al., 2022). 3.3 Algorithmic vs. Human—Based Distribution of Support Measures Nevertheless, higher distributive
fairness is also attested to ADM in university admission processes (Marcinkowski et al.,
2020) and without reference to a specific use case (Helberger et al., 2020). On the other
hand, there are some use cases where HDM systems are attested to have higher distribu-
tive fairness than ADM, such as resource allocation in social division tasks (Lee & Baykal,
2017) or criminal justice (Harrison et al., 2020). Another example is the decision on skin
cancer detection, where the fairness perception of ADM is also lower than those in HDM,
which is also reflected in the level of trust towards the decision. Nevertheless, this is only
true for people with low mistrust in human systems, whereas a high mistrust leads to
equally (un-)fair evaluations of ADM and HDM (Lee & Rich, 2021). In this context, the
fairness perception of ADM and HDM also depends on the nature of the decision. For
example, it plays a role in whether mechanical or human-attributable skills are required
(Lee, 2018). Furthermore, whether the decision’s impact is high or low is equally impor-
tant (Araujo et al., 2020). However, there are also reports of ambivalent results, where no preference is given to
the decision taken by an algorithmic system or a human decision-maker, for example, in
the already mentioned denial of a vacation request (Schlicker et al., 2021) or the allocation
process of COVID-19 vaccines (Lünich & Kieslich, 2022). All of these inconsistent find-
ings underscore the importance of reflecting on and examining ADM fairness perceptions
on a case-by-case basis, not just using ADM systems because the data are accessible and
it is technically possible (boyd, danah, and Kate Crawford., 2012). In line with the ideas
of human-in-the-loop (Holzinger, 2016; Starke & Lünich, 2020), the decision to use or
not to use ADM systems offers a range of possible hybrid courses of action, for example,
introducing cooperation between humans and algorithms. Thus, appropriate cooperation
may also lead to higher perceived fairness, although it is still preferred that humans retain
control over the decision (de Cremer & McGuire, 2022). Therefore, our RQ2 asks: To what
extent do perceptions of fairness APP systems using ADM differ from those using HDM? 3.4 Differences in Perceived Fairness Based on Students’ Study Subject Focusing on the specific context of AIEd, we also need to consider the varying percep-
tions of computer applications within different study fields and thus ask whether the
study subject of students impacts their fairness perception of the APP system. Even
though it can be assumed that today’s students all have grown up with computers and
are familiar with their use, there are different digital literacy levels between study sub-
jects, which may shape perceptions of computer applications (Gibson & Mourad, 2018). As Lee and Baykal (2017) show, fairness perceptions of ADM systems vary depending
on the respondents’ computer programming knowledge. Contrary to their hypothesis, 1 3 Fairness of Academic Performance Prediction for the Distribution… 1087 their findings indicate that individuals with lower programming skills perceive ADM
systems as fairer than respondents with higher knowledge. The authors assume that this
evaluation corresponds with a feeling of loss of control over the ADM as students with
more pronounced programming skills presumably know more about the algorithm’s
limitations. Logg et al. (2019) come to a similar conclusion, as their results indicate
that experts relied less on algorithmic advice than laypeople. Jang et al. (2022) also
point out that “students who had prior experience with AI education had a more sensi-
tive attitude toward the fairness of AI compared to students without experience” (p. 20). In addition, and as mentioned before, Kasinidou et al. (2021) point to differences in the
evaluation of different distributional norms, with computer science students preferring
a ratio distribution to an equal distribution. However, they do not examine differences
between different fields of study. Instead, they find marginal differences between under-
graduate and postgraduate students, with the former viewing a given decision on loan
allocation as more unfair than the latter when sociodemographic factors are considered. To our knowledge, however, there is no evidence yet on the fairness perceptions of
different fields of study towards AIEd and APP, respectively. Against this background,
when it comes to APP, it seems to be fruitful to take a closer look at the comparison
of students from so-called STEM (science, technology, engineering, and mathematics)
and SHAPE (social sciences, humanities, the arts for people and the economy) fields
of study (Jones et al., 2020). 4.1 Procedure For screening purposes, respondents had to indicate their occupational status to deter-
mine if they could participate in the student survey. Afterward, they were informed
about the nature and aim of the study, the duration of approximately 15 min to answer
the questionnaire, and the use and protection of their data. After giving informed con-
sent, respondents started to answer the questionnaire that included the vignettes of the
factorial survey design, the ensuing fairness perception of a university’s APP procedure,
questions regarding their self-assessed propensity for dropout, their expected probabil-
ity that an ADM would assign to their dropout, and their sociodemographics–i.e., age,
gender, study subject, numbers of semesters of study, their intended degree, and the
type of their recent HEI. Lastly, participants were thanked, debriefed, and redirected to the provider of the
online access panel (OAP), where they received monetary compensation for participa-
tion. The average time to complete the questionnaire was 13.72 min (SD = 5.6). 4 Method We conducted a cross-sectional factorial survey using a questionnaire with standardized
response options to address the research questions. Assessing the results, we performed
the data analysis in R (version 4.0.3) using the packages lavaan (Rosseel, 2012) and
semTools (Jorgensen et al., 2019). The data and code of our analyses are shared online
via OSF (https://osf.io/djt39/). Due to the exploratory nature of the study, there was
no pre-registration. Accordingly, for our analysis, in which we sought to identify even
small effect sizes that may hold consequential implications, we did not perform an a
priori power analysis. First, we still needed sufficient information regarding the fit of
our measurement models and the distribution of the underlying variables. On the other
hand, against this background, the power analysis of covariance-based structural equa-
tion modeling (SEM) is also rather intricate. Given this, we turned to G*Power 3.1.9.7
for a post hoc sensitivity analysis targeting the effect size (Faul et al., 2009). With an
alpha error probability set at 0.05 and a high power (1-beta err probability) of 0.999, our
sample size in the smallest group was 453, and for the whole sample was 1378. First,
our latent means model compared means for three groups. With a numerator degrees of
freedom (df) of 2 and a denominator df of 1384, the smallest effect we could have reli-
ably detected with our sample size was determined to be a f ~ 0.15 (i.e., a small effect). Second, our regression model encompassed six predictors. This analysis rendered the
smallest effect we could have reliably detected with our sample size to be a f 2 of ~ 0.08
(i.e., a small effect). These parameters guide our inquiry, emphasizing our interest in
detecting even subtle effects that may be overlooked in less rigorously designed analy-
ses. Remember that due to the different statistical approaches of SEM, our results below
may deviate from this sensitivity analysis. 3.4 Differences in Perceived Fairness Based on Students’ Study Subject On the one hand, we reason that STEM students are more
likely to be familiar with the mathematical and engineering foundations of computer
systems, algorithms, and AI techniques such as machine learning, which may shape
their perspective on APP. On the other hand, we assume that SHAPE students have a
greater familiarity and sensitivity with social questions of human nature and society that
may reflect in their perceptions of a sociotechnical system such as APP.l l
That this distinction between study subjects may have an important influence on fair-
ness perceptions is supported by evidence concerning additional differences in traits
between the respective student groups. Such factors and their consequences for individ-
ual approaches to social questions in the application of data and prediction systems may
influence distinct evaluations of data-driven prediction systems. For instance, accord-
ing to the literature, personality traits and cognitive skills predict study choice. While
the former mainly refers to the Big Five personality traits (eg., Humburg 2017; Vedel
2016), the latter is, for example, focused on mathematical skills but also on expected
future earnings (eg., Arcidiacono et al., 2012). In this sense, it shows that higher
expected future earnings, compared to current prospects, would also increase the prob-
ability of choosing SHAPE (Arcidiacono et al., 2012). Regarding personality traits, it
can be stated that compared to STEM students, students of SHAPE subjects are more
often willing to experiment and show higher levels of emotionality but are less consci-
entious (Verbree et al., 2021). Next to this, however, neuroticism also is attested to these
students (Vedel, 2016), and they also seem to show more flexibility than students of
STEM (Sherrick et al., 1971), who are instead less extrovert and more emotionally sta-
ble (Coenen et al., 2021; Humburg, 2017; Pozzebon et al., 2014). Nevertheless, Coenen
et al., (2021) find that openness to experience also positively influences the study choice
for STEM. Besides, the most interesting finding for our case is that SHAPE students
show a higher preference for altruistic views compared to other study fields (Pozzebon
et al., 2014). Following this, a study of German students shows that students who con-
sider social engagement particularly important are underrepresented in STEM subjects
(Stegmann, 1980). Accordingly, we ask RQ3: To what extent does the field of study of
students have an impact on the fairness perception of the APP system? 1 1 3 M. Lünich et al. 1088 4.2 Sample Participants were recruited with the online access panel (OAP) of the market research insti-
tute INNOFACT AG. Survey field time was between January 27 and February 1, 2022. i
All in all, 43776 respondents from the OAP were invited to participate in the survey.1
The questionnaire was accessed 3906 times, and 3680 persons started answering the ques-
tionnaire. Of those respondents, 1806 persons were screened out as they were not eligible
for our survey as they did not belong to the investigated population of German students. The survey’s non-completion rate was 6.1%, and dropouts were equally distributed over all
questionnaire pages. Additionally, in the middle of the questionnaire, there was an attention
check where respondents were asked to click on a specific scale point (“Please click the
‘tend to disagree’ item here to confirm your attention.”). Altogether, 333 respondents did
not answer correctly and were screened out. The final sample consists of 1378 participants. i
The average respondent age was 23.22 (SD = 3.32). All in all, 1012 (73.4%) respond-
ents identified as women, 358 (26%) as men, and 8 (0.6%) identified as non-binary. 4.1.1 Vignettes of the Factorial Survey Design Concerning the nature of the factorial survey, following a 2 × 3 design, each partici-
pant was randomly presented with one of six possible scenarios (see the wording of all
vignettes in the appendix). In all vignettes, participants first received information that 1 3 Fairness of Academic Performance Prediction for the Distribution… 1089 an HEI offers support measures for students but, due to limited resources, cannot offer
those to every student. The institution either deploys 1) an ADM system or 2) university
staff (HDM) to analyze student performance data, aiming to assess and predict students’
performance. an HEI offers support measures for students but, due to limited resources, cannot offer
those to every student. The institution either deploys 1) an ADM system or 2) university
staff (HDM) to analyze student performance data, aiming to assess and predict students’
performance. In the experimental condition with the vignette regarding the use of AI for the distribu-
tion of support measures, participants were given a brief explanation of the term AI, in
which ADM was explained as a specific form of AI. Next, the use case was introduced
in which an ADM system was used to assess and predict student performance based on
student performance data. Regarding the use of university staff, the vignette read that
human members of the university were in charge of this assessment and the prediction of
performance. Eventually, in each scenario, respondents learned that the university decides to use or
not use this information to either 1) allow all students to sign up for support measures irre-
spective of the available APP outcome (i.e., the equality norm), 2) allow only students with
a good APP outcome to sign up for support measures (i.e., the equity norm), or 3) allow
only students with a poor APP outcome to sign up for support measures (i.e., the need
norm). 1 We note that the high number of invitations results from the highly dynamic and often short durations
of academic careers of panel members. According to the panel provider, respondent information on edu-
cational attainment is regularly updated. However, it cannot be guaranteed that a person who signed up as
a student in higher education is necessarily enrolled at the field time of the survey. Hence, invitations were
distributed extensively, and the first question was then used for screening purposes. Study subject Study subject To inquire about the study subject of the respondents, we followed the study subject classi-
fication of the German Statistical Office (Destatis, 2021). Respondents could choose up to the
second level of the classification from a list of 79 study subjects for brevity. On the first level of
the classification, there were eight fields of study, of which we counted four as STEM-related
(i.e., mathematics and natural sciences; human medicine and health sciences; agricultural, for-
estry, and nutritional sciences, veterinary medicine; engineering) and five as SHAPE-related
(humanities; sports; law, economics, and social sciences; arts; as well as the category “other”). Eventually, based on the indicated study subject, respondents were assigned to either a
group that studies a STEM subject or a SHAPE subject. This procedure results in a dummy
variable coded as 0 ‘SHAPE’ and 1 ‘STEM’. Self-assessment of the individual expected probability that an ADM would assign to
one’s own dropout The self-assessment individual expected probability that an ADM would assign to one’s
own dropout was used as a control variable for the analysis of RQ3. Respondents were
asked the following question: “Thinking about your own current studies, what do you think
is the likelihood that an AI would predict that you would drop out?”. Respondents could
indicate a percentage from 0% to 100% using a slider. The average expected probability
that an ADM would assign to one’s dropout was 32.54% (SD = 29.33). For the analysis
and interpretation, the variable was divided by ten, so an increase of one unit reflects an
increase of ten percent in a respondent’s self-assessment. 4.3 Measurements Perceived Distributive Fairness Assessing the fairness involved respondents rating four statements on a five-point Likert
scale ranging from 1 = do not agree at all to 5 = totally agree. 1 We note that the high number of invitations results from the highly dynamic and often short durations
of academic careers of panel members. According to the panel provider, respondent information on edu-
cational attainment is regularly updated. However, it cannot be guaranteed that a person who signed up as
a student in higher education is necessarily enrolled at the field time of the survey. Hence, invitations were
distributed extensively, and the first question was then used for screening purposes. 3 M. Lünich et al. 1090 The four statements translated from German to English are as follows:
Table 1 Reliability values of
perceived fairness
Per-
ceived
Fairness
alpha
0.89
omega
0.90
omega2
0.90
omega3
0.89
avevar
0.68 Table 1 Reliability values of
perceived fairness • “A fair balance of interests between different concerns has been achieved.”
• “The distribution of support measures is fair.”
• “No one is unduly disadvantaged by the distribution of support measures.”
• “The distribution of funding measures is just.” The four indicators suggest good factorial validity (see Table 1). 5 Results To address RQ1 and RQ2, we ran a latent factor analysis for the perceived fairness in each
condition, and to address RQ3, we estimated a structural regression model with perceived
fairness as the dependent variable. In all analyses, effect coding was used for factor scaling,
a procedure that “constrains the set of indicator intercepts to sum to zero for each construct 1 3 Fairness of Academic Performance Prediction for the Distribution… 1091 Table 2 Measurement invariance of perceived fairness
Chisq (df)
CFI
RMSEA
comp
Chisq diff (df)
CFI diff
RMSEA
diff
M1: Configural
Invariance
24.32* (12)
1.00
0.07 (0.03−0.11)
M2: Metric Invari-
ance
58.21* (27)
0.99
0.07 (0.05−0.10)
M1
33.88* (15)
−0.01
0.00
M3: Scalar Invari-
ance
80.76* (42)
0.99
0.06 (0.04−0.08)
M2
22.56 (15)
0.00
−0.01
M4: Residual
Invariance
115.65* (62)
0.98
0.06 (0.04−0.08)
M3
34.89* (20)
0.00
0.00 and the set of loadings for a given construct to average 1.0” (Little et al., 2006, p. 62). The
estimated latent factor model is scaled like the indicators, which helps with interpretation. Before assessing the results, it is essential to test for measurement invariance between
our analysis groups. A test for measurement invariance suggests strong factorial invariance
of the latent factor in each condition of the factorial survey (see Table 2). 5.1 Mean Differences of Perceived Fairness Between the Distribution Norms
and Between ADM and HDM Subsequently, the model was estimated for each of the six experimental groups of the
factorial survey, estimating six means. The model shows good fit ( 휒2 (62) = 115.65, p =
< 0.001 ; RMSEA = 0.06 CI [0.04, 0.08]; TLI = 0.99). Perceived fairness of the distribution of support measures for students is highest in
the equality condition, followed by the need condition (see Table 3). In the condition that
equity is the distributive justice norm of choice, perceived fairness is lowest. Accordingly,
the mean difference between equality and need and equality and equity suggest a medium-
sized significant effect, irrespective of whether APP was based on ADM or HDM. Con-
cerning the mean difference between equity and need, results suggest a significant differ-
ence in the ADM condition but not in the HDM condition.f Concerning the differences in perceived fairness of the distinct distributive justice
norms between ADM and HDM, as seen on the right-hand side of Table 3, the results sug-
gest no difference. 5.2 Effect of Study Subject on Perceived Fairness To address RQ3, we estimated a structural regression model in which the perceived fair-
ness as the dependent variable is regressed on the study subject of a respondent as the
independent variable among control variables such as their age, gender, and the expected
probability that an ADM would assign to their drop out (i.e., the self-assessed likelihood
of AI drop out prediction). For a more accessible overview of the effects in light of the
results of RQ1 and RQ2, the condition of whether ADM or HDM performed APP was also
included as a dummy variable (0 = ‘ADM’ and 1 = ‘HDM’). Furthermore, a moderator
variable was included to assess whether the study subject had a different effect depending
on the condition of ADM or HDM (i.e., the scaled and mean-centered product of the two 1 3 3 M. Lünich et al. 1092 e 3 Latent means and mean differences for the perceived fairness
ADM
HDM
Mean Differences Between ADM
& HDM
B
SE
95% CI LL
95% CI UL
B
SE
95% CI LL
95% CI UL
Δ Mean
p
Cohen’s d
ality
3.26
0.05
3.15
3.37
3.31
0.05
3.20
3.41
−0.05
0.541
0.01
ty
2.72
0.06
2.61
2.84
2.69
0.06
2.58
2.80
0.03
0.685
0.00
d
2.88
0.05
2.78
2.98
2.82
0.06
2.71
2.93
0.06
0.464
−0.04
n Differences Between Norms
Δ Mean
p
Cohen’s d
Δ Mean
p
Cohen’s d
uality vs. Equity
0.54
0.000
0.64
0.62
0.000
0.79
uality vs. Need
0.38
0.000
0.48
0.48
0.000
0.57
Equity vs. Need
−0.16
0.044
−0.19
−0.13
0.087
−0.30 Table 3 Latent means and mean differences for the perceived fairness
ADM
HDM
Mean Differences Between ADM
& HDM
B
SE
95% CI LL
95% CI UL
B
SE
95% CI LL
95% CI UL
Δ Mean
p
Cohen’s d
Equality
3.26
0.05
3.15
3.37
3.31
0.05
3.20
3.41
−0.05
0.541
0.01
Equity
2.72
0.06
2.61
2.84
2.69
0.06
2.58
2.80
0.03
0.685
0.00
Need
2.88
0.05
2.78
2.98
2.82
0.06
2.71
2.93
0.06
0.464
−0.04
Mean Differences Between Norms
Δ Mean
p
Cohen’s d
Δ Mean
p
Cohen’s d
Equality vs. Equity
0.54
0.000
0.64
0.62
0.000
0.79
Equality vs. Need
0.38
0.000
0.48
0.48
0.000
0.57
Equity vs. 6 Discussion This study aimed to explore students’ perceptions of the deployment of APP as a basis for
the distribution of student support measures in an attempt of HEIs to improve student per-
formance and retention using AIEd. The study adds to the body of empirical evidence of
fairness perceptions of ADM, an important FAIR ML research field. More specifically, the
factorial survey design addressed German students’ distributive fairness perceptions of the
application of different distributive justice norms (i.e., equality, equity, and need) based on
student performance assessments by either ADM or HDM. The results of the factorial survey show that equality appears to be the favored distribu-
tive justice norm when it comes to the distribution of student support measures. Compared
to the distributive justice norms of equity and need, students perceive unqualified access
to resources as substantially more fair. This is in line with the literature that suggests that
in situations where collectives need to distribute limited resources, the low-threshold heu-
ristic suggests to allocate an equal-sized share to each member (Allison & Messick, 1990;
Allison et al., 1992; Morand et al., 2020). In addition, it can be assumed that students see
themselves as part of a group and, in this sense, are concerned with social cohesion (Cro-
panzano & Ambrose, 2015). It may also be speculated that some loss aversion of students
may contribute to their preference for equality as the distribution norm of choice (Smith
et al., 2019; Tversky & Kahneman, 1991). After all, students prefer a situation in which
everyone–at least in theory–has the same chance to receive support measures, irrespec-
tive of support measures being a limited resource that not everyone can draw from. Thus,
despite having additional information on the individual’s performance from APP, German
students remained in this ‘fallback option’ of equal distribution. As a result, the results
of our study are consistent with some previous research demonstrating the preference for
altruistic and equal distributions (Lee & Baykal, 2017; Uhde et al., 2020). On the other
hand, some disagreement with other studies was revealed that either indicates a preference
for proportional distributions, for example, in the context of loan decisions (Saxena et al.,
2020), or no preference at all (Schlicker et al., 2021). 5.2 Effect of Study Subject on Perceived Fairness Need
−0.16
0.044
−0.19
−0.13
0.087
−0.30 1 3 Fairness of Academic Performance Prediction for the Distribution… 1093 Table 4 Structural regression model for the relationship between study subjext and the perceived fairness of support measure distribution
Equality
Equity
Need
Estimate (Std.Err.)
p
Estimate (Std.Err.)
p
Estimate (Std.Err.)
p
Regression Slopes
Perceived Fairness
Condition of decision-making: ADM vs. HDM (1 = HDM)
0.04 (0.08)
0.560
−0.05 (0.08)
0.553
−0.05 (0.08)
0.468
Age
−0.00 (0.01)
0.826
−0.03 (0.01)
0.021
−0.01 (0.01)
0.220
Gender (1 = female)
−0.04 (0.08)
0.641
−0.32 (0.09)
0.000
0.04 (0.09)
0.631
Self-assessed likelihood of AI drop out prediction
−0.03 (0.01)
0.026
0.03 (0.01)
0.032
0.02 (0.01)
0.060
Study subject: SHAPE vs. STEM (1 = STEM)
0.02 (0.08)
0.801
0.07 (0.09)
0.384
0.16 (0.08)
0.039
Moderation Effect: ’ADM vs. HDM’*’SHAPE vs. STEM’
−0.00 (0.04)
0.980
−0.01 (0.04)
0.900
0.02 (0.04)
0.649
Fit Indices
휒2(df)
112.93 (84)
0.019
CFI
0.99
RMSEA
0.03
RMSEA.CI.LOWER
0.01
RMSEA.CI.UPPER
0.04 1 3 M. Lünich et al. 1094 dummy variables study subject and the respective condition of decision-making). The esti-
mated model shows a good fit (see Table 4). i
The results suggest that in the Equality condition (B = 0.02, SE = 0.08, p = 0.8, beta
= 0.01) and the Equity condition (B = 0.07, SE = 0.09, p = 0.38, beta = 0.04) the study
subject did not affect the perceived fairness of the distribution decision. Only in the Need
condition there was small significant effect (B = 0.16, SE = 0.08, p = 0.04, beta = 0.10). Whether ADM or HDM was used, the condition did not moderate this effect (B = 0.02, SE
= 0.04, p = 0.65, beta = 0.02). Accordingly, students studying a STEM subject judged the
latter scenario, in which students in need were eligible for support measures as fairer than
students studying a SHAPE subject. There was no difference in whether ADM or HDM
served as a basis for decision-making. 1 3 6 Discussion As of now, even though decision-makers on the university side have high hopes for
the use of AI systems and computer scientists go to great lengths to develop and improve
them, the idea of APP using ADM (as well as HDM) does not fall on fertile grounds when
proposed to students in higher education. After all, one does not need APP to distribute
support measures when equality is demanded of the students affected by APP. The same
distribution can be ensured by straightforward means, even if, in the end, not everyone 1 3 Fairness of Academic Performance Prediction for the Distribution… 1095 can access and benefit from support measures because of limited resources, especially
those students who might well deserve additional assistance according to the equity norm
or those who could genuinely use it under the need norm. Such evidence points to a con-
flict of interest between more or less well-intentioned aspirations for improving student
performance by administrators and the affected students that may not favor the ultimately
suggested solution. It remains to be seen whether more detailed information on the goals
and effects of APP or other additional communicative measures may improve preferences
for distributions that differentiate between students based on their performance. Further
research should thus investigate under what specific conditions students may judge the dis-
tribution logic based on assessing performance as more or less fair. Altogether, students were ambivalent regarding the distributive fairness perceptions of
the distribution norms of equity and need. While the reported fairness perception in the
equality condition was above the center of the scale, for these two conditions, the results
suggest a more negative fairness perception below the center of the scale. Accordingly, not
only were distribution norms of equity and need perceived as less fair than equality but
their overall assessment suggests that they were deemed somewhat unfair. From this find-
ing, one might conclude that those situations are evaluated more negatively, in which some
students that–based on a distributive justice norm–are deemed worthy of support benefit
more than all others. However, it remains to be seen whether such fairness perceptions will
change if, for example, a combination of different norms focusing on the most effective
distribution of support measures or fine-tuning APP to suggest only those measures with
the highest individual payoff in terms of increasing student performance are implemented. 6 Discussion f
Moreover, results show that students are similar in their fairness perception when con-
trasting HDM and ADM. As it is usually suggested within the literature on AIEd that there
is a qualitative difference between humans and AI systems concerning the assessment of
vast amounts of input data, our findings suggest that students do not draw a distinction
when it comes to their perceived fairness. This finding raises the question, why students are
the same in their assessment. In aggregate, do the individual strengths and weaknesses of
the respective approach employing HDM and ADM even out so that they are both eventu-
ally perceived as equally fair or unfair? Moreover, what are the specific drivers of per-
ceptions of (un)fairness concerning APP if this were the case? Are the underlying prefer-
ences of students concerning the application of systematic student performance assessment
opposed to the introduction of further means of bureaucratic management and organiza-
tional control? While the inclusion of sociodemographic variables attests that there are inconclusive
effects of individual characteristics on perceived fairness, the presumed central predictor of
the students’ study subject suggests that the field of study has hardly any effect on fairness
perceptions. Only in the need condition did STEM students perceive the proposed distribu-
tion as more fair than students from SHAPE studies. However, this effect was not moder-
ated by whether humans or algorithms were the basis of decision-making. While we can
only speculate about reasons for this effect limited to the norm of need, the absence of the
effects of interest suggests that there is no systematic difference with respect to the field of
study when it comes to APP in general. The study adds to the literature in two important regards. First, it contributes empiri-
cally to the understanding of how students perceive different distributive justice norms,
particularly focusing on the application of ADM and HDM in distributing student support
measures. This addresses a gap in existing research, uncovering nuanced insights into the
preference for equality over other norms like equity and need in the context of higher edu-
cation. Second, the study challenges commonly held assumptions about the potential for 1 3 3 M. Lünich et al. 1096 technological solutions like APP to enhance fairness in educational support allocation. 6 Discussion It
emphasizes the complexity of human perception and the importance of social cohesion,
altruism, and the potential conflict of interest between administrative aspirations and stu-
dent preferences. These findings not only underline the need for careful consideration and
transparent communication in implementing AI systems in education but also pave the way
for future research exploring how to align technological advancements with societal values
and expectations. Limitations of our study include the choice of the empirical design and the sampling of
students. First, using vignettes to illustrate the possible scenario of the deployment of APP
comes with limited room for the depiction of a usually detailed sociotechnical distribution
process. It may also be detached from the current study situation of the surveyed students
at their respective universities. After all, only three distinct distributive justice norms were
used for illustration. The systems and the decision-making processes HEIs deploy are more
varied and elaborate than can be easily depicted and understood in a brief written para-
graph within the factorial design. Further research should thus use an actual APP system
and give a vivid display of its inner procedural workings, especially in inviting students’
evaluation of ADM. Moreover, non-standardized empirical approaches may prove help-
ful in assessing the nuances and points of reference of students’ fairness perceptions more
closely than our standardized design and measure of fairness perceptions. Second, the sam-
ple was limited to German students and was skewed towards students who identified as
female. While this sample bias is less of an issue concerning the experimental logic within
a factorial survey design, we suggest that additional studies broaden the scope and include
a more diverse set of students with a special focus on cross-country comparisons, consid-
ering the diverse academic systems and institutions across nations. Such an approach is
critical given that previous research on FAIR ML has focused almost exclusively on study
samples from Western countries (Starke et al., 2022). As delineated by Lee et al. (2017),
these cultural subtleties can be discerned when contrasting American and Indian respond-
ents regarding their distinct fairness perceptions. Furthermore, the heterogeneous expec-
tations of students, shaped by the differing academic systems and sociocultural contexts
they hail from, warrant further exploration to ensure the comprehensive applicability of
any model or application. 7 Conclusion and Implications The deployment of AIEd is an ongoing process that aims to introduce sociotechnical AI
systems such as ADM for APP within HEIs. Consequently, it may change the social fab-
ric of educational organizations and the life trajectories of generations of students. While
the intentions to use APP to improve student performance and retention arguably appear
understandable from the perspective of university administrators, it is suggested to also
focus on the people affected by introducing sociotechnical AI systems. Research on the
social consequences and evaluation of the affected students thus deems necessary, and our
study answers the call for a deeper understanding of the distributive fairness perceptions
of distinct norms of distributive justice. Our findings show that German students prefer a
distribution of support measures based on an APP system according to the equality norm. In this regard, there are no differences in fairness perceptions of an APP comparing HDM
and ADM and concerning the students’ study subject. 1 3 1 1097 Fairness of Academic Performance Prediction for the Distribution… Eventually, the results of our study link to the literature on AIEd and thus contribute
essential findings to the research on perceptions of distributive justice in this field. Conse-
quently, these findings have implications for HEIs and administrators and future research
in this area. In light of evidence that students prefer a distribution according to the equality
norm when distributing support measures based on an algorithmic APP system, the ques-
tion arises as to whether it makes sense to use an APP system at all since support measures
that are–in theory–intended to be open to all students do not require particular classifica-
tion by an algorithm in the first place. Should individual universities nevertheless decide to
use an APP system to benefit from its presumed potential–for example, to enable individual
feedback or to increase the graduation rates (Attaran et al., 2018; Mah, 2016)–this use of
AIEd could evoke students’ disapproval. In light of potential negative consequences for the
university’s reputation with students and student retention (Marcinkowski et al., 2020), the
pros and cons of using APP must be carefully weighed. Should universities make plans to
introduce APP, we suggest that they conduct research that investigates the evaluation of
APP by its current and future student body. 7 Conclusion and Implications This evaluation may take the form of an ongo-
ing consultation process with all stakeholders within the university to navigate potential
fairness issues and their respective contextuality.i Further research in the field of AIEd, particularly regarding the deployment of APP
within HEIs, beckons multidimensional exploration. Firstly, studies should delve deeper
into understanding the various norms of distributive justice across diverse student popula-
tions. While our study focused on German students’ preferences towards the equality norm,
it remains to be seen if such preferences resonate similarly across different cultural and
educational contexts. Moreover, as suggested above, the practical ramifications of these
findings for universities worldwide need empirical substantiation. A targeted examina-
tion of whether supposedly innovative APP systems align with the core ethos and strategic
visions of HEIs could provide more clarity on the matter. Similarly, the potential for nega-
tive repercussions for universities, in terms of reputation and student retention, stemming
from student perceptions of AIEd, necessitates a comprehensive risk assessment. Lastly,
a co-creative approach, wherein students and other key stakeholders are involved in APP
deployment’s developmental and decision-making processes, might offer a blueprint for
future AIEd endeavors. Investigations into such participatory methodologies, gauging their
feasibility, efficiency, and efficacy, could shape the future discourse on AI in education. Text Vignette ADM ‑ German Original “Derzeit wird in der Öffentlichkeit viel über, Künstliche Intelligenz‘ (KI) gesprochen. Gemeint sind hier Computeranwendungen, die automatisiert digitale Daten auswerten. Die
Auswertung großer Datenmengen stellt für KI einen Lernprozess dar, aus dem sie laufend
neue Informationen verarbeitet und so im Zeitverlauf immer präzisere Muster erkennt. Auf
Grundlage dieser Analyse können Sachverhalte festgestellt sowie zukünftige Entwicklun-
gen prognostiziert werden. Systeme mit Künstlicher Intelligenz können dem Menschen
Handlungsempfehlungen vorschlagen oder autonom Entscheidungen treffen und diese auch 3 3 3 M. Lünich et al. 1098 direkt ausführen. KI gewinnt mit der Zeit auch an Hochschulen immer mehr an Bedeutung
und Relevanz. Versetzen Sie sich bitte einmal in die folgende Situation, zu der wir Ihnen auf der näch-
sten Seite ein paar Fragen stellen möchten: Eine Hochschule bietet Ihren Studierenden ein
Angebot an Fördermaßnahmen, kann jedoch, aufgrund begrenzter Ressourcen, nicht allen
Studierenden eine Teilnahme ermöglichen. Eine KI-Anwendung sichtet und analysiert systematisch alle vorliegenden Leistungs-
daten (z. B. Prüfungsergebnisse) der Studierenden. Auf dieser Basis erstellt die KI eine
Leistungsvorhersage für alle Studierenden.f Vor diesem Hintergrund trifft die Hochschule folgende Entscheidung für die Verteilung
der Fördermaßnahmen:” Text Vignette HDM ‑ German Original “Versetzen Sie sich bitte einmal in die folgende Situation, zu der wir Ihnen auf der näch-
sten Seite ein paar Fragen stellen möchten: Eine Hochschule bietet Ihren Studierenden ein
Angebot an Fördermaßnahmen, kann jedoch, aufgrund begrenzter Ressourcen, nicht allen
Studierenden eine Teilnahme ermöglichen. Beschäftigte der Hochschule sichten und analysieren systematisch alle vorliegenden
Leistungsdaten (z. B. Prüfungsergebnisse) der Studierenden. Auf dieser Basis erstellen die
Hochschulbeschäftigten Leistungsvorhersagen für die Studierenden. Vor diesem Hintergrund trifft die Hochschule folgende Entscheidung für die Verteilung
der Fördermaßnahmen:” Vignette Addendum ‑ Group Equity ‑ German Original “Auf Grundlage der Vorhersage der KI, können nur Studierende, die eine gute
Leistungsvorhersage erhalten haben, sich für das Angebot an Fördermaßnahmen
anmelden.” (emphasis in original) Vignette Addendum ‑ Group Need ‑ German Original “Auf Grundlage der Vorhersage der KI, können nur Studierende, die eine schlechte
Leistungsvorhersage erhalten haben, sich für das Angebot an Fördermaßnahmen
anmelden.” (emphasis in original) Vignette Addendum ‑ Group Equality ‑ German Original “Alle Studierenden können sich für Fördermaßnahmen anmelden, ungeachtet des
Ergebnisses der Vorhersage der KI.” (emphasis in original) “Alle Studierenden können sich für Fördermaßnahmen anmelden, ungeachtet des
Ergebnisses der Vorhersage der KI.” (emphasis in original) “Alle Studierenden können sich für Fördermaßnahmen anmelden, ungeachtet des
Ergebnisses der Vorhersage der KI.” (emphasis in original) Vignette Addendum ‑ Group Equity ‑ German Original Vignette Addendum ‑ Group Equity ‑ German Original Vignette Addendum ‑ Group Equality ‑ English Translation “All students are eligible to apply for remedial measures, regardless of the outcome of
the AI’s prediction.” (emphasis in original) Text Vignette HDM ‑ English Translation “Please put yourself in the following situation, about which we would like to ask you a few
questions on the next page: A university offers its students a range of support measures but,
due to limited resources, cannot enable all students to participate. University employees systematically review and analyze all available student perfor-
mance data (e.g., exam results). Based on this data, the university employees create perfor-
mance predictions for the students. With this in mind, the university makes the following decision for the distribution of
funding:” Text Vignette ADM ‑ English Translation “Currently, there is a lot of public talk about ‘artificial intelligence’ (AI). This refers to
computer applications that automatically evaluate digital data. The evaluation of large vol-
umes of data represents a learning process for AI, from which it continuously processes
new information and thus recognizes increasingly precise patterns over time. Based on this
analysis, facts can be established and future developments can be predicted. Systems with 1 Fairness of Academic Performance Prediction for the Distribution… 1099 artificial intelligence can suggest courses of action to humans or make decisions autono-
mously and also execute them directly. Over time, AI is also becoming increasingly impor-
tant and relevant at universities. artificial intelligence can suggest courses of action to humans or make decisions autono-
mously and also execute them directly. Over time, AI is also becoming increasingly impor-
tant and relevant at universities. Please put yourself in the following situation, about which we would like to ask you a
few questions on the next page: A university offers its students a range of support measures
but, due to limited resources, cannot allow all students to participate. An AI application systematically sifts through and analyzes all available student perfor-
mance data (e.g., exam results). On this basis, the AI generates a performance prediction
for all students. Against this background, the university makes the following decision for
the distribution of support measures:” Vignette Addendum ‑ Group Equity ‑ English Translation “Based on the prediction of the AI, only students who received a good prediction of
performance can register for remedial services.” (emphasis in original) References Adams, J. S. (1965). Inequity in social exchange. Advances in Experimental Social Psychology, 2, 267–299. https://doi.org/10.1016/S0065-2601(08)60108-2 p
g
(
)
Adams Becker, S., Cummins, M., Davis, A., Freeman, A., Hall Giesinger, C., & Ananthanarayanan, V. (2017). NMC Horizon Report: 2017 Higher Education Edition. Austin, Texas: The New Media Con-
sortium. https://www.sconul.ac.uk/sites/default/files/documents/2017-nmc-horizon-report-he-EN.pdf. pi
p
p
Adams, Richard, & McIntyre, Niamh. (2020). England a-Level Downgrades Hit Pupils from Disadvantaged
Areas Hardest: Analysis Also Shows Pupils at Private Schools Benefited Most from Algorithm. The
Guardian. https://www.theguardian.com/education/2020/aug/13/england-a-level-downgrades-hit-
pupils-from-disadvantaged-areas-hardest#. Ahmed, SA., & Khan, SI. (2019). A machine learning approach to predict the engineering students at risk of
dropout and factors behind: bangladesh perspective. In 10th International Conference on Computing,
Communication and Networking Technologies (ICCCNT) (pp. 1–6). https://doi.org/10.1109/ICCCN
T45670.2019.8944511. Allison, S. T., & Messick, D. M. (1990). Social decision heuristics in the use of shared resources. Journal of
Behavioral Decision Making, 3(3), 195–204. https://doi.org/10.1002/bdm.3960030304l Allison, S. T., McQueen, L. R., & Schaerfl, L. M. (1992). Social decision making processes and the equal
partitionment of shared resources. Journal of Experimental Social Psychology, 28(1), 23–42. https://
doi.org/10.1016/0022-1031(92)90030-N g
Alturki, S., Hulpuş, I., & Stuckenschmidt, H. (2022). Predicting academic outcomes: A survey from
2007 till 2018. Technology, Knowledge and Learning, 27(1), 275–307. https://doi.org/10.1007/
s10758-020-09476-0 Alyahyan, E., & Düştegör, D. (2020). Predicting academic success in higher education: Literature review
and best practices: Literature review and best Practices. International Journal of Educational Tech-
nology in Higher Education, 17(1), 1–21. https://doi.org/10.1186/s41239-020-0177-7 Ananny, M., & Crawford, K. (2018). Seeing without knowing: Limitations of the transparency ideal and its
application to algorithmic accountability. New Media & Society, 20(3), 973–989. https://doi.org/10.
1177/1461444816676645 Angwin, J., Larson, J., Mattu, S., & Kirchner, L. (2016). Machine bias: There’s software used across the
country to predict future criminals. And It’s Biased Against Blacks. ProPublica. https://www.propu
blica.org/article/machine-bias-risk-assessments-in-criminal-sentencing. g
g
Araujo, T., Helberger, N., Kruikemeier, S., & de Vreese, C. H. (2020). Seeing without knowing: Limita-
tions of the transparency ideal and its application to algorithmic accountability. AI & Society, 35(3),
611–23. https://doi.org/10.1007/s00146-019-00931-w p
g
Arcidiacono, P., Hotz, V. J., & Kang, S. (2012). Modeling college major choices using elicited measures
of expectations and counterfactuals. Journal of Econometrics, 166(1), 3–16. https://doi.org/10.3386/
w15729 Armatas, C., Kwong, T., Chun, C., Spratt, C., Chan, D., & Kwan, J. (2022). Learning analytics for pro-
gramme review: Evidence, analysis, and action to improve student learning outcomes. Technology,
Knowledge and Learning, 27(2), 461–478. https://doi.org/10.1007/s10758-021-09559-6 Arnold, K. Vignette Addendum ‑ Group Need ‑ English Translation “Based on the AI’s prediction, only students who received a poor performance predic-
tion can enroll in the range of remedial services.” (emphasis in original) “Based on the AI’s prediction, only students who received a poor performance predic-
tion can enroll in the range of remedial services.” (emphasis in original) Acknowledgements The authors thank Albina Maxhuni for her support and feedback on the first version of
the paper, as well as Cara Schönmüller and the students of the teaching research project Predicting student
performance through artificial intelligence: what are the perceptions and attitudes of the affected students? at Heinrich Heine University in the winter term 2021/2022. Funding Open Access funding enabled and organized by Projekt DEAL. This study was conducted as part
of the project Responsible Academic Performance Prediction (RAPP) project. The project is funded by the
German Federal Ministry of Education and Research [grant number 16DHB4020]. Funding Open Access funding enabled and organized by Projekt DEAL. This study was conducted as part
of the project Responsible Academic Performance Prediction (RAPP) project. The project is funded by the
German Federal Ministry of Education and Research [grant number 16DHB4020]. Data Availability The data and code for data analysis can be accessed via the project’s Open Science Foun-
dation repository: (https://osf.io/djt39/). Data Availability The data and code for data analysis can be accessed via the project’s Open Science Foun-
dation repository: (https://osf.io/djt39/). Data Availability The data and code for data analysis can be accessed via the project’s Open Science Foun-
dation repository: (https://osf.io/djt39/). 1 3 3 M. Lünich et al. 1100 Conflict of interest The authors declare no competing interests. Conflict of interest The authors declare no competing interests. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Com-
mons licence, and indicate if changes were made. The images or other third party material in this article
are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the
material. If material is not included in the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly
from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. References E., & Pistilli, M. D. (2012). Course Signals at Purdue: Using Learning Analytics to Increase Stu-
dent Success. In S. Dawson, C. Haythornthwaite, S. Buckingham Shum, D. Gašević, & R. Ferguson
(Eds.), Proceedings of the 2nd International Conference on Learning Analytics and Knowledge (LAK
2012) (pp. 267–270). https://doi.org/10.1145/2330601.2330666. 1 3 Fairness of Academic Performance Prediction for the Distribution… 1101 Asaria, M., Costa-Font, J., & Cowell, F. (2023). How does exposure to COVID-19 influence health and
income inequality aversion? Social Choice and Welfare, 61, 625–647. https://doi.org/10.1007/
s00355-023-01460-8 Attaran, M., Stark, J., & Stotler, D. (2018). Opportunities and challenges for big data analytics in US higher
education. Industry and Higher Education, 32(3), 169–182. https://doi.org/10.1177/0950422218
770937 Bai, B., Dai, H., Zhang, D., Zhang, F., & Hu, H. (2021). The impacts of algorithmic work assignment on
fairness perceptions and productivity: Evidence from field experiments. In Academy of Management
Proceedings: 81st Annual Meeting of the Academy of Management 2021: Bringing the Manager Back
in Management, AoM 2021, edited by Sonia Taneja, 2021:12335. https://doi.org/10.5465/AMBPP.
2021.175. Bai, B., Dai, H., Zhang, D. J., Zhang, F., & Hu, H. (2022). The Impacts of Algorithmic Work Assignment
on Fairness Perceptions and Productivity: Evidence from Field Experiments. Manufacturing & Ser-
vice Operations Management, 24(6), 3060–3078. https://doi.org/10.1287/msom.2022.1120i p
g
( )
p
g
Baker, R. S., & Hawn, A. (2022). Algorithmic Bias in education. International Journal of Artificial Intel-
ligence in Education, 32(4), 1052–1092. https://doi.org/10.1007/s40593-021-00285-9f Berens, J., Schneider, K., Gortz, S., Oster, S., & Burghoff, J. (2019). Early detection of students at risk -
predicting student dropouts using administrative student data from German universities and machine
learning methods. Journal of Educational Data Mining, 11(3), 1–41. https://doi.org/10.5281/zenodo.
3594771 Bettinger, E. P., & Baker, Rachel B. (2014). The effects of student coaching: An evaluation of a randomized
experiment in student advising. Educational Evaluation and Policy Analysis, 36(1), 3–19. https://doi.
org/10.3102/0162373713500523 Boyd, D., & Crawford, K. (2012). Critical questions for big data: Provocations for a cultural, technological,
and scholarly phenomenon. Information, Communication & Society, 15(5), 662–679. https://doi.org/
10.1080/1369118X.2012.678878 Brade, R., Himmler, O., & Jäckle, R. (2018). Normatively framed relative performance feedback – Field
experiment and replication. Munich: MPRA Paper No. 88830. https://mpra.ub.uni-muenchen.de/
88830/. Burton, J. W., Stein, M.-K., & Blegind Jensen, T. (2020). A systematic review of algorithm aversion in aug-
mented decision making. Journal of Behavioral Decision Making, 33(2), 220–239. https://doi.org/10.
1002/bdm.2155i Busuioc, M. (2021). References Accountable artificial intelligence: Holding algorithms to account: Holding algorithms
to account. Public Administration Review, 81(5), 825–836. https://doi.org/10.1111/puar.13293 Cheng, H-F, Stapleton, L., Wang, R., Bullock, P., Chouldechova, A., Wu, ZSS., & Zhu, H. (2021). Soliciting
stakeholders’ fairness notions in child maltreatment predictive systems. In Proceedings of the 2021
CHI Conference on Human Factors in Computing Systems (CHI ’21), edited by Yoshifumi Kitamura,
Aaron Quigley, Katherine Isbister, Takeo Igarashi, Pernille Bjørn, and Steven Drucker, pp. 1–17. https://doi.org/10.1145/3411764.3445308. Coenen, J., Borghans, L., & Diris, R. (2021). Personality traits, preferences and educational choices: A focus
on STEM. Journal of Economic Psychology, 84, 1-16. https://doi.org/10.1016/j.joep.2021.102361
Cropanzano, R. S., & Ambrose, M. L. (2015). The Oxford handbook of justice in the workplace. Oxford
University Press. https://doi.org/10.1093/oxfordhb/9780199981410.001.0001 Cropanzano, R., Rupp, D. E., Mohler, C. J., & Schminke, M. (2001). Three Roads to Organizational Jus-
tice. In G. R. Ferris (Ed.), Research in Personnel and Human Resources Management (Vol. 20, pp. 1–113). Emerald Group Publishing Limited. https://doi.org/10.1016/S0742-7301(01)20001-2 Dai, J., Fazelpour, S., & Lipton, Z. (2021). “Fair Machine Learning Under Partial Compliance.” In M. Four-
cade, B. Kuipers, S. Lazar, and D. Mulligan (Eds.), Proceedings of the 2021 AAAI/ACM Conference
on AI, Ethics, and Society (AIES ’21) (pp. 55–65). https://doi.org/10.1145/3461702.3462521 Daniel, B. (2015). Big data and analytics in higher education: Opportunities and challenges. British Journal
of Educational Technology, 46(5), 904–920. https://doi.org/10.1111/bjet.12230 de Cremer, D., & McGuire, J. (2022). Human-algorithm collaboration works best if humans lead (Because
It Is Fair!). Social Justice Research, 35(1), 33–55. https://doi.org/10.1007/s11211-021-00382-z
d Fi
Li h
K & d Fi
Li h
J (2020) A ifi i l i
lli
d
bli d
i i
k de Cremer, D., & McGuire, J. (2022). Human algorithm collaboration works best if humans lead (Because
It Is Fair!). Social Justice Research, 35(1), 33–55. https://doi.org/10.1007/s11211-021-00382-z
de Fine Licht, K., & de Fine Licht, J. (2020). Artificial intelligence, transparency, and public decision-mak- de Fine Licht, K., & de Fine Licht, J. (2020). Artificial intelligence, transparency, and public decision-mak-
ing: Why explanations are key when trying to produce perceived legitimacy. AI & Society, 35(4),
917–926. https://doi.org/10.1007/s00146-020-00960-w Gomes de Sousa, W., Pereira de Melo, E. R., De Souza Bermejo, P. H., Sousa Farias, R. A., & Oliveira
Gomes, A. (2019). How and where is artificial intelligence in the public sector going? A literature
review and research agenda. Government Information Quarterly, 36(4), 1–14. https://doi.org/10.
1016/j.giq.2019.07.004 1 3 M. Lünich et al. 1102 Destatis (2021). References Bildung Und Kultur: Studierende an Hochschule. Statistisches Bundesamt (Ed.), (Fachserie
11, Reihe 4.1). https://www.destatis.de/DE/Themen/Gesellschaft-Umwelt/Bildung-Forschung-Kultur/
Hochschulen/Publikationen/Downloads-Hochschulen/studierende-hochschulen-endg-2110410217
004.html. Deutsch, M. (1975). Equity, equality, and need: What determines which value will be used as the basis
of distributive justice? Journal of Social Issues, 31(3), 137–149. https://doi.org/10.1111/j.1540-4560.
1975.tb01000.x Diakopoulos, N. (2016). Accountability in algorithmic decision making. Communications of the ACM,
59(2), 56–62. https://doi.org/10.1145/2844110 Dietvorst, B. J., & Bharti, S. (2020). People reject algorithms in uncertain decision domains because
they have diminishing sensitivity to forecasting error. Psychological Science, 31(10), 1302–1314. https://doi.org/10.1177/0956797620948841 Dietvorst, B. J., Simmons, J. P., & Massey, C. (2015). Algorithm aversion: People erroneously avoid
algorithms after seeing them Err. Journal of Experimental Psychology: General, 144(1), 114–126. https://doi.org/10.1037/xge0000033 p
g
g
Dwork, C., Hardt, M., Pitassi, T., Reingold, O., & Zemel, R. (2012). Fairness through awareness. In S. Goldwasser (Ed.), Proceedings of the 3rd Innovations in Theoretical Computer Science Confer-
ence on - ITCS ’12 (pp. 214–226). https://doi.org/10.1145/2090236.2090255. (pp
)
p
g
Edwards, C. (2021). Let the algorithm decide? Communications of the ACM, 64(6), 21–22. https://doi.
org/10.1145/3460216 Ekowo, M., & Palmer, I. (2016). The promise and peril of predictive analytics in higher education: A
landscape analysis.” https://www.newamerica.org/education-policy/policy-papers/promise-and-
peril-predictive-analytics-higher-education/. p
p
y
g
European Commission. (2019). Ethics guidelines for trustworthy AI. https://digital-strategy.ec.europa.
eu/en/library/ethics-guidelines-trustworthy-ai-trustworthy-ai. Faul, F., Erdfelder, E., Buchner, A., & Lang, A. G. (2009). Statistical power analyses using g*power
3.1: Tests for correlation and regression analyses. Behavior Research Methods, 41(4), 1149–1160. https://doi.org/10.3758/BRM.41.4.1149 p
g
Fazelpour, S., & Danks, D. (2021). Algorithmic bias: Senses, sources, solutions. Philosophy Compass,
16(8), 1–16. https://doi.org/10.1111/phc3.12760 Folger, R., & Cropanzano, R. (2001). Fairness theory: Justice as accountability. In J. Greenberg & R. Cropanzano (Eds.), Advances in Organizational Justice (pp. 3–55). Stanford: Stanford University
Press. Friedler, S. A., Scheidegger, C., & Venkatasubramanian, S. (2021). The (Im)possibility of fairness. Communications of the ACM, 64(4), 136–143. https://doi.org/10.1145/3433949 p
g
Gibson, J. P., & Mourad, T. (2018). The growing importance of data literacy in life science education. American Journal of Botany, 105(12), 1953–1956. https://doi.org/10.1002/ajb2.1195 Greenberg, J. (1987). A taxonomy of organizational justice theories. Academy of Management Review,
12(1), 9–22. https://doi.org/10.5465/amr.1987.4306437 Greenberg, J. (1990). Organizational justice: Yesterday, today, and tomorrow. Journal of Management,
16(2), 399–432. https://doi.org/10.1177/014920639001600208 Greenberg, J. (1993). The social side of fairness: Interpersonal and informational classes of organiza-
tional justice. In R. Cropanzano (Ed.), Justice in the Workplace (pp. 79–103). Lawrence Erlbaum
Associates Inc.f Hagendorff, T, & Wezel, K. (2020). References Drucker (Eds.), Proceedings of the 2021 chi conference on human factors in computing systems
(CHI ’21) (pp. 1–15). https://doi.org/10.1145/3411764.3445424i pp
p
g
Humburg, M. (2017). Personality and field of study choice in university. Education Economics, 25(4), 366–
378. https://doi.org/10.1080/09645292.2017.1282426 Huppert, E., Cowell, J. M., Cheng, Y., Contreras-Ibáñez, C., Gomez-Sicard, N., Gonzalez-Gadea, M. L.,
Huepe, D., et al. (2019). The development of children’s preferences for equality and equity across 13
individualistic and collectivist cultures. Developmental Science, 22(2), 1–16. https://doi.org/10.1111/
desc.12729 Jang, Y., Choi, S., & Kim, H. (2022). Development and validation of an instrument to measure undergrad-
uate students’ attitudes toward the ethics of artificial intelligence (AT-EAI) and analysis of its dif-
ference by gender and experience of AI education. Education and Information Technologies, 27(8),
11635–11667. https://doi.org/10.1007/s10639-022-11086-5 Jiang, W., & Pardos, Z. A. (2021). Towards equity and algorithmic fairness in student grade prediction. In
M. Fourcade, B. Kuipers, S. Lazar, & D. Mulligan (Eds.), Proceedings of the 2021 AAAI/ACM Con-
ference on AI, Ethics, and Society (AIES ’21) (pp. 608–617). https://doi.org/10.1145/3461702.34626
23. Jones, M., Molly, D. A., & Lahiri, A. (2020). Shape the future: How the social sciences, humanities and the
arts can SHAPE a positive, post-pandemic future for peoples, economies and environments. Journal
of the British Academy, 8, 167–266. https://doi.org/10.5871/jba/008.167 Jorgensen, T. D., Pornprasertmanit, S., Schoemann, A. M., & Rosseel, Y. (2019). SemTools: Useful tools
for structural equation modeling. R Package Version 0.5-2.” https://CRAN.R-project.org/package=
semTools. Kaibel, C., Koch-Bayram, I., Biemann, T., & Mühlenbock, M. (2019). Applicant perceptions of hiring algo-
rithms - Uniqueness and discrimination experiences as moderators. Academy of Management Pro-
ceedings, 2019(1), 18172. https://doi.org/10.5465/AMBPP.2019.210 Kasinidou, M., Kleanthous, S., Barlas, P., & Otterbacher, J. (2021). I agree with the decision, but they didn’t
deserve this: Future developers’ perception of fairness in algorithmic decisions. In Proceedings of the
2021 ACM Conference on Fairness, Accountability, and Transparency (pp. 690–700). https://doi.org/
10.1145/3442188.3445931. Keller, B., Lünich, M., & Marcinkowski, F. (2022). How is socially responsible academic performance pre-
diction possible? Insights from a Concept of Perceived AI Fairness. In F. Almaraz-Menéndez, A. Maz-Machado, C. López-Esteban, & C. Almaraz-López (Eds.), Strategy, Policy, Practice, and Gov-
ernance for AI in Higher Education Institutions (pp. 126–155). IGI Global. https://doi.org/10.4018/
978-1-7998-9247-2.ch006.f Kemper, L., Vorhoff, G., & Wigger, B. U. (2020). Predicting student dropout: A machine learning approach. European Journal of Higher Education, 10(1), 28–47. https://doi.org/10.1080/21568235.2020.17185
20 Knox, J., Hoel, T., & Yuan, L. (2022). References 15 challenges for AI: Or What AI (Currently) Can’t Do. AI & Soci-
ety, 35(2), 355–365. https://doi.org/10.1007/s00146-019-00886-y Hannan, J., Chen, H.-Y. W., & Joseph, K. (2021). Who gets what, according to whom? An analysis of
fairness perceptions in service allocation. In M. Fourcade, B. Kuipers, S. Lazar, & D. Mulligan
(Eds.) Proceedings of the 2021 AAAI/ACM Conference on AI, Ethics, and Society (AIES ’21) (pp. 555–565). https://doi.org/10.1145/3461702.3462568. Harrison, G., Hanson, J., Jacinto, C., Ramirez, J., & Ur, B. (2020). An Empirical Study on the Perceived
Fairness of Realistic, Imperfect Machine Learning Models. In M. Hildebrandt, C. Castillo, E. Celis, S. Ruggieri, L. Taylor, & G. Zanfir-Fortuna (Eds.) Proceedings of the 2020 Conference on
Fairness, Accountability, and Transparency (FAT* ’20), (pp. 392–402). https://doi.org/10.1145/
3351095.3372831.i Hausmann, L. R. M., Ye, F., Schofield, J. W., & Woods, R. L. (2009). Sense of belonging and persis-
tence in white and African American first-year students. Research in Higher Education, 50(7),
649–669. https://doi.org/10.1007/s11162-009-9137-8 p
g
Helberger, N., Araujo, T., & de Vreese, C. H. (2020). Who is the fairest of them all? Public attitudes and
expectations regarding automated decision-making: Public attitudes and expectations regarding 1 3 Fairness of Academic Performance Prediction for the Distribution… 1103 automated decision-making. Computer Law & Security Review, 39, 1-16. https://doi.org/10.
1016/j.clsr.2020.105456 automated decision-making. Computer Law & Security Review, 39, 1-16. https://doi.org/10.
1016/j.clsr.2020.105456 automated decision-making. Computer Law & Security Review, 39, 1-16. https://doi.org/10.
1016/j.clsr.2020.105456 j
Holzinger, A. (2016). Interactive machine learning for health informatics: When do we need the human-
in-the-loop? Brain Informatics, 3(2), 119–131. https://doi.org/10.1007/s40708-016-0042-6
H
ll J A
R b
L D
S
K
& Gib
D C (2018) A
b
i j
Holzinger, A. (2016). Interactive machine learning for health informatics: When do we need the human-
in-the-loop? Brain Informatics, 3(2), 119–131. https://doi.org/10.1007/s40708-016-0042-6
ll
A
b
S
& Gib
C (2018) A
b
i j
Holzinger, A. (2016). Interactive machine learning for health informatics: When do we need the human-
in-the-loop? Brain Informatics, 3(2), 119–131. https://doi.org/10.1007/s40708-016-0042-6 Howell, J. A., Roberts, L. D., Seaman, K., & Gibson, D. C. (2018). Are we on our way to becoming
a ‘helicopter university’? Academics’ views on learning analytics. Technology, Knowledge and
Learning, 23(1), 1–20. https://doi.org/10.1007/s10758-017-9329-9 Hsu, S., Li, TW., Zhang, Z., Fowler, M., Zilles, C., & Karahalios, K. (2021). Attitudes surrounding
an imperfect AI autograder. In Y. Kitamura, A. Quigley, K. Isbister, T. Igarashi, P. Bjørn, & S. References From principles to processes: Lessons from higher education from
the development of AI ethics. In F. Almaraz-Menéndez, A. Maz-Machado, C. López-Esteban, & C. Almaraz-López (Eds.), Strategy, Policy, Practice, and Governance for AI in Higher Education Insti-
tutions (pp. 101–125). IGI Global. https://doi.org/10.4018/978-1-7998-9247-2.ch005. pp
p
g
König, P. D., & Wenzelburger, G. (2022). Between technochauvinism and human-centrism: Can algorithms
improve decision-making in democratic politics? European Political Science, 21(1), 132–149. https://
d i
/10 1057/ 41304 020 00298 3 doi.org/10.1057/s41304-020-00298-3
Chiteng Kot, F. (2014). The impact of centralized advising on first-year academic performance and second-
year enrollment behavior. Research in Higher Education, 55(6), 527–563. https://doi.org/10.1007/
s11162-013-9325-4 Kotsiantis, S. B. (2012). Use of machine learning techniques for educational proposes: A decision support
system for forecasting students’ grades. Artificial Intelligence Review, 37(4), 331–344. https://doi.org/
10.1007/s10462-011-9234-x 1 3 1 3 M. Lünich et al. 1104 Kusner, M., Loftus, J., Russell, C., & Silva, R. (2017). Counterfactual Fairness. In U. von Luxburg, I. Guyon, S. Bengio, H. Wallach, R. Fergus, S. V. N. Vishwanathan, & R. Garnett (Eds.), NIPS’17: Pro-
ceedings of the 31st International Conference on Neural Information Processing Systems (pp. 1–11). Lee, M. K. (2018). Understanding perception of algorithmic decisions: Fairness, trust, and emotion in
response to algorithmic management. Big Data & Society, 5(1), 1-16. https://doi.org/10.1177/20539
51718756684 Lee, M. K., & Baykal, S. (2017). Algorithmic mediation in group decisions: Fairness perceptions of algo-
rithmically mediated Vs. discussion-based social division. In Proceedings of the 2017 ACM Confer-
ence on Computer Supported Cooperative Work and Social Computing (pp. 1035–1048). https://doi.
org/10.1145/2998181.2998230. Lee, M. K., & Rich, K. (2021). Who is included in human perceptions of AI?: Trust and perceived fairness
around healthcare AI and cultural mistrust: Trust and perceived fairness around healthcare AI and
cultural mistrust. In Proceedings of the 2021 CHI Conference on Human Factors in Computing Sys-
tems (pp. 1–14). https://doi.org/10.1145/3411764.3445570. pp
p
g
Lee, M. K., Kim, J. T., & Lizarondo, L. (2017). A human-centered approach to algorithmic services: Con-
siderations for fair and motivating smart community service management that allocates donations
to non-profit organizations. In G. Mark, S. Fussell, C. Lampe, M. C. Schraefel, J. P. Hourcade, C. Appert, & D. Wigdor (Eds.), Proceedings of the 2017 CHI Conference on Human Factors in Comput-
ing Systems (pp. 3365–3376). https://doi.org/10.1145/3025453.3025884. Lee, M. K., Jain, A., Cha, H. J., Ojha, S., & Kusbit, D. (2019). References Procedural justice in algorithmic fairness:
Leveraging transparency and outcome control for fair algorithmic mediation. Proceedings of the ACM
on Human-Computer Interaction, 3, 1–26. https://doi.org/10.1145/3359284 p
g
Lee, M. K., Kusbit, D., Kahng, A., Kim, J. T., Yuan, X., Chan, A., See, D., et al. (2019). WeBuildAI:
Participatory framework for algorithmic governance. Proceedings of the ACM on Human-Computer
Interaction, 3, 1–35. https://doi.org/10.1145/3359283 Legault, L., Green-Demers, I., & Pelletier, L. (2006). Why do high school students lack motivation in the
classroom? Toward an understanding of academic amotivation and the role of social support. Journal
of Educational Psychology, 98(3), 567–582. https://doi.org/10.1037/0022-0663.98.3.567 Little, T. D., Slegers, D. W., & Card, N. A. (2006). A non-arbitrary method of identifying and scaling latent
variables in SEM and MACS models. Structural Equation Modeling: A Multidisciplinary Journal,
13(1), 59–72. https://doi.org/10.1207/s15328007sem1301_3 Logg, J. M., Minson, J. A., & Moore, D. A. (2019). Algorithm appreciation: People prefer algorithmic to
human judgment. Organizational Behavior and Human Decision Processes, 151, 90–103. https://doi.
org/10.1016/j.obhdp.2018.12.005 g
j
p
Lünich, M., & Kieslich, K. (2022). Exploring the roles of trust and social group preference on the legiti-
macy of algorithmic decision-making Vs. Human decision-making for allocating COVID-19 vaccina-
tions. AI & SOCIETY, 1–19. https://doi.org/10.1007/s00146-022-01412-3 Mah, D.-K. (2016). Learning analytics and digital badges: Potential impact on student retention in
higher education. Technology, Knowledge and Learning, 21(3), 285–305. https://doi.org/10.1007/
s10758-016-9286-8 Makhlouf, K., Zhioua, S., & Palamidessi, C. (2021). Machine learning fairness notions: Bridging the gap
with real-world applications. Information Processing & Management, 58(5), 1-32. https://doi.org/10.
1016/j.ipm.2021.102642i Mäntymäki, M., Minkkinen, M., Birkstedt, T., & Viljanen, M. (2022). Defining organizational AI govern-
ance. AI and Ethics, 2(4), 603–609. https://doi.org/10.1007/s43681-022-00143-x Marcinkowski, F., Kieslich, K., Starke, C., & Lünich, M. (2020). Implications of AI (Un-)fairness in Higher
Education Admissions: The Effects of Perceived AI (Un-)Fairness on Exit, Voice and Organizational
Reputation. In M. Hildebrandt, C. Castillo, E. Celis, S. Ruggieri, L. Taylor, & G. Zanfir-Fortuna
(Eds.), Proceedings of the 2020 Conference on Fairness, Accountability, and Transparency (FAT*
’20) (pp. 122–130). https://doi.org/10.1145/3351095.3372867. Martinez Neda, B., Zeng, Y., & Gago-Masague, S. (2021). Using machine learning in admissions: Reduc-
ing human and algorithmic bias in the selection process. In M. Sherriff, L. D. Merkle, P. Cutter, A. Monge, & J. Sheard (Eds.), Proceedings of the 52nd ACM Technical Symposium on Computer Sci-
ence Education (SIGCSE ’21) (p. 1323). https://doi.org/10.1145/3408877.3439664. Morand, D., Merriman, K., & Deckop, J. (2020). References https://doi.org/10.18637/jss.v048.i02f Rosseel, Y. (2012). Lavaan: An r package for structural equation modeling. Journal of Statistical Soft-
ware, 48(2), 1–26. https://doi.org/10.18637/jss.v048.i02f Abu Saa, A., Al-Emran, M., & Shaalan, K. (2019). Factors affecting students’ performance in higher
education: A systematic review of predictive data mining techniques. Technology, Knowledge and
Learning, 24(4), 567–598. https://doi.org/10.1007/s10758-019-09408-7 p
g
Saha, D., Schumann, C., McElfresh, D. C., Dickerson, J. P., Mazurek, M. L., & Tschantz, M. C. (2020). Human comprehension of fairness in machine learning. In A. Markham (Ed.), Proceedings of the
AAAI/ACM Conference on AI, Ethics, and Society (p. 152). https://doi.org/10.1145/3375627.3375819. Saxena, N. A., Huang, K., DeFilippis, E., Radanovic, G., Parkes, D. C., & Liu, Y. (2020). How do fair-
ness definitions fare? Testing public attitudes towards three algorithmic definitions of fairness in
loan allocations. Artificial Intelligence, 283, 1-15. https://doi.org/10.1016/j.artint.2020.103238
Ö i
Schlicker, N., Langer, M., Ötting, S., Baum, K., König, C. J., & Wallach, D. (2021). What to expect
from opening up ‘black boxes’? Comparing perceptions of justice between human and automated
agents. Computers in Human Behavior, 122, 1-16. https://doi.org/10.1016/j.chb.2021.106837i Sclater, N., & Mullan, J. (2017). Jisc briefing: Learning analytics and student success - assessing
evidence.” Jisc. https://repository.jisc.ac.uk/6560/1/learning-analytics_and_student_success.pdf evidence. Jisc. https://repository.jisc.ac.uk/6560/1/learning analytics_and_student_success.pdf. Sen, A. K. (2009). The Idea of Justice. Harvard University Press. https://doi.org/10.4159/9780674054578
Sherrick, M. F., Davenport, C. A., & Colina, T. L. (1971). Flexibility and satisfaction with college major. Journal of Counseling Psychology, 18(5), 487–489. https://doi.org/10.1037/h0031509
Shi
D (2019) T
d f i
t bl
d t
t
l
ith
C
t di
l
ith
i i Sen, A. K. (2009). The Idea of Justice. Harvard University Press. https://doi.org/10.4159/9780674054578
Sherrick M F Davenport C A & Colina T L (1971) Flexibility and satisfaction with college major Sen, A. K. (2009). The Idea of Justice. Harvard University Press. https://doi.org/10.4159/9780674054578
Sherrick, M. F., Davenport, C. A., & Colina, T. L. (1971). Flexibility and satisfaction with college major. Journal of Counseling Psychology, 18(5), 487–489. https://doi.org/10.1037/h0031509 Shin, D. (2019). Toward fair, accountable, and transparent algorithms: Case studies on algorithm ini-
tiatives in Korea and China. Javnost - The Public, 26(3), 274–290. https://doi.org/10.1080/13183
222.2019.1589249 Shin, D. (2019). Toward fair, accountable, and transparent algorithms: Case studies on algorithm ini-
tiatives in Korea and China. Javnost - The Public, 26(3), 274–290. https://doi.org/10.1080/13183
222.2019.1589249 Shin, D. (2020). User perceptions of algorithmic decisions in the personalized AI system: Perceptual
evaluation of fairness, accountability, transparency, and explainability. References Making sense of egalitarian pay: Reconceptualising its
treatment in organisations. Human Resource Management Journal, 30(2), 180–197. https://doi.org/
10.1111/1748-8583.12264i Morosanu, L., Handley, K., & O’Donovan, B. (2010). Seeking support: Researching first-year students’
experiences of coping with academic life. Higher Education Research & Development, 29(6), 665–
678. https://doi.org/10.1080/07294360.2010.487200 1 3 Fairness of Academic Performance Prediction for the Distribution… 1105 Muñoz, C., Megan, S., & Patil, D. J. (2016). Big data: A report on algorithmic systems, opportunity, and
civil
rights. https://obamawhitehouse.archives.gov/sites/default/files/microsites/ostp/2016_0504_
data_discrimination.pdf. p
Murphy, S. P. (2011). Fairness. In D. K. Chatterjee (Ed.), Encyclopedia of Global Justice (pp. 336–340). Springer Netherlands. Neugebauer, M., Heublein, U., & Daniel, A. (2019). Studienabbruch in Deutschland: Ausmaß, Ursachen,
Folgen, Präventionsmöglichkeiten. Zeitschrift für Erziehungswissenschaft, 22(5), 1025–1046. https://
doi.org/10.1007/s11618-019-00904-1 Nguyen, M.-T., Tran-Tien, M., Viet, A. P., Vu, H.-T., & Nguyen, V.-H. (2021). Building a chatbot for
supporting the admission of universities. In T. Theeramunkong (Ed.), Proceedings of 2021 13th
International Conference Knowledge and Systems Engineering (KSE 2021) (pp. 1–6). https://doi.
org/10.1109/KSE53942.2021.9648677. Nyarko, J., Goel, S., & Sommers, R. (2021). Breaking taboos in fair machine learning: An experimental
study: An experimental study. In Equity and Access in Algorithms, Mechanisms, and Optimization
(EAAMO ’21) (pp. 1–11). https://doi.org/10.1145/3465416.3483291. pp
p
g
Olaya, D., Vásquez, J., Maldonado, S., Miranda, J., & Verbeke, W. (2020). Uplift Modeling for pre-
venting student dropout in higher education. Decision Support Systems, 134, 1-11. https://doi. org/10.1016/j.dss.2020.113320 g
j
Ornelas, F., & Ordonez, C. (2017). Predicting student success: A Naïve Bayesian application to commu-
nity college data. Technology, Knowledge and Learning, 22(3), 299–315. https://doi.org/10.1007/
s10758-017-9334-z Orwat, C. (2020). Risks of Discrimination Through the Use of Algorithms: A Study Compiled with a
Grant from the Federal Anti-Discrimination Agency. Berlin, Germany: Federal Anti-Discrimina-
tion Agency. Pelletier, K., Brown, M., Brooks, D. C., McCormack, M., Reeves, J., Arbino, N., Bozkurt, A et al. (2021). 2021 EDUCAUSE Horizon Report: Teaching and Learning Edition. Boulder, CO: EDU-
CAUSE. https://library.educause.edu/resources/2021/4/2021-educause-horizon-report-teaching-
and-learning-edition. g
Pistilli, M. D., & Arnold, K. E. (2010). Purdue signals: Mining real-time academic data to enhance stu-
dent success. About Campus, 15(3), 22–24. https://doi.org/10.1002/abc.20025 Pozzebon, J. A., Ashton, M. C., & Visser, B. A. (2014). Major changes: Personality, ability, and congru-
ence in the prediction of academic outcomes. Journal of Career Assessment, 22(1), 75–88. https://
doi.org/10.1177/1069072713487858 g
Rawls, J. (1999). A Theory of Justice (Revised). Harvard University Press. Rosseel, Y. (2012). Lavaan: An r package for structural equati
ware, 48(2), 1–26. References Journal of Broadcasting &
Electronic Media, 64(4), 541–565. https://doi.org/10.1080/08838151.2020.1843357 Shin, D. (2020). User perceptions of algorithmic decisions in the personalized AI system: Perceptual
evaluation of fairness, accountability, transparency, and explainability. Journal of Broadcasting &
Electronic Media, 64(4), 541–565. https://doi.org/10.1080/08838151.2020.1843357 Shin, D. (2020). User perceptions of algorithmic decisions in the personalized AI system: Perceptual
evaluation of fairness, accountability, transparency, and explainability. Journal of Broadcasting &
Electronic Media, 64(4), 541–565. https://doi.org/10.1080/08838151.2020.1843357 1 3 M. Lünich et al. 1106 Shin, D. (2021). The effects of explainability and causability on perception, trust, and acceptance: Impli-
cations for explainable AI. International Journal of Human-Computer Studies, 146, 1–10. https://
doi.org/10.1016/j.ijhcs.2020.102551 g
j j
Shin, D., Zhong, B., & Biocca, F. A. (2020). Beyond user experience: What constitutes algorithmic
experiences? International Journal of Information Management, 52(3), 1-11. https://doi.org/10.
1016/j.ijinfomgt.2019.102061 Shneiderman, B. (2020). Bridging the gap between ethics and practice: Guidelines for reliable, safe,
and trustworthy human-centered AI systems. ACM Transactions on Interactive Intelligent Systems,
10(4), 1–31. https://doi.org/10.1145/3419764 ( ),
p
g
Slade, S., & Prinsloo, P. (2013). Learning analytics: Ethical issues and dilemmas. American Behavioral
Scientist, 57(10), 1510–1529. https://doi.org/10.1177/0002764213479366 Smith, H. (2020). Algorithmic bias: Should students pay the price? AI & Society, 35(4), 1077–1078. https://
doi.org/10.1007/s00146-020-01054-3 g
Smith, B. O., Shrader, R., White, D. R., Wooten, J., Dogbey, J., Nath, Steve, O’Hara, Michael, Nan, X., &
Rosenman, R. (2019). Improving student performance through loss aversion. Scholarship of Teaching
and Learning in Psychology, 5(4), 278–288. https://doi.org/10.1037/stl0000149 Srivastava, M., Heidari, H., & Kraus, A. (2019). Mathematical notions Vs. Human perception of fairness: A
descriptive approach to fairness for machine learning. In A. Teredesai, V. Kumar, Y. Li, R. Rosales,
E. Terzi, & G. Karypis (Eds.), Proceedings of the 25th ACM SIGKDD International Conference on
Knowledge Discovery & Data Mining (KDD ’19) (pp. 2459–2468). https://doi.org/10.1145/3292500.
3330664.i Starke, C., & Lünich, M. (2020). Artificial intelligence for political decision-making in the European Union:
Effects on Citizens’ perceptions of input, throughput, and output legitimacy. Data & Policy, 2, 1–17. https://doi.org/10.1017/dap.2020.19 Starke, C., Baleis, J., Keller, B., & Marcinkowski, F. (2022). Fairness perceptions of algorithmic decision-
making: A systematic review of the empirical literature. Big Data & Society, 9(2), 1–16. https://doi.
org/10.1177/20539517221115189 g
Stegmann, H. (1980). Abiturient Und Studium: Bestimmungsfaktoren für Die Studienaufnahme Und Die
Wahl Des Studiengangs. Mitteilungen Aus Der Arbeitsmarkt- Und Berufsforschung, 13, 531–542. Sun, L., & Tang, Y. (2021). Data-driven discrimination, perceived fairness, and consumer trust-the perspec-
tive of consumer attribution. References Frontiers in Psychology, 12, 1-13. https://doi.org/10.3389/fpsyg.2021.
748765 Thurman, N., Moeller, J., Helberger, N., & Trilling, D. (2019). My Friends, Editors, Algorithms, and I:
Examining audience attitudes to news selection. Digital Journalism, 7(4), 447–469. https://doi.org/
10.1080/21670811.2018.1493936 Tinto, V. (1975). Dropout from higher education: A theoretical synthesis of recent research. Review of Edu-
cational Research, 45(1), 89–125. Tversky, A., & Kahneman, D. (1991). Loss aversion in riskless choice: A reference-dependent model. The
Quarterly Journal of Economics, 106(4), 1039–1061. https://doi.org/10.2307/2937956 Uhde, A., Schlicker, N., Wallach, D. P., & Hassenzahl, M. (2020). Fairness and decision-making in collabo-
rative shift scheduling systems. In R. Bernhaupt, F. Mueller, D. Verweij, J. Andres, J. McGrenere, A. Cockburn, I. Avellino, et al. (Eds.), Proceedings of the 2020 CHI Conference on Human Factors in
Computing Systems (pp. 1–13). https://doi.org/10.1145/3313831.3376656.f van Hootegem, A., Abts, K., & Meuleman, B. (2020). Differentiated Distributive Justice Preferences? Configurations of preferences for equality, equity and need in three welfare domains. Social Justice
Research, 33(3), 257–283. https://doi.org/10.1007/s11211-020-00354-9 p
g
Veale, M., & Binns, R. (2017). Fairer machine learning in the real world: Mitigating discrimination with-
out collecting sensitive data: Mitigating discrimination without collecting sensitive data. Big Data &
Society, 4(2), 1-17. https://doi.org/10.1177/2053951717743530if Vedel, A. (2016). Big five personality group differences across academic majors: A systematic review. Per-
sonality and Individual Differences, 92, 1–10. https://doi.org/10.1016/j.paid.2015.12.011 y
ff
p
g
j p
Verbree, A.-R., Maas, L., Hornstra, L., & Wijngaards-de Meji, L. (2021). Personality predicts academic
achievement in higher education: Differences by academic field of study? Learning and Individual
Differences, 92, 1-11. https://doi.org/10.1016/j.lindif.2021.102081i ff
p
g
j
Verma, S., & Rubin, J. (2018). Fairness Definitions Explained. In Y. Brun, B. Johnson, & A. Meliou (Eds.),
Proceedings of the 40th International Workshop on Software Fairness (ICSE ’18) (pp. 1–7). https://
doi.org/10.1145/3194770.3194776. g
Vieira, D., Chinchilla, J. R., Liu, B. L., Yerovi, C., & Morales, D. (2022). Beyond the chatbot: How are
universities using ai nowadays? In F. Almaraz-Menéndez, A. Maz-Machado, C. López-Esteban, & C. 1 1 3 Fairness of Academic Performance Prediction for the Distribution… 1107 Almaraz-López (Eds.), Strategy, policy, practice, and governance for AI in higher education institu-
tions (pp. 1–22). IGI Global. https://doi.org/10.4018/978-1-7998-9247-2.ch001l Almaraz-López (Eds.), Strategy, policy, practice, and governance for AI in higher education institu-
tions (pp. 1–22). IGI Global. https://doi.org/10.4018/978-1-7998-9247-2.ch001l Almaraz-López (Eds.), Strategy, policy, practice, and governance for AI in higher education institu-
tions (pp. 1–22). IGI Global. https://doi.org/10.4018/978-1-7998-9247-2.ch001l Wang, R., Maxwell H. References F., & Zhu, H. (2020). Factors influencing perceived fairness in algorithmic decision-
making. In R. Bernhaupt, F. Mueller, D. Verweij, J. Andres, J. McGrenere, A. Cockburn, I. Avellino,
et al. (Eds.), Proceedings of the 2020 CHI Conference on Human Factors in Computing Systems (pp. 1–14). https://doi.org/10.1145/3313831.3376813. Webb, H., Koene, A., Patel, M., & Vallejos, E. P. (2018). Multi-stakeholder dialogue for policy recommen-
dations on algorithmic fairness. In Proceedings of the 9th International Conference on Social Media
and Society (pp. 395–399). https://doi.org/10.1145/3217804.3217952. y pp
p
g
Wenzelburger, G., & Hartmann, K. (2021). Explaining the rise and the fall of a policy: Investigating the
introduction and the dismantling of a French University admission policy through the lens of the mul-
tiple streams framework. p
Wenzelburger, G., & Hartmann, K. (2022). Policy formation, termination and the multiple streams frame-
work: the case of introducing and abolishing automated university admission in France. Policy Stud-
ies, 43(5), 1075–1095. https://doi.org/10.1080/01442872.2021.1922661i p
g
Wirtz, BW., & Müller, WM. (2019). An integrated artificial intelligence framework for public management. Public Management Review, 21(7), 1076–1100. https://doi.org/10.1080/14719037.2018.1549268i Wirtz, B. W., Weyerer, J. C., & Geyer, C. (2019). Artificial intelligence and the public sector-applications
and challenges. International Journal of Public Administration, 42(7), 596–615. https://doi.org/10.
1080/01900692.2018.1498103 Wong, P.-H. (2020). Democratizing algorithmic fairness. Philosophy & Technology, 33(2), 225–244. https://
doi.org/10.1007/s13347-019-00355-w g
Yilmaz, K., Gizem, F., & Yilmaz, R. (2020). Student opinions about personalized recommendation and
feedback based on learning analytics. Technology, Knowledge and Learning, 25(4), 753–768. https://
doi.org/10.1007/s10758-020-09460-8 Zawacki-Richter, O., Marín, V. I., Bond, M., & Gouverneur, F. (2019). Systematic review of research
on artificial intelligence applications in higher education - Where are the educators? International
Journal of Educational Technology in Higher Education, 16(1), 1–27. https://doi.org/10.1186/
s41239-019-0171-0 Zhou, J., Verma, S., Mittal, M., & Chen, F. (2021). Understanding relations between perception of fair-
ness and trust in algorithmic decision making. In Proceedings of the 8th International Conference on
Behavioral and Social Computing (BESC 2021) (pp. 1–5). https://doi.org/10.1109/BESC53957.2021.
9635182. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations. Marco Lünich1 · Birte Keller1 · Frank Marcinkowski1 Marco Lünich1 · Birte Keller1 · Frank Marcinkowski1 * Marco Lünich
marco.luenich@hhu.de
Birte Keller
birte.keller@hhu.de
Frank Marcinkowski
frank.marcinkowski@hhu.de 1
Department of Social Sciences, Heinrich Heine University, Düsseldorf, Germany 3 1
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Marc Schwaerzel1,2, Dominik Brunner1, Fabian Jakub3, Claudia Emde3,4, Brigitte Buchmann1, Alexis Berne2, and
Gerrit Kuhlmann1 Dominik Brunner1, Fabian Jakub3, Claudia Emde3,4, Brigitte Buchmann1, Alexis Berne2, an 1Empa, Swiss Federal Laboratories for Materials Science and Technology, Dübendorf, Switzerland
2Environmental Remote Sensing Laboratory, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
3Meteorological Institute, Ludwig Maximilian University of Munich, Munich, Germany
4German Aerospace Center (DLR), Oberpfaffenhofen, Germany Correspondence: Gerrit Kuhlmann (gerrit.kuhlmann@empa.ch) Received: 5 May 2021 – Discussion started: 2 July 2021
Revised: 1 September 2021 – Accepted: 18 September 2021 – Published: 8 October 2021 Received: 5 May 2021 – Discussion started: 2 July 2021 Abstract. Airborne imaging remote sensing is increasingly
used to map the spatial distribution of nitrogen dioxide (NO2)
in cities. Despite the small ground-pixel size of the sen-
sors, the measured NO2 distributions are much smoother than
one would expect from high-resolution model simulations
of NO2 over cities. This could partly be caused by 3D ra-
diative transfer effects due to observation geometry, adja-
cency effects and effects of buildings. Here, we present a
case study of imaging a synthetic NO2 distribution for a dis-
trict of Zurich using the 3D MYSTIC (Monte carlo code for
the phYSically correct Tracing of photons In Cloudy atmo-
spheres) solver of the libRadtran radiative transfer library. We computed NO2 slant column densities (SCDs) using
the recently implemented 3D-box air mass factors (3D-box
AMFs) and a new urban canopy module to account for the
effects of buildings. We found that for a single ground pixel
(50 m × 50 m) more than 50 % of the sensitivity is located
outside of the pixel, primarily in the direction of the main
optical path between sun, ground pixel, and instrument. Con-
sequently, NO2 SCDs are spatially smoothed, which results
in an increase over roads when they are parallel to the optical
path and a decrease otherwise. When buildings are included,
NO2 SCDs are reduced on average by 5 % due to the reduced
sensitivity to NO2 in the shadows of the buildings. The ef-
fects of buildings also introduce a complex pattern of vari-
ability in SCDs that would show up in airborne observations
as an additional noise component (about 12 µmolm−2) sim-
ilar to the magnitude of typical measurement uncertainties. The smearing of the SCDs cannot be corrected using 1D- layer AMFs that assume horizontal homogeneity and thus
remains in the final NO2 map. Marc Schwaerzel1,2, Dominik Brunner1, Fabian Jakub3, Claudia Emde3,4, Brigitte Buchmann1, Alexis Berne2, and
Gerrit Kuhlmann1 The 3D radiative transfer ef-
fects by including buildings need to be considered to com-
pute more accurate AMFs and to reduce biases in NO2 ver-
tical columns obtained from high-resolution city-scale NO2
remote sensing. Atmos. Meas. Tech., 14, 6469–6482, 2021
https://doi.org/10.5194/amt-14-6469-2021
© Author(s) 2021. This work is distributed under
the Creative Commons Attribution 4.0 License. Atmos. Meas. Tech., 14, 6469–6482, 2021
https://doi.org/10.5194/amt-14-6469-2021
© Author(s) 2021. This work is distributed under
the Creative Commons Attribution 4.0 License. 1
Introduction Nitrogen oxides (NOx = NO + NO2) are key air pollutants
mainly emitted by fuel combustion, traffic, heating systems,
industrial facilities and power plants. Their short lifetime and
localized sources result in a high spatial and temporal vari-
ability especially in urban areas (Berchet et al., 2017). An at-
tractive possibility to create high-resolution maps (< 100 m)
of the NO2 distribution in cities is to use an airborne imag-
ing spectrometer. This was first demonstrated for measure-
ments from the Airborne Prism Experiment (APEX) of a
Swiss–Belgium consortium (Popp et al., 2012; Tack et al.,
2017) and the Geostationary Trace gas and Aerosol Sen-
sor Optimization (GeoTASO) spectrometer (Nowlan et al.,
2016) developed in the United States. A comprehensive com-
parison between four airborne NO2 imaging spectrometers
flown over the city of Berlin was performed during the
AROMAPEX campaign (Tack et al., 2019), which included
APEX (Schaepman et al., 2015), AirMAP (Airborne imaging
DOAS instrument for Measurements of Atmospheric Pollu-
tion) (Schönhardt et al., 2015), SWING (Small Whiskbroom M. Schwaerzel et al.: 3D radiative transfer over cities with buildings M. Schwaerzel et al.: 3D radiative transfer over cities with buildings 6470 Imager for atmospheric compositioN monitorinG) (Merlaud
et al., 2013) and SBI (Spectrolite Breadboard Instrument)
(de Goeij et al., 2017; Vlemmix et al., 2017). airborne imaging spectrometers. The study builds on the
work by Schwaerzel et al. (2020), where we highlighted the
importance of 3D RT effects on trace gas remote sensing for
ground-based and airborne instruments. In this study, we also
use the Monte carlo code for the phYSically correct Tracing
of photons In Cloudy atmospheres (MYSTIC) solver of the
library for radiative transfer (libRadtran), which is a Monte
Carlo solver that traces individual photons in a 3D model
grid (Mayer, 2009; Emde and Mayer, 2007). These photon
paths are converted to 3D-box AMFs that describe the sensi-
tivity of an instrument to a trace gas (here NO2) in each grid
box (Schwaerzel et al., 2020). To account for the effect of
buildings, we additionally implemented a new urban canopy
feature that represents the full 3D building structure of a city
with assigned optical properties of the different surfaces. The
3D radiative transfer calculations in the urban canopy are
not new but so far focused only on applications not related
to trace gas observations such as the computation of radi-
ation budgets and broadband landscape imaging (Gastellu-
Etchegorry et al., 2015). The present study is the first to use
such a model to investigate 3D effects on trace gas retrievals
over a city. In order to isolate different effects on 3D-box
AMFs and on the spatial smoothing of the information re-
trieved from an airborne instrument, we use a comparatively
simple setup with 3D buildings representative of a district in
the city of Zurich with uniform optical properties of roofs,
walls and streets. The model, however, is able to describe the
optical properties of each single surface separately. Airborne imaging spectrometers are passive remote sens-
ing instruments from which we can retrieve NO2 slant col-
umn densities (SCDs) from backscattered solar radiance
spectra. SCDs are defined as the integrated trace gas con-
centrations along the optical path from the sun, through the
atmosphere and towards the instrument. The optical path and
therefore the SCD depend on illumination conditions; view-
ing geometry; the scattering and absorption by air molecules,
aerosols, and clouds; and the reflectance of the surface. M. Schwaerzel et al.: 3D radiative transfer over cities with buildings To
obtain a quantity that is independent of illumination and
viewing conditions, SCDs are divided by air mass factors
(AMFs) to compute vertical column densities (VCDs) de-
fined as the vertically integrated concentrations from the
ground to the top of the atmosphere. g
p
p
NO2 retrieval algorithms applied to airborne instruments
have been derived from algorithms developed for satellite
instruments with much lower spatial resolution (> 5 km). These algorithms rely on 1D radiative transfer simulations,
which reach their limits at high resolution and in the pres-
ence of spatially variable properties of the atmosphere and
the surface, as they assume horizontal homogeneity of all pa-
rameters within the optical path. A strong indication for the
importance of 3D radiative transfer effects is that NO2 maps
obtained from airborne imaging spectrometers over cities are
spatially much smoother than one would expect from the in-
strument resolution and compared to maps obtained from
high-resolution city-scale dispersion models, which show,
for example, strong gradients in the NO2 field along major
roads (Kuhlmann et al., 2017). A likely explanation is that
the measurements are not only sensitive to NO2 in the verti-
cal column above the observed ground pixels but also to the
atmosphere surrounding the pixels, which is known as the
horizontal smoothing error or the adjacency effect (Cracknell
and Varotsos, 2012; Lyapustin and Kaufman, 2001; Richter,
1990). The adjacency effect has been described especially in
the context of high-resolution land-surface remote sensing,
but it has also been discussed in the context of atmospheric
measurements (e.g., Richter, 1990; Minomura et al., 2001;
de Graaf et al., 2016). Evidence for 3D radiation effects
due to the presence of clouds in the vicinity of an observed
ground pixel has recently been reported for CO2 observa-
tions from the OCO-2 satellite (Massie et al., 2017). Such ef-
fects are expected to become increasingly important with the
increasing resolution of satellite observations (Schwaerzel
et al., 2020). An additional complexity over cities to be ac-
counted for is the effects of buildings on the photon paths
due to multiple reflections and shielding of the main optical
path. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. 2.1.1
Air mass factors AMFs are obtained by dividing the mean photon path length
in each layer or box by its height. For a layer or box i with
given concentration ci and optical properties, the 1D-layer or
3D-box AMF is given by AMFi = SCDi
VCDi
=
R
pathcidl
R zi+1
zi
cidz =
R
pathdl
hi
= Li
hi
,
(1) (1) where Li is the mean optical path in the layer/box i of all
photons that reach the instrument, and hi is the height of the
layer/box i. To calculate a total AMF that can be used to convert mea-
sured SCDs to VCDs, an a priori VCD (VCDi) is needed for
every layer/box i. For the 1D case, the total AMF is com-
puted as In MYSTIC, each photon is traced stepwise along its op-
tical path from one interaction to the other. To interact with
the urban canopy, a ray-tracing code searches for hits with
one of the triangles during each step. We use the Star-3D li-
brary (https://gitlab.com/meso-star/star-3d, last access: 5 Oc-
tober 2021) (Villefranque et al., 2019), which is a convenient
wrapper for Intel® Embree (https://www.embree.org, last ac-
cess: 5 October 2021), to facilitate efficient ray/triangle in-
tersection tests using a bounding volume hierarchy (BVH). AMF =
Pnz
k=1AMFkVCDk
Pnz
k=1VCDk
=
Pnz
k=1SCDk
Pnz
k=1VCDk
,
(2) (2) where nz is the number of vertical layers. For the 3D case,
the total AMF is computed as AMF =
Pnx
i=1
Pny
j=1
Pnz
k=1AMFi,j,kVCDi,j,k
Pnz
k=1VCDk
=
Pnx
i=1
Pny
j=1
Pnz
k=1SCDi,j,k
Pnz
k=1VCDk
,
(3) In the case that a ray hits a surface, a Lambertian reflec-
tion happens, and a new direction is attributed to the pho-
ton, which will continue its path until its next interaction. Absorption on surfaces is accounted for by reducing a pho-
ton weight by 1−albedo. Currently, the urban canopy mod-
ule only supports Lambertian reflections but an extension,
e.g., to specular reflections, would be straightforward. For an
even more realistic description of the surface reflectance for
real applications, BRDF (bidirectional reflectance distribu-
tion function) would be a well suited method. (3) where nx and ny are the number of grid cells in x and y
directions. In the 1D case, the AMF depends on the shape of
the vertical profile of the trace gas (but not on its abundance)
(Palmer et al., 2001). M. Schwaerzel et al.: 3D radiative transfer over cities with buildings shapefile into triangles. A building consists of one or sev-
eral flat roofs and several vertical walls. To create tri-
angles for the roof, we connect the polygon centroid to
each polygon corner. Wall triangles are created by split-
ting each wall diagonally into two triangles. Each trian-
gle surface carries information about its albedo and skin
temperature. The skin temperatures are used for thermal
simulations, a feature that is not used in this publication. The triangular mesh is stored in a NetCDF file readable
by MYSTIC. An example file layout is shown in the Sup-
plement. The file contains the variable vertices (shape:
Nv × 3, type: double), which is a list of x, y and z co-
ordinates. A mesh of Nt triangles is built from these ver-
tices using the triangles variable (shape: Nt × 3, type:
int) by storing the indices of the three vertices that create
the triangles. The variable materials_of_triangles
(shape: Nt, type: int) is used to assign each triangle the
index of a material type. The material types are defined
using the variables material_type (shape: Nm, type:
string), material_albedo (shape: Nm, type: double) and
temperature_of_triangle (shape: Nm, type: double)
to assign a name, albedo and temperature to each material. boxes with different properties and saves the mean photon
path length within each layer or box. A plane parallel geom-
etry is used for 3D AMF calculations, while spherical geom-
etry is possible for 1D calculations. 2.1.1
Air mass factors In the 3D case, it additionally depends
on the horizontal distribution of the trace gas. 2.1
The MYSTIC radiative transfer solver MYSTIC is operated as a RT solver of the libRadtran pack-
age (Mayer and Kylling, 2005; Emde et al., 2016). The
MYSTIC RT solver is based on the Monte Carlo principle
to calculate different radiative quantities such as irradiance,
radiance, absorption, emission, actinic flux, photon’s path
length and air mass factors (Mayer, 2009; Emde and Mayer,
2007; Schwaerzel et al., 2020). A Monte Carlo-based radia-
tive transfer model (RTM) traces individual photons from the
source (e.g., sun) to the instrument (e.g., airborne spectrom-
eter) accounting for ground and atmospheric interactions. Photon paths are treated as a combination of random deci-
sions with a given probability distribution for each interac-
tion (e.g., probability distribution of the scattering direction). By tracing several thousand photons, the averaged photon
paths reaching the instrument become representative of the
actual photon paths in the atmosphere. To simulate RT quan-
tities such as 3D-box AMFs, photons are traced backwards
from the instrument to the sun, which is equivalent to the
forward mode but greatly enhances computational efficiency
(Marchuk et al., 1980; Emde and Mayer, 2007). MYSTIC
divides the atmosphere into 1D vertical layers or 3D grid The aim of this study is to quantify, for the first time, the
impact of these 3D radiative transfer (RT) effects in the pres-
ence of spatially variable surface properties and NO2 concen-
trations over cities on NO2 retrievals from high-resolution https://doi.org/10.5194/amt-14-6469-2021 Atmos. Meas. Tech., 14, 6469–6482, 2021 6471 https://doi.org/10.5194/amt-14-6469-2021 2.2.2
Synthetic NO2 field We created a simple but quite realistic 3D NO2 concentra-
tion field based on the traffic emission inventory of the city
of Zurich (see Table 1 in Berchet et al., 2017). For this pur-
pose, the road emissions available as line sources were ras-
terized at 5 m × 5 m spatial resolution and normalized by the
maximum value in the rasterized field. The normalized field
was then multiplied with a NO2 concentration of 110 µgm−3,
which is a typical high concentration measured next to busy
streets in Zurich (Bär, 2016). A background NO2 concentra-
tion of 15 µgm−3 was added using a typical low value ob-
served in Zurich (Bär, 2016) (see the Supplement for de-
tails). The maximum concentration is thus 125 µgm−3 or
2.7 µmolm−3. Finally, we smoothed the concentration field
with a Gaussian filter with a standard deviation of 5 m to
mimic the effect of turbulent dispersion. The synthetic field
of the NO2 near surface concentrations is shown in Fig. 2a
binned on a 50 m × 50 m grid, which is the typical resolution
of trace gas measurements from an airborne instrument like
APEX. To resolve the spatial variability of surfaces and eleva-
tions within each 50 m × 50 m ground pixel, we specified an
instrument opening zenith angle corresponding to the 50 m
pixel size in x direction and moved the aircraft in 10 discrete
steps of 5 m along the y direction. The 10 different 3D-box
AMF fields computed in this way were then averaged to a
single field per pixel. The 3D-box AMFs were then used to-
gether with the synthetic NO2 field to compute total SCDs
that would be observed from an airborne instrument (see nu-
merator in Eq. 3). Then, the total AMFs were calculated di-
viding the total SCDs by the total VCDs. In the same way we
also computed SCDs ignoring buildings and SCDs based on
1D-layer AMFs (Eq. 2). Differences between the three types
of SCDs can be attributed to 3D and building effects. Since in
real applications VCDs would be computed from SCDs, we
also use the SCD field calculated with 3D-box AMFs includ-
ing buildings as the ”true” SCD measured by the airborne
spectrometer and calculate the VCD field using total AMF
calculated from 1D-layer AMFs and 3D-box AMFs without
considering buildings to show the errors caused by these sim-
plifications. M. Schwaerzel et al.: 3D radiative transfer over cities with buildings M. Schwaerzel et al.: 3D radiative transfer over cities with buildings 2.2.1
Buildings the whole domain in across-flight direction (i.e., in east–west
direction). For simplicity, the sun was placed at an azimuth
angle (SAA) of either 90◦(i.e., west) or 0◦(i.e., south). The
solar zenith angle (SZA) was set to 60◦, which corresponds
to values in the morning or afternoon in summer over Zurich. Simulations with a SZA of 30◦, which correspond to typical
summer noon measurements over Zurich are shown in the
Supplement. We used 3D building data from a shapefile obtained from the
Swiss Federal Office of Topography (swisstopo). Each build-
ing is defined as a polygon with x and y coordinates for the
edges and the centroid (Swiss LV03 coordinates system) with
a single height. As described above, the data were converted
to a triangular mesh and saved to a netCDF file. For simplic-
ity, we either applied albedos of 0.1 for walls (identical to
the used ground albedo) and 0.2 for roofs or used the same
albedo of 0.1 for all surfaces (albedos chosen after Mussetti
et al., 2020). Figure 1b shows the buildings colored by their
heights in the selected model domain. The simulations were conducted for a standard atmo-
sphere and for a wavelength of 490 nm, which is the cen-
ter of the NO2 fitting window used for the APEX instru-
ment (Kuhlmann et al., 2016). NO2 absorption features at
shorter wavelengths typically used in satellite retrievals are
less suitable for APEX due to the high instrument noise at
those wavelengths. Note that 3D effects would be stronger
at shorter wavelengths due to enhanced Rayleigh scatter-
ing. Aerosols were only included in the simulations as a
case study to analyze the footprint of the spectrometer (see
Sect. 2.3). Table S1 in the Supplement provides an overview
of the MYSTIC input parameters used in the simulations. 2.3
Footprint of an airborne spectrometer To study the sensitivity of a single measurement to the sur-
rounding of the ground pixel, we simulated the horizontal
distribution of 3D-box AMFs close to the ground for a sin-
gle observation scenario. The simulations were conducted
without aerosols and for a typical urban aerosol scenario
with an aerosol optical depth (AOD) of 0.1. For the sce-
nario, the sensor was placed at x = 675 and y = 75 m, point-
ing at a ground pixel with the lower left corner located at
x = 650 and y = 50 m. The corresponding viewing zenith
angle (VZA) was 0.72◦in the center of the pixel, and the 2.2.2
Synthetic NO2 field The vertical distribution was modeled as a linear de-
crease from the surface value to the background value of
0.65 µmolm−3 at 100 m. Above 100 m, all profiles decrease
exponentially with altitude with an e-folding vertical length
scale of 720 m, which was obtained by fitting a function to
a measured vertical profile (see the Supplement). Figure 2b
shows a NO2 background profile (blue) and a NO2 profile
over the road (red). Figure 2c shows the total VCDs over the
selected region in Zurich calculated from the 3D NO2 con-
centration field. For more details on the generation of the 3D
NO2 field, please refer to the Supplement. 2.2
Study case in Zurich The urban canopy was implemented in libRadtran as a trian-
gular mesh, where each triangle can be assigned different op-
tical and physical properties. Information on vertex positions
and optical properties are read from an input file that has to
be generated from a 3D building data set prior to the simula-
tion. Using a ray-tracing algorithm newly integrated into the
model, MYSTIC detects if and where a photon hits a triangle. The interaction of the photon with the surface (absorption
or reflection) is then simulated using preexisting MYSTIC
functions. To study radiative transfer effects on airborne measure-
ments for a realistic scene, we selected a 1 km × 1 km re-
gion in Zurich, Switzerland (see Fig. 1a). The low-rise build-
ings (10–15 m) with simple geometries are rather typical for
Swiss cities. The scene includes roads of different widths and
orientations and two open areas without buildings. To create a triangular mesh for each building, a Python
script was written that converts buildings stored in an ESRI https://doi.org/10.5194/amt-14-6469-2021 Atmos. Meas. Tech., 14, 6469–6482, 2021 6472 2.2.3
MYSTIC simulations Our scenario corresponds to an airborne imaging spectrome-
ter flying across the model domain from south to north (y di-
rection) slightly to the east of the center (x = 600 m) at an
altitude of 6 km, which represents the flight altitude of an
airborne spectrometer (dashed black line in Fig. 2c). The
viewing zenith angle was varied in discrete steps to cover Atmos. Meas. Tech., 14, 6469–6482, 2021 https://doi.org/10.5194/amt-14-6469-2021 M. Schwaerzel et al.: 3D radiative transfer over cities with buildings 6473 g
Figure 1. (a) Map of Zurich with the location of the study area in red. Map information was obtained from OpenStreetMap (© OpenStreetMap
contributors 2021 distributed under the Open Data Commons Open Database License (ODbL) v1.0.). (b) Building heights and major roads in
the study area with the lower left pixel at x = 678918 and y = 247040 m in the Swiss LV03 coordinate system. The black square represents
the location of the subdomain and the cyan square the location of a single pixel observation both used in Sect. 3.3. Figure 1. (a) Map of Zurich with the location of the study area in red. Map information was obtained from OpenStreetMap (© OpenStreetMap
contributors 2021 distributed under the Open Data Commons Open Database License (ODbL) v1.0.). (b) Building heights and major roads in
the study area with the lower left pixel at x = 678918 and y = 247040 m in the Swiss LV03 coordinate system. The black square represents
the location of the subdomain and the cyan square the location of a single pixel observation both used in Sect. 3.3. Figure 2. (a) NO2 ground concentration map. (b) NO2 vertical profile at a background location (blue line) and over a road at x = 5, y = 55 m
(red line). (c) Field of vertically integrated NO2 column densities (VCD) with aircraft flight track overlaid as dashed black line. Figure 2. (a) NO2 ground concentration map. (b) NO2 vertical profile at a background location (blue line) and over a road at x = 5, y = 55 m
(red line). (c) Field of vertically integrated NO2 column densities (VCD) with aircraft flight track overlaid as dashed black line. 3
Results viewing azimuth angle (VAA) was 270◦(instrument pointing
eastwards) (for details, see also table S1 in the Supplement). ) (
pp
)
To obtain a map of the close-to-ground NO2 sensitivity of
a single ground pixel measured by an airborne imaging spec-
trometer during a flight overpass, we simulated AMFs with
a 5 m × 5 m horizontal resolution and a 5 m vertical resolu-
tion in the lowest 45 m above ground for 10 equally spaced
instrument opening zenith angles corresponding to the 50 m
pixel size in x direction and moved the aircraft in 10 discrete
steps of 5 m along the y direction. We averaged the obtained
100 AMFs fields, integrated them vertically for the first 45 m
and scaled the result by the sum of all pixels to finally ob-
tain a 2D map of the fraction of the sensitivity within one
5 m × 5 m grid cell. In the following, this will be called the
NO2 footprint of an airborne spectrometer. In this section we first show the instrument incoming radi-
ance calculated with MYSTIC over the selected study area. Second, we analyze the 3D-box AMFs for a single NO2 ob-
servation both in the vertical and in the horizontal and com-
pare results with and without buildings. Finally, we compare
total AMFs for the complete image obtained from an air-
borne instrument flying over the study domain as illustrated
in Fig. 2c between the solution obtained with the 3D and the
1D RTM to illustrate the importance of 3D radiative transfer
effects on the SCD measurements. Atmos. Meas. Tech., 14, 6469–6482, 2021 3.1
Incoming radiance The surface re-
flectance was set to 0.10, while the reflectance of roofs and
walls was set to 0.20 in this example. Figure 4. (a) Integrated 3D-box AMFs in y direction for a ground
pixel at x = 650 and y = 50 m (lower left corner) for an instrument
placed at x = 600, y = 75 and z = 6000 m. The sun is at SAA = 90◦
(west) with a SZA of 60◦. (b) Vertical profile of horizontally inte-
grated AMFs (1D-layer AMFs) and (c) horizontal profile of verti-
cally integrated AMFs (column AMFs). Since building surfaces have higher reflectance in this ex-
ample, the bright building roofs clearly stand out. Some
bright walls illuminated by the sun can be seen in the east
of the domain. Shadows are also clearly visible in the east of
the buildings with taller buildings producing longer shadows. This radiance field is closely related to the close-to-ground
NO2 sensitivity. NO2 over a bright surface can more easily
be detected than NO2 over a dark surface. In the case of a
pixel covered by both bright and dark surfaces, the retrieved
signal will be more strongly affected by NO2 above the bright
parts. lowing simulations all surfaces have a constant albedo of 0.1. Most photons follow the geometric path from the sun to a
reflection on the ground or the roof and to the instrument
as indicated by the high 3D-box AMFs along this path. Fig-
ure 4b shows the 1D-layer AMFs obtained by integrating the
3D-box AMFs in x and y directions. The decrease close to
the ground due to the buildings is clearly visible. Figure 4c
shows column AMFs along the x axis, i.e., 3D-box AMFs in-
tegrated in y and z directions. AMFs in the column directly
below the instrument are highest, because the collected pho-
tons cross at least one of the column boxes when they are
scattered into the direction of the instrument. The column
AMFs decrease in the x direction with distance to the instru-
ment due to atmospheric absorption and scattering. 3.1
Incoming radiance An airborne imaging spectrometer measures radiance from
back-scattered and reflected solar irradiance. Figure 3 shows https://doi.org/10.5194/amt-14-6469-2021 Atmos. Meas. Tech., 14, 6469–6482, 2021 M. Schwaerzel et al.: 3D radiative transfer over cities with buildings 6474 erzel et al.: 3D radiative transfer over cities with buildings
Figure 4. (a) Integrated 3D-box AMFs in y direction for a ground
pixel at x = 650 and y = 50 m (lower left corner) for an instrument
placed at x = 600, y = 75 and z = 6000 m. The sun is at SAA = 90◦
(west) with a SZA of 60◦. (b) Vertical profile of horizontally inte-
grated AMFs (1D-layer AMFs) and (c) horizontal profile of verti-
cally integrated AMFs (column AMFs). Figure 3. Radiance seen by a downward-viewing instrument placed
in the center of the 1 km × 1 km region at an altitude of 6 km. SZA
and SAA are 60 and 90◦, respectively. Figure 3. Radiance seen by a downward-viewing instrument placed
in the center of the 1 km × 1 km region at an altitude of 6 km. SZA
and SAA are 60 and 90◦, respectively. Figure 3. Radiance seen by a downward-viewing instrument placed
in the center of the 1 km × 1 km region at an altitude of 6 km. SZA
and SAA are 60 and 90◦, respectively. the instrument incoming radiance at 490 nm for the selected
region with the urban canopy. Note that for this example the
instrument was placed in the center of the domain at an alti-
tude of 6 km, observing the scene with a very wide opening
angle of about 10◦to cover the full 1 km × 1 km domain. The
sun is located in the west with a SZA of 60◦. The surface re-
flectance was set to 0.10, while the reflectance of roofs and
walls was set to 0.20 in this example. the instrument incoming radiance at 490 nm for the selected
region with the urban canopy. Note that for this example the
instrument was placed in the center of the domain at an alti-
tude of 6 km, observing the scene with a very wide opening
angle of about 10◦to cover the full 1 km × 1 km domain. The
sun is located in the west with a SZA of 60◦. 3.3.1
Effects of 3D radiative transfer and buildings on
slant column densities Figure 6 compares total AMFs computed from 1D-layer
AMFs (Eq. 2) with total AMFs computed from 3D-box
AMFs (Eq. 3) without and with the urban canopy. The corre-
sponding SCDs are presented in the lower row of the figure,
which are related to the total AMFs by a division with VCDs
shown in Fig. 2c (see Eqs. 1 and 3). In the following we refer
to the SCDs calculated from 1D-layer AMFs as SCD1D and
to the SCDs calculated from 3D-box AMFs without and with
the urban canopy as SCD3D and SCD3D-UC, respectively. Figure 5a shows the footprint for a flat surface without
buildings without including aerosols. An important part of
the sensitivity (51.4 %) is located outside the ground pixel. The instrument will thus not only “see” near-surface NO2
above the ground pixel but also NO2 outside. A major part
of the sensitivity is located in the direction of the sun along
the main optical path. The main optical path is dominated by
photons that are either reflected from the ground pixel sur-
face or scattered in the atmosphere above the ground pixel
one single time upward into the direction of the instrument. A much smaller fraction is located outside this main path and
is caused by photons experiencing at least one more scatter-
ing (or reflection) event before being scattered (or reflected)
into the main optical path. AMFs derived from 1D-layer AMFs (Fig. 6a) are almost
horizontally homogeneous. They vary only slightly with
the instrument-viewing zenith angle and the NO2 distribu-
tion. The contribution of the a priori NO2 profile is only
significant, when high values collocate with high 1D-layer
AMFs values, but as our a priori NO2 profiles only dif-
fer in the lower 100 m, the effect is smaller than the noise
of the Monte Carlo simulations (σAMF = 0.014 and σSCD =
1.46 µmolm−2). The corresponding SCDs (Fig. 6d), com-
puted as the sum of the product of the 1D-layer AMFs with
the 1D NO2 profile (see Eq. 2) in Fig. 2d show the same pat-
tern as the VCDs with sharply elevated values above roads
and a homogeneous background aside. The SCDs are higher
than the VCDs because the AMFs are larger than 1. Figure 5b presents the same situation but with buildings
added to the simulation. Buildings affect the sensitivity both
within and outside the ground pixel. 3.3.1
Effects of 3D radiative transfer and buildings on
slant column densities The footprint is reduced
in the shadows of buildings but may be enhanced over the
sunlit sections of streets due to multiple reflections, as seen
near the upper right and lower left corners of the ground
pixel. In this example, the part of the sensitivity located out-
side the ground pixel is 52.6 %, comparable to the simulation
without buildings. g
AMFs calculated with 3D-box AMFs but without build-
ings (Fig. 6b) are lower over the roads and slightly larger
just beside the roads, because the 3D optical path crosses
neighboring columns with decreased or increased concen-
trations, respectively. This results in a spatial smearing of
SCDs (Fig. 6e), mainly in the direction of the main optical
path from the sun in the west to the ground pixel and to the
instrument in the east of the center. Far from the roads, the
SCDs are homogeneous and more or less identical to the 1D
solution. The difference SCD3D minus SCD1D is presented
in Fig. 7. The smearing effect in the 3D solution is visible as
negative differences over the roads and positive differences
aside especially on the eastern side of the roads opposite
to the sun. As a result, the individual roads show up much
less prominently in the SCD3D except for roads parallel to
the optical path. The reason for this pattern is illustrated in
Fig. 8, where the main 3D optical path of the photons col-
lected when viewing directly at the road (red path 1 in Fig. 8)
misses some of the enhanced NO2 concentrations in the ele- Figure S7 in the Supplement shows the footprint for the
aerosol scenario. The aerosol scattering increases the sensi-
tivity contribution from outside the ground pixel to 55 % for
both the scenario without and with buildings. The contribu-
tion from outside the main optical path is increased to 25 %
and 32 % without and with buildings, respectively, compared
to 18 % and 24 %, when not including aerosols. The main effect of the 3D optical path of the photons is
thus to smear out the sensitivity of a measurement into the di-
rection of the main optical path, which is determined by the
viewing and illumination geometry. 3.2.2
Horizontal footprint In the previous section we have shown how 3D radiative
transfer effects and buildings smear out and modify the sen-
sitivity of a single observation. Here we demonstrate how a
complete image of NO2 slant columns is affected by these
effects. Note that for simplicity in the following simulations,
the ground and building reflectance was set to 0.1. To further illustrate the horizontal sensitivity of an airborne
spectrometer to layers close to the ground, Fig. 5 shows the
near-surface footprint defined as the vertically integrated 3D-
box AMFs from 0–45 m above ground (see Sect. 2.3). The
same scene was simulated as in Sect. 3.2.1 but with a higher
horizontal resolution of 5 m × 5 m to illustrate the spatial pat-
tern of the sensitivity in greater detail. The figure is normal-
ized to show the fraction of the sensitivity represented by
each 5 m × 5 m pixel. M. Schwaerzel et al.: 3D radiative transfer over cities with buildings
ground pixel is 121 µmolm−2, and the SCD computed as
3.3
Im ground pixel is 121 µmolm−2, and the SCD computed as
VCD times total AMF is 239 µmolm−2. 3.2.1
Vertical distribution along the main optical path 3D-box AMFs have a distinct 3D distribution for each
ground pixel. Figure 4a shows an example of the 3D-box
AMFs projected onto a 2D (x–z) plane by integrating in y di-
rection for an instrument pointing almost in nadir direction
at the ground pixel centered at x = 675 and y = 75 m (cyan
square in Fig. 1b). Since the pixel is partly covered by build-
ings, 3D-box AMFs close to the ground (0–10 m) are smaller
than those above roof level. Note that in this and all the fol- Since 3D-box AMFs inform about the sensitivity to NO2,
we can conclude that the measurement is mainly sensitive to
NO2 along the geometrical optical path. It is not very sen-
sitive to adjacent pixels and also not to NO2 at the surface
because of the blocking of the photon path by buildings. The total AMF for the observation presented in this ex-
ample computed with Eq. (3) is 1.98. The VCD above the https://doi.org/10.5194/amt-14-6469-2021 Atmos. Meas. Tech., 14, 6469–6482, 2021 6475 https://doi.org/10.5194/amt-14-6469-2021 Atmos. Meas. Tech., 14, 6469–6482, 2021 3.3.1
Effects of 3D radiative transfer and buildings on
slant column densities The presence of build-
ings further modifies the sensitivity by adding a complex pat-
tern of enhancements and reductions due to the shielding ef-
fects of buildings and multiple reflections in street canyons. https://doi.org/10.5194/amt-14-6469-2021 Atmos. Meas. Tech., 14, 6469–6482, 2021 M. Schwaerzel et al.: 3D radiative transfer over cities with buildings 3.3.2
3D effects at higher spatial resolution To investigate the reduced SCDs over buildings observed in
the former paragraphs, we analyze the spatial distribution of
SCDs within the 50 m × 50 m pixel in more detail. For this
purpose, we ran additional simulations at higher spatial res-
olution (5 m × 5 m) for a 100 m × 100 m subdomain located
at x = 600 and y = 0 m (lower left corner of the subdomain)
of the original domain. SCDs were calculated from 3D-box
AMFs with and without buildings (Fig. 10a and b). Figure 8. Sketch of two main optical paths for an airborne spec-
trometer pointing at (1) a road in grey and (2) at a pixel aside the
road. At this resolution, we can better resolve the spatial distri-
bution of NO2, and SCDs remain high above roads. Since we
can also resolve individual buildings and their shadows, the
simulations makes it possible to investigate how buildings
reduce SCDs. over regions with many buildings. On average, SCD3D-UC
values are 12 % lower than SCDs without buildings. The
standard deviation of the difference between SCD3D-UC and
SCD3D is 12.3 µmolm−2 for the whole domain. Not includ-
ing the urban canopy would therefore underestimate VCDs
by 12 % and would add a source of noise in the image of
about 5 % for this particular scenario with rather low build-
ings. In areas with background NO2 and no buildings (e.g.,
lower left region), SCD1D, SCD3D and SCD3D-UC closely
agree (Fig. 7c). At 5 m resolution, we distinguish four types of ground pix-
els. 1. The ground pixel is on the surface and the geometric
path to the sun and the instrument is not obscured by
buildings. In this case, the 3D-box AMFs are only af-
fected by buildings through multiple scattering either
increasing the AMF when buildings reflect photons to-
wards the ground pixel or reducing the AMF when
buildings block photons from reaching the ground pixel. In our example, the effect results in a small but hardly
visible reduction of AMFs (see Fig. 10c). The results presented above were obtained for the spe-
cial situation where viewing and illumination directions were
along the same east–west direction. In this case, the smearing
effects are most prominent along this axis but relatively small
in the perpendicular direction. M. Schwaerzel et al.: 3D radiative transfer over cities with buildings 6477 Figure 7. (a) Difference plot between SCDs calculates with 3D-box AMFs and the SCDs calculated with 1D-layer AMFs. (b) Difference
plot between SCDs calculated with 3D-box AMFs including the urban canopy and SCDs calculated with 1D-layer AMFs. (c) Difference plot
between SCDs calculated with 3D-box AMFs with and without including the urban canopy. Figure 7. (a) Difference plot between SCDs calculates with 3D-box AMFs and the SCDs calculated with 1D-layer AMFs. (b) Difference
plot between SCDs calculated with 3D-box AMFs including the urban canopy and SCDs calculated with 1D-layer AMFs. (c) Difference plot
between SCDs calculated with 3D-box AMFs with and without including the urban canopy. Figure 8. Sketch of two main optical paths for an airborne spec-
trometer pointing at (1) a road in grey and (2) at a pixel aside the
road. to the north instead of the east of the buildings. Furthermore,
the SCDs tend to be lower, because the main optical path and
the corresponding smearing are both in N–S (sun to ground
pixel) and E–W (ground pixel to the instrument) orientations. The mean difference between SCD3D-UC and SCD3D is 12 %,
and the standard deviation is 13 %. M. Schwaerzel et al.: 3D radiative transfer over cities with buildings M. Schwaerzel et al.: 3D radiative transfer over cities with buildings 6476 Figure 5. (a) Footprint without buildings; 51.4 % of the signal is located outside the ground pixel (i.e., outside the red frame). (b) Footprint
with buildings; 52.6 % of the instrument sensitivity is located outside the ground pixel. Building contours are in white. Figure 5. (a) Footprint without buildings; 51.4 % of the signal is located outside the ground pixel (i.e., outside the red frame). (b) Footprint
with buildings; 52.6 % of the instrument sensitivity is located outside the ground pixel. Building contours are in white. Figure 6. AMFs for a simulation with SAA of 90◦with 1D-layer AMFs simulation (a), 3D-box AMFs without (b) and with (c) buildings. The respective SCDs are shown in the lower row (d, e, f). Roads are drawn in white and building contours in black for the simulation with
buildings. Figure 6. AMFs for a simulation with SAA of 90◦with 1D-layer AMFs simulation (a), 3D-box AMFs without (b) and with (c) buildings. The respective SCDs are shown in the lower row (d, e, f). Roads are drawn in white and building contours in black for the simulation with
buildings. vated levels above the road. In contrast, these enhanced NO2
values are “seen” by photons collected when viewing to the
east of the road (green path 2 in Fig. 8). In the 1D solution,
the SCDs are only affected by NO2 in the vertical column
directly above the observed ground pixel. measured NO2 is almost conserved. A small difference is to
be expected because of statistical noise from the Monte Carlo
method (about 1.3 µmolm−2 for 50 000 photons). AMFs calculated with the 3D-box AMFs module includ-
ing the urban canopy (Fig. 6c) also show lower values over
roads compared to AMFs calculated with 1D-layer AMFs. In
addition, they also show lower values over areas with build-
ings. As a consequence, the SCDs shown in Fig. 6f are lower The mean difference between SCD1D and SCD3D is
around 1 µmolm−2 (relative difference of 0.02 %), which
suggests that the signal is smeared, but the total amount of https://doi.org/10.5194/amt-14-6469-2021 Atmos. Meas. Tech., 14, 6469–6482, 2021 M. Schwaerzel et al.: 3D radiative transfer over cities with buildings 3.3.2
3D effects at higher spatial resolution Here, we also analyze the sit-
uation where the sun is in the south and thus viewing and
illumination directions are perpendicular to each other. As
shown in Fig. 9, the results are generally similar, but building
shadows and correspondingly reduced SCDs are now found 2. The reflecting point of the geometric path is located on
top of a building. The case is similar to the first case,
but 3D-box AMFs are smaller, because photons cannot 2. The reflecting point of the geometric path is located on
top of a building. The case is similar to the first case,
but 3D-box AMFs are smaller, because photons cannot https://doi.org/10.5194/amt-14-6469-2021 Atmos. Meas. Tech., 14, 6469–6482, 2021 M. Schwaerzel et al.: 3D radiative transfer over cities with buildings 6478 Figure 9. SCDs for simulations with a SAA of 0◦with 1D-layer AMFs (a) and 3D-box AMFs simulation without (b) and with (c) buildings. The roads are drawn in white and the building contours in black for the simulation with buildings. Figure 9. SCDs for simulations with a SAA of 0◦with 1D-layer AMFs (a) and 3D-box AMFs simulation without (b) and with (c) buildings. The roads are drawn in white and the building contours in black for the simulation with buildings. reach the ground. Since we assume that a priori VCDs
are fixed regardless of the presence of a building, SCDs
are reduced above buildings. However, the effect is very
small, because only about 3 % of the total VCD is below
a 10 m building. of buildings are not corrected, resulting in additional noise
introduced by the shielding effect of the buildings and sig-
nificantly lower VCDs with a field average of 90.1 µmolm−2
compared to the 108.4 µmolm−2 of the true VCDs. When
VCDs are computed using 3D-box AMFs without buildings,
the spatial smearing is corrected, but the noise component
and lower VCDs (mean: 91.4 µmolm−2) would remain in the
retrieved VCDs. 3. In the third case, the ground pixel is located on the sunlit
side of a building, but the direct path to the instrument
is blocked by the buildings. Since the VZAs of the sim-
ulated instrument are very small, we do not find these
cases in our example with rather small buildings. 3.4
Application to real observations 4. In the final case, the ground pixel is located in the
shadow of the buildings blocking the direct path towards
the sun. In this case, photons can only reach the in-
strument after multiple scattering or simple atmospheric
scattering directly into the direction of the instrument,
which drastically reduces the 3D-box AMFs near the
surface. As a result, SCDs are significantly lower in the
shadows of buildings (Fig. 10b). For example, SCDs are
about 35 % lower when increased ground NO2 concen-
trations (e.g., road) are located in the shadow of a build-
ing. The codes developed for this study can also be applied to real
observations, e.g., to the campaigns conducted with APEX
imaging spectrometer. A major challenge is to obtain the
required input data. The 3D building data are available for
many cities, but albedos for ground, roof and walls are gen-
erally not available. In addition, to compute the total AMFs,
realistic 3D NO2 fields from a building-resolving dispersion
model are required, which requires high-resolution emission
inventories and additional model development, because most
building-resolving models are not optimized for providing
realistic vertical distributions of trace gases or cannot be ap-
plied to a full city at high resolution (Berchet et al., 2017). We can therefore conclude that the reduction of SCDs over
buildings (Fig. 7c) is mainly caused by the building shielding
effect (case 4), while building height has a minor effect on the
SCDs (case 2) in our study case with rather small buildings. To minimize 3D effects when using 1D-layer AMFs, it
would be recommendable to obtain the airborne spectrom-
eter measurement around local noon when the SZA is low-
est and avoid large viewing zenith angles. However, around
noon, turbulent atmospheric mixing will be strong, and the
NO2 distributions would be smoothed as well. M. Schwaerzel et al.: 3D radiative transfer over cities with buildings 6479 Figure 10. SCDs over a small subdomain computed from high-resolution 3D-box AMFs simulated without (a) and with (b) buildings as
well as the difference between both (c). In the simulation, the sun was located in the west at a SZA of 60◦. The albedo of roofs, walls and
streets was set to 0.1. Roads are included as black dots and building contours as black lines. Figure 10. SCDs over a small subdomain computed from high-resolution 3D-box AMFs simulated without (a) and with (b) buildings as
well as the difference between both (c). In the simulation, the sun was located in the west at a SZA of 60◦. The albedo of roofs, walls and
streets was set to 0.1. Roads are included as black dots and building contours as black lines. Figure 11. (a) True VCD field and VCDs computed from SCDs using 3D-box AMFs and buildings assuming (b) 1D-layer AMFs and (c) 3D-
box AMFs without buildings. The scenario uses a SAA of 90◦. Figure 11. (a) True VCD field and VCDs computed from SCDs using 3D-box AMFs and buildings assuming (b) 1D-layer AMFs and (c) 3D-
box AMFs without buildings. The scenario uses a SAA of 90◦. complex 3D radiative transfer effects in cities. To study the
latter point, we implemented an urban canopy module into
the 3D MYSTIC solver of the libRadtran radiative transfer
model. We set up a case study for a 1 km × 1 km domain in
Zurich, for which 3D-box AMFs and NO2 slant column den-
sities were computed for a realistic field of NO2 concentra-
tions. lating AMFs for an APEX campaign would not require sim-
ulation for a 1 km × 1 km domain. Nonetheless, computing
3D-box AMFs is significantly more expensive than comput-
ing 1D-layer AMFs and reducing computation time, e.g., by
finding suitable parameterizations using machine learning,
which would make it possible to calculate the 3D-box AMFs
on smaller hardware, for larger campaigns, or to run simula-
tions with more details and at higher spatial resolution. Our case study shows that the footprint of a single obser-
vation is only partly located over the observed ground pixel
and that there is a “tail” in the direction of the main optical
path. M. Schwaerzel et al.: 3D radiative transfer over cities with buildings In the presented simulations with a SZA of 60◦and a
50 m × 50 m resolution, about 50 % of the sensitivity is lo-
cated outside the ground pixel for a nearly nadir-viewing in-
strument. Only a small but non-negligible number of photons
are from outside the main optical path, with 19 % for a simu-
lation without aerosols and without buildings and 24 % for a
simulation without aerosols and with buildings. The effect
becomes more important when aerosols are included with
25 % and 32 % of the sensitivity located outside the main op-
tical path for scenarios without and with buildings, respec- 3.3.3
The retrieval of vertical column densities In imaging remote sensing, NO2 VCDs are retrieved from
SCDs using AMFs. Here SCD3D-UC values are considered
the “true” SCDs measured by an airborne imaging spectrom-
eter, and VCDs were calculated using either 1D-layer AMFs
or 3D-box AMFs without buildings (Fig. 11). The computation of 3D-box AMFs with buildings is com-
putationally quite expensive but still manageable for cur-
rent airborne campaigns. For example, the computation of
the 3D-box AMF field for a single APEX pixel (e.g., in
Fig. 6f) takes about 280 s on a single core of our Linux ma-
chine (Intel® Xeon® W-2175 CPU at 2.50 GHz). Process-
ing a full campaign consisting of about 100 000 pixels takes
about 23 d using all 14 cores on the system. However, simu- The VCDs computed with 1D-layer AMFs fail to correct
for the spatial smoothing induced by the complex 3D opti-
cal path of the photons (Fig. 11b). In additions, the effects https://doi.org/10.5194/amt-14-6469-2021 Atmos. Meas. Tech., 14, 6469–6482, 2021 M. Schwaerzel et al.: 3D radiative transfer over cities with buildings Atmos. Meas. Tech., 14, 6469–6482, 2021 4
Conclusions Airborne imaging spectrometers are increasingly used for
high-resolution mapping of NO2 concentrations in cities. The NO2 maps obtained in this way were usually found to
be rather smooth and seemingly inconsistent with the much
more rapidly varying near-surface NO2 concentration fields
seen, for example, in city-scale dispersion model simula-
tions. The observed difference may partly be explained by
atmospheric mixing more strongly affecting total columns
than near-surface concentrations, but could also be caused by https://doi.org/10.5194/amt-14-6469-2021 M. Schwaerzel et al.: 3D radiative transfer over cities with buildings tively. The footprint fine structure is further modified with
the presence of buildings, but the general shape is conserved. Code availability. The libRadtran package including the 1D ver-
sion of MYSTIC is freely available at http://www.libradtran.org,
last access: 5 October 2021; the 3D MYSTIC code is available
upon request to Claudia Emde (mailto:claudia.emde@lmu.de). The
Python script used to create a triangular mesh from ESRI shape-
files is available in the Supplement, and the other used codes are
available upon request to the corresponding author. The 3D radiative transfer simulations show that 3D effects
introduce significant spatial smearing of high NO2 concen-
trations, e.g., over roads, which 1D-layer AMFs do not in-
clude. This results in increasing SCDs when roads are paral-
lel to the main optical path and decreasing SCDs otherwise. When buildings are included, NO2 SCDs are generally lower
due to the shielding effect of buildings. The buildings also
introduce a variability in the SCD field with a standard devi-
ation of 12.9 µmolm−2 (5.5 %) that would show up as addi-
tional noise component of airborne imagers. The magnitude
is, however, slightly smaller than the current NO2 SCD un-
certainty (about 20 µmolm−2 for the APEX instrument) but
could be noticeable for instruments dedicated to NO2 map-
ping that have lower SCD uncertainties. We also applied 1D-
layer and 3D-box AMFs without building to SCDs computed
with 3D-box AMFs with buildings showing that 3D radiative
transfer simulations are required to correct the smearing ef-
fect and that buildings are required to avoid an underestima-
tion of the VCDs. Data availability. The data used for this study are available on
https://doi.org/10.5281/zenodo.5519616 (Schwaerzel et al., 2021). Supplement. The supplement related to this article is available on-
line at: https://doi.org/10.5194/amt-14-6469-2021-supplement. Author contributions. MS designed and implemented the urban
canopy module, designed and simulated the 3D scenarios, and wrote
the article with input from all co-authors. FJ designed and imple-
mented the urban canopy module together with MS and CE. DB,
BB and AB provided critical feedback to the study and reviewed
the article; GK supervised the study, designed (together with MS)
the case studies and reviewed the article. Generalizing our results to other cities is challenging, be-
cause many relevant parameters such as building shapes, sur-
face reflectances and a priori NO2 distributions vary strongly
between different cities. M. Schwaerzel et al.: 3D radiative transfer over cities with buildings In our case study, we used a surface
reflectance of 0.1, which is a realistic value for Zurich but
not necessarily for other cities. In general, a higher surface
reflectance of the observed ground pixel implies less atmo-
spheric scattering and a higher sensitivity of the instrument
to the main optical path, and higher albedo of neighboring
pixels increases the sensitivity to this neighboring pixel. In
this study, the simulations were conducted at 490 nm, which
corresponds to the center of the fitting window used for NO2
retrieval from the APEX airborne spectrometer. At shorter
wavelength, used by other instruments, scattering increases,
which decreases the instrument sensitivity to the main optical
path. The footprint simulated with a wavelength of 420 nm
(without buildings) shows the increase in scattering and the
sensitivity to neighboring pixels, as 56 % of the sensitivity is
located outside of the ground pixel. Competing interests. The contact author has declared that neither
they nor their co-authors have any competing interests. Disclaimer. Publisher’s note: Copernicus Publications remains
neutral with regard to jurisdictional claims in published maps and
institutional affiliations. Acknowledgements. We obtained building data from the Swiss Fed-
eral Office of Topography (swisstopo) and the traffic emission in-
ventories from the city of Zurich. Financial support. This
research
has
been
supported
by
the
Schweizerischer
Nationalfonds
zur
Förderung
der
Wis-
senschaftlichen Forschung (SNSF, grant no. 172533). In conclusion, our case study demonstrates that 3D effects
explain the smooth NO2 field observed by airborne imag-
ing spectrometers, at least partly. Atmospheric mixing can
still result in additional smoothing that has not been studied
here. Furthermore, buildings reduce SCDs due to the light
shielding effect of buildings and add an additional noise com-
ponent that is difficult to generalize due to the complexity
and the heterogeneity of the buildings. The smearing in the
sun direction can result in features in the maps that are dif-
ficult to interpret when the sun position is not known. The
3D radiative transfer effects therefore need to be considered
when studying NO2 maps obtained from airborne imagers
and might become relevant with future NO2 satellite instru-
ments that measure NO2 at spatial resolutions down to 2 km. Review statement. This paper was edited by Lok Lamsal and re-
viewed by Frederik Tack and two anonymous referees. https://doi.org/10.5194/amt-14-6469-2021 Atmos. Meas. Tech., 14, 6469–6482, 2021 6480 M. Schwaerzel et al.: 3D radiative transfer over cities with buildings 6481 and examples of use, Atmos. Chem. Phys., 5, 1855–1877,
https://doi.org/10.5194/acp-5-1855-2005, 2005. umwelt_energie/luftqualitaet/publikationen/luftbilanz-2016. html (last access: 5 October 2021), 2016. umwelt_energie/luftqualitaet/publikationen/luftbilanz-2016. html (last access: 5 October 2021), 2016. Cracknell, A. P. and Varotsos, C.: Remote sensing and at-
mospheric ozone: Human activities versus natural variability,
Springer Science & Business Media, Berlin/Heidelberg, Ger-
many, https://doi.org/10.1007/978-3-642-10334-6, 2012. Merlaud, A., Constantin, D., Mingireanu, F., Mocanu, I., Fayt,
C., Maes, J., Murariu, G., Voiculescu, M., Georgescu, L., and
Van Roozendael, M.: Small whiskbroom imager for atmospheric
composition monitoring (SWING) from an unmanned aerial ve-
hicle (UAV), in: Proceedings of the 21st ESA Symposium on
European Rocket & Balloon Programmes and related Research,
Thun, Switzerland, 9–13, 2013. y
p
g
de Goeij, B. T. G., Otter, G. C. J., van Wakeren, J. M. O., Veefkind,
J. P., Vlemmix, T., Ge, X., Levelt, P. F., Dirks, B. P. F., Toet,
P. M., van der Wal, L. F., and Jansen, R.: First aircraft test results
of a compact, low cost hyperspectral imager for earth observa-
tion from space, in: International Conference on Space Optics –
ICSO 2016, edited by: Cugny, B., Karafolas, N., and Sodnik, Z.,
Vol. 10562, International Society for Optics and Photonics, SPIE,
Biarritz, France, 465–475, https://doi.org/10.1117/12.2296068,
2017. Minomura, M., KUzE, H., and Takeuchi, N.: Adjacency effect in the
atmospheric correction of satellite remote sensing data: Evalua-
tion of the influence of aerosol extinction profiles, Optical Rev.,
8, 133–141, https://doi.org/10.1007/s10043-001-0133-2, 2001. Mussetti, G., Brunner, D., Henne, S., Allegrini, J., Krayenhoff,
E. S., Schubert, S., Feigenwinter, C., Vogt, R., Wicki, A., and
Carmeliet, J.: COSMO-BEP-Tree v1. 0: a coupled urban cli-
mate model with explicit representation of street trees, Geosci. Model Dev., 13, 1685–1710, https://doi.org/10.5194/gmd-13-
1685-2020, 2020. de Graaf, M., Sihler, H., Tilstra, L. G., and Stammes, P.: How
big is an OMI pixel?, Atmos. Meas. Tech., 9, 3607–3618,
https://doi.org/10.5194/amt-9-3607-2016, 2016. Emde, C. and Mayer, B.: Simulation of solar radiation during a total
eclipse: a challenge for radiative transfer, Atmos. Chem. Phys.,
7, 2259–2270, https://doi.org/10.5194/acp-7-2259-2007, 2007. Nowlan, C. R., Liu, X., Leitch, J. W., Chance, K., González Abad,
G., Liu, C., Zoogman, P., Cole, J., Delker, T., Good, W., Mur-
cray, F., Ruppert, L., Soo, D., Follette-Cook, M. B., Janz,
S. J., Kowalewski, M. G., Loughner, C. P., Pickering, K. E.,
Herman, J. R., Beaver, M. R., Long, R. W., Szykman, J. J.,
Judd, L. M., Kelley, P., Luke, W. T., Ren, X., and Al-Saadi,
J. M. Schwaerzel et al.: 3D radiative transfer over cities with buildings A.: Nitrogen dioxide observations from the Geostationary
Trace gas and Aerosol Sensor Optimization (GeoTASO) air-
borne instrument: Retrieval algorithm and measurements dur-
ing DISCOVER-AQ Texas 2013, Atmos. Meas. Tech., 9, 2647–
2668, https://doi.org/10.5194/amt-9-2647-2016, 2016. Emde, C., Buras-Schnell, R., Kylling, A., Mayer, B., Gasteiger, J.,
Hamann, U., Kylling, J., Richter, B., Pause, C., Dowling, T.,
and Bugliaro, L.: The libRadtran software package for radia-
tive transfer calculations (version 2.0.1), Geosci. Model Dev., 9,
1647–1672, https://doi.org/10.5194/gmd-9-1647-2016, 2016. Gastellu-Etchegorry, J.-P., Yin, T., Lauret, N., Cajgfinger, T., Gre-
goire, T., Grau, E., Feret, J.-B., Lopes, M., Guilleux, J., Dedieu,
G., Malenovský, Z., Cook, D. B., Morton, D., Rubio, J., Dur-
rieu, S., Cazanave, G., Martin, E., and Ristorcelli, T.: Discrete
Anisotropic Radiative Transfer (DART 5) for Modeling Air-
borne and Satellite Spectroradiometer and LIDAR Acquisitions
of Natural and Urban Landscapes, Remote Sens., 7, 1667–1701,
https://doi.org/10.3390/rs70201667, 2015. Palmer, P. I., Jacob, D. J., Chance, K., Martin, A. V., Robert J., D.,
Kurosu, T. P., Bey, I., Yantosca, R., Fiore, A., and Li, Q.: Air
mass factor formulation for spectroscopic measurements from
satellites: Application to formaldehyde retrievals from the global
ozone monitoring experiment, J. Geophys. Res., 106, 14–539,
https://doi.org/10.1029/2000JD900772, 2001. Kuhlmann, G., Hueni, A., Damm, A., and Brunner, D.: An Algo-
rithm for In-Flight Spectral Calibration of Imaging Spectrome-
ters, Remote Sens., 8, 1017, https://doi.org/10.3390/rs8121017,
2016. Popp, C., Brunner, D., Damm, A., Van Roozendael, M., Fayt, C.,
and Buchmann, B.: High-resolution NO2 remote sensing from
the Airborne Prism EXperiment (APEX) imaging spectrometer,
Atmos. Meas. Tech., 5, 2211–2225, https://doi.org/10.5194/amt-
5-2211-2012, 2012. Kuhlmann, G., Berchet, A., and Brunner, D.: High-resolution
remote
sensing
and
modelling
of
NO2
air
pollution
over the city of Zurich, in: 10th EARSeL SIG Imag-
ing
Spectroscopy
Workshop
2017,
Zurich,
Switzerland,
https://doi.org/10.5281/zenodo.5220909, 2017. Richter,
R.:
A
fast
atmospheric
correction
algorithm
ap-
plied to Landsat TM images, Remote Sens., 11, 159–166,
https://doi.org/10.1080/01431169008955008, 1990. Lyapustin, A. and Kaufman, Y.: Role of adjacency effect in the re-
mote sensing of aerosol, J. Geophys. Res.-Atmos., 106, 11909–
11916, https://doi.org/10.1029/2000JD900647, 2001. Schaepman, M. E., Jehle, M., Hueni, A., D’Odorico, P., Damm,
A., Weyermann, J., Schneider, F. D., Laurent, V., Popp, C.,
Seidel, F. C., Lenhard, K., Gege, P., Küchler, C., Brazile, J.,
Kohler, P., De Vos, L., Meuleman, K., Meynart, R., Schläpfer, D.,
Kneubühler, M., and Itten, K. I.: Advanced radiometry measure-
ments and Earth science applications with the Airborne Prism
Experiment (APEX), Remote Sens. Environ., 158, 207–219,
https://doi.org/10.1016/j.rse.2014.11.014, 2015. References Berchet, A., Zink, K., Muller, C., Oettl, D., Brunner, J.,
Emmenegger, L., and Brunner, D.: A cost-effective method
for simulating city-wide air flow and pollutant dispersion
at building resolving scale, Atmos. Environ., 158, 181–196,
https://doi.org/10.1016/j.atmosenv.2017.03.030, 2017. Bär, P.: Luftbilanz 2016: Resultate der flächendeckenden Messkam-
pagne, available at: https://www.stadt-zuerich.ch/gud/de/index/ https://doi.org/10.5194/amt-14-6469-2021 Atmos. Meas. Tech., 14, 6469–6482, 2021 https://doi.org/10.5194/amt-14-6469-2021 M. Schwaerzel et al.: 3D radiative transfer over cities with buildings Marchuk, G., Mikhailov, G., Nazaraliev, M., Dacbinjan, R., Kargin,
B., and Elepov, B.: Monte Carlo methods in atmospheric optics,
Springer, Berlin/Heidelberg, Germany, 1980. Massie, S. T., Sebastian Schmidt, K., Eldering, A., and Crisp,
D.: Observational evidence of 3-D cloud effects in OCO-
2 CO2 retrievals, J. Geophys. Res.-Atmos., 122, 7064–7085,
https://doi.org/10.1002/2016JD026111, 2017. Schönhardt, A., Altube, P., Gerilowski, K., Krautwurst, S., Hart-
mann, J., Meier, A. C., Richter, A., and Burrows, J. P.: A wide
field-of-view imaging DOAS instrument for two-dimensional
trace gas mapping from aircraft, Atmos. Meas. Tech., 8, 5113–
5131, https://doi.org/10.5194/amt-8-5113-2015, 2015. Mayer, B.: Radiative transfer in the cloudy atmosphere, in: EPJ Web
of Conferences, Vol. 1, EDP Sciences, Les Ulis, France, 75–99,
https://doi.org/10.1140/epjconf/e2009-00912-1, 2009. Mayer, B. and Kylling, A.: Technical note: The libRadtran soft-
ware package for radiative transfer calculations – description Schwaerzel, M., Emde, C., Brunner, D., Morales, R., Wagner, T.,
Berne, A., Buchmann, B., and Kuhlmann, G.: Three-dimensional https://doi.org/10.5194/amt-14-6469-2021 Atmos. Meas. Tech., 14, 6469–6482, 2021 Atmos. Meas. Tech., 14, 6469–6482, 2021 https://doi.org/10.5194/amt-14-6469-2021 M. Schwaerzel et al.: 3D radiative transfer over cities with buildings 6482 radiative transfer effects on airborne and ground-based trace
gas remote sensing, Atmos. Meas. Tech., 13, 4277–4293,
https://doi.org/10.5194/amt-13-4277-2020, 2020. Villefranque, N., Fournier, R., Couvreux, F., Blanco, S., Cornet, C.,
Eymet, V., Forest, V., and Tregan, J.-M.: A Path-Tracing Monte
Carlo Library for 3-D Radiative Transfer in Highly Resolved
Cloudy Atmospheres, J. Adv. Model. Earth Syst., 11, 2449–
2473, https://doi.org/10.1029/2018MS001602, 2019. Schwaerzel, M., Brunner, D., Jakub, F., Emde, C., Buch-
mann, B., Berne, A., and Kuhlmann, G.: Dataset – Im-
pact of 3D radiative transfer on airborne NO2 imaging re-
mote sensing over cities with buildings, Zenodo [Data set],
https://doi.org/10.5281/zenodo.5519616, 2021. Vlemmix, T., Ge, X. (., de Goeij, B. T. G., van der Wal, L. F., Otter,
G. C. J., Stammes, P., Wang, P., Merlaud, A., Schüttemeyer, D.,
Meier, A. C., Veefkind, J. P., and Levelt, P. F.: Retrieval of tropo-
spheric NO2 columns over Berlin from high-resolution airborne
observations with the spectrolite breadboard instrument, Atmos. Meas. Tech. Discuss. [preprint], https://doi.org/10.5194/amt-
2017-257, in review, 2017. Tack, F., Merlaud, A., Iordache, M.-D., Danckaert, T., Yu, H., Fayt,
C., Meuleman, K., Deutsch, F., Fierens, F., and Van Roozendael,
M.: High-resolution mapping of the NO2 spatial distribution over
Belgian urban areas based on airborne APEX remote sensing, At-
mos. Meas. Tech., 10, 1665–1688, https://doi.org/10.5194/amt-
10-1665-2017, 2017. Tack, F., Merlaud, A., Meier, A. C., Vlemmix, T., Ruhtz, T., Ior-
dache, M.-D., Ge, X., van der Wal, L., Schuettemeyer, D., Arde-
lean, M., Calcan, A., Constantin, D., Schönhardt, A., Meuleman,
K., Richter, A., and Van Roozendael, M.: Intercomparison of four
airborne imaging DOAS systems for tropospheric NO2 mapping
– the AROMAPEX campaign, Atmos. Meas. Tech., 12, 211–236,
https://doi.org/10.5194/amt-12-211-2019, 2019. Atmos. Meas. Tech., 14, 6469–6482, 2021 https://doi.org/10.5194/amt-14-6469-2021
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Intensity in Spirituality of High-Ability Students
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International journal of academic research in progressive education and development
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International Journal of Academic Research in Progressive Education and
Development
Vol. 8, No. 4, 2019, E-ISSN: 2226-6348 © 2019 HRMARS
Intensity in Spirituality of High-Ability Students
Rosadah Abd Majid & Aliza Alias
To Link this Article: http://dx.doi.org/10.6007/IJARPED/v8-i4/6778 DOI:10.6007/IJARPED/v8-i4/6778
Received: 10 November 2019, Revised: 29 November 2019, Accepted: 16 December 2019
Published Online: 30 December 2019
In-Text Citation: (Majid & Alias, 2019)
To Cite this Article: Majid, R. A., & Alias, A. (2019). Intensity in Spirituality of High-Ability Students. International
Journal of Academic Research in Progressive Education and Development, 8(4), 877–885.
Copyright: © 2019 The Author(s) International Journal of Academic Research in Progressive Education and
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Vol. 8, No. 4, 2019, E-ISSN: 2226-6348 © 2019 HRMARS
Intensity in Spirituality of High-Ability Students
Rosadah Abd Majid & Aliza Alias
To Link this Article: http://dx.doi.org/10.6007/IJARPED/v8-i4/6778 DOI:10.6007/IJARPED/v8-i4/6778
Received: 10 November 2019, Revised: 29 November 2019, Accepted: 16 December 2019
Published Online: 30 December 2019
In-Text Citation: (Majid & Alias, 2019)
To Cite this Article: Majid, R. A., & Alias, A. (2019). Intensity in Spirituality of High-Ability Students. International
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http://hrmars.com/index.php/pages/detail/publication-ethics 877 877 Intensity in Spirituality of High-Ability Students
Rosadah Abd Majid & Aliza Alias
Universiti Kebangsaan Malaysia, Malaysia
Email: rosadah@ukm.edu.my Rosadah Abd Majid & Aliza Alias
Universiti Kebangsaan Malaysia, Malaysia
Email: rosadah@ukm.edu.my Introduction Spirituality is regarded as the essence on an individual (Phenwan, Peerawong, Tulathamkij, 2019);
an aspect that requires a deep understanding of faith and belief for the person’s wellbeing. Being
aware of the morality and faithfulness to one’s belief would develop one’s personality to be
closely influenced and guided by spirituality. Yearning for knowledge and wanting to embrace
faith to its core has the propensity to lead one to continuous questions on the matter. Palmer
(1999) defined spirituality as a quest for connectedness with self, with others, with history and
nature, and with the mystery of being alive. In searching for divine answers on spirituality, one
might get derailed from the meaningful quest of spirituality without constant guidance from a
person who is not only knowledgeable in the belief itself but also wisely uses different
approaches in tapping the interest and needs of the believer’s quest. Abstract The spirituality of an individual is primarily determined by the awareness, faith and morality
pertaining to one’s beliefs. The capacity to use one’s multisensory approach and experiences to
access inner knowledge in understanding life and belief in religion marks the nurturing of
curiosity, and development of awareness of the spiritual domain for achieving wellbeing in life. Being blessed with high intensity in learning and cognition, high-ability students’ curiosity has the
potential to lead them into deep and meaningful understanding of faith and belief, far ahead of
their peers. Therefore, religious education should address the needs of these students in
understanding the morality and spiritual aspects in religious education. tional Journal of Academic Research in Progressive Education
ment
2019, E-ISSN: 2226-6348 © 2019 HRMARS Internat
Developm
Vol. 8, No. 4, Intensity in Spirituality of High-Ability Students
Rosadah Abd Majid & Aliza Alias
Universiti Kebangsaan Malaysia, Malaysia
Email: rosadah@ukm.edu.my International Journal of Academic Research in Progressive Education and
Development Vol. 8, No. 4, 2019, E-ISSN: 2226-6348 © 2019 HRMARS development that distinguish the characteristics of high-ability students. Among these are
psychosocial, creative, kinaesthetic, communicative capacity, self-identity, and spirituality. Students who are going through asynchronous development experience uneven development
within various domains. One may be very advanced within the cognitive domain but may not be
equally developed in the emotional domain of development. Thus, this type of students are
capable of comprehending abstract concepts but may not have developed the necessary
emotional, holistic skills entailed in coping with these cerebral demands. The United Kingdom’s National Curriculum (2004) stated that students who have deep interest
in faith and beliefs will show high levels of insight into the obvious and ordinary. Apart from that,
they are able to comprehend and infer meaning from symbols, texts, metaphors and practices. These students also are sensitive to the numinous and are aware of how it can be explored and
expressed. Furthermore, they are able to transfer ideas and concepts from one unit of work to
another. Finally, they are capable of two further and more generic factors, namely having highly
developed skills of comprehension, analysis and research, and secondly, having the ability to
think quickly and deeply about a given topic. These students are defined as “those who can easily,
quickly and at an expert level, apply specific skills and processes of religious education, and who
demonstrate high levels of understanding, insight, maturity and achievement”. High-ability Student There are diverse learners enrolling in our school system in line with the efforts towards
establishing inclusive education (Zalizan & Manisah, 2014; Dollah, Zabit, & Omar, 2018; Onyali,
Okerekeoti, 2018) these include students with special needs as well as students with high ability. Students who have high ability in cognitive aspects showed certain characteristics that distinguish
them from their peers. Even though they are just teenagers, their cognitive ability is beyond the
grasp of their peers or even adults. Their development in certain areas may exceed that of their
peers; this is called asynchrony development. There are many aspects of asynchrony 878 Religious Education Religious education in Malaysia is taught as one of the subjects in the national curriculum. It is a
subject that has a responsibility to instil religious values in the students’ spiritual development. This subject is taught in school from as early as Year One (seven years old) to Form Five (17 years
old). One of the outcome of religious education is shaping the students’ attitudes in their daily
lives (Tamuri, Othman, Dakir, Ismail, Stapa, 2013). At the end of each school year, students are
tested on their knowledge of what they have learned. According to Withall (2006), religious
education has the capacity to open new realms of thought and experiences for students in the
classroom. However, teaching a mixed ability class, by treating the students as one homogenous
group does not cater to the unique needs of certain students in the class. Those with high ability
needs would find the lessons inadequate to satisfy their yearning for knowledge of spirituality. Therefore, religious education which is taught as a subject, has failed to engage the diverse needs
of students in the classroom. For students who have deeper interest in spirituality, they would
go further by asking questions on matters that intrigue or challenge their inquisitive minds. They
need more than just knowing about religious facts and values. There is a need for them to be
have a congruent understanding so as to deeply appreciate and honour the concept of spirituality
pertaining to the teaching of belief and faith. Philosophical Thinking p
g
Philosophical thinking is an impetus to pursue wisdom. It engages in provocative thoughts about
the reality and significance of all existence. Intellectually, high- ability students are capable of,
and enjoy exercising their minds in philosophical thoughts. This sometimes brings the mind to
extend beyond the mundane world which we live in, to a transcendental world of another
dimension. Realizing one’s existence in this universe leads one’s thought to question and ponder
the very reasons behind all of creations. Philosophical thinking is a common characteristic in
students who are talented in writing (Piirto, 1992). According to Erickson’s Psychosocial Theory,
teenagers have an urge to search for personal identities that could lead to formation of their own
philosophy of life by actualizing their core belief and faith. n yearning for spirituality in life, these young minds need guidance in making the right choices as
it could influence the values that they uphold in the future. This could be carried out in religious
education classes, especially in the case of teenage students. The teachers responsible need to
possess necessary facilitating skills to conduct meaningful discussions. Traits such as tactfulness,
honesty, and sincerity are necessary in handling delicate philosophical issues, especially when
dealing with teenagers. Thus teachers need to understand and appreciate these students’ unique
learning needs. It is important to know how these students think and appraise matters and
issues pertaining to the meaning of life and existence. n yearning for spirituality in life, these young minds need guidance in making the right choices as
it could influence the values that they uphold in the future. This could be carried out in religious
education classes, especially in the case of teenage students. The teachers responsible need to
possess necessary facilitating skills to conduct meaningful discussions. Traits such as tactfulness,
honesty, and sincerity are necessary in handling delicate philosophical issues, especially when
dealing with teenagers. Thus teachers need to understand and appreciate these students’ unique
learning needs. It is important to know how these students think and appraise matters and
issues pertaining to the meaning of life and existence. High Intensity High-ability students display distinctive behaviours indicating the intensity in their thoughts,
emotion and interest. They display unique observable behavioural characteristics (Rosadah,
Zalizan &, Noriah, 2004). These behaviours reflect personal values of the students in developing
their understanding on how life should be lived. Some individuals showed evidence of heightened
spiritual attributes: profound sensitivity and compassion, depth of empathy, integrity, honesty,
intuitive inner guidance, living in accordance with equality and justice, and an ability to move
beyond everyday reality into oneness with God, nature and the universe (Gatto-Walden, 2009). Theory of potential development by Dabrowski, who was a psychologist, addresses the core of
personal characteristics that resonate with behaviour of many gifted children. Dabrowski
believed that it was the nature of these students to uphold the principles they believe in, and to
attempt to be individuals who are true to themselves (Daniels & Piechowski, 2009). In searching
for the inner self, intensive questioning could lead them further to more questions rather than
answers. The questions frequently asked include "Who am I?" and "Who am I to become?". Being teenagers, these students were also going through a difficult phase of their lives. They need
support and guidance in understanding the intensity they experience, especially in spirituality. Dealing with these intense urges to understand the concept of faith and belief could affect their
wellbeing in the future. High ability students also face risks in their development (Rosadah &
ALliza, 2010). Piechowski (1999) explained that the difference in intensity is an expanded field of
subjective experiences. These experiences could increase the spiritual yearnings in order to fulfil
the need of understanding the purpose of existence in this world that is bound by morality and
wholesome values of one’s belief. 879 International Journal of Academic Research in Progressive Education and
Development
Vol. 8, No. 4, 2019, E-ISSN: 2226-6348 © 2019 HRMARS Vol. 8, No. 4, 2019, E-ISSN: 2226-6348 © 2019 HRMARS teachers or instructors to internalize appropriate attitudes in line with the understanding of the
lessons learned. Religious education is intended to mould students into acquiring appropriate
attitudes and values. Young adolescents at this stage of life are going through a difficult time of
seeking identity and sense of belonging according to Erickson’s psychosocial theory. The
development of their social and emotional aspects during this phase is critical in giving them a
balanced emotional state in regards to belief of the faith concerned. Stricker (2009) claimed that
affective learning involves the melding of thinking and feeling in how people learn. Phelan (2018)
mentioned that high-ability learners tend to have unique learning needs. Thus, it is important
that the social learning environment is taken into consideration in the process of gaining
knowledge on spirituality. A deeper awareness and understanding of this affective learning is
crucial, as it will help educators in choosing the suitable approach in teaching. Guiding and
teaching mental dispositions in how a person views, engages, and values learning can result in
better understanding and use of knowledge and skills. Learning outcomes are focused on
enculturation of norms, values, skilful practices, and dispositions for lifelong learning. Being gifted, these students are drawn to challenge their minds to understand issues and values
of what is going on around them. As their curiosity is triggered in their minds, a vast array of
questions needs to be answered succinctly. According to Sisk (2008), gifted youth have spiritual
yearning that needs to be addressed properly so as to build the personality of the individuals. The intensity of these youths in searching for answers to global issues can lead to
misunderstanding and confusion, which then may lead them to question and doubt their faith
and beliefs. Thus, religious education needs to nurture gifted students’ characters, in helping
them to develop constructive values. Affective Learning Teaching religious education is not merely focusing on the cognitive aspects of learning; it has to
be deeply rooted to its affective domain. Affective domains of teaching and learning necessitates 880 Methodology This research focused on assessing gifted students’ thinking and learning in order to reflect their
philosophy and religious inclination. In assessing the intensity of the characteristics of high-ability
students in spirituality, eight items from Piechowsk’s Questionnaire on Dabrowski’s over-
excitability concept of heightened intensity were chosen. The items were used to identify the
intensity of the students on characteristics that are closely related in spiritual development. These items were divided into philosophical thinking and affective learning, which are related to
the development of spiritual yearning. Each item in the survey used 5-Likert scale (1 = Not at all
like me, to 5 = Very much like me). The sample of this research was a group of participants in a school holiday camp for high- ability
students. There were 310 15-year-old students selected for this study. The students, who came
from various different schools, were selected by the organizer of the camp after completing two
screening tests. These screening tests were on cognitive abilities of the individual to identify high-
ability students for the program. 881 International Journal of Academic Research in Progressive Education and
Development
l
E ISSN 2226 6348 © Vol. 8, No. 4, 2019, E-ISSN: 2226-6348 © 2019 HRMARS Findings g
The data was analysed to get the mean score on the level of intensity in the aspects of
philosophical thinking, and in the aspects of affective learning. The level of intensity of the sample
in both aspects was above three (3), which is considered high for these high-ability students. Construct
No. Statement
Mean
item
Mean
construct
Philosophical
thinking
1
I am an independent thinker,
3.45
3.86
2
I question everything – how things work, what
things mean, why things are the way they are,
3.67
3
I am moved by beauty in nature,
4.44
4
Theories gets my mind going,
3.90
Affective
learning
5
I can form new concepts by putting together a
number of different things,
3.60
3.62
6
I observe and analyse everything,
3.87
7
I can take everything to heart,
3.67
8
I like to dig beneath the surface of issues,
3.35 Discussion From the findings, it is shown that the participants’ level of intensity is distinctively high in both
philosophy thinking and affective learning. This intensity combined with their interest in
searching for the meaning of life, could lead them to explore the spiritual aspect of life. This
showed that high-ability students who have high intensity in these behaviours would be able to
discuss and synthesise the concept of spirituality at higher levels than that of their their peers. Combined with their high sensitivity in learning regarding wellbeing in life, this then would spur
them to be aware of the need for self-development and betterment of one’s self. As affective
learning is a way to understand the interpretation of belief, students with high curiosity need
guidance to comprehend the richness and depth of faith and beliefs. Religious education needs an appropriate pedagogy to allow the mind of these inquisitive
students to be challenged intellectually in order to fulfil their yearning for wellbeing in life. Teaching and learning process in the classroom should be able to nurture these students’ mind
to explore existential questions, to evaluate their own and others’ responses, and subsequently
reassess their own understanding of the matter. It is no longer sufficient to provide these
students with knowledge without extensive explanation and discussion of what is being taught
(Withall 2006). In philosophy thinking aspect, the mean of 3.86 showed that these students have provocative
thoughts about reality and the significance of all existence. Being moved by beauty in nature has
the highest mean (4.4), which showed that the students innately feel the greatness of the creator. They comprehend and appreciate what life is offering them in order to find fulfilment and the
wellbeing in living. Tapping this emotion in religious education could be an eye-opener for
students regarding the purpose of living in this world that is in accordance with the teaching of 882 International Journal of Academic Research in Progressive Education and
Development Vol. 8, No. 4, 2019, E-ISSN: 2226-6348 © 2019 HRMARS one’s belief. If their minds are constantly thinking about theories independently and only in a
cerebral manner, this could push them into greater confusion if they are not guided carefully. These students also highlighted the need to be given the opportunity to think and explore their
curious minds. They question everything, seeking to find how things work, what things mean and
why things are the way they are. They crave to challenge their own thoughts, trying to find
answers to issues and ironies happening around them that are related to values and beliefs. Being affective learners, the students delve into melding the thinking process and feelings, in
attempts to understand how people around them are learning. The social learning environment
played an important role in gaining knowledge on spirituality. The students’ tendency to dig
beneath the surface of issues related to morality and spirituality surrounding them, calls for
religious education teachers to encourage students to readily express themselves and discuss
meaningfully certain topics of interests. The students cannot help being curious and striving to
get to the bottom of certain perplexing questions, be they of social, mundane or existential
nature. White (2010) had observed that high-ability learners tend to be critical in their thinking. Therefore, the teachers should avoid being judgemental of students’ suggestions or thoughts,
but rather help them to see it from the point of the values that should be incorporated in their
spirituality. The students have the capability to observe and analyse everything and eventually
form new concepts. This could be an alarming sign because without the guidance of educators in
religious education, they could be led away from the pillars of the faith that they are constantly
trying to comprehend. Therefore, to cater for the needs of high-ability students who have deep interest in spirituality,
religious teachers need to internalize humanistic character traits and embrace virtues and noble
values so as to enhance the delivery of religious education, thus further enhancing the wisdom
of teaching positive attitudes and values. Religious education teachers are the role models of
good conduct and fine characters or being referred to in Arabic as “muaddib and murobbi”
(Jaafar, Raus, Muhamad, Ghazalid, Amat, Hassan, Hashim, Ab. Halim Tamuri, Norshidah
Mohamed Salleh & Mohd Isa Hamzah, 2014). International Journal of Academic Research in Progressive Education and
Development Pine and Boy (1997) asserted that the self-
actualized humanistic teacher needs to have these characteristics: 1. thinks well of himself/herself and has a good self-concept 2. honest and genuine, there is no conflict between the real inner person and the role-
playing outer person 3. likes and accept others 3
es a d accept ot e s
4. lives by humanistic values, is honestly concerned with the welfare of fellow humans and
the improvement of human society
5
is sensitive and responsive to the needs and feelings of others 4. lives by humanistic values, is honestly concerned with the welfare of fellow humans and
the improvement of human society 5. is sensitive and responsive to the needs and feelings of others 5. is sensitive and responsive to the needs and feelings of others 6. is open to the view point of others, to new information and experiences, and to his or her
own inner feelings 7. exercises control over his or her life and environment 8. initiates needed changes 9. is responsive, vibrant, and spontaneous, and tries to live optimistically, and energetically. (Davis and Rimm, 2004) 883 Acknowledgement The study was supported by the grant from the Faculty of Education, Universiti Kebangsaan
Malaysia, GG-2019-003. Conclusion Being intensely interested in spirituality, high-ability students’ minds and interests should be
challenged positively and guided to the teaching of core spiritual beliefs. Yearning for further
knowledge in faith and belief, should be paralleled by the moral values and virtues being taught
in religious classes. Therefore, there is a need for suitable pedagogical approaches to teach
religious education to these highly intense students. These approaches should stress practising
and embracing the teachings of the essential truths of religious beliefs, which are ultimately for
the wellbeing of the students, both in current times as well as in the future. Phelan, D. A. (2018). Social and Emotional Learning Needs of Gifted Students. Ed.D Dissertation.
Walden University. USA. Walden Dissertations and Doctoral Studies References Tamuri, A. H., Othman, M. Y., Dakir, J., Ismail, A. M., Stapa, Z. (2013). Religious education and
ethical attitude of Muslim adolescents in Malaysia. Multicultural Education and
Technology Journal, 7(4), 257-274. https://doi.org/10.1108/METJ-03-2013-0008
Daniels, S., & Piechowski, M. M. (2009). Living with intensity. Great Potential Press : USA
Davis G A & Rimm S B (2004) Education of the gifted and talented (5th ed ) Boston: Pearson Tamuri, A. H., Othman, M. Y., Dakir, J., Ismail, A. M., Stapa, Z. (2013). Religious education and
ethical attitude of Muslim adolescents in Malaysia. Multicultural Education and
Technology Journal, 7(4), 257-274. https://doi.org/10.1108/METJ-03-2013-0008 Technology Journal, 7(4), 257-274. https://doi.org/10.1108/METJ-03-2013-0008
Daniels, S., & Piechowski, M. M. (2009). Living with intensity. Great Potential Press : USA Daniels, S., & Piechowski, M. M. (2009). Living with intensity. Great Potential Press : USA Davis, G. A., & Rimm, S. B. (2004). Education of the gifted and talented (5th ed.). Boston: Pearson
Education. Dollah, M. U., Zabit, M. N. M., & Omar, T. Z. Z. (2018). Comparing the Value of Control with
Characteristic of Prediction Conveyed in Mathematics Textbooks in Malaysia and
Australia. International Journal of Academic Research in Progressive Education and
Development, 7(4), 387–396. Gatto-Walden, P. (2009). Living one’s spirit song: Transcendent experiences in counselling gifted
adults. In S. Daniels & M. M. Piechowski (Eds.), Living with intensity: Understanding the
sensitivity, excitability,and emotional development of gifted children, adolescents, and
adults(pp. 203–223). Scottsdale, AZ: Great Potential Press. Hosseini, M., Elias, H., Krauss, S. E., & Aishah, S. (2010). A Review Study on Spiritual Intelligence,
Adolescence and Spiritual Intelligence, Factors that may Contribute to Individual
Differences in Spiritual Intelligence and the Related Theories. Journal of Social Sciences. 6(3): 429-438. Jaafar, N., Raus, N. M., Muhamad, N. A. F., Ghazalid, N. M., Amat, R. A. M., Hassan, S. S., Hashim,
M., Tamuri, A. H., Salleh, N. M., Hamzah, M. I. (2014). Quran Education for Special
Children: Teacher as Murabbi. Psychology. 2014. DOI:10.4236/ce.2014.57053 Onyali, C. I., Okerekeoti, C. U. (2018). Board Heterogenity and Corporate Performance of Firms
in Nigeria, International Journal of Academic Research in Accounting, Finance and
Management Sciences 8 (3): 103- 117. Phelan, D. A. (2018). Social and Emotional Learning Needs of Gifted Students. Ed.D Dissertation. Walden University. USA. Walden Dissertations and Doctoral Studies 884 International Journal of Academic Research in Progressive Education and
Development Vol. 8, No. 4, 2019, E-ISSN: 2226-6348 © 2019 HRMARS Phenwan, T., Peerawong, T., Tulathamkij, K. (2019). The Meaning of Spirituality and Spiritual
Well-Being among Thai Breast Cancer Patients: A Qualitative Study. Indian Journal of
Palliative Care. 2019 Jan-Mar; 25(1): 119–123. doi: 10.4103/IJPC.IJPC_101_18 Piechowski, M. M. (1999). Overexcitabilities. In M. Runco & S. Pritzker (Eds) Encyclopadea of
creativity (vol 2 pp 325-334) New York: Academic Press. J. (1992). Understanding those who create. Tempe, AZ: Gifted Psychology Press. Pine, G. J., & Boy, A. V. (1997). Learner Centered Teaching: A Humanistic View in Davis, G. A., &
Rimm, S. B. (2004). Education of the gifted and talented (5th ed.). Boston: Pearson
Education. Majid, R. A., Jelas, Z. M., & Ishak, N. M. (2004). Behavioral Characteristics Prototypes of
Academically Talented Students: Implications on Educational Interventions, Paper
presented at Gifted Education Conference, NIE, Nanyang Technology University,
Singapore. Majid, R. A., Alias, A. (2010). Consequences of Risk Factors in the Development of Gifted Children. Procedia Social and Behavioral Sciences 7(C) (2010) 63–69. Sisk, D. (2008). Engaging to Spiritual Intelligence of Gifted Students to Build Global Awareness in
the Classroom. Roeper Review 30: 24-30. Stricker, A. G. (2009). Why Affective Learning in a Situated Place Matters for the Millennial
Generation. http://www.au.af.mil/au/a46i/documents/Why-Affective-Learning-in-a-
Situated-Place.pdf p
The National Curriculum United Kingdom’s website (2004). (www.nc.uk.net/gt/re/index.htm). White, D. A. (2010). Thinking (With Help from Aristotle) About Critical Thinking. Gifted Child
Today. Summer 2010. 33(3): 14-19. Whittall, A. (2006). Using the Interpretive approach for teaching RE to gifted and talented pupils
at Key Stage 3. http://www.farmington.ac Zalizan, M. J., Ali, M. M. (2014). Inclusive education in Malaysia: Policy and practice. International
Journal of Inclusive Education, 18(10), 991-1003. https://doi.org/10.1080/13603116.2012.693398 885
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Comunicação social e saúde: Conscientização e incentivo a imunização
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ARTIGO DE REVISÃO SANTOS, Gustavo Gonçalves dos. SANTOS, Vitória Gonçalves dos. Comunicação
social e saúde: Conscientização e incentivo a imunização. Revista Científica
Multidisciplinar Núcleo do Conhecimento. Ano 04, Ed. 10, Vol. 06, pp. 64-78. Outubro
de
2019. ISSN:
2448-0959,
Link
de
acesso: https://www.nucleodoconhecimento.com.br/saude/incentivo-a-imunizacao REVISTA CIENTÍFICA MULTIDISCIPLINAR NÚCLEO DO
CONHECIMENTO ISSN: 2448-0959
https://www.nucleodoconhecimento.com.br REVISTA CIENTÍFICA MULTIDISCIPLINAR NÚCLEO DO
CONHECIMENTO ISSN: 2448-0959
https://www.nucleodoconhecimento.com.br REVISTA CIENTÍFICA MULTIDISCIPLINAR NÚCLEO DO
CONHECIMENTO ISSN: 2448-0959 Biblioteca Virtual: SciELO, LILACS, BDEFN e Google Acadêmico, utilizando os
descritores: Calendário Vacinal, Imunização, Comunicação Social. Resultados: A
Comunicação Social em saúde oferece condições significantes e relevantes para as
boas práticas de promoção à saúde, favorecendo o estímulo do autocuidado e o
cuidado dos usuários do serviço de saúde. Conclusões: Mobilizar profissionais da
saúde e os usuários dos serviços de saúde no que se diz respeito à conscientização
e incentivo da imunização. Descritores: calendário vacinal, imunização, comunicação social. RC: 39116
Disponível em: https://www.nucleodoconhecimento.com.br/saude/incentivo-a-imunizacao RC: 39116
Disponível em: https://www.nucleodoconhecimento.com.br/saude/incentivo-a-imunizacao RESUMO Objetivo: Descrever sobre o poder Comunicação Social na Enfermagem em relação
ao incentivo e conscientização em relação às campanhas de vacinação. Método:
Trata-se de uma revisão bibliográfica, realizada no período de Fevereiro à Julho de
2019. Foram utilizados 10 estudos como referencial teórico relativo à Saúde e
Comunicação Social com ênfase na Publicidade e Propaganda por meio de fontes
como publicações do Ministério da Saúde, Organização Mundial da Saúde e artigos
de periódicos científicos publicados nos últimos dez anos, disponibilizados na 1 Enfermeiro e Docente. Graduado em Enfermagem pela Escola de Ciências da Saúde
da Universidade Anhembi Morumbi. Pós-graduando Enfermagem Obstétrica e
Ginecológica pela Faculdade Israelita de Ciências da Saúde do Hospital Albert
Einstein – Instituo Israelita de Ensino e Pesquisa. Pós-graduando Enfermagem em
Saúde Pública pela Escola Paulista de Enfermagem da Universidade Federal de São
Paulo. 2 Graduanda em Comunicação Social – Publicidade e Propaganda pela Escola de
Comunicação da Universidade Anhembi Morumbi. RC: 39116
Disponível em: https://www.nucleodoconhecimento.com.br/saude/incentivo-a-imunizacao C 39
6
Disponível em: https://www.nucleodoconhecimento.com.br/saude/incentivo-a-imunizacao REVISTA CIENTÍFICA MULTIDISCIPLINAR NÚCLEO DO
CONHECIMENTO ISSN: 2448-0959
https://www.nucleodoconhecimento.com.br INTRODUÇÃO Imunização é uma ação de caráter preventiva, reconhecida pela redução nas taxas
de mortalidade. No Brasil, houve a criação do Programa Nacional de Imunizações,
instituído pela Lei nº 6.59/75, lei essa que regulamenta as ações de vigilância,
imunização e notificação compulsória de doenças. As vacinas são praticadas de
maneira sistemática e ofertadas de forma gratuita por entidades públicas, mas
também por entidades privadas. (1) O Art. 27 da Lei nº 6.59/75 regulamenta que as vacinas são obrigatórias em todo o
território nacional, as vacinas como tal que são definidas pelo Programa Nacional de
Imunizações. O decreto ainda dispõe no Art. 29 que é dever de todo cidadão
submeter-se a imunização, juntamente com os menores dos quais tenha guarda e/ou
responsabilidade. (1) A imunização consiste em uma das ações preventivas de Saúde Pública mais segura,
econômica e efetiva capaz de prevenir mortalidade e oferecer qualidade de vida, em
especial às populações de maior vulnerabilidade. (1,2) Tratar com descuido o Calendário Vacinal pode causar agravos à saúde, agravos
estes que podem se reverter em maiores problemas de Saúde Pública. Um apoio no
que se diz respeito à atuação de primeira linha em relação à imunização, é a Atenção
Primária à Saúde que é considerada um importante meio de atuação no que se refere RC: 39116
Disponível em: https://www.nucleodoconhecimento.com.br/saude/incentivo-a-imunizacao RC: 39116
Disponível em: https://www.nucleodoconhecimento.com.br/saude/incentivo-a-imunizacao REVISTA CIENTÍFICA MULTIDISCIPLINAR NÚCLEO DO
CONHECIMENTO ISSN: 2448-0959
https://www.nucleodoconhecimento.com.br
à maneira segura e efetiva o controle e/ou erradicação de doenças agravantes à
saúde. (3) à maneira segura e efetiva o controle e/ou erradicação de doenças agravantes à
saúde. (3) à maneira segura e efetiva o controle e/ou erradicação de doenças agravantes à
saúde. (3) à maneira segura e efetiva o controle e/ou erradicação de doenças agravantes à
saúde. (3) À medida que aumenta o número de vacinas e o seu uso pelos Programas de Saúde
Pública, cresce também a quantidade de pessoas e grupos que declaram
preocupações com a segurança e a necessidade da aplicação das vacinas. Pais e/ou
responsáveis, cuidadores, pacientes e os próprios profissionais da saúde que atuam
diretamente nesta perspectiva. No Brasil, o Programa Nacional de Imunizações conta
com a credibilidade e respeitabilidade da população, da comunidade científica e
também da comunidade da Comunicação Social. (4) A vacinação é considerada um custo-efetivo e segura, favorece proteção individual e
coletiva, estando diretamente ligada a promoção de saúde, proteção e manutenção
da saúde. O Ministério de Saúde tem oferecido ações, como o Dia D, com intuito de
imunizar o maior número de indivíduos, principalmente crianças e idosos que se
enquadram como indivíduos mais suscetíveis a doenças. (5) Diante do que foi exposto surgi a seguinte indagação: Qual a finalidade da
Comunicação Social em Saúde no que se diz respeito à conscientização e incentivo
para imunização? Respondendo tal questionamento este estudo tem como objetivo
descrever sobre a Comunicação Social em Saúde, em relação ao incentivo e
conscientização em relação vacinação e descrever a relevância da Comunicação
Social na Enfermagem no que se diz respeito à imunização da população. MÉTODO Trata-se de uma revisão integrativa, método este utilizado desde 1980 permitindo
buscar, avaliar e elaborar uma síntese de evidências utilizando variados estudos sobre
o tema estudado nesta presente revisão, possibilitando então o levantamento de
lacunas que precisam ser preenchidas com publicação de novos estudos. (13) O percurso metodológico seguiu as seguintes etapas: Identificação do tema,
elaboração da pergunta norteadora; definição dos critérios para inclusão e exclusão RC: 39116
Disponível em: https://www.nucleodoconhecimento.com.br/saude/incentivo-a-imunizacao Disponível em: https://www.nucleodoconhecimento.com.br/saude/incentivo-a-imunizaca REVISTA CIENTÍFICA MULTIDISCIPLINAR NÚCLEO DO
CONHECIMENTO ISSN: 2448-0959
https://www.nucleodoconhecimento.com.br (13,14) Fonte: Dados do estudo, 2019. REVISTA CIENTÍFICA MULTIDISCIPLINAR NÚCLEO DO
CONHECIMENTO ISSN: 2448-0959
https://www.nucleodoconhecimento.com.br REVISTA CIENTÍFICA MULTIDISCIPLINAR NÚCLEO DO
CONHECIMENTO ISSN: 2448-0959 de estudos; busca de artigos científicos na literatura; avaliação dos estudos incluídos
na presente revisão e interpretação dos resultados. No que se diz respeito à questão
norteadora, foi indagado: Qual a finalidade da Comunicação Social em Saúde no que
se diz respeito à conscientização e incentivo para imunização? Realizou-se o presente estudo no período de Fevereiro à Julho de 2018, buscando
artigos científicos com metodologia de Revisões sistemáticas da literatura, estudos
exploratório-descritivos, estudos qualitativos e estudos quantitativos nas bases de
dados da Literatura Latino-Americana e do Caribe em Ciências da Saúde (LILACS),
Scientific Eletronic Library Online (SciELO), Base de dados de Enfermagem (BDENF)
e Google Acadêmico, utilizando os seguintes descritores: Calendário Vacinal,
Imunização, Comunicação Social, Publicidade e Propaganda, Educação em
Enfermagem. Após a busca em bases de dados, foi selecionado um total de 20 (vinte)
artigos. Posteriormente foi realizada leitura na íntegra dos artigos selecionados, sendo
incluídos na presente revisão 10 (dez) artigos, que abordavam com relevância a
temática deste estudo e se enquadrava dentro dos critérios de inclusão estabelecidos. Para os critérios de inclusão dos estudos foram estabelecidos os seguintes: período
das publicações entre os anos de 2010 a 2018; publicações no idioma Português e
Espanhol e artigos indexados na íntegra nas bases de dados selecionadas. E para os critérios de exclusão foram adotados os seguintes: artigos repetidos em
mais de uma base de dados, artigos que não foram publicados nos últimos dez anos
e artigos no idioma Inglês. Foi elaborado um quadro, contendo referência e ano, autores, título do artigo e
objetivos. Os dados incluídos neste estudo foram analisados de forma descritiva, a
partir de trechos extraídos dos estudos incluídos nesta pesquisa. Utilizando a análise
temática de Conteúdo de Bardin, onde se encontram variados tipos de investigação,
apoiados em diferentes técnicas metodológicas. A análise temática adotada neste
estudo indica que a mesma já era utilizada desde as primeiras tentativas da
humanidade de interpretar os livros sagrados, tendo sido sistematizada como método
apenas na década de 20, por Leavel. Ou seja, utilizando essa análise, o pesquisador RC: 39116
Disponível em: https://www.nucleodoconhecimento.com.br/saude/incentivo-a-imunizacao RC: 39116
Disponível em: https://www.nucleodoconhecimento.com.br/saude/incentivo-a-imunizacao C
Disponível em: https://www.nucleodoconhecimento.com.br/saude/incentivo-a-imunizaca busca compreender as características, estruturas ou modelos que estão por trás dos
fragmentos da literatura tornados em consideração. (13,14) busca compreender as características, estruturas ou modelos que estão por trás dos
fragmentos da literatura tornados em consideração. RESULTADOS E DISCUSSÃO Na presente revisão integrativa foram analisados 10 (dez) artigos, pois atenderam aos
critérios de inclusão, conforme apresentado no Quadro 1. As fontes de publicação
foram extraídas de diversos periódicos, os quais destacaram a área da Enfermagem,
Comunicação em Saúde e Comunicação Social com ênfase na Publicidade e
Propaganda. RC: 39116
Disponível em: https://www.nucleodoconhecimento.com.br/saude/incentivo-a-imunizacao
Quadro 1 – Identificação dos estudos segundo referência, autores, título e objetivos. São Paulo – SP, Brasil. 2019. Referência Autores
Título
Objetivos
1
Barbieri CLA et
al
A
(não)
vacinação
infantil entre a cultura e
a lei: os significados
atribuídos por casais de
Compreender como pais e
mães
pertencentes
às
camadas médias de São
Paulo
significam
as RC: 39116
Disponível em: https://www.nucleodoconhecimento.com.br/saude/incentivo-a-imunizacao
Quadro 1 – Identificação dos estudos segundo referência, autores, título e objetivos. São Paulo – SP, Brasil. 2019. Referência Autores
Título
Objetivos
1
Barbieri CLA et
al
A
(não)
vacinação
infantil entre a cultura e
a lei: os significados
atribuídos por casais de
Compreender como pais e
mães
pertencentes
às
camadas médias de São
Paulo
significam
as Quadro 1 – Identificação dos estudos segundo referência, autores, título e objetivos. São Paulo – SP, Brasil. 2019. Referência Autores
Título
Objetivos
1
Barbieri CLA et
al
A
(não)
vacinação
infantil entre a cultura e
a lei: os significados
atribuídos por casais de
Compreender como pais e
mães
pertencentes
às
camadas médias de São
Paulo
significam
as Categoria Temática 1. Conscientização e incentivo à imunização 1. Comunicação Social em Saúde em prol da imunização Fonte: Dados do estudo, 2019. RC: 39116
Disponível em: https://www.nucleodoconhecimento.com.br/saude/incentivo-a-imunizacao REVISTA CIENTÍFICA MULTIDISCIPLINAR NÚCLEO DO
CONHECIMENTO ISSN: 2448-0959 camadas
médias
de
São Paulo, Brasil
normatizações
da
vacinação no país, a partir
de
suas
vivências
de
vacinar, selecionar ou não
vacinar os filhos
2
Silva FS et al
Incompletude
vacinal
infantil
de
vacinas
novas
e
antigas
e
fatores
associados:
coorte de nascimento
BRISA,
São
Luís,
Maranhão, Nordeste do
Brasil
Analisar
fatores
associados
para
a
incompletude de vacinas
de introdução recente. 3
Ramos CF et
al
Cumprimento
do
calendário
de
vacinação de crianças
em uma unidade de
saúde da família
Analisar o cumprimento do
calendário de vacinação
de crianças pelos usuários
da Unidade de Saúde da
Família Água bem como
possíveis razões para o
seu
eventual
não
cumprimento
4
Mizuta AH et al Percepções acerca da
importância das vacinas
e da recusa vacinal
numa
Escola
de
Medicina
Identificar a percepção da
importância das vacinas e
os riscos da recusa vacinal
entre alunos de Medicina
e médicos
5
Martins
KM,
Santos
WL,
Álvares ACM
A
importância
da
imunização:
revisão
integrativa
Proteger a gestante e o
recém-nascido,
pois
a
transmissão
perinatal
representa uma das vias
mais
convincentes
de REVISTA CIENTÍFICA MULTIDISCIPLINAR NÚCLEO DO
CONHECIMENTO ISSN: 2448-0959 https://www.nucleodoconhecimento.com.br
transmissão de doenças,
e
a
que
mais
repentinamente
leva
a
sequelas e óbitos
6
Maia
JÁ,
Menezes
FA,
Santos PAM
Percepção
dos
pais
sobre a importância de
acompanhamento
do
crescimento
e
desenvolvimento infantil
Explicar a importância do
acompanhamento
do
crescimento
e
do
desenvolvimento
infantil
de crianças de um mês a
cinco anos
7
De
Morais
Júnior SLA et. al
A evolução histórica do
calendário
vacinal
brasileiro infantil. Apresentar a evolução do
calendário vacinal infantil
no Brasil
8
Coriolano-
Marinus MWL
et al
Comunicação
nas
práticas
em
saúde:
revisão integrativa da
literatura
Descrever os principais
eixos
temáticos
explorados no campo da
comunicação nas práticas
em saúde nos cenários do
Sistema Único de Saúde
(SUS)
9
Vasconcellos-
Silva PR et al
A sociedade de risco
midiatizada,
o
movimento
antivacinação e o risco
do autismo
Observar
modificações
epidemiológicas
de
doenças infecciosas entre
famílias de classe média
de países industrializados
por
força
de
crenças
ligadas
aos
riscos
da
vacinação
10
Nardi ACF et. al
Comunicação
em
saúde: um estudo do
perfil e da estrutura das
Investigar
as
características
dos
serviços de comunicação assessorias
de
comunicação
municipais em 2014-
2015
em saúde, a partir da
Rede dos Conselhos das
Secretarias Municipais de
Saúde
(COSEMS)
no
Brasil Fonte: Dados do estudo, 2019. RC: 39116
Disponível em: https://www.nucleodoconhecimento.com.br/saude/incentivo-a-imunizacao Quadro 2 - Categorias temáticas segundo as referências dos estudos incluídos. São
Paulo – SP, Brasil. 2019. Quadro 2 - Categorias temáticas segundo as referências dos estudos incluídos. São
Paulo – SP, Brasil. 2019. Ao analisar os estudos, emergiram duas categorias conforme apresentado no Quadro
2. Categoria Temática
1. Conscientização e incentivo à imunização
1. Comunicação Social em Saúde em prol da imunização Categoria Temática REVISTA CIENTÍFICA MULTIDISCIPLINAR NÚCLEO DO
CONHECIMENTO ISSN: 2448-0959
https://www.nucleodoconhecimento.com.br REVISTA CIENTÍFICA MULTIDISCIPLINAR NÚCLEO DO
CONHECIMENTO ISSN: 2448-0959
https://www.nucleodoconhecimento.com.br irregulares se dão por atrasos e perda do dia da vacina, argumentos esses
considerados sem relevância. (3) irregulares se dão por atrasos e perda do dia da vacina, argumentos esses
considerados sem relevância. (3) Visto que a conscientização e incentivo à imunização são extremamente relevantes e
significantes, a Estratégia Saúde da Família está o nível da Atenção da Primária à
Saúde, tendo a imunização como um de seus princípios e desejos como foco de
trabalho. Ação essa de conscientização e incentivo dada pela atuação dos Agentes
Comunitários de Saúde, que alertam as famílias e suas microáreas sobre a
importância das vacinas. É considerado por tanto, com destaque, o espaço da
Comunicação Social (Publicidade e Propaganda) veiculado ao Ministério da Saúde
nos meios de comunicação de massa. Os agentes comunitários de saúde vêm
desenvolvendo atividades de promoção da saúde e prevenção de doenças e agravos
à saúde dos indivíduos, por meio de visitas domiciliares e de ações educativas
individuais e coletivas nos domicílios e na comunidade, mantendo a equipe informada,
principalmente a respeito daquelas em situação de risco. (3,6) A Atenção Primária à Saúde está diretamente ligada às ações de Educação em
Saúde, principalmente no que se diz respeito ao incentivo e conscientização da
população em relação à imunização. A imunização deve ser entendida como um
agente de modificação do processo de saúde-doença, pois garante a promoção e
proteção à saúde e proteção da saúde dos indivíduos. A imunização tem sido a forma
de prevenção de doenças. (5,6,7) Em ações de conscientização e incentivo a imunização destaca-se o Enfermeiro em
atuar diretamente em atividades e ações educativas com a população. O Enfermeiro
é quem consegue ter maior vínculo com os usuários dos serviços de saúde,
evidenciando a grande responsabilidade desse profissional no que se trata à
imunização, ou seja, neste sentido o Enfermeiro e a equipe multiprofissional que atua
em salas de vacina e ações educativas tem a autonomia de intervir no processo de
saúde-doença de forma eficiente, contribuindo para que os usuários adotem o serviço
de saúde e busquem sempre pela promoção e manutenção da saúde em todo o
processo do cuidar no ciclo vital. (5.6,7) CONSCIENTIZAÇÃO E INCENTIVO À IMUNIZAÇÃO Em estudos com abordagem qualitativos os indivíduos expressam diferentes
significações sobre as normas brasileiras de imunização. Os pais que vacinam seus
filhos revelam que imunizar os seus filhos é uma ação de responsabilidade. Pode-se
observar o valor simbólico de responsabilidade parental, agregando a ideia de
continuidade com o valor cultuado pelas tradições familiares e da sociedade. Sem
minimizar o protagonismo da sociedade e a influência dos indivíduos no processo de
imunização remetem uma ideia de que a sustentação e ênfase dado do ato de vacinar
é a valoração cultural que está relacionada ao fato de ser obrigatório pela lei. (1,2) Em estudos perspectivos, transversais e qualitativos apontam que cerca de 87,9% dos
cartões de vacinação encontram-se atualizados, uma vez que as condições Disponível em: https://www.nucleodoconhecimento.com.br/saude/incentivo-a-imunizacao REVISTA CIENTÍFICA MULTIDISCIPLINAR NÚCLEO DO
CONHECIMENTO ISSN: 2448-0959
https://www.nucleodoconhecimento.com.br Ocorrem falhas em que a imunização não ocorre por falta de aperfeiçoamento dos
profissionais, gerando a diminuição no aperfeiçoamento profissional qualificação. E
para garantir a imunização, os profissionais e usuários do serviço de saúde devem
sempre estar informados quanto às indicações, contraindicações e efeitos da vacina,
com o intuito de reduzir casos de atraso ou perda da vacina. Desta forma, devem-se
reorganizar os serviços de saúde, proporcionando o planejamento das unidades. (5,6,7) RC: 39116
Disponível em: https://www.nucleodoconhecimento.com.br/saude/incentivo-a-imunizacao RC: 39116
Disponível em: https://www.nucleodoconhecimento.com.br/saude/incentivo-a-imunizacao REVISTA CIENTÍFICA MULTIDISCIPLINAR NÚCLEO DO
CONHECIMENTO ISSN: 2448-0959
https://www.nucleodoconhecimento.com.br A comunicação terapêutica é capaz de ajudar os indivíduos a enfrentar situações
temporárias, ajustando-os à realidade, favorecendo o tratamento e o desenvolvimento
dos usuários, tornando-os ativos no processo de cuidar. Porém, acredita-se que os
aspectos de comunicação entre trabalhadores de saúde que integram as equipes,
tanto no ambiente hospitalar como nos cenários de atenção primária, precisam ser
investigados na procura dos problemas que permeiam as inter-relações dos
profissionais com os usuários dos serviços de saúde. (8,9) A Comunicação Social, em prol de campanha publicitária trás de forma impactante
alerta para a importância de manter sempre a vacinação em dia. O objetivo é mostrar
que as baixas coberturas vacinais podem ser extremamente perigosas, já que abrem
caminho para a reintrodução de doenças já eliminadas no país e que podem até
aumentar as taxas de mortalidade. As campanhas publicitárias são consideradas
impactantes, pois mostram casos reais de pessoas que sofrem até hoje pela não
vacinação. (8,10,11,12) No que se diz respeito a Comunicação Social ligada diretamente a Publicidade e
Propaganda é possível expor alguns cenários onde pela primeira vez, a mascote das
campanhas de vacinação do Ministério, o Zé Gotinha, aparece em um tom sério e
preocupado. Disseminar informação para o grande público é sempre um desafio para
a Saúde Pública. Por isso, a Saúde Brasil desenvolve campanhas educativas
diferenciadas na área da saúde, cidadania e meio ambiente, com o objetivo de orientar
a população divulgando informações para a promoção da saúde e qualidade de vida. As campanhas são de interesse social, desenvolvidas em uma linguagem simples e
acessível e por promoverem temas relevantes à sociedade ganham destaque em toda
mídia sendo reconhecidas como ferramenta de utilidade pública em saúde. (8,10,11,12) OMUNICAÇÃO SOCIAL EM SAÚDE EM PROL DA IMUNIZAÇÃO A Comunicação Social em Saúde pode ser compreendida como a prática social que
advém a interação dos seres humanos, por meio de falas, escritas sendo classificadas
como verbal e não verbal. No Sistema Único de Saúde (SUS) nota-se uma dificuldade
entre o preceptor e o emissor, dificuldades essas recorrentes a linguagens e saberes
diferentes, que nem sempre são compartilhados. Esses fatores diferenciam-se por
ordem sociocultural e estágios de desenvolvimentos cognitivos. (8) Procura-se entender a inteligência, racionalidade e o desempenho do indivíduo. O
princípio geral da inteligência é auxiliar a adaptação ao ambiente, podendo variar de
acordo com o ambiente, hábito e reflexos. Ação comunicativa trata-se de um contexto
dinâmico no qual podemos encaixar valores éticos e morais. A partir da relevância da
Comunicação Social como um instrumento principal, os trabalhadores do Sistema
Único de Saúde necessitam de suportes e implementações para a comunicação
efetiva dialogada. (8) A Comunicação Social proporciona um processo de compartilhamento e ajuda entre
o profissional de saúde e o usuário, estabelecendo um processo de ajuda ao indivíduo
e à família. Este processo de compartilhamento é caracterizado por atitudes de
sensibilidade, aceitação e empatia entre os profissionais e usuários do serviço de
saúde. A comunicação oferece condições significantes e relevantes para as boas
práticas de promoção da saúde, tornando o estímulo do autocuidado e o cuidado dos
usuários do serviço de saúde. (8) RC: 39116
Disponível em: https://www.nucleodoconhecimento.com.br/saude/incentivo-a-imunizacao RC: 39116
Disponível em: https://www.nucleodoconhecimento.com.br/saude/incentivo-a-imunizacao RC: 39116
Disponível em: https://www.nucleodoconhecimento.com.br/saude/incentivo-a-imunizacao CONSIDERAÇÕES FINAIS Os resultados levantados neste estudo estão associados diretamente com a atuação
direta da Atenção Primária à Saúde, principalmente no que se diz respeito a Estratégia
Saúde da Família que aproxima a comunidade com os serviços em saúde, sendo
capaz então de ofertar através de ações educativas campanhas publicitárias para o RC: 39116
Disponível em: https://www.nucleodoconhecimento.com.br/saude/incentivo-a-imunizacao REVISTA CIENTÍFICA MULTIDISCIPLINAR NÚCLEO DO
CONHECIMENTO ISSN: 2448-0959
https://www.nucleodoconhecimento.com.br incentivo e conscientização à imunização, ressaltando a importância da vacina em
todo o processo do ciclo vital. incentivo e conscientização à imunização, ressaltando a importância da vacina em
todo o processo do ciclo vital. RC: 39116 Disponível em: https://www.nucleodoconhecimento.com.br/saude/incentivo-a-imunizaca REFERÊNCIAS 1. Barbieri CLA et al. A (não) vacinação infantil entre a cultura e a lei: os
significados atribuídos por casais de camadas médias de São Paulo, Brasil. Cad. Saúde Pública 2017; 33(2):e00173315. Acesso em: 21 de Julho de 2019. Disponível
em:
http://www.scielo.br/pdf/csp/v33n2/1678-4464-csp-33-02-
e00173315.pdf 2. Silva FS et al. Incompletude vacinal infantil de vacinas novas e antigas e fatores
associados: coorte de nascimento BRISA, São Luís, Maranhão, Nordeste do
Brasil. Cad. Saúde Pública 2018; 34(3):e00041717. Acesso em: 21 de Julho de
2019. Disponível em: http://www.scielo.br/pdf/csp/v34n3/1678-4464-csp-34-
03-e00041717.pdf 3. Ramos CF, et al. Cumprimento do calendário de vacinação de crianças em uma
unidade de saúde da família. Rev Pan-Amaz Saude 2010; 1(2):55-60. Acesso
em:
21
de
Julho
de
2019. Disponível
em:
http://scielo.iec.gov.br/pdf/rpas/v1n2/v1n2a06.pdf 4. Mizuta AH et al. PERCEPÇÕES ACERCA DA IMPORTÂNCIA DAS VACINAS
E DA RECUSA VACINAL NUMA ESCOLA DE MEDICINA. Rev Paul Pediatr. 2019;37(1):34-40. Acesso em: 21 de Julho de 2019. Disponível em:
http://www.scielo.br/pdf/rpp/v37n1/0103-0582-rpp-2019-37-1-00008.pdf 5. Martins KM, Santos WL, Álvares ACM. A importância da imunização: revisão
integrativa. Rev Inic Cient Ext. 2019; 2(2): 28-33. Acesso em: 21 de Julho de
2019. Disponível
em:
https://revistasfacesa.senaaires.com.br/index.php/iniciacao-
cientifica/article/view/153 6. Maia JÁ, Menezes FA, Santos PAM. PERCEPÇÃO DOS PAIS SOBRE A
IMPORTÂNCIA
DE
ACOMPANHAMENTO
DO
CRESCIMENTO
E
DESENVOLVIMENTO INFANTIL. DêCiência em Foco. ISSN: 2526-5946. 2017; 1(2): 53-63. Acesso em: 21 de Julho de 2019. Disponível em: 6. Maia JÁ, Menezes FA, Santos PAM. PERCEPÇÃO DOS PAIS SOBRE A
IMPORTÂNCIA
DE
ACOMPANHAMENTO
DO
CRESCIMENTO
E
DESENVOLVIMENTO INFANTIL. DêCiência em Foco. ISSN: 2526-5946. 2017; 1(2): 53-63. Acesso em: 21 de Julho de 2019. Disponível em: RC: 39116
Disponível em: https://www.nucleodoconhecimento.com.br/saude/incentivo-a-imunizacao 7. De Morais Júnior SLA et. al. A evolução histórica do calendário vacinal
brasileiro infantil. REVISTA ENFERMAGEM ATUAL | 2018; 85. Acesso em: 21
de
Julho
de
2019. Disponível
em:
https://revistaenfermagematual.com.br/index.php/revista/article/view/247/146 8. Coriolano-Marinus MWL et al. Comunicação nas práticas em saúde: revisão
integrativa da literatura. Saúde e Sociedade [online]. 2014, v. 23, pp. 1356-
1369. Acesso
em:
21
de
Julho
de
2019. Disponível
em:
https://doi.org/10.1590/S0104-12902014000400019. 9. Vasconcellos-Silva PR et al. A sociedade de risco midiatizada, o movimento
antivacinação e o risco do autismo. Ciência & Saúde Coletiva, 20(2):607-616,
2015. Acesso
em:
21
de
Julho
de
2019. Disponível
em:
http://www.scielo.br/pdf/csc/v20n2/1413-8123-csc-20-02-0607.pdf 10. Nardi ACF et. al. Comunicação em saúde: um estudo do perfil e da estrutura
das assessorias de comunicação municipais em 2014-2015* Epidemiol. Serv. Saude, Brasília, 27(2):e2017409, 2018. Acesso em: 21 de Julho de 2019. Disponível
em:
https://www.scielosp.org/article/ssm/content/raw/?resource_ssm_path=/media/
assets/ress/v27n2/2237-9622-ress-27-02-e2017409.pdf 11. Toscano C. Cartilha de vacinas: para quem quer mesmo saber das coisas /
Cristina Toscano, Ligia Kosim . - Brasília: Organização Pan-Americana da
Saúde, 2003. 40p. ISBN: 85-87943-29-4. Acesso em: 21 de Julho de 2019. Disponível em: http://bvsms.saude.gov.br/bvs/publicacoes/cart_vac.pdf 12. Brasil. Ministério da Saúde. Departamento de Vigilância das Doenças
Transmissíveis. Manual de Normas e Procedimentos para Vacinação. Brasília
: Ministério da Saúde, 2014. 176 : il. ISBN 978-85-334-2164-6. Acesso em: 21
de
Julho
de
2019. Disponível
em:
http://bvsms.saude.gov.br/bvs/publicacoes/manual_procedimentos_vacinacao
.pdf 12. Brasil. Ministério da Saúde. Departamento de Vigilância das Doenças
Transmissíveis. Manual de Normas e Procedimentos para Vacinação. Brasília
: Ministério da Saúde, 2014. 176 : il. ISBN 978-85-334-2164-6. Acesso em: 21
de
Julho
de
2019. Disponível
em:
http://bvsms.saude.gov.br/bvs/publicacoes/manual_procedimentos_vacinacao
.pdf 13. Mendes KDS, Silveira RCCP, Galvão CM. REVISÃO INTEGRATIVA: MÉTODO
DE PESQUISA PARA A INCORPORAÇÃO DE EVIDÊNCIAS NA SAÚDE E NA 13. Mendes KDS, Silveira RCCP, Galvão CM. REVISÃO INTEGRATIVA: MÉTODO
DE PESQUISA PARA A INCORPORAÇÃO DE EVIDÊNCIAS NA SAÚDE E NA RC: 39116
Disponível em: https://www.nucleodoconhecimento.com.br/saude/incentivo-a-imunizacao REVISTA CIENTÍFICA MULTIDISCIPLINAR NÚCLEO DO
CONHECIMENTO ISSN: 2448-0959
https://www.nucleodoconhecimento.com.br
ENFERMAGEM. Texto Contexto Enferm, Florianópolis, 2008 Out-Dez; 17(4):
758-64.
Acesso
em:
08
de
Agosto
de
2019.
Disponível
em:
http://www.scielo.br/pdf/tce/v17n4/18.pdf
14. Câmara RH. Análise de conteúdo: da teoria à prática em pesquisas sociais
aplicadas às organizações. Gerais: Revista Interinstitucional de Psicologia, 6
(2), jul - dez, 2013,179-191. Acesso em: 08 de Agosto de 2019. Disponível em:
http://pepsic.bvsalud.org/pdf/gerais/v6n2/v6n2a03.pdf
i d
A
t
2019 ENFERMAGEM. Texto Contexto Enferm, Florianópolis, 2008 Out-Dez; 17(4):
758-64. Acesso
em:
08
de
Agosto
de
2019. Disponível
em:
http://www.scielo.br/pdf/tce/v17n4/18.pdf 14. Câmara RH. Análise de conteúdo: da teoria à prática em pesquisas sociais
aplicadas às organizações. Gerais: Revista Interinstitucional de Psicologia, 6
(2), jul - dez, 2013,179-191. Acesso em: 08 de Agosto de 2019. Disponível em:
http://pepsic.bvsalud.org/pdf/gerais/v6n2/v6n2a03.pdf 14. Câmara RH. Análise de conteúdo: da teoria à prática em pesquisas sociais
aplicadas às organizações. Gerais: Revista Interinstitucional de Psicologia, 6
(2), jul - dez, 2013,179-191. Acesso em: 08 de Agosto de 2019. Disponível em:
http://pepsic.bvsalud.org/pdf/gerais/v6n2/v6n2a03.pdf Enviado: Agosto, 2019. Aprovado: Outubro, 2019. RC: 39116
Disponível em: https://www.nucleodoconhecimento.com.br/saude/incentivo-a-imunizacao RC: 39116
Disponível em: https://www.nucleodoconhecimento.com.br/saude/incentivo-a-imunizacao
|
https://openalex.org/W4283795046
|
https://zenodo.org/records/6792979/files/KBI_2010421009_Angga%20Kurnia%20Illahi_Tugas%20Pengganti%20UAS%20PAI.pdf
|
English
| null |
Potential of herpetofauna in traditional medicine, geographic distribution and new record of endemic Ichthyophis elongatus in West Sumatra.
|
Zenodo (CERN European Organization for Nuclear Research)
| 2,022
|
cc-by
| 1,699
|
Angga Kurnia Illahi (2010421009) - Department of Biology, FMIPA, Andalas
University, Padang. Angga Kurnia Illahi (2010421009) - Department of Biology, FMIPA, Andalas
University, Padang. Mapping and the potential distribution of animals are closely related to the field of
animal ecology, through an ecological approach we can determine how the habitat or niche is
suitable for an animal. so that its natural availability is maintained. fields that are suitable for
collaborating with this field are phylogeographic, ethnozoology and taxonomy. The results
obtained by mixing these fields are that some animals from herpetofauna are used for
traditional medicine in West Sumatra by looking at and reviewing the geographic potential and
new records in West Sumatra, especially endemic animals. This study is not very common,
especially in today's era where many have turned to modern medicine. However, with the
richness of ethnicity and biodiversity in Indonesia, it is necessary to pay attention to it. it is also
very useful to be the basis or basis for modern medical research that refers to traditional
medicine. In this article, the phylogeographic, taxonomic and ethnozoological studies are
herpetofauna, some of which are endemic to the region of West Sumatra. Herpetofauna comes from corn, the word herpeton, which means reptiles. In the past,
before taxonomy developed, Reptiles are included in one group of animals because they are
considered to be both reptiles from amphibians. With the development of science, they are now
two separate groups. These two groups belong to one field, namely herpetology because they
have a way of life from almost similar habitats, both are ectothermal vertebrates (requires an
external heat source), and methods for the observer's arms (Kusrini, et al., 2008). Reptiles have
long been used by the community. For example, snakes are fauna resources that are widely
used as one of the most common commodes have high economic value. Snakes are used, among
others, as material for medical experiments, pets, craft materials (bags, shoes, belts, etc.) and
for consumption (Situngkir, 2009). Furthermore, the questionnaire used for In West Sumatra
can find traditional medicine at each quota, but there is not much information that is different
from the one selected. In terms of traditional medicine in other areas, such as the results of the
1995 Susenas which explained the use of traditional medicine by the community for treatment
in DKI Jakarta 2.76%, DI Yogyakarta 3.19%, East Java 5.59%. Angga Kurnia Illahi (2010421009) - Department of Biology, FMIPA, Andalas
University, Padang. The results of the Susenas can
be evidence that traditional medicine is still widely used by people from certain circles,
including intellectuals (Santoso, 1998). Amphibians also provide benefits to humans, such as
as a source of animal protein or direct benefits as part of the food chain. Millet skin secretions
of several types of amphibians have also been developed as antibiotics from painkillers
(Stebbins and Cohen, 1995). One type of species from the herpetofauna group which is endemic and found in West
Sumatra and is rarely known, namely Ichthyophis elongatus. Ichthyophis elongatus Taylor,
1965, is only known from the type locality in Padang, West Sumatra. This species is considered
Data Deficient by the International Union for the Conservation of Nature (IUCN SSC
Amphibian Specialist Group 2018). In addition, no single occurrence is available at the Global
Biodiversity Information Facility (https://www.gbif.org/) and publications on the species do
not have geographic coordinates (Teynie et al. 2010). To support sampling efforts in the field,
the maximum entropy algorithm (Phillips et al. 2006; Elith et al. 2011) was used to identify the potential distribution of Ichthyophis elongatus. MaxEnt software was chosen because it
provides satisfactory predictions with a small number of events (Yi et al. 2016; Ramos and
Torres 2011; Adhikari et al. 2012; Yang et al. 2013; Padalia et al. 2014; Remya et al. 2015;
According to Yuan et al. 2015; Pranata et al. 2019). The map generated from MaxEnt can be
useful for further studies on I. elongatus in particular and other Sumatran in general. Predicted
distribution based on entropy modelling maximum indicates a highly suitable habitat for the
species throughout the Barisan Mountains at 500–1000 m above sea level. Our model suggests
swamps, rice fields, and secondary forest 100–150 m from the river as the highest habitat for
the species. Further surveys in the two regions predicted by the model found new species
population. The research method used in researching the potential of herpetofauna in traditional
medicine is the survey method from questionnaires and direct documents. Traditional
medicines are ingredients or ingredients of plant materials, animal ingredients, mineral
ingredients, field extract preparations (Sugiyono, 2007). The survey was conducted to see the
presence of informants to obtain information about the use of herpetofauna as ingredients of
traditional medicine. Furthermore, questionnaires were used to obtain information on millet
informants who used herpetofauna as ingredients of traditional medicine (Sundari, 2011). Angga Kurnia Illahi (2010421009) - Department of Biology, FMIPA, Andalas
University, Padang. The
informants selected to be interviewed are traditional medicine sellers in shops from traditional
medicine sellers at street vendors. Meanwhile, research on new records and potential
geographic distribution, especially Ichthyophis elongatus and elongated caecelian endemic to
West Sumatra, was carried out with field surveys and recording of ecological parameters. We
conducted field surveys in four areas in West Sumatra: Padang (5–8 August 2017), Solok (20
December 2018), Tanah Datar (6–9 March 2019), and Pasaman (20–24 February 2020). We
actively search for caecilians by digging the soil with a hoe, turning rocks, leaf litter, and rotting
plants, during the day (09:00 – 16:00). The target habitats are garbage dumps, rice fields,
riverbanks, and secondary forests. and also the method used. Morphological measurements. Specimens euthanized with tricaine methanesulfonate (MS-222), fixed with 10% formalin
andstored in eta 70%. following the morphological measurements and diagnosis established by
Taylor (1965) to identify the species as I. elongatus. After that, the species distribution
modeling (HR) was carried out. by using the GeoCat Redlisting tool (Bachman et al. 2011) to
assess the extinction risk of I. elongatus based on our combined field survey data. then make a
distribution map and finally validate the field survey. It can be concluded by looking at the existing data and literacy that the species
Ichthyophis elongatus has little study on the geographical distribution. Thus, it is difficult to
study the potential possessed by Ichthyophis elongatus and conservation. The potential of
herpetofauna as an ingredient of traditional medicine varies from region to region West
Sumatra, each region has its own beliefs. West Sumatran people who still use traditional
medicine with ingredients from one of the genus Ichthyophis groups which are included in the
herpetofauna group become a separate problem. Especially endemic animal species that are
very easy to be threatened with extinction. Their survival is very dependent on the habitat which
is an important factor for these animals. the two methods and approaches used to determine the
distribution of geographic potential and its potential as traditional medicine are both carried
out in the field and in the same area, namely West Sumatra and mutual fallows are important
factors for these animals. The two methods and approaches used to determine the distribution
of geographic potential and its potential as traditional medicine are both carried out in the field and in the same area, namely West Sumatra and are interconnected. Angga Kurnia Illahi (2010421009) - Department of Biology, FMIPA, Andalas
University, Padang. knowing the potential for
geographic distribution and the record of new endemic animals and the potential of these
animals as traditional medicines in the community. conservation efforts and interior studies on
traditional medicine can be carried out without harming any aspect and will produce solutions
for the conservation and potential of herpetofauna in traditional medicine. BIBLIOGRAPHY Adhikari D, Barik SK, Upadhaya K (2012) Pemodelan distribusi habitat untuk introduksi
kembali Ilex khasiana Purk., spesies pohon paling berbahaya di timur laut India. Rekayasa Ekologi. 40: 37–43. https://doi.org/10.1016/j.ecoleng.2011.12.04 Bachman S, Moat J, Hill AW, De La Torre J, Scott B (2011) Mendukungn penilaian ancaman
Daftar Merah dengan geocat: alat konservasi penilaian geospasial. ZooKeys 150:
117–126. https://zookeys.pensoft.net/articles.php?id=3037 Elith J, Phillips SJ, Hastie T, Dudik M, Chee YE, Yates CJ (2011). Penjelasan statistik MaxEnt
untuk
ahli
ekologi. Keanekaragaman
dan
Distribusi
17
(1):
43–57. https://doi.org/10.1111/j.1472-4642.2010. Kusrini, M. D., A.U.Ul-Hasanah dan W. Endarwin. (2008). Pengenalan Herpetofauna -
Disampaikan Pada Pekan Ilmiah Kehutanan Nasional. Institut Pertanian Bogor. Bogor. Padalia H, Srivastava V, Kushwaha SPS (2014) Pemodelan kisaran invasi potensial spesies
invasif asing, Hyptis suaveolens (L.) titik. di India: Perbandingan MaxEnt dan GARP. Ekologis Dalam format 22: 36–43. https://doi.org/10.1016/j.ecoinf.2014.04.002 Phillips SJ, Anderson RP, Schapire RE (2006) Pemodelan en tropi maksimum dari distribusi
geografis spesies. Pemodelan ekologi 190 (3–4): 231–259.https://doi.org/10.1016/j Pranata S, Sulistijorini S, Chikmawati T (2019) Ekologi Rafflesia arnoldii (Rafflesiaceae) di
Pandam Gadang Sumatera Barat. Jurnal Ilmu Kehidupan Tropis 9 (3): 243–251. https://www.doi.org/ Ramos LT, Torres AM (2011) Mengembangkan database georeferensi darispesies pohon hutan
terpilih yang terancam di Filipina. Jurnal Sains Filipina 141 (2): 165–177. Santoso, SS (1998). Keuntungan Penderita Diabetes mellitus Yang Berobat ke Tradisional di
DKI Jakarta. DIYogyakarta. Situngkir, S. (2009). Pemanfaatan Secara Tradisional Wilayah Bogor dari Perdagangan. FakultasKehutanan. Institut PertanianTerima kasih kepada dari Alan Handru Bogor. Bogor Stebbins, RC dan NW Cohen. (1995). Sejarah Alam Amfibi Pers Universitas Princeton. Jersey
baru. Sundari, WS (2011). Etnobotani UpacaraAdat Batagak Minangkabau Panghulu Masarakat
Sumatera di Barat. Universitas Andalas. padang Sugiyono (2007). Memahami Penelitian Kualitatif Alfabet, Bandung. Teynie A, David P, Ohler A (2010) Catatan tentang koleksi amfibi dan reptil dari Sumatera
Barat (Indonesia), dengan deskripsi spesies baru dari genus Bufo. Zootaxa 2416 (1): 1–
43. https://doi.org/10.11646/zootaxa.2416.1.1 Remya K, Ramachandran A, Jayakumar S (2015) Memprediksi distribusihabitat yang cocok
saat ini dan masa depan dari Myristica dacty loides Gaertn. menggunakan model
MaxEnt
di
Ghats
Timur,
India. Rekayasa
Ekologi
82:
184–188. https://doi.org/10.1016/j. Yang XQ, Kushwaha SPS, Saran S, Xu J, Roy PS (2013) Pemodelan maksimaluntuk
memprediksi distribusi potensi tanaman obat, Justicia adhatoda L. dikaki bukit
Himalaya Kecil. Rekayasa Ekologi 51: 83–87. https://doi.org/10.1016/j.ecoleng. Yi Y, Cheng X, Yang ZF, Zhang SH (2016) Pemodelan maksimal untuk memprediksi
distribusi potensi tanaman obat yang terancam punah (H.riparia Lour) di Yunnan, Cina. Rekayasa Ekologi 92: 260–269. BIBLIOGRAPHY https://doi.org/10.1016/j.ecoleng.2016.04.010 Yuan HS, Wei YL, Wang XG (2015) Pemodelan maksimal untuk memprediksi distribusi
potensial Sanghuang, kelompok penting jamur obat di Cina. Ekologi Jamur 17: 140–
145.https://www.sciencedirect.com/science/article/abs/pii/S1754504815000793?via%
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A case report of successful treatment of pyoderma gangrenosum in a patient with autoimmune hepatitis, and review of the literature
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© 2015 Androutsakos et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. CASE REPORT Open Access Androutsakos et al. BMC Gastroenterology (2015) 15:149
DOI 10.1186/s12876-015-0376-1 Androutsakos et al. BMC Gastroenterology (2015) 15:149
DOI 10.1186/s12876-015-0376-1 Abstract Background: Pyoderma Gangrenosum (PG) is a cutaneous condition, its diagnosis suggested by the presence of a
painful cutaneous ulcer showing rapid progression. Pyoderma gangrenosum is associated with a concomitant systemic
disease in 50 to 70 % of cases, including inflammatory bowel disease (IBD), rheumatoid arthritis, and lymphoproliferative
disorders. Although PG has also been reported with viral hepatitis, it is rarely associated with autoimmune hepatitis. Case presentation: A 19-year-old Caucasian female, with a prior diagnosis of autoimmune hepatitis (AIH) in remission,
presented with bilateral lower limb ulcers 4 years after the diagnosis of AIH. She was diagnosed with PG and treated
with high-dose prednisolone, methotrexate and cyclosporine. One year later she was well, the ulcers completely healed,
and with the autoimmune hepatitis still in remission. Conclusion: We report a case of autoimmune hepatitis and the subsequent, rarely occurring, extra-hepatic onset of
pyoderma gangrenosum, with the AIH in remission, strengthening the association between the two conditions. Since
both the AIH and the PG can present serious diagnostic challenges, thus delaying vital therapy, it is important that the
development of either prompts us to consider the possibility of the other developing in the future or if already present
facilitate its diagnosis, such considerations making the case for a systematic follow up. * Correspondence: T_Androutsakos@yahoo.gr
1Pathophysiology Department, Laiko General Hospital, National and
Kapodistrian University of Athens, Medical School, Athens, Greece
Full list of author information is available at the end of the article Case presentation
Clinical aspects azathioprine was reintroduced at 150 mg, while the ste-
roids were increased from 5 to 20 mg prednisolone, daily. The patient was discharged pending the pathology and
bacteriology results. A 19-year-old Caucasian female presented to us with bilat-
eral lower limb ulcers. She reported a scratch on her left
ankle 3 months earlier that worsened over time into an
ulcer, in spite of antibiotic treatment. A week later, new
lesions appeared on the left leg and similar ones were seen
on her right leg. The patient denied fever, weight loss, or
other signs or symptoms of systemic illness. Four years
earlier she had been admitted at another hospital with
transaminasemia and jaundice and was diagnosed with
autoimmune hepatitis based on serological (ANA and
ASMA) positivity and compatible histopathological fea-
tures, in the absence of viral markers. Treatment with
prednisolone and azathioprine brought the disease into
remission. The patient was on 5 mg prednisolone upon
presentation. She returned a week later with arthritis of both ankles
and worsening of the ulcers (Fig. 1b). Further cultures were
again negative. The biopsy was interpreted as being com-
patible with pyoderma gangrenosum (Fig. 2a, b), hence
prednisolone 1 mg/kg was started, azathioprine was
switched to cyclosporine at 150 mg that was further
increased to 250 mg daily, and methotrexate 10 mg/week
was added to the regimen. Given the rare combination of
pyoderma gangrenosum with autoimmune hepatitis, upper
and lower endoscopies, as well as upper abdominal
MRI and Magnetic Resonance Cholangio-pancreatography
(MRCP) were done to exclude more common associated
diseases. All of the investigations were negative. A review
of the initial liver biopsies confirmed the diagnosis of auto-
immune hepatitis, showing dense portal and periportal
lymphocytic and plasmacytic infiltrates, as well as mild
periportal fibrosis. The patient was again discharged, with
slow tapering of the steroids. On admission, the patient was afebrile, bearing purulent,
painful ulcers in both legs (Fig. 1a). Lab results showed
the inflammation markers just exceeding the upper nor-
mal limits (Table 1). Transaminases were within a normal
range, while anti-nuclear antibodies (ANA) were positive
at a titer of 1/320 and anti-smooth muscle antibodies
(ASMA) were positive at a low titer (1/80). Cryoglobulin
test was negative. The lesions were cultured for bacteria
and Mycobacterium tuberculosis, and empiric therapy
was started with clindamycin and moxifloxacin. Background gangrenosum is associated with a concomitant disease in
50 to 70 % of cases, including inflammatory bowel disease
(IBD) in 10 to 15 % [6], rheumatoid arthritis [7], and lym-
phoproliferative disorders [8–10]. Although PG has been
encountered with viral hepatitis [11], only a small number
of cases have been reported with autoimmune hepatitis
(AIH) [12–15]—one associated with concurrent primary
sclerosing cholangitis and ulcerative colitis [13]. Pyoderma Gangrenosum (PG), classified as a neutro-
philic dermatosis, is an ulcerative cutaneous condition,
first described in 1930 by Brusting and colleagues [1, 2]. Commonly, the lesions present as tender pustules that
evolve into enlarging suppurative ulcers, with a tendency
to last for months or years. Although of non-infectious etiology, the pathogenesis of
the disease is unclear, but evidence suggests an underlying
defective neutrophilic function [1, 3]. After excluding
more common causes of cutaneous ulcerations, the diag-
nosis of PG is suggested by the presence of a painful,
necrolytic cutaneous ulcer, with an irregular undermined
border, showing rapid progression [1]. Histologically, there
is lymphocytic infiltration in the early stages followed by
neutrophilic infiltration and hemorrhage [4, 5]. Pyoderma Treatment of PG is non-surgical and consists of a
combination of local wound care and systemic therapy,
the latter centered on the use of high-dose corticoste-
roids [16]. Other treatment agents include immunosup-
pressants, intravenous immunoglobulin, and biologic
agents—such as tumor necrosis factor alpha inhibitors
[16]. We report a 19-year-old patient, with a history of
AIH in remission, who presented with pyoderma gang-
renosum of the lower extremities, and was treated suc-
cessfully with cortisone, cyclosporine and methotrexate. * Correspondence: T_Androutsakos@yahoo.gr
1Pathophysiology Department, Laiko General Hospital, National and
Kapodistrian University of Athens, Medical School, Athens, Greece
Full list of author information is available at the end of the article Page 2 of 4 Page 2 of 4 Androutsakos et al. BMC Gastroenterology (2015) 15:149 Case presentation
Clinical aspects Magnetic
Resonance Imaging (MRI) of the region of ulcers in both
legs revealed diffuse subcutaneous oedema and subcuta-
neous nodular lesions on the dorsal surface of the feet. With a working diagnosis of pyoderma gangrenosum, a
skin biopsy was acquired, her previous medication of On follow up a month later she was feeling well, with no
ankle swelling, and with all ulcers healing satisfactorily
(Fig. 1c). Six months hence she was on 4 mg methylpred-
nisolone, 100 mg cyclosporine, and 7.5 mg methotrexate,
with normal liver enzymes, and with the ulcers nearly
healed (Fig. 1d). Cyclosporine was discontinued at
8 months, and a year later the patients was on 4 mg
methylprednisolone and 7.5 mg methotrexate, with
further improvement and complete healing of the ul-
cers (Fig. 1e). Fig. 1 Images of ulcerated areas. a On admission. b A week later. The swelling and the inflammation are noted. c One month later. Significant
improvement of the ulcerating areas. d At 6 months. The inflammation has subsided, with further improvement of the ulcerating areas. e At one
year. No new ulcers and healed old ones Fig. 1 Images of ulcerated areas. a On admission. b A week later. The swelling and the inflammation are noted. c One month later. Significant
improvement of the ulcerating areas. d At 6 months. The inflammation has subsided, with further improvement of the ulcerating areas. e At one
year. No new ulcers and healed old ones Androutsakos et al. BMC Gastroenterology (2015) 15:149 Page 3 of 4 Page 3 of 4 Table 1 Patient’s lab results
Lab test
Normal values
Result
Hgb (g/dl)
12,0–16,0
12,4
Hct (%)
38–47
37,7
WBC (K/μL)
4,5–11,0
12,300
Neut/Lymph/Mono/Eos (%)
69/25/5/0
PLT (K/μL)
140–440
357
Glu (mg/dl)
74–106
75
Urea (mg/dl)
15–40
25
Creatinine (mg/dl)
0,6–1,1
0,7
AST (U/L)
5–31
13
ALT (U/L)
5–34
11
γGT (U/L)
7–36
12
ALP (U/L)
48–141
59
INR
0,9–1,15
1
HBA1c (%)
4,8–6
5,5
TSH (mU/L)
0,3–4
3
FT4 (pmol/L)
10–25
17
Total Protein (g/dl)
6,4–8,3
7
Albumin (g/dl)
3,5–5,2
4,1
Total Bilirubin (mg/dl)
0,3–1,2
0,47
Direct Bilirubin (mg/dl)
0–0,3
0,14
Sodium (mMol/L)
136–145
144
Potassium (mMol/L)
3,5–5,1
4,2
CRP (mg/dl)
0–5
15,3
ESR (mm)
0–20
38 (IgG) and autoantibodies, in the absence of viral markers
[17]. Case presentation
Clinical aspects Based on the autoantibody profile, two types of AIH
are recognized: type 1 (AIH-1), positive for ANA and/or
anti-smooth muscle antibody (SMA), and occurring at
any age, and type 2 (AIH-2), positive for anti-liver-kidney
microsomal antibody (anti-LKM) or for anti-liver cytosol
type-1 antibody (anti-LC-1), with a peak incidence in chil-
dren and adolescents [17–19]. AIH generally responds to
immunosuppressive treatment, but if left untreated usually
progresses to liver failure requiring transplantation [17]. Extra-hepatic disorders, mainly autoimmune condi-
tions, are common in autoimmune hepatitis and occur
in all stages of liver disease, including ulcerative colitis,
Crohn’s disease, vasculitis, arthritis, thyroiditis, diabetes
mellitus, autoimmune hemolytic anemia, glomeruloneph-
ritis, fibrosing alveolitis, systemic lupus erythematosus,
coeliac disease, sicca syndrome, vitiligo, or lymphoprolifer-
ative syndromes [20, 21]. On the other hand, PG is associated with a variety of
mainly autoimmune diseases in 50–70 % of cases, with in-
flammatory bowel disease (IBD) topping the list at 10–15 %
[6]. However, there is a dearth of data on the association of
PG with AIH, as a search of the English-language literature
unearthed only fifteen other cases [15]. Our patient had AIH-1, as the majority of reported
cases when the type of AIH was identified, although
AIH-2 may be underestimated [15]. Our patient’s gender,
the age at diagnosis of AIH, and the interval to the sub-
sequent onset of PG concur with those of the other re-
ported cases, as does the development of PG during a
quiescent stage of the AIH. Our patient remained in re-
mission subsequently, even with the use of potentially
hepatotoxic agents. Although a fulminant presentation
of AIH has been proposed as a possible risk factor for
the subsequent development of PG [15], our patient’s
hepatitis course was not severe, emphasizing our poor
understanding of the pathogenesis of PG in relation to
AIH. Finally, pathergy, although rare [15], is a hallmark
of PG, and our patient’s first appearance of an ulcer was Commentary Autoimmune hepatitis is a chronic self-perpetuating in-
flammatory disease occurring in all ages and races [17]. Besides its diverse presentation and heterogeneity of the
clinical features, AIH is characterized biochemically by el-
evated transaminases, histologically by interface hepatitis,
and serologically by increased levels of immunoglobulin G Fig. 2 Images of skin biopsy. a Abscess formation in the epidermis (red arrow) with accumulation of neutrophils and granuloma (green arrows)
formation in the dermis. b The edge of an ulcer: deposits of eosinophilic material on the wall of the small blood vessels (blue arrow) Fig. 2 Images of skin biopsy. a Abscess formation in the epidermis (red arrow) with accumulation of neutrophils and granuloma (green arrows)
formation in the dermis. b The edge of an ulcer: deposits of eosinophilic material on the wall of the small blood vessels (blue arrow) Page 4 of 4 Page 4 of 4 Androutsakos et al. BMC Gastroenterology (2015) 15:149 at a previously “scratched” area, raising the distinct pos-
sibility of such event. 2. Powell FC, Su WP, Perry HO. Pyodermagangrenosum classification and
management. J Am Acad Dermatol. 1996;34:395–409. doi:10.1016/S0190-
9622(96)90428-4. PMID: 8609250. 2. Powell FC, Su WP, Perry HO. Pyodermagangrenosum classification and
management. J Am Acad Dermatol. 1996;34:395–409. doi:10.1016/S0190-
9622(96)90428-4. PMID: 8609250. 3. Wollina U. Pyodermagangrenosum—a review. Orphanet J Rare Dis. 2007;2:19. doi:10.1186/1750-1172-2-19. 3. Wollina U. Pyodermagangrenosum—a review. Orphanet J Rare Dis. 2007;2:19. doi:10.1186/1750-1172-2-19. Author details
1 1Pathophysiology Department, Laiko General Hospital, National and
Kapodistrian University of Athens, Medical School, Athens, Greece. 2Second
Department of Propedeutic Surgery, Laiko General Hospital, National and
Kapodistrian University of Athens, Medical School, Athens, Greece. 3Department of Dermatopathology, Medical School, National and
Kapodistrian University of Athens, Athens, Greece. 21. Teufel A, Weinmann A, Kahaly GJ, Centner C, Piendl A, Worns M, et al. Concurrent autoimmune diseases in patients with autoimmune hepatitis. J Clin Gastroenterol. 2010;44:208–13. doi:10.1097/MCG.0b013e3181c74e0d. PMID: 20087196. Received: 20 July 2015 Accepted: 14 October 2015 Received: 20 July 2015 Accepted: 14 October 2015 Authors’ contribution 16. Miller J, Yentzer BA, Clark A, Jorizzo JL, Feldman SR. Pyoderma
gangrenosum: A review and update on new therapies. J Am Acad
Dermatol. 2010;62:646–54. doi:10.1016/j.jaad.2009.05.030. PMID: 20227580. TA took care of the patient, collected all data and clinical images, and
drafted the manuscript. GH was the hepatologist in charge of the patient
and contributed in the drafting of the manuscript. PS provided surgical
expertise about the patient’s ulcers and took the biopsies from the lesions. KA was the pathologist that examined the biopsy and provided the biopsy
pictures. All authors read and approved the final manuscript. 17. Manns MP, Lohse AW, Vergani D. Autoimmune hepatitis – Update 2015. J Hepatol. 2015;62(1 Suppl):S100–11. doi:10.1016/j.jhep.2015.03.005. PMID:
25920079. 18. Liberal R, Grant CR, Mieli-Vergani G, Vergani D. Autoimmune hepatitis: a
comprehensive review. J Autoimmun. 2013;41:126–39. doi:10.1016/
j.jaut.2012.11.002. PMID: 23218932. Consent 11. Keane FM, MacFarlane CS, Munn SE, Higgins EM. Pyoderma gangrenosum
and hepatitis C virus infection. Br J Dermatol. 1998;139:924–5. doi:10.1046/
j.1365.1998.02531.x. PMID: 9892972. Written consent was obtained from the patient for publica-
tion of this Case report and any accompanying images. A
copy of the written consent is available for review by the
Editor of this journal. 12. Goldenberg G, Jorizzo JL. Use of etanercept in treatment of pyoderma
gangrenosum in a patient with autoimmune hepatitis. J Dermatol Treat. 2005;16(5-6):347–9. doi:10.1080/09546630500424722. PMID: 16428159. 13. Koskinas J, Raptis I, Manika Z, et al. Overlapping syndrome of
autoimmune hepatitis and primary sclerosing cholangitis associated with
pyoderma gangrenosum and ulcerative colitis. Eur J Gastroenterol
Hepatol. 1999;11:1421–4. doi:10.1097/00042737-199912000-00014. PMID:
10654805. Conclusion 4. Su WP, Schroeter AL, Perry HO, Powell FC. Histopathologic and
immunopathologic study of pyodermagangrenosum. J CutanPathol. 1986;13:323–30. PMID: 3537032. 4. Su WP, Schroeter AL, Perry HO, Powell FC. Histopathologic and
immunopathologic study of pyodermagangrenosum. J CutanPathol. 1986;13:323–30. PMID: 3537032. In summary, we report a patient with autoimmune hepatitis
and pyoderma gangrenosum treated successfully with corti-
sone, cyclosporine, and methotrexate. Thorough testing
excluded concurrent autoimmune diseases, confirming the
association of pyoderma gangrenosum with autoimmune
hepatitis, even when the latter is inactive. Clearly, the diag-
nosis of PG should raise the possibility of concomitant
AIH, since its diagnosis can be challenging and the results
of failure to treat it devastating. On the other hand, AIH
should sensitize us to the possible future development of
PG. Overall, our case adds to the pool of knowledge
about the presentation and management of PG in asso-
ciation with AIH. 5. Callen JP. Pyodermagangrenosum. Lancet. 1998;351:581–5. doi:10.1016/
S0140-6736(97)10187-8. PMID: 9492798. 5. Callen JP. Pyodermagangrenosum. Lancet. 1998;351:581–5. doi:10.1016/
S0140-6736(97)10187-8. PMID: 9492798. 6. Bernstein CN, Blanchard JF, Rawsthorne P, et al. The prevalence of extraintestinal
diseases in inflammatory bowel disease: a population based study. Am J
Gastroenterol. 2001;96:1116–22. doi:10.1111/j.1572-0241.2001.03756.x. PMID:
11316157. 7. Stolman LP, Rosenthal D, Yaworsky R, et al. Pyoderma gangrenosum and
rheumatoid arthritis. Arch Dermatol. 1975;111:1020–3. doi:10.1001/
archderm.1975.01630200080009. PMID: 786171. 8. Powell FC, Schroeter AL, Su WP, et al. Pyoderma gangrenosum: a review of
86 patients. Q J Med. 1985;55:173–86. PMID: 3889978. 7. Stolman LP, Rosenthal D, Yaworsky R, et al. Pyoderma gangrenosum and
rheumatoid arthritis. Arch Dermatol. 1975;111:1020–3. doi:10.1001/
archderm.1975.01630200080009. PMID: 786171. 7. Stolman LP, Rosenthal D, Yaworsky R, et al. Pyoderma gangrenosum and
rheumatoid arthritis. Arch Dermatol. 1975;111:1020–3. doi:10.1001/
archderm.1975.01630200080009. PMID: 786171. 8. Powell FC, Schroeter AL, Su WP, et al. Pyoderma gangrenosum: a review of
86 patients. Q J Med. 1985;55:173–86. PMID: 3889978. 9. Török L, Kirschner A, Gurzo M, et al. Bullous pyoderma gangrenosum as a
manifestation of leukemia cutis. Eur J Dermatol. 2000;10:463–5. PMID:
10980469. 10. Wayte JA, Rogers S, Powell FC. Pyoderma gangrenosum, erythema elevatum
diutinum and IgA monoclonal gammopathy. Australas J Dermatol. 1995;36:21–3. doi:10.1111/j.1440- 0960.1995.tb00919.x. PMID: 7763217. Abbreviations PG: Pyoderma gangrenosum; IBD: Inflammatory bowel disease;
AIH: Autoimmune hepatitis; ANA: Anti-nuclear antibodies; ASMA: Anti-
smooth muscle antibodies; MRI: Magnetic resonance imaging;
MRCP: Magnetic resonance cholangiopancreatography; Anti-LKM: Anti-liver
kidney microsomal antibodies; Anti-LC-1: Anti-liver cytosol type 1 antibodies;
IgG: Immunoglobulin G. 14. Halac U, Dhaybi RA, Powell J, Soglio DB-D, Alvarez F. Unusual association
between autoimmune hepatitis and severe pyoderma gangrenosum. JPGN. 2010;50:219–21. doi:10.1097/MPG.0b013e31819a62f5. PMID: 19590450. 15. Wat H, Haber RM. Trends in the association between Pyoderma
Gangrenosum and Autoimmune Hepatitis: Case report and Review of the
Literature. J Cutan Med Surg. 2014;18(5):345–52. doi:10.2310/
7750.2013.13177. PMID: 25186997. Acknowledgements
h
h
h
k The authors thank our patient for her participation and her consent to
publish these data. We also wish to thank Professor Athanasios Tzioufas,
Director of Department of Pathophysiology of Laiko General Hospital, for his
help in the diagnosis and treatment of the patient. 19. Vierling JM. Autoimmune hepatitis and overlap syndromes: Diagnosis and
Management. Clin Gastroenterol Hepatol 2015; PMID: 26284592 DOI:
10.1016/j.cgh.2015.08.012 19. Vierling JM. Autoimmune hepatitis and overlap syndromes: Diagnosis and
Management. Clin Gastroenterol Hepatol 2015; PMID: 26284592 DOI:
10.1016/j.cgh.2015.08.012 20. Muratori P, Fabbri A, Lalanne C, Lenzi M, Muratori L. Autoimmune liver
disease and concomitant extrahepatic autoimmune disease. Eur J
Gastroenterol Hepatol. 2015;27(10):1175–9. doi:10.1097/
MEG.0000000000000424. PMID: 26148248. 20. Muratori P, Fabbri A, Lalanne C, Lenzi M, Muratori L. Autoimmune liver
disease and concomitant extrahepatic autoimmune disease. Eur J
Gastroenterol Hepatol. 2015;27(10):1175–9. doi:10.1097/
MEG.0000000000000424. PMID: 26148248. Competing interests p
g
The authors declare that they have no competing interests. 1.
Su WP, Davis MD, Weenig RH, et al. Pyoderma gangrenosum: clinicopathologic
correlation and proposed diagnostic criteria [review]. Int J Dermatol.
2004;43:790–800. doi:10.1111/j.1365-4632.2004.02128.x. PMID: 15533059. References 1. Su WP, Davis MD, Weenig RH, et al. Pyoderma gangrenosum: clinicopathologic
correlation and proposed diagnostic criteria [review]. Int J Dermatol. 2004;43:790–800. doi:10.1111/j.1365-4632.2004.02128.x. PMID: 15533059.
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Exploring a Novel Approach to Establishing a Geoscience 3D Printing Lab in the Era of Digital Earth
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International journal of education and humanities
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2. 3D printing and Digital Earth In the global wave of digitalization, emerging technologies
such as the metaverse and artificial intelligence are rapidly
advancing, bringing profound impacts to various industries. Consequently, there is an increasing demand for scientific and
technological innovation talents, especially in emerging fields. Among them, 3D printing technology, as an interdisciplinary
emerging technology, has become a highly sought-after
innovation frontier in various industries, and its practical
applications
in
the
education
sector
have
already
demonstrated its power. At the same time, the concept of the
digital Earth in the field of Earth sciences provides us with a
new research objective and methodology. Therefore,
combining 3D printing technology with the concept of the
digital Earth, it becomes an important task to construct a new
type of university laboratory to cultivate innovative talents
that meet the needs of the times. 3D printing technology encompasses the integration of
virtual simulation, three-dimensional mathematical modeling,
computer-aided manufacturing, and other information
technologies with material sciences. It serves as a remarkable
outcome of interdisciplinary integration and has significantly
impacted various fields, including military aerospace,
healthcare, education, fashion, and jewelry design. However,
the application of 3D printing technology in Earth sciences,
particularly within the domain of geoscience research in the
context of the digital Earth, requires further exploration. The concept of the digital Earth entails a comprehensive
digital representation and simulation of spatial data related to
the Earth's surface, made possible through the utilization of
information technology. Digital Earth technology empowers
us to observe, study, and predict intricate details about our
planet. University laboratories are important places for teaching
and scientific research, bearing the responsibilities of
experimental teaching, scientific research, and practical
production. Laboratories not only provide experimental
equipment and space but also cultivate students' rigorous
thinking, inventiveness, and scientific work attitude. The profound integration of 3D printing technology with
the realm of geoscience has unlocked new avenues for
laboratory research and teaching. By exploring innovative
models for geoscience 3D printing laboratories within the
digital Earth framework, this paper aims to optimize the
design of laboratory layouts, establish open laboratory
courses, facilitate the formation of 3D printing associations,
and guide participation in various levels of competitions. These approaches are designed to cultivate a new generation
of adaptable talents who possess innovative thinking,
multidisciplinary perspectives, and the ability to thrive in a
rapidly evolving world. 2. 3D printing and Digital Earth In the context of digitalization, the core issue regarding the
construction of a geoscience 3D printing laboratory is the
optimization of the hardware and soft environments. This
includes aspects such as laboratory layout design, open course
settings, 3D printing associations, competitions at various
levels and in various disciplines. By establishing a new model
for geoscience 3D printing laboratories in the context of the
digital Earth, we aim to cultivate high-quality talents with
innovative
thinking,
innovative
capabilities,
and
a
multidisciplinary perspective. This will provide new insights
and directions for our higher education industry, driving it
towards a more innovative, open, and high-quality direction. Qinghua Song*, Zhenhua Wang Qinghua Song*, Zhenhua Wang School of information and Engineering, China University of Geosciences Beijing, China
* Corresponding author: song@cugb.edu.cn School of information and Engineering, China University of Geosciences Beijing, China
* Corresponding author: song@cugb.edu.cn Abstract: The rapid development of Digital China necessitates the cultivation of millions of individuals equipped with digital
knowledge structures and practical abilities. The establishment of digital laboratories represents a significant innovation in
teaching and management. This research endeavors to explore the construction of a geoscience 3D printing laboratory within the
framework of the digital Earth, employing a well-designed integration of hardware and soft environments. The laboratory offers
a comprehensive platform for teaching, research, innovation, and learning for both educators and students, leveraging state-of-
the-art computer technologies. By incorporating 3D modeling and 3D printing technologies into laboratory teaching, a seamless
connection between theoretical instruction and practical application is achieved, fostering reciprocal reinforcement between
teaching and learning. With the support of the geoscience 3D printing laboratory, students have accomplished notable
achievements, such as patent publications and research papers, as well as outstanding performance in various competitions at all
levels. These accomplishments demonstrate that this model has effectively stimulated students' enthusiasm for learning,
enhanced their problem-solving skills, and unveiled a promising pathway for the development of geoscience 3D printing
laboratories in nurturing digital talents. eywords: Digital Earth, Innovative talents, 3D printing, Laboratory construction. Keywords: Digital Earth, Innovative talents, 3D printing, Laboratory construction. International Journal of Education and Humanities
ISSN: 2770-6702 | Vol. 9, No. 3, 2023 International Journal of Education and Humanities
ISSN: 2770-6702 | Vol. 9, No. 3, 2023 3.1. Hardware Equipment and Layouts The laboratory space is structured into a printing area and
a modeling area. The printing area serves as the workspace
for 3D printers, while the modeling area is where students
build their models. As shown in Figure 2, the layout design Figure 2. Space structure of 3D printing laboratory
3.1.1. Printing Area
In the printing area of the laboratory, an Intel i7 series
personal computer with a dedicated graphics card is installed. This computer serves as the control center for managing and
operating multiple advanced 3D printing devices. The
computer is connected to the Projet 660 Pro full-color 3D
printer from 3D Systems, which utilizes a fixed IP address for
connection to enable efficient data transfer and operational
control. Additionally, the Hori Z500 3D printer and Uniz Plus
LCD 3D printer are also equipped. Figure 2. Space structure of 3D printing laboratory Figure 2. Space structure of 3D printing laboratory Figure 2. Space structure of 3D printing laboratory computer is connected to the Projet 660 Pro full-color 3D
printer from 3D Systems, which utilizes a fixed IP address for
connection to enable efficient data transfer and operational
control. Additionally, the Hori Z500 3D printer and Uniz Plus
LCD 3D printer are also equipped. 3. Content of Establishing Geoscience
3D Printing Lab Laboratory construction is a complex and important task. In this paper, we will discuss it in terms of both the hardware 51 and soft function aspects. In terms of hardware construction,
the laboratory is physically divided into a printing area and a
modeling area [7]. In terms of soft function construction, we
create a vibrant and innovative learning atmosphere by
implementing open courses, supporting entrepreneurship and
innovation competitions, organizing science and technology club activities, promoting collaboration between universities
and industry and military. These initiatives aim to cultivate
students' innovative spirit and practical abilities, and
ultimately contribute to society by producing more highly
skilled and innovative talents. Figure 1. Frame diagram of the laboratory construction Figure 1. Frame diagram of the laboratory construction separates the printers, aiming to provide a more convenient
environment for the daily operation and teaching research of
the laboratory, while ensuring the safety of the experimenters
through rational spatial planning and equipment deployment
[8]. separates the printers, aiming to provide a more convenient
environment for the daily operation and teaching research of
the laboratory, while ensuring the safety of the experimenters
through rational spatial planning and equipment deployment
[8]. 3.1. Hardware Equipment and Layouts 3.1.1. Printing Area In the printing area of the laboratory, an Intel i7 series
personal computer with a dedicated graphics card is installed. This computer serves as the control center for managing and
operating multiple advanced 3D printing devices. The 52 b) Hori Z500 c)Uniz Plus LCD
Figure 3. 3D printing equipment
nd Management of
missions" provides
y associated with
D printing process
alysis, the study
these particles to
o induce moderate
e planning of the
ve implemented a
series of measures to effectively mitigate the potential hazards
associated with 3D printing emissions on human health. Firstly, we have selected 3D printers equipped with efficient
filtration systems, ensuring that the exhaust is directed
outdoors to minimize the dispersion of particles within the
laboratory. Secondly, we have strictly adhered to the principle
of separating humans from the machines by establishing
dedicated printing areas furnished with high-performance
ventilation systems, as depicted in Figure 4. a) Projet 660 Pro b) Hori Z500 c)Uniz Plus LCD
Figure 3. 3D printing equipment a) Projet 660 Pro b) Hori Z500 c)Uniz Plus LCD
Figure 3. 3D printing equipment
h paper titled "Assessment and Management of
Impacts from 3D Printing Emissions" provides
ive evaluation of the toxicity associated with
tter generated during the 3D printing process
ucting experiments and analysis, the study
potential risks posed by these particles to
specifically their capacity to induce moderate
ng cells Consequently in the planning of the
series of measures to effectively mitigate the potential hazards
associated with 3D printing emissions on human health. Firstly, we have selected 3D printers equipped with efficient
filtration systems, ensuring that the exhaust is directed
outdoors to minimize the dispersion of particles within the
laboratory. Secondly, we have strictly adhered to the principle
of separating humans from the machines by establishing
dedicated printing areas furnished with high performance a) Projet 660 Pro b) Hori Z500
Figure 3. 3D printing equipment a) Projet 660 Pro a) Projet 660 Pro c)Uniz Plus LCD b) Hori Z500
Figure 3. 3D printing equipment Figure 3. 3D printing equipment series of measures to effectively mitigate the potential hazards
associated with 3D printing emissions on human health. Firstly, we have selected 3D printers equipped with efficient
filtration systems, ensuring that the exhaust is directed
outdoors to minimize the dispersion of particles within the
laboratory. 3.1.1. Printing Area Secondly, we have strictly adhered to the principle
of separating humans from the machines by establishing
dedicated printing areas furnished with high-performance
ventilation systems, as depicted in Figure 4. The research paper titled "Assessment and Management of
Human Health Impacts from 3D Printing Emissions" provides
a comprehensive evaluation of the toxicity associated with
particulate matter generated during the 3D printing process
[9]. By conducting experiments and analysis, the study
highlights the potential risks posed by these particles to
human health, specifically their capacity to induce moderate
toxicity in lung cells. Consequently, in the planning of the
laboratory's physical environment, we have implemented a Figure 4. Ventilation system diagram of forming area Figure 4. Ventilation system diagram of forming area wisdom and facilitate the exchange of knowledge among
students [10]. On the other hand, the individual creation zone
offers students a dedicated space for independent work and
personal expression. It is furnished with suitable desks and
chairs, creating a comfortable working environment
conducive to individual creativity [10]. Furthermore, we maintain rigorous compliance with the
laboratory's safety regulations when operating the printing
area. To ensure optimal conditions, we prioritize excellent
ventilation within the laboratory and limit the time spent by
personnel in the printing area, adhering to the principle of
swift entry and exit. When utilizing the 3D printer, it is
mandatory for all participating faculty and students to employ
industrial-grade masks of N95 or higher, as well as wear
goggles, anti-static clothing, and nitrile gloves. These safety
measures are strictly enforced to safeguard the physical well-
being of all individuals involved in the experiment [10]. Furthermore, the modeling area is organized into five
distinct working groups based on the method of model
creation. These groups encompass the Popular Software
Modeling Team, Mobile Photogrammetry Modeling Team,
Reusable Model Library Maintenance Team, Existing Data
Conversion Team, and Complex Model Modeling Team. Each
group tackles crucial stages of the modeling process, ranging
from geological data collection and processing to modeling
and design, material selection and optimization, printing and
post-processing, as well as quality control and evaluation. 3.1.2. Modeling Area The modeling area within the laboratory is thoughtfully
designed to cater to the diverse needs and working styles of
different students. It is subdivided into a public discussion
zone and an individual creation zone, each serving distinct
purposes. In the public discussion zone, there are 7-10 fully
equipped PC workstations provided to foster collective a. Popular Software Modeling Team: Equipped with high-
performance PC workstations, this team utilizes popular
modeling software such as Maya, Blender, SolidWorks, 53 area is responsible for the regular collection and organization
of printed models within the laboratory. The models are
carefully classified and stored based on geological categories,
graduation theses, competitions, innovation projects, and
post-class assignments. This systematic approach enables
teachers and students to easily locate and utilize existing
model resources, eliminating duplication of efforts and saving
valuable time and resources in teaching and research activities. d. Existing Data Conversion Team: To ensure compatibility
between geological data and 3D printers, the Existing Data
Conversion Team focuses on three-dimensional modeling of
geological data[13]. The PC in the work area is equipped with
software such as Surpac, GoCAD, and Petrel to facilitate the
conversion into file formats readable by 3D printers. This
conversion process ensures seamless integration of geological
data with 3D printing technology, providing students with
greater creative possibilities[14]. Rhino, SketchUp, among others, to carry out geological data
modeling and design tasks. Students can leverage these tools
to process data and create precise geological models, as
depicted in Figure 5. area is responsible for the regular collection and organization
of printed models within the laboratory. The models are
carefully classified and stored based on geological categories,
graduation theses, competitions, innovation projects, and
post-class assignments. This systematic approach enables
teachers and students to easily locate and utilize existing
model resources, eliminating duplication of efforts and saving
valuable time and resources in teaching and research activities. Figure 5. Geoscience terrain model d. Existing Data Conversion Team: To ensure compatibility
between geological data and 3D printers, the Existing Data
Conversion Team focuses on three-dimensional modeling of
geological data[13]. The PC in the work area is equipped with
software such as Surpac, GoCAD, and Petrel to facilitate the
conversion into file formats readable by 3D printers. This
conversion process ensures seamless integration of geological
data with 3D printing technology, providing students with
greater creative possibilities[14]. Figure 5. Geoscience terrain model Figure 5. Geoscience terrain model Figure 7. 3.2.1. Designing Open Course Open laboratory courses are exploratory experimental
courses offered to students of all majors and grades based on
the requirements of the teaching syllabus. The design of these
courses aims to create an academic environment for students
to explore and cultivate their practical abilities, stimulate their
innovative thinking, and foster their independent learning
skills. Through open laboratory courses, students have the
opportunity to autonomously select experimental topics,
develop experimental plans, and engage in practical
operations and data analysis. 3.1.2. Modeling Area DEM conversion of high-resolution remote
sensing images Figure 5. Geoscience terrain model Figure 5. Geoscience terrain model Figure 7. DEM conversion of high-resolut
sensing images b. Mobile Photogrammetry Modeling Team: This team
focuses on capturing images of field rock, mineral, and fossil
specimens using mobile phones and utilizing software such as
Agisoft PhotoScan and ColMap for modeling. Students
capture images of on-site samples by taking photos and then
use the software to generate accurate three-dimensional
models, as shown in Figure 6. Figure 6. Modeling diagram of sample shooting Figure 7. DEM conversion of high-resolution remote
sensing images e. Complex Modeling Team: To address the requirements
of complex model modeling, a dedicated team called the
Complex Modeling Team has been established, consisting of
relevant faculty and students. This team primarily handles
modeling tasks that involve special complexity and
challenges, aiming to fulfill the advanced modeling needs of
teachers and students. Figure 8. Diagram of a curved cylinder Figure 6. Modeling diagram of sample shooting Figure 6. Modeling diagram of sample shooting c. Reusable Model Library Maintenance Team: This work Figure 8. Diagram of a curved cylinder 54 3.2. Establishing Lab Soft Functions education and teaching, cultivates innovative talents, and
fosters strategic scientific and technological talents,
technology leaders, and innovative teams with international
standards. As the main platform for developing practical skills and
fostering innovative thinking, the Geoscience 3D Printing
Laboratory implements the work of cultivating virtue and
talents in the new era. It responds to the trend of educational
informatization, promotes the deep integration of modern
information technology and experimental teaching, expands
the breadth and depth of experimental teaching content, and
improves the quality and level of experimental teaching[13-
15]. As the main platform for developing practical skills and
fostering innovative thinking, the Geoscience 3D Printing
Laboratory implements the work of cultivating virtue and
talents in the new era. It responds to the trend of educational
informatization, promotes the deep integration of modern
information technology and experimental teaching, expands
the breadth and depth of experimental teaching content, and
improves the quality and level of experimental teaching[13-
15]. By
participating
in
innovation
projects,
various
competitions, the 3D Printing Association, industry-
academia-research collaboration projects, and military-
civilian integration projects, students are provided with
opportunities to enter the laboratory and research teams early
on. High-level scientific research supports high-quality As a representative of modern information technology, 3D
printing technology is an important innovation tool. The
Geological 3D Printing Laboratory provides research
directions and technical support for students to participate in
innovation projects, various competitions, the 3D Printing
Association,
industry-academia-research
collaboration
projects, and military-civilian integration projects. Through
the continuous update and construction of the laboratory's
software environment to accommodate various projects,
students are gradually trained to become innovative, skilled,
and application-oriented talents who can respond to the
demands of the times[16]. ]
By
participating
in
innovation
projects,
various
competitions, the 3D Printing Association, industry-
academia-research collaboration projects, and military-
civilian integration projects, students are provided with
opportunities to enter the laboratory and research teams early
on. High-level scientific research supports high-quality Figure 9. Construction structure diagram of soft environment Figure 9. Construction structure diagram of soft environment classmates from different academic backgrounds. This design
facilitates collaboration and communication among students,
while fostering teamwork and interdisciplinary thinking
abilities. 3.2.2. Supporting Entrepreneurship and Innovation
Competition Based on the laboratory's innovation and entrepreneurship
projects, as well as various competitions, students have
achieved fruitful results in the field of scientific and
technological innovation. These results are presented in Table
1. Innovation and entrepreneurship projects, along with
competitions, are important components of students'
innovative practices. They not only provide a platform to
showcase talent and innovative achievements but also inspire
students' motivation for independent learning. Additionally,
they foster the ability to collaborate in interdisciplinary teams. Furthermore,
the
course
design
emphasizes
interdisciplinary cross-collaboration and cooperative learning. Students can transcend disciplinary boundaries by choosing
experimental courses relevant to their own major or ones they
are interested in, and participate in experimental projects with 55 Table 1. Situation of the Entrepreneurship and Innovation competition
Category
2019
2020
2021
2022
Results
Number of innovation
and entrepreneurship
projects
6
8
11
9
Utility model: 2 items
Invention patents: 2 items
CPCI paper: 1 article
MDP paper: 1 article
National first prize: 3 items
National second prize: 1 item
National third prize: 2 items
Number of participants
in each category
6
10
10
12 into the latest industry trends and development. Through the
collaboration between the university and the company, we are
able to fully leverage industry resources and establish a bridge
for industry-academia collaboration. This provides students
with opportunities for field visits and on-site learning,
enabling them to gain in-depth understanding of the
manufacturing processes and working principles of various
components. It also offers internship and employment
opportunities, laying a solid foundation for their professional
development. [2]. into the latest industry trends and development. Through the
collaboration between the university and the company, we are
able to fully leverage industry resources and establish a bridge
for industry-academia collaboration. This provides students
with opportunities for field visits and on-site learning,
enabling them to gain in-depth understanding of the
manufacturing processes and working principles of various
components. It also offers internship and employment
opportunities, laying a solid foundation for their professional
development. [2]. 3.2.3. Establishing 3D Printing Technology Association g
g
gy
Relying on the laboratory, the 3D Printing Technology
Association is established. The association regularly
organizes training seminars and sharing sessions on popular
software and technical advancements. Members from
different grades and majors form teams to participate in daily
learning, innovation projects, and various competitions. By
applying a multidisciplinary and cutting-edge scientific
approach, they address scientific problems within their
respective fields of expertise. 3.2.2. Supporting Entrepreneurship and Innovation
Competition Figure 11. Production practice diagram of production,
education and research enterprises Figure 10. 3D printing popular science publicity diagram
3.2.4. Building Industrial Collaboration
By fully utilizing the advantages of enterprises and industry
resources, we have established close cooperation with Beijing
Space Intelligent Construction Technology Co., Ltd. Through
the implementation of industry-education integration, we
have personally experienced the actual working environment
and the production practices of the company, gaining insights Figure 10. 3D printing popular science publicity diagram
2.4. Building Industrial Collaboration Figure 11. Production practice diagram of production,
education and research enterprises 4.1. Printed product: After the coordinated construction of both the hardware
and soft environment in the laboratory, significant results
have been achieved. The following are examples of student
classroom works, competition awards, and innovation
projects Figure 13. Students' classroom works display Figure 13. Students' classroom works display Figure 14. Entries and Awards Figure 14. Entries and Awards
Figure 15. Prize-winning results display diagram Figure 14. Entries and Awards Figure 15. Prize-winning results display diagram Figure 15. Prize-winning results display diagram 3.2.5. Building Military Collaboration Figure 10. 3D printing popular science publicity diagram
3.2.4. Building Industrial Collaboration Figure 10. 3D printing popular science publicity diagram
3.2.4. Building Industrial Collaboration Military-civilian integration and collaborative construction
between the military and civilian sectors: In terms of military-
civilian integration, we actively promote collaborative
construction and cooperation between the military and
civilian sectors. This includes the joint construction of our 3D
printing laboratory with a certain engineering university
laboratory, aiming to achieve resource sharing, mutual
exchange of experiences, and facilitate the sharing and
communication of research achievements. By fully utilizing the advantages of enterprises and industry
resources, we have established close cooperation with Beijing
Space Intelligent Construction Technology Co., Ltd. Through
the implementation of industry-education integration, we
have personally experienced the actual working environment
and the production practices of the company, gaining insights Figure 12. Architecture diagram of civil-military fusion 56 The collaboration of military-civilian integration is not
only reflected in the joint construction of laboratories but also
extends to the joint development of experimental and
internship courses. Together, we formulate and implement
experimental and internship courses to enhance students'
practical abilities and overall qualities through practical
activities. Students have the opportunity to visit and exchange
ideas with each other among different laboratories,
broadening their academic perspectives and improving their
research and innovation capabilities[17-18]. References The laboratory is a distinctive place that embodies the spirit,
culture, and academic level of a university. It is also one of
the main indicators of a school's academic and research
capabilities. It is undeniable that compared to world-class
universities abroad, there is still a significant gap in the
conditions of our experiments. We must catch up. Therefore,
we need to broaden our thinking in terms of laboratory
infrastructure, be bold in exploration and experimentation,
and, at the same time, ensure laboratory safety through the
responsible system of experimental teachers. We should adopt
a flexible and autonomous laboratory management approach,
providing a platform and technical support for students and
teachers to participate in various competitions, innovation
projects, and other activities. This will safeguard the
continuous growth of students and explore a geological 3D
printing laboratory construction model that nurtures talents
with adaptability, exploratory mindset, creativity, and
pioneering spirit to meet the developmental needs of the times. [1] Mo Yuandong, Lian Haishan, Mo Deyun, et al. Discussion on
the application of university-industry co-built 3D printing
laboratory in practical teaching. Journal of Laboratory
Research and Exploration, 2020, 39(08): 232-235. [2] Wu Gaomiaoxian. Discussion on the multi-level digital
technology talent system under the background of Digital
China. Journal of Longyan University, vol. 41, no. 1, pp. 117-
121, 2023. [3] Jing Minchang, Liu Zhenfeng. Digital Earth, Digital China, and
Digital Oilfield. Journal of Xi'an Shiyou University (Natural
Science Edition), vol. 06, pp. 82-85+93, 2003. [4] Yang Hui. "Three Innovations" Talents in the Era of "Internet
Plus": Connotation, Characteristics, and Training Path - Taking
Digital Media Art Major as an Example. Journal of Educational
Theory and Practice. [5] Wang Deli, Tang Haifeng, Meng Wei, et al. Construction of
digitalized experimental teaching platform. Experimental
Technology and Management, 2014, 31(12): 152-154. 57 [6] Zu Qiang, Wei Yongjun. Analysis of the construction status of
national virtual simulation experimental teaching centers. Experimental Technology and Management, 2015(11): 156-
158. [13] Gao Jie, Hu Hua. Discussion on the application of 3D modeling
and 3D printing technology in basic geological laboratory. Science and Technology Vision, 2022, No. 389(23): 37-39. DOI: 10.19694/j.cnki.issn2095-2457.2022.23.11. [7] Feng Jing, Li Zhenzhen. Preliminary exploration of 3D printing
laboratory management. Southern Agricultural Machinery,
2017, 48(01): 145+148. [14] Dong Qiujing, Luo Chunhua, Wang Chang, et al. Preliminary
exploration of "new materials + 3D printing" college student
maker laboratory. Guangdong Chemical Industry, 2020, 47(15):
206-207. References [8] Zhou Shufang, Song Aili, Ma Yuhua. Analysis of the
construction plan of 3D printing laboratory. China Information
Technology Education, 2015, No.222(19): 133-135. [15] Li Cuichao, Fu Yuzhuo, Xu Wei. Exploration of the
construction and service model of the 3D printing service
center in the national innovation and entrepreneurship
demonstration base. Journal of Laboratory Research and
Exploration, 2020, 39(05): 143-148. [9] Yousef Mohammadian and Nafiseh Nasirzadeh. Toxicity risks
of occupational exposure in 3D printing and bioprinting
industries: A systematic review. [16] Liu Desheng, Yin Ruojun, Deng Jiaxuan, et al. Research on the
construction of 3D printing innovation laboratory based on
university innovation education. Science and Technology
Wind, 2019, No. 394(26): 71-72. DOI: 10.19392/j.cnki.1671-
7341.201926060. [10] Wang Chao, Li Dasheng, Wei Tianlu, Fan Hengliang, Zhang
Jie. Laboratory construction and curriculum design for 3D
printing large-scale instruments and equipment. Journal of
Wuhan University of Light Industry, 2022, 41(02): 107-112. [11] Zhang Hai, Fu Wei. Construction and exploration of laboratory
open platform supported by Internet Plus 3D printing
technology. Education and Teaching Forum, 2018, No. 391(49):
139-141. [17] Qiu Haifei. Research on the talent training system of 3D
printing major. Modern Manufacturing Engineering, 2018, No. 452(05): 62-67. DOI: 10.16731/j.cnki.1671-3133.2018.05.012. [18] He Yangbo, Chen Jian, Fang Feiyu, et al. Construction of
undergraduate teaching laboratory for 3D printing technology
under the background of the new engineering. Journal of
Laboratory Research and Exploration, 2020, 39(01): 275-
278+306. [12] Guo Yanjun, Zhang Jinjiang, Chen Bin, et al. Immersive
visualization and interaction method of multi-scale geological
data based on VR technology. Earth Science Frontiers, 2019,
26(04): 146-158. 58
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English
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Knowledge implementation in health care management: a qualitative study
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BMC health services research
| 2,020
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cc-by
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© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if
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The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the
data made available in this article, unless otherwise stated in a credit line to the data. Roohi et al. BMC Health Services Research (2020) 20:188
https://doi.org/10.1186/s12913-020-5043-8 Roohi et al. BMC Health Services Research (2020) 20:188
https://doi.org/10.1186/s12913-020-5043-8 (2020) 20:188 Open Access Knowledge implementation in health care
management: a qualitative study G. Roohi1, G. Mahmoodi2* and H. Khoddam3 Abstract Background: The gap between knowledge and practice is a global issue, which increases wasteful spending in
healthcare. There are several models and frameworks to address this gap and try to solve the challenge. Promoting
Action on Research Implementation in Health Services (PARIHS) framework highlights the interaction of three main
elements: evidence, context and facilitation, to implement research into practice, successfully. This framework can
use as a tool to evaluate the situation and guide the changing. This study conducted to explain the status of
knowledge implementation in Iran’s healthcare management system. Methods: This qualitative study was done by using a directive content analysis approach through conducting in-
depth, structured interviews with 15 health managers based on the PARIHS framework. Guiding questions were
based on the three main elements of the framework: evidence, context and facilitation. The content of the
interviews entered into the Qualitative Data Analysis software (MAXQDA version 10) and, then, analyzed. Results: The most common source of evidence used by managers for decision-making was local information and
previous experience. Evaluation more emphasized compared to other sub-elements of context, i.e. culture and
leadership. In terms of facilitation, performing tasks by others was the dominant opinion. Conclusion: Our results showed that managers in the healthcare system of Iran use their own and other manager’s
experience and the local information for decision-making and have no ideas about facilitation. Keywords: Research implementation, PARIHS framework, Healthcare management system, Iran, Content analysis * Correspondence: ghahraman.mahmoodi@gmail.com
2Associated professor, hospital administration research center, Sari branch,
Islamic Azad University, Sari, Iran
Full list of author information is available at the end of the article Background between knowledge and performance of health system is
indeed a gap between knowledge and practice [6, 7]. Promotion of public health requires attention to a know-
ledge- and evidence-based decision making system [8]
and human development is possible by storing, using
and sharing knowledge [9]. Nowadays, improving the quality of treatment and
achieving the highest standard of care is one of the key
goals of any healthcare system [1]. Recent rapid ad-
vances in medical sciences and technology have not only
raised public awareness and improved economic status,
but also increased people’s expectations of health ser-
vices [2, 3]. Despite the point that millions of dollars are
spent every year on healthcare, quality of health services
is still poor and unfavorable [4]. Decision making in the health system has a significant
relationship with the research findings and knowledge
available in this field [10]. Until a common understand-
ing between owners of knowledge and the health system
is developed, the research findings are merely the dis-
semination of results and will not be effective for the pa-
tients and health system [11]. Lack of using knowledge
in the health management system will waste financial re-
sources, time and energy [12] and increase the costs im-
posed on the patients. It may be even detrimental to Knowledge is an important source of wisdom and effi-
cient actions in any organization [5]. Lack of synergy * Correspondence: ghahraman.mahmoodi@gmail.com
2Associated professor, hospital administration research center, Sari branch,
Islamic Azad University, Sari, Iran
Full list of author information is available at the end of the article Roohi et al. BMC Health Services Research (2020) 20:188 Page 2 of 9 Page 2 of 9 policy. It means, the Ministry of Health and Medical Edu-
cation (MOHME) is responsible for policy making and
managing the whole health system and the executive pro-
cesses accomplished by the medical and educational uni-
versities in each province. For this reason, the university’s
deans do the selection and appointment of the middle and
top-level managers of hospitals and health care canters. These managers can be a general physician or medical
specialist with or without an education in health care
management. Therefore, the interviews carried out on 15
executive and top-level health managers with enough ex-
periences in health and treatment sectors of Medical Sci-
ences Universities in 2018–2019 (Table 2). The inclusion
criteria were having at least 5 years of management ex-
perience in the field of healthcare and willingness to par-
ticipate in the study. After obtaining the informed
consent, the participants interviewed individually in a
semi-structured manner. Since, the study based on the
PARiHS framework; we use the diagnostic and evaluative
questions in terms of the main elements (Evidence, Con-
text and facilitation) of this model as an interview guide
[25]. The interviews began with open and general ques-
tions about their managerial experiences, manner of
decision-making, resource allocation and system evalu-
ation. Then, to obtain the rich and specified data, the
interview was guided to take examples and detailed expla-
nations related to the framework’s elements. patients [13]. In addition, it will hinder implementation
of the recommended healthcare services in terms of pre-
vention, treatment and management [10, 11, 14]. g
Implementing knowledge into practice is a dynamic and
interactive process that includes production, dissemin-
ation, exchange and application of knowledge to improve
service delivery [15]. Barriers to knowledge utilization in-
clude lack of or inability to access knowledge resources,
indifference towards the knowledge gained through re-
search findings [16] and lack of time to find evidence that
can help managers and policy makers [17]. Moreover,
managers generally make decisions based on the informa-
tion gained through recommendations and results of rou-
tine organizational measurements rather than research
findings [18]. In recent decades, several models have been
proposed on how to apply knowledge in practice or facili-
tate this process [19, 20]. Methods The approach followed in this qualitative study was di-
rected content analysis. In doing so, we traced the steps
described by Hsieh and Shannon [24] to conduct the in-
terviews and analyse the emerged content. In fact, this
deductive approach could help form the study categories
and subcategories before starting the data gathering cor-
responding with three main elements of PARIHS frame-
work, i.e. Evidence, context and facilitation. The “Promoting Action on Re-
search Implementation in Health Services” (PARIHS) is a
conceptual framework that developed by Kitson et al. (1998) with the aim of promoting research implementa-
tion in practice. This model highlights the interaction of
evidence, context and facilitation for successful implemen-
tation. These elements made up of some sub-elements,
which define them. Evidence is defined by sub-elements of
research findings, experiences of service providers and re-
cipients as well as local information (i.e. Organizational
knowledge); culture refers to the sub-elements of leader-
ship and evaluation methods and the last element, facilita-
tion, is defined by goals, roles and skills of individuals in/
out of the organization that helps others make things eas-
ier [21]. Based on the framework, each sub-element is
placed on a continuum from low to high (Table 1) [22]
and can be used as a tool to evaluate and describe the
current status of an organization in terms of research im-
plementation in practice [23]. Considering the differences
between various disciplines and organizations and the
shortcomings in the implementation of management
knowledge in the health system of Iran, we are aiming to
use the above-mentioned framework to demonstrate the
position of this discipline among other members of the
healthcare service providers in terms of translating know-
ledge into practice. On average, each interview lasted 60–90 min and the in-
terviewees invited for extra meeting if needed. The con-
tent of each interview was transcribed verbatim and read
several times to find the general content of the partici-
pants’ speech. Then, the content entered into the data
management software, MAXQDA ver. 10, to identify and
label the meaning units represented the main elements
and sub-elements of PARIHS frameworks. Eventually, the
coded segments categorized under the pre-determined
categories, i.e. evidence, context and facilitation. To confirm the rigor of the study, the extracted data
shared with the participants in a two-hour focused
group discussion. The participants were asked to identify
the position of Iran’s healthcare management on the
continuum of evidence, context and facilitation based on
the PARIHS framework and rate it as weak or strong
(Table 1). Results Analysis of the qualitative data showed that, in terms of
evidence, most managers used the local information, ser-
vice providers and recipients’ experience and local infor-
mation as a main source of knowledge for decision
making and managing the organization. In terms of con-
text, most of the codes were relate to the sub-element of
evaluation. Among the facilitators, most managers used
others as facilitators. This study conducted in Iran health care system. The
healthcare system in Iran managed with a centralized Page 3 of 9 Roohi et al. Results BMC Health Services Research (2020) 20:188 Patient experience
• Not valued as evidence
• Seen as only type of evidence
• Patient not involved • Conclusion drown Local data/ information
• Not valued as evidence
• Lack of systematic methods for
collection and analysis
• Not reflected upon
• Not Conclusion drown
Context
Culture
• Unclear valued and beliefs
• Low regard for individuals
• Task driven organization
• Lake of consistency
• Resources not allocated
• Well integrated with strategic goals • Valued as evidence • Collected and analysis systematically rigorously • Evaluated and reflected upon • Conclusion drown • Values individual staff and clients • Promotes learning organization • Consistency of individuals role/experience to value
l ti
hi • Power and authority • Rewards /recognition • Resources-human, financial, equipment, allocated Leadership
• Traditional, command, and control leadership
• Lack of role clarity
• Lack of teamwork
• Poor organizational structures
• Autocratic decision-making processes
• Didactic approaches to learning/
teaching/managing
Evaluation
• Absence of any form of feedback
• Narrow use of performance information sources
• Evaluations rely on single rather than multiple methods
Facilitation
Purpose
Task
Doing for others
• Episodic contact
• Practical/technical help
• Didactic, traditional approach
to teaching
• External agents
• Low intensity—extensive coverage
Skills and attributes
Task/doing for others
• Project management skills
• Technical skills
• Marketing skills
• Subject/technical/clinical credibility Leadership
• Traditional, command, and control leadership
• Lack of role clarity
• Lack of teamwork
• Poor organizational structures
• Autocratic decision-making processes
• Didactic approaches to learning/
teaching/managing
Evaluation
• Absence of any form of feedback
• Narrow use of performance information sources
• Evaluations rely on single rather than multiple methods y
• Effective teamwork cilitation
Purpose
Task
Doing for others
• Episodic contact
• Practical/technical help
• Didactic, traditional approach
to teaching
• External agents
• Low intensity—extensive coverage
Skills and attributes
Task/doing for others
• Project management skills
• Technical skills
• Marketing skills
• Subject/technical/clinical credibility • Cocounseling • Critical reflection • Giving meaning g
g
• Flexibility of role g
• Subject/technical/clinical credibility y
• Realness/authenticity Roohi et al. Results BMC Health Services Research (2020) 20:188 Page 4 of 9 Table 2 Demographic characteristics of the participants
Subject Work experience
(years)
Gender Expertise/Specialty
Level of education
Main experiences
1
30
Male
Laboratory sciences
Doctorate
Deputy of cultural and students affairs
2
20
Male
Medical and health services
management
PhD
Health Services Manager
3
30
Male
Nursing
MSc
Director of education
4
22
Male
Pediatric neurology
Medical doctor-
Fellowship
Hospital CEO and deputy of treatment
5
21
Male
Pharmacology
Doctorate
Deputy of food and drug
6
20
Male
Nutrition
PhD
Health Services Manager
7
30
Male
Anesthesiology
MSc
Head of college and Vice-chancellor for cultural
affairs
8
20
Male
General practitioner
Medical doctor
Director of treatment monitoring
9
21
Male
Reproductive health
PhD
Health center manager
10
29
Female Otorhinolaryngology
Medical doctor-
specialist
Health center manager
11
26
Male
Virology
PhD
Deputy of development and Director of
graduate studies
12
28
Male
Physiotherapy
PhD
Health clinic center manager
13
21
Male
Cardiology
Medical doctor-
specialist
Hospital CEO
4
30
Male
Medical and health services
management
PhD
Director general of health insurance
15
28
Male
General practitioner
Medical doctor
Hospital CEO However, most of them were using the comments that
took in meetings and councils: Evidence Analysis of the data in terms of evidence and its sub-
element, i.e. research, experience of service providers,
experience of service recipients and local information,
reported below. "I created a small group, called the scientific and ex-
ecutive committee, and used the comments of ex-
perts in the fields of environmental health and
occupational health and diseases." Research Some
participants
used
related
websites
to
obtain
information. Results of the councils usually considered for decision-
making: "At least once or twice a week, I visit the WHO
website to check the latest findings on my subject of
interest." "In weekly meetings, we discussed issues and, then,
prepared them for decision making by the university
executives." They did not often use databases as a source of research
findings for decision-making: Some of the managers used the experiences they had
gained elsewhere: "It’s the last thing I would do if I wanted to improve
my knowledge about a subject." "One of the tasks is to empirically use other places
as a template; for example, I used some of the works
that were beneficial at other universities." Experiences of service providers (clinical experience):
Most managers were using their own and their col-
leagues’ experiences: Comments of service recipients (patient preferences)
The participants in our study rarely took into account
service recipients’ viewpoints for decision-making and
did not have a regular planning on this matter: "One of my tasks was to call successful individuals
and ask about their viewpoints." Page 5 of 9 Page 5 of 9 Page 5 of 9 Roohi et al. BMC Health Services Research (2020) 20:188 Roohi et al. BMC Health Services Research (2020) 20:188 "Getting feedback and comments from service recipi-
ents was not systematic, but because I am usually in
the workplace environment, I ask for comments and
decide accordingly." Context Analysis of the data in terms of context and its sub-
element, i.e. culture, leadership and evaluation reported
below. This approach also followed by another participant: This approach also followed by another participant: "I have to hear and know about problems of a nurse
and his/her motivation; meeting with a manager is
pleasing to some of them and motivates them." "For example, when we have a problem in our hos-
pital concerning payment of salaries, I ask my
trusted advisors before decision making." The participants also believed in the system of encour-
agement and some of them used qualitative tools that
designed with the staff’s consent: Organizational/local information In the construct of evidence, most of the codes belonged
to the sub-element of local information, most of which
included the regulations, council minutes and upstream
documents. "In insurance, payments are based on ratings that
employees receive and any creativity or capability is
considered when assessing their work." "Upstream documents itself cannot play a role in
the executive field, but can guide the executive sys-
tem through goal setting." Some believed in defining organizational culture based on
maintaining values and beliefs and were pleased to provide
services, but this feeling was fading over time since indi-
viduals were paying more attention to personal gain: However, some of the participants considered the in-
structions as obstacles to their maneuverability: “There
are so many regulations and instructions in the ministry
of health that have made it impossible to form the struc-
ture that a manager has to have on the mind in order to
get things done.” "Healthcare is the holiest area of activity because its
main concern is humans’ well-being and health, and
the look of satisfaction of a patient after receiving
the treatment is a joyful experience." Based on the findings, the level of applying the system
data and care provider experiences as a source of evi-
dence in health care managers’ decision making pro-
cesses rated as high and conversely the level of research
findings and service recipients’ experiences utilization
rated as low (Table 3). Nevertheless, some of the participants believed that the
health system lacked coherence, integrity and strategic
objectives: "In this field, sometimes, decisions are made indi-
vidually and without harmony; for instance, a plan
is instructed at the deputy of health level, but not at
the ministerial level, which requires modifications
for implementation at the workplace." Culture Most of the participants believed that communicating
with others has a key role in the organization. One of
the participants regarded communication as a motivat-
ing factor: Facilitation The study findings about the facilitation, the third main
element of implementation process, presented below in
terms
of
skills,
attributes,
purpose,
and
roles
of
facilitators. Skills and attributes It seems that managers pay great attention to the con-
text due to the lack of resources and pay little attention
to facilitation because of the inaccurate identification of
obstacles and lack of structure and processes in the
organization. In addition, managers rarely use research
findings, mainly because of the heavy workload and lack
of access to exploitable results. Gagnon and Bergeron re-
ported that despite the interest in evidence-based deci-
sion making, individuals and organizations create some
obstacles in this regard [29]. This issue is not just limited
to managers and nurses are also not familiar with
evidence-based performance and do not implement re-
search findings in practice [30]. Despite all the efforts,
unfortunately, the utilization of knowledge has not yet The majority of the participants used consultation, part-
nership and lobbying to remove obstacles, which are not
formal, except in the case of Health Charity Assembly. "For example, there was a problem with a hospital
project. We used consultants in the field of con-
tracting and, even sometimes, asked for the help of
technical engineers at Ministry of Intelligence." Evaluation According to most participants, evaluation is a major
component of the context core element, which is at a
high level in the current health system. The participants
claimed that they used various methods and multiple re-
sources to evaluate employees: "For example, we had a problem on building a hos-
pital; we negotiated with the Parliament and the
Plan and Budget Organization to get the job done." Using the PARHIS framework to determine the level of
facilitation in health care management showed that all
sub-elements of facilitation rated as low (Table 3). "We have a triangular model; electronic monitoring,
in person and in the system, which utilizes a check-
list to determine whether the employee really per-
formed a task or not." Leadership Table 3 Status of knowledge implementation healthcare
management in Iran
Core
elements
Sub-elements
Rating
Low
high
Evidence
Research
✓
Clinical experience
✓
patient preferences
✓
Local information
✓
Context
Culture
✓
Leadership
✓
Evaluation
✓
Facilitation
Skills and attributes
✓
Role and purpose
✓ Table 3 Status of knowledge implementation healthcare
management in Iran
Core
elements
Sub-elements
Rating
Low
high
Evidence
Research
✓
Clinical experience
✓
patient preferences
✓
Local information
✓
Context
Culture
✓
Leadership
✓
Evaluation
✓
Facilitation
Skills and attributes
✓
Role and purpose
✓ Table 3 Status of knowledge implementation healthcare
management in Iran A number of participants expressed that clarifying the
role of staff was an effective factor in organizational
progress: "Designing a task-based framework in line with the
objectives of the organization can help us become
well-organized." However, some of them believed that lack of structure,
efficiency and flexibility of the organization prevents the
creation of a suitable platform for leadership in the
organization: "One day, we had a university with a limited num-
ber of students and faculty members. But after 10 Roohi et al. BMC Health Services Research (2020) 20:188 Roohi et al. BMC Health Services Research (2020) 20:188 Page 6 of 9 Page 6 of 9 Page 6 of 9 "The health system-related works are based on the
bargaining and lobbying power. Hence, we use this
solution when facing problems." years, the number of faculty members has been
doubled; our students have quadrupled and diversity
of courses has increased. Nevertheless, nothing has
changed in our organizational structure, which
forces utilization of employees against the law." The participants also benefited more from the technical
facilitation capabilities of politics, such as the Parliament
members, provincial officials and headquarters of the
ministry: Discussion The healthcare system of Iran often manage by the spe-
cialized or general physicians. In some cases, however, at
the executive level, general practitioners are responsible
for administering hospitals. Since they are most likely to
manage the healthcare, system based on their experi-
ences and participation in short management courses
and workshops on management. Therefore, considering
the above conditions and the viewpoints of the partici-
pants in this study, the status of knowledge utilization in
the health system is not favorable, and the managers
mainly run the system based on their specialty and ex-
perience as well as relying on medical specialty. As our
results show. Moreover, the results indicated that feedbacks are often
individual and non-systematic: "I myself send results of the evaluations to col-
leagues and managers at different levels to check
the issues with their employees." Rating the status of context sub- elements indicated a
high level of application of evaluation among healthcare
managers as an effective elements to prepare the context
for implementing knowledge (high level), while attention
to other sub-elements i.e. culture and leadership rated as
low level (Table 3). Our results indicated the considerable emphasis on
context and less attention to evidence and facilitation. This finding is in line with findings of Janson and Fors-
berg [26]. According to Ward et al., facilitation and con-
text are the most influential factors in decision making
[27]. The utilization of knowledge requires the availabil-
ity of the best evidence, a correct understanding of the
structure and goals, culture of change and utilization of
effective strategies [28]. Role and purpose In this sub-element, great attention paid to occasional
contacts with people outside the organization for facili-
tation, which was not teamwork: Page 7 of 9 Page 7 of 9 Roohi et al. BMC Health Services Research (2020) 20:188 Roohi et al. BMC Health Services Research (2020) 20:188 been institutionalized in Iran’s health system [31]. In
other studies, lack of sufficient time has been regarded as
a barrier to the implementation of research results [32,
33] and some managers claim that not enough research
findings are available in areas that are important to them
[34]. Moreover, policymakers generally rely on informa-
tion other than research findings such as recommenda-
tions and routine measurements for decision making [18]. Knowledge utilization is a nonlinear process that begins
with needs assessment, situational assessment and needs-
based knowledge production, and continues with the
evaluation of knowledge transferred to policymakers,
peers and public users as well as monitoring and providing
feedback [35]. Therefore, it seems that factors such as un-
certainty, lack of consensus on research findings and un-
availability of brief results for routine decision making by
managers may affect this process. healthcare managers ought to apply a structured en-
couragement and punishment system with objective in-
dices for the employees. Moreover, designing and
implementing a performance-based paying system can
motivate employees and improve their efficiency. Our
results indicated that managers generally put emphasis
on evaluation and utilize multiple methods and re-
sources for this purpose. Other studies have also eluci-
dated that evaluation is a complex, but necessary,
component of the environments that seek to implement
changes [27, 36, 40, 43]. In Iran, health managers seem to be interested in non-
systematic monitoring, use the results as a basis for
judgment and fail to make corrective actions. Neverthe-
less, in recent years, the health system has sought to
resolve these issues by using the operational plan moni-
toring system and performing accreditation. In order to utilize knowledge, all three elements of the
PARIHS model (evidence, context and facilitation) must be
available [27]. Our findings indicate that the health system
of Iran focused mainly on the subject of organizational
culture and evaluation, and managers believe that the
current culture of the health system lacks coherence. Role and purpose Meanwhile, it is believed that organizational integrity and
establishment of coordination between experts and policy-
makers is essential for implementing changes [18, 36] and
encouraging teamwork spirit [37, 38] in the organization. Senge suggested that successful implementation of theories
in practice could be only achieved by establishing a collab-
orative culture via education [39]. It seems that, the lack of
organizational integrity and poor team working are the
result of dominant culture of the society, prioritizing the
personal gain over the organizational success. Furthermore,
organizational processes are incoherent and predominately
based on individual taste and bargaining. This highlights
the need for a reformation in the current management
model
and
comprehensive
planning
to
help
build
organizational structures based on the actual needs of the
health system. Our results demonstrated that the element of facilita-
tion has received less attention and the individual skills
inside and outside the organization have emphasized
more. Some researchers believe that facilitation should
be carried out by doing work by others and enabling
others [44], while others suggest these two methods of
facilitation are mostly carried out in one group [38]. Ac-
cording to Harvey et al., in order to facilitate the change
process, first, an interface should be created between the
internal staff and external facilitators, which requires
specific infrastructure and planning [45]. In addition, ac-
cess to resources can facilitate knowledge utilization
[46]. Thus, for proper facilitation, it is crucial to create a
suitable context and allocate resources to this issue [47]. However, it is thought that if clinical managers play their
leadership role as internal facilitators, they can success-
fully implement changes in the organization despite the
influence of complex and, sometimes, contradictory ele-
ments and environmental fluctuations [48]. It seems that insufficient understanding of the existing
problems and lack of a systematic structure have led to
the use of lobbying for facilitation, which can be resolved
by creating a systematic structure and process. Managers also believe that they should define values
and beliefs for employees. In this regard, Ward stated that
personnel rating should be carried out in a coherent man-
ner, so that employees become involved in the change
process and prioritize the interests of the organization
over their own [27]. Role and purpose Other studies have also demonstrated
that the interest of individuals, valuing the goals of the
organization, belief in change and paying attention to
inter-disciplinary activities are effective factors in imple-
menting changes in an organization [40, 41]. Terminating
employees who are effective in the implementation of
change is dangerous for the organization and these indi-
viduals should be encouraged and rewarded [42]. Conclusion Based on the result. It seems necessary to develop a
structure in healthcare system for easy and applicable
access to research findings, experiences of colleagues
and information. Moreover, we need to train managers
to accept of the role of insider or outsider facilitators of
the organization in healthcare system. Abbreviations
MAXQDA: Qualitative & mixed methods data analysis tool;
PARIHS: Promoting action on research implementation in health services;
W.H.O: World health organization Abbreviations Abbreviations
MAXQDA: Qualitative & mixed methods data analysis tool;
PARIHS: Promoting action on research implementation in health services;
W.H.O: World health organization It seems that paying attention to values and beliefs in-
fluenced by the dominant culture of the society, but Page 8 of 9 Page 8 of 9 Page 8 of 9 Roohi et al. BMC Health Services Research (2020) 20:188 Roohi et al. BMC Health Services Research (2020) 20:188 References References
1. Jahani M, Naghavian M, Naghshine A. The evaluation of performance
indicatores in the health care centers of babol university of medical
sciences by the pabn lasoo model 2011–2014. J Babol Univ Med Sci. 2015;
17:76–82. 2. Sharifi A, Jalali R, Shahbazi N. Evaluation of awareness on the patient bill of
rights and observing rate on the patient’s perspective in imam Reza
Hospital in Kermanshah in 2012. Iranian J of Med Law. 2013;6:125–35. 3. Basiri MK, Basiri MM, Moslem A, Ajam ZH, Jamal F. Health Providers and
Patients'awareness on Patient Bill Of Rights and its Observing Rate in an
Educational Hospital in Gonabad. Horizon Med Sci. 2011;17:45–54. 4. Grimshaw JM, Eccles MP, Lavis JN, Hill SJ, Squires JE. Knowledge translation
of research findings. Implement Sci. 2012;7:50. 5. Rocha ESB, Nagliate P, Furlan CEB, Rocha K Jr, Trevizan MA, Mendes IAC. Knowledge management in health: a systematic literature review. Revista
latino-americana de enfermagem. 2012;20:392–400. 6. Ellen ME, Lavis JN, Horowitz E, Berglas R. How is the use of research
evidence in health policy perceived? A comparison between the reporting
of researchers and policy-makers. Health Res Policy Syst. 2018;16:64. 7. Rubio DM, Schoenbaum EE, Lee LS, Schteingart DE, Marantz PR, Anderson
KE, Platt LD, Baez A, Esposito K. Defining translational research: implications
for training. Academic Med. 2010;85:470. 23. Rycroft-Malone J, Seers K, Chandler J, Hawkes CA, Crichton N, Allen C,
Bullock I, Strunin L. The role of evidence, context, and facilitation in an
implementation trial: implications for the development of the PARIHS
framework. Implement Sci. 2013;8:28. 23. Rycroft-Malone J, Seers K, Chandler J, Hawkes CA, Crichton N, Allen C,
Bullock I, Strunin L. The role of evidence, context, and facilitation in an
implementation trial: implications for the development of the PARIHS
framework. Implement Sci. 2013;8:28. 1. Jahani M, Naghavian M, Naghshine A. The evaluation of performance
indicatores in the health care centers of babol university of medical
sciences by the pabn lasoo model 2011–2014. J Babol Univ Med Sci. 2015;
17:76–82. 1. Jahani M, Naghavian M, Naghshine A. The evaluation of performance
indicatores in the health care centers of babol university of medical
sciences by the pabn lasoo model 2011–2014. J Babol Univ Med Sci. 2015;
17:76–82. 24. Hsieh H-F, Shannon SE. Three approaches to qualitative content analysis. Qual Health Res. 2005;15:1277–88. 2. Sharifi A, Jalali R, Shahbazi N. Received: 10 July 2019 Accepted: 26 February 2020 Received: 10 July 2019 Accepted: 26 February 2020 22. Rycroft-Malone J. The PARIHS framework—a framework for guiding the
implementation of evidence-based practice. J Nurs Care Qual. 2004;19:297–304 22. Rycroft-Malone J. The PARIHS framework—a framework for guiding the
implementation of evidence-based practice. J Nurs Care Qual. 2004;19:297–304. Acknowledgements
ld l k
h 8. Schloemer T, Schröder-Bäck P. Criteria for evaluating transferability of health
interventions: a systematic review and thematic synthesis. Implement Sci. 2018;13:88. We would like to thank all the healthcare system manager and staffs who
kindly cooperated for the interviews. In addition, we would like to thank
Islamic Azad University- Sari Branch, Sari, Iran FOR supported this research. 9. Litvaj I, Stancekova D. Decision-making, and their relation to the knowledge
management, use of knowledge management in decision-making. Procedia
Econ Finance. 2015;23:467–72. Consent for publication
Not applicable. 18. Rosenbaum SE, Glenton C, Wiysonge CS, Abalos E, Mignini L, Young T,
Althabe F, Ciapponi A, Marti SG, Meng Q. Evidence summaries tailored to
health policy-makers in low-and middle-income countries. Bull World Health
Organ. 2011;89:54–61. Not applicable. Availability of data and materials 13. Ellen ME, Lavis JN, Shemer J. Examining the use of health systems and
policy research in the health policymaking process in Israel: views of
researchers. Health Res Policy Syst. 2016;14(1):66. Since the present study is a qualitative study based on the statements of the
participants as pointed out in Section 4 of the informed consent form, the
participants only allowed the researchers to publish the findings as a whole. Therefore, we do not have permission to provide research data and
materials in public or in an appendix. However, the research team is
committed to providing personal data (IPDs) if requested by other
researchers by email to the responsible author. 14. Cameron D, Lavis JN, Guindon GE, Akhtar T, Posada FB, Ndossi GD, Boupha
B. Bridging the gaps among research, policy and practice in ten low-and
middle-income countries: development and testing of a questionnaire for
researchers. Health Res Policy Syst. 2010;8:4. 15. Graham ID, Kothari A, McCutcheon C. Moving knowledge into action for more
effective practice, programmes and policy: protocol for a research programme
on integrated knowledge translation. Implement Sci. 2018;13:22. 15. Graham ID, Kothari A, McCutcheon C. Moving knowledge into action for more
effective practice, programmes and policy: protocol for a research programme
on integrated knowledge translation. Implement Sci. 2018;13:22. Competing interests The authors declare that they have no competing interests. 19. M. Dobbins, D. Ciliska, R. Cockerill, J. Barnsley, A. DiCenso, A framework for
the dissemination and utilization of research for health-care policy and
practice, Worldviews on Evidence-based Nursing presents the archives of
Online Journal of Knowledge Synthesis for Nursing, 9 (2002) 149–160. Authors’ contributions 10. Uzochukwu B, Onwujekwe O, Mbachu C, Okwuosa C, Etiaba E, Nyström ME,
Gilson L. The challenge of bridging the gap between researchers and policy
makers: experiences of a health policy research group in engaging policy
makers to support evidence informed policy making in Nigeria. Glob Health. 2016;12:67. The research was conception and designed by M GH, R GH and H KH in
collaboration. M GH conducted the interview and took main role in drafting
and revising the manuscript. R GH and H KH contributed to supervision in
drafting and revising. All authors have approved the submitted version of
the manuscript, and agreed to be personally accountable for the author’s
own contributions and to ensure that questions related to any part of the
work appropriately investigated and resolved. 11. Shroff Z, Aulakh B, Gilson L, Agyepong IA, El-Jardali F, Ghaffar A. Incorporating research evidence into decision-making processes: researcher
and decision-maker perceptions from five low-and middle-income
countries. Health Res Policy Syst. 2015;13:70. Funding
No founding. 12. Cossette B, Bergeron J, Ricard G, Éthier JF, Joly-Mischlich T, Levine M, Sene
M, Mallet L, Lanthier L, Payette H. Knowledge translation strategy to reduce
the use of potentially inappropriate medications in hospitalized elderly
adults. J Am Geriatr Soc. 2016;64:2487–94. 12. Cossette B, Bergeron J, Ricard G, Éthier JF, Joly-Mischlich T, Levine M, Sene
M, Mallet L, Lanthier L, Payette H. Knowledge translation strategy to reduce
the use of potentially inappropriate medications in hospitalized elderly
adults. J Am Geriatr Soc. 2016;64:2487–94. Author details
1 1Department of Health Services Management, Sari Branch, Islamic Azad
University, Sari, Iran. 2Associated professor, hospital administration research
center, Sari branch, Islamic Azad University, Sari, Iran. 3Nursing Research
Center, Golestan University of Medical Sciences, Gorgan, Iran. 20. Ward V, House A, Hamer S. Developing a framework for transferring
knowledge into action: a thematic analysis of the literature. J Health Serv
Res Policy. 2009;14:156–64. 21. Harvey G, Kitson A. PARIHS revisited: from heuristic to integrated framework
for the successful implementation of knowledge into practice. Implement
Sci. 2015;11:33. Ethics approval and consent to participate The research approved by the research ethics Committee of Islamic Azad
University- Sari branch. Before beginning the interviews, each participant
provided with necessary information by principal investigator about the
study and interview began after obtaining written informed consent. 16. Banja JD, Eisen A. Ethical perspectives on knowledge translation in
rehabilitation. Arch Phys Med Rehabil. 2013;94:S55–60. 16. Banja JD, Eisen A. Ethical perspectives on knowledge translation in
rehabilitation. Arch Phys Med Rehabil. 2013;94:S55–60. 17. Yousefi-Nooraie R, Rashidian A, Nedjat S, Majdzadeh R, Mortaz-Hedjri S,
Etemadi A, Salmasian H. Promoting development and use of systematic
reviews in a developing country. J Eval Clin Pract. 2009;15:1029–34. 17. Yousefi-Nooraie R, Rashidian A, Nedjat S, Majdzadeh R, Mortaz-Hedjri S,
Etemadi A, Salmasian H. Promoting development and use of systematic
reviews in a developing country. J Eval Clin Pract. 2009;15:1029–34. Consent for publication
Not applicable. References Role of “sexternal facilitation” in
implementation of research findings: a qualitative evaluation of facilitation
experiences in the veterans health administration. Implement Sci. 2006;1:23. 45. Harvey G, Llewellyn S, Maniatopoulos G, Boyd A, Procter R. Facilitating the
implementation of clinical technology in healthcare: what role does a
national agency play? BMC Health Serv Res. 2018;18:347. 45. Harvey G, Llewellyn S, Maniatopoulos G, Boyd A, Procter R. Facilitating the
implementation of clinical technology in healthcare: what role does a
national agency play? BMC Health Serv Res. 2018;18:347. 46. Léon G, Ouimet M, Lavis JN, Grimshaw J, Gagnon M-P. Assessing availability
of scientific journals, databases, and health library services in Canadian
health ministries: a cross-sectional study. Implement Sci. 2013;8:34. 46. Léon G, Ouimet M, Lavis JN, Grimshaw J, Gagnon M-P. Assessing availability
of scientific journals, databases, and health library services in Canadian
health ministries: a cross-sectional study. Implement Sci. 2013;8:34. 47. Holroyd-Leduc J, Resin J, Ashley L, Barwich D, Elliott J, Huras P, Légaré F,
Mahoney M, Maybee A, McNeil H. Giving voice to older adults living with
frailty and their family caregivers: engagement of older adults living with
frailty in research, health care decision making, and in health policy. Res
Involv Engagem. 2016;2:23. 47. Holroyd-Leduc J, Resin J, Ashley L, Barwich D, Elliott J, Huras P, Légaré F,
Mahoney M, Maybee A, McNeil H. Giving voice to older adults living with
frailty and their family caregivers: engagement of older adults living with
frailty in research, health care decision making, and in health policy. Res
Involv Engagem. 2016;2:23. 48. Mekki TE, Øye C, Kristensen B, Dahl H, Haaland A, Nordin KA, Strandos M,
Terum TM, Ydstebø AE, McCormack B. The inter-play between facilitation
and context in the promoting action on research implementation in health
services framework: a qualitative exploratory implementation study
embedded in a cluster randomized controlled trial to reduce restraint in
nursing homes. J Adv Nurs. 2017;73:2622–32. References Evaluation of awareness on the patient bill of
rights and observing rate on the patient’s perspective in imam Reza
Hospital in Kermanshah in 2012. Iranian J of Med Law. 2013;6:125–35. 25. Rycroft-Malone J, Bucknall T. Models and frameworks for implementing
evidence-based practice: linking evidence to action: John Wiley & Sons; 2010. 3. Basiri MK, Basiri MM, Moslem A, Ajam ZH, Jamal F. Health Providers and
Patients'awareness on Patient Bill Of Rights and its Observing Rate in an
Educational Hospital in Gonabad. Horizon Med Sci. 2011;17:45–54. 26. Jansson I, Forsberg A. How do nurses and ward managers perceive that
evidence-based sources are obtained to inform relevant nursing
interventions?–an exploratory study. J Clin Nurs. 2016;25:769–76. 4. Grimshaw JM, Eccles MP, Lavis JN, Hill SJ, Squires JE. Knowledge translation
of research findings. Implement Sci. 2012;7:50. 27. Ward MM, Baloh J, Zhu X, Stewart GL. Promoting action on research
implementation in health services framework applied to TeamSTEPPS
implementation in small rural hospitals. Health Care Manag Rev. 2017;42:2–13. 5. Rocha ESB, Nagliate P, Furlan CEB, Rocha K Jr, Trevizan MA, Mendes IAC. Knowledge management in health: a systematic literature review. Revista
latino-americana de enfermagem. 2012;20:392–400. 28. Snelgrove-Clarke E, Davies B, Flowerdew G, Young D. Implementing a fetal
health surveillance guideline in clinical practice: a pragmatic randomized
controlled trial of action learning. Worldviews Evid-Based Nurs. 2015;12:281–8. 6. Ellen ME, Lavis JN, Horowitz E, Berglas R. How is the use of research
evidence in health policy perceived? A comparison between the reporting
of researchers and policy-makers. Health Res Policy Syst. 2018;16:64. 29. F. Gagnon, P. Bergeron, Les conditions de mise à profit des connaissances
par les acteurs de santé publique lors de la formulation des politiques
publiques: L’apport de la littérature sur le transfert des connaissances,
Chaire d’étude sur l’application des connaissances, UQÀM, (2013). 7. Rubio DM, Schoenbaum EE, Lee LS, Schteingart DE, Marantz PR, Anderson
KE, Platt LD, Baez A, Esposito K. Defining translational research: implications
for training. Academic Med. 2010;85:470. Page 9 of 9 Roohi et al. BMC Health Services Research (2020) 20:188 Roohi et al. BMC Health Services Research (2020) 20:188 30. Grove SK, Burns N, Gray J. The practice of nursing research: appraisal,
synthesis, and generation of evidence, Elsevier Health Sciences; 2012. 31. Majdzadeh R, Sadighi J, Nejat S, Mahani AS, Gholami J. Knowledge
translation for research utilization: design of a knowledge translation model
at Tehran University of Medical Sciences. References J Contin Educ Health Prof. 2008;28:
270–7. 32. Mehrdad N, Salsali M, Kazemnejad A. The spectrum of barriers to and
facilitators of research utilization in Iranian nursing. J Clin Nurs. 2008;17:
2194–202. 33. Yava A, Tosun N, Çiçek H, Yavan T, Terakye G, Hatipoğlu S. Nurses'
perceptions of the barriers to and the facilitators of research utilization in
Turkey. Appl Nurs Res. 2009;22:166–75. 34. Ciliska D, Hayward S, Dobbins M, Brunton G, Underwood J. Transferring
public-health nursing research to health-system planning: assessing the
relevance and accessibility of systematic reviews, Canadian journal of
nursing research archive, 31; 1999. 35. Azimi A, Fattahi R, Asadi-Lari M. Knowledge translation status and barriers. J
Med Libr Assoc. 2015;103:96. 36. Suter E, Armitage G. Use of a knowledge synthesis by decision makers and
planners to facilitate system level integration in a large Canadian provincial
health authority. Int J Integr Care. 2011;11:e011. 37. Von Krogh G, NÅden D. Implementation of a documentation model
comprising nursing terminologies–theoretical and methodological issues. J
Nurs Manag. 2008;16:275–83. 38. Ritchie MJ, Kirchner JE, Parker LE, Curran GM, Fortney JC, Pitcock JA, Bonner
LM, Kilbourne AM. Evaluation of an implementation facilitation strategy for
settings that experience significant implementation barriers. In:
Implementation Science, BioMed Central; 2015. p. A46. 39. Senge PM, Cambron-McCabe N, Lucas T, Smith B, Dutton J. Schools that
learn (updated and revised): a fifth discipline fieldbook for educators,
parents, and everyone who cares about education, Crown Business; 2012. 40. Lessard S, Bareil C, Lalonde L, Duhamel F, Hudon E, Goudreau J, Lévesque L. External facilitators and interprofessional facilitation teams: a qualitative study
of their roles in supporting practice change. Implement Sci. 2015;11:97. 41. Dogherty EJ, Harrison MB, Baker C, Graham ID. Following a natural
experiment of guideline adaptation and early implementation: a mixed-
methods study of facilitation. Implement Sci. 2012;7:9. 42. Kirchner JE, Parker LE, Bonner LM, Fickel JJ, Yano EM, Ritchie MJ. Roles of
managers, frontline staff and local champions, in implementing quality
improvement: stakeholders' perspectives. J Eval Clin Pract. 2012;18:63–9. 43. Lövestam E, Orrevall Y, Koochek A, Karlström B, Andersson A. Evaluation of
N utrition C are P rocess documentation in electronic patient records: need
of improvement. Nutr Diet. 2015;72:74–80. 44. Stetler CB, Legro MW, Rycroft-Malone J, Bowman C, Curran G, Guihan M,
Hagedorn H, Pineros S, Wallace CM. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in
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Musical ability is associated with enhanced auditory and visual cognitive processing
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© 2015 Faßhauer et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/
publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Faßhauer et al. BMC Neurosci (2015) 16:59
DOI 10.1186/s12868-015-0200-4 Faßhauer et al. BMC Neurosci (2015) 16:59
DOI 10.1186/s12868-015-0200-4 Open Access Abstract Background: Musical ability has always been linked to enhanced cognitive and intellectual skills. We were interested
in the relation between musical ability and short-time cognitive processing as measured by event-related potentials,
in particular in visual processing, since previous studies have already suggested such a link for acoustic cognitive
processing. We measured auditory and visual event-related potentials as elicited by an oddball paradigm in 20 healthy
subjects (10 musicians and 10 non-musicians; 10 female; mean age 24 ± 2 years). In addition, the Seashore test and a
test developed by the authors to detect relevant amusia, the latter one with a high ceiling effect, were also applied. Results: The most important finding was that there is a significant linear correlation between musical ability as
measured by these tests and the P3 latencies of both the auditory and visual event-related potentials. Furthermore,
musicians showed shorter latencies of the event-related potentials than non-musicians. Conclusions: We conclude that musical ability as measured by neuropsychological tests is associated with improved
short-time cognitive processing both in the auditory and, surprisingly, also in the visual domain. Keywords: Music, Musical ability, Event-related potentials, Visual cognition, Auditory cognition Conclusions: We conclude that musical ability as measured by neuropsychological tests is associated with improved
short-time cognitive processing both in the auditory and, surprisingly, also in the visual domain. Keywords: Music, Musical ability, Event-related potentials, Visual cognition, Auditory cognition (ERP), magnetic resonance imaging (MRI) to the differ-
ent methods of functional imaging. The processing of
auditory stimuli and the differences in such processing
between musicians and non-musicians have been inves-
tigated especially by means of auditory ERP which can
objectively quantify latencies of stimulus processing. This evaluation is regarded as a measure for the quality
of some aspects of cognitive processing such as stimu-
lus evaluation, cognitive processing speed and internal
short-term memory functions. Musical ability is associated
with enhanced auditory and visual cognitive
processing Caroline Faßhauer1, Achim Frese1,2 and Stefan Evers1,3* *Correspondence: everss@uni‑muenster.de
3 Department of Neurology, Krankenhaus Lindenbrunn, Lindenbrunn 1,
31863 Coppenbrügge, Germany
Full list of author information is available at the end of the article Background Musical ability as a term is used to describe the sensitiv-
ity for music, the ability to understand music, and/or the
ability to produce music. There is no standard definition,
and it is hard to measure musical ability. One can only
measure how well a person can perceive musical stimuli
such as small changes in pitch, loudness, rhythm, and
other sub-domains of music processing. It is generally
accepted that some people show higher musical abil-
ity than others. But what are the correlates for the term
‘musical ability’ in the human brain? What are the differ-
ences between people with and without musical ability?h In general, rare target stimuli and frequent non-target
stimuli are presented to subjects in ERP studies. The
latencies and amplitudes of the potentials evoked by the
target stimuli are analyzed such as the Mismatch Nega-
tivity (MMN or N2a) in auditory ERP studies and the
P3 in both visual and auditory ERP studies. In previous
music research studies, the subjects were either profes-
sional and non-professional musicians, people with abso-
lute pitch, or non-musicians. These questions and many others about music and
music processing in the human brain have been a subject
of interest in neuroscience research. The methods used in
this field range widely from EEG, event-related potentials *Correspondence: everss@uni‑muenster.de
3 Department of Neurology, Krankenhaus Lindenbrunn, Lindenbrunn 1,
31863 Coppenbrügge, Germany
Full list of author information is available at the end of the article Faßhauer et al. BMC Neurosci (2015) 16:59 Page 2 of 9 Looking at the results of different studies [1–5], one
can sum up that musicians with absolute pitch ability and
musicians with relative pitch seem to have a shorter P3
latency in the auditory evoked ERP and also a smaller P3
amplitude than non-musicians. The P3 could even not
be found at all in some musicians with absolute pitch [3]
when the method that led to a P3 signal in all musicians
with relative pitch was the discrimination between major
thirds as non-target and minor thirds as target stimuli. In a timbre discrimination study [6], subjects with abso-
lute pitch showed a shorter P3 latency and a smaller P3
amplitude than other musicians and non-musicians. The subjects had to differentiate between the sound of a
tone with the same pitch played on a viola or a cello, on
two different kinds of flutes and on two different kinds
of tubas. Results As shown in Table 1, there were no significant differ-
ences between the two groups with respect to age and the
scores of the Zerssen scale. All subjects showed a normal
mental well-being. This is important since feeling unwell
could impair the results of the musical testing. Musical abilityh The scores in the self-developed amusia tests and in the
Seashore test are shown in Table 1. In average, the group
of musicians scored 83.5 ± 1.2 out of 86 points in the
amusia test as opposed to the group of non-musicians
with a score of 67.2 ± 5.7 (p < 0.001). In the Seashore
test, the results were significantly different between both
groups for each of the six categories. The musicians’ total
score was 233.4 ± 6.6, the non-musicians’ total score was
197.9 ± 22.7 (p < 0.001). All in all, the studies mentioned above suggest a faster
discrimination of auditory stimuli in musicians (with or
without absolute pitch). None of the studies had analyzed
the latencies of visually evoked ERP, and only two [1, 10]
have measured the amplitudes of the visually evoked P3
latency in subjects with absolute pitch, which was not
found to be changed. Background In the last (tuba) part, the musicians showed a
significantly shorter P3 latency than the non-musicians. similar study, our group was able to show a significantly
decreased auditory and visually evoked P3 latency in
musicians as well as a larger amplitude of the P3 in the
auditory domain [12]. In the present study, we were interested in aspects of
the specific visual cognitive processing in musicians ver-
sus non-musicians. In particular, we aimed to correlate
the ERP results of visual and auditory stimulus processing
with musical ability as evaluated by a psychometric meas-
ure. We chose an oddball paradigm since this is a very
easy task not detracting the probands too much. Further-
more, oddball paradigms are often used in research on
musical cognitive processing in the past. Since a specific
cognitive processing of ERP has been shown for auditory
stimuli, a similar result for visual stimuli would suggest
that musical ability is associated with a specific cognitive
processing in all modalities. i
In a different approach, a piano phrase by Bach was
used for eliciting ERP [7]. They presented the original
phrase (non-target) or the same phrase with an inhar-
monic note (target) or the same phrase with an unex-
pected mordent (target) to a group of musicians and a
group of non-musicians. A shorter P3 latency for musi-
cians could not be shown, but instead of that and among
other results, a shorter N2 latency was detected. In
a study in 1996 [8], the reaction time of musicians was
shorter in comparison to non-musicians when the sub-
jects had to discriminate between small differences in
frequency. Further, it was incidentally found a signifi-
cantly decreased P3a latency in musicians (i.e., vocalists
and instrumentalists) compared to non-musicians, evok-
ing the P3 by means of pitch deviants [9]. The P3a signal
was suggested to be a sensitive index of musical expertise. Visually and auditory evoked ERP Table 2 shows the data of the visual ERP (elicited by an
oddball paradigm with red light as target), Table 3 shows
the data of the auditory ERP (elicited by an oddball para-
digm with a high tone as target), presented separately for
both groups. The analysis of the visual ERP resulted in
significant differences in the P3 latency and the P3 habit-
uation (i.e., difference of P3 latency in two different trials)
after the target stimuli: the P3 latency was 390 ± 33 ms
in the musician group and 411 ± 22 ms in the non-musi-
cian group (p = 0.043). The P3 latency habituation was
0.6 ± 7.8 ms (musicians) and 7.3 ± 11.0 ms (non-musi-
cians) with a significance of p = 0.018. The evaluation
of the mean choice reaction time, the P3 amplitude, and
the signals occurring after the frequent stimulus did not
result in any significant differences. We raised the question whether people with absolute
pitch have a different cognitive stimulus processing as
measured by ERP, and not just a different processing
of auditory stimuli, as most of the studies mentioned
above suggest. In an experiment with three participat-
ing groups (musicians with absolute pitch, musicians
without absolute pitch, and non-musicians), our group
was able to show that both people with absolute pitch
compared to non-musicians and musicians compared
to non-musicians show a significantly decreased P3
latency in the auditory and visually evoked ERP [11]. The P3 amplitudes were not significantly different. In a Faßhauer et al. BMC Neurosci (2015) 16:59 Page 3 of 9 Table 1 Data at baseline of the two subject groups
Comparison between groups by Mann–Whitney U test
Musicians (n = 10)
Non-musicians (n = 10)
Significance
Age
23 ± 2
24 ± 2
ns (p = 0.436)
Sex
5 male/5 female
5 male/5 female
–
Amusia test
1a. Rhythm
15.5 ± 0.7
9.8 ± 1.8
p < 0.001
1b. Metrum
16.0 ± 0.0
11.4 ± 2.9
p < 0.001
2. Comparison of melodies
15.5 ± 0.7
12.6 ± 1.8
p < 0.001
3. Emotion
11.2 ± 0.9
11.0 ± 0.9
ns (p = 0.684)
4. Pitch
12.0 ± 0.0
10.5 ± 1.3
p = 0.007
5. Identification of melodies
13.3 ± 0.7
11.9 ± 1.6
ns (p = 0.052)
Total score
83.5 ± 1.2
67.2 ± 5.7
p < 0.001
Seashore test
1. Visually and auditory evoked ERP Pitch
46.1 ± 2.9
34.6 ± 8.6
p = 0.001
2. Loudness
45.4 ± 1.8
42.4 ± 3.0
p = 0.011
3. Rhythm
28.6 ± 1.0
25.7 ± 3.0
p = 0.005
4. Duration of a tone
45.0 ± 2.8
42.2 ± 2.3
p = 0.029
5. Timbre
42.3 ± 3.1
37.1 ± 6.0
p = 0.043
6. Tonal memory
26.0 ± 2.3
15.9 ± 6.7
p = 0.001
Total score
233.4 ± 6.6
197.9 ± 22.7
p < 0.001
Zerssen 1
29.9 ± 5.6
32.8 ± 6.8
ns (p = 0.315)
Zerssen 2
31.3 ± 9.9
32.2 ± 6.0
ns (p = 0.393) Table 1 Data at baseline of the two subject groups Table 2 Data of visually evoked event-related potentials (oddball paradigm) recording presented separately for both
subjects group The latencies in the auditory ERP showed similar dif-
ferences between the two groups: the P3 latency in
the musician group was 328 ± 34 ms as opposed to
360 ± 10 ms in the non-musician group (p = 0.019). The
P3 latency habituation differed by 23.2 ms between the
two groups with a P3 habituation of −5.8 ± 19.6 ms in
the group of musicians (p = 0.009). Correlation of P3 latency and musical ability
We further calculated the correlation between the results
in the Seashore test and the P3 latencies. As shown in
Figs. Visually and auditory evoked ERP 1 and 2, there was a negative correlation between the
P3 latency of the visual ERP and the total score of the Sea-
shore test (r = −0.470 and p = 0.036; Spearman-rank-coef-
ficient), as well as between the P3 latency of the auditory
Table 2 Data of visually evoked event-related potentials (oddball paradigm) recording presented separately for both
subjects group
Musicians (n = 10)
Non-musicians (n = 10)
Significance
Frequent stimulus
P1 latency (ms)
122 ± 11
81 ± 0
ns (p = 0.500)
N1 latency (ms)
165 ± 23
169 ± 24
ns (p = 0.604)
P2 latency (ms)
252 ± 14
251 ± 16
ns (p = 0.837)
N2 latency (ms)
305 ± 19
301 ± 18
ns (p = 0.755)
P3 latency (ms)
394 ± 23
383 ± 52
ns (p = 0.902)
Infrequent stimulus
P1 latency (ms)
94 ± 27
116 ± 0
ns (p = 0.667)
N1 latency (ms)
156 ± 23
168 ± 20
ns (p = 0.243)
P2 latency (ms)
228 ± 9
239 ± 15
ns (p = 0.105)
N2 latency (ms)
272 ± 16
277 ± 15
ns (p = 0.631)
P3 latency (ms)
390 ± 33
411 ± 22
p = 0.043
P3 amplitude (µV)
14 ± 6
12 ± 4
ns (p = 0.253)
P3 habituation (ms)
0.6 ± 7.8
7.3 ± 11.0
p = 0.018
Mean choice reaction time (ms)
381 ± 45
405 ± 58
ns (p = 0.436) lly evoked event-related potentials (oddball paradigm) recording presented separately for both
Musicians (n = 10)
Non-musicians (n = 10)
Significance lly evoked event-related potentials (oddball paradigm) recording presented separately for both Table 2 Data of visually evoked event-related potentials (oddball paradigm) recording presente
subjects group Correlation of P3 latency and musical ability The latencies in the auditory ERP showed similar dif-
ferences between the two groups: the P3 latency in
the musician group was 328 ± 34 ms as opposed to
360 ± 10 ms in the non-musician group (p = 0.019). The
P3 latency habituation differed by 23.2 ms between the
two groups with a P3 habituation of −5.8 ± 19.6 ms in
the group of musicians (p = 0.009). We further calculated the correlation between the results
in the Seashore test and the P3 latencies. As shown in
Figs. Visually and auditory evoked ERP 2 Correlation between P3 latency of auditory evoked event-
related potentials and results in the Seashore-test (r = −0.434;
p = 0.038; Spearman-rank-coefficient) Table 3 Data of auditory evoked event-related potentials (oddball paradigm) recording presented separately for both
subjects group
Musicians (n = 10)
Non-musicians (n = 10)
Significance
Frequent stimulus
P1 latency (ms)
60 ± 2
63 ± 3
ns (p = 0.229)
N1 latency (ms)
125 ± 22
112 ± 6
ns (p = 0.247)
P2 latency (ms)
229 ± 27
199 ± 17
p = 0.021
N2 latency (ms)
302 ± 11
259 ± 43
ns (p = 0.190)
P3 latency (ms)
360 ± 29
332 ± 48
ns (p = 0.400)
Infrequent stimulus
P1 latency (ms)
54 ± 5
71 ± 29
ns (p = 0.413)
N1 latency (ms)
109 ± 10
109 ± 9
ns (p = 0.631)
P2 latency (ms)
171 ± 20
180 ± 13
ns (p = 0.165)
N2 latency (ms)
218 ± 22
230 ± 15
ns (p = 0.123)
P3 latency (ms)
328 ± 34
360 ± 10
p = 0.019
P3 amplitude (µV)
13 ± 5
12 ± 4
ns (p = 0.971)
P3 habituation (ms)
−5.8 ± 19.6
17.4 ± 11.2
p = 0.009
Mean choice reaction time (ms)
345 ± 43
349 ± 56
ns (p = 1.000) Table 3 Data of auditory evoked event-related potentials (oddball paradigm) recording presented separately for both
subjects group
Musicians (n = 10)
Non-musicians (n = 10)
Significance Fig. 2 Correlation between P3 latency of auditory evoked event-
related potentials and results in the Seashore-test (r = −0.434;
p = 0.038; Spearman-rank-coefficient) Fig. 1 Correlation between P3 latency of the visual event-related
potentials and the total score of the Seashore-test (r = −0.470 and
p = 0.036; Spearman-rank-coefficient) Fig. 1 Correlation between P3 latency of the visual event-related
potentials and the total score of the Seashore-test (r = −0.470 and
p = 0.036; Spearman-rank-coefficient) Fig. 2 Correlation between P3 latency of auditory evoked event-
related potentials and results in the Seashore-test (r = −0.434;
p = 0.038; Spearman-rank-coefficient) Fig. 2 Correlation between P3 latency of auditory evoked event-
related potentials and results in the Seashore-test (r = −0.434;
p = 0.038; Spearman-rank-coefficient) ERP and the total score of the Seashore-test (r = −0.434
and p = 0.038). Visually and auditory evoked ERP 1 and 2, there was a negative correlation between the
P3 latency of the visual ERP and the total score of the Sea-
shore test (r = −0.470 and p = 0.036; Spearman-rank-coef-
ficient), as well as between the P3 latency of the auditory Faßhauer et al. BMC Neurosci (2015) 16:59 Page 4 of 9 ERP and the total score of the Seashore-test (r = −0.434
and p = 0.038). This means that a higher musical ability is
correlated to a shorter visual and auditory P3 latency. Discussion
Testing musical ability
In the amusia test, the musicians scored significantly
higher than the non-musicians, except for the categories
‘emotion’ and ‘identification of melodies’. The purpose
of this amusia test was to rule out a clinically relevant
amusia in the non-musician group. The average score
of 67 is within the normal range of >60 [13]. Therefore,
the non-musician group did not have a relevant impair-
ment of musical ability. One has to take into account that
this test has a high ceiling effect and was not designed
Table 3 Data of auditory evoked event-related potentials (oddball paradigm) recording presented separately for both
subjects group
Musicians (n = 10)
Non-musicians (n = 10)
Significance
Frequent stimulus
P1 latency (ms)
60 ± 2
63 ± 3
ns (p = 0.229)
N1 latency (ms)
125 ± 22
112 ± 6
ns (p = 0.247)
P2 latency (ms)
229 ± 27
199 ± 17
p = 0.021
N2 latency (ms)
302 ± 11
259 ± 43
ns (p = 0.190)
P3 latency (ms)
360 ± 29
332 ± 48
ns (p = 0.400)
Infrequent stimulus
P1 latency (ms)
54 ± 5
71 ± 29
ns (p = 0.413)
N1 latency (ms)
109 ± 10
109 ± 9
ns (p = 0.631)
P2 latency (ms)
171 ± 20
180 ± 13
ns (p = 0.165)
N2 latency (ms)
218 ± 22
230 ± 15
ns (p = 0.123)
P3 latency (ms)
328 ± 34
360 ± 10
p = 0.019
P3 amplitude (µV)
13 ± 5
12 ± 4
ns (p = 0.971)
P3 habituation (ms)
−5.8 ± 19.6
17.4 ± 11.2
p = 0.009
Mean choice reaction time (ms)
345 ± 43
349 ± 56
ns (p = 1.000)
Fig. 1 Correlation between P3 latency of the visual event-related
potentials and the total score of the Seashore-test (r = −0.470 and
p = 0.036; Spearman-rank-coefficient)
Fig. Visually and auditory evoked ERP This means that a higher musical ability is
correlated to a shorter visual and auditory P3 latency. ‘emotion’ and ‘identification of melodies’. The purpose
of this amusia test was to rule out a clinically relevant
amusia in the non-musician group. The average score
of 67 is within the normal range of >60 [13]. Therefore,
the non-musician group did not have a relevant impair-
ment of musical ability. One has to take into account that
this test has a high ceiling effect and was not designed Visually and auditory evoked ERP 2 Correlation between P3 latency of auditory evoked event-
related potentials and results in the Seashore-test (r = −0.434;
p = 0.038; Spearman-rank-coefficient) ERP and the total score of the Seashore-test (r = −0.434
and p = 0.038). This means that a higher musical ability is
correlated to a shorter visual and auditory P3 latency. ‘emotion’ and ‘identification of melodies’. The purpose
of this amusia test was to rule out a clinically relevant
h
Th
Table 3 Data of auditory evoked event-related potentials (oddball paradigm) recording presented separately for both
subjects group
Musicians (n = 10)
Non-musicians (n = 10)
Significance
Frequent stimulus
P1 latency (ms)
60 ± 2
63 ± 3
ns (p = 0.229)
N1 latency (ms)
125 ± 22
112 ± 6
ns (p = 0.247)
P2 latency (ms)
229 ± 27
199 ± 17
p = 0.021
N2 latency (ms)
302 ± 11
259 ± 43
ns (p = 0.190)
P3 latency (ms)
360 ± 29
332 ± 48
ns (p = 0.400)
Infrequent stimulus
P1 latency (ms)
54 ± 5
71 ± 29
ns (p = 0.413)
N1 latency (ms)
109 ± 10
109 ± 9
ns (p = 0.631)
P2 latency (ms)
171 ± 20
180 ± 13
ns (p = 0.165)
N2 latency (ms)
218 ± 22
230 ± 15
ns (p = 0.123)
P3 latency (ms)
328 ± 34
360 ± 10
p = 0.019
P3 amplitude (µV)
13 ± 5
12 ± 4
ns (p = 0.971)
P3 habituation (ms)
−5.8 ± 19.6
17.4 ± 11.2
p = 0.009
Mean choice reaction time (ms)
345 ± 43
349 ± 56
ns (p = 1.000)
Fig. 1 Correlation between P3 latency of the visual event-related
potentials and the total score of the Seashore-test (r = −0.470 and
p = 0.036; Spearman-rank-coefficient)
Fig. Testing musical ability In the amusia test, the musicians scored significantly
higher than the non-musicians, except for the categories Page 5 of 9 Faßhauer et al. BMC Neurosci (2015) 16:59 to auditory stimuli in form of practising and listening to
music, this result does not seem surprising. for measuring small interindividual differences in musi-
cal ability, although different categories of musical abil-
ity are tested. This explains why in two of the categories
the results were not significantly different between musi-
cians and non-musicians; and this is also the reason why
we used the Seashore test results for further analysis of
musical ability and the correlation analysis with the P3
latencies. Even though the mechanisms that determine latency of
the P3 response have not yet been fully understood, there
are several studies which have shown differences in the
(auditory) brain structures of musicians. Several stud-
ies suggest a practice-induced brain plasticity and thus
a functional and structural difference in auditory brain
areas of musicians versus non-musicians [14–17, 19]. The
fact that musicians show an earlier P3 latency than non-
musicians and thus have a faster auditory stimulus pro-
cessing may be associated with those results. Supporting
this notion, a negative correlation between P3 latency
and the years of musical training was found [12]. The musicians scored significantly higher in the Sea-
shore-test. This confirms that the group of musicians
has a higher musical ability than the group of non-musi-
cians. But what are the reasons for this? Several studies
have investigated the question whether a higher degree
of musical ability in musicians could be a result of prac-
tice-induced cortical plasticity or an innately given talent
[14–18]. In our study, not only the auditory P3 latency was ear-
lier in musicians but also the visual P3 latency. Musical
training and/or musical ability therefore seem to affect
also the visual perceptive system. This also goes along
with the results of other studies concerning the musi-
cians’ visual abilities. It was shown that musicians out-
performed non-musicians in the ability of perceptual
speed, which requires quick visual information process-
ing [20]. Visual memory was also reported to be better in
musicians than in non-musicians [21–24]; even primary
visual perception [25] and somatosensory cognitive pro-
cessing [26] were reported to be enhanced in musicians. However, these studies used psychometric measures and
did not evaluate cognitive processing in an objective way. Testing musical ability A difference in grey matter volume in visual-spatial areas
was found [15], going along with other results [27] indi-
cating an improvement of visual-spatial tasks in musi-
cally trained children. In two studies [16, 17], non-musicians who had learned
to play a certain rhythmical sequence or different melodic
sequences showed an increased MMN in response to
rhythm or pitch incongruities. Another group of non-
musicians, who had just listened to the other group
practising, did not show an increased MMN response. The authors interpret this finding as a sign of practice-
induced cortical plasticity. Therefore, we assume that the
musicians’ higher score in the Seashore test is a result of
the musical training of 14 ± 2 years on average, although
parts of the musical ability could also be a genetically
driven talent. It was shown that professional musicians compared
to amateur and non-musicians have an increase in grey
matter volume in several brain regions such as motor, vis-
ual-spatial, and especially auditory regions [15]. Because
of the associations found between structural differences,
musician status, and practice intensity, the authors inter-
pret their results as practice-induced adaptations of the
brain. In 2009, changes induced by private keyboard les-
sons in young children who had no prior musical training
were studied [18]: the results showed that instrumental
children (versus children in a control group) showed a
greater size of certain motor and auditory areas after
15 months of training and that their outcomes in motor
and melody/rhythm tests also improved significantly. Again, this supports the hypothesis of practice-induced
musical skills going along with structural brain changes. Correlation of Seashore test results and P3 latency
In our study, we were also able to link the early P3
responses in the auditory and visual paradigms to the
results of the Seashore-test and found a negative cor-
relation between the data. In studies on musicians, a
musician is usually defined as a subject who has learned
to play an instrument for a certain amount of years and
musicians can be classified as ‘professional’ or ‘amateur’. These definitions usually are the criteria for the participa-
tion of musicians in studies, but having had music lessons
for a certain amount of years does not necessarily yield in
a high musical ability. Testing musical ability With our results, we showed that
not just the status ‘musicianship’, but the amount of musi-
cal ability as measured by a psychometric test is related
to the improved cognitive processing of musicians versus
non-musicians. P3 latency and musical abilityh The P3 response to the target stimuli of both the visual
and auditory ERP was significantly earlier in the group
of musicians. This result confirms the results of previ-
ous studies [11, 12]. An early P3 response indicates fast
stimulus processing and discrimination. Thus, musicians
seem to be able to discriminate faster between auditory
stimuli. Considering their long and special exposition All in all, people with high musical ability seem to have
a faster auditory and visual cognitive processing. The
question is, however, whether this connection between
musical ability and cognitive processing is inherited or Faßhauer et al. BMC Neurosci (2015) 16:59 Page 6 of 9 whether the acquisition of musical ability (i.e. long-term
practice of an instrument) leads to an enhancement of
cognitive processing. At this time, one can only specu-
late about the answer: on the one hand, fast cognitive
processing is probably helpful for learning an instru-
ment, which requires fast interaction of auditory, motor,
and visual stimuli. One could even conjecture that musi-
cal ability is a congenital ability of cognitive processing. Some studies have shown evidence that the conditions
upon which musical ability develops are innately pre-
determined. Hassler for example showed that a certain
androgynous level of testosterone is characteristic of
musicians, and that differences between musicians and
non-musicians are possibly formed prenatally under the
influence of hormones [28]. the ability of keeping the processing of stimuli faster for a
longer time than normal subjects. This could be an indi-
cator of an improved working memory in musicians, as
it has been reported by some authors [12, 39, 40]. Keep-
ing the results from migraine patients in mind, it would
be interesting to study whether in musicians a similar
effect like the normalization of the habituation during
a migraine attack could be observed after an interval of
increased alertness. A loss of cognitive habituation (or higher cognitive
excitability) also means that stimuli on different levels
might be processed faster and more accurate for a cer-
tain amount of time. This can be regarded as an advan-
tage for musicians, e.g. when playing in an orchestra or
playing both complex rhythmic and complex melodic
music. P3 latency and musical abilityh l
On the other hand it also seems plausible that by prac-
ticing an instrument the processing of all these stimuli
becomes faster, which then might lead to the improve-
ment of auditory and visual abilities, as shown in the
studies mentioned above. In an MEG study, an enhanced
auditory evoked magnetic field response concerning vio-
lin tones was shown in children who had been musically
trained for 1 year [29]. There has also been a study not
connected to music, which showed alterations in adults’
ERP in response to an auditory discrimination task [30]. Hence, it has been shown before that training can have
an influence on stimulus processing and therefore it is
plausible that musical training which leads to a higher
amount of musical ability could have an effect on auditive
and visual processing [31, 32]. Limitations
d h Our study has some limitations which have to be consid-
ered when interpreting our results. First of all, the con-
cept of musical ability is still under discussion. There is
no clear definition and no commonly accepted measure
to evaluate musical ability. The sub-domains of musi-
cal ability as measured in both the Seashore test and the
amusia test only include some aspects of musical ability
which are commonly accepted as basic skills of musical
ability (e.g., rhythm, loudness, pitch, duration of a tone,
tonal memory); other aspects such as creativity, emo-
tional perception of music, the ability of interpreting
music etc. are not included because these aspects are
hard to measure in an objective way. Nonetheless, they
determine the amount of one’s musical ability. We accept
that our study did not comprise all aspects of musical
ability. All in all we suppose that there might be certain inher-
ited conditions such as fast cognitive processing upon
which musical ability develops but that a stimulating
environment as well as long-term musical practice also
leads to an improvement of cognitive processing and/or
aptitude. This is also important for disturbances of cog-
nition e.g. after stroke, after brain injury, or in different
types of amusia [33, 34]. A second problem is the procedure of testing musical
ability. The test results depend strongly on the coopera-
tion and motivation of the subjects, which in turn is hard
to evaluate. This leads to a reduced objectivity of the test-
ing of musical ability. P3 latency habituation Third, when looking at the P3 latency results one has to
consider that they only apply to our testing range. Since
we looked at P3 latencies that were within in the nor-
mal range and only included healthy subjects one cannot
transfer the results of the correlation analysis to subjects
with pathological P3 latencies, i.e. pathological P3 laten-
cies certainly do not yield in a total lack of musical ability. Fourth, it is not clear whether or to which extent a
decreased P3 latency has an impact on everyday life. A
decreased P3 latency implies a faster cognitive process-
ing. Some authors suggest an improved working memory,
but there is still an ongoing discussion about this (e.g.,
[12]). Third, when looking at the P3 latency results one has to
consider that they only apply to our testing range. Since
we looked at P3 latencies that were within in the nor-
mal range and only included healthy subjects one cannot
transfer the results of the correlation analysis to subjects
with pathological P3 latencies, i.e. pathological P3 laten-
cies certainly do not yield in a total lack of musical ability. In our study, we calculated the P3 latency habitua-
tion and found that musicians showed a habituation for
both the visual and the auditory latencies close to zero,
whereas in non-musicians it was significantly higher. There is evidence that, in normal subjects, the visual and
auditory P3 latency increases over trial blocks [35–38]. A loss of habituation in the visual P3 latency of migraine
patients during the migraine interval was shown [37],
similar to our findings of a loss of habituation in musi-
cians. We believe that the loss of habituation and the
generally decreased P3 latency in musicians indicate that
musicians have a remarkable cognitive processing with Fourth, it is not clear whether or to which extent a
decreased P3 latency has an impact on everyday life. A
decreased P3 latency implies a faster cognitive process-
ing. Some authors suggest an improved working memory,
but there is still an ongoing discussion about this (e.g.,
[12]). Page 7 of 9 Page 7 of 9 Faßhauer et al. BMC Neurosci (2015) 16:59 Then, two tests of musical ability were conducted. The
first one was a test on amusia developed by the authors
[13]. P3 latency habituation In this test, the participants had to perform different
tasks in the following five different categories: (1) produc-
tion of rhythm/metrum by repeating different rhythms
(knocking with a pin; (2) comparison of melodies (listening
to two short melodies and deciding whether they are dif-
ferent or not); (3)emotional impression of music (listening
to short pieces of new music which had to be categorized
as happy, angry, frightening etc.; the impression could be
said to the examiner or shown by different pictures show-
ing the emotion); (4) pitch (comparison of two tones and
deciding whether they have the same pitch or a different
pitch); (5) identification of 14 commonly known melodies
such as “Frère Jaques” or “Yesterday”. The maximum score
of this test is 86 points, the higher the score the better the
musical ability. This test was originally designed to detect
amusia in neurological patients. It has a high ceiling effect,
which means that a score of less than 90 % of the maximum
denotes a relevant impairment. Therefore, the test is or
poor value to detect small differences between individuals. Participants We compared a group of musicians (N = 10; 5 male) to
a group of non-musicians (N = 10; 5 male). The mean
age of the two groups was 23 ± 2 and 24 ± 2 years,
respectively. Musicians were defined by having received
at least 10 years of private music lessons (mean
14 ± 2 years) and being active musicians (i.e., mem-
bers of orchestras, chamber music groups, or choirs)
at the time of the study. The non-musician group had
no or very few (less than 2 years) music lessons in their
lives. Exclusion criteria for the participation in the study
were psychiatric or neurological disorders including
migraine and epilepsy and medication on a regular basis
except for oral contraceptives. Consumption of alcohol,
nicotine, or caffeine was not allowed on the day of the
experiment.h f
In order to evaluate the subjects’ musical ability in a
previously designed test, we used the “Seashore test for
Musical Ability” [42] as a second test. It contains six dif-
ferent categories: (1) pitch; (2) loudness; (3) rhythm; (4)
duration of a tone; (5) timbre; (6) tonal memory. The
maximum score is 260. The subjects have to differenti-
ate between small changes in pitch, loudness, rhythm etc. In the category pitch, for example, 50 pairs of tones are
presented, differing in frequency from/between 17 and
2 Hz. The subjects are asked whether the second tone
is lower or higher in frequency than the first. This test
shows a nearly parametric distribution of scores and has
no defined scores for amusia. ERP recordingh The experiment was always performed in the afternoon
in a room without daylight. The room temperature was
always the same. For the last part of the experiment, the
recording of the ERP, the lights were turned off. All par-
ticipants gave written informed consent. The study was
approved by the local ethics committee. The second part of the experiment was the recording of
visually and auditory evoked ERP. The subjects had to sit
in a comfortable chair in a dark room. For the visually
evoked ERP the subjects were looking at a 30 cm × 30 cm
video screen, standing approximately 150 cm in front of
them. Two trains of 200 flashes of light each with a 3 min
break in between the trains were presented in a random
order. We used 15 % red (target) and 85 % white (non-
target) flashes of light. The duration of a single flash
was 100 ms and the interval between two flashes was
1800 ms. Subjects were asked to press a button with their
dominant hand whenever they observed a red flash. For
the auditory evoked ERP, two trains of 200 tones each
(3 min break between the two trains) were presented
to the subjects. The target tones (15 %) had a pitch of
600 Hz, the non-target tones had a pitch of 325 Hz, they Conclusion Our most important finding is that measures in tests of
musical ability are associated with decreased latencies
in auditory and visual ERP suggesting that musical abil-
ity is associated with a general enhancement of cognitive
processing. Regarding the auditory P3 latency, we were able to
reproduce findings of previous studies [1–5]. Additionally,
we were able to show a significant correlation between the
level of musical ability as measured by the Seashore test
and both the visual and the auditory P3 latency. Also, a
significant difference in habituation of the visual and audi-
tory P3 latencies between non-musicians and musicians
could be observed. These results cannot be attributed to
a different mental state of the two groups, as this would
have lead to significantly different results between the two
groups in the Zerssen self-rating Scale. References Music training and working memory: an ERP study. Neuropsychologia. 2011;49:1083–94. 12. George EM, Coch D. Music training and working memory: an ERP study. Neuropsychologia. 2011;49:1083–94. Acknowledgements There was no funding and no other sources of support for this manuscript. There was no funding and no other sources of support for this manuscript. Authors’ contributions were also randomly mixed. Again, the subjects had to
press a button whenever a target stimulus occurred. The
duration of a tone was 100 ms, and the interval between
two stimuli was 1800 ms. All authors contributed to the design of the study, to the drafting of the
manuscript and to the statistical analysis and discussion. SE and CF conducted
the experiments. All authors read and approved the final manuscript. Competing interests
h
h
d
l
h The authors declare that they have no competing interests. The authors declare that they have no competing interests. Received: 21 June 2015 Accepted: 8 September 2015 Received: 21 June 2015 Accepted: 8 September 2015 The evaluation of the ERP latencies was performed
by a physician who did not conduct the experiment and
who did not know which subjects were musicians and
which ones were not. The components of the ERP fol-
lowing the red/high and white/low stimuli were evalu-
ated. We determined the latencies of the P1, N1, P2, N2,
and P3 components and the amplitude of the P3 com-
ponent, further we measured the mean choice reaction
time (i.e., the time between onset of the target stimulus
and pressing of the button) according to international
recommendations [19]. For both the visual and the audi-
tory ERP, the curves of the first and of the second train
(200 stimuli), separated in target and non-target stimuli,
were averaged and then the latencies, the P3 amplitude
and the mean choice reaction time were measured. By
calculating the difference of P3 latency between the
first and the second train, the P3 habituation could also
be evaluated. The P3 component of the ERP was cho-
sen as the main parameter since it is associated with
endogenous (mainly stimulus independent) cognitive
processing whereas the P2 and the N2 components are
exogenous (i.e., mainly dependent from the physical
properties of the stimulus). Author details
1 In both ERP measurements (auditory and visual), the
EEG was recorded by an amplifier using Ag/AgCl sur-
face electrodes, which were placed at centroparietal (Pz),
centrocentral (Cz), and frontocentral (Fz) (=recording
electrodes) according to the international 10–20 system. They were linked to the mastoid (=reference electrode). EOG was also registered in order to exclude EEG periods
with eye movement artifacts from the ensuing averaging
process. A high-frequency filter was set at 70 Hz and a
low-frequency filter at 0.1 Hz. The EEG was stored dig-
itally. EEG periods of 300 ms before and 1100 ms after
onset of the stimulus were averaged separately for the
target and non-target stimuli. 1 Department of Neurology, University of Münster, Münster, Germany. 2 Acad-
emy of Manual Medicine, Münster, Germany. 3 Department of Neurology,
Krankenhaus Lindenbrunn, Lindenbrunn 1, 31863 Coppenbrügge, Germany. Psychometric testing
h
b
h In the beginning, the participants had to complete the
Zerssen self-rating scale [41]. The Zerssen scale measures
the actual mental well-being with a score between 0 and
56. In the validation sample, a mean score of 12 ± 10 was
described for healthy subjects. Only subjects with a score
within the normal mean plus/minus one standard devia-
tion in both measures were included in the study. Feeling
mentally not well would impair the results of neuropsy-
chological testing in an unappropriate way. Faßhauer et al. BMC Neurosci (2015) 16:59 Page 8 of 9 Page 8 of 9 12. George EM, Coch D. Music training and working memory: an ERP study.
Neuropsychologia. 2011;49:1083–94. y
y
11. Evers S. Musikperzeption und visuelle Reizverarbeitung. Aachen: Shaker
Verlag; 2003. References 1. Klein M, Coles MGH, Donchin E. People with absolute pitch process tones
without producing a P300. Science. 1984;223:1306–9. g
2. Schütz K. P300 bei Musikern mit und ohne absolutes Gehört im Vergleich
zu zwei Kontrollgruppen. Med Diss Freiburg 1988. 3. Hantz E, Crummer G, Wayman J, Walton J, Frisina R. Effects of musical
training and absolute pitch on the neural processing of melodic intervals:
a P3 event-related potential study. Music Percept. 1992;10:25–42. 4. Hantz EC, Kreilick KG, Kananen W, Swartz KP. Neural responses to melodic
and harmonic closure: an event-related potential-study. Music Percept. 1997;15:69–98. 4. Hantz EC, Kreilick KG, Kananen W, Swartz KP. Neural responses to melodic
and harmonic closure: an event-related potential-study. Music Percept. 1997;15:69–98. 5. Wayman JW, Frisina RD, Walton JP, Hantz EC, Crummer GC. Effects of
musical training and absolute pitch ability on event-related activity in
response to sine tones. J Acoust Soc Am. 1992;34:131–56. 5. Wayman JW, Frisina RD, Walton JP, Hantz EC, Crummer GC. Effects of
musical training and absolute pitch ability on event-related activity in
response to sine tones. J Acoust Soc Am. 1992;34:131–56. 6. Crummer GC, Walton JP, Wayman JW, Hantz EC, Frisina RD. Neural
processing of musical timbre by musicians, nonmusicians and musicians
possessing absolute pitch. J Acoust Soc Am. 1994;95:2720–7. 6. Crummer GC, Walton JP, Wayman JW, Hantz EC, Frisina RD. Neural
processing of musical timbre by musicians, nonmusicians and musicians
possessing absolute pitch. J Acoust Soc Am. 1994;95:2720–7. 7. Levett C, Martin F. The relationship between complex music stimuli and
the late components of the event-related potential. Psychomusicology. 1992;11:125–40. 8. Baribeau J, Roth RM, Velikonja K. Auditory brain potentials in musicians:
effects of attention, task difficulty, and ear of stimulation. Brain Cogn. 1996;32:296–9. 9. Nikjeh DA, Lister JJ, Frisch SA. Hearing of a note: an electrophysiologic
and psychoacoustic comparison of pitch discrimination between vocal
and instrumental musicians. Psychophysiology. 2008;45:994–1007. 9. Nikjeh DA, Lister JJ, Frisch SA. Hearing of a note: an electrophysiologic
and psychoacoustic comparison of pitch discrimination between vocal
and instrumental musicians. Psychophysiology. 2008;45:994–1007. 10. Barnea A, Granot R, Pratt H. Absolute pitch—electrophysiological evi-
dence. Int J Psychophysiol. 1994;16:29–38. 10. Barnea A, Granot R, Pratt H. Absolute pitch—electrophysiological evi-
dence. Int J Psychophysiol. 1994;16:29–38. y
y
11. Evers S. Musikperzeption und visuelle Reizverarbeitung. Aachen: Shaker
Verlag; 2003. 11. Evers S. Musikperzeption und visuelle Reizverarbeitung. Aachen: Shaker
Verlag; 2003. 12. George EM, Coch D. 10. Barnea A, Granot R, Pratt H. Absolute pitch—electrophysiological evi-
dence. Int J Psychophysiol. 1994;16:29–38. 15. Gaser C, Schlaug G. Brain structures differ between musicians and non-
musicians. J Neurosci. 2003;23:9240–5. Statistical analysis 13. Lorenz C. Entwicklung eines klinischen Musikalitätstests: Normwerte,
klinischer Einsatz und Vergleich von Patienten mit Hirninfarkten. Med Diss
Münster. 2000. We used non-parametric tests to analyze the correlation
between the different test scores and the ERP parameters
and to analyze group differences between musicians and
non-musicians. For correlation analysis, the Spearman
rank coefficient was calculated. For group comparisons,
we used the Mann–Whitney U test. Multiple compari-
sons were corrected by Bonferroni correction. All cal-
culations were performed by the program SPSS version
18.0. Significance level was set at p = 0.05. 14. Pantev C, Oostenveld R, Engelien A, Ross B, Roberts LE, Hoke M. Increased
auditory cortical representation in musicians. Nature. 1998;392:811–4. auditory cortical representation in musicians. Nature. 1998;392:811 4. 15. Gaser C, Schlaug G. Brain structures differ between musicians and non-
musicians. J Neurosci. 2003;23:9240–5. 15. Gaser C, Schlaug G. Brain structures differ between musicians and non-
musicians. J Neurosci. 2003;23:9240–5. 16. Lappe C, Herholz S, Trainor L, Pantev C. Cortical plasticity induced by
short-term unimodal and multimodal musical training. J Neurosci. 2008;28:9632–9. 17. Lappe C, Trainor L, Herholz S, Pantev C. Cortical plasticity induced by
short-term multimodal musical rhythm training. PLoS One. 2011;6:21493. 17. Lappe C, Trainor L, Herholz S, Pantev C. Cortical plasticity induced by
short-term multimodal musical rhythm training. PLoS One. 2011;6:21493. Faßhauer et al. BMC Neurosci (2015) 16:59 Page 9 of 9 30. Reinke KS, He Y, Wang C, Alain C. Perceptual learning modulates sensory
evoked response during vowel segregation. Brain Res Cogn Brain Res. 2003;17:781–91. 18. Hyde KL, Lerch J, Norton A, Forgeard M, Winner E, Evans AC, Schlaug
G. Musical training shapes structural brain development. J Neurosci. 2009;29:3019–25. 19. Goodin D, Desmedt J, Maurer K, Nuwer MR. IFCN recommended
standards for long-latency auditory event-related potentials. Report of
an IFCN committee. International Federation of Clinical Neurophysiolog
Electroencephalogr Clin Neurophysiol. 1994;91:18–20. 31. Moreno S, Bialystok E, Barac R, Schellenberg EG, Cepeda NJ, Chau T. Short-
term music training enhances verbal intelligence and executive function. Psychol Sci. 2011;22:1425–33. 32. Moreno S, Wodniecka Z, Tays W, Alain C, Bialystok E. Inhibitory control
in bilinguals and musicians: event related potential (ERP) evidence for
experience-specific effects. PLoS One. 2014;9:e94169. Electroencephalogr Clin Neurophysiol. 1994;91:18–20. y
20. Helmbold N, Rammsayer T, Altenmüller E. Differences in primary
mental abilities between musicians and nonmusicians. J Individ Differ. 2005;26:74–85. 33. Chen MC, Tsai PL, Huang YT, Lin KC. Statistical analysis Pleasant music improves visual
attention in patients with unilateral neglect after stroke. Brain Inj. 2013;27:75–82. 21. Zafranas N. Piano keyboard training and the spatial-temporal develop-
ment of young children attending kindergarten classes in Greece. Early
Child Dev Care. 2004;174:199–211. 34. Schlesiger C, Evers S. Amusien. Nervenheilkunde. 2013;32:675–80. 22. Kalakoski V. Effect of skill level on recall of visually presented patte
musical notes. Scand J Psychol. 2007;48:87–96. 35. Lammers W, Badia C. Habituation of P300 to target stimuli. Psychol Behav
1989;45:595–601. 36. Geisler M, Polich J. P300 habituation from visual stimuli? Physiol Behav. 1994;56:511–6. 23. Bugos J, Perlstein W, McCrae C, Brophy T, Bedenbaugh P. Individualized
piano instruction enhances executive functioning and working memory
in older adults. Aging Ment Health. 2007;11:464–71. 37. Evers S, Bauer B, Suhr B, Husstedt IW, Grotemeyer KH. Cognitive process-
ing in primary headache: a study on event-related potentials. Neurology. 1997;48:108–13. 24. Jakobson L, Cuddy L, Kilgour A. Time tagging: a key to musicians’ superior
memory. Music Percept. 2003;20:307–13. 38. Pan J, Takeshita T, Morimoto K. P300 habituation from auditory single-
stimulus and oddball paradigms. Int J Psychophysiol. 2000;37:149–53. 25. Jomori I, Hoshiyama M, Uemura J, Nakagawa Y, Hoshino A, Iwamoto Y. Effects of emotional music on visual processes in inferior temporal area. Cogn Neurosci. 2013;4:21–30. 39. Gunter T, Schmidt BH, Besson M. Let’s face the music: a behavioral and
electrophysiological exploration of score reading. Psychophysiology. 2003;40:742–51. 26. Pantev C, Lappe C, Herholz SC, Trainor L. Auditory-somatosensory
integration and cortical plasticity in musical training. Ann N Y Acad Sci. 2009;1169:143–50. 40. Lee Y, Lu M, Ko H. Effects of skill training on working memory capacity. Learn Instr. 2007;17:336–44. 27. Hetland L. Learning to make music enhances spatial reasoning. J Aesthet
Educ. 2000;34:179–238. 41. von Zerssen D. Die Befindlichkeitsskala-Skala: Manual. Weinheim: Beltz
Test; 1976. 28. Hassler M. Music medicine. A neurobiological approach. Neuro Endo-
crinol Lett. 2000;21:101–6. 42. Lewis D, Saetveit J. Seashore Measures of Musical Talent’s Manual
(Revised). New York: Psychological Corporation; 1960. 29. Fujioka T, Ross B, Kakigi R, Pantev C, Trainor LJ. One year of musical
training affects development of auditory cortical-evoked fields in young
children. Brain. 2006;129:2593–608. Statistical analysis Submit your next manuscript to BioMed Central
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A governança no comércio de gado em cidades do interior de Alagoas
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Diversitas Journal
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cc-by
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KEYWORD: Auctions. Governance. Institutionalism. Alagoas. Fair. Governance in the trade of cattle in cities of the countryside of
Alagoas Página | 655
Página | 655 Página | 655
Página | 655 José Adeilton da Silva Filho(1), André Maia Gomes Lages(2) Diversitas Journal
ISSN 2525-5215
DOI: 10.17648/diversitas-journal-v3i3.666
Volume 3, Número 3 (set./dez. 2018) pp: 655-674.
www.kentron.ifal.edu.br/index.php/diversitas_journal
© Diversitas Journal Diversitas Journal
ISSN 2525-5215
DOI: 10.17648/diversitas-journal-v3i3.666
Volume 3, Número 3 (set./dez. 2018) pp: 655-674. www.kentron.ifal.edu.br/index.php/diversitas_journal
© Diversitas Journal Diversitas Journal Volume 3, Número 3 (set./dez. 2018) pp: 655-674. www.kentron.ifal.edu.br/index.php/diversitas_journal
© Diversitas Journal DIVERSITAS JOURNAL. Santana do Ipanema/AL. vol. 3, n. 3, p.655-674, set./dez. 2018. (1)Mestre em Economia – Programa de Mestrado em Economia Aplicada – UFAL. E-mail:
adeilltonfilho@gmail.com.
(2)Prof. Dr. Adjunto 4. Programa de Mestrado em Economia Aplicada – UFAL. E-mail:
andre_lages@msn.com. (1)Mestre em Economia – Programa de Mestrado em Economia Aplicada – UFAL. E-mail:
adeilltonfilho@gmail.com. (2)Prof. Dr. Adjunto 4. Programa de Mestrado em Economia Aplicada – UFAL. E-mail:
andre_lages@msn.com. (1)Mestre em Economia – Programa de Mestrado em Economia Aplicada – UFAL. E-mail:
adeilltonfilho@gmail.com. (2)Prof. Dr. Adjunto 4. Programa de Mestrado em Economia Aplicada – UFAL. E-mail:
andre_lages@msn.com. Todo o conteúdo expresso neste artigo é de inteira responsabilidade dos seus autores. Recebido em: 11 de outubro de 2018; Aceito em: 15 de novembro de 2018; publicado em 15 de 12 de 2018. Copyright© Autor, 2018. RESUMO: Este trabalho consiste na discussão da existência de ineficiência no mercado de animais
denominado como feiras de gado, para o desenvolvimento do sistema agropecuário de bovinos no interior
do Estado de Alagoas. Apresentando, desta forma, o leilão como alternativa mais eficiente para o sistema,
baseando-se a partir do referencial teórico da Nova Economia das Instituições. Para esse objetivo,
utilizou-se o modelo desenvolvido com base no atributo das transações, que tem como objetivo mostrar a
relação entre a especificidade dos ativos e o aumento nos custos de transação. Também foram coletados
dados de uma pesquisa de campo com aplicação de questionário nas duas principais feiras do interior de
Alagoas no ano de 2015. Ao final concluiu-se que as feiras de gado, não podem, por si só, desenvolver o
sistema de comércio de bovinos no interior de Alagoas, onde os custos de transação se fazem presentes,
necessitando, assim, da intervenção das prefeituras para a mudança da estrutura de governança, onde os
leilões conseguiriam reduzir os custos de transação, dado um aumento na especificidade dos ativos, que é
inibido pela estrutura atual das feiras, o que viabilizaria o desenvolvimento da produção de bovinos,
acarretando o provável desenvolvimento dos demais participantes dessa cadeia produtiva do interior de
Alagoas. PALAVRAS-CHAVE: Leilões. Governança. Institucionalismo. Alagoas. Feira. ALAVRAS-CHAVE: Leilões. Governança. Institucionalismo. Alagoas. Feira. PALAVRAS-CHAVE: Leilões. Governança. Institucionalismo. Alagoas. Feira. ABSTRACT: This paper consists of the discussion of the existence of inefficiency in the market of
animals denominated like feira de gado, for the development of the agricultural system of bovines in the
interior of the Alagoas Brazilian State. Thus, presenting the auction as a more efficient alternative for the
system, based on the theoretical reference of the New Economy of the Institutions. For this purpose, the
model was developed based on the attribute of the transactions, which aims to show the relationship
between the specificity of the assets and the increase in transaction costs. We also collected data from a
field survey with questionnaire application in the two main fairs in fairs in inland of Alagoas in the year
2015. In the end, it was concluded that livestock fairs, by themselves, cannot develop the system cattle
trade in the interior of Alagoas, where transaction costs are present. The change of the governance
structure, where auctions could reduce transaction costs, given an increase in the specificity of assets,
which is inhibited by the current structure of the fairs, which would enable the development of cattle
production, leading to in the probable development of the other participants of this productive chain in
inland of Alagoas. KEYWORD: Auctions. Governance. Institutionalism. Alagoas. Fair. DIVERSITAS JOURNAL. Santana do Ipanema/AL. vol. 3, n. 3, p.655-674, set./dez. 2018. A GOVERNANÇA NO COMÉRCIO DE GADO EM CIDADES DO INTERIOR DE ALAGOAS
SILVA, FILHO, JOSÉ ADEILTON DA, LAGES, ANDRÉ MAIA GOMES INTRODUÇÃO Este trabalho discute a ineficiência das feiras de gado para o desenvolvimento do
sistema agropecuário de bovinos no interior do Estado de Alagoas, onde apresenta o
papel dos leilões como alternativa de instituição de governança mais eficiente para o
sistema de vendas de animais, dentro do ambiente institucional existente, portanto,
baseando-se a partir do referencial teórico da Nova Economia das Instituições. Os municípios do interior de Alagoas ainda têm na pecuária uma de suas
principais atividades econômicas que segundo Lira (2007) adveio desde a concepção do
estado, quando os senhores de engenho precisavam criar animais, dentre os quais os
bovinos, para auxiliar na produção de açúcar e na pecuária. “Os senhores de engenho,
necessitando de muitos animais para exercerem várias tarefas nos canaviais, passaram a
criar cavalos, burros e bovinos [...]” (LIRA, 2007, p. 14, grifo nosso). DIVERSITAS JOURNAL. Santana do Ipanema/AL. vol. 3, n. 3, p.655-674, set./dez. 2018. Página | 656 PALAVRAS-CHAVE: Leilões. Governança. Institucionalismo. Alagoas. Feira. As tradicionais feiras de animais, conhecidas também como feira de gado, é um
comércio popular presente em grande parte desses municípios interioranos do Estado de
Alagoas. Duas feiras são as que mais se destacam, por conta do grande número de
comercializações, são as feiras localizadas no município de Dois Riachos, e no povoado de
Canafístula do município de Palmeira dos Índios. O primeiro município está localizado
na mesorregião do sertão alagoano. Nele, prevalece o bioma caatinga, tipo de savana
somente existente no Brasil. O segundo está localizado na mesorregião agreste
alagoano, onde prevalece uma região de transição entre o bioma caatinga e o bioma da
mata atlântica. O enfoque moderno com respeito ao papel das instituições, consequentemente
das estruturas de governança e dos custos de transação, traz evidências sobre a
fragilidade do mercado, sendo o sistema de preços insuficiente para responder a todas as
mudanças que a dinâmica econômica provoca em algumas situações. Deste modo, as
relações complexas presentes nas atividades econômicas, como as feiras de animais dos
municípios do interior de Alagoas, possivelmente estariam fadadas a ineficiência e
consequentemente as limitações para o seu desenvolvimento, dado o seu grau de
evolução institucional. Segundo Fiani (2011), a ação dos mercados é altamente questionável, não
existindo eficiência sem o papel da cooperação entre os agentes, assim, a adaptação do
mercado não se sustenta sem a promoção da redução dos conflitos, e deste modo à
possibilidade do desenvolvimento da economia, onde para entender o funcionamento do
sistema econômico é preciso ressaltar o problema da coordenação, que, encontra-se A GOVERNANÇA NO COMÉRCIO DE GADO EM CIDADES DO INTERIOR DE ALAGOAS
SILVA, FILHO, JOSÉ ADEILTON DA, LAGES, ANDRÉ MAIA GOMES presente na atividade econômica. Isso implica a existência de um ambiente de confiança,
e de capital social conforme analisa Locke (2001), baseado na contribuição crítica das
principais referências sobre o tema. O mercado da feira de gado tem resistido a mudanças muito por conta do papel
das prefeituras na coordenação de tais eventos. Mas diante de uma situação ineficiente
desse mercado, até que ponto as prefeituras estariam se beneficiando com a sua
manutenção? E os pecuaristas, como estariam sendo restringidos no desenvolvimento da
sua atividade econômica? PALAVRAS-CHAVE: Leilões. Governança. Institucionalismo. Alagoas. Feira. O trabalho está dividido, além da introdução conclusão e referências, em mais seis
tópicos, em um total de nove, onde no segundo tópico abordará os mercados e sua
eficiência para o setor agrícola; no terceiro tópico adentrará especificamente nas teorias
institucionais relatando os custos de transação, com o viés para o mercado agrícola; no
quarto apresentará um modelo com base no atributo das transações; no quinto os leilões
do tipo inglês como estrutura eficiente de governança; no sexto é destinado ao modo de
pesquisa realizado com seus materiais e métodos; por fim o sétimo apresenta os
resultados e discussões. Na busca de elucidar esses dois questionamentos principais, o trabalho
apresentará através das obras pioneiras de Zylbersztajn e Machado Filho (1999), que
estuda o sistema de vendas de animais no Brasil baseados nas obras Williamson (1975,
1985, 1991), apresentando uma modelagem, onde os leilões representam uma estrutura
de governança apropriada para o mercado de bovinos, por meio da sua eficiência com
relação aos custos de transações, diante de um ambiente institucional, no qual, acredita-
se poder ser aplicado, com as devidas ressalvas, também ao ambiente existente no
comércio de bovinos no interior de Alagoas. DIVERSITAS JOURNAL. Santana do Ipanema/AL. vol. 3, n. 3, p.655-674, set./dez. 2018. Página | 657 ABORDAGEM DOS MERCADOS PARA O SETOR AGRÍCOLA. O mercado está no cerne dos estudos econômicos e seu estudo faz parte da
evolução da economia capitalista, onde a sua compreensão passa inicialmente pelo pai da
economia moderna Adam Smith e o teórico do equilíbrio geral Léon Walras, assim como
do equilíbrio parcial Alfred Marshall, os dois últimos dentro do escopo da teoria
neoclássica. Outros estudiosos desenvolveram a ideia do mercado, como sendo o
eficiente alocador de recursos do sistema econômico, mas também ficou claro ao longo
do tempo suas imperfeições. A GOVERNANÇA NO COMÉRCIO DE GADO EM CIDADES DO INTERIOR DE ALAGOAS
SILVA, FILHO, JOSÉ ADEILTON DA, LAGES, ANDRÉ MAIA GOMES DIVERSITAS JOURNAL. Santana do Ipanema/AL. vol. 3, n. 3, p.655-674, set./dez. 2018. OS CUSTOS DE TRANSAÇÃO PARA O MERCADO AGRÍCOLA Os fluxos, portanto de cadeias produtivas, onde um ativo flui de uma atividade
econômica para outra, nem sempre acontecem de modo harmonioso, logo na visão de
Williamson (1985) resultam em custos, denominados como custos de transação, assim
quanto maior e diversificada é a economia, maiores são as dificuldades de coordenação e
maiores os custos de transação, que tem impactos muitas vezes negligenciados em
outras teorias nos rumos do desenvolvimento. Página | 659 To be sure, complex organizations commonly serve a variety of economic and
noneconomic purposes. That is plainly true of the economic institutions of
capitalism, which are numerous, subtle, and continuously evolving. My
emphasis on transaction cost aspects is not meant to suggest that transaction
cost economizing is the only purpose served; but its importance has hitherto
been neglected and/or undervalued. (WILLIAMSON, 1985, p. 2). To be sure, complex organizations commonly serve a variety of economic and
noneconomic purposes. That is plainly true of the economic institutions of
capitalism, which are numerous, subtle, and continuously evolving. My
emphasis on transaction cost aspects is not meant to suggest that transaction
cost economizing is the only purpose served; but its importance has hitherto
been neglected and/or undervalued. (WILLIAMSON, 1985, p. 2). Quando se realizam as trocas, por exemplo, entre os agentes da cadeia, os
agentes engajam-se em transações, tais transações podem ser diagnosticadas por três
características básicas, definidas por Williamson (1975) são: A Frequência, Incerteza e a
Especificidades de Ativos, onde a Frequência está associada à quantidade de vezes que os
agentes realizam as transações, enquanto a Incerteza remete a comportamento
oportunístico na quebra de contratos diante da racionalidade limitada dos agentes1, e a
Especificidades de Ativos ao custo oriundo da perda, caso a transação não se concretize,
por não haver uso alternativo para esse ativo na mesma magnitude, assim Williamson
(1975) definiu os ativos em três classes: Não Específicos, Mistos ou Altamente
Específicos. Com relação à frequência das transações, é preciso ainda acrescentar, que esta,
segundo Williamson (1975) é descriminada em três tipos: Únicas, Ocasionais ou
Recorrentes. Quanto maior a frequência das transações, maiores as vantagens de se
manter estruturas especializadas, com menores custos fixos médios, sendo assim, para
reduzir o custo de se recorrer a esse bem ou esse serviço, que se faz necessário de forma
não ocasional, não haverá problema em incorporá-lo a estrutura da organização. 1Pois, para Williamson os agentes desejam ser racionais, porém só conseguem ser de modo parcial. DIVERSITAS JOURNAL. Santana do Ipanema/AL. vol. 3, n. 3, p.655-674, set./dez. 2018. A GOVERNANÇA NO COMÉRCIO DE GADO EM CIDADES DO INTERIOR DE ALAGOAS
SILVA, FILHO, JOSÉ ADEILTON DA, LAGES, ANDRÉ MAIA GOMES Segundo Ganem (2015), o processo evolutivo para a formação dos
ideais do liberalismo econômico, que consiste na noção hegemônica do
mercado quanto alocador de recursos, advém de Walras, e percorre a
seguinte trajetória teórica: Página | 658 Walras mantém a ambição smithiana do mercado como uma teoria da
sociedade e procede às reduções necessárias à demonstração. O sujeito
smithiano, movido pelo autointeresse, cortado por paixões, dá lugar ao homem
econômico racional herdado de Mill, cujo cálculo maximizador traz como
resultado a ordem racional. O fundamento microeconômico dessa ordem é um
ser abstrato, atomizado e movido pelo cálculo, e que, através de um
mecanismo de ajuste automático, produz o equilíbrio, uma noção (precisa)
física que substitui a noção (vaga) de bem-estar smithiana. (GANEM, 2015,
p.151). Figura 1– Sistema agroindustrial, seus sistemas e fluxos de suprimentos. Fonte: adaptada de BATALHA (2009). Figura 1– Sistema agroindustrial, seus sistemas e fluxos de suprimentos. Fonte: adaptada de BATALHA (2009). Quando se observa o mercado da feira de gado, sabe-se que engloba outros
mercados, dentro de uma estrutura de cadeias produtivas e seus macrossegmentos. Segundo a definição de Batalha (2009) o primeiro macrossegmento, corresponde às
iniciativas empreendidas para a formação de toda a atividade agropecuária, a produção. O segundo macrossegmento é correspondente a toda transformação de matéria-prima do
setor agrícola em produtos agroindustriais. O terceiro macrossegmento configura-se nas
ações de compra e venda realizadas nos moldes do varejo ou atacado. Como mostra a
figura 01, onde o primeiro macrossegmento é denominado dentro da porteira. O
segundo e o terceiro são pós-porteira. DIVERSITAS JOURNAL. Santana do Ipanema/AL. vol. 3, n. 3, p.655-674, set./dez. 2018. A GOVERNANÇA NO COMÉRCIO DE GADO EM CIDADES DO INTERIOR DE ALAGOAS
SILVA, FILHO, JOSÉ ADEILTON DA, LAGES, ANDRÉ MAIA GOMES VERSITAS JOURNAL. Santana do Ipanema/AL. vol. 3, n. 3, p.655-674, set./dez. 2018. VERSITAS JOURNAL Santana do Ipanema/AL vol 3 n 3 p 655-674 set /dez 2018 When bilaterally dependent parties are unable to respond quickly and easily,
because of disagreements and self-interested bargaining, maladaptation costs
are incurred. Although the transfer of such transactions from market to
hierarchy creates added bureaucratic costs, those costs may be more than
offset by the bilateral adaptive gains that result. (WILLIAMSON, 1991,
p.282). DIVERSITAS JOURNAL. Santana do Ipanema/AL. vol. 3, n. 3, p.655-674, set./dez. 2018. 2Nota-se que a visão de incerteza, aqui exposta, difere da propalada na literatura por Frank Knight.
3Modalidade de negócios onde são realizados à vista com a entrega imediata sendo muito usado nas feiras 2Nota-se que a visão de incerteza, aqui exposta, difere da propalada na literatura por Frank Knight. OS CUSTOS DE TRANSAÇÃO PARA O MERCADO AGRÍCOLA Para a Incerteza, tem-se uma situação, que, segundo ZYLBERSZTAJN (1995)
tem um enorme campo ainda para ser discutido e estudado, pois a teoria ainda necessita
de desenvolvimento conceitual. Porém ela está dividia em no mínimo duas formas 2Nota-se que a visão de incerteza, aqui exposta, difere da propalada na literatura por Frank Knight.
3Modalidade de negócios, onde são realizados à vista com a entrega imediata, sendo muito usado nas feiras
livres. A GOVERNANÇA NO COMÉRCIO DE GADO EM CIDADES DO INTERIOR DE ALAGOAS
SILVA, FILHO, JOSÉ ADEILTON DA, LAGES, ANDRÉ MAIA GOMES A GOVERNANÇA NO COMÉRCIO DE GADO EM CIDADES DO INTERIOR DE ALAGOAS
SILVA, FILHO, JOSÉ ADEILTON DA, LAGES, ANDRÉ MAIA GOMES principais: a incerteza2 proveniente do comportamento estratégico dos agentes, e a
incerteza de contingência. UM MODELO COM BASE NO ATRIBUTO DAS TRANSAÇÕES. O trabalho de Machado Filho, C. A.; Zylbersztajn (1999) tentam diante dessa
realidade encontrar uma alternativa ao mercado para a maior eficiência no comércio de
bovinos, mais precisamente nos bovinos para corte. Para isso trabalharam com três
tipos de estruturas de governança, conforme a obra de Williamson para assim, tentar
encontrar o melhor mecanismo. No presente trabalho, as características das transações serão utilizadas para a
análise da maneira mais eficiente de governança. Na comercialização de
animais, duas das formas de governança a serem consideradas no presente
estudo estão dentro da alternativa via mercado, embora no sentido puro strictu
sensu do mercado– laissez-faire –, apenas a venda direta (do tipo mercado spot,
contrato clássico) possa ser considerada mercado, uma vez que, no caso do
leilão, esta instituição de governança age como catalisadora do encontro de
oferta e demanda, atuando como uma mão visível (portanto não mais mercado
na forma estrita) no processo de troca de direito de propriedade. A outra
opção de governança a ser considerada é a venda direta por relações
contratuais do tipo mista, quer sejam contratos neoclássicos ou relacionais. (MACHADO FILHO, C. A.; ZYLBERSZTAJN, 1999, p. 275). Deste modo foi desenvolvido um modelo com base no atributo das transações,
onde segundo Machado Filho, C. A.; Zylbersztajn (1999) tem como objetivo mostrar,
através da obra de Williamson (1985), a relação entre a especificidade dos ativos e o
aumento nos custos de transação (CT), onde na medida em que as especificidades do
ativo aumentam (K), os custos de transação também se elevam, porém de formas
diferentes entre os três tipos de estruturas de governança, logo comparando-se a
alternativa entre três vias de governança; via venda direta (mercado spot), leilões e de
relação contratual mista. A figura 2 apresenta três curvas de custos de transação. A curva (L) que
representa a alternativa de governança via leilão; a curva (M) que representa a
alternativa de governança via negociação direta (mercado spot); e a terceira curva (X)
que representa um tipo de negociação pela via contratual mista. Quando há um aumento
da especificidade de atributos dos animais (K), os leilões são uma forma redutora de
custos, pois facilita a obtenção de informações, que quando comparado ao mercado spot
serve de referencial de preços mais eficiente. principais: a incerteza2 proveniente do comportamento estratégico dos agentes, e a
incerteza de contingência. Basicamente, pode-se considerar que existam dois tipos de incerteza: a
incerteza proveniente do comportamento estratégico dos agentes, isto é, a
incerteza decorrente da impossibilidade de saber as ações que os agentes irão
colocar em prática; e a incerteza de contingência (estado da natureza), ou seja,
a incerteza inerente à própria atividade e a impossibilidade de se prever todas
as possibilidades ou contingências, que ocorrerão num tempo futuro, que
afetem a atividade. (MACHADO FILHO, C. A.; ZYLBERSZTAJN, 1999, p. 274). Página | 660 A incorporação da Economia dos Custos de Transação passa a ser importante
para entender as limitações do mercado, analisado até aqui. Vendo-se por exemplo a
impossibilidade de se compreender na teoria do equilíbrio geral sua capacidade de
explicar o equilíbrio múltiplo dos mercados. Embora colocada na visão walrasiana,
parece pouco realista para muitos. Então a teoria do custo de transação revela um
avanço na compreensão de certos mercados e/ou governanças. Deste modo, quando a
economia se diversifica e desenvolve os custos transacionais, tornam-se extremamente
importantes, de forma que não há razões para que os mercados permaneçam sempre
eficientes, por todo tempo. Diante de tal realidade, surgem as opções além do mercado puro, ou seja, além do
mercado tradicional das feiras, também conhecido no âmbito do agronegócio como
mercado spot3, logo vale a descrição teórica das demais estruturas de negociações
existentes, logo, das estruturas de governança, tais estruturas têm sido estudadas por
meio da abordagem da Nova Economia Institucional (NEI). Uma alternativa para a estrutura do mercado puro é a estrutura de governança
hierárquica, ou ainda, a estrutura híbrida, que, no caso apresenta uma mistura das duas
estruturas, para Williamson (1991), embora também haja custos para transladar de uma
estrutura para a outra, muitas vezes tais custos ainda sãomais eficientes diante de um
ambiente, onde as partes estejam com problemas de oportunismo e conflito nas suas
negociações bilaterais. DIVERSITAS JOURNAL. Santana do Ipanema/AL. vol. 3, n. 3, p.655-674, set./dez. 2018. A GOVERNANÇA NO COMÉRCIO DE GADO EM CIDADES DO INTERIOR DE ALAGOAS
SILVA, FILHO, JOSÉ ADEILTON DA, LAGES, ANDRÉ MAIA GOMES DIVERSITAS JOURNAL. Santana do Ipanema/AL. vol. 3, n. 3, p.655-674, set./dez. 2018. Página | 661 UM MODELO COM BASE NO ATRIBUTO DAS TRANSAÇÕES. Como também diante de um aumento da
especificidade, maiores são as dificuldades de firmar em uma só negociação a venda de
todos os animais para um determinado comprador, afinal, este selecionaria somente
aqueles de seu interesse. Logo, os custos de transação associados aos leilões são menores DIVERSITAS JOURNAL. Santana do Ipanema/AL. vol. 3, n. 3, p.655-674, set./dez. 2018. A GOVERNANÇA NO COMÉRCIO DE GADO EM CIDADES DO INTERIOR DE ALAGOAS
SILVA, FILHO, JOSÉ ADEILTON DA, LAGES, ANDRÉ MAIA GOMES que os custos de transação via mercado spot. (MACHADO FILHO, C. A.;
ZYLBERSZTAJN 1999). que os custos de transação via mercado spot. (MACHADO FILHO, C. A.;
ZYLBERSZTAJN 1999). Página |
Figura 2 – Relação entre alternativas de Governança e o aumento nos Custos de
Transação. Fonte: adaptada de MACHADO FILHO, C. A.; ZYLBERSZTAJN (1999). Figura 2 – Relação entre alternativas de Governança e o aumento nos Custos de
Transação. Figura 2 – Relação entre alternativas de Governança e o aumen
Transação. Fonte: adaptada de MACHADO FILHO, C. A.; ZYLBERSZTAJN (1999). Página | 662 Página | 662 Fonte: adaptada de MACHADO FILHO, C. A.; ZYLBERSZTAJN (1999). Deveria estar subjacente a isso, a relevância da qualidade genética dos animais,
quanto maior for a especificidade de ativo. Então, diante de uma relação, onde exista um
nível de monitoramento maior, quando os compradores exigem uma rastreabilidade da
especificação maior do animal, os leilões talvez não possam garantir toda essa
informação, o que infere em maiores custos transacionais, cabendo assim uma via de
mercado mista, com contratos firmados, que apresentaria custos de transação menores,
ou ainda, em casos mais específicos uma verticalização da produção. (MACHADO
FILHO, C. A.; ZYLBERSZTAJN 1999). Assim, para k=0 tem-se que M(0) < (0) < X(0). À medida que k aumenta,
(k>0), e a partir de um determinado nível de especificidade variando no
intervalo A - B, tem-se que L(k) < X(k) < M(k), podendo ainda ocorrer a
possibilidade de L(k) < M(k) < X(k). A partir de um nível de especificidade
maior, após o ponto B, tem-se que X(k) < L(k) < M(k). (MACHADO FILHO,
C. A.; ZYLBERSZTAJN, 1999, p.279). DIVERSITAS JOURNAL. Santana do Ipanema/AL. vol. 3, n. 3, p.655-674, set./dez. 2018. O LEILÃO INGLÊS COMO ESTRUTURA EFICIENTE DE GOVERNANÇA O leilão pode ser tratado como uma estrutura de governança, que encontrar-se-ia
entre o mercado spot e a estrutura de venda mista, como apresentada por Machado Filho
e Zylbersztajn. O leilão tem característica, portanto, que fogem do mercado puro, pois há A GOVERNANÇA NO COMÉRCIO DE GADO EM CIDADES DO INTERIOR DE ALAGOAS
SILVA, FILHO, JOSÉ ADEILTON DA, LAGES, ANDRÉ MAIA GOMES a figura do leiloeiro como agente responsável pela coordenação das negociações, que
fazem os leilões um tipo de estrutura singular. Os bens que podem ser leiloados são divididos em dois tipos de natureza de bens. Os leilões para valor privado correspondem àqueles leilões em que a natureza do bem
tem diversas atribuições de valoração, onde cada participante do leilão atribui um valor
privado para aquele bem. O outro tipo de natureza de bens leiloados é correspondente
aos leilões comuns. Leilões comuns têm bens com uma valoração que é comungada por
todos os participantes de um leilão, pois são muito próximas, embora seus valores
possam variar segundo as estimativas de cada licitante. (VARIAN, 2016) Independentemente de ser para bens de valor privado ou comum os leilões
oferecem várias formas de arremates desses bens, dentre esses mecanismos existem os
leilões denominados de Vickrey, Holandês e o Inglês, todos distintos em suas regras de
arremates, porém cada um específico para o ambiente de negociações que se pretende
estabelecer. O trabalho contempla a estrutura de leilões do tipo inglês, sendo o mais popular
entre os tipos de leilões, esse tipo de leilão segundo Pindyck; Rubinfeld (2006) também é
conhecido como tradicional ou oral, seu mecanismo se dá através de solicitações ativas
de lances mais altos por parte do vendedor a um grupo de potenciais compradores, onde
busca-se, assim, o comprador vencedor. Sendo esse o tipo de leilão utilizado para a venda
de bovino em todo o território nacional. Dentre as características que fazem com que os leilões sejam estruturas de
governança eficientes, diante do mercado puro, pode-se citar no mínimo três
consequências de sua prática que empregam tendências de custos menores, são as
tendências de menores custos de Informação, Negociação e Monitoramento. Tais custos,
que foram abordados anteriormente, estão dentro da análise da NEI com relação aos
custos de transação. 4Mesmo nas feiras, embora exista uma centralidade, os currais ficam dispersos, conforme presenciados em
visitas, que, assim, dificultam o comprador descobrir o animal que melhor lhe satisfaça, além da
aleatoriedade existente, onde enquanto procura o animal de sua preferência, este possa ser comprado por
outro, que talvez não avaliasse o animal da mesma forma. DIVERSITAS JOURNAL. Santana do Ipanema/AL. vol. 3, n. 3, p.655-674, set./dez. 2018. Página | 663 A GOVERNANÇA NO COMÉRCIO DE GADO EM CIDADES DO INTERIOR DE ALAGOAS
SILVA, FILHO, JOSÉ ADEILTON DA, LAGES, ANDRÉ MAIA GOMES A GOVERNANÇA NO COMÉRCIO DE GADO EM CIDADES DO INTERIOR DE ALAGOAS
SILVA, FILHO, JOSÉ ADEILTON DA, LAGES, ANDRÉ MAIA GOMES potenciais compradores. O leilão tende a reduzir os Custos de Monitoramento, através
da figura do leiloeiro, como por exemplo, em situações em que o leiloeiro se torna fiador
da transação (ZYLBERSZTAJN, et al. 2000). potenciais compradores. O leilão tende a reduzir os Custos de Monitoramento, através
da figura do leiloeiro, como por exemplo, em situações em que o leiloeiro se torna fiador
da transação (ZYLBERSZTAJN, et al. 2000). Página | 664 Página | 664 5Vide apêndice. MATERIAL E MÉTODOS A metodologia utilizada para o estudo do mercado da Feira de Gado para
resolução da problemática desse trabalho seguiu as seguintes etapas: • Realização de visitas in loco para o conhecimento das feiras de gado de Dois
Riachos e Canafístula, e as suas práticas comerciais; • Realizou-se a aplicação de questionários5 aos compradores e vendedores de gado
das feiras de gado de Dois Riachos e Canafístula; • Foi realizado um levantamento de dados sobre as feiras do Gado de Dois Riachos
e Canafístula, no Sistema de Defesa Agropecuária de Alagoas (SIDAGRO-AL); Este trabalho é um estudo de caráter descritivo, exploratório e estudo de caso. Nesse quadro, o estudo preserva certa dose pioneirismo em nível local. Os aspectos
culturais das localidades induzem a um certo tipo de ambiente institucional
principalmente no aspecto informal, onde a palavra empenhada pelas partes ganha a
força de um contrato, surgindo assim aspectos peculiares assinalados no texto. Isso
implica também uma natureza de um capital social, mas que foge ao escopo desse
trabalho maior aprofundamento. DIVERSITAS JOURNAL. Santana do Ipanema/AL. vol. 3, n. 3, p.655-674, set./dez. 2018. O LEILÃO INGLÊS COMO ESTRUTURA EFICIENTE DE GOVERNANÇA Os leilões tendem a reduzir os Custos de Informação, que o aproxima de um
mercado competitivo, reduz a assimetria de informação, logo reduz a incerteza,
consequentemente reduz os custos de transação oferecendo, assim, importante fonte de
informação de mercado. Os leilões reduzem os Custos de Negociação, pois as
propriedades rurais são fisicamente dispersas4, enquanto que, o leilão oferece a
possibilidade de reunir em um mesmo espaço físico um número elevado de ofertantes e DIVERSITAS JOURNAL. Santana do Ipanema/AL. vol. 3, n. 3, p.655-674, set./dez. 2018. 5Vide apêndice. 6Pois se desconhecia os possíveis valores que seriam encontrados. A GOVERNANÇA NO COMÉRCIO DE GADO EM CIDADES DO INTERIOR DE ALAGOAS
SILVA, FILHO, JOSÉ ADEILTON DA, LAGES, ANDRÉ MAIA GOMES compra e venda. Os períodos de dados conseguidos no SIDAGRO para a feira de
Canafístula são de 23 de março até 31 de agosto de 2015, já para a feira de Dois Riachos
os dados obtidos são de 01 de abril até 23 de setembro de 2015. Para se chegar ao número de amostra necessária para aplicar o questionário,
extraiu-se o número médio de acordos fechados nas duas feiras, e depois a utilização de
95% de nível de confiança, 50% de proporção de acerto esperado6 e 5% para o erro
amostral. Segundo os dados coletados no SIDAGRO e tratados em planilha eletrônica, a
média de negociações confirmadas, ou seja, a quantidade de vendedores e compradores
que fecharam acordo de compra e venda no período de aproximadamente seis meses nas
duas principais feiras no interior de Alagoas são de 127,3617021, logo considera-se 128
negociações por feira. O que solicita uma amostra mínima de 97 entrevistas. Como trata-se de uma população finita, a amostra foi obtida através da seguinte
fórmula: n = (1)
onde: N = tamanho da população, Z = nível de confiança, P = proporção de acerto
esperado, Q = (1 – P) e d = erro amostral. Z² x P x Q x N
d² x (N-1) + Z² x P x Q (1) onde: N = tamanho da população, Z = nível de confiança, P = proporção de acerto
esperado, Q = (1 – P) e d = erro amostral. onde: N = tamanho da população, Z = nível de confiança, P = proporção de acerto
esperado, Q = (1 – P) e d = erro amostral. DIVERSITAS JOURNAL. Santana do Ipanema/AL. vol. 3, n. 3, p.655-674, set./dez. 2018. AMOSTRA As informações a serem descritas foram coletadas com a utilização de
questionário e dados do SIDAGRO nas duas principais feiras de gado do interior de
Alagoas segundo os responsáveis pela Agência de Defesa e Inspeção Agropecuária de
Alagoas (ADEAL). Os dados coletados no SIDAGRO são a respeito do número de negociações
confirmadas, ou seja, a quantidade de vendedores e compradores que fecharam acordo de A GOVERNANÇA NO COMÉRCIO DE GADO EM CIDADES DO INTERIOR DE ALAGOAS
SILVA, FILHO, JOSÉ ADEILTON DA, LAGES, ANDRÉ MAIA GOMES Página | 665 Página | 665 A GOVERNANÇA NO COMÉRCIO DE GADO EM CIDADES DO INTERIOR DE ALAGOAS
SILVA, FILHO, JOSÉ ADEILTON DA, LAGES, ANDRÉ MAIA GOMES A GOVERNANÇA NO COMÉRCIO DE GADO EM CIDADES DO INTERIOR DE ALAGOAS
SILVA, FILHO, JOSÉ ADEILTON DA, LAGES, ANDRÉ MAIA GOMES preços, com a mensuração em percentual, para a comparação entre os seus objetivos
finais de cada compra (Abate, Comércio, Recria) e os tipos de animais adquiridos
classificados em sexo e meses de idade. Página | 666 Página | 666 DIVERSITAS JOURNAL. Santana do Ipanema/AL. vol. 3, n. 3, p.655-674, set./dez. 2018. PROCEDIMENTOS A pesquisa, com a utilização dos questionários, foi realizada nos dias 13 de junho
e 16 de novembro de 2015 em Canafístula, e nos dias 15 de junho e 18 de novembro em
Dois Riachos. Dentre as perguntas do questionário aplicado, as que serão utilizadas para
fins desse estudo são a respeito do tipo da finalidade por parte do comprador, divididos
em três tipos de fins: Abate, Comércio, Recria. E do lado da oferta o tipo de sexo é idade
em meses de cada animal negociado na feira, como também seus respectivos valores de
compra. Deste modo será possível ver qual é a especificidade de cada consumidor, seus
valores dispendidos, para assim, compará-los. Por fim, foram utilizados programas de computador como planilhas eletrônicas
(Excel, Calc, Gnumeric), para quantificar os dados, extrair as médias, as quantidades e CARACTERIZAÇÃO DO OBJETO DE ESTUDO Localizada na margem esquerda da BR-316 sentido Maceió / Santana do
Ipanema, no povoado de Canafístula de Frei Damião no Município de Palmeira dos
Índios/AL. Encontram-se os currais da Feira de Gado de Canafístula. Localizada
também na margem esquerda e no mesmo sentido da BR-316, no município de Dois
Riachos, encontram-se os currais da Feira de Gado de Dois Riachos. A feira de Canafístula ocorre todas as segundas-feiras, já a de Dois Riachos
ocorre todas as quartas-feiras. As feiras começam de madrugada por volta das 4 horas e
30 minutos, e se estendem até as 13 horas, embora segundo os vendedores, após as 11
horas as chances de vender um animal cai muito, o ponto alto da feira é às 9 horas, é
quando o fluxo de compra e venda está mais intenso. Os animais são negociados, como na maioria das feiras, no mercado spot, embora
também possam ser vendidos a crédito. Na verdade, o crédito segundo alguns
vendedores é uma das modalidades mais utilizadas na feira, principalmente entre
compradores que negociam também com gado, portanto compradores que compram para
comercializar. Esse é um elemento de confiança (trust), conforme é analisado em outros
contextos por Locke, (2001). Isso acontece e mostra uma menor incerteza no sentido
comportamental em função da recorrência (frequência) da feira e desses atores sempre
presentes. É uma tradição do interior nordestino o acordo apalavrado, assim acontece a
compra a crédito sem nenhum contrato formal estabelecido. A Feira de Gado não se resume apenas a venda de bovinos, encontra-se também
o comércio de caprinos, suínos, ovinos e equinos. Em alguns momentos na feira, existem
alguns que se arriscam também a vender aves de diversos tipos, em seus arredores, ainda
bastante próximo, existe a venda de roupas e utensílios para vaqueiro, como também
diversos outros tipos de produtos que seriam ofertados em qualquer comércio, há
também a oferta de refeições e lanches, é portanto um evento que movimenta bastante a
economia das suas localidades. A GOVERNANÇA NO COMÉRCIO DE GADO EM CIDADES DO INTERIOR DE ALAGOAS
SILVA, FILHO, JOSÉ ADEILTON DA, LAGES, ANDRÉ MAIA GOMES 7Os animais são avaliados de modo visual; não existindo balança para aferir a arrobação do animal. Por
isso, as tabelas pertinentes somente fazem referência a idade. Esse procedimento acontece nas duas feiras. DIVERSITAS JOURNAL. Santana do Ipanema/AL. vol. 3, n. 3, p.655-674, set./dez. 2018. Página | 667 RESULTADOS E DISCUSSÕES Os compradores segundo aplicação do questionário são divididos em três grupos,
com três finalidades distintas, são elas: Abate, Comércio e Recria, embora cada grupo
deseje animais com especificidades distintas, alguns animais têm idades e sexo que
abrangem a necessidade dos três grupos, geralmente animais de 12 a 24 meses sendo
machos e fêmeas, como mostram as tabelas7 01, 02 e 03. Tabela 01– Percentual dos tipos de animais para recria. <12
12 a 24
25 a 36
> 36
Total
M
F
M
F
M
F
M
F
97
6,19% 1,03% 30,93% 13,40% 29,90% 7,75% 4,65% 17,05% 100%
Fonte: Autores, 2017. Tabela 01– Percentual dos tipos de animais para recria. <12
12 a 24
25 a 36
> 36
Total
M
F
M
F
M
F
M
F
97
6,19% 1,03% 30,93% 13,40% 29,90% 7,75% 4,65% 17,05% 100%
Fonte: Autores, 2017. Fonte: Autores, 2017. A tabela 01 apresenta os animais comprados com a finalidade de recria, onde
estão tipificados em categorias de sexo, sendo M para macho e F para fêmea, como
também, estão definidos por idade, onde < 12 representam os animais menores de doze
meses; 12 a 24 os animais de um até dois anos de idade, 25 a 36 são os animais de vinte e
cinco meses até três anos de idade; e >36 representa os animais com mais de três anos de
idade. O total representa em unidades o total de bovinos vendidos para tal finalidade,
como também, a tabela a apresenta a discriminação em percentual desse total para cada
tipo de animal, sendo distinguidos por seu sexo e idade. Logo a Tabela 01 mostra, que
dos 97 animais para recria 30,93 % são machos de 24 a 36 meses, e 13,40% são fêmeas da
mesma idade. Quando somados fêmeas e machos de 12 a 24 meses temos 44,33% do total
de vendas para de animais destinados para recria. DIVERSITAS JOURNAL. Santana do Ipanema/AL. vol. 3, n. 3, p.655-674, set./dez. 2018. A GOVERNANÇA NO COMÉRCIO DE GADO EM CIDADES DO INTERIOR DE ALAGOAS
SILVA, FILHO, JOSÉ ADEILTON DA, LAGES, ANDRÉ MAIA GOMES A GOVERNANÇA NO COMÉRCIO DE GADO EM CIDADES DO INTERIOR DE ALAGOAS
SILVA, FILHO, JOSÉ ADEILTON DA, LAGES, ANDRÉ MAIA GOMES Tabela 02– Percentual dos tipos de animais para comércio. <12
12 a 24
24 a 36
> 36
Total
M
F
M
F
M
F
M
F
125
1,60%
2,40% 12,00% 68,80% 1,60% 8,00% 3,20% 2,40% 100%
Fonte: Autores, 2017. 668 A Tabela 02 nos mostra que dos 125 animais para recria 12,00 % são machos de
12 a 36 meses e 68,8% são fêmeas da mesma idade. Quando somados fêmeas e machos de
12 a 24 meses, temos 80,8% do total de vendas dessa idade para animais destinados com
fins de comércio. Tabela 02 – Percentual dos tipos de animais para abate. <12
12 a 24
24 a 36
> 36
Total
M
F
M
F
M
F
M
F
129
0,00%
0,00% 23,26% 36,44% 4,65% 7,75%
4,65% 17,05% 100%
Fonte: Autores, 2017. A tabela 03 nos mostra que dos 129 animais para recria 23,26 % são machos de
12 a 24 meses e 36,44% são fêmeas da mesma idade. Quando somados fêmeas e machos
de 12 a 24 meses temos 59,69% do total de vendas para de animais destinados para o
abate. Tabela 03 – Valor pago em média pelos três tipos de compradores. Tipo
de
Comprador
Idade do animal
(meses)
Média
do
valor
pago
(Reais)
Finalidade Abate 12 a 24
4.086,11
Finalidade
Comércio
12 a 24
7.707,78
Finalidade Recria 12 a 24
2.405,88
Fonte: Autores, 2015. Tabela 03 – Valor pago em média pelos três tipos de compradores. DIVERSITAS JOURNAL. Santana do Ipanema/AL. vol. 3, n. 3, p.655-674, set./dez. 2018. 8Preços correntes do período da pesquisa. DIVERSITAS JOURNAL. Santana do Ipanema/AL. vol. 3, n. 3, p.655-674, set./dez. 2018. Página | 669 11Esse tipo de melhoramento genético nas feiras é bastante limitado, basicamente pode ser desde um
animal, que embora seja mestiço, apresente cruzamentos com animais de raça, ou qualquer outra forma de 9É bem verdade, que esses agentes possam comprar carne nos tais matadouros, porém eles são uma parcela
ínfima diante do vasto número de outros consumidores, fluxo, que não exibe grande representatividade
para o estudo da cadeia produtiva, onde não podem interferir nos preços. p
p
p
p
ç
10Esse personagem pode ser desde um comerciante de carnes, até mesmo um representante de alguma
churrascaria ou Buffet entre outros. A GOVERNANÇA NO COMÉRCIO DE GADO EM CIDADES DO INTERIOR DE ALAGOAS
SILVA, FILHO, JOSÉ ADEILTON DA, LAGES, ANDRÉ MAIA GOMES Ao se analisar os valores pagos 8em cada negociação, pelos três tipos de
compradores, que apresentam suas distintas e possíveis finalidades de compra (Abate,
Comércio e Recria) gerou-se a tabela 04. Que mostra a diferença paga em média pelos
três tipos possíveis de compradores, que foram identificados na pesquisa, para os animais
com as idades iguais entre 12 a 24 meses. Página | 669 Página | 669 Diante desses resultados, sabe-se que as feiras de gado do interior de Alagoas
têm três possíveis tipos de compradores, que são classificados para fins de Abate,
Comércio e Recria, e com esse dado é possível traçar um esboço docanal de
comercialização, onde se permite entender os mercados e o desenvolvimento desse
sistema econômico, como nos mostra a figura 03. Figura 03 – Canal de comercialização dos bovinos no interior de Alagoas Figura 03 – Canal de comercialização dos bovinos no interior de Alagoas Figura 03 – Canal de comercialização dos bovinos no interior de Alagoas
Fonte: Autores, 2017 Fonte: Autores, 2017 A figura 03 apresenta um dos tipos de canal de comercialização de bovinos
popular no interior do estado de Alagoas, onde pode-se observar a presença dos três
tipos de personagens diagnosticados na pesquisa, e seus fluxos de venda e compra de
animais. O primeiro ponto importante é mostrar que para os personagens que utilizam
as feiras de animais, tem na feira sua função central, ou seja, esta se encontra no cerne de
toda transação econômica, e dos fluxos decorrentes de cada mercado, logo, trata-se de
uma representação, onde os agentes envolvidos, tem apenas essa rotina de
comercialização, pois, poderia haver outro tipo de canal, por exemplo, entre criador e
matadouro ou frigorífico de modo direto, porém esse não é o caso, embora isso também
aconteça em solo alagoano. A venda em feira é típica do mercado spot. E os animais
vendidos são do tipo geral, conforme tipifica Machado Filho e Zylbersztajn (1999). Nota-se, também na figura 03, que o ambiente favorece a redução do custo de
transação pela alta frequência que precisa estar atrelada no caso a reputação. E baixa DIVERSITAS JOURNAL. Santana do Ipanema/AL. vol. 3, n. 3, p.655-674, set./dez. 2018. Página | 670 ínfima diante do vasto número de outros consumidores, fluxo, que não exibe grande representativid
para o estudo da cadeia produtiva, onde não podem interferir nos preços. 9É bem verdade, que esses agentes possam comprar carne nos tais matadouros, porém eles são uma par
ínfima diante do vasto número de outros consumidores, fluxo, que não exibe grande representativid melhoramento genético. Essa limitação, como será mostrada durante o trabalho, é decorrente do próprio
sistema de comércio.
12A manutenção e a organização do espaço e do evento é papel preponderante das prefeituras dos dois
municípios. DIVERSITAS JOURNAL. Santana do Ipanema/AL. vol. 3, n. 3, p.655-674, set./dez. 2018. A GOVERNANÇA NO COMÉRCIO DE GADO EM CIDADES DO INTERIOR DE ALAGOAS
SILVA, FILHO, JOSÉ ADEILTON DA, LAGES, ANDRÉ MAIA GOMES incerteza comportamental. O segundo ponto importante, é notar, que muitas vezes a
figura do atravessador e do criador se confunde, afinal pode se tratar do mesmo
indivíduo, embora em tempo e situações distintas, que muitas vezes se alternam, como
também, existe entre eles um mercado bilateral. O terceiro é último ponto para início de
análise do canal de comercialização está na presença do matadouro, que tem seu fluxo
apenas de modo unilateral9, embora esse possa necessitar, também, de um atravessador,
não se trata do mesmo10, que já consta no esquema. A figura 03 exibem quatro mercados e seus fluxos bilaterais e unilateral, girando
entorno da feira, onde o criador produz e vende seu animal utilizando-se desta
instituição, que é mantida pelas prefeituras de cada município, ou então, vendendo a
algum atravessador que utilizará da feira para finalizar a comercialização, ou poderá
vender diretamente para outro criador. Os atravessadores, muitas vezes, também criam
animais, embora dediquem como atividade principal, em algum período, apenas a
comercialização de bovinos, que como abordado, comercializam para algum criador ou
então utilizam-se da feira. O matadouro na ponta final tem apenas o papel unilateral,
onde é representado pelos compradores para abate, que se utilizam da estrutura da feira
apenas para a compra dos animais. Os quatro mercados existentes e relatados; ente criador e feira; matadouro e feira;
atravessador e feira; e criador e atravessador, exibem às vezes demandas para um mesmo
grupo de animais, em sua maioria animais machos ou fêmeas de 12 a 24 meses como
apresentado nas tabelas 01, 02 e 03, onde cada um dos três tipos de compradores exibem
pagamentos com valores distintos para esse mesmo grupo de animais, conforme
apresenta a tabela 04. A diferença dos valores médios pago por cada tipo de comprador ocorre por
diversos motivos, sendo o comprador com finalidade de comércio (atravessador) o que
em média paga mais pelos animais, de forma consecutiva vem o pagamento com
finalidade de abate e recria. Isso se faz extremamente relevante, porque mostra que os
interessados pelos animais têm avaliações distintas, onde a estrutura de mercado clássica
não consegue fazer a dissociação, correta, como exemplo, um criador que investiu no
melhoramento genético11 de seu animal de 12 a 24 meses e resolveu vender na feira, DIVERSITAS JOURNAL. Santana do Ipanema/AL. vol. 3, n. 3, p.655-674, set./dez. 2018. A GOVERNANÇA NO COMÉRCIO DE GADO EM CIDADES DO INTERIOR DE ALAGOAS
SILVA, FILHO, JOSÉ ADEILTON DA, LAGES, ANDRÉ MAIA GOMES A GOVERNANÇA NO COMÉRCIO DE GADO EM CIDADES DO INTERIOR DE ALAGOAS
SILVA, FILHO, JOSÉ ADEILTON DA, LAGES, ANDRÉ MAIA GOMES Página | 671 CONSIDERAÇÕES FINAIS Diante do que foi apresentado, concluiu-se que os mercados, como os
representados pelas feiras de gado, não podem, por si só, desenvolver o sistema de
comércio de bovinos no interior de Alagoas, conforme a teoria apresentada pela NEI,
onde os custos de transação se fazem presente, necessitando assim, de uma “mão visível”
para coordenar esses mercados, onde os leilões como uma nova estrutura de governança
traria maior eficiência para todo o sistema. Os leilões conseguiriam reduzir os custos de transação, dado que haveria espaço
para um aumento na especificidade dos ativos, que é inibido pela estrutura atual das
feiras, o que viabilizaria o desenvolvimento da produção de bovinos, acarretando o
provável desenvolvimento dos demais participantes desse(s) tipo(s) de canal (is) de
comercialização do interior de Alagoas. Há, portanto, uma restrição imposta aos
pecuaristas advinda dos custos de transação, com isso a manutenção da estrutura de
mercado existente, infligi na permanência do menoscabo dos custos de transação,
subestimando seus efeitos negativos dentro dessa economia. Enfim, pode-se dizer que
mesmo o gado considerado geral e o que carrega mais especificidades poderiam ter
espaço no leilão com a coordenação das prefeituras nos casos em análise, mas com menor
grau de assimetria de informação, mais sinalização em função do comprador saber de
várias informações sobre a origem do animal. (cf SILBERBERG & SUEN, 2001). O trabalho, por fim, diagnosticou ser necessária a intervenção das prefeituras
para a mudança da estrutura de governança, pois as mesmas reforçam um ambiente
institucional, que corrobora para a manutenção desse sistema de comércio, que
impendem à troca, espontânea ou não, das feiras de gado para a estrutura de leilões do
tipo inglês. Que diante da possibilidade do desenvolvimento desse tipo de canal
propiciaria um incentivo para aumento da qualidade do rebanho comercializado, pois
levaria incentivos reais aos produtores agropecuários e geraria um aumento da
arrecadação municipal. A GOVERNANÇA NO COMÉRCIO DE GADO EM CIDADES DO INTERIOR DE ALAGOAS
SILVA, FILHO, JOSÉ ADEILTON DA, LAGES, ANDRÉ MAIA GOMES onde sua intenção é encontrar um criador que veja tal qualidade, porém isso é bastante
difícil no ambiente da feira, pois a aleatoriedade e a baixa sinalização fazem-se presentes,
deste modo poderá vender pra um atravessador, como também é possível que não
encontre-o, e por fim acabe vendendo para um comprador com finalidade de abate. Dessa
forma, poderá ser vítima de uma assimetria de informação entre seu produto e a
avaliação do comprador. Página | 671 Os leilões tornam-se a forma mais correta para a venda de animais, diante de tal
ambiente, ao proporcionar para vendedor uma avaliação de muitos compradores ao
mesmo tempo, reduzindo problemas da aleatoriedade presente nas feiras, como também
garantido uma melhor sinalização dos atributos do animal, assim reduzindo a assimetria
de informação para os consumidores. Deste modo os leilões reduziriam ainda mais a
incerteza do tipo comportamental e garantiria uma recompensa pelo aumento no
atributo do animal, como apresenta a figura 2, torna-se assim, uma estrutura mais
eficiente que as feiras. O que garante o desenvolvimento do sistema, como também
apresenta um papel importante no monitoramento facilitando a identificação da origem
da carne, agregando assim mais valor, aos preços pagos pelo matadouro, que
consequentemente poderia abastecer clientes mais refinados. Deste modo poderia haver
um desenvolvimento em todos os mercados abrangendo desde o início até o final do
canal de comercialização pertinente. O mercado da feira de gado tem resistido a mudanças muito por conta do papel
das prefeituras na coordenação de tais eventos, como apresentado por Williamson, no
tópico 02. Logo, é preciso, que, os custos sejam mais do que compensados pelos
ganhosadaptativos bilaterais que resultam diante de uma situação ineficiente desse
mercado, ou seja, o poder público mantêm ainda descompensados os custos, tornando-os
ainda altos para a adaptação de uma nova instituição, diante da incerteza de uma nova
estrutura de governança com suas novas rotinas e mecanismos de manutenção12, além de
tecnologias já predefinidas, o que impede à transição. Deste modo, não existe ambiente
institucional que favoreça a troca, espontânea ou não, das feiras para os leilões, com isso
o sistema não se desenvolve e consequentemente, há uma possível perda de aumento da
arrecadação, como também de umlimitantepara o bem-estar dessa população. A GOVERNANÇA NO COMÉRCIO DE GADO EM CIDADES DO INTERIOR DE ALAGOAS
SILVA, FILHO, JOSÉ ADEILTON DA, LAGES, ANDRÉ MAIA GOMES DIVERSITAS JOURNAL. Santana do Ipanema/AL. vol. 3, n. 3, p.655-674, set./dez. 2018. Página | 672 DIVERSITAS JOURNAL. Santana do Ipanema/AL. vol. 3, n. 3, p.655-674, set./dez. 2018. REFERÊNCIAS 1. BATALHA, M.O. (Coord.) Gestão do agronegócio: Textos selecionados. São
Carlos: EdUFSCar, 2009. DIVERSITAS JOURNAL. Santana do Ipanema/AL. vol. 3, n. 3, p.655-674, set./dez. 2018. A GOVERNANÇA NO COMÉRCIO DE GADO EM CIDADES DO INTERIOR DE ALAGOAS
SILVA, FILHO, JOSÉ ADEILTON DA, LAGES, ANDRÉ MAIA GOMES 2. FIANI, Ronaldo. Cooperação e conflito: instituições e desenvolvimento
econômico. Rio de Janeiro: Elsevier Brasil, 2011. 2. FIANI, Ronaldo. Cooperação e conflito: instituições e desenvolvimento
econômico. Rio de Janeiro: Elsevier Brasil, 2011. Página | 673
3. GANEM, Angela. O mercado como ordem social em Adam Smith, Walras e
Hayek: uma perspectiva crítico-filosófica. Economia e Filosofia: Controvérsias e
Tendências Recentes. Rio de Janeiro: Editora UFRJ, 2012. Página | 673 4. LIRA, Fernando José. Formação da Riqueza e da Pobreza de Alagoas. Maceió: Edufal, 2007. 5. LOCKE, Richard M. Construindo confiança. Niterói, Econômica, v. 3, n. 2,
2001. 6. MACHADO FILHO, C. A.; ZYLBERSZTAJN, D. Os Leilões sob a ótica da
economia institucional. São Carlos, Gestão & Produção, v. 6, n. 3, 1999. 7. SILBERBERG, Eugene; SUEN, Wing. The Structure of Economics. Third
Edition, Singapore: McGraw-Hill Higher Education, 2001. 8. VARIAN, HAL R. Microeconomia. 9ª Edição. Rio de Janeiro: Elsevier, 2016. 9. WILLIAMSON, Oliver E. Markets and hierarchies. New York: The Free
Press, 1975. 10. WILLIAMSON, Oliver E. The Economic Institutions of capitalism. Lon
ZYLBERSZTAJN, Decio don: The Free Press, 1985. 11. WILLIAMSON, Oliver E. Comparative economic organization: The analysis of
discrete structural alternatives. Administrative science quarterly, p. 269-296,
1991. 12. ZYLBERSZTAJN, Decio. Estruturas de governança e coordenação do
agribusiness: uma aplicação da nova economia das instituições. 1995. Tese
de Doutorado. Universidade de São Paulo. 13. ZYLBERSZTAJN, Decio; NEVES, Marco Fava (org.) Economia e gestão dos
negócios agroalimentares. São Paulo: Pioneira, 2000. A GOVERNANÇA NO COMÉRCIO DE GADO EM CIDADES DO INTERIOR DE ALAGOAS
SILVA, FILHO, JOSÉ ADEILTON DA, LAGES, ANDRÉ MAIA GOMES APÊNDICE – Questionários para os vendedores e compradores de gado na feira do
município de Dois Riachos/AL e do povoado de Canafístula no município de Palmeira
dos Índios/AL APÊNDICE – Questionários para os vendedores e compradores de gado na feira do
município de Dois Riachos/AL e do povoado de Canafístula no município de Palmeira
dos Índios/AL DIVERSITAS JOURNAL. Santana do Ipanema/AL. vol. 3, n. 3, p.655-674 Página | 674 Página | 674 DIVERSITAS JOURNAL. Santana do Ipanema/AL. vol. 3, n. 3, p.655-674, set./dez. 2018. DIVERSITAS JOURNAL. Santana do Ipanema/AL. vol. 3, n. REFERÊNCIAS 3, p.655-674, set./dez. 2018. DIVERSITAS JOURNAL. Santana do Ipanema/AL. vol. 3, n. 3, p.655-674, set./dez. 2018.
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KEPASTIAN HUKUM PENDAFTARAN TANAH MELALUI PENDAFTARAN TANAH SISTEMATIS LENGKAP DI KOTA BATU
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Jurnal Mimbar Hukum
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KEPASTIAN HUKUM PENGUASAAN TANAH MELALUI PENDAFTARAN
TANAH SISTEMATIS LENGKAP DI KOTA BATU Isdiyana Kusuma Ayu∗ Fakultas Hukum Universitas Islam Malang,
Jalan MT Haryono Nomor 193, Kota Malang *
Alamat korespondensi: isdiyana@unisma.ac.id. Abstract Systematic and Complete Land Registration (PTSL) is a government program to implement single land
mapping in Indonesia. The target of PTSL is only for uncertified land in a village or suburb. One of the
areas implementing PTSL program is Batu City that had previously applied National Agrarian Operation
Project (PRONA). As a new registration system, PTSL is worthy to discuss and analyze whether or not
systematic and complete land registration has already provided legal certainty for land owners. According
to the research result and analysis, it can be concluded that there are many hindering factors experienced
by Agrarian Office of Batu City, PTSL participants and village officials in Batu City. Land registration
through PTSL program is, however, still substantial in providing legal certainty for all participants. Keywords: rule of law, land registration, systematic. Intisari Pendaftaran Tanah Sistematis Lengkap (PTSL) merupakan program pemerintah untuk mewujudkan
peta tunggal di Indonesia. Sasaran PTSL yaitu bidang tanah yang belum memiliki sertifikat dalam satu
kelurahan atau desa. Salah satu daerah yang mengikuti Program PTSL yaitu Kota Batu yang sebelumnya
juga mengikuti Proyek Operasi Nasional Agraria (PRONA). Sebagai sistem pendaftaran yang baru,
PTSL menarik untuk dikaji dan dianalisis agar dapat diketahui faktor penghambat dan pendukung dalam
pelaksanaan, perlu pula dikaji dan dianalisis apakah pendaftaran tanah sistematis lengkap telah memberikan
kepastian hukum kepada pemilik tanah. Berdasarkan hasil kajian dan analisis dapat disimpulkan bahwa
banyak ditemukan faktor penghambat yang muncul dari pihak Kantor Pertanahan Kota Batu, Peserta PTSL,
dan pihak Desa atau Kelurahan di Kota Batu. Pendaftaran tanah melalui Program PTSL tetap memberikan
kepastian hukum kepada pesertanya. p
p
p
y
Kata Kunci: kepastian hukum, pendaftaran tanah, sistematis. p
p
y
nci: kepastian hukum, pendaftaran tanah, sistematis. Pokok Muatan
A. Latar Belakang Masalah..................................................................................................................... 339
B. Metode Penelitian............................................................................................................................... 340
C. Hasil Penelitian dan Pembahasan...................................................................................................... 340
1. Pendaftaran Tanah dan Pendaftaran Tanah Sistematis Lengkap................................................... 340
2. Pelaksanaan Pendaftaran Tanah Sistematis Lengkap di Kota Batu............................................... 342
3. Kepastian Hukum Penguasaan Tanah melalui Pendaftaran Tanah Sistematis Lengkap
di Kota Batu................................................................................................................................... 346
D. Kesimpulan ........................................................................................................................................ 350 A.
Latar Belakang Masalah wilayah desa/kelurahan, atau nama lainnya yang
setingkat dengan itu, yang meliputi pengumpulan
dan penetapan kebenaran data fisik dan data yuridis
mengenai satu atau beberapa obyek pendaftaran
tanah untuk keperluan pendaftarannya.2 Pendaftaran tanah merupakan kegiatan yang
dilakukan oleh Pemerintah untuk memberikan
kepastian hukum dan perlindungan hukum terhadap
pemegang hak atas tanah. Kegiatan pendaftaran
tanah terdiri dari pendaftaran tanah pertama kali
dan pemeliharaan data. Kegiatan pendaftaran
tanah pertama kali dapat dilakukan secara sporadis
maupun sistematis. Pendaftaran tanah pertama kali
melalui sporadis berarti pendaftaran tanah tersebut
dilakukan oleh sekelompok orang yang akan
mendaftarkan tanah yang masih berstatus hak lama. Pendaftaran tanah pertama kali melalui sistematis
merupakan pendaftaran tanah pertama kali yang
dilaksanakan oleh Pemerintah terhadap satu
kelurahan yang masih belum bersertifikat tanah. Latar belakang pelaksanaan program PTSL
yaitu Pemerintah masih menemukan tanah di
Indonesia yang belum bersertifikat atau tidak
memiliki sertifikat. Sertifikat tanah merupakan
alat bukti yang kuat terhadap penguasaan tanah
oleh masyarakat. Pemerintah tidak membebankan
biaya kepada masyarakat untuk proses pendaftaran
tanah, melainkan hanya membebankan biaya
administrasinya saja, seperti penyediaan surat
tanah untuk tanah yang belum memiliki surat
tanah, pembuatan dan pemasangan tanda batas,
pajak peralihan, serta biaya materai, fotokopi bukti
kepemilikan tanah seperti letter C, ataupun biaya
saksi. Kegiatan pendaftaran tanah pertama kali
yaitu Proyek Operasi Nasional Agraria (PRONA)
yang dilaksanakan sejak tahun 1981 berdasarkan
Keputusan Menteri Dalam Negeri Nomor 189
Tahun 1981 tentang Proyek Operasi Nasional
Agraria. Tujuan PRONA yaitu upaya dalam
melaksanakan Garis-garis Besar Haluan Negara
dan Catur Tertib di bidang pertanahan, khususnya
dalam pemberian sertifikat kepada masyarakat yang
belum memilikinya. Kelemahan dari PRONA yaitu
sejak 1981 hingga 2016 ternyata hanya berhasil
menyertifikatkan tanah sebanyak 44% saja, sehingga
masih kurang 56% di seluruh Indonesia.1 Upaya
percepatan pendaftaran tanah melalui PRONA
tidak mencapai target sehingga digagas pada tahun
2015, program pemerintah yang juga melaksanakan
pendaftaran tanah pertama kali yaitu Pendaftaran
Tanah Sistematis Lengkap (PTSL). Salah satu daerah di Indonesia yang dapat
obyek dari PTSL yaitu Kota Batu. Kota Batu
yang terletak di Provinsi Jawa Timur dengan luas
wilayah 19.908,72 Ha dengan jumlah 3 kecamatan,
4 kelurahan, dan 24 desa3. Berdasarkan data Kantor
Pertanahan Kota Batu, tanah yang sudah terdaftar
sampai 2017 lalu, kurang lebih sebanyak 42.482
bidang. Sementara tanah yang belum terdaftar
sebanyak 54.708 bidang. Ditargetkan, semua tanah
yang belum terdaftar itu sudah terdaftar pada 2025
mendatang. Kota Batu mendapatkan bagian 514 sertifikat
gratis dari PTSL tahun 2016. Pokok Muatan Ayu, Kepastian Hukum Penguasaan Tanah Melalui Pendaftaran Tanah Sistematis Lengkap Ayu, Kepastian Hukum Penguasaan Tanah Melalui Pendaftaran Tanah Sistematis Lengkap 339 1
Ihsanuddin. “Jokowi: Prona Sudah 35 Tahun, baru 44 Persen Tanah Warga Bersertifikat”, https://nasional.kompas.com/
read/2016/10/16/12474581/jokowi.prona.sudah.35.tahun.baru.44.persen.tanah.warga.bersertifikat, diakses 12 November 2018.
2
Lihat Pasal 1 angka 2 Peraturan Menteri Agraria dan Tata Ruang/Kepala Badan Pertanahan Nasional Republik Indonesia Nomor 6 Tahun 2018
tentang Pendaftaran Tanah Sistematis Lengkap (Berita Negara Republik Indonesia Tahun 2018 Nomor 501),
3
Badan Pusat Statistik Kota Batu, “Statistik Daerah Kota Batu 2016”, Publikasi Statistik Daerah Kota Batu 2016, September 2016, hlm. 1.
4
Roy Mastur, “BPN Beri 514 Sertifikat Gratis Kota Batu”, http://www.rri.co.id/post/berita/248262/daerah/bpn_beri_514_sertifikat_gratis_
kota_batu.html, diakses 17 Juni 2017. Badan Pusat Statistik Kota Batu, Statistik Daerah Kota Batu 2016 , Publikasi Statistik Daerah Kota Batu 2016, September 2016, hlm. 1.
4
Roy Mastur, “BPN Beri 514 Sertifikat Gratis Kota Batu”, http://www.rri.co.id/post/berita/248262/daerah/bpn_beri_514_sertifikat_gratis_
kota_batu.html, diakses 17 Juni 2017. g
g
p (
g
p
),
3
Badan Pusat Statistik Kota Batu, “Statistik Daerah Kota Batu 2016”, Publikasi Statistik Daerah Kota Batu 2016, Septembe
4
Roy Mastur, “BPN Beri 514 Sertifikat Gratis Kota Batu”, http://www.rri.co.id/post/berita/248262/daerah/bpn_beri_514_
k
b
h
l di k
17 J
i 2017 2
Lihat Pasal 1 angka 2 Peraturan Menteri Agraria dan Tata Ruang/Kepala Badan Pertanahan Nasional Republik Indonesia Nom
tentang Pendaftaran Tanah Sistematis Lengkap (Berita Negara Republik Indonesia Tahun 2018 Nomor 501), 1
Ihsanuddin. “Jokowi: Prona Sudah 35 Tahun, baru 44 Persen Tanah Warga Bersertifikat”, https://nasional.kompas.com/
read/2016/10/16/12474581/jokowi.prona.sudah.35.tahun.baru.44.persen.tanah.warga.bersertifikat, diakses 12 November 2018. B.
Metode Penelitian Jenis penelitian yang digunakan dalam
penelitian ini adalah yuridis empiris dengan
menggunakan metode pendekatan yuridis sosiologis. Penelitian ini meninjau dan menganalisis obyek
penelitiannya terhadap implementasi Peraturan
Menteri Agraria dan Tata Ruang/ Kepala Badan
Pertanahan Nasional Nomor 6 Tahun 2018 tentang
Pendaftaran
Sistematis
Lengkap
(selanjutnya
disebut Permen ATR/BPN No 6/2018), untuk
mengetahui kepastian hukum penguasaan tanah
melalui PTSL di Kota Batu. Lokasi penelitian
di Kota Batu, khususnya di Desa Oro-Oro Ombo
yang merupakan desa yang memiliki jumlah tempat
wisata yang sering dikunjungi oleh masyarakat dan
Kelurahan Dadaprejo yang merupakan kelurahan
yang menjadi tempat pelaksanaan PTSL Kota Batu
2018. Jumlah wilayah Kota Batu yang sudah
bersertifikat tidak sampai 60% ini menyebabkan
Kota Batu menjadi daerah yang dipilih untuk
menjalankan PTSL. Tujuan dari PTSL yaitu untuk
mempercepat pemetaan bidang tanah di Kota
Batu. Selain itu, PTSL yang dilaksanakan di Kota
Batu, pada dasarnya sebagai bentuk perhatian dari
Pemerintah untuk memberikan kepastian hukum
dan perlindungan hukum hak atas tanah masyarakat,
sehingga dapat meningkatkan kesejahteraan dan
kemakmuran masyarakat dan ekonomi negara, serta
mengurangi dan mencegah sengketa dan konflik
pertanahan yang terjadi di masyarakat Kota Batu. Pelaksanaan PTSL di Kota Batu merupakan
kegiatan
yang
bertujuan
untuk
memberikan
kepastian dan perlindungan hukum terhadap
masyarakat. PTSL merupakan kegiatan yang
dilaksanakan secara sistematis, artinya kegiatan
pendaftaran pertama kali yang dilakukan secara
serentak yang meliputi semua obyek pendaftaran
tanah yang belum terdaftar dalam wilayah atau
bagian wilayah suatu desa/kelurahan. Namun,
pelaksanaan PTSL di Indonesia tidak berjalan
dengan mulus karena banyak kendala yang dihadapi
oleh petugas di lapangan. Seperti bidang tanah yang
belum memenuhi syarat untuk diterbitkan sertifikat,
bidang tanah yang masih dalam keadaan sengketa
atau perkara di Pengadilan, subyek bidang tanah
yang tidak diketahui, tidak jelas, atau tidak berada
di tempat, dan batas tanah yang tidak jelas.5 Sumber data yang digunakan dibedakan
menjadi dua, yaitu sumber data primer dan sumber
data sekunder. Sumber data primer diperoleh dari
lapangan melalui wawancara dengan pihak yang
bersangkutan, seperti perangkat desa dengan Kepala
Desa Oro-Oro Ombo, Bapak Wiweko, Lurah
Dadaprejo, Bapak Abdul Salam, Kasi Hubungan
Hukum Pertanahan Kantor Pertanahan Kota Batu
yaitu Agus Harianto, dan Wira Santyani, S.SiT,
sebagai Kaur Umum dan Kepegawaian Kantor
Pertanahan Kota Batu dan Peserta PTSL. Sumber
data sekunder yaitu data-data yang diperoleh dari
kajian pustaka, peraturan perundang-undangan, dan
buku-buku literatur. Berdasarkan
uraian
tersebut,
rumusan
masalah dalam penelitian ini adalah, Pertama,
pengertian pendaftaran tanah dan pendaftaran tanah
sistematis lengkap. Kedua, pelaksanaan PTSL di
Kota Batu. B.
Metode Penelitian Ketiga, kepastian hukum penguasaan
tanah melalui pendaftaran tanah sistematis lengkap
di Kota Batu. 5
Erwin Hutapea, Tak Selalu Berjalan Mulus, Pendaftaran Tanah Terkendala Empat Hal, https://properti.kompas.com/read/2018/12/17/183000721/
tak-selalu-berjalan-mulus-pendaftaran-tanah-terkendala-empat-hal?page=all, diakses pada tanggal 10 April 2019. A.
Latar Belakang Masalah Sertifikat tersebut
oleh Kantor Pertanahan Kota Batu diberikan
kepada empat desa, yaitu Desa Sumberejo, Desa
Pesanggrahan, Desa Oro-oro Ombo, dan Desa
Tulungrejo4, sedangkan pada tahun 2017, Kepala
Kantor Pertanahan Kota Batu menyebutkan terdapat
1000 bidang yang masuk dalam PTSL 2017. Pada PTSL
merupakan
kegiatan
pendaftaran
tanah untuk pertama kali yang dilakukan secara
serentak bagi semua obyek pendaftaran tanah di
seluruh wilayah Republik Indonesia dalam satu MIMBAR HUKUM Volume 31, Nomor 3, Oktober 2019, Halaman 338-351 340 tahun 2018 ditargetkan 10.000 bidang tanah telah
bersertifikat melalui program PTSL. tahun 2018 ditargetkan 10.000 bidang tanah telah
bersertifikat melalui program PTSL. B. Metode Penelitian
Jenis penelitian ya C.
Hasil Penelitian dan Pembahasan
1.
Pendaftaran Tanah dan Pendaftaran
Tanah Sistematis Lengkap Pendaftaran tanah merupakan serangkaian
kegiatan yang dilakukan oleh Pemerintah secara
terus menerus, berkesinambungan, dan teratur. Asas
pendaftaran tanah yang dilaksanakan di Indonesia Ayu, Kepastian Hukum Penguasaan Tanah Melalui Pendaftaran Tanah Sistematis Lengkap 341 yaitu asas sederhana, aman, terjangkau, mutakhir,
dan terbuka. Asas yang melandasi kegiatan
pendaftaran tanah di Indonesia lebih mengutamakan
kepentingan masyarakat dalam hal penguasaan atas
suatu bidang tanah atau satuan rumah susun yang
sesuai dengan ketentuan yang ada dengan biaya
yang terjangkau. Pendaftaran tanah diatur dalam
Pasal 19 Undang-Undang Nomor 5 Tahun 1960
tentang Pokok-Pokok Agraria (UUPA). Pasal 19
ayat (1) UUPA menyebutkan bahwa:6 obyek tanah yang belum didaftarkan berdasarkan
Peraturan Pemerintah Nomor 10 Tahun 1961 tentang
Pendaftaran Tanah atau Peraturan Pemerintah
Nomor 24 Tahun 1997 tentang Pendaftaran Tanah
(PP No 24/1997). Bentuk kegiatan pendaftaran
tanah pertama kali dibagi menjadi dua yaitu: a. Pendaftaran tanah secara sistematis,
artinya
pendaftaran
tanah
untuk
pertama kali yang dilakukan secara
serentak
meliputi
semua
obyek
pendaftaran tanah yang belum didaftar
dalam wilayah atau bagian wilayah
suatu desa/kelurahan. (1)
Untuk menjamin kepastian hukum,
oleh Pemerintah diadakan pendaftaran
tanah di seluruh wilayah Republik
Indonesia, menurut ketentuan yang
diatur dengan Peraturan Pemerintah. Pendaftaran tanah secara sporadis,
artinya
pendaftaran
tanah
untuk
pertama kali mengenai satu atau
beberapa obyek pendaftaran tanah
dalam wilayah/bagian wilayah suatu
desa/kelurahan secara individual atau
masal. Pendaftaran tanah yang dilaksanakan di Indonesia
memiliki tujuan untuk Pemerintah, Masyarakat, dan
pihak ketiga, tujuan tersebut antara lain:7 a. Memberikan
kepastian
hukum
dan perlindungan hukum kepada
pemegangan hak atas suatu bidang
tanah
dalam
bentuk
pemberian
sertifikat hak atas tanah Pendaftaran tanah untuk pertama kali telah
dilaksanakan oleh Pemerintah Indonesia melalui
Program Pendaftaran Tanah Sistematis Lengkap
(PTSL). Dasar hukum PTSL dapat dilihat dalam
Peraturan Menteri Agraria dan Tata Ruang/Kepala
Badan Pertanahan Nasional Nomor 35 Tahun
2016. Peraturan tersebut diubah dengan Peraturan
Menteri Agraria dan Tata Ruang/Kepala Badan
Pertanahan Nasional Nomor 1 Tahun 2017 tentang
Perubahan atas Peraturan Menteri Agraria dan Tata
Ruang/Kepala Badan Pertanahan Nasional Nomor
35 Tahun 2016 tentang Percepatan Pelaksanaan
Pendaftaran Tanah Sistematis Lengkap. b. Menyediakan informasi kepada pihak-
pihak
yang
berkepentingan
agar
dengan mudah untuk memperoleh
data, baik data fisik dan data yuridis
yang sudah terdaftar terbuka untuk
umum. c. Terselenggaranya tertib administrasi,
baik dalam hal peralihan, pembebanan,
dan hapusnya hak atas bidang tanah
dan hak milik satuan rumah susun
yang wajib didaftarkan di Kantor
Pertanahan setempat. c. 6
Pasal 19 ayat (1) Undang-Undang Nomor 5 Tahun 1960 tentang Pokok-Pokok Agraria (Lembaran Negara Republik Indonesia Tahun 1960
Nomor 104, Tambahan Lembaran Negara Nomor 2043).
7
Pasal 3 dan Pasal 4 Peraturan Pemerintah Nomor 24 Tahun 1997 tentang Pendaftaran Tanah (Lembaran Negara Republik Indonesia Nomor 59
Tahun 1997, Tambahan Lembaran Negara Republik Indonesia Nomor 3696). 6
Pasal 19 ayat (1) Undang-Undang Nomor 5 Tahun 1960 tentang Pokok-Pokok Agraria (Lembaran Negara Republik Indonesia Tahun 1960
Nomor 104, Tambahan Lembaran Negara Nomor 2043).
7
Pasal 3 dan Pasal 4 Peraturan Pemerintah Nomor 24 Tahun 1997 tentang Pendaftaran Tanah (Lembaran Negara Republik Indonesia Nomor 59 Nomor 104, Tambahan Lembaran Negara Nomor 2043).
7
Pasal 3 dan Pasal 4 Peraturan Pemerintah Nomor 24 Tahun 1997 tentang Pendaftaran Tanah (Lembaran Negara Republik Indonesia Nomor 59
Tahun 1997, Tambahan Lembaran Negara Republik Indonesia Nomor 3696). 6
Pasal 19 ayat (1) Undang-Undang Nomor 5 Tahun 1960 tentang Pokok-Pokok Agraria (Lembaran Negara Republik Indonesia
Nomor 104, Tambahan Lembaran Negara Nomor 2043). C.
Hasil Penelitian dan Pembahasan
1.
Pendaftaran Tanah dan Pendaftaran
Tanah Sistematis Lengkap Terselenggaranya tertib administrasi,
baik dalam hal peralihan, pembebanan,
dan hapusnya hak atas bidang tanah
dan hak milik satuan rumah susun
yang wajib didaftarkan di Kantor
Pertanahan setempat. Peraturan tersebut di atas diubah kembali
menjadi Peraturan Menteri Agraria dan Tata Ruang/
Kepala Badan Pertanahan Nasional Nomor 6 Tahun
2018 (Permen ATR/BPN No 6/2018). Pembaruan
dari aturan terhadap PTSL terjadi karena aturan-
aturan yang terkait dengan PTSL sebelumnya masih Salah satu kegiatan pendaftaran tanah yaitu
kegiatan pendaftaran tanah untuk pertama kali. Kegiatan pendaftaran tanah untuk pertama kali
merupakan kegiatan yang dilakukan terhadap MIMBAR HUKUM Volume 31, Nomor 3, Oktober 2019, Halaman 338-351 342 yaitu Kecamatan Batu, Kecamatan Bumiaji,
dan Kecamatan Junrejo. Kecamatan Batu terdiri
dari Desa Ngaglik, Desa Songgo Kerto, Desa
Pesanggrahan, Desa Sisir, Desa Temas, Desa Oro-
Oro Ombo, Desa Sidomulyo, dan Desa Sumberejo. Kecamatan Bumiaji terdiri dari Desa Bumiaji, Desa
Pandan Rejo, Desa Giripurno, Desa Bulukerto, Desa
Sumber Gondo, Desa Tulung Rejo, Desa Gunung
Sari, Desa Punten, dan Desa Sumber Brantas. Kecamatan Junrejo terdiri dari Desa Junrejo, Desa
Mojorejo, Kelurahan Dadaprejo, Desa Pendem,
Desa Torongrejo, Desa Beji, dan Desa Tlekung. memerlukan penyempurnaan substansi atau materi
untuk menyesuaikan pada ketentuan peraturan
perundang-undangan terkait pendaftaran tanah
maupun ketentuan pertanahan lainnya.8 Dasar hukum PTSL yang berlaku saat ini yaitu
Instruksi Presiden Nomor 2 Tahun 2018 tentang
Percepatan Pendaftaran Tanah Sistematis Lengkap,
Peraturan Menteri Agraria dan Tata Ruang/Badan
Pertanahan Nasional Nomor 6 Tahun 2018 tentang
Pendaftaran Tanah Sistematis Lengkap, Keputusan
Bersama Menteri Agraria dan Tata Ruang/ Kepala
Badan Pertanahan Nasional, Menteri Dalam Negeri,
Menteri Desa, Pembangunan Daerah Tertinggal
dan Transmigrasi Nomor 25/SKB/V/2017, Nomor
590-3167A Tahun 2017, Nomor 34 Tahun 2017
tentang Pembiayaan Persiapan Pendaftaran Tanah
Sistematis. Landasan pelaksanaan yang digunakan oleh
Kantor Pertanahan Kota Batu dalam melaksanakan
Program PTSL telah diatur dalam Permen ATR/
BPN No 6/2018. Pasal 4 ayat (4) Permen ATR/
BPN No 6/2018 menyebutkan tahapan pelaksanaan
PTSL sebagai berikut:9 PTSL merupakan kegiatan pendaftaran tanah
untuk pertama kali yang dilakukan secara serentak
bagi semua obyek pendaftaran tanah di seluruh
wilayah Republik Indonesia dalam satu wilayah
desa/kelurahan. Kegiatan ini bertujuan untuk
mewujudkan pemberian kepastian hukum dan
perlindungan hukum hak atas tanah masyarakat. Asas yang digunakan dalam pelaksanaan PTSL
yaitu sederhana, cepat, lancar, aman, adil,
merata dan terbuka, serta akuntabel. C.
Hasil Penelitian dan Pembahasan
1.
Pendaftaran Tanah dan Pendaftaran
Tanah Sistematis Lengkap PTSL yang
dilaksanakan oleh Pemerintah pada dasarnya untuk
meningkatkan kesejahteraan dan kemakmuran
rakyat dan ekonomi negara, selain itu juga untuk
mengurangi dan mencegah sengketa dan konflik
pertanahan akibat tidak adanya alat bukti yang kuat
terhadap penguasaan suatu bidang tanah. a. Perencanaan;
b. Penetapan lokasi;
c. Persiapan;
d. Pembentukan dan penetapan panitia
ajudikasi PTSL dan satuan tugas;
e. Penyuluhan;
f. Pengumpulan
data
fisik
dan
pengumpulan data yuridis;
g. Penelitian
data
yuridis
untuk
pembuktian hak;
h. Pengumuman data fisik dan data
yuridis serta pengesahannya;
i. Penegasan konversi, pengakuan hak
dan pemberian hak;
j. Pembukuan hak;
k. Penerbitan sertifikat hak atas tanah;
l. Pendokumentasian dan penyerahan
hasil kegiatan;
m. Pelaporan. a. Perencanaan; b. Penetapan lokasi; c. Persiapan; g. Penelitian
data
yuridis
untuk
pembuktian hak;i h. Pengumuman data fisik dan data
yuridis serta pengesahannya; Pembukuan hak;i k. Penerbitan sertifikat hak atas tanah; g
g
p (
g
p
)
9
Pasal 4 ayat (4) Peraturan Menteri Agraria dan Tata Ruang/ Kepala Badan Pertanahan Nasional Nomor 6 Tahun 2018 tentang Pendaftaran
Tanah Sistematis Lengkap (Berita Negara Republik Indonesia Tahun 2018 Nomor 501), 8
Dasar Menimbang huruf b Peraturan Menteri Agraria dan Tata Ruang/Kepala Badan Pertanahan Nasional Republik Indonesia Nomor 6 Tahun
2018 tentang Pendaftaran TanahSistematis Lengkap (Berita Negara Republik Indonesia Tahun 2018 Nomor 501). 8
Dasar Menimbang huruf b Peraturan Menteri Agraria dan Tata Ruang/Kepala Badan Pertanahan Nasional Republik Indonesia Nomor 6 Tahun
2018 tentang Pendaftaran TanahSistematis Lengkap (Berita Negara Republik Indonesia Tahun 2018 Nomor 501).
9
Pasal 4 ayat (4) Peraturan Menteri Agraria dan Tata Ruang/ Kepala Badan Pertanahan Nasional Nomor 6 Tahun 2018 tentang Pendaftaran 8
Dasar Menimbang huruf b Peraturan Menteri Agraria dan Tata Ruang/Kepala Badan Pertanahan Nasional Republik Indonesia Nomor 6 Tahun
2018 tentang Pendaftaran TanahSistematis Lengkap (Berita Negara Republik Indonesia Tahun 2018 Nomor 501).
9
Pasal 4 ayat (4) Peraturan Menteri Agraria dan Tata Ruang/ Kepala Badan Pertanahan Nasional Nomor 6 Tahun 2018 tentang Pendaftaran
Tanah Sistematis Lengkap (Berita Negara Republik Indonesia Tahun 2018 Nomor 501), 2. Setiap kegiatan PTSL ditujukan kepada
seluruh obyek pendaftaran tanah di seluruh wilayah
Republik Indonesia, baik yang sudah ada tanda
batasnya ataupun yang akan ditetapkan tanda
batasnya dalam kegiatan PTSL. Perencanaan Kota Batu merupakan salah satu kota di
Jawa Timur yang mengikuti Program PTSL pada
tahun 2018. Kota Batu terdiri dari 3 kecamatan, Ayu, Kepastian Hukum Penguasaan Tanah Melalui Pendaftaran Tanah Sistematis Lengkap 343 Setelah
persiapan
selesai,
langkah
selanjutnya yang dilakukan adalah Kepala Kantor
Pertanahan Kota Batu membentuk dan menetapkan
Panitia Ajudikasi melalui Surat Keputusan Kepala
Kantor Pertanahan Kota Batu. Kegiatan PTSL
Kota Batu tahun 2018 diketuai oleh Bapak Agus
Harianto, Kepala Seksi Bagian Hubungan Hukum
Pertanahan, sebagai Ketua Panitia Ajudikasi yang
bertugas dan berwenang untuk memimpin dan
bertanggung jawab terhadap seluruh pelaksanaan
program kegiatan ajudikasi. dilakukan oleh setiap kantor pertanahan khususnya
Kepala Kantor Pertanahan untuk menetapkan lokasi
penyebaran target PTSL yang dikhususkan pada
beberapa desa/kelurahan, dan atau kecamatan. PTSL
Kota Batu telah direncanakan oleh Kepala Kantor
Pertanahan Kota Batu antara tanggal 20 hingga
25 Januari 2018 untuk menerbitkan SK Lokasi
Ajudikasi PTSL, yaitu pada Desa Pesanggrahan,
Desa Oro-Oro Ombo, Desa Torongrejo, dan Desa
Sumberejo.10 Kriteria untuk penentuan lokasi yaitu
ketersediaan anggaran PTSL dalam APBN/APBD,
PNBP, CSR, atau sumber dana lainnya.11 Penetapan
lokasi tersebut lebih diprioritaskan terhadap daerah
yang sebelumnya telah melaksanakan PRONA
seperti Desa Oro-Oro Ombo, dan pertimbangan
kemampuan petugas pelaksana PTSL pada masing-
masing Kantor Pertanahan. Letak dari keempat desa
tersebut juga berdekatan satu dengan lainnya. Panitia Ajudikasi dalam bertugas juga dibantu
oleh Satuan Tugas (Satgas) yang terdiri dari Satgas
Fisik, Satgas Yuridis, dan Satgas Administrasi yang
akan tetap saling berkoordinasi dengan Panitia
Ajudikasi. Ketua Panitia Ajudikasi PTSL Kota
Batu, Bapak Agus Harianto, bersama tim melakukan
penyuluhan secara bertahap di masing-masing desa
yang berpartisipasi dalam PTSL tahun 2018. Setelah perencanaan dan penetapan lokasi,
langkah selanjutnya adalah melakukan persiapan. Persiapan berdasarkan Pasal 4 ayat (1) Permen
ATR/BPN No 6/2018, pertama harus dilakukan
oleh Kepala Kantor Pertanahan yaitu terkait sarana
dan prasarana pelaksanaan Kegiatan PTSL, seperti
alat ukur dan lokasi tempat kerja Panitia Ajudikasi. Kedua, sumber daya manusia yang perlu disiapkan
oleh Kepala Kantor Pertanahan dengan menetapkan
Panitia
Ajudikasi
sebagai
pelaksana
PTSL. Ketiga, kebutuhan transportasi, PTSL merupakan
pendaftaran pertama kali dimana pihak dari Kantor
Pertanahan Kota Batu harus datang langsung ke
lokasi untuk melakukan pengukuran, sehingga
perlu adanya akomodasi untuk mempermudah
kegiatan tersebut. Keempat, koordinasi dengan
aparat pemerintah lainnya. 11 Pasal 7 ayat (4) Peraturan Menteri Agraria dan Tata Ruang/ Kepala Badan Pertanahan Nasional Nomor 6 Tahun 2018 tentang Pendaftaran
Tanah Sistematis Lengkap (Berita Negara Republik Indonesia Tahun 2018 Nomor 501), 0 Hasil wawancara dengan Ibu Wira Sentyani, S.SiT, Kaur Umum Kepegawaian Kantor Pertanahan Kota Batu. 2. Aparat pemerintah
yang berkaitan dengan pelaksanaan PTSL yaitu
perangkat desa atau kelurahan, Perangkat Desa
Oro-oro Ombo, dan Pegawai Kelurahan Dadaprejo,
yang juga masuk dalam bagian Panitia Ajudikasi. Penyuluhan yang dilakukan tidak hanya
melibatkan kantor pertanahan Kota Batu, melainkan
turut mengundang pihak kepolisian, kejaksaan, dan
perbankan. Tujuan melibatkan pihak-pihak tersebut
yaitu agar terjadi keterbukaan dalam pelaksanaan
PTSL sehingga tidak ada pihak yang dirugikan. Sasaran penyuluhan yaitu masyarakat yang sudah
memiliki atau belum memiliki sertifikat tanah. Dalam kegiatan penyuluhan ini, penyuluhan di Desa
Oro-Oro Ombo dan Kelurahan Dadaprejo berjalan
dengan baik. Hal ini terlihat dari antusiasme warga
dalam mengikuti penyuluhan, dengan mengajukan
beberapa pertanyaan khususnya dengan biaya yang
dikeluarkan saat PTSL berlangsung. Namun, tidak
semua warga dapat hadir dalam penyuluhan karena
bersamaan dengan waktu bekerja. Selain itu, masih
terdapat warga yang sudah lanjut usia dan berada di
tingkat perekonomian yang rendah, sehingga tidak
mudah untuk memahami apa yang dipaparkan oleh
Panitia Ajudikasi, sehingga perlu adanya pendekatan 344 MIMBAR HUKUM Volume 31, Nomor 3, Oktober 2019, Halaman 338-351 langsung antara Panitia Ajudikasi dengan warga. Ajudikasi Kota Batu akan meneliti terkait riwayat
tanah berdasarkan bukti kepemilikan yang telah
dikumpulkan oleh peserta PTSL di Desa Oro-Oro
Ombo dan Kelurahan Dadaprejo. Tujuan penelitian
ini yaitu untuk keperluan pembuktian hak. Ajudikasi Kota Batu akan meneliti terkait riwayat
tanah berdasarkan bukti kepemilikan yang telah
dikumpulkan oleh peserta PTSL di Desa Oro-Oro
Ombo dan Kelurahan Dadaprejo. Tujuan penelitian
ini yaitu untuk keperluan pembuktian hak. angsung antara Panitia Ajudikasi dengan warga. langsung antara Panitia Ajudikasi dengan warga. Pengumpulan data fisik dan data yuridis
dilakukan
oleh
Panitia
Ajudikasi. Dalam
pelaksanaan PTSL di Kota Batu, pihak yang paling
berperan dalam pengumpulan data yuridis yaitu dari
pihak desa. Pihak-pihak yang berperan dalam PTSL
di lingkungan Desa Oro-oro Ombo dan Kelurahan
Dadaprejo dilakukan oleh Panitia Desa yang terdiri
dari Karang Taruna, Tokoh Pemuda, dan Tokoh
Masyarakat, khususnya Ketua RT dan RW. Ketiga
elemen tersebut mendapatkan Surat Keputusan dari
Kepala Desa Oro-oro Ombo dan Lurah Dadaprejo
untuk memperkuat tugas Panitia Desa/Kelurahan
ketika melakukan pendataan di masyarakat. Peran Ketua RT dan RW yaitu mengumpulkan
berkas, mendampingi pada saat pengukuran, dan
menyelesaikan masalah yang ada masyarakat
desa. Ketika permasalahan tersebut tidak dapat
diselesaikan, maka akan dibawa ke Kantor Desa
untuk diselesaikan. g
y
p g
13 Sudikno Mertokusumo, 2006, Hukum Acara Perdata Indonesia, Liberty, Yogyakarta, hlm. 127. 12 Hasil wawancara dengan Ibu Wira Sentyani, S.SiT, Kaur Umum Kepegawaian Kantor Pertanahan Kota Batu. 2. pantai, sungai, dan pembatasan penggunaan
tanah hak dalam kawasan lindung. Ketika sudah
ditandatangani dan sudah memenuhi syarat untuk
diberikan tanda bukti haknya, sertifikat hak atas
tanah akan diterbitkan dan diberikan kepada
masyarakat. Ketentuan 14 hari kalender untuk melakukan
pengumuman berbeda dengan ketentuan dalam
Pasal 26 PP NO 24/1997, yang menentukan
bahwa pengumuman pendaftaran pertama kali
secara sistematik dilakukan selama 30 hari kerja. Alasan Pemerintah mempersingkat waktu tersebut
karena PTSL merupakan pendaftaran tanah secara
massal yang diketahui oleh masyarakat umum dan
merupakan kegiatan percepatan pendaftaran tanah
antara 4 hingga 6 bulan sejak sosialisasi kegiatan,
sehingga pengumumannya lebih singkat dan ketika
ada sengketa tanah akan segera diketahui.14 Hambatan
yang
dialami
oleh
Kantor
Pertanahan Kota Batu dapat diselesaikan dengan
menambah Pegawai Kantor Pertanahan Kota
Batu, khususnya petugas ukur. petugas ukur dari
Kantor Pertanahan Kota Batu yang masih kurang
menyebabkan pengukuran tanah warga menjadi
lebih lama dan tidak segera selesai, karena satu
hari hanya dapat dilakukan pengukuran sebanyak
10 bidang. Cara yang dapat segera dilakukan oleh
Kantor Pertanahan adalah bekerja sama dengan
surveyor berlisensi milik swasata. Ketika pengumuman selama 14 hari kalender
tidak ada pihak yang keberatan atau data sudah
lengkap maka hal yang harus dilakukan lagi
yaitu penegasan konversi, pengakuan hak, atau
pemberian hak. Perbedaan dari ketiga kegiatan
tersebut ketika penegasan konversi yaitu diberikan
pada bidang tanah yang alat bukti tertulisnya
lengkap atau alat bukti tertulisnya tidak lengkap,
tetapi terdapat keterangan saksi maupun pernyataan
yang bersangkutan. Pengakuan hak akan diberikan
kepada tanah yang dibuktikan dengan kenyataan
penguasaaan fisiknya selama 20 tahun secara terus
menerus. Sedangkan pemberian hak diberikan
kepada tanah yang termasuk tanah Negara dengan
mengusulkan secara kolektif oleh Kepala Kantor
Pertanahan. Pelaksanaan PTSL di Kota Batu lebih
didominasi oleh penyelesaian kegiatan dengan
penegasan konversi karena memang mayoritas
tanah di Kota Batu masih belum bersertifikat. Hal ini juga diikuti dengan merekrut petugas
ukur baru agar dapat mengurangi biaya kerja sama
tersebut. Selain itu, rangkap jabatan antara Petugas
Harian Kantor Pertanahan dengan Panitia Ajudikasi
juga dapat memperlambat jalannya kegiatan PTSL
dan kinerja pelayanan lainnya. Kantor Pertanahan
Kota Batu dapat menentukan Panitia Ajudikasi yang
merupakan Pegawai Kantor Pertanahan Kota Batu
yang tidak berperan langsung dengan masyarakat,
seperti bagian loket yang bertemu langsung dengan
masyarakat untuk melakukan pendaftaran tanah
atau pemeliharaan data. 2. Kemudahan dari pendaftaran melalui PTSL
yaitu ketika riwayat kepemilikan tanah peserta PTSL
tidak lengkap, maka dapat dilengkapi dan dibuktikan
dengan surat pernyataan tertulis bermaterai cukup
dengan tentang kepemilikan dan/atau penguasaan
fisik bidang tanah dengan itikad baik dari pihak yang
bersangkutan. Indikator itikad baik yaitu tidak ada
pihak yang keberatan atas tanah yang dimiliki atau
tidak dalam keadaan sengketa, tidak termasuk asset
pemerintah, dan bukan kawasan hutan. Meskipun
terdapat kemudahan tersebut, kekuatan pembuktian
dari surat pernyataan bermaterai yang dibuat oleh
Peserta PTSL sama dengan akta di bawah tangan. Kekuatan pembuktian akta di bawah tangan
adalah orang terhadap siapa akta di bawah tangan
tersebut digunakan diwajibkan membenarkan
atau
memungkiri
tanda
tangannya. Ketika
tandatangan tersebut dipungkiri, maka hakim harus
memerintahkan agar kebenaran akta diperiksa. Namun, ketika tanda tangan diakui oleh yang
bersangkutan maka akta di bawah tangan tersebut
mempunyai kekuatan dan menjadi bukti yang
sempurna.13 Sehingga, dapat disimpulkan bahwa
kekuatan pembuktian dari surat pernyataan yang
dibuat oleh Peserta PTSL adalah tidak otomatis
memiliki kekuatan pembuktian yang sempurna. Setelah data yuridis sudah terkumpul,
maka pengumpulan data fisik akan dilakukan oleh
petugas ukur dari Kantor Pertanahan Kota Batu. Namun, jumlah petugas ukur di Kantor Pertanahan
Kota Batu masih kurang dan tidak sampai 20
petugas ukur, dengan setiap tim terdiri dari 2
orang. Rata-rata pengukuran yang dilakukan oleh
setiap tim yaitu 10 bidang tanah dalam satu hari.12
Akibat perbandingan antara luas tanah yang diukur
dengan jumlah petugas ukur yang tidak sebanding,
maka penyelesaian pengukuran di Kota Batu untuk
kegiatan PTSL berlangsung lama. Selain itu, faktor
penghambat lain yaitu, jarak satu bidang tanah
dengan bidang tanah lainnya yang jauh dan cuaca
yang tidak mendukung di Kota Batu. Langkah selanjutnya yaitu pengumuman
data fisik dan data yuridis serta pengesahannya. Pengumuman dalam PTSL hanya dilakukan selama
14 hari kalender dan diumumkan di Kantor Panitia
Ajudikasi PTSL dan Kantor Kepala Desa/Kelurahan. Hal ini juga diberlakukan dalam pelaksanaan PTSL
di Kota Batu. Lama waktu 14 hari kalender tersebut
digunakan untuk diadakannya pengajuan keberatan
atau kekuranglengkapan data. Ketika terdapat
pihak yang keberatan terhadap data fisik atau data Setelah bidang tanah sudah diukur untuk
memenuhi data fisik, langkah selanjutnya adalah
pembuktian hak dengan melakukan penelitian data
yuridis. Penelitian tersebut terdiri dari pemeriksaan
riwayat kepemilikan tanah yang dituangkan
dalam Risalah Penelitian Data Yuridis. Panitia Ayu, Kepastian Hukum Penguasaan Tanah Melalui Pendaftaran Tanah Sistematis Lengkap 345 yuridis yang diumumkan tersebut, maka pihak yang
keberatan wajib memberitahukan secara tertulis
agar segera mengajukan gugatan ke Pengadilan. 14 Hasil wawancara dengan Bapak Agus Harianto, Kasi Hubungan Hukum Pertanahan Kota Batu. 2. Masyarakat yang ikut sebagai peserta PTSL
lebih didominasi oleh masyarakat yang memiliki
tingkat perekonomian yang rendah dan sudah
lanjut usia, sehingga ketika diberikan sosialisasi
terkait program PTSL dengan menggunakan
Bahasa Indonesia, terkadang tidak mengerti
dan perlu diberikan pemahaman satu persatu. Ketika dilakukan sosialisasi mengenai program
ini, Kantor Pertanahan Batu seharusnya dapat
menggunakan bahasa yang biasa digunakan oleh
masyarakat setempat. Kantor Pertanahan Batu juga
dapat meminta bantuan perangkat desa/kelurahan Tahap selanjutnya yaitu pembukuan hak
yang merupakan kegiatan dimana data fisik dan
data yuridis yang dibukukan dalam buku tanah
yang ditandatangani oleh Ketua Panitia Ajudikasi
atas nama Kepala Kantor Pertanahan. Pembukuan
hak ini berisikan pembatasan dalam pemindahan
hak, penggunaan tanah menyangkut garis sempadan MIMBAR HUKUM Volume 31, Nomor 3, Oktober 2019, Halaman 338-351 346 Kepastian hukum merupakan hal yang
menjadi harapan subyek hukum untuk mengetahui
hal yang dilarang ataupun tidak dilarang oleh
hukum, sehingga perlu adanya suatu penormaan
yang baik dan jelas dalam suatu peraturan
perundang-undangan, serta jelas juga penerapannya
di masyarakat. Kepastian hukum bukanlah suatu
hal yang mutlak untuk memenuhi tujuan hukum,
melainkan pada sebagai saran yang digunakan sesuai
dengan situasi dan kondisi dengan memperhatikan
asas manfaat dan efisiensi. Kepastian hukum
apabila dikaitkan dengan pendaftaran tanah yang
secara umum diatur dalam pasal 19 ayat (1) UUPA
yang menyebutkan bahwa:16 setempat untuk menjelaskan program tersebut. Hal ini disebabkan karena perangkat desa telah
mengenal budaya masyarakat, sehingga dapat lebih
dipahami oleh masyarakat. Pihak selanjutnya yang memiliki hambatan
dalam pelaksanaan PTSL yaitu Perangkat Kantor
Desa/Kelurahan. Kantor Desa/Kelurahan memiliki
peran dalam pengumpulan dokumen masyarakat
yang ikut program PTSL. Namun, ketika sudah
terjun ke masyarakat, hambatan-hambatan yang
terjadi bersifat teknis, seperti pemilik tanah yang
tidak diketahui keberadannya, obyek menjadi
jaminan utang, klaim batas tanah yang tidak jelas,
masyarakat tidak segera mengumpulkan dokumen. Permasalahan tersebut dapat diselesaikan ketika
perangkat desa/kelurahan bersikap persuasif dan
menjelaskan pentingnya sertifikat tanah. Perangkat
Desa/Kelurahan melakukan sosialisasi pentingnya
bukti kepemilikan tanah sehingga masyarakat dapat
menjaga tanah mereka. “Untuk menjamin kepastian hukum oleh
Pemerintah diadakan pendaftaran tanah di
seluruh wilayah Republik Indonesia menurut
ketentuan-ketentuan yang diatur dengan
Peraturan Pemerintah.” “Untuk menjamin kepastian hukum oleh
Pemerintah diadakan pendaftaran tanah di
seluruh wilayah Republik Indonesia menurut
ketentuan-ketentuan yang diatur dengan
Peraturan Pemerintah.” Berdasarkan pasal tersebut, Pemerintah telah
memberikan
jaminan
kepastian
hukum
dan
perlindungan hukum bagi masyarakat untuk
menguasai tanah yaitu melalui pendaftaran tanah. Berdasarkan pasal tersebut, Pemerintah telah
memberikan
jaminan
kepastian
hukum
dan
perlindungan hukum bagi masyarakat untuk
menguasai tanah yaitu melalui pendaftaran tanah. 15 Amran Suadi, et al., 2016, Politik Hukum: Perspektif Hukum Perdata dan Pidana Islam Serta Ekonomi Syariah, Kencana, Jakarta, hlm. 295.
16 Pasal 19 ayat (1) Undang-Undang Nomor 5 Tahun 1960 tentang Pokok-Pokok Agraria (Lembaran Negara Republik Indonesia Tahun 1960
Nomor 104, Tambahan Lembaran Negara Nomor 2043).
17 Urip Santoso, 2005, Hukum Agraria dan Hak-Hak Atas Tanah, Kencana, Jakarta, hlm. 73. ,
g
)
17 Urip Santoso, 2005, Hukum Agraria dan Hak-Hak Atas Tanah, Kencana, Jakarta, hlm. 73. ,
p
f
16 Pasal 19 ayat (1) Undang-Undang Nomor 5 Tahun 1960 tentang Pokok-Pokok Agraria (Lem
Nomor 104, Tambahan Lembaran Negara Nomor 2043). 3.
Kepastian Hukum Penguasaan Tanah
melalui Pendaftaran Tanah Sistematis
Lengkap di Kota Batu Penguasaan tanah memiliki dua arti, yaitu
arti fisik dan arti yuridis. Penguasaan tanah
dalam arti yuridis merupakan penguasaan yang
dilandasi oleh hak yang dilindungi oleh hukum
dan memberikan kewenangan kepada pemegang
hak untuk menguasai secara fisik tanah yang
dihaki, seperti pemilik tanah menggunakan atau
mengambil manfaat dari tanah yang dihaki, tidak
diserahkan kepada pihak lain.17 Pasal 1 angka
2 Peraturan Pemerintah Nomor 16 Tahun 2004
tentang Penatagunaan Tanah menyebutkan bahwa
penguasaan tanah merupakan hubungan hukum
antara orang perorangan, kelompok masyarakat
atau badan hukum dengan tanah sesuai dengan
UUPA. Berdasarkan uraian tersebut maka dapat
diketahui bahwa hak penguasaan tanah merupakan Hukum dapat ditegakkan jika memperhatikan
tiga unsur yaitu kepastian hukum, kemanfaatan, dan
keadilan. Namun, dari ketiga unsur tersebut tidak
dapat dititikberatkan pada salah satu unsur saja,
karena tidak selalu mudah untuk mengusahakan
timbulnya keseimbangan antara ketiga unsur
tersebut. Kepastian
hukum
secara
normatif
merupakan suatu peraturan yang dibuat dan
diundangkan secara pasti karena mengatur secara
jelas dan logis.15 Tanpa adanya kepastian hukum,
seseorang tidak tahu hal apa yang akan dilakukan
dan akhirnya timbul perasaan tidak nyaman, namun
ketika terlalu menitikberatkan pada suatu kepastian
hukum, terlalu ketat mentaati suatu aturan akibatnya
akan kaku dan menimbulkan ketidakadilan. Ayu, Kepastian Hukum Penguasaan Tanah Melalui Pendaftaran Tanah Sistematis Lengkap 347 Kota Batu yang merupakan salah satu wilayah
yang menjalankan program ini juga menggunakan
kedua aturan tersebut. Pemerintah menginginkan
bahwa seluruh wilayah di Indonesia terdaftar dalam
peta tunggal, sehingga seluruh wilayah khususnya
di Kota Batu diukur dan didata. Tujuan utama
pelaksanaan PTSL yaitu memberikan kepastian
hukum kepada masyarakat dalam hal kepemilikan
tanah dalam bentuk sertifikat tanah. serangkaian wewenang, kewajiban, dan atau
larangan untuk berbuat sesuatu atau tidak berbuat
sesuatu bagi pemegang haknya. Salah satu hak penguasaan atas tanah menurut
Hukum Tanah Nasional yaitu hak perseorangan
atas tanah yang meliputi hak-hak atas tanah, wakaf
tanah hak milik, hak tanggungan, dan hak milik atas
satuan rumah susun.18 Hak perseorangan atas tanah
merupakan hak atas tanah sebagai hak individual
yang semuanya secara langsung ataupun tidak
langsung bersumber pada hak bangsa.19 Perbedaan pendaftaran tanah pertama kali
melalui PTSL dengan pendaftaran tanah pertama
kali berdasarkan PP No 24/1997 yaitu terdapat pada
pembayaran pajak peralihan tanah dan bangunan
dan jangka waktu pengumuman. Ibid, hlm 11.
19 Lihat Pasal 16 dan Pasal 51 Undang-Undang Nomor 5 Tahun 1960 tentang Pokok-Pokok Agraria (Lembaran Negara Republik Indonesia
Tahun 1960 Nomor 104, Tambahan Lembaran Negara Nomor 2043). 22 Lihat Pasal 33 Peraturan Menteri Agraria dan Tata Ruang/Kepala Badan Pertanahan Nasional Nomor 6 Tahun 2018 tentang Pendaftaran Tanah
Sistematis Lengkap (Berita Negara Republik Indonesia Tahun 2018 Nomor 501). j ,
,
f
p
g,
p
g,
sal 103 Peraturan Menteri Negara Agraria Nomor 3 Tahun 1997 tentang Pelaksanaan Peraturan Pemerintah Nomor 24 Tahun 1997
endaftaran Tanah. ,
g
)
arja, 2010, Hukum Pendaftaran Tanah, Universitas Lampung, Bandar Lampung, hlm. 41. 18 Ibid, hlm 11.
19 Lihat Pasal 16 dan Pasal 51 Undang-Undang Nomor 5 Tahun 1960 tentang Pokok-Pokok Agraria (Lembaran Negara Republik Indonesia
Tahun 1960 Nomor 104, Tambahan Lembaran Negara Nomor 2043).
20 FX. Sumarja, 2010, Hukum Pendaftaran Tanah, Universitas Lampung, Bandar Lampung, hlm. 41.
21 Lihat Pasal 103 Peraturan Menteri Negara Agraria Nomor 3 Tahun 1997 tentang Pelaksanaan Peraturan Pemerintah Nomor 24 Tahun 1997
tentang Pendaftaran Tanah.
22 Lihat Pasal 33 Peraturan Menteri Agraria dan Tata Ruang/Kepala Badan Pertanahan Nasional Nomor 6 Tahun 2018 tentang Pendaftaran Tanah
Sistematis Lengkap (Berita Negara Republik Indonesia Tahun 2018 Nomor 501). ,
g
)
20 FX. Sumarja, 2010, Hukum Pendaftaran Tanah, Universitas Lampung, Bandar Lampung, hlm. 41. FX. Sumarja, 2010, Hukum Pendaftaran Tanah, Universitas Lampung, Bandar Lampung, hlm. 41.
21 Lihat Pasal 103 Peraturan Menteri Negara Agraria Nomor 3 Tahun 1997 tentang Pelaksanaan Peraturan Pemerintah Nomor 24
tentang Pendaftaran Tanah.
22
Lih
P
l 33 P
M
i A
i d
T
R
/K
l B d
P
h
N
i
l N
6 T h
2018
P
d Ibid, hlm 11.
19 Lihat Pasal 16 dan Pasal 51 Undang-Undang Nomor 5 Tahun 1960 tentang Pokok-Pokok Agraria (Lembaran Negara Republik Indonesia
Tahun 1960 Nomor 104, Tambahan Lembaran Negara Nomor 2043).
20 FX. Sumarja, 2010, Hukum Pendaftaran Tanah, Universitas Lampung, Bandar Lampung, hlm. 41.
21 Lihat Pasal 103 Peraturan Menteri Negara Agraria Nomor 3 Tahun 1997 tentang Pelaksanaan Peraturan Pemerintah Nomor 24 Tahun 1997
tentang Pendaftaran Tanah 3.
Kepastian Hukum Penguasaan Tanah
melalui Pendaftaran Tanah Sistematis
Lengkap di Kota Batu Alasan
Pemerintah mempersingkat waktu pengumuman
karena PTSL merupakan pendaftaran tanah secara
massal yang diketahui oleh masyarakat umum
sehingga pengumumannya lebih singkat dan ketika
ada sengketa tanah akan segera diketahui. Obyek PTSL di Kota Batu didominasi
kepemilikan tanah yang termasuk kluster 1, yaitu
tanah-tanah yang belum dilekati oleh hak atas tanah
sesuai dengan ketentuan UUPA. Tanah tersebut
masuk dalam obyek konversi hak. Konversi hak
merupakan penyesuaian hak-hak atas tanah yang
tunduk kepada sistem hukum yang lama, yaitu hak-
hak atas tanah menurut KUH Perdata dan hukum
adat menjadi hak atas tanah menurut UUPA.23
Contoh bekas tanah milik ada sebelum diundangkan
UUPA dibuktikan dengan petuk pajak bumi, girik,
pipil, kekitir, dan verponding Indonesia, yang
digantikan dengan Kutipan Letter C yang dibuat
oleh Kepala Desa/Kelurahan.24 Konversi hak dapat
didaftarkan dengan pendaftaran tanah sistematik
maupun sporadis dengan melengkapi alat-alat
bukti mengenai adanya hak tersebut berupa bukti-
bukti tertulis, keterangan saksi dan atau pernyataan
yang bersangkutan yang kadar kebenarannya oleh
Panitia Ajudikasi dalam pendaftaran tanah secara
sistematik atau oleh Kepala Kantor Pertanahan
dalam pendaftaran tanah secara sporadis, dianggap
cukup untuk mendaftar hak, pemegang hak, dan
hak-hak pihak lain yang membebaninya.25 Luaran dari PTSL yang dilakukan oleh
Kantor Pertanahan Kota Batu ternyata tidak hanya
menerbitkan sertifikat tanah, melainkan juga peta
bidang tanah dan daftar tanah. Tujuan dan target
utama Program PTSL yaitu mengukur keseluruhan
tanah-tanah yang meskipun tidak dapat diterbitkan
sertifikat karena tidak dapat memenuhi persyaratan
yang ada. Hal ini bertujuan agar dapat mewujudkan
peta tunggal pertanahan di Indonesia. Pelaksanaan
yang dilaksanakan oleh Kantor Pertanahan Kota
Batu telah sesuai dengan Poin Kedua dari Instruksi
Presiden Republik Indonesia Nomor 2 Tahun 2018
tentang Percepatan Pendaftaran Tanah Sistematis
Lengkap di Seluruh Wilayah Republik Indonesia
bahwa luaran dari pelaksanaan PTSL yaitu: PTSL
dilakukan
untuk
mendapatkan
kepastian hukum bagi pemegang hak atas tanah
maupun pihak lain yang memiliki kepentingan
dengan tanah yang dikuasainya tersebut. Kepastian
hukum kepemilikan tanah dapat diwujudkan
dengan penerbitan sertifikat tanah. Undang-Undang
Pokok Agraria mengatur bahwa Pemerintah
mengadakan pendaftaran tanah di seluruh wilayah
Republik Indonesia yang bertujuan untuk menjamin
kepastian hukum atas hak-hak atas tanah.26 Upaya
mewujudkan kepastian hukum dalam pendaftaran
tanah yaitu penerbitan sertifikat tanah. Sertifikat
menurut PP No 24/1997 adalah berupa satu lembar a. Kluster 1 yaitu bidang tanah yang
memenuhi syarat yang diterbitkan
sertifikat. i
b. Kluster 2 yaitu bidang tanah yang
hanya dicatat dalam buku tanah karena
sedang dalam keadaan sengketa atau
berperkara di Pengadilan c. 3.
Kepastian Hukum Penguasaan Tanah
melalui Pendaftaran Tanah Sistematis
Lengkap di Kota Batu Pendaftaran tanah
pertama kali secara sistematik yang di atur dalam
PP No 24/1997 jika terdapat pemindahan hak
atas tanah, maka kewajiban para pihak juga perlu
melampirkan bukti pelunasan pembayaran Bea
Perolehan Hak atas Tanah dan Bangunan (BPHTB)
dan PPh.21 Contoh dari hak perseorangan yaitu hak milik,
hak guna usaha, hak guna bangunan, hak pakai,
hak sewa, hak membuka tanah, hak memungut
hasil hutan, hak-hak lain yang diatur dalam Pasal
53 UUPA. Upaya pemerintah untuk memberikan
kepastian hukum terhadap penguasaan tanah
kepada individu yaitu melalui pendaftaran tanah
yang diatur dalam Peraturan Pemerintah Nomor 10
Tahun 1961 tentang Pendaftaran Tanah dan telah
diperbarui dengan PP NO 24/1997. Bagi tanah yang
belum melakukan pendaftaran tanah berdasarkan
aturan tersebut maka tanah tersebut akan menjadi
obyek pendaftaran tanah untuk pertama kali, baik
secara sistematik maupun sporadis. Ketika bukti pembayaran pajak peralihan
tersebut tidak dapat dilampirkan, maka sertifikat
hak atas tanah masih belum bisa diterbitkan. Namun, hal ini berbeda dengan pendaftaran tanah
melalui PTSL karena peserta PTSL yang masih
memiliki tunggakan pembayaran BPHTB dan PPh
maka masih tetap dapat diterbitkan sertifikat hak
atas tanah dengan catatan peserta PTSL membuat
surat pernyataan BPHTB dan/atau PPh terhutang.22
Apabila peserta PTSL tidak bersedia membuat surat
pernyataan terhutang BPHTB dan/atau PPh, maka
bidang tanah tersebut tidak dapat dibukukan dan
diterbitkan sertifikat dan masuk dalam kluster 3. Pendaftaran tanah secara sistematik lebih
diutamakan
karena
mempercepat
perolehan
data mengenai bidang-bidang tanah yang akan
didaftar daripada melalui pendaftaran tanah secara
sporadis.20 Implementasinya yaitu Program PTSL
yang merupakan program percepatan pendaftaran
tanah yang terlihat dari aturannya yaitu Permen
ATR/BPN No 6/2018 dan Instruksi Presiden Nomor
2 Tahun 2018 tentang Percepatan Pendaftaran Tanah
Sistematis Lengkap di Seluruh Wilayah Republik
Indonesia. Kedua aturan tersebut merupakan
landasan pelaksanaan PTSL di Indonesia. Perbedaan lain dapat ditemukan mengenai
jangka waktu pengumuman. Pengumuman yang
dilakukan oleh Panitia Ajudikasi jika berdasarkan
Pasal 26 PP No 24/1997 menyebutkan bahwa hasil MIMBAR HUKUM Volume 31, Nomor 3, Oktober 2019, Halaman 338-351 348 terdaftar belum terpetakan dalam Peta
Pendaftaran Aplikasi KKP. pengukuran diumumkan selama 30 (tiga puluh)
hari dalam pendaftaran tanah secara sistematik. Sedangkan pengumuman yang dilakukan dalam
PTSL diatur dalam Pasal 24 Permen ATR/BPN
No 6 Tahun 2018 yang menyebutkan bahwa
pengumuman hanya dilakukan selama 14 (empat
belas) hari kalender di Kantor Panitia Ajudikasi
PTSL dan Kantor Kepala Desa/Kelurahan. ,
g
p
)
26 Urip Santoso, 2010, Pendaftaran dan Peralihan Hak atas Tanah, Prenada Media Group, Jakarta, hlm. 248. 23 A.P Parlindungan, 1990, Konversi Hak-Hak Atas Tanah, Mandar Maju, Bandung, hlm. 5. p
25 Pasal 24 ayat (1) Peraturan Pemerintah Nomor 24 Tahun 1997 tentang Pendaftaran Tanah (Lembaran Negara Republik Indonesia Nomor 59
Tahun 1997, Tambahan Lembaran Negara Republik Indonesia Nomor 3696). p
,
,
,
,
25 Pasal 24 ayat (1) Peraturan Pemerintah Nomor 24 Tahun 1997 tentang Pendaftaran Tanah (Lembaran Negara Republik Indonesia Nomor 59
Tahun 1997, Tambahan Lembaran Negara Republik Indonesia Nomor 3696).
26 Urip Santoso, 2010, Pendaftaran dan Peralihan Hak atas Tanah, Prenada Media Group, Jakarta, hlm. 248. g
j
g
24 Urip Santoso, 2015, Perolehan Hak Atas Tanah, Kencana, Jakarta, hlm. 92. 24 Urip Santoso, 2015, Perolehan Hak Atas Tanah, Kencana, Jakarta, hlm. 92. p
25 Pasal 24 ayat (1) Peraturan Pemerintah Nomor 24 Tahun 1997 tentang Pendaftaran Tanah (Lem
Tahun 1997, Tambahan Lembaran Negara Republik Indonesia Nomor 3696). 3.
Kepastian Hukum Penguasaan Tanah
melalui Pendaftaran Tanah Sistematis
Lengkap di Kota Batu Kluster 3 yaitu bidang tanah yang
hanya didaftarkan dalam daftar tanah
karena subyek atau obyeknya belum
memenuhi syarat PTSL atau tidak
diketahui keberadaannya. y
d. Kluster 4 yaitu bidang tanah yang Ayu, Kepastian Hukum Penguasaan Tanah Melalui Pendaftaran Tanah Sistematis Lengkap 349 dokumen yang memuat informasi tentang data
yuridis dan data fisik yang diperlukan terhadap
suatu bidang tanah yang didaftarkan haknya. menemukan masalah. Permasalahan muncul ketika
sertifikat telah diterbitkan, maka terhadap sertifikat
tersebut masih ada kemungkinan untuk dibatalkan
karena ada bukti kepemilikan yang sah sebelumnya. Pembatalan sertifikat juga dapat melalui putusan
Pengadilan Negeri. Sertifikat tanah menghubungkan antara
kepastian hukum bidang tanah dan pemegang hak. Sifat sertifikat tanah yaitu alat bukti yang kuat, tetapi
hal yang ditegaskan dalam Pasal 19 ayat (2) PP No
24/1997 belum menjamin sepenuhnya kepastian
dan perlindungan hukum bagi pemegang hak atas
tanah. Hal ini disebabkan karena sistem pendaftaran
tanah yang dianut Indonesia adalah sistem stelsel
negatif bertendensi positif. Sehingga, segala yang
tercantum dalam buku tanah dan sertifikat hak atas
tanah berlaku sebagai tanda bukti yang kuat sampai
dapat dibuktikan suatu keadaan sebaliknya yang
tidak benar.27 Terhadap obyek PTSL yang telah diketahui
namun telah terjadi sengketa saat proses PTSL
berlangsung, maka akan penerbitan sertifikatnya
akan ditunda dan sengketanya diselesaikan sesuai
dengan kluster tanah tersebut. Ketika obyek
tersebut masih dalam jaminan perbankan, maka
akan dibuatkan Surat Pernyataan oleh Kantor
Pertanahan
dengan
ditandatangani
Pemohon,
bahwa ketika sertifikat tanah sudah diterbitkan
oleh Kantor Pertanahan Kota Batu akan diserahkan
kepada Bank terkait. Dalam hal ini perbankan tidak
perlu khawatir apabila obyek jaminannya akan
hilang karena program PTSL, melainkan bank akan
diuntungkan karena penerbitan sertifikat tanah akan
menambah nilai dari tanah tersebut. Berdasarkan penelitian ini, bahwa PTSL
memberikan kemudahan pada masyarakat untuk
memperoleh sertifikat tanah dan Pemerintah akan
memiliki peta tunggal terkait tanah di Indonesia. Selain itu, meskipun menyimpangi beberapa
ketentuan dalam PP No 24/1997, khususnya
berkaitan dengan penangguhan pembayaran pajak
peralihan (BPHTB dan/atau PPh) dan jangka waktu
pengumuman, kegiatan ini tetap memberikan
kepastian
hukum
pada
masyarakat
dalam
penguasaan tanah yang berasal dari kegiatan PTSL. Hal ini disebabkan karena kegiatan PTSL di Kota
Batu dilakukan sesuai dengan ketentuan dalam
Permen ATR/BPN No 6 Tahun 2018. Permasalahan-permasalahan
yang
terjadi
lebih kepada persoalan teknis pelaksanaan PTSL. Meskipun persyaratan dalam Program PTSL dapat
dikatakan sederhana, namun tetap saja ketelitian
Panitia Ajudikasi dalam pengecekan data yuridis
dan fisik harus ditingkatkan. 28 Arifin Bur, et al., “Sertifikat sebagai Alat Pembuktian yang Kuat dalam Hubungannya dengan Sistem Publikasi Pendaftaran Tanah”, VIR Law
Review, Vol. 01, No. 02, Oktober 2017, hlm. 127-136. 27 Adrian Sutedi, 2012, Sertifikat Hak Atas Tanah, Sinar Grafika, Jakarta, hlm. vi.ii Sutedi, 2012, Sertifikat Hak Atas Tanah, Sinar Grafika, Jakarta, hlm D.
Kesimpulan PTSL di Kota Batu khususnya di Desa
Oro-Oro Ombo dan Kelurahan Dadaprejo telah
dilaksanakan sesuai dengan Permen ATR/BPN No
6/2018. Hambatan yang ditemui hanya berkaitan
dengan yang bersifat teknis, seperti administrasi
yang belum lengkap, alat bukti kepemilikan tanah
masih dijaminkan, pemilik yang tidak ada saat
pengukuran, dan jumlah petugas ukur yang masih
sedikit. Terdapat perbedaan pada pelaksanaan PTSL
di beberapa ketentuan dalam PP No 24/1997, 3.
Kepastian Hukum Penguasaan Tanah
melalui Pendaftaran Tanah Sistematis
Lengkap di Kota Batu Obyek tanah yang
ikut dalam program PTSL di Kota Batu ketika
terdapat sengketa seharusnya penerbitannya dapat
ditunda terlebih dahulu hingga sengketa tersebut
selesai. Hal ini akan berkaitan dengan pemberian
jaminan kepastian hukum kepemilikan tanah yang
diwujudkan dengan penerbitan sertifikat. Meskipun
sertifikat merupakan alat bukti yang kuat dan dapat
dibatalkan ketika ada pihak yang menggugatnya,
namun ketika hal tersebut dapat dihindari, maka
masyarakat dapat memegang sertifikat tersebut
dengan tenang dan Pemerintah dapat mewujudkan
cita-citanya untuk memiliki peta tunggal. Kantor Pertanahan Kota Batu tidak ragu
untuk menerbitkan sertifikat dari hasil PTSL,
karena Indonesia menganut sistem publikasi
negatif yang mengandung unsur positif. Artinya,
semua keterangan yang terdapat dalam sertifikat
tersebut
mempunyai
kekuatan
pembuktian
yang kuat sepanjang tidak ada bukti lain yang
mengingkarinya.28 Ketika proses PTSL berlangsung
hingga diterbitkannya sertifikat, Panitia tidak MIMBAR HUKUM Volume 31, Nomor 3, Oktober 2019, Halaman 338-351 350 khususnya yang berkaitan dengan penangguhan
pembayaran pajak peralihan (BPHTB dan/atau
PPh) dan jangka waktu pengumuman. Walaupun
demikian, kegiatan ini tetap memberikan kepastian
hukum pada masyarakat dalam penguasaan tanah
yang berasal dari kegiatan PTSL khususnya di Kota
Batu. Hal ini disebabkan karena kegiatan PTSL
di Kota Batu dilakukan sesuai dengan ketentuan
dalam Permen ATR/BPN 6/2018. Selain itu, tujuan
utama dari pelaksanaan PTSL yaitu agar Pemerintah
memiliki peta tunggal terkait pertanahan di
Indonesia. D.
Internet Hutapea, Erwin, “Tak Selalu Berjalan Mulus,
Pendaftaran Tanah Terkendala Empat Hal”,
https://properti.kompas.com/read/2018/
12/17/183000721/tak-selalu-berjalan-
mulus-pendaftaran-tanah-terkendala-empat-
hal?page=all, diakses pada tanggal 10 April
2019. Suadi, Amran, et al., 2016, Politik Hukum:
Perspektif Hukum Perdata dan Pidana Islam
Serta Ekonomi Syariah, Kencana, Jakarta. Sumarja, FX, 2010, Hukum Pendaftaran Tanah,
Universitas Lampung, Bandar Lampung. Santoso, Urip, 2005, Hukum Agraria dan Hak-Hak
Atas Tanah, Kencana, Jakarta. Ihsanuddin, “Jokowi: Prona Sudah 35 Tahun,
baru 44 Persen Tanah Warga Bersertifikat”,
https://nasional.kompas.com/read/2016/
10/16/12474581/jokowi.prona.sudah.35. tahun.baru.44.persen.tanah.warga. bersertifikat, diakses 12 November 2018. Santoso, Urip, 2010, Pendaftaran dan Peralihan
Hak atas Tanah, Prenada Media Group,
Jakarta. Santoso, Urip, 2015, Perolehan Hak Atas Tanah,
Kencana, Jakarta. Mastur, Roy, “Bpn Beri 514 Sertifikat Gratis
Kota
Batu”,
http://www.rri.co.id/post/
berita/248262/daerah/bpn_beri_514_
sertifikat_gratis_kota_batu.html, diakses 17
juni 2017. C.
Artikel Majalah Badan Pusat Statistik Kota Batu, “Statistik Daerah
Kota Batu 2016”, Publikasi Statistik Daerah
Kota Batu 2016, September 2016. Mertokusumo, Sudikno, 2006, Hukum Acara
Perdata Indonesia, Liberty, Yogyakarta. Parlindungan, A.P, 1990, Konversi Hak-Hak Atas
Tanah, Mandar Maju, Bandung. Suteri, Adrian, 2012, Sertifikat Hak Atas Tanah,
Sinar Grafika, Jakarta. DAFTAR PUSTAKA C. Artikel Majalah
Badan Pusat Statistik Kota Batu, “Statistik Daerah
Kota Batu 2016”, Publikasi Statistik Daerah
Kota Batu 2016, September 2016. B.
Artikel Jurnal Bur, Arifin, et al., “Sertifikat sebagai Alat
Pembuktian yang Kuat dalam Hubungannya
dengan Sistem Publikasi Pendaftaran Tanah”,
VIR Law Review, Vol. 01, No. 02, Oktober
2017. E. Peraturan Perundang-undangan
Undang-Undang Nomor 5 Tahun 1960 tentang Ayu, Kepastian Hukum Penguasaan Tanah Melalui Pendaftaran Tanah Sistematis Lengkap 351 Peraturan Pemerintah Nomor 24 Tahun 1997
tentang
Pendaftaran
Tanah
(Lembaran
Negara Republik Indonesia Nomor 59 Tahun
1997, Tambahan Lembaran Negara Republik
Indonesia Nomor 3696). Peraturan Pemerintah Nomor 24 Tahun 1997
tentang
Pendaftaran
Tanah
(Lembaran
Negara Republik Indonesia Nomor 59 Tahun
1997, Tambahan Lembaran Negara Republik
Indonesia Nomor 3696). Peraturan Pemerintah Nomor 24 Tahun 1997
tentang
Pendaftaran
Tanah
(Lembaran
Negara Republik Indonesia Nomor 59 Tahun
1997, Tambahan Lembaran Negara Republik
Indonesia Nomor 3696). Peraturan Menteri Agraria dan Tata Ruang/Kepala
Badan
Pertanahan
Nasional
Republik
Indonesia Nomor 6 Tahun 2018 tentang
Pendaftaran
Tanah
Sistematis
Lengkap
(Berita Negara Republik Indonesia Tahun
2018 Nomor 501). Peraturan Menteri Negara Agraria Nomor 3 Tahun
1997 tentang Pelaksanaan PP 24 Tahun 1997
tentang Pendaftaran Tanah. Peraturan Menteri Negara Agraria Nomor 3 Tahun
1997 tentang Pelaksanaan PP 24 Tahun 1997
tentang Pendaftaran Tanah.
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Behavioural reactions of consumers to economic recession
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Verslas: teorija ir praktika
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Department of Economics, Faculty of Operation and Economics of Transport and Communications,
University of Žilina, Univerzitná 1,010 26, Žilina, Slovakia E-mails: 1katarina.valaskova@fpedas.uniza.sk (corresponding author); 2tomas.kliestik@fpedas.uniza.sk Received 05 August 2014; accepted 11 June 2015 Abstract. The negative effects caused by the economic recession of the recent year, led to the necessity to recognize the changes
in the consumer behaviour that may have a lasting impact, and they definitely will. The situation in the Slovak consumer market
is described and characterized by the survey which was conduced in 2013. The article also raises three hypotheses considering
the basic fact about consumers and their impact on the intensity of behavioural changes. In order to verify our results surveys
and studies carried out by many companies in various countries are analyzed in the last chapter. Keywords: consumer, consumer policy, behavioural changes, trends, consumer policy, recession. JEL Classification: C12, D11. Copyright © 2015 The Authors. Published by VGTU Press.
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 (CC BY-NC 4.0) license, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The material cannot be
used for commercial purposes.
To link to this article: http://dx.doi.org/10.3846/btp.2015.515 Verslas: Teorija ir praktika / Business: Theory and Practice
Issn 1648-0627 / eIssn 1822-4202
http://www.btp.vgtu.lt
2015 16(3): 290–303
doi:10.3846/btp.2015.515 Verslas: Teorija ir praktika / Business: Theory and Practice
Issn 1648-0627 / eIssn 1822-4202
http://www.btp.vgtu.lt
2015 16(3): 290–303
doi:10.3846/btp.2015.515 py gh
y
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 (CC BY-NC 4.0) license, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The material cannot be
used for commercial purposes.
To link to this article: http://dx.doi.org/10.3846/btp.2015.515 behavioural reactions of consumers to economic recession Katarína VALÁŠKOVÁ1, Tomáš KLIEŠTIK2 Department of Economics, Faculty of Operation and Economics of Transport and Communications,
University of Žilina, Univerzitná 1,010 26, Žilina, Slovakia
E-mails: 1katarina.valaskova@fpedas.uniza.sk (corresponding author); 2tomas.kliestik@fpedas.uniza.sk 1. Characteristics of consumer behaviour Consumer policy and the related consumer protection
are parts of the economic policy of every state. They can
significantly improve the functioning of the market by
providing the information about goods and services and
so help consumers make rational selection of the goods or
services (Hawkins et al. 1991). Consumer policy protects
consumers from the market imperfection due to which
consumers are in disadvantage compared to suppliers or
retailers of goods or services. In order to describe changes in behaviour of Slovak con
sumers we conducted a survey which was aimed to describe
the key changes in behaviour. The aim was not only to define
new trends in consumer behaviour but also to determine
the possible correlation between the selected descriptive va
riables of consumers. For the purposes of this paper, we set
three hypotheses and we searched the relationship between
changes in consumer behaviour and the age structure of
consumers, the length of crisis and disposable income. The
intensity of changes in consumer behaviour is classified into
five categories: very low intensity, low intensity, no changes,
high intensity and very high intensity of changes. Each of
them is characterized by different parameters. Very low in
tensity of changes is described by unstable consumption and
conscious savings. Postponing the purchase is typical of low
intensity of changes. At high level of intensity consumers
seek the shopping benefits and purchase mostly the private
label products. The last group is characterized by buying
the cheapest products in the cheapest business sales points. Introduction to introduce the European consumer policy of the
current period 2014–2020, 2. to introduce the European consumer policy of the
current period 2014–2020, changes in shopping behaviour of Slovak consumers. The
methods used are: logical and comparative analysis of litera
ture, analysis, synthesis, deduction, induction, description,
excerption and statistical hypothesis testing. 3. to present the results of the survey conducted in the
Slovak consumer market, 4. to verify the results of the survey by various studies
and searches realized in other countries to portray
the main changes in consumer shopping behaviour. Hypothesis 2 H0: There is no statistically significant dependence
between the changes in consumer buying behaviour and
the disposable income of consumers.hi H1: There is statistically significant dependence between
the changes in consumer buying behaviour and the dispo
sable income of consumers. Hypothesis 1 H0: There is no statistically significant dependence
between the changes in consumer buying behaviour and
the age structure of the population. H1: There is statistically significant dependence betwe
en the changes in consumer buying behaviour and the age
structure of the population. 1. Characteristics of consumer behaviour Consumer policy monitors and tries to specify objecti
ves, which are also considered to be the key activities: en
sure and provide the possibility of supplying food, housing,
energy and health services for the best prices; enhance the
freedom of consumer choice, improve market transparency
by providing necessary information on a given product;
protect consumers from adverse effects and health risks of
products and production processes; protect the economic
interests of consumers in the stores, in e-commerce, financi
al institutions, etc.; improve the legal position of consumers,
protect them from unfair or fraudulent business conditions;
ensure competitive supplying by public goods and services;
provide consumer information and advice on consumer
rights and responsibilities, sustainable consumption, nutri
tion, rational and energy-efficient household management;
improve consumers position in the market by providing
functional competitiveness on domestic and foreign mar
kets; defend and promote the interests of consumers in the
private sector as well as at all political levels; develop ins
titutional framework of a country consumer policy using
consumer information network and various other institutes
and improve coordination and cooperation between the
EU, consumer organizations and individual countries in
law implementing, monitoring and control (Steffens 2006). According to the Economic encyclopaedia (Šíbl 2002), con
sumer policy defines two main objectives. Firstly, it informs
consumers about goods and services, appropriate buying
decision can contribute to the functioning of competitive
markets and enable consumers to make positive selection. On the other hand, it protects consumer where their posi
tion on the market in not strong enough to allow to protect
themselves from those who have the power to detriment
them. A consumer is generally thought of as a person who
identifies a need or desire, makes a purchase and then dis
poses of the product during the three stages in the con
sumption process. A typical consumer’s utility is dependent
on the consumption of agricultural and industrial goods, Hypothesis 1 Introduction more) and on head-to-silver generation, people in the age of
45–54 (Higham 2010). Not only these consumer, but all of
them, are oriented on purchasing products which they need
and their purchasing behaviour has changed significantly. Consumption of luxury goods is very often postponed or
simply deferred. The economic shocks of the past years,
since 2008, have created greater requirements on retailers
and their suppliers to understand and respond to the new
marketplace realities, especially to the new trends in con
sumer behaviour. They are analysed in details in the fourth
chapter also with references. The economic cycle can have an important impact on con
sumption patterns, with expenditure usually rising during
the periods of economic upturn. When an economy is boo
ming, there is often an upturn in the consumption of luxury
goods and consumer durables. And vice versa, during the
periods of recession, disposable income is reduced and con
sumer confidence usually falls. A slowdown or contraction
in economic activity often results in fewer luxury purchases
being made, for example, lower sales of jewellery, holidays
abroad, or other big-ticket purchase, such as cars or furniture
(Eurostat European Commission 2009). The novelty of the study is that the article analyses a variety
of published surveys and determines new trends in consumer
shopping behaviour caused by the economic crisis which puts
and important foundation for further research. European consumers, as well as consumers of every
other market, are considered to be a driving force of the
market competitiveness, if they seek out the best offers. Indeed, consumer spending is often cited as a key driver
for growth and economic integration. Within the parti
cular market there is a huge amount of consumer. In the
middle 60th or 70th the population in Europe was quite
young, but it is not the same these days. The market is mos
tly focused on so-called silver generation (55 years old and The object of this article is changed shopping behaviour
of the European consumers. The main aim of the study is to
analyze the post- recession consumer behaviour. To achieve
the main aim, partial aims were set: 1. to analyse consumer behaviour and its characteristics
from a literature perspective, 1. to analyse consumer behaviour and its characteristics
from a literature perspective, Business: Theory and Practice, 2015, 16(3): 290–303 291 2. to introduce the European consumer policy of the
current period 2014–2020, 2. Hypothesis 3 On the other hand, it protects consumer where their posi
tion on the market in not strong enough to allow to protect
themselves from those who have the power to detriment
them. A consumer is generally thought of as a person who
identifies a need or desire, makes a purchase and then dis
poses of the product during the three stages in the con
sumption process. A typical consumer’s utility is dependent
on the consumption of agricultural and industrial goods, H0: There is no statistically significant dependence
between the changes in consumer buying behaviour and
the length of the economic recession.hi H1: There is statistically significant dependence between
the changes in consumer buying behaviour and the length
of the economic recession. Finding the relation between the selected descriptive va
riables is crucial to be able to recognize the most important K. Valášková, T. Klieštik. Behavioural reactions of consumers to economic recession 292 services, housing and wealth (Gregová 2009). Consumers
may be also organizations or groups in which one person
may make the decisions involved in purchasing products
that will be used by many, as when a purchasing agent or
ders the company’s office supplies. In other organizational
situations, purchase decisions may be made by a large group
of people (Solomon et al. 2006). a huge assortment of products was at the disposal
as well as a huge variety of stores. Every consumer
prefers something else, mostly the stated measures
and their components: supplying: product manipu
lation (transport, purchase in the group), optimal
rate of price and quality, extra services provided in
the store; professional assistance: professional servi
ces (trustworthiness), formal professionalism, huge
assortment in one product category, availability;
speed and comfort: speed (orientation, size, servi
ce), prompt availability, opening time, specific kinds
of assortment (fresh, pre prepared, spirits, sweets),
congenial atmosphere, low price sensibility; grocery
purchase: taste, price, smell, aesthetics, ingredients,
nutrition value, genetic modification, trademark,
producer, type of production, package, recyclability,
fashion, etc. (Zamazalová 2009). Consumers represent the bearers of consumption. No
one of them is the same, as everyone is influenced by diffe
rent factors which form the consumer behaviour. It can be
perceived at the macro and micro level. Hypothesis 3 The macro consu
mer behaviour is created by social issues but to reach the
factors of micro consumer behaviour, so called individual
factors, the following indicators have to be considered: cul
tural anthropology, history, demography, semiotic/ literary
criticism, macroeconomics, sociology, social psychology,
microeconomics, human ecology, developmental psycho
logy, clinical psychology and experimental psychology
(Solomon et al. 2006). The internationalization of the market structures ma
kes increasingly necessary for business people to acquire a
clear perspective and understanding of cultural differences
and similarities among consumers from various countries. Culture, education, purchasing power and other factors (e.g. labelling, information) considerably influence consumer
habits, and hence the percentage of income spent on pro
ducts and services. All social groups do not purchase the
same kind of products, the extent to which price affects
acceptance will vary according to the type of product and
the type of consumer motivation. Better knowledge of the
space behaviour of a customer enables the retailers to un
derstand the attractiveness of the sales point and plan the
future implantation of a new point of sale. That is why it
is of a vital importance for traders and manufacturers to
recognize all the factors, trends and conditions which affect
consumers in order to meet their needs (Howells, Weatherill
2005). Consumer behaviour is an important and constant de
cision-making process of searching, purchasing, using, eva
luating and disposing of products and services (Stašenková
2006). The economists nowadays regard the appropriate
consumer behaviour as one of the most important fields of
economy. People usually purchase those products and servi
ces which are of maximal utility. Samuleson and Nordhaus
(2000) claim consumers try to maximize their utility, satis
faction or joy by purchasing consumer goods. Taking the
lifestyle and its factors into consideration, consumers are
able to create some opinion and attitudes to particular pro
ducts. The combination of one´ s lifestyle, different factors
and attitudes affect the consumer decision- making process
as well as the consumer behaviour. Approaches defined in
the consumer behaviour can be divided into the following
three groups (Dzurová 2007): –– psychical approach: it highlights the relation betwe
en the psyche and behaviour of the consumer. The
consumer behaviour is possible to describe by the
consumer reactions to the external stimuli, so it
may be claimed that the essence of this behaviour
are experience and learning. Hypothesis 3 Consumer protection and consumer policy are also the
priorities of the European Union. Consumer policy is a part
of the internal market. If the market works smoothly, it cre
ates favourable conditions for entrepreneurship and com
petition. The single consumer policy brings many benefits,
but on the other hand, also some risks (more stringent and
effective market control). –– sociological approach: it is devoted to the reactions
of consumers in different situations or how the beha
viour is influenced by various social occasions. 2. European consumer policy –– enforcement: CPC Network (network of national en
forcement authorities) not used to its full potential;
low awareness about ECC-Net (network of European
Consumer Centres) among consumers and need to
strengthen its effectiveness. f
At the same time, the new Programme, with its four
priorities of Safety, Information and education, Rights and
redress, and Enforcement, needs to take into account new
societal challenges which have grown in importance in re
cent years. They include: the increased complexity of con
sumer decision-making, the need to move towards more
sustainable patterns of consumption, the opportunities
and threats that digitalisation brings, an increase in social
exclusion and the number of vulnerable consumers and an
ageing population.h The problems which should be tackled by consumer
policy are of wide range and so the accepted actions have
to be targeted on areas where intervention at the EU level
can make a difference and add value. The selected areas
for actions are actions corresponding to legal obligations
imposed by the Treaty and Member states (e.g. providing
and operating of RAPEX system), actions which are not or
could not be undertaken at national level because of their
EU-level character and actions complementing and enhan
cing the efficiency of measures undertaken at national level. The new Consumer Programme, with a budget of 175
million Euros for the period 2014–2020, will support the
general objective of future consumer policy placing the
empowered consumer at the centre of the Single Market. European consumer policy supports and complements na
tional policy by seeking to ensure that EU citizens can fully
feel the benefits of the Single Market and their safety and
economic interests are properly protected. fi
This Consumer Programme was designed only at EU
level. At the level of the Slovak Republic, as it was in the
case of the previous programming period 2007–2013, it
absent. Most of the European rules and directives are based
on the principle of minimum harmonization, which means
that they can be (but do not have to be) more stringent
at the level of individual countries. The Slovak Republic
implements consumer legislation in accordance with the
principle of minimum harmonization. Therefore we must
hope that sufficient attention will be given to the consumer
policy at the national level. 2. European consumer policy In the new period the main problems were addressed
and they can be grouped in the following categories:f –– safety: differences between Member States in enfor
cing product safety legislation, the presence of unsafe
products on the Single Market; risks linked to the
globalisation of the production chain; services safety;
lack of an adequate coordination structure at EU level
to ensure the best value of EU co-financing. i
–– information and education: lack of robust and good
quality data and analysis on the functioning of the
Single Market for consumers; insufficient capacity of
consumer organisations, including lack of resources
and expertise, notably in the new Member States; lack
of transparent, comparable, reliable and user-friendly
information for consumers, particularly for cross-
border cases; poor knowledge and understanding
of key consumer rights and protection measures by
consumers and retailers alike; inadequacy of current
EU consumer education tools, especially with regards
to the developments in the digital environment. –– information and education: lack of robust and good
quality data and analysis on the functioning of the
Single Market for consumers; insufficient capacity of
consumer organisations, including lack of resources
and expertise, notably in the new Member States; lack
of transparent, comparable, reliable and user-friendly
information for consumers, particularly for cross-
border cases; poor knowledge and understanding
of key consumer rights and protection measures by
consumers and retailers alike; inadequacy of current
EU consumer education tools, especially with regards
to the developments in the digital environment. 2. European consumer policy –– economic approach: it uses the basic knowledge of
micro economy, consumers define their require
ments and, on the other hand, companies present
the sources for satisfying the needs. Subsequently,
the consumer interests are confronted and traded
on the market. In the course of time, the behaviour
of consumer has changed excessively. In the pur
chasing process many criteria are considered by
consumers which were not in the past, as not such At the time when Europe needs new sources of growth,
consumer policy is the field which can significantly contri
bute to meet the Europe 2020 objectives. In Europe the
re are 500 million consumers and consumer expenditure
amounts to 56% of EU GDP. The more consumers are able
to make informed and rational decisions, the greater the
impact they can have on strengthening the Single Market
and stimulating growth. Empowered consumers that are Business: Theory and Practice, 2015, 16(3): 290–303 293 well-protected and able to benefit from the Single Market
can thus drive innovation and growth by demanding value,
quality and service (European Commission 2013b). The
businesses which respond will be the best-placed to cope
with the pressures of the global market. Empowerment is
not only a question of consumer rights but of building an
overall environment that enables consumers to make use
of those rights and benefit from them. It means building a
framework wherein consumers can rely on the basic pre
mise that safety is assured and that tools are in place to
detect failings in standards and practices and to address
them effectively across Europe. It means building an envi
ronment where consumers through education, information
and awareness know how to navigate the Single Market
to benefit from the best offers on products and services. Finally empowerment requires that consumers can con
fidently exercise their EU rights across Europe and that,
when something goes wrong, they can count both on the
effective enforcement of those rights and on easy access to
efficient redress (European Commission 2013a).h –– enforcement: CPC Network (network of national en
forcement authorities) not used to its full potential;
low awareness about ECC-Net (network of European
Consumer Centres) among consumers and need to
strengthen its effectiveness. –– enforcement: CPC Network (network of national en
forcement authorities) not used to its full potential;
low awareness about ECC-Net (network of European
Consumer Centres) among consumers and need to
strengthen its effectiveness. 2.1. Ways of consumer protection at the EU level The European Commission is trying by various measures
and rules to protect consumers not only in their Member
State but also out of their borders. In addition, for the free
movement of goods is essential to ensure that Member
States are properly informed about the situations in other
countries, both in terms of the products and provision of
adequate services even after the purchase. Two indices are
used to measure the level of consumer policy within the
European market – Consumer Condition Index (CCI) and
Market Performance Index (MPI). –– rights and redress: sub-optimal protection of consu
mer rights, in particular in cross-border situations;
need to further improve the integration of consumer
interests in EU policies; problems faced by consumers
when trying to secure redress, notably cross-border. –– rights and redress: sub-optimal protection of consu
mer rights, in particular in cross-border situations;
need to further improve the integration of consumer
interests in EU policies; problems faced by consumers
when trying to secure redress, notably cross-border. European Commission issues an annual statistic report,
based on political directions and regulations that assign
better understanding of consumer policy. It helps the po
licy makers make sure that the policy they adopt is in tune
with the expectations of consumers and identify priorities to K. Valášková, T. Klieštik. Behavioural reactions of consumers to economic recession 294 focus on in order to improve consumer conditions. Taking
the total consumption into consideration, the household
expenditures amounts to 56% of GDP in the EU (European
Commission 2013c), it is therefore necessary to focus on
improving consumer conditions which increase the eco
nomic growth. of overall Slovakian market performance since 2012, which
continues a downward trend observed from 2011 to 2012. In terms of goods markets as a whole, Slovakia experienced
a decline in performance in comparison to 2012, so that
Slovakian goods markets are currently 2.3 points below the
EU28 average and 1.1 points below 2012’s score. The average
MPI on services markets in Slovakia is relatively unchanged
since 2012 and it is currently close to that observed for the
EU28. When comparing goods markets to the EU28 avera
ge, Slovakia performs best in terms of fuel for vehicles, and
worst in the markets for clothing and footwear, meat and
meat products, fruit and vegetables, second-hand cars, and
non-prescription medicines. All of these markets have lower
component scores for comparability, trust and expectations. 2.1. Ways of consumer protection at the EU level Overall, the top three goods markets in Slovakia are the
books, magazines and newspapers, spectacles and lenses
and entertainment goods markets. The bottom three are the
second-hand cars, clothing and footwear and meat and meat
products markets. The non-prescription medicines market
is rated in 26th position across the EU28. The clothing and
footwear market is in 27th position. Looking at Slovakia’s
services markets since 2012, there has been an improvement
in the performance of mobile telephone services, and ve
hicle maintenance and repair. Overall, the top three services
markets in Slovakia are the personal care services, culture
and entertainment, and fixed telephone services markets. The bottom three are the investment products, private pen
sions and securities, real estate services, and maintenance
services markets (European Commission 2013b). If consumers feel to be a full member of the consumer
market, they can make right decisions based on perfect
information and appreciate the efficient and innovative
business, contribute to competitiveness stimulation and
economic growth. But on the other hand, markets in which
consumers are confused and deceived are mostly markets,
where there is low competitiveness and frequent clash of
consumers with some form of injury. Therefore it is neces
sary to identify which parts of the European market do not
work in favour of consumers. And that is the function of
the Consumer Markets Scoreboard. It does not only identify
the problem markets it also carries out in-depth market
research on sectors which seem to be inefficient. The main
aim is to recognize the problem easily and define possible
solutions. Most of the data comes from the annual marke
ting research which measures consumers’ experiences and
their perception of consumers´ conditions in 21 markets of
goods and 30 markets of services which amount to 60% of
all household expenditures (European Commission 2013c). Conditions in each market are valued on the basis of six cri
teria: comparability, trust, problems and complaints, overall
satisfaction, choice and switching. The research includes all
the Member States, Iceland and Norway. Evaluation of all
51 markets is based on MPI Index which takes into account
four aspects of consumer experience: p
The Consumer Conditions Scoreboard shows how the
single market is working for European consumers and
warns them of possible problems. 2.1. Ways of consumer protection at the EU level The Consumer Condition
Scoreboard takes into account data from all EU Member
States, Iceland and Norway and consists of 12 indicators
(together they form CCI index – Consumer Conditions
Index), which relate to consumer trust, economic and pro
duct safety enforcement, consumer complaints and redress. The total possible score achieved is 100, and its value varies
considerably depending on the individual Member States. CCI index is positively correlated with gross disposable
household income per capita in the Member States and
negatively correlated with the amount of the most econo
mically vulnerable consumers in the population. As the
countries with the best consumer conditions are conside
red Finland, Great Britain, the Netherlands, Luxembourg,
Ireland, Denmark, Austria, Sweden, Belgium, Germany and
Portugal (CCI is in all of them above the European average
55%). Consumer conditions seem to be the less favoura
ble in the Member States on the east and south part of the
Union, in Greece, Croatia, Bulgaria and Cyprus, where the
index reaches the lowest value. CCI index depends also on
the socio-demographic groups. In general, the value of the
index worsens the older the population, the lower levels of
education, the higher the number of unemployed people,
blue collars and those who do not have internet at home
(European Commission 2013c).h are ascertained by taking into consideration the evaluation
indices of the condition of the enterprise and various in
dices; they allow selecting the prevention measures out of
the possible leading set of internal and external measures. In the time of crisis consumers purchase products of diffe
rent prices and usually there are several reasons to decide
about the purchase. They refuse to buy at higher prices if
the product does not have qualitative advantage. So the role
of e-commerce has increased since the beginning of the
last recession. If consumers are loyal to a particular brand
name they are willing to wait to the price cut of these pro
ducts (Korčoková 2006). However, it is essential to follow
not only the consumer´ s perspective and their changes in
purchasing behaviour but it is also necessary to monitor
the development of business enterprises. The companies
on the market should realize that the situation will never
be as good as before, at the time before the recession, and
they have to accept changes in consumer behaviour if they
want to succeed in the market. 2.1. Ways of consumer protection at the EU level The data available in the
annual reports allow political leaders of both the EU and
the Member States to estimates the impacts of their poli
cies on consumer welfare and to compare the performance
of consumer policy over time. Consumer-oriented single
market contributes to economic growth and, moreover,
demand stimulation is essential to get the EU out of the
current economic crisis (European Commission 2013c). If
there are informed and assertive consumers in the market
who understand their rights and are sufficiently protected,
they improve not only their own welfare but also stimulate
competitiveness, innovation and integration of the internal
market. Consumer Conditions Scoreboard primarily looks
at the integration of the European retail market to see if con
sumers are willing to purchase anywhere in the EU with the
same level of confidence and protection as at the national le
vel. Secondly, it focuses on the quality of national consumer
conditions. Key indicators are related to the enforcement of
consumer and product safety legislation, the effectiveness of
redress as well as consumer awareness, empowerment and
information. The data are derived from a large-scale EU
wide research of consumers and retailers, data by Eurostat –– the ease of comparing goods and services on offer, –– consumers´ trust in retailers / suppliers to comply
with consumer protection rules, –– problems experienced and the degree to which they
have led to complaints, –– consumers´ satisfaction (the extent to which the mar
ket lives up to what consumers expect).h These components of the index are equally weighted
and total possible score achieved is on a scale from 0 to
100 which allows to compare the country markets easily. Consumers’ overall assessment of market performance im
proved slightly between 2012 and 2013 (by 0.3 points). This
continues the positive trend observed since 2010. While the
performance of goods markets has remained stable since
2012, services markets have improved by 0.5 point. As a
consequence the performance gap between the two market
groups has slightly narrowed. In general, it may be noted
that markets of goods are doing much better than markets
of services. Overall market performance in Slovakia is below the
EU28 average, with a difference of 0.9 points. There has
been a slight, non-significant, decline by 0.3 points in terms Business: Theory and Practice, 2015, 16(3): 290–303 295 and the information received by the Member States and the
European Consumers Centres. 3.1. Methodology of the research In 2009 the economic crisis fully hit the Slovak trade. The
commodity prices stagnated, consumer demand decrea
sed and in addition the behaviour of shoppers and overall
structure of the retail chains changed. Consumer sentiment
had never been worse on the Slovak market than in 2012. Relatively stable development of Slovak companies was
enacted on the background of turbulent changes in pur
chasing behaviour of the Slovak customers. As shown in
the studies of INCOMA GfK, in 2012 few short-term and
long-term trends were blended together. The strong impact
of the economic crisis, evident in the field of buying psycho
logy, influenced mainly the buying behaviour. Consumers´
doubts about the future meet the long-term behavioural
changes – with greater emphasis placed on quality than on
quantity of food and the overall reasonableness of purcha
sing decisions. Sales (in particular the ones communicated
directly at the store but also the leaflet ones) become one of
the strongest impulses for selection of products and stores. 2.1. Ways of consumer protection at the EU level The economic crisis has
significantly changed consumer behaviour: in the recent
research 72% of consumers claimed that their buying be
haviour changed as a result of the new economic environ
ment, only 7% did not register any changes (Kantar Retail
and... 2010). Consumers try to have minimum expenditures
and maximum savings. A number of tools, techniques and
programs can be used to achieve these goals, for instance
prepare a shopping list, plan meals, use coupons, vouchers
and compare prices in different stores. Here is the scope
for traders because they can build loyalty by knowing the
customers´ needs and adapt them which lead to an increase
in their own income. The CCI index in Slovakia reaches 57% and it can still
be described as good and consumer-oriented, although it
is only slightly above the EU average (55%). The Slovak
Republic has the second highest EU percentage of con
sumers´ correct answers to all questions relating to basic
consumer rights and Slovak consumers are considered the
second best informed about the guarantee validity right. Therefore, an increase in consumer confidence should
translate into higher rates of consumption in the future, le
ading to a possible rise in economic growth (Celik, Ozerkek
2009). On the contrary, awareness of retailers of prohibited
commercial practices is the third lowest in the EU and in
the period 2009–2012 decreased significantly the know
ledge of retailers about the legal period to have a defective
product repaired. Slovaks are the fourth most likely in the
EU to take action in case of any problems. The percentage of
consumers whose behaviour is affected by the information
from the media has gradually grown since 2009. The level
of consumer policy annually increases which shows that the
level of consumer policy improves. But there still is much
room for improvement compared to the best EU countries. 3. Research on behavioural changes
of Slovak consumers Consumers make purchases even in times of economic
recession. The size of the crisis, the crisis factors of activity In order to describe changes in behaviour of Slovak con
sumers we conducted a survey which was aimed to describe K. Valášková, T. Klieštik. Behavioural reactions of consumers to economic recession 296 the key changes in behaviour. The aim was not only to define
new trends in consumer behaviour but also to determine
the possible correlation between the selected descriptive
variables of consumers. contingency represent the categories of one attribute and
the columns of the other one. The field presents the number
of objects which belong simultaneously into both categories
(Luha 2006). Set of statistics consists of consumers and as every ci
tizen of a country is a kind of consumer, we focused the
survey on the age group of 15+. Statistical unit is a Slovak
consumer who is over 15 are realizes its shopping behaviour
in one of the Slovak stores. Statistic features is the shopping
activity in which the statistical units meet their needs, the
needs of their family members, friends and acquaintances. To obtain the necessary data the method of questionnaire
was used. The survey was conducted in the period from
1.9.2013 to 31.12.2013. To find the relevant sample size,
we used the formula for determining sample size (Creative
Research Systems 2013). According to the last census in
2011 (Statistical office... 2012) there are 5,397,036 inhabit
ants in Slovakia. If we subtract the number of those under
15, the total number of inhabitants is 4,570,520 inhabitants,
which are the potential consumers. Using the mentioned
formula we calculated the necessary sample size. The for
mula considers three key variables: confidence level, confi
dence interval (permissible margin of error) and population
size. The confidence level was determined at 95%, which
means that 95% of the survey results are valid for a given
population. Permissible margin of error was determined
at 5%. Necessary statistical sample size represents 384 res
pondents. As the questionnaire was conducted largely in
electronic form, we received 458 completed questionnaires. Table 1. 3. Research on behavioural changes
of Slovak consumers Contingency table of the hypotheses
Length of the economic recession
Intensity of
behavioural
changes
0–1
2–3
4–5
5 and
more
∑
Very low
5
22
19
36
82
Low
30
17
3
0
50
No
15
2
3
3
23
High
60
55
30
11
156
Very high
58
53
23
13
147
∑
168
149
78
63
458
Disposable income
Intensity of
behavioural
changes
< 250€
251–
500 €
501–
750 €
751–
1000 €
> 1000
€
∑
Very low
13
17
20
21
19
90
Low
42
3
1
0
0
46
No
0
0
4
0
16
20
High
50
53
38
6
0
147
Very high
59
54
24
14
4
155
∑
164
127
87
41
39
458
Age structure
Intensity of
behavioural
changes
15–30
31–45
46–60
61–75
over
75
∑
Very low
7
5
21
39
0
72
Low
4
28
45
0
0
77
No
34
30
0
0
0
64
High
6
33
27
7
14
87
Very high
26
36
35
24
37
158
∑
77
132
128
70
51
458 3.2. Statistical hypotheses testing The results of the statistical hypotheses testing
Measures
of ordinal
association
Hypothesis 1
Hypothesis 2
Hypothesis 3
Kendall’s
tau-b
0.018 598 196
–0.164 292 587
–0.170 542 548
95% CI
–0.062 472 089
to
0.099 668 481
–0.243 724 487 to
–0.084 860 688
–0.250 505 289
to
–0.090 579 807
Two-sided
P-value
0.652 969 854 0.000 050 358 567 0.00 002 912 024
Kendall’s
tau-c
0.017 984 497
–0.151 491 676
–0.164 400 628
95% CI
–0.060 410 649
to
0.096 379 643
–0.224 734 613 to
–0.07 824 874
–0.24 148 359
to
–0.087 317 666
Two-sided
P-value
0.652 969 854
0.000 050 358 567 0.00 002 912 024
Goodman-
Kruskal’s
gamma
0.023 405 872
–0.216 545 427
–0.231 515 629
95% CI
–0.078 621 268
to
0.125 433 013
–0.321 240 439
to
–0.111 850 414
–0.340 066 981
to
–0.122 964 277
Two-sided
P-value
0.652 969 854
5.03 586E-05
2.91 202E-05 disposable income and the length of the economic reces
sion. In the second hypothesis we also follow the P-value
of Kendall tau-b. The P-value is lower than the chosen
significance level (0.000 050 358 567 < 0.05), so we reject
the null hypothesis. Alternative hypothesis is therefore ac
cepted, and we can claim that the changes in consumer
buying behaviour and the disposable income have statisti
cally significant dependence, and the mutual correlation is
low. The changes in consumer behaviour are dependent on
the income of consumers. The last hypothesis, searching
the relation between the intensity of changes of consumer
buying behaviour and the length or the crisis, follows the
Kendall’s tau-c. The P-value is lower than the chosen signifi
cance level (0.00 002 912 024 < 0.05), so we reject the null
hypothesis. Alternative hypothesis is therefore accepted,
and we can claim that the changes in consumer buying
behaviour and the length of the crisis have statistically sig
nificant dependence, and the mutual correlation is again
low. The changes in consumer behaviour are dependent on
the length of the economic recession. To sum up, the longer the crisis, the more vigilant the
consumers. They focus strongly on the purchase of pro
ducts, whether food or non-food, on sale and discounts
and consistently compares prices. That’s why the huge
amount of consumers prefers the purchase of private label
products. 3.2. Statistical hypotheses testing Statistical hypothesis testing procedure is one of the most
important statistical inference. To accept or reject the hy
potheses it is appropriate to follow statistical hypothesis
testing methodology, which consists of the following steps: 1) Formulation of the null hypothesis (H0) 2) Formulation of the alternative hypothesis (H1), i 3) Determination of the significance level (α), 4) Calculate of the test statistics and P-value, We used the software program Microsoft Excel with its
complement Contingency Table (v. 2010).xlam for the most
relevant statistical tests used in the analysis of contingency
tables (Table 1). For the calculation of the test statistics and
of probability we used Kendall correlation coefficient which
is designed to measure the dependence between two ordinal
variables. If one variable takes a different number of unique
values than the other (contingency table m×n), we use the
tau-c, which is also called Stuart’s tau-c or Kendall-Stuart
tau-c (Table 2).fi 5) Decision about the acceptance or rejection of the
null hypothesis (Rimarčík 2007).h The null hypothesis (H0) is a statement that expresses
the independence of variables. The alternative hypothesis
(H1) is the logical opposite of the null hypothesis and it
explains the existence of dependence between variables. The
third step in testing statistical hypotheses is to determine
the level of significance. This is the probability of errors
which we accept if we reject the null hypothesis, which is
in fact true. Traditionally it is set at the level of 5% (or 1%). Subsequently, the statistical hypothesis is tested and on that
basis it can be decided about it acceptance or rejection. In
our case we examine two ordinal variables with qualita
tive attributes, so we use contingency tables. The rows of Kendall’s ordinal correlation coefficient allows to test
the significance of the tau coefficient, which we use in the
last step of statistical testing. If the P-value is lower than the
chosen significance level, the null hypothesis is rejected. Business: Theory and Practice, 2015, 16(3): 290–303 297 Table 2. 3.2. Statistical hypotheses testing Regarding the disposable income of consumers,
large purchases are made once a month, the number of
items per purchase in the long term decreases (conside
ring the consumers with the low level of income, below
500€). The average number of items per purchase is 30–40
items in a case of big purchase and up to 20 items of smal
ler one (Lehutová, Gregová 2012). Consumer purchases
thoughtfully, mostly with shopping lists to avoid impulsi
ve buying behaviour. Consumer purchases are planned to
be able to save some financial sources for the future. Based
on the survey three new types of consumer behaviour can
be defined: benefit-seeking shopping behaviour (typical
for age category from 15 to 45, with disposable income
at the level 501–750€ and if the recession has lasted for
more than 4 years), limited shopping behaviour (as for
the age category over 61, with the income lower than
500€ and in the first phase of crisis) and saving shopping
behaviour (it describes the age category 46 to 60, with the
highest income and if the length of the crisis is about three
years). Benefit-seeking shopping behaviour is defined by
consumers using sales, coupons, comparison of prices
and purchase of products at the place where they can
get the best price for best quality (e.g. standard price for
products of higher quality and low price for products of
normal quality). It can be assumed that this type of consu
mer behaviour persists even after the recession in a form
of some trend. Limited shopping behaviour is presented
by consumers who only buy necessary items, postpone This means that the difference between the coefficients cal
culated from the sample and the zero is too high to be only
the result of the selection, therefore it is statistically signi
ficant – there is the relationship between variables. If the
P-value equals to or is greater than the chosen significance
level, the null hypothesis cannot be rejected. This means that
the difference can be only the result of the selection, there
fore it is not statistically significant – there is no relationship
between variables. In 1988 Cohen created a simple tool for
the interpretation of the correlation coefficients: the corre
lation below 0.1 is considered as trivial, 0.1 to 0.3 small, 0.3
to 0.5 medium and large above 0.5 (Rimarčík 2007). 4. Recent studies on behavioural changes Various researches were carried out by many compa
nies to find the main changes in shopping behaviour of
consumers. For the purposes of this paper, the following
studies were analyzed: statistical data from Eurostat,
articles by Ang, Leong and Kotler (2000), Consumer
Behaviour Report (Rodrigues 2009), Sellsumers trend
watching report (Trend Briefings 2009), Understanding
the Post-recession consumer (Flatters, Willmott 2009),
Retail & Consumer Insight (Kantar Retail and... 2010),
McKinsey research (Bohlen et al. 2010), Survey of POPAI
CE Association (POPAI Central Europe 2011), Retail
Systems Research (Cunningham 2011), Post recession
Retailing (Raidoo et al. 2011), new trends that shape
consumer behaviour (Barkworth 2014), the research
on social effects of recession (Kay 2010), the study on
keeping customers in a recession (Latham, Braun 2010)
or study by Zurawicki and Braidot (2005). g
p
g
g
Traditional consumers were usually not involved in
production, defined as conformists and most often unin
formed. Their behaviour was largely motivated by a need
for comfort. Bohlen et al. in research done for McKinsey in
August 2009 found that, in any given category, an average of
18 percent of consumer-packaged-goods consumers bought
lower-priced brands in the past two years. Of the consumers
who switched to cheaper products, 46 percent said they
performed better than expected, and the large majority of
these consumers said the performance of such products
was much better than expected. As a result, 34 percent of
the switchers said they no longer preferred higher-priced
products, and an additional 41 percent said that while they
preferred the premium brand, it was not worth the money
(Bohlen et al. 2010). Most of them were realized in different countries at the
beginning of the economic recession as it was necessary to
describe the key changes in the consumer market. Since
then, no more relevant studies have been carried out (in
Slovakia no research was carried out on changes in con
sumer behaviour caused by recession) and the economy
is on the next wave of the economic downturn, the above
mentioned researches, surveys and reports can be consi
dered as crucial and relevant also in the current economic
conditions.hi As it may be seen in the Figure 1, if consumers perceive
enough value in a premium-brand product (Product A)
they will favour it over the product of a more basic brand
(Product B), despite the premium product’s higher price. 3.3. Results of the research Discussing the first hypothesis, we follow the P-value of
Kendall tau-b (as there is the same number of attributes
of both variables).The P-value is greater than the chosen
significance level (0.65> 0.05), so we accept the null hy
pothesis. Alternative hypothesis is therefore rejected, and
we can claim that the changes in consumer buying be
haviour and the age structure of the population have no
statistically significant dependence. It means that changes
in consumer behaviour are not dependent on the age of
the population and the economic recession has affected
the consumers of all age categories. More important is the
results of the other ordinal variables; the amount of the K. Valášková, T. Klieštik. Behavioural reactions of consumers to economic recession 298 in retail), in which it was found that the current buying
behaviour is characterized by the following variables: the
group of consumers older than 35 realizes more than 69%
of all consumer spending. Seven out of ten purchasing de
cisions happens in the shop, despite the fact that the con
sumer made a decision before entering the store, in 47%
is the behaviour changed due to various factors in the sto
re – communications, affordability, news, special offer, pro
duct placement, offer private brand products, etc. (POPAI
Central Europe 2011). The point of sale is, was and will be
an effective mean which may confirm, modify and change
buying behaviour and also provide a unique opportunity to
influence consumers´ behaviour. The question is whether
it is possible to work effectively with the point of sale and
communicate with customers so that they are willing to
buy products and listen to traders. The customer is willing
to devote 30 seconds to move down the aisle and choose
goods, which they want to buy. It means that in 30 seconds
they find the right place in the store, assess the relevance of
the offer, identify the brand and decide to purchase. During
this process the customer is willing to pay two seconds to
any advertisements so it is very important what and how
companies present and whether it is consistent with what
the customer wants. Understanding the consumer is the key
to success. The average purchase in the supermarket takes
about 30 minutes, a customer purchases 10 to 11 items on
average of 6–7 product categories (Stahlberg, Malta 2010). 3.3. Results of the research purchases, buy fewer things (the number of items per
purchase decreases), and in some cases the frequency
of purchases increases. This form of behaviour is typical
for the lower income consumers. Saving shopping beha
viour is typical for consumers who choose carefully and
responsibly the trader, product and the brand in advance. This behaviour is typical for consumers who prefer private
label products. Any change in social and economic situation chan
ges also the way of consumer behaviour, but especially
what and why consumers buy. Slovak consumers started
to purchase purposefully, economically and responsibly. They consider various possible aspects of the products,
their design, packaging, safety, cost, etc. Thus, consumers
have become cautious and their behaviour influenced
by the regime of savings in the time of economic reces
sion will certainly continue in the post- recession time. Demonstration of rational behaviour is that the consumer
with the given income and given prices purchases the
volume of goods, which taking their budget into account,
brings the maximum utility. 4. Recent studies on behavioural changes In a recession, though, consumers become less willing to
pay more and the preferences of some consumers begin to
shift from Product A to Product B (Bohlen et al. 2010). For
companies attempting to address the change in consumer
behaviour, understanding the economic theory can help to
inform decision making. Changes in the relationship betwe
en how much consumers are willing to pay, on the one hand, The facts are confirmed by one of the last surveys of
POPAI CE Association (Global Association for Marketing Business: Theory and Practice, 2015, 16(3): 290–303 299 and their perception of the value they are receiving, on the
other, supports behavioural changes. “sellsuming” – it means that the increased need for cash
forced them to sell their own extra space unwanted private
services or product. Not surprisingly, information is the for
ce that operated the consumers. Technology adoption and
the recession are two key factors that have likely impacted
consumer´ s definition of necessity (Rodrigues 2009). Due
to expansion of information technology and the Internet,
information is now cheaper and available to almost all con
sumers. It is no wonder, that more and more consumers are
buying online and online services and social media have
become the main source of information. Smart shopping
needs to be mentioned as many consumers more often look
for bargains. On the other hand, consumers are less likely
to visit entertainment centres, eat out, etc. and they incline
to stay at home and spend time there. It can be declared by the recent report by Retail Systems
Research (Cunningham 2011) listed the biggest changes in
purchasing behaviour retailers are facing nowadays. They
can be summarized as following: keeping up with evolving
consumers shopping patterns, getting consumers to enga
ge online, maintaining growth rates, managing the online
assortment, uncertain consumer demand being difficult to
anticipate and plan for, providing more ways for consumers
to connect to their brand, stemming cart abandonment and
balancing online growth against poor store sales. h The information found on the Eurostat proves that the
latest figures in the Central and Eastern Europe has im
proved and this region starts to recover from the global
financial crisis. Despite that, the consumer behaviour did
not change and consumers continue in the trends acquired
in the recession time. 4. Recent studies on behavioural changes As a consequence of all the economic changes, several
new trends in shopping behaviour were set in Retail&
Consumer Insight (Kantar Retail and... 2010). They conclu
ded that the economic recession has caused many changes
that led to a new type of consumer behaviour. The wasteful
and impulsive behaviour was replaced by the rational, res
ponsible and socially acceptable one. The research published in Consumer Behaviour Report
noted some changes in American consumer behaviour
due to changed economic conditions. The most significant
ones are to be named. Some consumers started to practise Fig. 1. Changes in consumer behaviour (Bohlen et al. 2010) K. Valášková, T. Klieštik. Behavioural reactions of consumers to economic recession K. Valášková, T. Klieštik. Behavioural reactions of consumers to economic recession 300 Fig. 2. Impact of recession on trends (Flatters, Willmott 2009) Fig. 2. Impact of recession on trends (Flatters, Willmott 2009) According to the research, which was by devoted by
Raidoo et al. (2011)1, consumers demand higher value. The
greatest challenge for traders is to understand consumer
trends and gain invaluable information for future planning. New market situation is characterized as the age of thrift. The mentioned research was conducted in South Africa
and it revealed that 69% of consumers is more cautious
about their spending than a year ago, while 58% say that
their financial situation is worse than in 2010. The trend
of more modest lifestyle of consumers can be claimed by
the fact that 55% of consumers purchase in cheaper stores
than before, while 68% of respondents state they consi
der several options before making a final decision about
purchase. However, consumers purchase less due to the
recession, but the frequency of purchases increases. More
frequent buying is typical for those consumers who pay less
to buy the same products than before and so they shift some
proportion of their consumption expenditure to savings. they want to be able to trust the company and its product
before purchase. Price is the easiest and relative cheep tool
to use in practice. Ang, Leong and Kotler determine in their
literature two different pricing strategies that can be used
in the recession. One of them is to maintain the quality
by charging normal prices to higher quality products. The
second strategy should help the company to maintain or
increase its market share by low prices for the products of
standard quality. Flatters and Willmott (2009) in their study proved that the
impact of the recession on consumer attitudes and trends is
critical. Some trends are advanced by the recession, while the
others are slowed or completely arrested. Figure 2 shows the
impact of the recession on trends. Dominant trends include:
the demand for simplicity, which indicates that consumers
seek uncomplicated and consumer-oriented products and
services that simplify their lives and focus on the pursuance of
a company (consumers are outraged by the unlawful conduct
and unethical company behaviour). Mercurial consumption
and discretionary thrift are trends that are driven by the reces
sion in a positive sense. Even those consumers are knowingly
thrifty who do not need to save. 1 Despite the fact that this research was conducted in South Africa, its
results confirm that consumers are more cautious in consumption,
purchase carefully and look for the most affordable products we quote
it in this paper. Fig. 1. Changes in consumer behaviour (Bohlen et al. 2010) Fig. 1. Changes in consumer behaviour (Bohlen et al. 2010) Fig. 1. Changes in consumer behaviour (Bohlen et al. 2010) K. Valášková, T. Klieštik. Behavioural reactions of consumers to economic recession Mercurial consumption is
characterized by easy access to information and shopping
without any disputes makes consumers more active and less
loyal. Arrested trends are formed by two trends, too. There are
ethical consumerism (altruistic consumption and expenditu
re, such as eating only cage free eggs or support any charity,
fall and consumers focus on solving their own life situation)
and extreme – experience seeking (expensive, frivolous and
risky recreational experience popular during the economic
growth is not so popular any more). The last group, slowed
trends, consist of green consumerism (consumers give up
expensive organic products and instead they limit waste che
aply and prudently) and decline of deference to institutions
and authorities. Ang, Leong and Kotler (2000) believe that the changes
that must happen at any financial crisis are focused on the
need of products of cheaper brands, price cuts and use of
information in the market. In the recession and in the post
recession period the most important business tools are pro
motion and products themselves. Private label products
combined with a well structured strategy based on lower
prices and aggressive promotion can help traders achieve
maximum profit. In the time of economic recession cons
tant communication with customers and various loyalty
programs play an important role. Communication must be
appropriate and thought-out to encourage the consumer to
purchase. People also prefer information about products Business: Theory and Practice, 2015, 16(3): 290–303 301 The Cultural Insight team (Barkworth 2014) conducted
a research to determine the 2014 global trends. Consumers
are increasingly expecting things to do more that involves
interacting with all their senses. It is no longer enough to
immerse the viewer in an experience, and people are rejec
ting the idea of passive on-looking, they are craving active
participation. One of the most important trends of the last
year is the era of the new industrial revolution. We are in
a new form of industrial revolution where technological
advances are enabling people to make the transition from
users to creators. Consumers expect more from every type
of experience. Due to the advanced technologies producers
and retailer are able to read consumers and give them what
they want. To sum up, the cultural shifts show a move to
wards the wholehearted and intense. People want all aspects
of their lives to be rich and full. Conclusions To sum up, changes that have occurred on the consumer
market – the use of new technologies, the creation of a
single European market and the economic recession–
have contributed to the changes in consumer behaviour. On the one hand, consumers have limited resources, but
on the other hand they can choose from a wide range of
products of many retailers. In the EU countries, a great
emphasis is given to the policy, programs and measures to
help consumers make right decisions. Consumer policy is
an important part of the EU policy. If the European market
wants to work properly and stimulate economic growth,
it needs informed and rational- behaved consumers who
believe traders and markets. Zurawicki and Braidot (2005) identify two types of
reactions among consumers; reactive and proactive. The
reactive reaction comprises the adaptation of the consump
tion. Consumers give up on or postpone some expenses that
are not necessary, they revaluate their own needs by chan
ging their priorities. Now the basic needs take the lead in
most of the consumers’ lives. The proactive reaction refers to
temporary measures such as the liquidation of assets, using
the savings, different types of credit or finding a second job
or, the extreme, taking in account illegal activities. Every change of the social or economic situation can
change the way the consumer purchases, bur especially
what do they buy and why. In the European countries
consumers´ purchases are more purposeful, economical
and responsible. This is confirmed by the results of both
primary and secondary research. In our survey we con
firmed that there is no statistically significant relationship
between the age of consumers and changes in consumer
behaviour, but there is a statistically significant relations
hip (small correlation coefficient) between the length of
recession and consumer behavioural changes as well as
between the disposable income and the intensity of chan
ges in consumer behaviour. Impulsive consumer beha
viour has been replaced by sophisticated, responsible and
socially acceptable, which is confirmed by the results of
surveys carried out in the world. The study published in 2019 in Journal of Business
Strategy (Latham, Braun 2010) reveals that changes in con
sumer behaviour happen very fast in times of recession. Also, they state that recessions do not affect people in the
same way. The effects can be direct and indirect or financial
and psychological. Conclusions The main reactions of the consumers
are that consumers defer major purchasing decisions, are
more price-sensitive, they can be resistant to the attempt
of the companies to reinstate pre-crisis prices and they can
temporary give up on buying from the usual place or person,
due to budget constraint. On the other hand, they evaluate all the possible aspects
of product, its design, package, safety, origin, price etc. Thus,
consumers have become more vigilant and their behaviour
influenced by thrifty regime in the time of recession will,
surely, continue in post-recession time. As their habit to
be accustomed to limited offers and simple demand will
continue, but they will ask only for greater utility. The main
difference is that the post-recession shopping behaviour will
more appropriately be characterized as purposeful rather
that panicked as it seemed in the recession. Recessions are certainly a crucial period for many peo
ple, requiring changes in habitual spending and consump
tion patterns (Kay 2010). They have changed consumers
not only economically but also mentally. They started to be
interested in their future, they do not want to spend money
on premium products any more. When shopping, consu
mers are switching to cheaper brand products and purchase
only what is necessary. That is the reason why the retailers
should start selling private label products. Especially hyper
and supermarkets, having a large number of products at
disposal, can offer cheaper alternatives of different brands
to their customers.hi K. Valášková, T. Klieštik. Behavioural reactions of consumers to economic recession These trends give us a clear
sense of where culture is heading and brands have to work
hard to meet consumers’ demands and expectations. behaviour. During the economic recession many changes
have made their presence, resulted in the new consumer
shopping behaviour. The one associated with wasteful, im
pulsive spending was replaced by thoughtful, responsible
spending which is and also will be more socially acceptable. References Ang, S. H.; Leong, S. M.; Kotler, P. 2000. The Asian apocalypse:
crisis marketing for consumers and businesses, Long Range
Planning 33(1): 97–119 [online], [cited 16 January 2014]. Available from Internet: http://www.sciencedirect.com/sci
ence/article/pii/S0024630199001004 Kay, M. J. 2010. Marketing during a recession: social effects
and marketing opportunities, Proceedings of the Northeast
Business & Economics Association: 587–589. Korčoková, M. 2006. Spotrebiteľská politika členských krajín
európskej únie a jej inštitucionálne zabezpečenie, Nová
ekoomika 5: 38–42. Barkworth, H. 2014. Six trends that will shape consumer
behaviour this year [online], [cited 16 February 2015]. Available from Internet: http://www.forbes.com/sites/on
marketing/2014/02/04/six-trends-that-will-shape-consumer-
behavior-this-year/ Latham, S. F.; Braun, M. R. 2010. Jilted? The manager´s little book
for keeping customers in a recession, Journal of Business Stra
tegy 31(1): 4–10. http://dx.doi.org/10.1108/02756661011012732 Lehutová, K.; Gregová, E. 2012. Impact of economic recession
on consumer behaviour, Journal on Law, Economy & Ma
nagement 2(1): 78–82. Bohlen, B.; Carlotti, S.; Mihas, L. 2009. How the recession has
changed US consumer behaviour [online], [cited 10 March
2012]. McKinsey&Company. Available from Internet:
https://www.mckinseyquarterly.com/How_the_recession_has_
changed_US_consumer_behavior_2477 Luha, J. 2006. Štatistické metódy analýzy kvantitatívnych zna
kov, Forum Statisticum Slovacum 2(2): 2–20. Celik, S.; Ozerkek, Y. 2009. Panel cointegration analysis of
consumer confidence and personal consumption in the
European Union, Journal of Business Economics and Ma
nagement 10(2): 161–168. http://dx.doi.org/10.3846/1611-1699.2009.10.161-168 POPAI Central Europe. 2011. Průvodce oborem marketing-at retail
2011 [online], [cited 10 February 2014]. POPAI Central Europe
Association. Available from Internet: http://www.popai.cz/1-
299/Pruvodce-oborem-marketing-at-retail-2011.aspx Raidoo, K. et al. 2011. Post-recession retailing [online], [cited 10
February 2014]. W&RSETA ILDP. Available from Internet:
http://www.wrseta.org.za/downloads/ILDP/Post_Recession_
Retailing_Master%20-%20Fast_Forward_ILDP_2011.pdf Creative Research Systems. 2013. Sample size calculator [online],
[cited 12 March 2013]. Available from Internet: http://www. surveysystem.com/sscalc.htm Rimarčík, M. 2007. Štatistika pre prax. Bratislava.ISBN 978-
80-969813-1-1 Cunningham, M. 2011. Adapting to the changes in online con
sumer behavior [online], [cited 10 February 2014]. Retail
Online Integration. Available from Internet: http://www. mytotalretail.com/article/adapting-changes-online-consumer-
behavior/1/ Rodrigues, S. 2009. Consumer behaviour report: economic trends
in consumer spending [online], [cited 10 March 2013]. Avai
lable from Internet: https://mr.pricegrabber.com/Economic_
Trends_in_Consumer_Spending_June_2009_CBR_1.pdf Dzurová, M. 2007. Spotrebiteľské teórie a reálie. Bratislava:
Vydavateľstvo Ekonóm, 164–175. Samuleson, P. A.; Nordhaus, W. D. 2000. Ekonómia 2. Bratislava:
Bradlo. 844 p. European Commission. 2013a. Programme consumers 2014–
2020. Luxembourg: Office for Official Publications of the
European Union, 13–52. Solomon, M. et al. 2006. Consumer behaviour: A European
perspective. England: Pearson Education Limited. 731 p. European Commission. 2013b. Consumer market scoreboard. Making markets work for consumers. Disclosure statement Mrs. Katarína Valášková and Mr. Tomáš Kieštik have no
relevant financial, nonfinancial or professional relations
hips to disclosure. The studies and researches mentioned above confirmed
the results of our survey about the changes in consumer K. Valášková, T. Klieštik. Behavioural reactions of consumers to economic recession 302 http://www.wpp.com/NR/rdonlyres/1BF0C6E8-C8AF-4B91-
93F2-0482526B6004/0/newconsumerbehaviorparadigm.pdf References Luxembourg: Office
for Official Publications of the European Union. Stahlberg, M.; Malta, V. 2010. Shopper marketing: How to in
crease purchase decision at the point of sale. London: Kogan
Page Limited. European Commission. 2013c. The consumer conditions sco
reboard consumers at home at the single market. Luxem
bourg: Office for Official Publications of the European
Union, 98–135. Statistical Office of the Slovak Republic. 2012. Základné údaje
zo sčítania obyvateľov, domov a bytov 2011: Obyvateľstvo
podľa veku a pohlavia. Bratislava: Statistical Office of the
Slovak Republic. 532 p. Eurostat European Commission. 2009. Consumers in Europe. Luxembourg: Office for Official Publications of the European
Communities. Stašenková, B. 2006. Prečo a ako vzdelávať spotrebiteľa. Bratis
lava: Metodicko-pedagogické centrum Bratislavského kraja. Steffens, H. 2006. Správanie spotrebiteľov a spotrebiteľská poli
tika. Bratislava: Vydavateľstvo Ekonóm. Flatters, P.; Willmott, M. 2009. Understanding the post recession
consumer, Harvard Business Review 87 (7/8): 64–72. Šíbl, D. 2002. Veľká ekonomická encyklopédia. Bratislava:
SPRINT. 967 p. Gregová, E. 2009. Mikroekonómia. Edis. 363 p. Hawkins, I.; Best, J.; Coney, A. 1991. Consumer behaviour. Im
plications for marketing strategy. USA: Irwin. Trend Briefings. 2009. Sellsumers: If saving is the new spending,
then selling is the new saving [online], [cited 10 February 2014]. Available from Internet: http://trendwatching.com/trends/
pdf/trendwatching%202009-04%20SELLSUMERS.pdf Higham, W. 2010. Trendológia The Next Big Thing: Ako rozpoznať
a predpokladať budúce trendy v správaní zákazníkov. Bra
tislava: Eastone Books, 12–18. Zamazalová, M. 2009. Marketing obchodní firmy. Praha: Grada
Publishing. 232 p. Howells, G.; Weatherill, S. 2005. Consumer protection law. UK:
Ashgate Publishing Limited. Zurawicki, L.; Braidot, N. 2005. Consumers during crisis:
responses from the middle class in Argentina, Journal of
Business Research 58: 1100–1109. http://dx.doi.org/10.1016/j.jbusres.2004.03.005 Kantar Retail and PricewaterhouseCoopers. 2009. The new
consumer behavior paradigm: permanent or fleeting? [on
line], [cited 10 February 2014]. Available from Internet: Business: Theory and Practice, 2015, 16(3): 290–303 303 Katarína VALÁŠKOVÁ, Ing. Phd., a university pedagogue at the Department of Economics, Faculty of Operation and Economics
of Transport and Communications, University of Žilina in Žilina, Slovakia. Scientific activities: more than 55 contributions and
presentations made at the conferences on national and international level. Research interests: consumer behaviour, fuzzy logic. Tomáš KLIEŠTIK, Prof. Ing. PhD., a professor at the Department of Economics, Faculty of Operation and Economics of Transport
and Communications, University of Žilina in Žilina, Slovakia. Scientific activities: scientific monograph, professional publications,
5 books, more than 160 national and international contributions and presentations. References Research interests: financial and investment
management and artificial intelligence such as fuzzy logic, artificial neural networks.
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Analysis of the 2020 Taal Volcano tephra fall deposits from crowdsourced information and field data
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Bulletin of volcanology
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Abstract After 43 years of dormancy, Taal Volcano violently erupted in January 2020 forming a towering eruption plume. The fall
deposits covered an area of 8605 km2, which includes Metro Manila of the National Capital Region of the Philippines. The
tephra fall caused damage to crops, traffic congestion, roof collapse, and changes in air quality in the affected areas. In a
tropical region where heavy rains are frequent, immediate collection of data is crucial in order to preserve the tephra fall
deposit record, which is readily washed away by surface water runoff and prevailing winds. Crowdsourcing, field surveys,
and laboratory analysis of the tephra fall deposits were conducted to document and characterize the tephra fall deposits of the
2020 Taal Volcano eruption and their impacts. Results show that the tephra fall deposit thins downwind exponentially with
a thickness half distance of about 1.40 km and 9.49 km for the proximal and distal exponential segments, respectively. The
total calculated volume of erupted fallout deposit is 0.057 km3, 0.042 km3, or 0.090 km3 using the exponential, power-law,
and Weibull models, respectively, and all translate to a VEI of 3. However, using a probabilistic approach (Weibull method)
with 90% confidence interval, the volume estimate is as high as 0.097 km3. With the addition of the base surge deposits
amounting to 0.019 km3, the volume translates to a VEI of 4, consistent with the classification for the observed height and
umbrella radius of the 2020 main eruption plume. VEI 4 is also consistent with the calculated median eruption plume height
of 17.8 km and sub-plinian classification based on combined analysis of isopleth and isopach data. Phreatomagmatic activity
originated from a vent located in Taal Volcano’s Main Crater Lake (MCL), which contained 42 million m3 of water. This
eruptive style is further supported by the characteristics of the ash grain components of the distal 12 January 2020 tephra fall
deposits, consisting dominantly of andesitic vitric fragments (83–90%). Other components of the fall deposits are lithic (7–
11%) and crystal (less than 6%) grains. Further textural and geochemical analysis of these tephra fall deposits contributes
to better understand the volcanic processes that occurred at Taal Volcano, one of the 16 Decade Volcanoes identified by the
International Association of Volcanology and Chemistry of the Earth’s Interior (IAVCEI) because of its destructive nature
and proximity to densely populated areas. Abstract The crowdsourcing initiative provided a significant portion of the data used for
this study while at the same time educating and empowering the community to build resilience. Keywords Taal 2020 · Crowdsourced · Tephra fall Keywords Taal 2020 · Crowdsourced · Tephra fall Keywords Taal 2020 · Crowdsourced · Tephra fall Editorial responsibility: M.H. Ort; Deputy Executive Editor: J. Tadeucci
A. M. F. Lagmay
aalagmay@up.edu.ph
1
UP National Institute of Geological Sciences, College
of Science, University of the Philippines, Diliman,
Quezon City, Metro Manila, Philippines
Extended author information available on the last page of the article. Editorial responsibility: M.H. Ort; Deputy Executive Editor: J. Tadeucci
A. M. F. Lagmay
aalagmay@up.edu.ph
1
UP National Institute of Geological Sciences, College
of Science, University of the Philippines, Diliman,
Quezon City, Metro Manila, Philippines
Extended author information available on the last page of the article. Analysis of the 2020 Taal Volcano tephra fall deposits
from crowdsourced information and field data M. I. R. Balangue-Tarriela1 · A. M. F. Lagmay1,2
· D. M. Sarmiento1 · J. Vasquez2 · M. C. Baldago1 · R. Yba˜nez1 ·
A. A. Yba˜nez2 · J. R. Trinidad1 · S. Thivet3 · L. Gurioli3 · B. Van Wyk de Vries3 · M. Aurelio1 · D. J. Rafael1 · A. Bermas2 ·
J. A. Escudero1 M. I. R. Balangue-Tarriela1 · A. M. F. Lagmay1,2
· D. M. Sarmiento1 · J. Vasquez2 · M. C. Baldago1 · R. Yba˜nez1 ·
A. A. Yba˜nez2 · J. R. Trinidad1 · S. Thivet3 · L. Gurioli3 · B. Van Wyk de Vries3 · M. Aurelio1 · D. J. Rafael1 · A. Bermas2 ·
J. A. Escudero1 Received: 13 September 2021 / Accepted: 14 January 2022
© The Author(s) 2022
/ Published online: 2 March 2022 https://doi.org/10.1007/s00445-022-01534-y
Bulletin of Volcanology (2022) 84: 35 https://doi.org/10.1007/s00445-022-01534-y
Bulletin of Volcanology (2022) 84: 35 RESEARCH ARTICLE Extended author information available on the last page of the article. 1
UP National Institute of Geological Sciences, College
of Science, University of the Philippines, Diliman,
Quezon City, Metro Manila, Philippines Ground and satellite observations Photos taken by residents around Taal Lake, weekend
tourists, and passengers of commercial airplanes were
reviewed to determine the type of eruption that took place
on 12–13 January. Most of these photographs are available
from the Internet but some were provided directly to the
authors. Time-lapse videos taken by the authors on 13
January from Tagaytay, north of TVI, were also reviewed. Many local tourists and residents around Taal Lake
witnessed the explosive eruption as the event happened
on a weekend. As news of the eruption spread, some
authors of this paper rushed towards TVI to document the
event through photographs and videos, and by sampling
tephra for further analysis. The lead authors from the
University of the Philippines (UP) have research and
public service mandates (Congress of the Philippines 2020). Some are also researchers of the UP Resilience Institute,
whose mission is to empower local communities through
multidisciplinary actions toward resilience. As such, an
independent quick response team was formed to solicit
crowdsourced information from the public through social
media outlets. Residents affected by the tephra fall, known
as ashfall to the layman, were requested through social
media to document and collect samples before they were
washed out by rain or swept away. In the weeks following
the main eruption of Taal Volcano, the team conducted
fieldwork to measure tephra fall thickness and collect
samples in different locations where tephra falls were
reported to have been deposited. The eruption plume was directly observed and pho-
tographed by some of the authors from a distance of about
22 km in the late afternoon of 12 January. The following
day, the eruption was observed from the northern ridge of
Taal Caldera. Time-lapse videos of the eruption were taken
during the observation periods (see Supplementary Files). Himawari satellite images (Bachmeier 2020) were
used to measure the umbrella cloud radius. These were
compared with estimates of the eruption cloud height
of the cataclysmic event on 12 January determined
by the Pacific Tropical Cyclone Center (PTCC) (Perttu
et al. 2020; PTCC 2020) and the Cooperative Institute
for Meteorological Satellite Studies (CIMSS) (Bachmeier
2020) and used to determine the Volcanic Explosivity Index
(VEI) (Constantinescu et al. 2021). Introduction According to the Philippine
Institute of Volcanology and Seismology (PHIVOLCS),
the agency mandated to mitigate disasters that may arise
from volcanic eruptions, progression from phreatic to a
phreatomagmatic eruption took place at about 5:00 p.m.,
when magma came into contact with groundwater or lake
water from the Main Crater Lake (MCL) of Taal Volcano
Island (TVI) (PHIVOLCS 2020a). The tall volcanic plume
and large associated umbrella cloud of the main eruption
resulted in tephra (ash) fall to the north–northeast as far
as the capital city of Manila, 65 km from the active vent. By 2:49 a.m. of the following day (13 January 2020), the
activity transitioned to lava fountaining. The change in
eruptive style was the start of waning activity, characterized
by discrete cannon-like explosions that generated 2-km-
high bent-over plumes during the rest of the second day
and eruption columns of various heights less than 1 km that
lasted until 22 January 2020. Steam-laden plumes persisted
in the next few weeks with decreasing intensity. of volcanic hazards phenomena. As one of the 16 Decade
Volcanoes identified by the International Association
of Volcanology and Chemistry of the Earth’s Interior
(IAVCEI), this work is of significant value, most notably in
light of Taal Volcano’s destructive nature and proximity to
densely populated areas (Torres et al. 1995), including the
country’s National Capital Region, a metropolis inhabited
by more than 13 million people (PSA 2021). Methodology To characterize Taal Volcano’s 2020 eruption through its
tephra deposits, ground observations, satellite remote sens-
ing, crowdsourcing, field validation, numerical simulations,
and laboratory analysis were performed. Each of these
methods are described below. Introduction Editorial responsibility: M.H. Ort; Deputy Executive Editor: J. Tadeucci After 43 years of repose, Taal Volcano violently erupted
on 12 January 2020, forming a plume that reached 17–
21 km above sea level (PTCC 2020; Perttu et al. 2020;
Bachmeier 2020). The eruption started at around 1 p.m. (all
times described in this manuscript are local times, which
correspond to UTC+8) as a series of phreatic explosions
involving heated ground water or lake water. The volcano
Alert Level was raised to 2 at 2:30 p.m. because of the
escalating volcanic activity. It was raised further on the Extended author information available on the last page of the article. 35 Page 2 of 22 Bull Volcanol (2022) 84: 35 same day to Alert Level 3 by 4:00 p.m. and then to Alert
Level 4 by 7:30 p.m. Alert Level 5, the highest category
on a scale of 0–5, which means that a hazardous eruption
is ongoing, was not raised. According to the Philippine
Institute of Volcanology and Seismology (PHIVOLCS),
the agency mandated to mitigate disasters that may arise
from volcanic eruptions, progression from phreatic to a
phreatomagmatic eruption took place at about 5:00 p.m.,
when magma came into contact with groundwater or lake
water from the Main Crater Lake (MCL) of Taal Volcano
Island (TVI) (PHIVOLCS 2020a). The tall volcanic plume
and large associated umbrella cloud of the main eruption
resulted in tephra (ash) fall to the north–northeast as far
as the capital city of Manila, 65 km from the active vent. By 2:49 a.m. of the following day (13 January 2020), the
activity transitioned to lava fountaining. The change in
eruptive style was the start of waning activity, characterized
by discrete cannon-like explosions that generated 2-km-
high bent-over plumes during the rest of the second day
and eruption columns of various heights less than 1 km that
lasted until 22 January 2020. Steam-laden plumes persisted
in the next few weeks with decreasing intensity. same day to Alert Level 3 by 4:00 p.m. and then to Alert
Level 4 by 7:30 p.m. Alert Level 5, the highest category
on a scale of 0–5, which means that a hazardous eruption
is ongoing, was not raised. Crowdsourced accounts and field observations This paper describes the collective work done to
document details of the eruption sequence and impacts of
tephra fall of the 2020 Taal Volcano eruption. A significant
amount of data comprising this paper is owed to the
efforts of the general public who immediately reported their
observations and contributed tephra samples around Taal
Volcano. It was a unique and significant opportunity to
engage the public in citizen science, with the crowdsourcing
initiative providing the authors with invaluable data while
raising awareness through engagement in the observation Recognizing that the Philippines remains the leader in social
media and Internet usage worldwide (Baclig 2020) and
the fact that tephra fall deposits are easily removed by
water erosion in tropical regions, an interactive Google Map
was created on the morning of 13 January (Fig. 1a) for
“netizens,” or citizens who are highly active on social media
and other online platforms, to quickly report the pin location
and estimated thickness of ashfall deposits in their area. The Bull Volcanol (2022) 84: 35 Page 3 of 22 35 Page 3 of 22 35 30 mm; and dark red, 30–100 mm. No report for ashfall with thickness
of 0.03–0.30 mm was received through the Google Map platform. (b)
Map showing the location of measurement and sampling points during
the field validation Fig. 1 (a) Interactive Google Map showing the locations of ashfall
occurrences as reported by netizens. Each pin corresponds to an ashfall
thickness in that site: dark green, 0.01 to 0.03 mm; light green, 0.30 to
1.00 mm; yellow, 1.00 to 3.00 mm; orange, 3 to 10 mm; light red, 10 to
30 mm; and dark red, 30–100 mm. No report for ashfall with thickness
of 0.03–0.30 mm was received through the Google Map platform. (b)
Map showing the location of measurement and sampling points during
the field validation Fig. 1 (a) Interactive Google Map showing the locations of ashfall
occurrences as reported by netizens. Each pin corresponds to an ashfall
thickness in that site: dark green, 0.01 to 0.03 mm; light green, 0.30 to
1.00 mm; yellow, 1.00 to 3.00 mm; orange, 3 to 10 mm; light red, 10 to
30 m
of 0
Map
the f 30 mm; and dark red, 30–100 mm. No report for ashfall with thickness
of 0.03–0.30 mm was received through the Google Map platform. Power-law model The power-law model is another fitting method that
describes the relation between thickness, T, as a function of
root isopach area, x, as follows (Eq. 2): T (x) = cx−m
(2) (2) T (x) = cx−m where c represents a linear scaling factor and m,
characterizes the rate of decrease in tephra deposit thickness
(Bonadonna et al. 1998; Bonadonna and Houghton 2005). Similar to the exponential model, this equation is linearized
by taking the logarithm of both sides of the equation and
least squares regression is applied to get the values for
parameters, c and m. The main disadvantage of using the
power-law model is that T(x) is not integrable between 0 and
∞. Thus, the proximal and distal limits of integration have
to be selected (e.g., Bonadonna and Costa 2012) (Daggitt
et al. 2014). Eruption parameter calculations TephraFits was used to model the Taal Volcano’s 2020
main eruptive episode based on the crowdsourced and
surveyed tephra fall data. It is a Matlab function, a collection
of codes dedicated to the characterization of tephra fall
deposits such as AshCalc (Daggitt et al. 2014) and TError
(Biass et al. 2014). The TephraFits program enables the
user to quickly compute eruption parameters including
the deposit volume and mass, VEI, magnitude, and rates
of thickness-decay away from the source and allows for
better eruption classification using deposit-based schemes
(Biass et al. 2019). Outputs generated by the TephraFits
algorithm follow three well-known and frequently used
fitting methods, which are the exponential, power-law, and
Weibull integration models. Weibull model Of the three fitting methods, the Weibull model was
proposed more recently as it combines the advantage of the
exponential model being integrable between 0 and ∞and
the power-law model’s use of variable rates of decrease in
deposit thickness (Bonadonna and Costa 2012). The model
describes the relationship between thickness, T, and root
isopach area, x, as follows (Eq. 2): T (x) = θ(x
λ)k−2e( x
λ )k
(3) (3) Crowdsourced accounts and field observations (b)
Map showing the location of measurement and sampling points during
the field validation Fig. 1 (a) Interactive Google Map showing the locations of ashfall
occurrences as reported by netizens. Each pin corresponds to an ashfall
thickness in that site: dark green, 0.01 to 0.03 mm; light green, 0.30 to
1.00 mm; yellow, 1.00 to 3.00 mm; orange, 3 to 10 mm; light red, 10 to The calls for crowdsourced data were retweeted by a
media outfit, a science organization (AGHAM, Advocates
of Science and Technology for the People) as well as local
and international scientists with one post garnering a total of
372,003 impressions, 86,206 engagements, 1954 likes, and
1436 retweets. The interactive Google Map reached over
3000 views and received 247 unique submissions. interactive map also allowed attachment of photos to help
visualize the ashfall deposit thickness. Social media posts
on Facebook and Twitter containing reports of ashfall were
integrated into the Google Map. The advantage of using
Google Maps is that contributors could visualize ashfall
reports and how their individual pins shaped the data and
the map. All reports were vetted to assess the quality of thickness
measurements and only those with associated photographs
were considered. Of the 247 reports, 167 thickness
measurements were deemed accurate. Contour lines were
drawn on points of similar tephra fall deposit thicknesses to
construct a GIS isopach map. The use of hashtags such as #TaalVolcano, #iTalaAng-
TaalAsh (translation: #RecordTheTaalAsh), and #TaalAsh-
fall were particularly useful in consolidating the posts,
tweets, and news regarding the event. Informative discus-
sions on the on-going state of Taal Volcano and its impli-
cations were formed in Twitter and Facebook, engaging
everyone to participate and spread awareness. In addition,
the hashtags were used to pinpoint the location of severely
affected areas needing donations, relief efforts, and rescue. A series of field surveys were conducted by the Quick
Response Team of the UP National Institute of Geologi-
cal Sciences and UP Resilience Institute in the following
municipalities: (1) Agoncillo, Batangas; (2) Calaca, Batan-
gas; (3) Laurel, Batangas; (4) Lemery, Batangas; (5) San
Nicolas, Batangas; (6) Taal, Batangas; (7) Talisay, Batan-
gas; (8) Tanauan, Batangas; (9) Silang, Cavite; and (10) The authors also focused on public communication,
leading forums, and producing infographics about the
eruption. The discussions on the original posts in social
media lasted for over a week on Twitter and Facebook. Crowdsourced accounts and field observations 35 Page 4 of 22 Bull Volcanol (2022) 84: 35 Tagaytay City, Cavite. Observation and measurement points
were chosen to show optimal preservation of the deposits
(Fig. 1b). Tephra fall thicknesses were most reliable when
measured on house and car rooftops, bridge columns and
railings, road and fence ledges, fallen trees, and tombs
in cemeteries. The total lockdown in some places due to
the eruption significantly reduced anthropogenic activity,
reducing reworking of the tephra and preserved the tephra
fall deposits in many areas for weeks. Tephra samples were
collected and stored in resealable plastic bags with several
samples solicited from colleagues residing in towns where
tephra fall occurred. Exponential model The exponential model is an integration strategy that
describes the relationship between the deposit thickness, T,
as a function of the root isopach area, x as follows (Eq. 1): The additional parameter, λ, allows the Weibull model to
capture variation in the deposit’s thinning rate, which in the
exponential model necessitates multiple segments (Daggitt
et al. 2014). (1) T (x) = ce−mx For the exponential, power-law, and Weibull fitting,
the implementation strategies recommended by Biass
et al. (2019) were used in this study. A 10% error was
applied to all thickness and diameter measurements and
a 20% error on the distal limit (C). Several iterative
runs were conducted while observing strict statistical
measures and ensuring critical interpretation that would
match geologically realistic values. For example, the θ
output for the Weibull Fit was ensured to agree with the
thickness (∼1 m) and maximum grain size values (∼11
cm) of tephra fall deposits observed near the crater rim
where there were no identified base surge deposits (Lagmay
et al. 2021). Values were compared to tephra fall deposits
from other volcanoes in the global dataset to ensure they
were realistic (Bonadonna and Costa 2013; Daggitt et al. 2014; Biass et al. 2019). Further sensitivity analyses were
performed for output values, especially for the distal limits where c represents the theoretical maximum thickness
located at the vent and m describes the rate of decrease in
tephra deposit thickness (Pyle 1989; Biass et al. 2019). This
equation is linearized by taking the logarithm of both sides
of the equation and least squares regression is applied to get
the values for parameters, c and m (Daggitt et al. 2014). Multiple exponential segments are applied to the data
so that the exponential law allows for modelling deposits
of varying thinning rates with distance away from the
vent as displayed by many well-constrained tephra fall
deposits (Pyle 1989; 1990; Fierstein and Nathenson 1992;
Bonadonna and Houghton 2005; Watt et al. 2009). This
integration approach is effective for simple approximations
such as determining the minimum volume of a deposit given
sparse data points (e.g., Pyle 1995;
1999; Legros 2000;
Sulpizio 2005) (Daggitt et al. 2014). Bull Volcanol (2022) 84: 35 Page 5 of 22 35 Page 5 of 22 35 Duration of peak activity where thickness values are harder to constrain (sub mm)
and preservation is short-lived. A list of the final selected
input parameters used for the results presented in this work
is shown in Table 1. where thickness values are harder to constrain (sub mm)
and preservation is short-lived. A list of the final selected
input parameters used for the results presented in this work
is shown in Table 1. The duration of peak activity was determined from the
review of proximal seismic data recorded by a station north
of TVI in Dasmarinas, Cavite. Data were retrieved from the
International Federation of Digital Seismograph Networks
(FDSN) (Fig. 2), which provides open access to seismic data
from the Philippines. The seismic records came from the
nearest station to Taal, which is part of a growing network
of low-cost but professional seismometers (Anthony et al. 2018; Bent et al. 2018; Holmgren and Werner 2021; Subedi
et al. 2020) in the country, owned and operated by private
entities and citizen scientists (Aurelio et al. 2020a; 2020b). The estimate of the duration of peak activity was compared
with the accounts of eruptive activity in official bulletins of
PHIVOLCS (Table 2), reports from the Global Volcanism
Program, news articles, social media posts, and airlines and
ground observations by the authors. Furthermore, this study used the Weibull method to
estimate the plume height according to equation 7 of
Bonadonna and Costa (2013). The modeled eruption height
values were then compared with those measured from satel-
lite data (Perttu et al. 2020; Bachmeier 2020). Magnitude-
related parameters such as thickness half-distance, bt and
λT H, as well as intensity-related parameters, in particular
thickness half-clast, bc and λML (Biass et al. 2014), were
used to classify the 2020 Taal eruption according to the
schemes of Pyle (1989) and Bonadonna and Costa (2013). Limitations Circumspection on the use of TephraFits is hereby noted. Our calculations are based on crowdsourced and field
survey data, which are relatively scarce point observations
of the entire tephra fall deposit. More thickness and
maximum grain-size measurements could have been added,
but lockdown restrictions due to the raised alert level for
Taal Volcano and later for COVID-19 prevented further
collection of data. Most of the crowdsourced data were
visually estimated from the photos submitted whereas field
surveys were delayed by 1–2 weeks. Erosion by wind and
surface water runoff may have reduced the thickness of the
fall deposits. Furthermore, the data points were subjectively
contoured to generate isopach and isopleth maps used to
constrain transport and depositional processes. As such,
many sources of uncertainties interacted and propagated to
the final results presented in this work (Biass et al. 2019). Nonetheless, these caveats guided us in the critical use
and interpretation of the models, ensuring that they reflect
geological reality and direct observations of Taal Volcano’s
main eruption. Laboratory analysis Samples used for grain-size analysis conducted at the UP
National Institute of Geological Sciences (UP NIGS) came
from those collected during fieldwork and those solicited
from colleagues (see Fig. 1b for locations). For grain size
analysis, the samples were oven dried for 6–8 h and reduced
to 100 g using coning and quartering to eliminate systematic
biases. The maximum grain sizes of predominantly equant
tephra grains utilized in the isopleth maps came from
twenty samples that were handpicked and measured using
a micrometer. Manual sieving was then carried out on three
samples using the No. 18 (1.00 mm, 0φ), No. 35 (0.50
mm, 1φ), No. 60 (0.25 mm, 2φ), No. 120 (0.125 mm,
3φ), and No. 230 (0.063 mm, 4φ) test sieves of the U.S.A. Standard Test Sieves ASTM E-11-20 (International ASTM
2014) to determine the size frequency distribution. Using an
analytical balance, size fractions were weighed, and those Table 1 List of input parameters used to calculate volume, VEI, eruption height, and eruption magnitude based on tephra fall thickness and grain
size parameters of tephra fall samples
Isopach
Isopleth
Exponential
Power-law
Weibull
Exponential
Power-law
Weibull
Isopachs used (cm)
45, 10, 1.5, 0.5, 0.3, 0.10, 0.05
—
Isopleths used (cm)
—
1.65, 0.325, 0.13, 0.075
Break in slope (BIS)
4
—
—
2
—
—
Distal limit (C)
—
130
—
—
—
—
λ range
—
—
5–20
—
—
2–10
n range
—
—
0.2–20
—
—
0.5–2
A 10% error was applied to all thickness and diameter measurements and a 20% error on distal limit (C). Values of λ and n are within the ranges
recommended by Bonadonna and Costa (2013) Table 1 List of input parameters used to calculate volume, VEI, eruption height, and eruption magnitude based on tephra fall thickness and grain
size parameters of tephra fall samples
Isopach
Isopleth
Exponential
Power-law
Weibull
Exponential
Power-law
Weibull
Isopachs used (cm)
45, 10, 1.5, 0.5, 0.3, 0.10, 0.05
—
Isopleths used (cm)
—
1.65, 0.325, 0.13, 0.075
Break in slope (BIS)
4
—
—
2
—
—
Distal limit (C)
—
130
—
—
—
—
λ range
—
—
5–20
—
—
2–10
n range
—
—
0.2–20
—
—
0.5–2
A 10% error was applied to all thickness and diameter measurements and a 20% error on distal limit (C). Laboratory analysis Values of λ and n are within the ranges
recommended by Bonadonna and Costa (2013) A 10% error was applied to all thickness and diameter measurements and a 20% error on distal limit (C). Values of λ and n are within the ranges
recommended by Bonadonna and Costa (2013) A 10% error was applied to all thickness and diameter measurements and a 20% error on distal limit (C). Values of λ and n are within the ranges
recommended by Bonadonna and Costa (2013) 35 Page 6 of 22 Bull Volcanol (2022) 84: 35 Fig. 2 Seismogram from a public seismic network (PSN) station
in Dasmarinas, Cavite, available from FDSN. Blue lines are seis-
mic waveforms whereas the red lines are clipped waveforms due
to their high amplitude. The estimated period of intense seismic
activity corresponding to the development of the 17–21-km-high
plume is approximately from 5 p.m. of 12 January to 3 a.m. the follow-
ing day. The inset circle is the wave panel showing a magnified view
of the recorded waveform with amplitude represented in counts Fig. 2 Seismogram from a public seismic network (PSN) station
in Dasmarinas, Cavite, available from FDSN. Blue lines are seis-
mic waveforms whereas the red lines are clipped waveforms due
to their high amplitude. The estimated period of intense seismic activity corresponding to the development of the 17–21-km-high
plume is approximately from 5 p.m. of 12 January to 3 a.m. the follow-
ing day. The inset circle is the wave panel showing a magnified view
of the recorded waveform with amplitude represented in counts The following textural and chemical analyses were then
performed on the sample grain-size modes (125–90 μm). Componentry was determined on the 2 samples from
epoxy-impregnated polished sections using a JEOL JSM-
5910 LV scanning electron microscope (SEM). The 2D
internal textures of the particles were investigated with
backscattered electron (BSE) imagery with an acceleration
voltage of 15 kV. At least 100 particles were counted for
each sample. The 3D particle surfaces were imaged using a
CARL ZEISS Supra 55/55 VP field emission (FE) SEM and
using secondary electron (SE) imagery, with an acceleration
voltage of 3 kV. data were plotted on a standard grain-size distribution graph. The manually sieved tephra samples were then sonicated
to separate aggregates for componentry analysis using a
Zeiss stereomicroscope. Laboratory analysis The relative abundance of each
component was assessed by grain counting at least 300
particles per size fraction (> 125–250 μm, 250–500 μm,
500 μm–1 mm, 1–2 mm, 2–4 mm, and > 4 mm) for each
sample. data were plotted on a standard grain-size distribution graph. The manually sieved tephra samples were then sonicated
to separate aggregates for componentry analysis using a
Zeiss stereomicroscope. The relative abundance of each
component was assessed by grain counting at least 300
particles per size fraction (> 125–250 μm, 250–500 μm,
500 μm–1 mm, 1–2 mm, 2–4 mm, and > 4 mm) for each
sample. Two additional samples were sent to the Laboratoire
Magmas et Volcans, University Clermont Auvergne (LMV-
UCA), for further analyses on size frequency distribution,
texture, morphology, and geochemistry. Both samples were
collected at the same location 20 km from the eruptive vent
(sample 4A and sample 4B) on 13 January (see Fig. 1b
for location). The first (sample 4A) was collected at 10:22
a.m. and the second (sample 4B) at 1:05 p.m. Grain sizes
for both samples were first measured by manual sieving
following the same procedure as described above. Particle
size distribution (PSD) was measured on size fractions
below 1 mm in diameter using a MALVERN Mastersizer
300 laser diffractometer, following the Thivet et al. (2020)
procedure. Finally, 2D ash morphology was quantified using a
MALVERN Morphologi G3 morpho-grainsizer following
the Thivet et al. (2020) procedure. Measurements were
performed on grain size modes of both samples. At least
1000 particles were analyzed for each sample and particle
shapes were automatically measured by the instrument. The
different shape parameters used in our study are described
in Thivet et al. (2020). In our study, we represented particle
morphology through two distinct roughness parameters,
solidity (SLD) and convexity (CVX), which represent the Page 7 of 22 35 Bull Volcanol (2022) 84: 35 Table 2 Timeline of events up to 22 January 2020 according to PHIVOLCS bulletins and cross-referenced with other sources
Date
Time
Alert level
Event description
Ashfall areas
12 Jan 2020
1:00p.m. 1
Increased steaming activities observed in
five places in the main crater with the
most active part exhibiting a phreatic
explosion with a 100-m-high plume
2:00p.m. 1
Booming sounds were heard in Talisay,
Batangas
2:04p.m. 1
Increase in activity within the MCL and
produced a 1-km-high eruption column
Ashfall observed in the southwest
portion of TVI
2:30p.m. Laboratory analysis 2
Alert level to 2
4:00p.m. 3
Alert level to 3
5:30p.m. 3
Activity intensified with sustained erup-
tion generating a steam-laden tephra col-
umn 10–15 km high
Wet ashfall fell to the north and
reaching as far as Quezon City
7:30p.m. 4
Alert level to 4
Ashfall was observed in several places
13 Jan 2020
2:49a.m. 4
Magmatic eruption with lava fountaining
observed
3:20a.m. 4
PHIVOLCS
bulletin
update
of
areas
affected by ashfall
4:28a.m. 4
Lava
fountaining
ceased. Eruption
quickly resumed with weak sporadic
fountaining and hydrovolcanic activity
within the main crater persisted with
2-km-high steam-laden plume
4:00p.m. 4
Newly formed lateral vents were discov-
ered in the northern flank of the main
crater island where 500-m lava fountains
emanated
Heavy ashfall to the southwest, expe
enced mainly in Cuenca, Lemery, a
Taal, Batangas
14 Jan 2020
8:00a.m. 4
Activity within the last 24 h has been
defined by sustained eruption in the main
crater due to hydrovolcanic and magmatic
activity
Heavy ashfall from the ongoing erupt
has fallen in Lemery, Talisay, Taal, a
Cuenca, Batangas
1:00p.m. 4
Activity produced a 800-m-high dark gray
steam-laden plume
Plume drifted southwest from the
main crater
15 Jan 2020
8:00a.m. 4
The sustained eruption produced a 1-km-
high dark gray steam-laden plum. Ash dispersed southwest of the m
crater
5:00p.m. 4
The continuous eruption produced a 700-
m-high dark gray steam-laden plume. “Drying-up” of portions of the Pansipit
River has been reported. Acquired satel-
lite images also show water from the
MCL has disappeared and new vent
craters have developed in the north flank
of the volcano
Plumes drifted southwest from the m
crater
16 Jan 2020
6:17a.m. 4
Eruptive activity produced a 500-m-high
dark gray ash plume
Ash dispersed west–southwest from
main crater
6:21a.m. 4
Eruptive activity produced a 800-m-high
dark gray ash plume
Ash dispersed west–southwest from
main crater
8:00a.m. 4
Activity within the last 24 h has dimin-
ished to weak emissions of steam-laden
plumes 700m high
Ash dispersed southwest from the m
crater Ashfall observed in the southwestern
portion of TVI Wet ashfall fell to the north and
reaching as far as Quezon City
Ashfall was observed in several places Lava
fountaining
ceased. Results morphological (particle scale) and the textural scale (smaller
scale) roughness of the particles, respectively. A perfectly
round or square particle has a SLD and CVX value of 1. On
the other hand, SLD and CVX decrease as soon as shape
irregularities appear. Laboratory analysis 4
Activity within the last 24 h was
by consistent steam emissions an
ular explosions that produced ash
500–1000m high
21 Jan 2020
8:00a.m. 4
Activity within the last 24 h was
by weak emissions of white stea
plumes 500–600m high
22 Jan 2020
8:00a.m. 4
Activity within the last 24 h was
by weak emissions of white stea
plumes 50–500m high
4:00p.m. 4
Since 5:00a.m., no ash emissio
observed based on visual obse
and seismic records. However,
35 Page 8 of 22 Bull Volcanol (2022) 84: 35 35 Page 8 of 22 35 Page 8 of 22 Activity within the last 24 h was defined
by consistent steam emissions and irregu-
lar explosions that produced white to dirty
white ash plumes 500–1000m Activity within the last 24 h was defined
by weak emissions of white steam-laden
plumes 500–600m high Activity within the last 24 h was defined
by weak emissions of white steam-laden
plumes 50–500m high Since 5:00a.m., no ash emissions were
observed based on visual observations
and seismic records. However, uncon-
solidated ash in Taal was remobilized
by strong-low level winds, and based
on reports by multiples airlines, reached
approximately 5800m. Elevations are above mean sea level. *Abridged Laboratory analysis Eruption
quickly resumed with weak sporadic
fountaining and hydrovolcanic activity
within the main crater persisted with
2-km-high steam-laden plume Heavy ashfall to the southwest, experi-
enced mainly in Cuenca, Lemery, and
Taal, Batangas Newly formed lateral vents were discov-
ered in the northern flank of the main
crater island where 500-m lava fountains
emanated Heavy ashfall from the ongoing eruption
has fallen in Lemery, Talisay, Taal, and
Cuenca, Batangas Activity within the last 24 h has been
defined by sustained eruption in the main
crater due to hydrovolcanic and magmatic
activity Plume drifted southwest from the
main crater
Ash dispersed southwest of the main
crater
Plumes drifted southwest from the main
crater Activity produced a 800-m-high dark gray
steam-laden plume The sustained eruption produced a 1-km-
high dark gray steam-laden plum. The continuous eruption produced a 700-
m-high dark gray steam-laden plume. “Drying-up” of portions of the Pansipit
River has been reported. Acquired satel-
lite images also show water from the
MCL has disappeared and new vent
craters have developed in the north flank
of the volcano 16 Jan 2020
6:17a.m. 4
6:21a.m. 4
8:00a.m. 4
5:00p.m. 4 Ash dispersed west–southwest from the
main crater
Ash dispersed west–southwest from the
main crater
Ash dispersed southwest from the main
crater Eruptive activity produced a 500-m-high
dark gray ash plume Eruptive activity produced a 800-m-high
dark gray ash plume Activity within the last 24 h has dimin-
ished to weak emissions of steam-laden
plumes 700m high Plumes drifted southwest from the main
crater Activity within the last 8 h has been
defined by weak emission of steam-laden
plumes 800m high. Nine distinct weak
explosions were recorded Table 2
(continued)
Date
Time
Alert level
Event description
Ashfall areas
17 Jan 2020
8:00a.m. 4
Activity within the last 24 h was defined
by consistent steam emissions and irregu-
lar explosions that produced dark gray ash
plumes 100–800m high
Ash dispersed west–southwest from the
main crater
5:00p.m. 4
Activity since 8:00a.m. was distinguished
by weak emissions of steam-laden plumes
800m high. Five distinct weak explosions
were recorded
Plume drifted southwest from the main
crater
18 Jan 2020
8:00a.m. 4
Activity within the last 24 h was defined
by consistent steam emissions and irregu-
lar explosions that produced white to dirty
white ash plumes 50–600m high
Ash dispersed southwest from the main
crater
6:00p.m. 4
Activity since 8:00a.m. Laboratory analysis was distinguished
by a weak emission of steam-laden
plumes 500–800m high. Two distinct
weak explosions were recorded
Plume drifted southwest from the main
crater
19 Jan 2020
8:00a.m. 4
Activity within the last 24 h was defined
by consistent steam emissions and irregu-
lar explosions that produced white to dirty
white ash plumes 500–1000m
Ash dispersed southwest from the main
crater
6:00p.m. 4
Activity since 8:00a.m. was distinguished
by a weak emission of steam-laden
plumes 300–500m high
Plume drifted southwest from the main
crater
20 Jan 2020
8:00a.m. 4
Activity within the last 24 h was defined
by consistent steam emissions and irreg-
ular explosions that produced ash plumes
500–1000m high
Ash dispersed southwest from the main
crater
21 Jan 2020
8:00a.m. 4
Activity within the last 24 h was defined
by weak emissions of white steam-laden
plumes 500–600m high
Ash dispersed southwest from the main
crater
22 Jan 2020
8:00a.m. 4
Activity within the last 24 h was defined
by weak emissions of white steam-laden
plumes 50–500m high
Ash dispersed southwest from the main
crater
4:00p.m. 4
Since 5:00a.m., no ash emissions were
observed based on visual observations
and seismic records. However, uncon-
solidated ash in Taal was remobilized
by strong-low level winds, and based
on reports by multiples airlines, reached
approximately 5800m. Remobilized unconsolidated ash drifted
southwest
affecting
Lemery
and
Agoncillo
35 Page 8 of 22
Bull Volcanol (2022) 84: 35 Table 2
(continued)
Date
Time
Alert level
Event description
17 Jan 2020
8:00a.m. 4
Activity within the last 24 h was
by consistent steam emissions and
lar explosions that produced dark
plumes 100–800m high
5:00p.m. 4
Activity since 8:00a.m. was distin
by weak emissions of steam-laden
800m high. Five distinct weak ex
were recorded
18 Jan 2020
8:00a.m. 4
Activity within the last 24 h was
by consistent steam emissions and
lar explosions that produced white
white ash plumes 50–600m high
6:00p.m. 4
Activity since 8:00a.m. was distin
by a weak emission of stea
plumes 500–800m high. Two
weak explosions were recorded
19 Jan 2020
8:00a.m. 4
Activity within the last 24 h was
by consistent steam emissions and
lar explosions that produced white
white ash plumes 500–1000m
6:00p.m. 4
Activity since 8:00a.m. was distin
by a weak emission of stea
plumes 300–500m high
20 Jan 2020
8:00a.m. Crowdsourced accounts The majority of crowdsourced reports came from Metro
Manila with fewer netizen reports received from the
CALABARZON (Cavite, Laguna, Batangas, Rizal, and
Quezon) Region. This can be attributed to the difference in
population density between the National Capital Region and
these adjacent provinces, and that most of CALABARZON
was heavily affected by the eruption (Fig. 1). All residents In situ glass compositions were determined from
microlite-free juvenile particles, using a Cameca SxFive-
Tactis electron probe micro-analyzer (EPMA), with an
acceleration voltage of 15 kV, current intensity of 8 nA,
and a laser beam of 10 microns, following the Gurioli et al. (2018) procedure. Elevations are above mean sea level. *Abridged Bull Volcanol (2022) 84: 35 Page 9 of 22 35 were also fully covered with thick tephra that required
clearing for safe use. Netizens from these areas reported
“smoking” ash outside of their homes as wind remobilized
and dispersed tephra particles. within 14 km of TVI were ordered to evacuate and many
other residents outside of this area voluntarily relocated
to safety. Partial power interruptions in the municipalities
of Cavite, Laguna, and Batangas lasting for days after
the eruption (Del Castillo et al. 2020) likely contributed
to fewer ashfall reports from these locations. Despite the
smaller number of data reported from proximal locations,
we consider the dataset robust enough when augmented
with additional field observations. The thickest tephra fall deposits were reported in
the municipalities and cities of Batangas immediately
surrounding Taal Lake. These include Agoncillo, Calaca,
Laurel, Lemery, Lipa, San Nicolas, Talisay, Taal, and
Tanauan. Deposits 40 to 50 mm thick heavily damaged
vegetation (Fig. 3). No netizen reports of ashfall from TVI
were received. The thinnest tephra fall deposits were reported north of
Metropolitan Manila as well as in the provinces of Rizal,
Bulacan, and Pampanga. These deposits were observed as
dispersed tephra particles with a thickness of < 1 mm that
were hardly visually discernible, particularly in urban to
suburban areas. Fig. 3 Representative photos of
tephra fall deposits with varying
thicknesses crowdsourced from
netizens and from our fieldwork:
(a) < 1 mm in Quezon City; (b)
1 mm in Quezon City; (c) 2.5
mm in Lipa City, Batangas; (d) 5
mm at Westgrove Heights,
Silang, Cavite; (e) 40 mm in
Talisay, Batangas (dashed lines
represent different layers in the
deposit); and (f) 110 mm in
Agoncillo, Batangas; (g)
400–500 mm at the north shore
of TVI. The two photos are of a
banca, a small boat, typically
used in the island. One photo
was taken before the eruption;
the other is a video screenshot
from Facebook posted by GO
Batangas on January 13.
Numerical values in the photos
refer to heights (in centimeters) Field observations On 12 January, tephra from the eruption reaching plume
heights of 17–21 km was first deposited southwest of Taal
Volcano (Lagmay et al. 2021). A few hours later, the plume
shifted north–northeastwards and wet tephra fall showered
cities and municipalities 70 km north–northeast of the main
crater. On 13 January, continuous activity at the volcano
generated plume heights up to 2 km above sea level and an
eruption cloud that drifted to the southwest of the volcano. Thicker deposits, with thickness of ≥1 mm, were more
apparent and easily documented by netizens. These could
be measured with a standard ruler or better estimated in
photographs, and were reported in southern Metropolitan
Manila and some portions of the provinces of Rizal, Cavite,
and Laguna. Tephra fall deposits progressively thicken
towards the eruption source. Deposits with thicknesses of at
least 1 up to 10 mm (Fig. 3c, d) fully blanketed the surface
of roads, roofs, car hoods, and leaves of plants. The tephra fall deposits exhibited parallel layerings made
distinct by changes in color and grain size. In proximal
areas, layers with ash-sized (<2 mm in diameter) to
lapilli-sized (>2 mm diameter) lithic, vitric, and crystal
fragments are observed in the lower layers. North of the
volcano island, in Silang, Tagaytay, Talisay, and Tanauan, Tephra fall deposits closer to the source in Cavite,
Laguna, and Batangas were much thicker with thicknesses
of 10 to 30 mm (Fig. 3e, f). Objects and structures situated
outdoors were heavily blanketed with tephra. Public roads Fig. 3 Representative photos of
tephra fall deposits with varying
thicknesses crowdsourced from
netizens and from our fieldwork:
(a) < 1 mm in Quezon City; (b)
1 mm in Quezon City; (c) 2.5
mm in Lipa City, Batangas; (d) 5
mm at Westgrove Heights,
Silang, Cavite; (e) 40 mm in
Talisay, Batangas (dashed lines
represent different layers in the
deposit); and (f) 110 mm in
Agoncillo, Batangas; (g)
400–500 mm at the north shore
of TVI. The two photos are of a
banca, a small boat, typically
used in the island. One photo
was taken before the eruption;
the other is a video screenshot
from Facebook posted by GO
Batangas on January 13. Numerical values in the photos
refer to heights (in centimeters) 35 Page 10 of 22 Bull Volcanol (2022) 84: 35 deposits have thicknesses from 1 to 4 cm with at least
four layers. Field observations In Talisay, Batangas, 9 km from the crater, a
deposit was observed to exhibit a light gray bottom layer
(layer 1) overlain by a dark gray ash deposit (layer 2)
with coarse accidental lithic lapilli, vitric grains, and free
crystals (Fig. 3e). The third layer (layer 3) is made up
of brown ash and is the thickest. Finally, light gray ash
makes up the topmost layer (layer 4). In Tagaytay, a similar
deposit was observed but with the bottom-most light gray
layer absent. Furthermore, most of the observed proximal
deposits contained accretionary lapilli. A total isopach map area of 8605 km2 was covered by
tephra fall after the 12 January eruption based on 0.05-
cm depth, crowdsourced data, and field observation. Within
this area, seven isopach contours were drawn (Fig. 4a)
corresponding to the reported thickness ranges of tephra fall
after the eruption. The isopleth contours were delineated based on the
maximum grain sizes measured from the samples (Fig. 4b)
gathered during field validation. A gradual decrease of
tephra grain sizes from the TVI to the surrounding
communities can be observed on the map, with the smallest
(0.02–0.10 cm) grain sizes sampled just within Metro
Manila. The trend of decreasing tephra grain size also
followed the north–northeast wind direction at the time. Deposit volume calculation Thickness reports and field data reveal the approximate
distribution of the tephra fall deposits of the 12–13 January
eruption of Taal Volcano (Fig. 4a). Ellipticity for all isopach
areas range from 0.15 to 0.56, which implies elongated
tephra fall deposition rather than an axial symmetry. The
ellipticity of the tephra deposit is attributed to winds
blowing toward the north–northeast at higher levels in the
atmosphere during the main part of the eruption (i.e., 1:00
p.m. 12 January to 4:28 a.m. 13 January). The thickness and maximum grain size values used for
the isopach and isopleth contours, respectively, are listed in
Table 3 with their corresponding area in square kilometers. The thickest isopach contour, 40 mm, approximately
encloses TVI and has a distance, r, of approximately 2 km
away from the main crater. At this distance, the thickness of
the tephra deposit was determined from the nearly complete
burial of the hull of a banca, a traditional boat used in
Taal Lake (Fig. 3g). The thinnest measurement is ≤1 mm, Fig. 4 Isopach (a) and isopleth (b) maps generated from vetted crowdsourced accounts and field data corresponding to the 12 January to early
morning (about 4:00 a.m.) of 13 January eruption of Taal Volcano Fig. Deposit volume calculation 4 Isopach (a) and isopleth (b) maps generated from vetted crowdsourced accounts and field data corresponding to the 12 January to early
morning (about 4:00 a.m.) of 13 January eruption of Taal Volcano Page 11 of 22 35 Bull Volcanol (2022) 84: 35 Page 11 of 22 35 Table 3 Thickness values of isopach contours and maximum grain size values of isopleth contours
Isopach
Maroon
Red
Red orange
Orange
Yellow
Light Green
Green
Thickness (cm)
45
10
1.50
0.50
0.30
0.10
0.05
Area (km2)
22.90
123.24
436.77
764.24
1107.91
2285.22
3864.89
Isopleth
Red
Orange
Yellow
Green
Max grain size (cm)
1.65
0.325
0.13
0.075
Area (km2)
216.57
699.98
1092.35
1307.47
The corresponding areas covered by the isopach and isopleth contours are correspondingly listed Table 3 Thickness values of isopach contours and maximum grain size values of isopleth contours The corresponding areas covered by the isopach and isopleth contours are correspondingly listed Using the probabilistic approach with 90% confidence
intervals or the 5–95th percentile intervals, the range of
volumes become 0.051–0.063 km3 (exponential), 0.036–
0.047 km3 (power-law), and 0.073–0.097 km3 (Weibull),
which shows that the Weibull fit is sensitive to uncertainties
regarding isopach thickness and the square root of the
isopach area. In the Weibull fit, the 95th percentile of the
distribution results in a volume of 0.097 km3, which gets
very close to the volume that corresponds to VEI 4 (0.1–1
km3). reported by netizens in Quezon City in Metro Manila and in
the Rizal/Quezon Province boundary. The isopach contours show exponential thinning of
tephra downwind with a 1.40-km thickness half-distance
for the proximal segment and 9.49-km half-distance for
the distal segment. Along the crosswind direction, the
tephra fall deposit also thins exponentially with a 1.28-
km thickness half-distance for the proximal segment and
1.50-km half-distance for the distal segment (Fig. 5). Results on the volume of Taal Volcano’s 2020 tephra
fall deposit differ based on the model used (Fig. 6). The
exponential model yields a volume of 0.057 km3, whereas
the power-law model calculates a value of 0.042 km3. The
Weibull model gives the largest volume of 0.090 km3. All are within the volume range of 0.01–0.1 km3, which
translates to a VEI of 3. TephraFits calculations using the Weibull method based
on isopleth data show a median eruption height of 17.8 km
above vent. Deposit volume calculation Considering typical uncertainties of 20% for
determining plume heights (Bonadonna and Costa 2013),
the range of plume heights determined for the main eruption
of Taal Volcano is 14.2–21.3 km above vent. In terms of the Fig. 5 Thickness half-distance exponential plot of the downwind and crosswind segments for Taal Volcano’s tephra fall deposits Fig. 5 Thickness half-distance exponential plot of the downwind and crosswind segments for Taal Volcano’s tephra fa 35 Page 12 of 22 Bull Volcanol (2022) 84: 35 Fig. 6 Results of the TephraFits model calculations using the exponential, power-law, and Weibull methods Fig. 6 Results of the TephraFits model calculations using the exponential, power-law, and Weibull methods with grain-size frequency plots also displaying a single
mode at 125–500 μm. classification of the eruption, we calculate λT H and λMC
λT H ,
as well as thickness half-distance bt and thickness half-
distance over thickness half-clast ( bt
bc ) values that plot in the
sub-plinian fields of Pyle (1989) and Bonadonna and Costa
(2013) (Fig. 7). Grain-size distributions of the samples collected south–
southwest of Taal Volcano (Fig. 9a) range between 300
μm (1.75 φ) and 1 μm (10 φ) and show a single mode at
125–190 μm (3–3.5 φ). These volume distributions (which
reflect mass distributions assuming a constant particle
density for each grain size bin) are asymmetric and skewed
toward relatively coarse grain sizes. Note that both bulk
samples contained aggregates that were crushed during the
analysis. These aggregates were irregular in shape with no
specific internal grain-size organization. Tephra sample characterization Grain-size distributions of the tephra fall deposits from
samples collected north–northeast of Taal Volcano show a
unimodal distribution (Fig. 8a). The grain-size frequency
plot for the most proximal deposits shows an asymmetrical
distribution, skewed towards the finer grain sizes. Coarser
tephra grains were absent for the two distal deposits
collected in Muntinlupa, Metro Manila, and Taytay, Rizal, Binocular microscope analysis showed that the 2020 Taal
Volcano tephra consists of volcanic glass (vitric), accidental
lithic fragments, crystals, and crystal fragments (Fig. 8c). Page 13 of 22 35 Page 13 of 22 35 Bull Volcanol (2022) 84: 35 Fig. 7 Results of the TephraFits calculations for isopleth data (a) and the classification schemes according to Bonadonna and Costa (2013) (b)
and Pyle (1989) (c). Both plots in the sub-plinian field Fig. 7 Results of the TephraFits calculations for isopleth data (a) and the classification schemes according to Bonadonna and Costa (2013) (b)
and Pyle (1989) (c). Both plots in the sub-plinian field Grain counting reveals that volcanic glass is the dominant
component in all the samples. Volcanic glass or the vitric
components consist of about 83–90%. Lithic grains only
represent 7–11%, whereas crystal fragments compose less
than 6% of the tephra material (Fig. 8b). of juvenile glassy particles are typically composed of
stepped features and microfractures. Tephra morphology measurements (Fig. 10) reflect
the coexistence of both irregular (glassy and transparent
textures) and blocky (microlite-rich and opaque textures)
juvenile particles, as well as blocky crystals (conchoidal
fractures and transparent textures). Vitric components are translucent light brown to black. The grains vary from blocky to fluted to scoriaceous forms. Lithic fragments, on the other hand, include whitish yellow
hydrothermally altered fragments, sub-rounded volcanic
rock fragments, and reddish-orange oxidized grains. Crystal
fragments of olivine, quartz, and plagioclase were observed
as free crystals embedded within glass. Sulfides were also
observed in some of the hydrothermal fragments (Fig. 8c). In
situ
glass
compositions
measured
on
juvenile
microlite-free tephra particles reveal that the erupted magma
is andesitic in composition, with no significant variations
within the two samples from Taal Volcano’s 12 January to
early morning of 13 January eruption (Fig. 11). Backscatter electron (BSE) image analysis of each of
the grain size modes of samples 4A and 4B (Fig. Tephra sample characterization 9b) was
used to identify different components, including juvenile
glassy (sideromelane) particles (80%), juvenile microlite-
rich (tachylite) particles (10%), scarce phenocrysts of
olivine and plagioclase (10%), and very scarce non-juvenile
hydrothermal fragments (<1%). Analysis of isopach and isopleth data The tephra fall of Taal Volcano during its 2020 unrest
was dispersed over a large region covering an approximate
area of at least 8605 km2. In a tropical environment where
rainfall is common, tephra deposits can be easily washed Scanning electron (SE) images (Fig. 9c) confirmed the
presence of abundant fine adhering particles. The surfaces Bull Volcanol (2022) 84: 35 35 Page 14 of 22 Fig. 8 Samples 1, 2, and 3
analyzed at UP-NIGS. (a)
Grain-size distribution for the
tephra fall deposit in different
localities. (b) Relative
percentage of the tephra
components. (c) Tephra
components including vitric,
lithic, and crystals (olivine,
quartz), as well as secondary
quartz (H quartz) and sulfides
adhering to the rock fragments
away and deliberately removed by residents, forever erasing
their depositional record. Using combined crowdsourcing
the day after the eruption and field surveys within weeks
after the event, tephra fall deposits were systematically
documented, revealing some insights on the characteristics
of the 12 January to early morning of 13 January eruption of
Taal Volcano. Ashfall deposit measurement was found to be
more accurate on flat horizontal and undisturbed surfaces
Using TephraFits to determine the thickness half-
distance of tephra fall deposits, we calculate 1.40 km and
9.49 km for the proximal and distal exponential segments,
respectively. The total calculated volume of erupted tephra
for the 12 January to early morning of 13 January main
eruption is 0.057 km3 (exponential model), 0.042 km3
(power-law model), and 0.090 km3 (Weibull model). All
these computed values translate to a VEI of 3 3
r the
rent
ric,
e,
ary
ides
ments Fig. 8 Samples 1, 2, and 3
analyzed at UP-NIGS. (a)
Grain-size distribution for the
tephra fall deposit in different
localities. (b) Relative
percentage of the tephra
components. (c) Tephra
components including vitric,
lithic, and crystals (olivine,
quartz), as well as secondary
quartz (H quartz) and sulfides
adhering to the rock fragments Using TephraFits to determine the thickness half-
distance of tephra fall deposits, we calculate 1.40 km and
9.49 km for the proximal and distal exponential segments,
respectively. The total calculated volume of erupted tephra
for the 12 January to early morning of 13 January main
eruption is 0.057 km3 (exponential model), 0.042 km3
(power-law model), and 0.090 km3 (Weibull model). All
these computed values translate to a VEI of 3. away and deliberately removed by residents, forever erasing
their depositional record. Fig. 8 Samples 1, 2, and 3
analyzed at UP-NIGS. (a)
Grain-size distribution for the
tephra fall deposit in different
localities. (b) Relative
percentage of the tephra
components. (c) Tephra
components including vitric,
lithic, and crystals (olivine,
quartz), as well as secondary
quartz (H quartz) and sulfides
adhering to the rock fragments Analysis of isopach and isopleth data Using combined crowdsourcing
the day after the eruption and field surveys within weeks
after the event, tephra fall deposits were systematically
documented, revealing some insights on the characteristics
of the 12 January to early morning of 13 January eruption of
Taal Volcano. Ashfall deposit measurement was found to be
more accurate on flat, horizontal, and undisturbed surfaces. Field sampling focused on such areas and would make better
observation points for netizens in future implementations of
crowdsourcing ashfall data. However, using the probabilistic approach (Weibull
method) with 90% confidence interval, the volume estimate
can be as high as 0.097 km3 which is close to the lower Page 15 of 22 35 Bull Volcanol (2022) 84: 35 . 9 Samples 4A and 4B analyzed at LMV-UCA. (a) Grain-size distribution from laser diffraction measurements, (b) BSE images, (c) SE im V-UCA (a) Grain-size distribution from laser diffraction measurements (b) BSE images (c) SE im g. 9 Samples 4A and 4B analyzed at LMV-UCA. (a) Grain-size distribution from laser diffraction measurements, (b) BSE images, (c) SE im Fig. 9 Samples 4A and 4B analyzed at LMV-UCA. (a) Grain-size distribution from laser diffraction measurements, (b) Fig. 9 Samples 4A and 4B analyzed at LMV-UCA. (a) Grain-size distribution from laser diffraction measurements, (b) BSE images, (c) SE images Discrepancies between the power-law, exponential, and
Weibull volumes which lead to different estimates of VEI
values have been documented for other volcanoes. For
example, in Layer 5 of the Cotopaxi (1180 ±80 years
B.P.) fall deposits, both power-law and exponential methods
yielded a VEI 4 whereas the Weibull method resulted in a
VEI 5 estimate (Biass et al. 2019). For Taal, all methods
have a resulting volume corresponding to a VEI 3. A VEI
4 volume can be reached if the largest end of the largest
estimate (Weibull method) is used with the addition of
the base surge deposits. This is more consistent with the
VEI classification of Constantinescu et al. (2021) for the end of the volume range of VEI 4 (0.1–1 km3). Adding the
volume of the base surge deposits amounting to 0.019 km3
(Lagmay et al. 2021), the total erupted volume translates
to a VEI of 4, consistent with the classification criteria
of Constantinescu et al. (2021), namely (1) volume of
ejecta; (2) eruption plume height; and (3) umbrella cloud
radius. Analysis of isopach and isopleth data The total erupted volume was calculated using
crowdsourced and field survey data, whereas the erupted
height and umbrella cloud radius are direct measurements
from satellite data of Taal Volcano’s 2020 main eruption
plume (Bachmeier 2020; Perttu et al. 2020; Lagmay et al. 2021). Bull Volcanol (2022) 84: 35 35 Page 16 of 22 Fig. 10 Tephra morphology
measurements, representing
convexity vs. solidity
parameters. Analysis conducted
at LMV-UCA
observed plume height of 17–21 km (Bachmeier 2020;
Perttu et al. 2020) and umbrella radius of 100 km (Lagmay
et al. 2021) of Taal Volcano’s main eruption in 2020. An eruption size at the low end of a VEI 4 perhaps
explains the smaller volume and limited distribution of
base surge deposits. Based on co-eruptive modeling of
Bato et al. (2021), the magma reservoir at ∼5–6 km depth
below TVI lost an estimated volume of −0.531 ±0.004
km3. Much of this withdrawn magma is believed to have
stalled at depth and emplaced as a northeast-striking dike
extending under the towns of Taal, Lemery, Agoncillo, and
San Nicolas, Batangas (Bato et al. 2021). Had most of the observed plume height of 17–21 km (Bachmeier 2020;
Perttu et al. 2020) and umbrella radius of 100 km (Lagmay
et al. 2021) of Taal Volcano’s main eruption in 2020. An eruption size at the low end of a VEI 4 perhaps
explains the smaller volume and limited distribution of
base surge deposits. Based on co-eruptive modeling of
Bato et al. (2021), the magma reservoir at ∼5–6 km depth
below TVI lost an estimated volume of −0.531 ±0.004
km3. Much of this withdrawn magma is believed to have
stalled at depth and emplaced as a northeast-striking dike
extending under the towns of Taal, Lemery, Agoncillo, and
San Nicolas, Batangas (Bato et al. 2021). Had most of the observed plume height of 17–21 km (Bachmeier 2020;
Perttu et al. 2020) and umbrella radius of 100 km (Lagmay
et al. 2021) of Taal Volcano’s main eruption in 2020. An eruption size at the low end of a VEI 4 perhaps
explains the smaller volume and limited distribution of
base surge deposits. Based on co-eruptive modeling of observed plume height of 17–21 km (Bachmeier 2020;
Perttu et al. 2020) and umbrella radius of 100 km (Lagmay
et al. 2021) of Taal Volcano’s main eruption in 2020. observed plume height of 17–21 km (Bachmeier 2020;
Perttu et al. Analysis of isopach and isopleth data 2020) and umbrella radius of 100 km (Lagmay
et al. 2021) of Taal Volcano’s main eruption in 2020. Bato et al. (2021), the magma reservoir at ∼5–6 km depth
below TVI lost an estimated volume of −0.531 ±0.004
km3. Much of this withdrawn magma is believed to have
stalled at depth and emplaced as a northeast-striking dike
extending under the towns of Taal, Lemery, Agoncillo, and
San Nicolas, Batangas (Bato et al. 2021). Had most of the An eruption size at the low end of a VEI 4 perhaps
explains the smaller volume and limited distribution of
base surge deposits. Based on co-eruptive modeling of An eruption size at the low end of a VEI 4 perhaps
explains the smaller volume and limited distribution of
base surge deposits. Based on co-eruptive modeling of Fig. 11 Plot of the geochemical analysis in this work against the
whole-rock geochemistry of lava flow deposits and loose rocks from
previous eruptions of Taal Volcano (Miklius et al. 1991). In situ glass
compositions measured on juvenile microlite-free particles show an
alkali-rich andesitic composition in the TAS diagram Fig 11 Plot of the geochemical analysis in this work against the
compositions measured on juvenile microlite free particles show an compositions measured on juvenile microlite-free particles show an
alkali-rich andesitic composition in the TAS diagram Fig. 11 Plot of the geochemical analysis in this work against the
whole-rock geochemistry of lava flow deposits and loose rocks from
previous eruptions of Taal Volcano (Miklius et al. 1991). In situ glass Bull Volcanol (2022) 84: 35 Page 17 of 22 35 Page 17 of 22 35 magma withdrawn from the reservoir been erupted in the
2020 event, it would still be classified as a VEI 4 with a
more extensive pyroclastic surge deposit. resulted in wet tephra to fall in clumps (Fig. 12). The excess
water included in the eruption may have contributed to
the dramatic display of thousands of lightning strokes and
bolts (Prata et al. 2020; Van Eaton et al. 2020) (Fig. 12b)
throughout the period of the sub-plinian eruption. Maximum particle size data of the tephra deposits
suggest that the 2020 eruption was larger than a “strong
to moderate” event classified by PHIVOLCS at the time
of eruption. Grain size distribution, morphology, and
composition The glass fraction of juvenile tephra is andesitic in
composition for the two samples that were analyzed. The
whole-rock geochemistry of lava flows and loose rocks from
the Volcano Island reported by Miklius et al. (1991) is
plotted on the Le Maitre et al. (2005) TAS diagram showing
most of the older eruption products were basalt to basaltic
andesite in composition (Fig. 11). The 2020 microlite-free
juvenile tephra materials are more andesitic and contain
higher SiO2 (55–56%) and Na2O + K2O (<5.5%) than most
of the older volcanic rocks. As a preliminary interpretation,
this suggests that the magma erupted during the 2020
eruption is slightly more evolved relative to the 1968–1969
lavas erupted from Mt Tabaro, located southwest of Taal
Volcano Island. However, for loose samples collected from
the Main Crater rim and lavas from the Bignay eruption
center, the composition is quite similar to the 2020 tephra
(Fig. 11). A more detailed study of the 2020 eruptive
products is the topic of another paper in preparation that will
compare whole-rock geochemistry from different eruptive
events. The volcanic glass component of the tephra deposits
represents newly erupted magma that came into contact
with the MCL. Recent studies suggest that explosive
magma-water interactions can be identified in some tephra
features, such as grain size, componentry, morphology,
and texture (e.g., Wohletz (2013), Jordan et al. (2014),
Thivet et al. (2020), and Ross et al. (2021)). Nevertheless,
phreatomagmatism fragmentation is still difficult to assess
without any direct observation of the eruption itself (White
and Valentine 2016). Present knowledge and monitoring
of the Taal Volcano undoubtedly confirm the presence
of a water lake (MCL) within the 2020 eruptive site. We also suggest that magma-water interaction favor (1)
the fragmentation of some lithic grains (10–17% of the
counted grains), (2) the occurrence of specific textural
signatures (ubiquitous hackle lines, stepped features, and
micro-fractures), and (3) the relatively fine grain size of
the deposit (grain modes ranging between 90 and 500 μm
depending on the deposit locations). Lithic grains, including
hydrothermally altered fragments, sub-rounded volcanic
rock fragments, and oxidized grains are those plucked from
the volcano’s conduit during the eruption. Free crystals
found in the tephra fall deposit were formed earlier in the
magma body’s ascent history and liberated from the glassy
matrix during eruption fragmentation. A phreatomagmatic
eruption starting at about 4:00–5:30 p.m. of 12 January Analysis of isopach and isopleth data We modeled an eruption height of 17.8
km using the Weibull method of TephraFits, which is
consistent with the range of maximum heights measured
from satellite images by Perttu et al. (2020) (16–17 km)
and Bachmeier (2020) (20–21 km). Typical uncertainties
of 20% for determining plume heights (Bonadonna and
Costa 2013) yield a maximum value of 21.26 km. This calculation matches the above-anvil cirrus plume
temperature measurement of ∼
60◦C, which translates
to approximately 21 km based on comparison of data
with 3 rawinsonde sites in Legaspi, Mactan, and Laoag,
Philippines (Bachmeier 2020). The calculations of thickness
half-distance bt and the ratio of thickness half-distance over
thickness half-clast ( bt
bc ) plot in the sub-plinian classification
scheme of Pyle (1989). A sub-plinian classification was also
calculated using the classification scheme of Bonadonna
and Costa (2013). Accretionary lapilli were abundant in the tephra fall
deposits, especially in the areas of Agoncillo, Laurel, and
San Nicolas Batangas (8–9 km away). In more distal areas,
including Metro Manila (65 km away), tephra fall deposits
include irregular-shaped aggregates with no specific internal
grain organization. The “wet” nature of the eruption can be
attributed to the interaction of the eruptive materials with
the 42 million m3 of water contained in the MCL (Bernard
A et al. 2020) as evidenced by lapilli-sized aggregates. This
is consistent with the interpretation of a phreatomagmatic
eruption starting at about 4:00–5:30 p.m. of 12 January,
which resulted in wet tephra to fall in clumps (Fig. 12). Base
surges, known to form from the interaction of magma and
water, are also reported to have cascaded down the slopes
of Taal Volcano. They may have travelled across the lake
up to about 600 m beyond the island’s shores based on the
energy-line model (Lagmay et al. 2021). Tephra fall (ashfall) hazards The eruption of Taal Volcano deposited heavy, corrosive ash
onto exposed surfaces such as house fittings (i.e., external
pipes and gutters) and roads (Fig. 13a). The interior of
buildings were also affected by wind-blown ash, causing
massive disruption of services (e.g., suspending work and 35 Page 18 of 22 Bull Volcanol (2022) 84: 35 Fig. 12 Evidence for a wet
tephra plume. (a) Tephra falling
in clumps on the car windshield
in Silang, Cavite, 22 km north of
Taal Volcano (inset shows
clumps of tephra falling on the
arm of author at 5:30 p.m. on 12
January). (b) Lightning during
the eruption CC-BY
Wikicommons. (c) Accretionary
lapilli in tephra fall deposit. (d)
Lightning produced during the
eruption of Taal Volcano
between 3:00 p.m. on 12 January
and 5:00 a.m. on 13 January
local time (0700–2100 UTC on
12 January). Red squares
indicate plume-to-ground
strokes; black circles indicate
in-plume pulses. Courtesy of
Chris Vagasky, Vaisala (Global
Volcanism Program 2020)
school) and posing hazards to the health of residents
(Fig. 13b) (Baxter et al. 1982). In more severe cases,
such as in the municipalities of Agoncillo and Laurel in
Batangas, houses made of light materials collapsed as wet
ash increased the load on roofs and walls (Fig. 13c). Ground transportation was heavily impacted as primary
and resuspended ash dispersed in air and caused reduced
visibility and traction of road networks (Fig. 13d). To
wash off ashfall, cars needed to automatically spray water
while wiping. Without spraying water, ash remained on the nce for a wet
(a) Tephra falling
he car windshield
te, 22 km north of
inset shows
ra falling on the
at 5:30 p.m. on 12
ightning during
C-BY
. (c) Accretionary
a fall deposit. (d)
duced during the
al Volcano
p.m. on 12 January
on 13 January
00–2100 UTC on
ed squares
-to-ground
circles indicate
s. Courtesy of
Vaisala (Global
gram 2020) Fig. 12 Evidence for a wet
tephra plume. (a) Tephra falling
in clumps on the car windshield
in Silang, Cavite, 22 km north of
Taal Volcano (inset shows
clumps of tephra falling on the
arm of author at 5:30 p.m. on 12
January). (b) Lightning during
the eruption CC-BY
Wikicommons. (c) Accretionary
lapilli in tephra fall deposit. (d)
Lightning produced during the
eruption of Taal Volcano
between 3:00 p.m. on 12 January
and 5:00 a.m. on 13 January
local time (0700–2100 UTC on
12 January). Tephra fall (ashfall) hazards For aviation,
several airports including the Ninoy Aquino International
Airport (NAIA) had to suspend over 240 flights (Rappler
2020; Reuters 2020; Chen 2020) as flying through ash
clouds (Fig. 13e) is known to cause significant damage to
aircraft engines (Casadevall 1992; Guffanti et al. 2010). distal exponential segments, respectively. In terms of tephra
fall volume, model results using the exponential, power-law,
and Weibull models, yield values of 0.057 km3, 0.042 km3,
and 0.090 km3, respectively; all of these values translate to
a VEI of 3. However, using the probabilistic approach (Weibull
method) with 90% confidence interval, the volume estimate
can be as high as 0.097 km3. With the addition of the
base surge deposits of 0.019 km3 (Lagmay et al. 2021),
the total eruption volume translates to a VEI of 4, which
is more consistent with the classification for the observed
plume height of 17–21 km (Perttu et al. 2020; Bachmeier
2020) and umbrella radius of Taal Volcano’s main eruption
in 2020. A VEI of 4 is also consistent with the calculated
median height of 17.8 km and sub-plinian classification
which are based on the combined analysis of isopleth and
isopach data derived from crowdsourced and field data. Agriculture areas were also damaged by extensive tephra
fall. Within the municipality of Agoncillo in Batangas,
the leaves of coconut trees sagged and were on occasion
felled by ashfall (Fig. 13f). Pineapple crops and trees in
Tagaytay, Cavite, were blanketed by ash for weeks. Taal
Volcano’s eruption caused severe damage to the Philippines’
agricultural sector, with losses climbing to PhP 3.06 billion
(USD 59.98 million/ EUR 54.19 million) according to the
Department of Agriculture (CNN Philippines Staff 2020). Affected were coffee, cacao, pineapple, rice, coconut, and
other high-value produce. The fisheries sector recorded the
highest value of damage after the eruption, as it lost PhP
1.6 billion (USD 31.36 million/EUR 28.33 million) for the
tilapia and milkfish (bangus) culture around the Taal Lake
(CNN Philippines Staff 2020). The eruptive vent, located at the MCL, and the
numerous fissures generated by ground deformation,
allowed the explosive interaction of magma and water. Analyses of the fall deposits using binocular and electron
microscopes reveal vitric (83–90%), lithic (7–11%), and
crystal components (<6%) that indicate a phreatomagmatic
eruption. This interpretation is supported by the presence of
accretionary lapilli in fall deposit layers and tephra falling
in wet clumps at the height of the eruptive event. Tephra fall (ashfall) hazards Red squares
indicate plume-to-ground
strokes; black circles indicate
in-plume pulses. Courtesy of
Chris Vagasky, Vaisala (Global
Volcanism Program 2020) Wikicommons. (c) Accretionary
lapilli in tephra fall deposit. (d)
Lightning produced during the
eruption of Taal Volcano
between 3:00 p.m. on 12 January
and 5:00 a.m. on 13 January
local time (0700–2100 UTC on
12 January). Red squares
indicate plume-to-ground
strokes; black circles indicate
in-plume pulses. Courtesy of
Chris Vagasky, Vaisala (Global
Volcanism Program 2020) Ground transportation was heavily impacted as primary
and resuspended ash dispersed in air and caused reduced
visibility and traction of road networks (Fig. 13d). To
wash off ashfall, cars needed to automatically spray water
while wiping. Without spraying water, ash remained on the school) and posing hazards to the health of residents
(Fig. 13b) (Baxter et al. 1982). In more severe cases,
such as in the municipalities of Agoncillo and Laurel in
Batangas, houses made of light materials collapsed as wet
ash increased the load on roofs and walls (Fig. 13c). school) and posing hazards to the health of residents
(Fig. 13b) (Baxter et al. 1982). In more severe cases,
such as in the municipalities of Agoncillo and Laurel in
Batangas, houses made of light materials collapsed as wet
ash increased the load on roofs and walls (Fig. 13c). school) and posing hazards to the health of residents
(Fig. 13b) (Baxter et al. 1982). In more severe cases,
such as in the municipalities of Agoncillo and Laurel in
Batangas, houses made of light materials collapsed as wet
ash increased the load on roofs and walls (Fig. 13c). Fig. 13 Tephra fall impacts. Ashfall (a) blanketing a garage
in Laguna Province; (b) covering
road surfaces; (c) causing the
collapse of roofs; (d) impairing
road visibility; (e) causing
danger to inbound flights to
Manila; and (f) damaging crops Fig. 13 Tephra fall impacts. Ashfall (a) blanketing a garage
in Laguna Province; (b) covering
road surfaces; (c) causing the
collapse of roofs; (d) impairing
road visibility; (e) causing
danger to inbound flights to
Manila; and (f) damaging crops Page 19 of 22 35 Bull Volcanol (2022) 84: 35 Page 19 of 22 35 windshield and scratched the glass, reducing visibility for
the car driver. The water tank for the wiper spray needed
to be replenished after only a few minutes. Tephra fall (ashfall) hazards Textural
signatures on juvenile particles, such as stepped features,
also support the occurrence of the interaction between
magma and external fluids at the fragmentation level, which
enhanced the formation of fine ash particles. The National Disaster Risk Reduction and Management
Council (NDRRMC) ordered the evacuation of at least
18,187 residents and housed them in 76 evacuation centers
(MMDA 2020) because of the threats posed by the eruption
and from ashfall. People were advised by the Department
of Health (DOH) to stay indoors and wear medical masks
to avoid inhalation of ash particles and skin exposure (Viray
2020). Although the primary eruption lasted for only about 10
h, the damage to infrastructure and crops, and effects on air
quality were significant. Eruptive activity continued for 7
days but was mainly characterized by discrete cannon-like
explosions that generated 2-km-high bent-over plumes on
13 January that drifted to the west and southwest. Waning
of activity generated much lower eruption heights of about
500 m until the eruptive activity ended on 22 January
(PHIVOLCS 2020a; 2020b). Conclusions On 12 January 2020, Taal Volcano erupted after 43 years of
dormancy. Within a few hours from the start of the event
at about 1 p.m., a towering 17–21-km-high eruption plume
formed with its umbrella cloud drifting towards the north–
northeast, affecting nearby provinces, including Metro
Manila, the National Capital Region of the Philippines. Tephra fall deposits mantled the ground surface, covering
an area of 8605 km2 as far as 70 km north–northeast of Taal
Volcano. This study is a culmination not only of the authors’ own
work, but also of the contributions of hundreds of members
of the general public who stepped up to the call of citizen
science, providing invaluable data and making themselves
part of this crucial scientific work. Through their work and
our own, we advance the knowledge on Taal Volcano, the
second most active eruptive center in the Philippines with a
long history of devastating eruptions. The growing density
of population and rapid development of areas surrounding
Taal Volcano make such understanding of its consequent
tephra fall hazards and their impacts more significant. Tephra fall deposits were mapped using crowdsourced
data collected during the 12–13 January main eruption,
and field surveys within weeks after the peak eruptive
period. This complementary technique for gathering data is
particularly useful for a tropical country where tephra fall
deposits are easily washed away by rain. The TephraFits model results show that the tephra
fall thins downwind exponentially with a thickness half-
distance of about 1.40 km and 9.49 km for the proximal and Supplementary Information The online version contains supplemen-
tary material available at https://doi.org/10.1007/s00445-022-01534-y. 35 Page 20 of 22 Bull Volcanol (2022) 84: 35 in taal volcanic lake: a simple gasometer for volcano monitoring,
vol 47 Acknowledgements We thank the citizen scientists who contributed
their records, donated samples of the ashfall deposits in their
neighborhood, helped in the processing of samples for analysis, and
participated in the editing of the draft manuscript. The authors greatly
appreciate the efforts of Dr. Peter Julian Cayton, who shared his
statistical expertise, and Mr. Eligio Obille Jr., who took time to review
the manuscript. We also thank Steve Self, Kristi Wallace, and Michael
Ort for their insightful reviews. Biass S, Bagheri G, Aeberhard WH, Bonadonna C (2014) Terror:
towards a better quantification of the uncertainty propagated dur-
ing the characterization of tephra deposits, statistics in volcanol-
ogy. Statistics in Volcanology 1(2). References Constantinescu R, Hopulele-Gligor A, Connor CB, Bonadonna C,
Connor LJ, Lindsay JM, Charbonnier S, Volentik AC (2021) The
radius of the umbrella cloud helps characterize large explosive vol-
canic eruptions. Communications Earth & Environment 2(1):1–8. https://doi.org/10.1038/s43247-020-00078-3 Anthony R, Ringler A, Wilson D, Wolin E (2018) Do low-
cost seismographs perform well enough for your network? an overview of laboratory tests and field observations of the
OSOP Raspberry Shake 4D. Seismol Res Lett 90(1):219–228. https://doi.org/10.1785/0220180251 Daggitt ML, Mather T, Pyle DM, Page S (2014) AshCalc–a new tool
for the comparison of the exponential, power-law and Weibull
models of tephra deposition. Journal of Applied Volcanology 3(7). https://doi.org/10.1186/2191–5040–3–7 Aurelio M, Lagmay A, Escudero J, Catugas S (2020a) Another
large earthquake strikes the southern Philippines. Temblor, p 1. https://doi.org/10.32858/temblor.196 Del Castillo M, Paraiso P, Vicente M, Jamero M, Narisma G (2020)
Impacts of Taal Volcano phreatic eruption (12 January 2020)
on the environment and population: satellite-based observations
compared with historical records. https://bit.ly/3kdAxCh Aurelio M, Lagmay A, Escudero J, Catugas S (2020b) Latest
Philippine earthquake reveals tectonic complexity. Temblor, p 1. https://doi.org/10.32858/temblor.191 Bachmeier S (2020) Eruption of the Taal Volcano in the Philippines. CIMSS Satellite Blog. https://bit.ly/3sm17Nn Fierstein J, Nathenson M (1992) Another look at the calcula-
tion of fallout tephra volumes. Bull Volcanol 54(2):156–167. https://doi.org/10.1007/BF00278005 Baclig C (2020) Out of 45 countries, PH ranks first as most obsessed
with social media. Philippine Daily Inquirer. https://bit.ly/3kkb627 Global Volcanism Program (2020) Report on Taal (Philippines)
Krippner JB, Venzke E (eds). Bulletin of the Global Vol-
canism
Network,
vol
45
No. 6. Smithsonian
Institution. https://doi.org/10.5479/si.GVP.BGVN202006-273070 Bato MG, Lundgren P, Pinel V, Jr RS, Daag A, Cahulogan M (2021)
The 2020 eruption and large lateral dike emplacement at Taal Vol-
cano, Philippines: insights from satellite radar data. Geophysical
Research Letters. https://doi.org/10.1029/2021GL092803 Guffanti M, Casadevall T, Budding K (2010) Encounters of aircraft
with volcanic ash clouds; A compilation of known incidents,
1953–2009. US Geological Survey Data 545:12. https://pubs.usgs. gov/ds/545/ Baxter
P,
Bernstein
R,
Falk
H,
French
J
(1982)
Medical
aspects of a volcanic disaster: an outline of the hazards
and emergency response measures. Disasters 6(4):268–276. https://doi.org/10.1111/j.1467-7717.1982.tb00549.x Gurioli L, Di Muro A, Vlast´elic I, Moune S, Thivet S, Valer M,
Villeneuve N, Boudoire G, Peltier A, Bach`elery P et al (2018)
Integrating field, textural, and geochemical monitoring to track
eruption triggers and dynamics: a case study from Piton de la Four-
naise. Solid Earth 9(2):431. Conclusions https://doi.org/10.5038/2163-
338X.1.2 Biass S, Bonadonna C, Houghton BF (2019) A step-by-step evaluation
of empirical methods to quantify eruption source parameters from
tephra-fall deposits. Journal of Applied Volcanology 8(1):1–16. https://doi.org/10.1186/s13617-018-0081-1 Funding This work is funded by the National Institute of Geological
Sciences and the UP Resilience Institute project on complex hazards. It
is part of the Geoheritage for Geohazard Resilience (UNESCO Project
692) which aims to empower areas with the capacity to communicate
and manage geohazards and to establish a global community for best
practice in Geoheritage for Geohazard Resilience. Bonadonna C, Costa A (2012) Estimating the volume of tephra
deposits: a new simple strategy. Geol 40(5):415–418. https://doi. org/10.1130/G32769.1 Bonadonna C, Costa A (2013) Plume height, volume, and classifica-
tion of explosive volcanic eruptions based on the Weibull function. Bull Volcanol 75(8):1–19. https://doi.org/10.1007/s00445-013-
0742-1 Bonadonna C, Houghton B (2005) Total grain-size distribution and
volume of tephra-fall deposits. Bull Volcanol 67(5):441–456. https://doi.org/10.1007/s00445-004-0386-2 Open Access This article is licensed under a Creative Commons
Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format, as
long as you give appropriate credit to the original author(s) and the
source, provide a link to the Creative Commons licence, and indicate
if changes were made. The images or other third party material in this
article are included in the article’s Creative Commons licence, unless
indicated otherwise in a credit line to the material. If material is not
included in the article’s Creative Commons licence and your intended
use is not permitted by statutory regulation or exceeds the permitted
use, you will need to obtain permission directly from the copyright
holder. To view a copy of this licence, visit http://creativecommons. org/licenses/by/4.0/. Bonadonna C, Ernst G, Sparks R (1998) Thickness variations and
volume estimates of tephra fall deposits: the importance of particle
Reynolds number. J Volcanol Geotherm Res 81(3):173–187. https://doi.org/10.1016/S0377-0273(98)00007-9 Casadevall T (1992) Volcanic hazards and aviation safety: lessons of
the past decade. Federal Aviation Administration Aviation Safety
Journal 2:3–11 Chen J (2020) Manila Airport partially reopens after Taal volcano
eruption leads to over 240 cancelled flights. Business Traveller
Asia Pacific https://bit.ly/3ma2f5q CNN Philippines Staff (2020) Agricultural damage from Taal eruption
hits Php 3.06-B. CNN Philippines https://bit.ly/3smrLWr Congress of the Philippines (2020) Republic Act 9500, The University
of the Philippines Charter of 2008. https://www.officialgazette. gov.ph/2008/04/19/republic-act-no-9500/ References https://doi.org/10.5194/se-9-431-2018 Bent A, Cassidy J, Pr´epetit C, Lamontagne M, Ulysse S (2018) Real-
time seismic monitoring in Haiti and some applications. Seismol
Res Lett 89(2A):407–415 Bernard A, Villacorte E, Maussen K, Caudron C, Robic J, Maximo R,
Rebadulla R, Bornas MA, Solidum Jr R (2020) Carbon dioxide Bull Volcanol (2022) 84: 35 Page 21 of 22 35 Page 21 of 22 35 Holmgren J, Werner M (2021) Raspberry shake instruments provide
initial ground-motion assessment of the induced seismicity
at the United Downs Deep Geothermal Power Project in
Cornwall, United Kingdom. The Seismic Record 1(1):27–34. https://doi.org/10.1785/0320210010 Pyle DM (1995) Assessment of the minimum volume of tephra
fall
deposits. J
Volcanol
Geotherm
Res
69(3-4):379–382. https://doi.org/10.1016/0377-0273(95)00038-0 Pyle DM (1999) Widely dispersed quaternary tephra in Africa. Glob
Planet Chang 21(1-3):95–112. https://doi.org/10.1016/S0921-
8181(99)00009-0 International ASTM (2014) Standard Specification for Woven Wire
Test Sieve Cloth and Test Sieves. ASTM International, West
Conshocken, PA Rappler (2020) NAIA flights ’on hold’ due to Taal volcano ash
eruption. Rappler. https://bit.ly/3xWvY46 Reuters (2020) Philippines suspends Manila airport flights as volcano
spews ash. Reuters. https://reut.rs/3yVxiFV Jordan S, D¨urig T, Cas R, Zimanowski B (2014) Processes controlling
the shape of ash particles: Results of statistical IPA. J Volcanol
Geotherm Res 288:19–27. https://doi.org/10.1016/j.jvolgeores. 2014.09.012 Ross P, D¨urig T, Comida P, Lefebvre N, White J, Andronico D,
Thivet S, Eychenne J, Gurioli L (2021) Standardized analysis
of juvenile pyroclasts in comparative studies of primary magma
fragmentation; 1. Overview and workflow. Bull Volcanol 84(13). https://doi.org/10.1007/s00445-021-01516-6 Lagmay A, Balangue-Tarriela R, Aurelio M, Ybanez R, Ybanez
A, Sulapas J, Baldago M, Sarmiento D, Cabria H, Rodolfo R,
Rafael D, Trinidad R, Obille E, Rosell N (2021) Hazardous base
surges of taal’s 2020 eruption. Scientific Reports, 11(15703). https://doi.org/10.1038/s41598-021-94866-2 p
g
Subedi S, Het´enyi G, Denton P, Sauron A (2020) Seismology at
school in Nepal: a program for educational and citizen seismology
through a low-cost seismic network. Frontiers in Earth Science
8:73. https://doi.org/10.3389/feart.2020.00073 Le Maitre RW, Streckeisen A, Zanettin B, Le Bas M, Bonin B,
Bateman P (2005) Igneous rocks: a classification and glossary of
terms: recommendations of the International Union of Geological
Sciences Subcommission on the Systematics of Igneous Rocks. Cambridge University Press, Cambridge Sulpizio R (2005) Three empirical methods for the calculation of
distal volume of tephra-fall deposits. J Volcanol Geotherm Res
145(3):315–336. https://doi.org/10.1016/j.jvolgeores.2005.03.001 Legros F (2000) Minimum volume of a tephra fallout deposit estimated
from a single isopach. J Volcanol Geotherm Res 96(1-2):25–32. Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations. References https://doi.org/10.1016/S0377-0273(99)00135-3 Thivet S, Gurioli L, Di Muro A, Eychenne J, Besson P, Ned-
elec JM (2020) Variability of ash deposits at Piton de la
Fournaise (La Reunion Island): insights into fragmentation pro-
cesses at basaltic shield volcanoes. Bull Volcanol 82(9):1–20. https://doi.org/10.1007/s00445-020-01398-0 Miklius A, Flower M, Huijmans J, Mukasa S, Samuel B, Castillo P
(1991) Geochemistry of lavas from Taal Volcano, southwestern
Luzon, Philippines: evidence for multiple magma supply sys-
tems and mantle source heterogeneity. J Petrol 32(3):593–627. https://doi.org/10.1093/petrology/32.3.593 Torres R, Self S, Punongbayan R (1995) Attention focuses on taal:
Decade volcano of the philippines. Eos, Transactions American
Geophysical Union 76(24):241–247 MMDA (2020) MMDA to Set Up Portable Water Purifiers in
Batangas. https://bit.ly/3xTccGE Van Eaton AR, Schneider DJ, Smith CM, Haney MM, Lyons JJ, Said
R, Fee D, Holzworth RH, Mastin LG (2020) Did ice-charging
generate volcanic lightning during the 2016–2017 eruption of
bogoslof volcano, alaska? Bull Volcanol 82(3):1–23 Perttu A, Taisne B, De Angelis S, Assink JD, Tailpied D, Williams
RA (2020) Estimates of plume height from infrasound for regional
volcano monitoring. J Volcanol Geotherm Res 106997:402. https://doi.org/10.1016/j.jvolgeores.2020.106997 Viray PL (2020) DOH cautions public on health effects of Taal ashfall. Philstar Global. https://bit.ly/2XAp0W7 PHIVOLCS (2020a) Taal Volcano Bulletin 12 January 2020 04:00 PM. https://bit.ly/3z7Fxil Watt SF, Pyle DM, Mather TA, Martin RS, Matthews NE (2009) Fall-
out and distribution of volcanic ash over Argentina following the
May 2008 explosive eruption of chait´en, Chile. Journal of Geo-
physical Research: Solid Earth, 114(B4). https://doi.org/10.1029/
2008JB006219 PHIVOLCS (2020b) Taal Volcano Bulletin 26 January 2020. https://
bit.ly/3AQY1nk Prata A, Folch A, Prata A, Biondi R, Brenot H, Cimarelli C, Corradini
S, Lapierre J, Costa A (2020) Anak krakatau triggers volcanic
freezer in the upper troposphere. Scientific Reports 10(1):1–13 White J, Valentine G (2016) Magmatic versus phreatomagmatic
fragmentation: absence of evidence is not evidence of absence. Geosphere 12:1478–1488. https://doi.org/10.1130/GES01337.1 PSA (2021) 2020 Census of Population and Housing (2020 CPH)
Population Counts Declared Official by the President. https://bit. ly/3xXPjBV Wohletz K (2013) Magma–water interactions. Cambridge University
Press, Cambridge. https://doi.org/10.1017/CBO9781139021562. 011 PTCC (2020) Are you affected? https://fb.watch/4mw1qqclqI/ Pyle DM (1989) The thickness, volume and grainsize of tephra
fall deposits. Bull Volcanol 51(1):1–15. https://doi.org/10.1007/
BF01086757 Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations. Pyle DM (1990) New estimates for the volume of the Minoan eruption. References Thera and the Aegean World III(2):113–121 35 Page 22 of 22 Bull Volcanol (2022) 84: 35 M. I. R. Balangue-Tarriela1 · A. M. F. Lagmay1,2
· D. M. Sarmiento1 · J. Vasquez2 · M. C. Baldago1 · R. Yba˜nez1 ·
A. A. Yba˜nez2 · J. R. Trinidad1 · S. Thivet3 · L. Gurioli3 · B. Van Wyk de Vries3 · M. Aurelio1 · D. J. Rafael1 · A. Bermas2 ·
J. A. Escudero1 3
Laboratoire Magmas et Volcans, Universit´e Clermont Auvergne,
CNRS, IRD, OPGC, F-63000 Clermont-Ferrand, France 2
UP Resilience Institute and NOAH Center, University of the
Philippines, Diliman, Quezon City, Metro Manila, Philippines 2
UP Resilience Institute and NOAH Center, University of the
Philippines, Diliman, Quezon City, Metro Manila, Philippines
3
Laboratoire Magmas et Volcans, Universit´e Clermont Auvergne,
CNRS, IRD, OPGC, F-63000 Clermont-Ferrand, France Affiliations M. I. R. Balangue-Tarriela1 · A. M. F. Lagmay1,2
· D. M. Sarmiento1 · J. Vasquez2 · M. C. Baldago1 · R. Yba˜nez1 ·
A. A. Yba˜nez2 · J. R. Trinidad1 · S. Thivet3 · L. Gurioli3 · B. Van Wyk de Vries3 · M. Aurelio1 · D. J. Rafael1 · A. Bermas2
J. A. Escudero1
2
UP Resilience Institute and NOAH Center, University of the
Philippines, Diliman, Quezon City, Metro Manila, Philippines
3
Laboratoire Magmas et Volcans, Universit´e Clermont Auvergne,
CNRS, IRD, OPGC, F-63000 Clermont-Ferrand, France M. I. R. Balangue-Tarriela1 · A. M. F. Lagmay1,2
· D. M. Sarmiento1 · J. Vasquez2 · M. C. Baldago1 · R. Yba˜nez1 ·
A. A. Yba˜nez2 · J. R. Trinidad1 · S. Thivet3 · L. Gurioli3 · B. Van Wyk de Vries3 · M. Aurelio1 · D. J. Rafael1 · A. Bermas2 ·
J. A. Escudero1 M. I. R. Balangue-Tarriela1 · A. M. F. Lagmay1,2
· D. M. Sarmiento1 · J. Vasquez2 · M. C. Baldago1 · R. Yba˜nez1 ·
A. A. Yba˜nez2 · J. R. Trinidad1 · S. Thivet3 · L. Gurioli3 · B. Van Wyk de Vries3 · M. Aurelio1 · D. J. Rafael1 · A. Bermas2 ·
J. A. Escudero1
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The Dutch language anterior cruciate ligament return to sport after injury scale (ACL-RSI) – validity and reliability
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Journal of sports sciences
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University of Groningen University of Groningen Journal of Sports Sciences Journal of Sports Sciences ISSN: 0264-0414 (Print) 1466-447X (Online) Journal homepage: http://www.tandfonline.com/loi/rjsp20 Date: 14 December 2017, At: 06:44 The Dutch language anterior cruciate ligament return to sport after injury scale (ACL-RSI) -
validity and reliability
Slagers, Anton J.; Reininga, Inge H. F.; van den Akker-Scheek, Inge IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from
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Slagers, A. J., Reininga, I. H. F., & van den Akker-Scheek, I. (2017). The Dutch language anterior cruciate
ligament return to sport after injury scale (ACL-RSI) - validity and reliability. Journal of Sports Sciences,
35(4), 393-401. https://doi.org/10.1080/02640414.2016.1167230 Copyright
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University Medical Center Groningen, PO Box 30.001 9700 RB Groningen, The Netherlands
The local Institutional Review Board approved the procedures employed in this study.
© 2016 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited. ABSTRACT Returning to the sport before the athlete is
psychologically ready may lead to anxiety, reoccurrence of
injury, injuries to other body parts, depression, and perfor-
mance degradation (Gobbi & Francisco, 2006; Tjong et al.,
2014; Tripp et al., 2007). It is suggested that attention to
psychological recovery, in addition to physical recovery, after
ACL injury and reconstruction surgery may be warranted Stanish, Ebel-Lam, Brewer, & Birchard, 2007). The athlete’s
psychological response before and after surgery was found
to be related to whether or not the athlete returned to pre-
injury level of sports at 12 months post-operatively (Ardern,
Webster, Taylor, & Feller, 2011; Langford et al., 2009; Webster
et al., 2008). Furthermore, fear of new injuries, reduced moti-
vation,
and
other
negative
psychological
reactions
are
described in athletes who did not return to the pre-injury
level of sports after ACL reconstruction (Gobbi & Francisco,
2006; Kvist et al., 2005; Langford et al., 2009; Tjong et al., 2014;
Tripp et al., 2007). Consequently, athletes should not only be
physically prepared to return to sports but they also have to
be psychologically ready (Ardern et al., 2012, 2013, 2014). Research showed that physical and psychological readiness
to return to the sports, do not always coincide (Quinn &
Fallon, 1999). Returning to the sport before the athlete is
psychologically ready may lead to anxiety, reoccurrence of
injury, injuries to other body parts, depression, and perfor-
mance degradation (Gobbi & Francisco, 2006; Tjong et al.,
2014; Tripp et al., 2007). It is suggested that attention to
psychological recovery, in addition to physical recovery, after
ACL injury and reconstruction surgery may be warranted ARTICLE HISTORY
Accepted 2 March 2016
KEYWORDS
ACL reconstruction; knee;
psychology; return to sport;
patient-reported measures Anton J. Slagersa,b, Inge H. F. Reiningac,d and Inge van den Akker-Scheeka,e aCenter for Sports Medicine, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands; bDepartment of
Rehabilitation, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands; cDepartment of Trauma Surgery,
University of Groningen, University Medical Center Groningen, Groningen, The Netherlands; dDepartment of Orthopaedics, Martini Hospital
Groningen, Groningen, The Netherlands; eDepartment of Orthopaedics, University of Groningen, University Medical Center Groningen, Groningen,
The Netherlands ARTICLE HISTORY
Accepted 2 March 2016
KEYWORDS
ACL reconstruction; knee;
psychology; return to sport;
patient-reported measures ABSTRACT ABSTRACT
The ACL-Return to Sport after Injury scale (ACL-RSI) measures athletes’ emotions, confidence in
performance, and risk appraisal in relation to return to sport after ACL reconstruction. Aim of this
study was to study the validity and reliability of the Dutch version of the ACL-RSI (ACL-RSI (NL)). Downloaded by [University of Groningen] at 06:44 14 December 2017 Total 150 patients, who were 3–16 months postoperative, completed the ACL-RSI(NL) and 5 other
questionnaires regarding psychological readiness to return to sports, knee-specific physical functioning,
kinesiophobia, and health-specific locus of control. Construct validity of the ACL-RSI(NL) was deter-
mined with factor analysis and by exploring 10 hypotheses regarding correlations between ACL-RSI(NL)
and the other questionnaires. For test–retest reliability, 107 patients (5–16 months postoperative)
completed the ACL-RSI(NL) again 2 weeks after the first administration. Cronbach’s alpha, Intraclass
Correlation Coefficient (ICC), SEM, and SDC, were calculated. Bland–Altman analysis was conducted to
assess bias between test and retest. Nine hypotheses (90%) were confirmed, indicating good construct validity. The ACL-RSI(NL) showed
good internal consistency (Cronbach’s alpha 0.94) and test–retest reliability (ICC 0.93). SEM was 5.5 and
SDC was 15. A significant bias of 3.2 points between test and retest was found. Downloaded by [University of Groningen] a Therefore, the ACL-RSI(NL) can be used to investigate psychological factors relevant to returning to
sport after ACL reconstruction. Stanish, Ebel-Lam, Brewer, & Birchard, 2007). The athlete’s
psychological response before and after surgery was found
to be related to whether or not the athlete returned to pre-
injury level of sports at 12 months post-operatively (Ardern,
Webster, Taylor, & Feller, 2011; Langford et al., 2009; Webster
et al., 2008). Furthermore, fear of new injuries, reduced moti-
vation,
and
other
negative
psychological
reactions
are
described in athletes who did not return to the pre-injury
level of sports after ACL reconstruction (Gobbi & Francisco,
2006; Kvist et al., 2005; Langford et al., 2009; Tjong et al., 2014;
Tripp et al., 2007). Consequently, athletes should not only be
physically prepared to return to sports but they also have to
be psychologically ready (Ardern et al., 2012, 2013, 2014). Research showed that physical and psychological readiness
to return to the sports, do not always coincide (Quinn &
Fallon, 1999). The Dutch language anterior cruciate ligament return to sport after injury scale
(ACL-RSI) – validity and reliability The Dutch language anterior cruciate ligament return to sport after injury scale
(ACL-RSI) – validity and reliability Anton J. Slagersa,b, Inge H. F. Reiningac,d and Inge van den Akker-Scheeka,e Anton J. Slagers, Inge H. F. Reininga & Inge van den Akker-Scheek To cite this article: Anton J. Slagers, Inge H. F. Reininga & Inge van den Akker-Scheek (2017)
The Dutch language anterior cruciate ligament return to sport after injury scale (ACL-RSI) – validity
and reliability, Journal of Sports Sciences, 35:4, 393-401, DOI: 10.1080/02640414.2016.1167230 To link to this article: https://doi.org/10.1080/02640414.2016.1167230 © 2016 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group
Published online: 15 Apr 2016. Submit your article to this journal
Article views: 1319
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Citing articles: 5 View citing articles © 2016 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
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Published online: 15 Apr 2016. Submit your article to this journal
Article views: 1319
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http://www.tandfonline.com/action/journalInformation?journalCode=rjsp20 Date: 14 December 2017, At: 06:44 Download by: [University of Groningen] JOURNAL OF SPORTS SCIENCES, 2017
VOL. 35, NO. 4, 393–401
http://dx.doi.org/10.1080/02640414.2016.1167230 JOURNAL OF SPORTS SCIENCES, 2017
VOL. 35, NO. 4, 393–401
http://dx.doi.org/10.1080/02640414.2016.1167230 Introduction Anterior cruciate ligament (ACL) rupture is a common injury in
athletes. An ACL rupture is often surgically treated with an
ACL reconstruction to restore the stability of the knee, aiming
to allow patients to return to sport after rehabilitation (Marx,
Jones, Angel, Wickiewicz, & Warren, 2003; Myklebust, 2005). After ACL reconstruction and the subsequent rehabilitation,
many patients report a good knee function but do not return
to their pre-injury level of sport participation (Ardern, Taylor,
Feller, & Webster, 2014; Ardern et al., 2014; Feller & Webster,
2003; Kvist, Ek, Sporrstedt, & Good, 2005; Langford, Webster, &
Feller, 2009; Webster, Feller, & Lambros, 2008). As surgery and
functional outcome were satisfactory in these patients, the
question remains which other factors may cause the low
return-to-sports rate. Previous research showed that the low rate of return to
sports might be caused by psychological reactions to the
initial injury, surgery, and following rehabilitation (Ardern,
Taylor, Feller, & Webster, 2012, 2013; Kvist et al., 2005;
Langford et al., 2009; Tjong, Murnaghan, Nyhof-Young, &
Ogilvie-Harris, 2014). The main reason is fear of re-rupture of
the ACL (Ardern et al., 2013, 2014; Tjong et al., 2014; Tripp, A. J. SLAGERS ET AL. 394 (Ardern et al., 2011, 2014; Langford et al., 2009; Tripp et al.,
2007; Webster et al., 2008). period from 1 January 2012 to 1 February 2013 were recruited. Patients were eligible for participation when they had under-
gone ACL reconstruction 3–16 months previous to the start of
the study. This time interval was chosen because (1) patients
start to perform sport-specific exercises and focus increasingly
on return to the sport from about 3 months after ACL recon-
struction, and (2) it was expected that 16 months after recon-
struction the patients have returned in the sport. Patients who
were unable to understand written Dutch were excluded. To evaluate various psychological factors associated with
return to sports, several questionnaires have been used (Gobbi
& Francisco, 2006; Kori, Miller, & Todd, 1990; Morrey, Stuart,
Smith, & Wiese-Bjornstal, 1999; Thomeé et al., 2008). However,
these scales were not developed specifically to evaluate the
psychological impact of returning to sport after ACL injury or
reconstruction. Therefore, Webster et al. developed the ACL Return to
Sport after Injury scale (ACL-RSI) that measures athletes’ emo-
tions, confidence in performance, and risk appraisal in relation
to return to sport after ACL injury (Webster et al., 2008). Introduction The
ACL-RSI has been shown to discriminate between athletes
who return and athletes who do not return to sport after
reconstruction surgery. The scale was recently validated in
Swedish and French (Bohu, Klouche, Lefevre, Webster, &
Herman, 2015; Kvist et al., 2013). In the Netherlands, there is
need for a questionnaire that measures the psychological
impact of returning to sport after ACL injury or reconstruction. Moreover, in general translated versions of the ACL-RSI in
different languages are needed in order to be able to compare
the results of different studies conducted in countries that
speak different languages. Translation The developers of the original ACL-RSI were informed and
gave their consent to a Dutch translation of the ACL-RSI
questionnaire (Webster, personal communication, 2013). The
English language ACL-RSI was translated according to the
method described by Beaton (Beaton et al., 2000). This
method recognises 5 stages: (1) translation, (2) synthesis, (3)
back translation, (4) expert committee review, and (5) pre-
testing. The expert committee consisting of 4 translators
from stages 1 and 3, 2 human movement scientist/epidemiol-
ogist, and a sports physical therapist drafted the final version
of the Dutch version of the ACL-RSI (ACL-RSI(NL)) (stage 4),
which was pre-tested on 15 patients (4–12 months after ACL
reconstruction). Demographic characteristics were retrieved from the elec-
tronic patient records (gender, height, weight, age, and sur-
gery date). To measure the current level of work and sports
activities, patients were asked to complete the Tegner score in
the first mailing. Methods The ACL-RSI was translated into Dutch according to interna-
tional guidelines (Beaton, Bombardier, Guillemin, & Ferraz,
2000). Then, the translated version was tested for clinimetric
properties. The local Medical Ethical Committee approved the
procedures employed in this study. Questionnaires The ACL-RSI(NL) scale consists of 12 questions regarding the
psychological impact of returning to sports in this population. The original ACL-RSI items were scored on a visual analogical
scale (VAS) from 0 to 100. Upon the advice of Webster
(Webster et al., 2008), we have used an adapted version of
the ACL-RSI, which is also used in several follow-up studies Procedure Eligible patients were sent 6 questionnaires in order to assess
construct validity:
the
ACL-RSI(NL), the
Multidimensional
Health Locus of Control Scale (MHLC) (Wallston, Stein, &
Smith, 1994), the Injury-Psychological Readiness to Return to
Sport scale (I-PRRS) (Glazer, 2009), the Tampa Scale of
Kinesiophobia (TSK) (Vlaeyen, Kole-Snijders, Boeren, & Van
Eek, 1995), the Knee injury and Osteoarthritis Outcome Score
(KOOS) (De Groot, Favejee, Reijman, Verhaar, & Terwee, 2008),
and the International Knee Documentation Committee Score
(IKDC) 2000 (Haverkamp, 2006). Questionnaires were sent by
mail with an accompanying letter explaining the study and
explaining that all data will be anonymised and that return of
the questionnaire will be considered as consent to participate. Patients were asked to fill in the questionnaires at home and
to return them by mail. Patients who did not respond after 1
week were reminded. Downloaded by [University of Groningen] at 06:44 14 December 2017 In order to be able to use the ACL-RSI by Dutch speaking
patients, the aim of this study was to translate the ACL-RSI into
Dutch and to study the validity and reliability of the ACL-
RSI(NL). Downloaded by [University of Groningen] a Patients who underwent ACL reconstruction 3–4 months
before the start of the study are not likely to be in a stable
state since these patients are starting at that phase of the
rehabilitation with sport-specific exercises and actual changes
in status are very likely (Van Grinsven, Van Cingel, Holla, & Van
Loon, 2010). Hence, we asked patients who underwent ACL
reconstruction at least 5 months previous to the start of the
study to complete the ACL-RSI(NL) questionnaire for a second
time after an interval of 2 weeks to explore test–retest relia-
bility. We confirmed our assumption by having these same
patients provide a Global Rating of Change (GRC) score to
quantify a change in the confidence regarding sports resump-
tion during the previous 2 weeks. The GRC was scored on a 5-
point Likert scale ranging from much more confident (+2) to
much less confident (−2). Participants who reported being
much more confident or less confident on the GRC were
excluded from our test–retest analysis. Reliability According to the COSMIN guidelines, reliability was assessed
in terms of internal consistency, test–retest reliability, and
measurement error (Mokkink et al., 2010). Reliability refers to
the extent to which an instrument consistently yields the same
scores on 2 successive occasions in stable persons and the
extent to which patients can be distinguished from each other
despite measurement errors. Internal consistency is a measure
of the extent to which items in a questionnaire’s (sub)scale are
correlated (homogeneous), thus measuring the same concept
(Terwee et al., 2007). Test–retest reliability concerns the extent
to which scores of patients are the same for repeated mea-
surements (Mokkink et al., 2010). The Bland and Altman
method was used to explore repeatability, which reflects the
amount of agreement in repeated measurements (Bland &
Altman, 1986). Additionally, measurement error was investi-
gated; measurement error is a measure of systematic error of a
patient’s score what is not caused by actual changes in the
measured construct (Mokkink et al., 2010). The IKDC Subjective Knee Form 2000 is an 18-item instru-
ment designed to measure pain, symptoms, function, and
sports activity in patients with a variety of knee conditions
(Haverkamp, 2006). A sum score was calculated, which is then
transformed to 100-point scale, with 100 indicating no restric-
tions in daily and sports activities and the absence of symp-
toms. The IKDC 2000 was translated to Dutch and validated for
a heterogeneous population of patients with knee complaints. The TSK measures fear for re-injury due to movement and
physical activity. It contains 17 items scored on a 4-point Likert
scale regarding the subjective experience of the injury and
physical activity. The sum of the items results in a score
between 17–68, where 68 indicates a high level of fear (Kori
et al., 1990). There is a Dutch version of the TSK available
(Vlaeyen et al., 1995). For this study, the modified Dutch
version, adapted for knee injuries was used. The MHLC measures different dimensions of the Health
Locus of Control (HLC), that is, persons’ believe whether
their health is determined by their behaviour or external
events
(Halfens
&
Philipsen,
1988;
Wallston,
Wallston,
Kaplan, & Maides,1976). Forms A and B are general health
locus of control scales. Form C is used in place of Form A/B
when studying people with an existing medical or health-
related problem (Wallston et al., 1994). Participants Patients who had undergone ACL reconstruction at the
department
of
Orthopaedics
of
the
Martini
Hospital
Groningen or at the department of Orthopaedics or Trauma
Surgery of the University Medical Center Groningen in the JOURNAL OF SPORTS SCIENCES 395 (Bohu et al., 2015; Kvist et al., 2005, 2013; Langford et al.,
2009). The items of the adapted ACL-RSI are identical to
those of the original version, but the VAS is replaced by an
11-point Numeric Rating Scale (NRS) in the form of boxes to
be ticked from 0 to 100. This version of the ACL-RSI was
recently validated in the Swedish and French language
(Bohu et al., 2015; Kvist et al., 2013) (Bohu et al., 2015; Kvist et al., 2005, 2013; Langford et al.,
2009). The items of the adapted ACL-RSI are identical to
those of the original version, but the VAS is replaced by an
11-point Numeric Rating Scale (NRS) in the form of boxes to
be ticked from 0 to 100. This version of the ACL-RSI was
recently validated in the Swedish and French language
(Bohu et al., 2015; Kvist et al., 2013) Floor and ceiling effects The presence of floor and ceiling effects may jeopardise the
validity and reliability of a questionnaire instrument. It is then
likely that extreme items are missing in the lower or upper
ends of the questionnaire (Terwee et al., 2007). Floor and
ceiling effects are defined as 15% of the participants achieving
the minimum or maximum score, respectively (McHorney &
Tarlov, 1995). Reliability The MHLC-C con-
sists of 4 subsections; Internal, Chance, Doctors, and Other
(powerful) People. The internal HLC subsection assesses the
extent to which one believes that internal factors are
responsible for health/illness. Persons with strong internal
HLC believe that the outcome is directly a result of their
own behaviour or action. In this validation study, only the
internal HLC subsection is used. We translated the MHLC-C
into Dutch following the international guidelines (Beaton
et al., 2000). For the current study, the word “condition”
was replaced by “knee problems”. Construct validity Validity of the ACL-RSI(NL) was expressed in terms of construct
validity. Construct validity refers to the extent to which scores
on a particular measure relate to other measures, consistent
with theoretically derived hypotheses concerning the con-
structs that are being measured (Terwee et al., 2007). Construct validity of the ACL-RSI(NL) was determined by eval-
uating its structural validity and by hypothesis testing. Structural validity was assessed to determine whether the
ACL-RSI(NL) is also 1 dimensional (i.e., it does not contain
subscales), like the original ACL-RSI. Additionally, according
to
the
guidelines
proposed
by
the
COSMIN
initiative
(Mokkink et al., 2010), we formulated 10 hypotheses about
the magnitude of relationships between the ACL-RSI(NL) and
the corresponding (sub)scales of the I-PRRS, KOOS, IKDC 2000,
TSK, and MHLC (Table 4). According to the COSMIN guidelines,
the construct validity of the ACL-RSI(NL) is considered good if
≥75% of the predefined hypotheses are confirmed (Terwee
et al., 2007). The I-PRRS was developed to assess an athlete’s psycholo-
gical readiness to return to sport participation after injury. The
I-PRRS is a valid and reliable questionnaire and consists of 6
items that are scored on a 100-point scale (Glazer, 2009). The
total score consists of the raw sum score of the 6 items,
divided by 10. A high score (max 60) implies a higher con-
fidence to return to sport. We translated the I-PRRS into Dutch
following the international guidelines (Beaton et al., 2000). The KOOS was developed to assess the patient’s opinion
about their knee and associated problems. The KOOS contains
42 questions and consists of 5 subscales; pain, other symp-
toms, function in daily living (ADL), function in sport and
recreation (Sport/Rec), and knee-related quality of life (QOL). The KOOS has been validated in Dutch (De Groot et al., 2008). Questions were answered using a 5-point Likert scale. For each
subscale, a sum score was calculated and converted into a
100-point scale. Higher scores reflect fewer symptoms and
limitations. Construct validity Internal consistency of the ACL-RSI(NL) was assessed with
Cronbach’s alpha using the data from the first administration
of the ACL-RSI(NL). It is widely accepted that Cronbach’s alpha
should be between 0.70 and 0.95 (Terwee et al., 2007). To
determine test–retest reliability of the ACL-RSI(NL), the intra-
class correlation coefficient (ICC: 2-way random, type agree-
ment) with corresponding 95% CIs were calculated between
the first and second administration of the ACL-RSI(NL). The ICC
is generally considered to be good at 0.70 and above (Terwee
et al., 2007). Internal consistency of the ACL-RSI(NL) was assessed with
Cronbach’s alpha using the data from the first administration
of the ACL-RSI(NL). It is widely accepted that Cronbach’s alpha
should be between 0.70 and 0.95 (Terwee et al., 2007). To
determine test–retest reliability of the ACL-RSI(NL), the intra-
class correlation coefficient (ICC: 2-way random, type agree-
ment) with corresponding 95% CIs were calculated between
the first and second administration of the ACL-RSI(NL). The ICC
is generally considered to be good at 0.70 and above (Terwee
et al., 2007). The factor analysis showed a 1-factor structure of the ACL-RSI
(NL), with an explained variance of 59% and an eigenvalue of
7.1. The Spearman correlations between the ACL-RSI(NL) and
the I-PRRS, the subscales of the KOOS, IKDC 2000, TSK, and
MHLC-C are shown in Table 3. The ACL-RSI(NL) showed a high correlation with the I-PRRS
(r = 0.79) and a moderate correlation with the 4 subscales of the
KOOS (r = 0.30–0.48). Only the subscale “activities of daily living”
has a low correlation with the ACL-RSI(NL) (r = 0.25). The KOOS
subscales “sport and function” (r = 0.48) and “knee-related
quality of life” (r = 0.40) had higher correlations than the other
KOOS subscales. Moderate correlation was found with the TSK
(r = −0.46) and a low correlation with the MHLC-C (r = −0.15). Of
the predefined hypotheses to determine construct validity, 9 of
the 10 were confirmed (90%) (Table 4). Only the hypothesis
regarding the correlation between the ACL-RSI(NL) and the
KOOS subscale “function in daily living” was rejected, since a
correlation of 0.25 instead of 0.30–0.60 was found. Downloaded by [University of Groningen] Additionally, a Bland and Altman analysis was performed; the
mean difference between the first and second administration of
the ACL-RSI(NL) with a 95% CI was calculated. Clinimetric properties Clinimetric properties assessing the construct validity of the ACL-RSI(NL), and a
sample size of at least 50 for establishing the test–retest
reliability of the ACL-RSI(NL). p
In total 241 patients were approached. Of these, 150 patients
returned the first questionnaires response rate 62%). Two
weeks after receiving the first questionnaires, all 122 respon-
dents who were treated with an ACL reconstruction 5–16
months prior to the second questionnaire start of the study,
were asked to complete the ACL-RSI(NL) once more. The
second questionnaire was returned by 107 patients (response
rate 88%). The mean time between receiving the first and
second questionnaire was 22.9 days (SD 7.9). A flow diagram
of inclusion of participants is shown in Figure 1. Demographic
characteristics of the participants are shown in Table 1. Construct validity When zero is lying
within the 95% CI of the mean difference, it can be seen as a
criterion for absolute agreement. When zero lies outside the
95% CI, a bias in the measurements is indicated (Bland &
Altman, 1986) The standard error of measurement (SEM), a
measure of the instrument’s absolute measurement error, was
calculated. The value of the SEM can be derived by dividing the
SD of the mean differences between 2 measurements (SDdiff)
by √2 (de Vet, Terwee, Knol, & Bouter, 2006). The smallest
detectable change (SDC) for the individual score and for the
group
was
calculated
according
to
Beaton
(SDCind = 1,96x√2xSEM; SDCgroep = SDCind/√n) (Beaton
et al., 2000). Construct validity To assess the structural validity of the ACL-RSI(NL), exploratory
factor analysis was conducted on all ACL-RSI(NL)items using
principal component analyses (PCA) with varimax rotation. Factor analyses with 1-, 2-, 3-factor solution were performed. The Spearman’s Rho was calculated between the ACL-RSI(NL)
and the other questionnaires. The Spearman’s Rho was inter-
preted according to Hinkle (Hinkle, Wiersma, & Jurs, 1998). Correlation coefficients above 0.6, 0.6–0.3, and less than 0.3
are considered to be high, moderate, and low, respectively. Description of the results
An overview of the scores on the various questionnaires is
shown in Table 2. Downloaded by [University of Groningen] at 06:44 14 December 2017 Reliability The Cronbach’s alpha of ACL-RSI(NL) was 0.94, which indicates
good internal consistency. Of the 107 participants who
returned the second administration of the ACL-RSI(NL), 74
(49.3%) participants indicated that no change had occurred
in the expectations regarding sport resumption in the 2 weeks
prior to completion of the retest. Seven participants (4.7%)
reported less confidence, 23 (15.3%) more confidence, and 3
participants (2%) reported to have much more confidence. These 3 participants were excluded from the test–retest relia-
bility analysis. The test–retest reliability analyses are presented
in Table 5. The mean score for the ACL-RSI(NL) was 56.1 (SD
22.3) at the first measurement and 59.3 (SD 21.9) at the
second measurement. The mean difference was 3.2 (95% CI
1.6–4.6; SD 7.8), with the 95% CI not containing zero, indicat-
ing a systematic bias (Figure 2). The ICC was 0.93 (95% CI 0.88–
0.96), indicating excellent test–retest reliability. SEM was 5.5;
SDCind was 15.3, and SDCgroup 1.5. All statistical analyses were performed using PASW statistical
package (version 20, SPSS Inc, Chicago). A p-value of <0.05 indi-
cated statistical significance. Descriptive statistics were used for
patient characteristics and to display outcomes of questionnaires. Statistical analysis A sample size of at least 100 is considered an adequate sample
size for studies regarding measurement properties of ques-
tionnaires, and a sample size of 50 is considered adequate for
determining test–retest reliability (Terwee et al., 2012). Hence,
we planned a sample size of at least 100 participants for A. J. SLAGERS ET AL. 396 Floor and ceiling effects investigate psychological factors relevant to returning to sport
after ACL reconstruction. The ACL-RSI(NL) showed good valid-
ity and reliability. investigate psychological factors relevant to returning to sport
after ACL reconstruction. The ACL-RSI(NL) showed good valid-
ity and reliability. The mean total score for the ACL-RSI was 56.5 (range 8.3–100,
SD 22.2). No significant floor and ceiling effects were found:
0.7% of participants scored below 10 and 10.7% of partici-
pants scored 90 or higher. Construct validity In accordance with the English and Swedish versions, the
factor analysis revealed that the ACL-RSI(NL) primarily evalu-
ates 1 dimension (Kvist et al., 2013; Webster et al., 2008). Therefore, the 3 psychological responses that are included in
the ACL-RSI, that is, emotions, confidence in performance, and Translation Mean
SD
Minimum Maximum
ACL-RSI
56.5
22.2
8.3
100
ACL-RSI test–retest
59.8
21.9
10.8
100
IPRRS
43.2
13.1
0
60
KOOS subscale “pain”
84.1
15.6
22.2
100
KOOS subscale “other symptoms”
75.9
16.5
32.1
100
KOOS subscale “function in daily living”
91.1
14.8
0
100
KOOS subscale “function in sport and
recreation”
65.9
23.3
0
100
KOOS subscale “knee-related Quality of
life”
49.7
13.0
18.8
81.3
IKDC 2000
76.9
16
6.9
100
TSK
36.6
6.5
17
56
MHLC-C
19.4
6.2
6
36
ACL-RSI, Anterior Cruciate Ligament scale – Return to Sports after Injury scale;
IKDC,
International
Knee
Documentation
Committee
Score;
MHLC-C,
Multidimensional Health Locus of Control, C form; TSK, Tampa Scale of
Kinesiophobia; KOOS, Knee injury and Osteoarthritis Outcome Score. Table 2. Descriptive statistics of the various measurements. Mean
SD
Minimum Maximum
ACL-RSI
56.5
22.2
8.3
100
ACL-RSI test–retest
59.8
21.9
10.8
100
IPRRS
43.2
13.1
0
60
KOOS subscale “pain”
84.1
15.6
22.2
100
KOOS subscale “other symptoms”
75.9
16.5
32.1
100
KOOS subscale “function in daily living”
91.1
14.8
0
100
KOOS subscale “function in sport and
recreation”
65.9
23.3
0
100
KOOS subscale “knee-related Quality of
life”
49.7
13.0
18.8
81.3
IKDC 2000
76.9
16
6.9
100
TSK
36.6
6.5
17
56
MHLC-C
19.4
6.2
6
36
ACL-RSI, Anterior Cruciate Ligament scale – Return to Sports after Injury scale;
IKDC,
International
Knee
Documentation
Committee
Score;
MHLC-C,
Multidimensional Health Locus of Control, C form; TSK, Tampa Scale of
Kinesiophobia; KOOS, Knee injury and Osteoarthritis Outcome Score. Table 2. Descriptive statistics of the various measurements. Translation The ACL-RSI was successfully translated into Dutch (ACL-RSI
(NL)) according to guidelines (see Appendix 1). No changes
were made after pretesting the ACL-RSI(NL); all questions were
clear. A detailed report of the translation process can be
provided by the authors. JOURNAL OF SPORTS SCIENCES
397 397 Eligible patients
3-16 months after ACL reconstruction
(n = 241)
Patients’ participation
(n = 150); 62%
Non-response
(n=91); 38%
Internal consistency and validity
3-16 months after ACL reconstruction
(n = 150)
Test-retest reliability
5-16 months after ACL reconstruction
(n = 122)
Questionnaires returned
(n=107) 88%
Figure 1. Flowchart of patients’ participation. Eligible patients
3-16 months after ACL reconstruction
(n = 241) Non-response
(n=91); 38% Patients’ participation
(n = 150); 62% Downloaded by [University of Groningen] at 06:44 14 December 2017 Test-retest reliability
5-16 months after ACL reconstruction
(n = 122) Downloaded by [University of Groningen] Figure 1. Flowchart of patients’ participation. Floor and ceiling effects
The mean total score for the ACL-RSI was 56.5 (range 8.3–100,
SD 22.2). No significant floor and ceiling effects were found:
0.7% of participants scored below 10 and 10.7% of partici-
pants scored 90 or higher. Discussion
The aim of this study was to translate the ACL-RSI scale from
English to Dutch and to examine whether the Dutch version of
the ACL-RSI (ACL-RSI(NL)) is a valid and reliable instrument to
investigate psychological factors relevant to returning to sport
after ACL reconstruction. The ACL-RSI(NL) showed good valid-
ity and reliability. Construct validity
In accordance with the English and Swedish versions, the
factor analysis revealed that the ACL-RSI(NL) primarily evalu-
ates 1 dimension (Kvist et al., 2013; Webster et al., 2008). Therefore, the 3 psychological responses that are included in
the ACL-RSI, that is, emotions, confidence in performance, and
Table 1. Demographic characteristics of the participants. Characteristics
ACL-RSI (NL) samplea
Age (years) (N = 150)
29.2 (10.8)
Time after reconstruction (months)
9.5 (4.0)
Gender (N = 150)
Male (%)
84 (56)
Female (%)
66 (44)
Length (cm)
178 (9.4)
Body weight (kg)
79 (17)
Graft type
Semitendinosis/gracilis (%)
143 (95)
Bone-tendon-bone (%)
7 (5)
Tegner activity-level score
5.5 (2.2)
a Age, time after reconstruction, length, body weight, and Tegner activity level
are given as the mean, with the standard deviation in parentheses. Other
values are given as the number of patients, with the percentage in
parentheses. Table 2. Descriptive statistics of the various measurements. Discussion ACL-RSI(NL) first administration (SD)
56.1 (SD 22.3)
ACL-RSI (NL) second administration (SD)
59.3 (SD 21.9)
Mean difference (95% CI; SD)
3.2 (95% CI 1.6–4.6; SD 7.8)
ICC (95% CI)
0.93 (95% CI 0.88–0.96)
SEM
5.5
SDC – individual
15.3
SDC – group
1.5
Abbreviations: SD = standard deviation; CI = confidence interval; ICC = intraclass
correlation coefficient; SEM = standard error of measurement; SDC = smallest
detectable change. yp
1
A high correlation between the ACL-RSI(NL) and the I-PRRS(NL)
(r > 0.6). +
2
A moderate correlation between the ACL-RSI(NL) and the KOOS(NL)
subscale “pain” (r = 0.6–0.3). +
3
A moderate correlation between the ACL-RSI(NL) and the KOOS(NL)
subscale “other symptoms” (r = 0.6–0.3). +
4
A moderate correlation between the ACL-RSI(NL) and the KOOS(NL)
subscale “function in daily living” (r = 0.6–0.3). -
5
A moderate correlation between the ACL-RSI(NL) and the KOOS(NL)
subscale “function in sport and recreation” (r = 0.6–0.3). +
6
A moderate correlation between the ACL-RSI(NL) and the KOOS(NL)
subscale “knee-related Quality of life” (r = 0.6–0.3). +
7
KOOS(NL) subscales “function in sport and recreation” and “knee-
related quality of life” have stronger correlation with ACL-RSI than
the other 3 subscales. +
8
A moderate correlation between the ACL-RSI(NL) and the IKDC 2000
(NL) (r = 0.6–0.3). +
9
A moderate correlation between the ACL-RSI(NL)
and TSK(NL)(r = 0.6–0.3). +
10 A low correlation between the ACL-RSI
and the MHLC-C(NL) (r < 0.3). +
+ = hypothesis is confirmed; – = hypothesis is rejected. Downloaded by [University of Groningen] at 06:44 14 December 2017 psychological readiness to return to sport participation after
injury respectively. However, the ACL-RSI is specifically devel-
oped for the ACL patient while the I-PRRS developed for
injured
athletes
in
general. The
different
psychological
responses measured in the ACL-RSI, such as emotions, con-
fidence in performance, and risk appraisal receive less atten-
tion in the I-PRRS. To our knowledge, the relationship between
the I-PRRS and the ACL-RSI has not been investigated
previously. Moderate correlations were found between the ACL-RSI(NL)
and the subscales of the KOOS. Only the subscale “activities of
daily living” had a low correlation with the ACL-RSI(NL). The
results of this study with regard to the degree of correlation of
the ACL-RSI(NL) with the KOOS are comparable to those of the
study of Kvist et al. (2013), who also found moderate correla-
tions. Discussion In accordance with Kvist et al. (2013), the ACL-RSI(NL)
showed higher correlations with KOOS subscales “sport and
function” and “knee-related quality of life” than with the other
KOOS subscales. Overall, Kvist et al. found slightly higher
correlations between the ACL-RSI and these KOOS subscales
compared with this study, which may be explained by the fact
that Kvist et al. included participants 2–5 year after ACL recon-
struction. We hypothesise that these patients, for those who
practice sports, good quality of life is strongly associated with
return to sports after ACL reconstruction. A significant part of
the participants of our study (3–16 months after reconstruc-
tion), still participate in a rehabilitation program after ACL
reconstruction. This might have led to less strong correlations. Probably, the quality of life is partly determined by the possi-
bility of being able to do sports. Possibly, patients still partici-
pating in a rehabilitation program, already indicate a higher
quality of life, while they still score low on the ACL-RSI. The
hypothesis that these 2 subscales had a stronger correlation
with ACL-RSI(NL) than the other 3 subscales, within the mod-
erate range, was confirmed. Downloaded by [University of Groninge + = hypothesis is confirmed; – = hypothesis is rejected. Table 5. Test–retest reliability measures of the ACL-RSI(NL) (N = 104). ACL-RSI(NL) first administration (SD)
56.1 (SD 22.3)
ACL-RSI (NL) second administration (SD)
59.3 (SD 21.9)
Mean difference (95% CI; SD)
3.2 (95% CI 1.6–4.6; SD 7.8)
ICC (95% CI)
0.93 (95% CI 0.88–0.96)
SEM
5.5
SDC – individual
15.3
SDC – group
1.5
Abbreviations: SD = standard deviation; CI = confidence interval; ICC = intraclass
correlation coefficient; SEM = standard error of measurement; SDC = smallest
detectable change. Table 5. Test–retest reliability measures of the ACL-RSI(NL) (N = 104). Abbreviations: SD = standard deviation; CI = confidence interval; ICC = intraclass
correlation coefficient; SEM = standard error of measurement; SDC = smallest
detectable change. risk appraisal cannot be separated and 1 total score should be
used. Construct validity was examined by testing pre-defined
hypotheses. Except for the correlation between the ACL-RSI
(NL) and the KOOS subscale “activities of daily living”, all
hypotheses described were confirmed (90%). As this was
above the 75% criterion set in the COSMIN guidelines, it can
be concluded that the ACL-RSI(NL) has good construct validity
Terwee et al., 2007). The ACL-RSI(NL) correlated moderately with the IKDC 2000. Discussion The aim of this study was to translate the ACL-RSI scale from
English to Dutch and to examine whether the Dutch version of
the ACL-RSI (ACL-RSI(NL)) is a valid and reliable instrument to 398
A. J. SLAGERS ET AL. A. J. SLAGERS ET AL. 398 Figure 2. The Bland and Altman graph of the test–retest. Table 3. Spearman correlation between ACL-RSI(NL) and other questionnaires. r
p-value
IPRRS
0.79
<0.001
KOOS subscale “pain”
0.36
<0.001
KOOS subscale “other symptoms”
0.30
<0.001
KOOS subscale “function in daily living”
0.25
0.002
KOOS subscale “function in sport and recreation”
0.48
<0.001
KOOS subscale “knee-related quality of life”
0.40
<0.001
IKDC 2000
0.51
<0.001
TSK
−0.46
<0.001
MHLC-C
−0.15
0.072
ACL-RSI, Anterior Cruciate Ligament scale – Return to Sports after Injury scale;
I-PRRS, Injury-Psychological Readiness to Return to Sport scale; KOOS, Knee
injury
and
Osteoarthritis
Outcome
Score;
IKDC,
International
Knee
Documentation Committee Score; TSK, Tampa Scale of Kinesiophobia;
MHLC-C, Multidimensional Health Locus of Control, C form. Table 3. Spearman correlation between ACL-RSI(NL) and other questionnaires. Figure 2. The Bland and Altman graph of the test–retest. Table 4. Predefined hypotheses and the confirmation or rejection of the
hypothesis. hypothesis. 1
A high correlation between the ACL-RSI(NL) and the I-PRRS(NL)
(r > 0.6). +
2
A moderate correlation between the ACL-RSI(NL) and the KOOS(NL)
subscale “pain” (r = 0.6–0.3). +
3
A moderate correlation between the ACL-RSI(NL) and the KOOS(NL)
subscale “other symptoms” (r = 0.6–0.3). +
4
A moderate correlation between the ACL-RSI(NL) and the KOOS(NL)
subscale “function in daily living” (r = 0.6–0.3). -
5
A moderate correlation between the ACL-RSI(NL) and the KOOS(NL)
subscale “function in sport and recreation” (r = 0.6–0.3). +
6
A moderate correlation between the ACL-RSI(NL) and the KOOS(NL)
subscale “knee-related Quality of life” (r = 0.6–0.3). +
7
KOOS(NL) subscales “function in sport and recreation” and “knee-
related quality of life” have stronger correlation with ACL-RSI than
the other 3 subscales. +
8
A moderate correlation between the ACL-RSI(NL) and the IKDC 2000
(NL) (r = 0.6–0.3). +
9
A moderate correlation between the ACL-RSI(NL)
and TSK(NL)(r = 0.6–0.3). +
10 A low correlation between the ACL-RSI
and the MHLC-C(NL) (r < 0.3). +
+ = hypothesis is confirmed; – = hypothesis is rejected. Table 5. Test–retest reliability measures of the ACL-RSI(NL) (N = 104). Conclusion The ACL-RSI is successfully translated into Dutch (ACL-RSI(NL)),
and the ACL-RSI(NL) is a valid and reliable questionnaire to
investigate athletes’ emotions, confidence in performance,
and risk appraisal in relation to return to sport after ACL
reconstruction. Reliability The ACL-RSI(NL) demonstrated good internal consistency. The
magnitude of the Cronbach’s alpha (0.94) was comparable
with the Cronbach’s alpha of the original ACL-RSI (0.92) and
the Swedish (0.94) and French versions (0.96) (Bohu et al.,
2015; Kvist et al., 2005; Webster et al., 2008). The test-retest
reliability of the ACL-RSI(NL) was high (ICC: 0.93), which corre-
sponds with the Swedish (ICC: 0.89) and French (ICC:0.90)
versions (Bohu et al., 2015; Kvist et al., 2013). The Bland and
Altman analysis showed a significant bias of 3.2 points
between the first and second administration of the ACL-RSI
(NL). However, this bias is less than the SEM (5.5). Therefore, it
cannot be distinguished from the measurement error. The
SEM (5.5) and SDCind (15.3) found in this study are in line
with those found in the Swedish ACL-RSI study (SEM 7.0 en
SDCind 19) (Kvist et al., 2013). Limitations and strengths Limitations and strengths A limitation of this study is that there is no information avail-
able with regard to validity and reliability of the Dutch lan-
guage version of the I-PRRS and the MHLC-C. The validity and reliability of the original questionnaires has
been demonstrated though and both are translated following
the international guidelines (Beaton et al., 2000; Glazer, 2009;
Wallston et al., 1994). A strength of this study is the study population used. In
contrast to the English and Swedish ACL-RSI studies, using a
population being a considerable period of time after ACL recon-
struction, the current study was performed in a patient popula-
tion partly still participating in a rehabilitation program after ACL
reconstruction. We have specifically chosen this patient group
because in these patients the return to sport is an issue. Moreover, if we want to implement interventions to modify
psychological factors based on the outcomes of the ACL-RSI,
these will take place in this phase of the rehabilitation. Downloaded by [University of Groningen] at 06:44 14 December 2017 Discussion This is in line with previous research into the validity of the
French version of the ACL-RSI. The IKDC 2000 partially mea-
sures aspects, which are related to psychological factors rele-
vant to returning to sport, but are primary developed to
measure other constructs, such as symptoms, limitations in
function, and sports due to impairment of the knee. Since As expected, we found a high correlation between the ACL-
RSI(NL) and the I-PRRS. Both questionnaires measure a com-
parable construct, namely the psychological impact of return-
ing to sport after ACL injury or reconstruction and athlete’s JOURNAL OF SPORTS SCIENCES 399 the IKDC 2000 has similarities with the KOOS, hence the
moderate correlation can also be explained from that point
of view. highest possible total score. Like the original English, Swedish
and French version the mean score is somewhere in the mid-
dle of the scale (56.5) and varies from 8–100. Also in these
studies, no floor or ceiling effects were found. The moderate correlation found between the ACL-RSI and
the TSK was comparable with a foregoing study (Kvist et al.,
2013). Previously, the TSK is used to assess the fear of re-injury
in relation to predicting return to sport in recreational athletes
with anterior cruciate ligament injuries (Kvist et al., 2005; Tripp
et al., 2007). Fear of re-injury has been shown to be a hin-
drance for return to sports. A higher score on the TSK has
previous been associated with not returning to sports after
ACL injury(Kvist et al., 2005). The TSK has not been tested for
responsiveness in patients with ACL injuries or after ACL
reconstruction. It seems that the underlying construct of the
TSK does not correspond entirely with the construct of the
ACL-RSI. Beside fear of re-injury, the ACL-RSI evaluates other
emotions (such as nervousness, frustration, and stress) and
confidence in performance and risk appraisal in relation to
return to sport after ACL reconstruction. Additionally, as
expected and in line with previous research, the correlation
between the ACL-RSI(NL) and the MHLC-C was low (Kvist et al.,
2013). Disclosure statement No potential conflict of interest was reported by the authors. References Statistical methods for assessing
agreement between two methods of clinical measurement. The
Lancet, 327(8476), 307–310. doi:10.1016/S0140-6736(86)90837-8 consensus on taxonomy, terminology, and definitions of measurement
properties for health-related patient-reported outcomes. Journal of
Clinical Epidemiology, 63(7), 737–745. doi:10.1016/j.jclinepi.2010.02.006 Bohu, Y., Klouche, S., Lefevre, N., Webster, K., & Herman, S. (2015). Translation, cross-cultural adaptation and validation of the French ver-
sion of the Anterior Cruciate Ligament-Return to Sport after Injury
(ACL-RSI) scale. Knee Surgery, Sports Traumatology, Arthroscopy, 23(4),
1192–1196. doi:10.1007/s00167-014-2942-4 Morrey, M. A., Stuart, M. J., Smith, A. M., & Wiese-Bjornstal, D. M. (1999). A
longitudinal
examination
of
athletesʼ
emotional
and
cognitive
responses to anterior cruciate ligament injury. Clinical Journal of Sport
Medicine, 9(2), 63–69. doi:10.1097/00042752-199904000-00004 Myklebust, G. (2005). Return to play guidelines after anterior cruciate
ligament surgery. British Journal of Sports Medicine, 39, 127–131. doi:10.1136/bjsm.2004.010900 De Groot, I. B., Favejee, M. M., Reijman, M., Verhaar, J. A., & Terwee, C. B. (2008). The Dutch version of the knee injury and osteoarthritis outcome
score: A validation study. Health and Quality of Life Outcomes, 6(1), 16. doi:10.1186/1477-7525-6-16 Quinn, A. M., & Fallon, B. J. (1999). The changes in psychological charac-
teristics and reactions of elite athletes from injury onset until full
recovery. Journal
of
Applied
Sport
Psychology,
11,
210–229. doi:10.1080/10413209908404201 de Vet, H. C. W., Terwee, C. B., Knol, D. L., & Bouter, L. M. (2006). When to
use
agreement
versus
reliability
measures. Journal
of
Clinical
Epidemiology, 59(10), 1033–1039. doi:10.1016/j.jclinepi.2005.10.015 Streiner, D. L., & Norman, G. R. (2008). Health measurement scales. A
practical guide to their development and use (4th ed.). New York, NY:
Oxford University Press. Feller, J. A., & Webster, K. E. (2003). A randomized comparison of patellar
tendon and hamstring tendon anterior cruciate ligament reconstruc-
tion. The American Journal of Sports Medicine, 31(4), 564–573. Terwee, C. B., Bot, S. D. M., de Boer, M. R., van der Windt, D. A. W. M., Knol,
D. L., Dekker, J., . . . de Vet, H. C. W. (2007). Quality criteria were
proposed for measurement properties of health status questionnaires. Journal
of
Clinical
Epidemiology,
60(1),
34–42. doi:10.1016/j. jclinepi.2006.03.012 Glazer, D. D. (2009). Development and preliminary validation of the Injury-
Psychological Readiness to Return to Sport (I-PRRS) scale. Journal of
Athletic Training, 44(2), 185–189. doi:10.4085/1062-6050-44.2.185 Downloaded by [University of Groningen] at 06:44 14 December 2017 Gobbi, A., & Francisco, R. (2006). References Factors affecting return to sports after
anterior cruciate ligament reconstruction with patellar tendon and
hamstring graft: A prospective clinical investigation. Knee Surgery,
Sports Traumatology, Arthroscopy, 14(10), 1021–1028. doi:10.1007/
s00167-006-0050-9 Terwee, C. B., Mokkink, L. B., Knol, D. L., Ostelo, R. W. J. G., Bouter, L. M., &
de Vet, H. C. W. (2012). Rating the methodological quality in systematic
reviews of studies on measurement properties: A scoring system for the
COSMIN checklist. Quality of Life Research, 21, 651–657. doi:10.1007/
s11136-011-9960-1 Halfens, R., & Philipsen, H. (1988). Een gezondheidsspecifieke beheersing-
sorienatieschaal: Validiteit en betrouwbaarheid van de MHLC. T Social
Gezondheidsz, 66, 399–403. Downloaded by [University of Groningen] at 06:44 14 Thomeé, P., Währborg, P., Börjesson, M., Thomeé, R., Eriksson, B. I., &
Karlsson, J. (2008). Self-efficacy of knee function as a pre-operative
predictor of outcome 1 year after anterior cruciate ligament reconstruc-
tion. Knee Surgery, Sports Traumatology, Arthroscopy, 16(2), 118–127. doi:10.1007/s00167-007-0433-6 Haverkamp, D. (2006). Translation and validation of the Dutch version of
the International Knee Documentation Committee Subjective Knee
Form. American
Journal
of
Sports
Medicine,
34(10),
1680–1684. doi:10.1177/0363546506288854 Tjong, V. K., Murnaghan, M. L., Nyhof-Young, J. M., & Ogilvie-Harris, D. J. (2014). A qualitative investigation of the decision to return to sport
after anterior cruciate ligament reconstruction: To play or not to play. The American Journal of Sports Medicine, 42(2), 336–342. doi:10.1177/
0363546513508762 Hinkle, D. E., Wiersma, W., & Jurs, S. G. (1998). Applied statistics for the
behavioral sciences. Boston, MA: Houghton MiZin. Kamper, S. J., Maher, C. G., & Mackay, G. (2009). Global rating of change
scales: A review of strengths and weaknesses and considerations for
design. Journal of Manual & Manipulative Therapy, 17(3), 163–170. doi:10.1179/jmt.2009.17.3.163 Tripp, D. A., Stanish, W., Ebel-Lam, A., Brewer, B. W., & Birchard, J. (2007). Fear of reinjury, negative affect and catastrophizing predicting return
to sport in recreational athletes with anterior cruciate ligament injuries
at 1 year postsurgery. Rehabilitation Psychology, 52, 74–81. doi:10.1037/
0090-5550.52.1.74 Kori, S. H., Miller, R. P., & Todd, D. D. (1990). Kinisophobia: A new view of
chronic pain behavior. Pain Management, 3(1), 35–43. Kvist, J., Ek, A., Sporrstedt, K., & Good, L. (2005). Fear of re-injury: A
hindrance for returning to sports after anterior cruciate ligament recon-
struction. Knee Surgery, Sports Traumatology, Arthroscopy, 13(5), 393–
397. doi:10.1007/s00167-004-0591-8 Van Grinsven, S., Van Cingel, R. E. H., Holla, C. J. M., & Van Loon, C. J. M. (2010). References It should be noted that the time period between the 2
measurements of the ACL-RSI(NL) may have affected the
results. The mean time between receiving the first and second
questionnaire was 22.9 days (SD 7.9). The COSMIN recommen-
dation is that the time interval should be long enough to
prevent recall bias and short enough to ensure that patient
characteristics have not changed regarding the construct to
be measured (Mokkink et al., 2010). A time interval of 2 weeks
is often used in repeatability studies (Streiner & Norman,
2008). To check whether the patients were not changed with
regard to the construct measured by the ACL-RSI during the
period between test and retest, a global rating of change
question was included in the second measurement (Kamper,
Maher, & Mackay, 2009). The 3 participants who indicated that
they received much more confidence during the time interval
between the first and second measurement were not included
in the analysis. Ardern, C. L., Taylor, N. F., Feller, J. A., & Webster, K. E. (2012). Fear of re-
injury in people who have returned to sport following anterior cruciate
ligament reconstruction surgery. Journal of Science and Medicine in
Sport, 15, 488–495. doi:10.1016/j.jsams.2012.03.015 Ardern, C. L., Taylor, N. F., Feller, J. A., & Webster, K. E. (2013). A systematic
review of the psychological factors associated with returning to sport
following injury. British Journal of Sports Medicine, 47(17), 1120–1126. doi:10.1136/bjsports-2012-091203 Ardern, C. L., Taylor, N. F., Feller, J. A., & Webster, K. E. (2014). Fifty-five per
cent return to competitive sport following anterior cruciate ligament
reconstruction surgery: An updated systematic review and meta-analy-
sis including aspects of physical functioning and contextual factors. British Journal of Sports Medicine, 48(21), 1543–1552. doi:10.1136/
bjsports-2013-093398 Ardern, C. L., Webster, K. E., Taylor, N. F., & Feller, J. A. (2011). Return to the
preinjury level of competitive sport after anterior cruciate ligament
reconstruction surgery: Two-thirds of patients have not returned by
12 months after surgery. The American Journal of Sports Medicine, 39,
538–543. doi:10.1177/0363546510384798 Beaton, D. E., Bombardier, C., Guillemin, F., & Ferraz, M. B. (2000). Guidelines
for the process of cross-cultural adaptation of self-report measures. Spine,
25(24), 3186–3191. doi:10.1097/00007632-200012150-00014 In this study, no floor or ceiling effects were observed, since
less than 15% of the respondents had achieved the lowest or A. J. SLAGERS ET AL. 400 Bland, J. M., & Altman, D. G. (1986). ACL-RSI-vragenlijst Instructies: Beantwoord de volgende vragen met betrekking tot uw hoofdsport die u voorafgaand aan
uw blessure beoefende. Kruis bij elke vraag een vakje aan tussen de twee beschrijvingen om aan te
geven hoe u zich op dit moment voelt ten opzichte van de twee uitersten. 1.
Bent u er zeker van dat u weer op uw oude niveau uw sport kunt beoefenen?
Helemaal
niet zeker
Helemaal
zeker
2.
Denkt u dat u waarschijnlijk uw knie opnieuw zal blesseren bij het beoefenen van uw sport?
Heel erg
Waarschijnlijk
Helemaal niet
waarschijnlijk
3.
Bent u zenuwachtig over het beoefenen van uw sport?
Heel erg
zenuwachtig
Helemaal niet
zenuwachtig
4.
Weet u zeker dat u niet door de knie gaat tijdens uw sportbeoefening?
Helemaal
niet zeker
Helemaal
zeker
5.
Weet u zeker dat u uw sport kan beoefenen zonder bezorgd te zijn over uw knie?
Helemaal
niet zeker
Helemaal
zeker
6.
Vindt u het frustrerend rekening te moeten houden met uw knie in uw sport?
Heel erg
frustrerend
Helemaal niet
frustrerend
7.
Bent u bang opnieuw geblesseerd te raken aan uw knie door het beoefenen van uw sport?
Heel erg
bang
Helemaal niet
bang
uw blessure beoefende. Kruis bij elke vraag een vakje aan tussen de twee beschrijvingen om aan te
geven hoe u zich op dit moment voelt ten opzichte van de twee uitersten.
8.
Bent u er zeker van dat uw knie belasting aan kan?
Helemaal
niet zeker
Helemaal
zeker
9.
Bent u bang voor het per ongeluk blesseren van uw knie door het beoefenen van uw sport?
Heel erg
bang
Helemaal niet
bang
10.
Houden de gedachten aan het weer opnieuw moeten ondergaan van een operatie en revalidatie u
tegen om weer uw sport te beoefenen?
Altijd
Nooit
11.
Bent u zeker van uw vermogen om goed te presteren in uw sport?
Helemaal
niet zeker
Helemaal
zeker
12.
Voelt u zich ontspannen over het beoefenen van uw sport?
Helemaal niet
ontspannen
Helemaal
ontspannen 1. Bent u er zeker van dat u weer op uw oude niveau uw sport kunt beoefenen? References Evidence-based rehabilitation following anterior cruciate liga-
ment reconstruction. Knee Surgery, Sports Traumatology, Arthroscopy,
18, 1128–1144. doi:10.1007/s00167-009-1027-2 Kvist, J., Osterberg, A., Gauffin, H., Tagesson, S., Webster, K., & Ardern, C. (2013). Translation and measurement properties of the Swedish version
of ACL-Return to Sports after Injury questionnaire. Scandinavian Journal
of Medicine & Science in Sports, 23(5), 568–575. Vlaeyen, J. W. S., Kole-Snijders, A. M. J., Boeren, R. G. B., & Van Eek, H. (1995). Fear of movement/(re)injury in chronic low back pain and its
relation to behavioral performance. Pain, 62(3), 363–372. doi:10.1016/
0304-3959(94)00279-N Langford, J., Webster, K. E., & Feller, J. A. (2009). A prospective longitudinal
study to assess psychological changes following anterior cruciate liga-
ment reconstruction surgery. British Journal of Sports Medicine, 43, 377–
378. doi:10.1136/bjsm.2007.044818 Wallston, B. S., Wallston, K. A., Kaplan, G. D., & Maides, S. A. (1976). Development and validation of the health locus of control (HLC)
scale. Journal of Consulting and Clinical Psychology, 44(4), 580–585. doi:10.1037/0022-006X.44.4.580 Marx, R. G., Jones, E. C., Angel, M., Wickiewicz, T. L., & Warren, R. F. (2003). Beliefs and attitudes of members of the American Academy of
Orthopaedic Surgeons regarding the treatment of anterior cruciate
ligament injury. Arthroscopy: The Journal of Arthroscopic & Related
Surgery, 19(7), 762–770. doi:10.1016/S0749-8063(03)00398-0 Wallston, K. A., Stein, M. J., & Smith, C. A. (1994). Form C of the MHLC
scales: A condition-specific measure of locus of control. Journal of
Personality
Assessment,
63(3),
534–553. doi:10.1207/
s15327752jpa6303_10 McHorney, C. A., & Tarlov, A. R. (1995). Individual-patient monitoring in
clinical practice: Are available health status surveys adequate? Quality
of Life Research, 4, 293–307. doi:10.1007/BF01593882 Webster, K. E., Feller, J. A., & Lambros, C. (2008). Development and pre-
liminary validation of a scale to measure the psychological impact of
returning to sport following anterior cruciate ligament reconstruction
surgery. Physical
Therapy
in
Sport,
9(1),
9–15. doi:10.1016/j. ptsp.2007.09.003 Mokkink, L. B., Terwee, C. B., Patrick, D. L., Alonso, J., Stratford, P. W., Knol,
D. L., . . . de Vet, H. C. W. (2010). The COSMINstudy reached international JOURNAL OF SPORTS SCIENCES
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DNA methylation patterns–based subtype distinction and identification of soft tissue sarcoma prognosis
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Medicine
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cc-by
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DNA methylation patterns–based subtype
distinction and identification of soft tissue sarcoma
prognosis Zhengyuan Wu
(
wuzhengyuan1995@163.com
)
Guangxi Medical University First Affiliated Hospital Zhen-pei Wen
Nanning, 530021, China Tian-yu Ren
Nanning, 530021, China Research Keywords: Soft tissue sarcomas, DNA methylation, Molecular subtype, Prognosis
Posted Date: May 6th, 2020
DOI: https://doi.org/10.21203/rs.3.rs-26298/v1
License:
This work is licensed under a Creative Commons Attribution 4.0 International
Lice
Read Full License Abstract Background: Soft tissue sarcomas (STSs) are heterogeneous at the clinical with a variable tendency of
aggressive behavior. Methods: In this study, we constructed a specific DNA methylation-based classification to identify the
distinct prognosis-subtypes of STSs based on the DNA methylation spectrum from the TCGA database. Results: Eventually, samples were clustered into four subgroups, and their survival curves were distinct
from each other. Meanwhile, the samples in each subgroup reflected differentially in several clinical
features. Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was
also conducted on the genes of the corresponding promoter regions of the above‐described specific
methylation sites, revealing that these genes were mainly concentrated in certain cancer‑associated
biological functions and pathways. In addition, we calculated the differences among clustered
methylation sites and performed the specific methylation sites with LASSO algorithm. The selection
operator algorithm was employed to derive a risk signature model, and a prognostic signature based on
these methylation sites performed well for risk stratification in STSs patients. At last, a nomogram
consisted of clinical features and risk score was developed for the survival prediction. Conclusion: In conclusion, this study declares that DNA methylation-based STSs subtype classification is
highly relevant for future development of personalized therapy as it identifies the prediction value of
patient prognosis. DOI: https://doi.org/10.21203/rs.3.rs-26298/v1 License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. R
d F ll Li License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Page 1/22 Background Soft tissue sarcomas (STSs), which arise predominantly from the embryonic mesoderm, are a set of
malignancies that account for 0.73–0.81% of all and 6% of pediatric cancers [1, 2]. In accordance with
the heterogeneity in histopathological features, clinical manifestations, and molecular signature,
approximately 50 different histological subtypes have been discovered within STSs patients [3]. Meanwhile, STSs are commonly presenting as a symptomless mass in almost every part of the human
body, including the retroperitoneum, viscera, and extremities [4, 2], with approximately 50% of five-year
overall survival (OS) [5]. Although STSs have some common morphologic features, its proper diagnosis
and treatment are still challenging for pathologists and physicians due to its extremely variable biology
and genetics and low incidence. Up to now, the most optimal management of STSs is still surgical
resection, although its less successful in the advanced STS in accompany with the high rate of local
recurrence [6, 7]. Consequently, molecular characteristics of these heterogeneous tumors are warranted to
be further explored and new classification systems should be elucidated for providing more potential
prognostic factors in the clinic. Previously, several genomic and transcriptome data have focused on exploring effective diagnostic or
prognostic markers in STSs, including alternative splicing [8], copy number variation [9], and genes
expression [10]. As one of the core elements in tumorigenesis progression, DNA methylation occurs early Page 2/22 Page 2/22 Page 2/22 and frequently in regulating a variety of genomic functions [11–13]. DNA methylation is a
posttranslational modification process, which are selectively occurred on the cytosines of 5′-CpG-3′ to
generates 5-methyldeoxycytidine. The aberrant of DNA methylation, especially in CpG-rich regions (CpG
islands), has been found closely related with physio-pathologic mechanisms underlying an array of
human diseases [14]. Moreover, CpG islands was frequently detected in the promoter regions of the
structural transcription gene [15], and abnormal CpG island hypermethylation of various tumor
suppressor genes and hypomethylation of oncogenes play vital role in carcinogenesis [16, 17]. At present,
as a promising molecular marker of STSs, abnormal DNA methylation appears in early detection,
prognosis prediction, molecular classification [18, 19]. Meanwhile, multiple biological studies have also
clarified that a series of methylations in gene promoter sequences is correlated with the prognosis and
progression of STSs patients [20–22]. Background Nevertheless, the prognostic value of these aberrantly methylation
sites in STSs subtypes and the complex role of DNA methylation in distinct gene regions are still largely
unclarified and require further validation in the prognostic role of DNA methylation in STSs. Therefore, in this study, we addressed a classification method by identifying specific prognosis-subtypes
which were based on DNA methylation profiles of STS from TCGA database, which may help to identify
new markers to accurately subdivide STSs patients. Moreover, our classification system provides a more
accurate prediction of clinical behavior and identifies higher risk assessment accuracy for clinicians on
personalized treatments Materials And Methods Consensus clustering of prognosis molecular subtypes and
clinical characteristic analyses The K-means clustering algorithm in the ConcensusClusterPlus R packet was utilized for consensus
clustering to determine STSs subgroups [28]. In this study, 80% of the STSs samples were carried out 100
times by adopting the resampling method, and Euclidean distance and κ-means algorithm was
calculated to measure the similarity in samples distance and determine the stability and reliability of
classification results, respectively. The optimal cluster number was then determined by using the
cumulative distribution function (CDF) and the delta area plot which should be with relative high
consistency, low variation coefficient, and no obviously increased area under the CDF curve, and the
cluster outcomes were applied for the following clinical characteristics analysis. The pheatmap R
package was generated to construct the corresponding consensus cluster heatmap which was plotted
based on the age, gender, tissue or organ of origin, and histological type of each sample. Afterward, the
overall survival analysis of STSs subgroups was construct by Kaplan–Meier method and log-rank test
with R Bioconductor survival package, and chi-square test was used to comprehensively determine the
associations between DNA methylation clusters and clinical characteristics. COX Proportional Risk Regression Models regarding
h l
i
i COX Proportional Risk Regression Models regarding
methylation sites univariate COX proportional risk regression models were constructed with the survival data and every
methylation site by utilizing the survival coxph function R package with the significant threshold was set
as P < 0.001 [27]. Subsequently, the obtained methylation sites were introduced into further multivariate
Cox proportional regression model analysis, where age, gender, tissue or organ of origin, and histological
type were selected as the covariates from the downloaded STSs clinical data, with P < 0.001 set as the
significance threshold. Finally, the CpG sites, which were identified as independent prognostic factors,
were chosen as the classification features. Date pre-processing and initial screening of DNA
methylation sites in STSs The latest DNA methylation data were downloaded from the Illumina Infinium HumanMethylation450
Bead-Chip array of TCGA Genomic Data Commons application programming interface
(https://portal.gdc.cancer.gov) [14] on April 6th, 2020, which containing a total of 269 STSs samples. The
corresponding clinical and prognostic parameters were acquired from the University of California Santa
Cruz Xena (UCSC Xena; http://xena.ucsc.edu/) [23] on April 5th, 2020, for further methylation profile
matching, ultimately, 258 STSs samples were selected for methylation analysis. Afterward, the RNA-
sequencing (RNA‐Seq) date included 265 samples were also collected from the TCGA public database
(accessed April 5th, 2020) which was normalized by the DESeq package in R platform [24]. Acquiring and
usage of all data in the current study are following the publication guidelines of TCGA
(https://cancergenome.nih.gov/publications/publicationguidelines). Moreover, the CpG sites with missing data over 70% from all samples were all removed. The k-nearest
neighbor (KNN) imputation method in the sva R package was utilized to estimate the missing values, with
the further removal of the unstable genomic methylation sites which contained CpG sites in sex
chromosomes and single nucleotide polymorphisms [25]. Subsequently, DNA methylation with strongly
genetic modulation effects was selected for the following exploration based on the annotation between Page 3/22 Page 3/22 CpG sites and gene promoter regions [26], and the promoter region was defined as the 2 kb upstream to
0.5 kb in the transcription start site. Yielding 206,636 methylation sites were obtained for further analysis. Identification of methylation sites annotated gene
expression and pathway enrichment analysis DNA methylation at the promoter site can modulate gene expression. In order to investigate the
association of previously obtained CpG sites with gene expression in the classified subgroups,
methylation sites were subjected to genomic annotations to determine the corresponding genes, and
then, these genes were identified for plotting the expression profile heat map. Meanwhile, to explore the
biological terms of specific gene lists, GO and KEGG was performed for enrichment analysis based on the
ClusterProfiler R package [29], where a P < 0.05 was set as the cut-off criteria. And all three aspects of GO
analysis were included for providing gene function, including, biological process (BP), cellular component
(CC), and molecular function (MF).fi Page 4/22 Page 4/22 Screening of intragroup-specific methylation sites, and
prognosis prediction model construction To identify the differences among the clustered methylation sites, Kolmogorov-Smirnov tests were
applied for each CpG site to determine its different distribution in the methylation level [30], and the
thresholds set at log2(fold change (FC)) > 1 and false discovery rate (FDR) < 0.05 were indicated as the
significant difference. Furthermore, the heatmap corresponding to the differential frequency of every CpG
site in each subgroup was further detected by ComplexHeatmap R package. In this exploration, the
specific methylation sites in Cluster1 with a great number of specific CpG sites and the best prognosis
were selected for the prognostic model construction. From this, the least absolute shrinkage and selection
operator (LASSO) algorithm was developed to construct a potential risk signature, finally, 13 methylation
sites were contained in the model. The formula of risk score was calculated as follows: (see Equation 1 in
the Supplementary Files) Based on the LASSO model, patients were categorized into high or low risk group, and the overall survival
analysis and clinical relevance of STSs samples at high and low risk were also generated as we
described before. Furthermore, the survivalROC packet in R Program was calculated to verify the stability
and reliability of the model [31], and Univariate and multivariate Cox regression analyses were counted to
validate the predictive accuracy of the clinical characteristics and risk score. Construction a predictive nomogram To provide an application tool for predicting STSs clinical outcome, the nomogram incorporated with age,
gender, tissue or organ of origin, histological type, and risk score was plotted by using R package [32]. Characteristics of DNA methylation sites based on the
prognosis results of STSs To cluster the STSs prognostic subtypes, a total of 485,577 CpG sites were successfully downloaded
from the TCGA methylation 450 k database in 269 STSs samples. After removing the missing values, sex
chromosomes and single-nucleotide polymorphisms, batch effects, methylation sites not located in the
gene promoter regions, and samples with insufficient information, univariate Cox proportional hazards
regression model was conducted to each methylation site in accompanied with the survival data, and
2,693 CpG sites were identified as significant survival correlated methylation sites (p < 0.001). Furthermore, these significant CpG sites were then introduced into the multivariate Cox proportional risk
regression models. As a result, 1,445 intersected independent prognostic CpG sites between two analysis
were chosen for the further prognosis analysis, and top 20 CpG sites were revealed in Table 1. To cluster the STSs prognostic subtypes, a total of 485,577 CpG sites were successfully downloaded
from the TCGA methylation 450 k database in 269 STSs samples. After removing the missing values, sex
chromosomes and single-nucleotide polymorphisms, batch effects, methylation sites not located in the
gene promoter regions, and samples with insufficient information, univariate Cox proportional hazards
regression model was conducted to each methylation site in accompanied with the survival data, and
2,693 CpG sites were identified as significant survival correlated methylation sites (p < 0.001). Furthermore, these significant CpG sites were then introduced into the multivariate Cox proportional risk
regression models. As a result, 1,445 intersected independent prognostic CpG sites between two analysis
were chosen for the further prognosis analysis, and top 20 CpG sites were revealed in Table 1. Page 5/22 Page 5/22 Page 5/22 Table 1 p
g
y
y
g
g
prognosis. Characteristics of DNA methylation sites based on the
prognosis results of STSs CpGs
HR
Lower 95% CI
Upper 95% CI
P
cg09347923
1.17E + 04
8.79E + 02
1.57E + 05
1.37E-12
cg00579036
4.92E + 02
7.91E + 01
3.06E + 03
3.02E-11
cg07058109
1.12E + 03
1.36E + 02
9.29E + 03
7.15E-11
cg16174121
1.58E + 02
3.44E + 01
7.27E + 02
7.93E-11
cg19357499
2.01E + 01
7.89E + 00
5.12E + 01
3.20E-10
cg19112957
1.56E + 02
3.21E + 01
7.57E + 02
3.70E-10
cg08508337
9.85E + 00
4.81E + 00
2.02E + 01
3.85E-10
cg07691531
2.61E + 01
9.34E + 00
7.28E + 01
4.80E-10
cg11491074
1.04E + 05
2.59E + 03
4.18E + 06
8.79E-10
cg22982767
6.23E + 01
1.64E + 01
2.36E + 02
1.25E-09
cg26132723
1.42E-03
1.70E-04
1.18E-02
1.34E-09
cg22271305
1.21E + 02
2.56E + 01
5.72E + 02
1.40E-09
cg12213680
2.11E-02
6.01E-03
7.42E-02
1.77E-09
cg10662943
1.77E-02
4.64E-03
6.77E-02
3.71E-09
cg19912470
3.00E-03
4.32E-04
2.09E-02
4.35E-09
cg17133388
9.94E + 05
9.75E + 03
1.01E + 08
4.84E-09
cg19853703
4.85E + 01
1.32E + 01
1.79E + 02
5.13E-09
cg08588180
1.13E + 04
4.75E + 02
2.69E + 05
7.83E-09
cg04217927
1.13E + 02
2.25E + 01
5.68E + 02
9.47E-09
cg12818699
4.38E-02
1.48E-02
1.30E-01
1.56E-08
Abbreviations: CI, confidence interval; HR, hazard ratio. sus clustering of STSs identified prognosis su
acteristic DNA methylation sites
optimal cluster subtypes, the obtained 1,445 CpGs sites of 258 STSs samples
the Consensus clustering, and the intercluster variation coefficient and average Consensus clustering of STSs identified prognosis subtypes
by characteristic DNA methylation sites Consensus clustering of STSs identified prognosis subtypes
by characteristic DNA methylation sites Clinical characteristics with DNA methylation clustering Kaplan–Meier and log-rank tests showed that the patients in four different molecular subtypes showed a
significant diversity OS (Fig. 4A). Among them, Cluster 1 had the best survival rate, whereas Clusters 2
and 3 had a dismal prognosis, indicating that lower DNA methylation levels may be connected with the
poorer OS in STSs patients. Indeed, these four molecular subtypes were also found significantly related to
some clinical characteristics of each sample. As shown in Fig. 4B, Cluster 1 was highly enriched with age
< = 65, and more older patients were enriched in Cluster 2 and 3. Meanwhile, Fig. 4D indicated Cluster 1
had more leiomyosarcoma and dedifferentiated liposarcoma, Cluster 3 had more fibromyxosarcoma and
undifferentiated sarcoma. Figure 4E implicated more sarcoma in Clusters 1 and 2 were original sited in
retroperitoneum, and sarcoma in Clusters 3 and 4 were mainly enriched in connective, subcutaneous and
other soft tissues. Figure 4C demonstrated no difference in gender among these four subgroups. These
results indicate that these DNA methylation profiles could serve as prognosis markers to better
understand the OS of STSs patients, more importantly, they may also demonstrate as clinical biomarkers
for some clinical features prediction. Consensus clustering of STSs identified prognosis subtypes
by characteristic DNA methylation sites To obtain the optimal cluster subtypes, the obtained 1,445 CpGs sites of 258 STSs samples were
employed for the Consensus clustering, and the intercluster variation coefficient and average cluster
consistency were both calculated for the number of each cluster. As shown in Fig. 2A and B, the area
under the CDF curve tended to be stable after 4 clusters. Meanwhile, according to the result of consistent Page 6/22 clustering (Fig. 3A), Cluster 4 had a stable clustering result with small variation coefficient, and there were
no extremely small sample sizes in Cluster 4. Therefore, k = 6 was regarded as the optimal cluster number
for the subsequent analysis. Heatmap annotated by labels of clinical characters was generated corresponded to the DNA methylation
classification (Fig. 3B). It was discovered that most CpG sites had high abundance in each sample,
besides, the DNA methylation profiles of the 4 categories were obviously different, while Cluster 3 has the
lowest methylation level and Cluster 1 has the highest methylation level. The evaluation of prognosis prediction model Overall, the predictive accuracy and stability was also analyzed to determine the function of the
prognostic prediction model. As shown in Fig. 7C, there was a significant inverse correlation between the
OS and risk valuation, indicating the remarkably better prognosis of low-risk samples as compared with
the higher one. And the area under the ROC curve all reached over 0.79 revealed the prediction model was
quietly precised to predict one-, three-, and five-year survival rates for STSs patients (Fig. 8B). Furthermore, the heatmap revealed the expression of the 13 specific CpG sites in each group (Fig. 8A),
and there were significant differences between the two prognosis model groups with respect to age,
tissue or organ of original, and histological type. The nomogram is another application tool for predicting
clinical outcomes. In this exploration, the nomogram of risk score and others clinical characters showed
that risk score will contribute to the most points in the model, compared to other traditional clinical
parameters (Fig. 8E). These results indicated that this prognostic predictor showed great promise for
predicting STSs outcomes and clinical features. Overall, the predictive accuracy and stability was also analyzed to determine the function of the
prognostic prediction model. As shown in Fig. 7C, there was a significant inverse correlation between the
OS and risk valuation, indicating the remarkably better prognosis of low-risk samples as compared with
the higher one. And the area under the ROC curve all reached over 0.79 revealed the prediction model was
quietly precised to predict one-, three-, and five-year survival rates for STSs patients (Fig. 8B). y
y
(
)
Furthermore, the heatmap revealed the expression of the 13 specific CpG sites in each group (Fig. 8A),
and there were significant differences between the two prognosis model groups with respect to age,
tissue or organ of original, and histological type. The nomogram is another application tool for predicting
clinical outcomes. In this exploration, the nomogram of risk score and others clinical characters showed
that risk score will contribute to the most points in the model, compared to other traditional clinical
parameters (Fig. 8E). These results indicated that this prognostic predictor showed great promise for
predicting STSs outcomes and clinical features. To determine whether the risk signature was the independent prognostic factor, the univariate and
multivariate Cox regression analyses were also performed in the next exploration (Fig. 8C and D). Identification of specific DNA methylation sites and
prognosis model construction To find the specific methylation sites, the differences of the 1,445 CpG sites in STSs subgroups were
further compared, resulting in 232 CpGs were identified as cluster specific methylation sites. The heat
map declared in Fig. 6A revealed that the most of the specific methylation sites were located in Clusters 1
and 3, and the most of which in Cluster 1 were hypomethylated sites as compared with other subgroups
(Fig. 6B). In addition, result of Cluster 1 was connected with the best prognosis among all clusters, so the
specific CpG sites in Cluster 1 were selected for the further construction. However, due to a large number
of CpG sites in Cluster 1 (81 CpG sites) was not good for clinical prediction, thus, the LASSO algorithm
was employed for the specific CpG site range shrinkage (Fig. 7A and B). Consequently, 13 CpG sites were
selected for the calculation of risk score, and all STSs patients were separated into low- and high-risk
groups. As Survival analysis indicated in Fig. 7D, the OS of STSs samples was gradually decreased with
an increased risk score, which declared that this predicting model was significantly correlated with the
prognosis of STSs patients. And Fig. 7D also discovered that most of the methylation levels were
increased in high-risk subgroup, except methylation sites cg15094605 and cg27321439. cytoskeleton formation, and so on. These results indicated that the CpG sites in this study were related
with Olfactory transduction and tumors progression of STSs. cytoskeleton formation, and so on. These results indicated that the CpG sites in this study were related
with Olfactory transduction and tumors progression of STSs. Identification of CpGs corresponded genes and pathway
enrichment analysis The 1,445 methylation sites in each subtype were subsequently subjected to genomic annotations and
1,268 corresponded genes were identified, meanwhile, the expression profile of thus CpG sites associated
genes were also extracted for plotting a heat map. As shown in Fig. 4F, each subgroup displayed a
variation in the gene expression levels, which indicating a partial consistency between the methylation
modifications and their corresponding genes expression. The enrichment analysis of corresponding gene functions by GO and KEGG enrichment were the
conventional means to investigate the molecular mechanism of these pathogenesis methylation sites in
STSs. The KEGG enrichment suggested that these genes were enriched in 16 KEGG pathways, especially
belonged to Olfactory transduction (Fig. 5D). GO enrichment analysis also draw the conclusion that these
genes were mainly related to Olfactory-related biological processes, like olfactory receptor activity,
sensory perception of smell, and cellular component of synaptic and postsynaptic membrane (Fig. 5A-C). In addition, these genes were also closely correlated with tumors progression, such as Cell adhesion
process, apoptosis, SMAD pathway, phosphatidylinositol 3 − kinase (PI3K) pathway, intermediate filament Page 7/22 Page 7/22 Discussion STSs is a rare group of malignancies with 50 histological subtypes and performs differing in behavior,
biology, and sensitivity to treatment [33]. However, therapies for each subtype remains similar in situ
STSs that surgical resection is the main method and supplemented with radiotherapy [2]. The application
of appropriate biomarkers is critical in tumor biology for prediction or risk stratification. For example,
genotyping methods based on genomics have been widely used to classify tumors, thus conducting the
clinical trials. Recent studies have confirmed that DNA methylation provide insights into various tumor
early diagnosis, molecular classification, and precise treatment [34, 14, 30]. Meanwhile, aberrant DNA
methylation has been regarded as one of the hallmarks of cancer tissues [35, 36], alterations in DNA
methylation also play a virtual role in the progression and development of STSs [20, 21]. In addition, S. Peter Wu et all had confirmed that methylation-based classifier could be used to provide diagnostic
assistance in bone sarcoma [18]. Therefore, we carried out this discovery to indicate the potential
application of DNA methylation in STSs epigenomes classification. The TCGA database is a publicly
available resource that contains more than 30 large cohorts of human tumors with a comprehensive
multidimensional analysis [37], these large sample sizes are absolutely the basis for us to provide an in-
depth understanding of the etiology of STSs. In this study, the whole genome DNA methylation sites
corresponding to 269 STSs samples were also obtained from TCGA database and methylation sites in
the gene promoter regions were first applied to select the prognosis associated CpG sites. Four specific
prognosis subgroups, classified by 1,445 intersected independent prognostic CpG sites, were developed
to present a molecular stratification for individual tumors, which has the significance of making
therapeutic decisions and exploring the biological mechanisms involved in the progression of RCC. STSs is not only a heterogeneous tumor, but also has been found presenting in almost every part of the
human body, and patients with different histological types are significantly distinct in clinical outcomes. Thus, analysis combined with the comprehensive clinical characteristics, such as age, gender, tissue or
organ of origin, and histological, might effectively improve the accuracy of prognosis in STSs patients
[38]. In this study, the distribution of the disease-specific OS in these 4 distinct prognostic subtypes of
STSs was seemed to be predicted, as well as original sites, histological classification, age, and gender
distribution. The evaluation of prognosis prediction model In the
univariate analysis, the risk score and age were both showed obviously connected with the STSs
prognosis, and similar results were also discovered in the multivariate analysis, thus indicating that the
risk score from specific CpG sites could be identified as an independently prognosis feature in STSs. Page 8/22 Discussion As the results suggested, the survival curves of these 4 specific subgroups were distinct from
each other. Meanwhile, our classification scheme also provided an accurate diagnosis for individual
tumors. For example, while patients were assigned to Cluster 1, they were found to have high chances to
be diagnosed as leiomyosarcoma primary from retroperitoneum and connected with better prognosis,
and these results might prompt clinicians to re-evaluate the treatment for STSs patients. Conclusion, our
classifications of 4 subtypes based on the DNA methylation sites can classify STSs more accurately and
guide clinicians in terms of clinical diagnosing, treating strategies and prognostic judgment of different
STSs patients. Previous researches have reported that CpG island methylation was shown to promote carcinogenesis by
disrupting the function of tumor suppressor genes or oncogenes [39]. Furthermore, it is also verified that
the hypomethylation of hub genes was significantly correlated to tumor proliferation and metastasis [40]. Page 9/22 In terms of tumor progression, methylation has also been identified significantly associated with multiple
biological processes and signaling pathways in cancers, including tumor stem cell growth [41], self-
renewal [42], ultraviolet-induced DNA damage response [43], focal adhesion pathway [30], and so on. So,
for comprehensively evaluating the mechanism of DNA methylation sites in STSs progression, the
enrichment analysis of CpG sites corresponded genes was also applied in this study. Based on the gene
expression profiles and CpG sites on 4 subgroups of STSs, we found that the levels of gene expression
and DNA methylation were consistent, which revealed that methylation sites might affected the
pathogenesis of STSs through modulating the expression of corresponded genes. Furthermore, the
functional analysis discovered that these annotated genes were enriched in several biological processes
and signaling pathways, like Cell adhesion process, apoptosis, SMAD pathway, phosphatidylinositol 3 −
kinase (PI3K) pathway, intermediate filament cytoskeleton formation, and others. All of these biological
processes and signaling pathway has been declared significantly correlated with tumorigenesis and
progression [44–46]. These explores could provide clues to emphasize the relationship between these
specific methylation sites and STSs biological processes and signaling pathways. Moreover, in this study, we identified whether these specific methylation sites could be used at the
prognostic level and followed by the construction of a STSs prognostic prediction model. Eventually, we
focused on the differential CpG sites among 4 clusters and developed a novel thirteen methylation sites
for prognostication. Discussion The constructed risk model was determined with a robust prognostic value and
demonstrated to be an independent prognostic factor for STSs. In addition, the risk score was also
confirmed with a major advantage of its biological implications for predicting STSs intrinsic histological
subtypes and its original sites. A similar scenario was also observed in the nomogram analysis that risk
signature played a virtual role in predicting the OS of STSs, which may be caused by the intensive
correlation between the risk signature and STSs pathogenesis. Conclusion In conclusion, our research identified a new classification of STSs into four different prognosis
subgroups based on the DNA methylation data. This classification will help to provide more accurate
subdivision of STSs and facilitate clinicians to choose a more individualized treatment. Furthermore, the
specific CpG sites and corresponding genes in each epigenetic subtype can be used as biomarkers for
early diagnosis, precise prognosis prediction, and biological processes and signaling pathways
exploration. Most importantly, our study provides a framework to construct a novel classification of
molecular subtypes associated with specific tumors. Funding This work has been financially supported by Innovation Project of Guangxi Graduate Education (Grant
No. YCSW2019117). Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. Ethics approval and consent to participate Not applicable. Acknowledgements Not applicable. Authors’ contributions Jun Yao and Jia Li designed the experiments. Zhengyuan Wu, Miao Yu, and Zhenpei Wen performed
research. Zhengyuan Wu and Tianyu Ren analyzed data. Zhengyuan Wu wrote the manuscript. All
authors reviewed the article. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author
on reasonable request. Abbreviations STSs: soft tissue sarcomas: TCGA: The Cancer Genome Atlas; GO: Gene Ontology; KEGG: Kyoto
Encyclopedia of Genes and Genomes; OS: overall survival; PI3K: phosphatidylinositol 3−kinase; UCSC
Xena: University of California Santa Cruz Xena; RNA‐Seq: RNA‐sequencing; KNN: k-nearest neighbor; CDF: Page 10/22 Page 10/22 cumulative distribution function; BP: biological process; CC: cellular component; MF: molecular function;
FC: fold change; FDR: false discovery rate; LASSO: least absolute shrinkage and selection operator. cumulative distribution function; BP: biological process; CC: cellular component; MF: molecular function;
FC: fold change; FDR: false discovery rate; LASSO: least absolute shrinkage and selection operator. References 1. Hoefkens F, Dehandschutter C, Somville J, Meijnders P, Van Gestel D. Soft tissue sarcoma of the
extremities: pending questions on surgery and radiotherapy. Radiation oncology (London, England). 2016;11:136. 1. Hoefkens F, Dehandschutter C, Somville J, Meijnders P, Van Gestel D. Soft tissue sarcoma of the
extremities: pending questions on surgery and radiotherapy. Radiation oncology (London, England). 2016;11:136. Page 11/22
2. von Mehren M, Randall RL, Benjamin RS, Boles S, Bui MM, Ganjoo KN, et al. Soft Tissue Sarcoma,
Version 2.2018, NCCN Clinical Practice Guidelines in Oncology. Journal of the National
Comprehensive Cancer Network : JNCCN. 2018;16:536-63. 2. von Mehren M, Randall RL, Benjamin RS, Boles S, Bui MM, Ganjoo KN, et al. Soft Tissue Sarcoma,
Version 2.2018, NCCN Clinical Practice Guidelines in Oncology. Journal of the National Version 2.2018, NCCN Clinical Practice Guidelines in Oncology. Journal of the National
Comprehensive Cancer Network : JNCCN. 2018;16:536-63. Page 11/22 3. Casali PG, Abecassis N, Aro HT, Bauer S, Biagini R, Bielack S, et al. Soft tissue and visceral sarcomas:
ESMO-EURACAN Clinical Practice Guidelines for diagnosis, treatment and follow-up. Annals of
oncology : official journal of the European Society for Medical Oncology. 2018;29:iv268-iv69. 4. Honoré C, Méeus P, Stoeckle E, Bonvalot S. Soft tissue sarcoma in France in 2015: Epidemiology,
classification and organization of clinical care. Journal of visceral surgery. 2015;152:223-30. 4. Honoré C, Méeus P, Stoeckle E, Bonvalot S. Soft tissue sarcoma in France in 2015: Epidemiology,
classification and organization of clinical care. Journal of visceral surgery. 2015;152:223-30. 5. Kotilingam D, Lev DC, Lazar AJ, Pollock RE. Staging soft tissue sarcoma: evolution and change. CA:
a cancer journal for clinicians. 2006;56:282-91; quiz 314-5. 5. Kotilingam D, Lev DC, Lazar AJ, Pollock RE. Staging soft tissue sarcoma: evolution and change. CA:
a cancer journal for clinicians. 2006;56:282-91; quiz 314-5. 6. [Chinese expert consensus on diagnosis and treatment of soft tissue sarcomas (Version 2015)]. Zhonghua zhong liu za zhi [Chinese journal of oncology]. 2016;38:310-20. 6. [Chinese expert consensus on diagnosis and treatment of soft tissue sarcomas (Version 2015)]. Zhonghua zhong liu za zhi [Chinese journal of oncology]. 2016;38:310-20. 7. Scheer M, Dantonello T, Hallmen E, Blank B, Sparber-Sauer M, Vokuhl C, et al. Synovial Sarcoma
Recurrence in Children and Young Adults. Annals of surgical oncology. 2016;23:618-26. 8. Yang X, Huang WT, He RQ, Ma J, Lin P, Xie ZC, et al. References Determining the prognostic significance of
alternative splicing events in soft tissue sarcoma using data from The Cancer Genome Atlas. Journal
of translational medicine. 2019;17:283. 9. Xie J, Lin D, Lee DH, Akunowicz J, Hansen M, Miller C, et al. Copy number analysis identifies tumor
suppressive lncRNAs in human osteosarcoma. International journal of oncology. 2017;50:863-72. 10. Zhu Z, Jin Z, Zhang H, Zhang M, Sun D. Integrative Clustering Reveals a Novel Subtype of Soft
Tissue Sarcoma With Poor Prognosis. Frontiers in genetics. 2020;11:69. 11. Koch A, Joosten SC, Feng Z, de Ruijter TC, Draht MX, Melotte V, et al. Author Correction: Analysis of
DNA methylation in cancer: location revisited. Nature reviews Clinical oncology. 2018;15:467. 12. Koch A, Joosten SC, Feng Z, de Ruijter TC, Draht MX, Melotte V, et al. Analysis of DNA methylation in
cancer: location revisited. Nature reviews Clinical oncology. 2018;15:459-66. 13. A. Koch, S. C. Joosten, Z. Feng, T. C. de Ruijter, M. X. Draht, V. Melotte, et al. Analysis of DNA
methylation in cancer: location revisited. Nat Rev Clin Oncol. 2018 Jul;15:459-66. 14. Chen W, Zhuang J, Wang PP, Jiang J, Lin C, Zeng P, et al. DNA methylation-based classification and
identification of renal cell carcinoma prognosis-subgroups. Cancer cell international. 2019;19:185. 15. Lai HC, Lin YW, Huang TH, Yan P, Huang RL, Wang HC, et al. Identification of novel DNA methylation
markers in cervical cancer. International journal of cancer. 2008;123:161-7. 16. Voisin S, Eynon N, Yan X, Bishop DJ. Exercise training and DNA methylation in humans. Acta
physiologica (Oxford, England). 2015;213:39-59. 16. Voisin S, Eynon N, Yan X, Bishop DJ. Exercise training and DNA methylation in humans. Acta
physiologica (Oxford, England). 2015;213:39-59. 17. Ferreira HJ, Esteller M. CpG Islands in Cancer: Heads, Tails, and Sides. Methods in molecular biology
(Clifton, NJ). 2018;1766:49-80. 17. Ferreira HJ, Esteller M. CpG Islands in Cancer: Heads, Tails, and Sides. Methods in molecular biology
(Clifton, NJ). 2018;1766:49-80. 18. Wu SP, Cooper BT, Bu F, Bowman CJ, Killian JK, Serrano J, et al. DNA Methylation-Based Classifier for
Accurate Molecular Diagnosis of Bone Sarcomas. JCO precision oncology. 2017;2017. 18. Wu SP, Cooper BT, Bu F, Bowman CJ, Killian JK, Serrano J, et al. DNA Methylation-Based Classifier for
Accurate Molecular Diagnosis of Bone Sarcomas. JCO precision oncology. 2017;2017. 19. Tian W, Li Y, Zhang J, Li J, Gao J. Combined analysis of DNA methylation and gene expression
profiles of osteosarcoma identified several prognosis signatures. Gene. References 2018;650:7-14. Page 12/22 Page 12/22 20. Peille AL, Brouste V, Kauffmann A, Lagarde P, Le Morvan V, Coindre JM, et al. Prognostic value of
PLAGL1-specific CpG site methylation in soft-tissue sarcomas. PloS one. 2013;8:e80741. 21. Knösel T, Altendorf-Hofmann A, Lindner L, Issels R, Hermeking H, Schuebbe G, et al. Loss of
p16(INK4a) is associated with reduced patient survival in soft tissue tumours, and indicates a
senescence barrier. Journal of clinical pathology. 2014;67:592-8. 22. Xie Y, Zong P, Wang W, Liu D, Li B, Wang Y, et al. Hypermethylation of potential tumor suppressor miR-
34b/c is correlated with late clinical stage in patients with soft tissue sarcomas. Experimental and
molecular pathology. 2015;98:446-54. 22. Xie Y, Zong P, Wang W, Liu D, Li B, Wang Y, et al. Hypermethylation of potential tumor suppressor miR-
34b/c is correlated with late clinical stage in patients with soft tissue sarcomas. Experimental and
molecular pathology. 2015;98:446-54. 23. Liu J, Li R, Liao X, Hu B, Yu J. Comprehensive investigation of the clinical significance and molecular
mechanisms of plasmacytoma variant translocation 1 in sarcoma using genome-wide RNA
sequencing data. Journal of Cancer. 2019;10:4961-77. 23. Liu J, Li R, Liao X, Hu B, Yu J. Comprehensive investigation of the clinical significance and molecular
mechanisms of plasmacytoma variant translocation 1 in sarcoma using genome-wide RNA
sequencing data. Journal of Cancer. 2019;10:4961-77. 24. Anders S, Huber W. Differential expression analysis for sequence count data. Genome biology. 2010;11:R106. 24. Anders S, Huber W. Differential expression analysis for sequence count data. Genome biology. 2010;11:R106. 25. Zhang S, Li X, Zong M, Zhu X, Wang R. Efficient kNN Classification With Different Numbers of
Nearest Neighbors. IEEE transactions on neural networks and learning systems. 2018;29:1774-85. 25. Zhang S, Li X, Zong M, Zhu X, Wang R. Efficient kNN Classification With Different Numbers of
Nearest Neighbors. IEEE transactions on neural networks and learning systems. 2018;29:1774-85. 26. Chen X, Zhao C, Zhao Z, Wang H, Fang Z. Specific Glioma Prognostic Subtype Distinctions Based on
DNA Methylation Patterns. Frontiers in genetics. 2019;10:786. 26. Chen X, Zhao C, Zhao Z, Wang H, Fang Z. Specific Glioma Prognostic Subtype Distinctions Based on
DNA Methylation Patterns. Frontiers in genetics. 2019;10:786. 27. Zhang Y, Li H, Zhang W, Che Y, Bai W, Huang G. LASSO‑based Cox‑PH model identifies an 11‑lncRNA
signature for prognosis prediction in gastric cancer. Molecular medicine reports. 2018;18:5579-93. 27. oncology. 2018;20:1616-24. Clinical & experimental metastasis. 2014;31:15-24. 45. Conti A, Espina V, Chiechi A, Magagnoli G, Novello C, Pazzaglia L, et al. Mapping protein signal
pathway interaction in sarcoma bone metastasis: linkage between rank, metalloproteinases turnover
and growth factor signaling pathways. Clinical & experimental metastasis. 2014;31:15-24. 46. Yang L, Shi P, Zhao G, Xu J, Peng W, Zhang J, et al. Targeting cancer stem cell pathways for cancer
therapy. Signal transduction and targeted therapy. 2020;5:8. 46. Yang L, Shi P, Zhao G, Xu J, Peng W, Zhang J, et al. Targeting cancer stem cell pathways for cancer
therapy. Signal transduction and targeted therapy. 2020;5:8. References Zhang Y, Li H, Zhang W, Che Y, Bai W, Huang G. LASSO‑based Cox‑PH model identifies an 11‑lncRNA
signature for prognosis prediction in gastric cancer. Molecular medicine reports. 2018;18:5579-93. 28. Wilkerson MD, Hayes DN. ConsensusClusterPlus: a class discovery tool with confidence
assessments and item tracking. Bioinformatics (Oxford, England). 2010;26:1572-3. 29. Yu G, Wang LG, Han Y, He QY. clusterProfiler: an R package for comparing biological themes among
gene clusters. Omics : a journal of integrative biology. 2012;16:284-7. 30. Li C, Ke J, Liu J, Su J. DNA methylation data-based molecular subtype classification related to the
prognosis of patients with cervical cancer. Journal of cellular biochemistry. 2020;121:2713-24. 31. Heagerty PJ, Zheng Y. Survival model predictive accuracy and ROC curves. Biometrics. 2005;61:92-
105. 32. Gu HY, Zhang C, Guo J, Yang M, Zhong HC, Jin W, et al. Risk score based on expression of five novel
genes predicts survival in soft tissue sarcoma. Aging. 2020;12:3807-27. 33. Sleijfer S, Gelderblom H. Current clinical trials for advanced osteosarcoma and soft tissue sarcoma. Current opinion in oncology. 2014;26:434-9. 34. Ma H, Zhao C, Zhao Z, Hu L, Ye F, Wang H, et al. Specific glioblastoma multiforme prognostic-
subtype distinctions based on DNA methylation patterns. Cancer gene therapy. 2019. 35. Klutstein M, Nejman D, Greenfield R, Cedar H. DNA Methylation in Cancer and Aging. Cancer research. 2016;76:3446-50. 36. Witt H, Gramatzki D, Hentschel B, Pajtler KW, Felsberg J, Schackert G, et al. DNA methylation-based
classification of ependymomas in adulthood: implications for diagnosis and treatment. Neuro- Page 13/22 Page 13/22 oncology. 2018;20:1616-24. oncology. 2018;20:1616-24. 37. Zhang Z, Li H, Jiang S, Li R, Li W, Chen H, et al. A survey and evaluation of Web-based
tools/databases for variant analysis of TCGA data. Briefings in bioinformatics. 2019;20:1524-41. 38. Huang R, Meng T, Chen R, Yan P, Zhang J, Hu P, et al. The construction and analysis of tumor-
infiltrating immune cell and ceRNA networks in recurrent soft tissue sarcoma. Aging. 2019;11:10116-
43. 39. Zhang Y, Zhang J. Identification of functionally methylated regions based on discriminant analysis
through integrating methylation and gene expression data. Molecular bioSystems. 2015;11:1786-93. 39. Zhang Y, Zhang J. Identification of functionally methylated regions based on discriminant analysis
through integrating methylation and gene expression data. Molecular bioSystems. 2015;11:1786-93. 39. Zhang Y, Zhang J. Identification of functionally methylated regions based on discriminant analysis
through integrating methylation and gene expression data. Molecular bioSystems. 2015;11:1786-93. 40. Hua S, Ji Z, Quan Y, Zhan M, Wang H, Li W, et al. Identification of hub genes in hepatocellular
carcinoma using integrated bioinformatic analysis. Aging. 2020;12:5439-68. 41. Pan Y, Ma P, Liu Y, Li W, Shu Y. Multiple functions of mA RNA methylation in cancer. Journal of
hematology & oncology. 2018;11:48. 41. Pan Y, Ma P, Liu Y, Li W, Shu Y. Multiple functions of mA RNA methylation in cancer. Journal of
hematology & oncology. 2018;11:48. 42. Cui Q, Shi H, Ye P, Li L, Qu Q, Sun G, et al. mA RNA Methylation Regulates the Self-Renewal and
Tumorigenesis of Glioblastoma Stem Cells. Cell reports. 2017;18:2622-34. 42. Cui Q, Shi H, Ye P, Li L, Qu Q, Sun G, et al. mA RNA Methylation Regulates the Self-Renewal and
Tumorigenesis of Glioblastoma Stem Cells. Cell reports. 2017;18:2622-34. 43. Xiang Y, Laurent B, Hsu CH, Nachtergaele S, Lu Z, Sheng W, et al. RNA mA methylation regulates the
ultraviolet-induced DNA damage response. Nature. 2017;543:573-76. 43. Xiang Y, Laurent B, Hsu CH, Nachtergaele S, Lu Z, Sheng W, et al. RNA mA methylation regulates the
ultraviolet-induced DNA damage response. Nature. 2017;543:573-76. 44. Omary MB. "IF-pathies": a broad spectrum of intermediate filament-associated diseases. The Journal
of clinical investigation. 2009;119:1756-62. 44. Omary MB. "IF-pathies": a broad spectrum of intermediate filament-associated diseases. The Journal
of clinical investigation. 2009;119:1756-62. 45. Conti A, Espina V, Chiechi A, Magagnoli G, Novello C, Pazzaglia L, et al. Mapping protein signal
pathway interaction in sarcoma bone metastasis: linkage between rank, metalloproteinases turnover
and growth factor signaling pathways. Figures Page 14/22 Page 14/22 Figure 1
Flowchart describing the schematic overview of the study design. Flowchart describing the schematic overview of the study design. Page 15/22 Page 15/22 Page 15/22 Figure 2
Consensus clustering of DNA methylation-based prognostic subgroups. (A) The consensus among
clusters for each category number k. (B) Delta area curve of consensus clustering, which indicates the
relative change in the area under the cumulative distribution function (CDF) curve for each category
number k compared with k‐1. The horizontal axis represents the category number k and the vertical axis
represents the relative change in the area under the CDF curve. Figure 2 Figure 2 Consensus clustering of DNA methylation-based prognostic subgroups. (A) The consensus among
clusters for each category number k. (B) Delta area curve of consensus clustering, which indicates the
relative change in the area under the cumulative distribution function (CDF) curve for each category
number k compared with k‐1. The horizontal axis represents the category number k and the vertical axis
represents the relative change in the area under the CDF curve. Page 16/22
p
g Page 16/22 Figure 3 Cluster Analysis of 4 subtypes with the corresponding heat map. (A) The heat map corresponding to the
consensus matrix for 4 molecular subtypes obtained by applying consensus clustering. (B) The heatmap
corresponding to the dendrogram in the figure A, which was generated using the pheatmap function in R
with DNA methylation classification, age, gender, histological type, and tissue or organ of original as the
annotations. Cluster Analysis of 4 subtypes with the corresponding heat map. (A) The heat map corresponding to the
consensus matrix for 4 molecular subtypes obtained by applying consensus clustering. (B) The heatmap
corresponding to the dendrogram in the figure A, which was generated using the pheatmap function in R
with DNA methylation classification, age, gender, histological type, and tissue or organ of original as the
annotations. Figure 4 Figure 4 Characterization of different features between each DNA methylation cluster. (A) The survival curves of
each sub-clusters indicate the prognostic differences among STSs patients. The distribution of age (B),
gender (C), histological type (D), and tissue or organ of original (E) in each DNA methylation subgroups. (F) The heatmap of methylation sites annotated genes distribution in 4 DNA methylation clusters. Characterization of different features between each DNA methylation cluster. (A) The survival curves of
each sub-clusters indicate the prognostic differences among STSs patients. The distribution of age (B),
gender (C), histological type (D), and tissue or organ of original (E) in each DNA methylation subgroups. (F) The heatmap of methylation sites annotated genes distribution in 4 DNA methylation clusters. Page 17/22 Page 17/22 Figure 5
Functional enrichment analysis of methylation sites annotated genes. (A-C) To
enrichment terms in biological process (BP), cellular component (CC), and mol
KEGG enrichment analysis of CpG sites. In each bubble plot, the size of the dot
enriched genes. Figure 5 Functional enrichment analysis of methylation sites annotated genes. (A-C) Top 30 classes of GO
enrichment terms in biological process (BP), cellular component (CC), and molecular function (MF). (D)
KEGG enrichment analysis of CpG sites. In each bubble plot, the size of the dot represents the number of
enriched genes. Page 18/22 Page 18/22 Figure 6
The specific CpG sites for each DNA methylation cluster. (A) The distribution of specific methylation sites
in each DNA methylation prognostic subtype. (B) The boxplot based on the specific methylation sites in
Cluster 1 for comparison the methylation level in each subgroup. Figure 6 The specific CpG sites for each DNA methylation cluster. (A) The distribution of specific methylation sites
in each DNA methylation prognostic subtype. (B) The boxplot based on the specific methylation sites in
Cluster 1 for comparison the methylation level in each subgroup. The specific CpG sites for each DNA methylation cluster. (A) The distribution of specific methylation sites
in each DNA methylation prognostic subtype. (B) The boxplot based on the specific methylation sites in
Cluster 1 for comparison the methylation level in each subgroup. The specific CpG sites for each DNA methylation cluster. (A) The distribution of specific methylation sites
in each DNA methylation prognostic subtype. (B) The boxplot based on the specific methylation sites in
Cluster 1 for comparison the methylation level in each subgroup. Page 19/22 Figure 7 Figure 7
Construction of the prognosis prediction model by LASSO. (A) The changing trajectory of each
independent variable. (B) Confidence intervals for each lambda. (C) The Kaplan–Meier overall survival
curves were assigned to compare the prognostic difference based on the risk score. (D) The relationship
between the methylation profile, overall survival, and risk scores. Figure 7 Construction of the prognosis prediction model by LASSO. (A) The changing trajectory of each
independent variable. (B) Confidence intervals for each lambda. (C) The Kaplan–Meier overall survival
curves were assigned to compare the prognostic difference based on the risk score. (D) The relationship
between the methylation profile, overall survival, and risk scores. Page 20/22 Page 20/22 Figure 8 Figure 8 Figure 8 Verifying the prognostic value of prediction model for patients with STSs. (A) The heatmap shows the
distribution of clinicopathological features and specific methylation sites expression level in the low- and
high-risk groups. *P < 0.05 and **P < 0.01. (B) ROC curves showed the predictive efficiency of the risk
signature on the one-, three-, and five-year survival rate. Univariate (C) and multivariate (D) Cox regression
analysis of the relationship between clinicopathological features (including the risk score) and overall Verifying the prognostic value of prediction model for patients with STSs. (A) The heatmap shows the
distribution of clinicopathological features and specific methylation sites expression level in the low- and
high-risk groups. *P < 0.05 and **P < 0.01. (B) ROC curves showed the predictive efficiency of the risk
signature on the one-, three-, and five-year survival rate. Univariate (C) and multivariate (D) Cox regression
analysis of the relationship between clinicopathological features (including the risk score) and overall Page 21/22 Page 21/22 survival of STSs patients. (E) Nomogram of risk score and other clinical factors for STSs 1-, 3-, and 5-year
event prediction Equation1.pdf Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Equation1.pdf Page 22/22
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Structural basis for non-genuine phenolic acceptor substrate specificity of Streptomyces roseochromogenes prenyltransferase CloQ from the ABBA/PT-barrel superfamily
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RESEARCH ARTICLE Structural basis for non-genuine phenolic
acceptor substrate specificity of Streptomyces
roseochromogenes prenyltransferase CloQ
from the ABBA/PT-barrel superfamily Carla Araya-Cloutier1, Bianca Martens1, Gijs Schaftenaar2, Franziska Leipoldt3,
Harry Gruppen1, Jean-Paul Vincken1* 1 Laboratory of Food Chemistry, Wageningen University, Wageningen, The Netherlands, 2 Nijmegen Centre
for Molecular Sciences, Radboud University Medical Centre, Nijmegen, The Netherlands, 3 Pharmaceutical
Biology, Pharmaceutical Institute, Eberhard Karls University Tu¨bingen, Tu¨bingen, Germany a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 * jean-paul.vincken@wur.nl * jean-paul.vincken@wur.nl * jean-paul.vincken@wur.nl OPEN ACCESS Citation: Araya-Cloutier C, Martens B, Schaftenaar
G, Leipoldt F, Gruppen H, Vincken J-P (2017)
Structural basis for non-genuine phenolic acceptor
substrate specificity of Streptomyces
roseochromogenes prenyltransferase CloQ from
the ABBA/PT-barrel superfamily. PLoS ONE 12(3):
e0174665. https://doi.org/10.1371/journal. pone.0174665 Abstract Acceptor substrate specificity of Streptomyces roseochromogenes prenyltransferase
SrCloQ was investigated using different non-genuine phenolic compounds. RP-UHPLC-
UV-MSn was used for the tentative annotation and quantification of the prenylated products. Flavonoids, isoflavonoids and stilbenoids with different types of substitution were prenylated
by SrCloQ, although with less efficiency than the genuine substrate 4-hydroxyphenylpyru-
vate. The isoflavan equol, followed by the flavone 7,4’-dihydroxyflavone, were the best non-
genuine acceptor substrates. B-ring C-prenylation was in general preferred over A-ring C-
prenylation (ratio 5:1). Docking studies of non-genuine acceptor substrates with the B-ring
oriented towards the donor substrate dimethylallyl pyrophosphate, showed that the carbonyl
group of the C-ring was able to make stabilizing interactions with the residue Arg160, which
might determine the preference observed for B-ring prenylation. No reaction products were
formed when the acceptor substrate had no phenolic hydroxyl groups. This preference can
be explained by the essential hydrogen bond needed between a phenolic hydroxyl group
and the residue Glu281. Acceptor substrates with an additional hydroxyl group at the C3’
position (B-ring), were mainly O3’-prenylated (> 80% of the reaction products). This can be
explained by the proximity of the C3’ hydroxyl group to the donor substrate at the catalytic
site. Flavones were preferred over isoflavones by SrCloQ. Docking studies suggested that
the orientation of the B-ring and of the phenolic hydroxyl group at position C7 (A-ring) of fla-
vones towards the residue Tyr233 plays an important role in this observed preference. Finally, the insights obtained on acceptor substrate specificity and regioselectivity for
SrCloQ were extended to other prenyltransferases from the CloQ/NhpB family. Introduction Prenylation is one of nature’s tools to modulate the bioactivity of primary [1] and secondary
metabolites [2, 3] by increasing their lipophilicity and, thereby, their interactions with biologi-
cal targets, such as proteins and membranes [4]. The enzymes responsible for transferring a
prenyl group from a donor substrate (e.g. dimethylallyl pyrophosphate) to an acceptor sub-
strate are known as prenyltransferases (PTs) [5]. Aromatic PTs catalyse the transfer reaction of
prenyl moieties onto aromatic acceptors, such as phenolic acids, (iso)flavonoids, coumarins,
naphthalenes, phenazines, and indole derivatives. These enzymes contribute substantially to
the large diversity of secondary metabolites present in plants, fungi, and bacteria [6, 7]. Plants from the Fabaceae family are well known for their production of prenylated isoflavo-
noids upon abiotic or biotic stress [8]. The prenyl group is most often added to a free aromatic
carbon (C-prenylation), but also to phenolic oxygens (O-prenylation) [9]. Several reports on
legume PTs are available. PTs responsible for the production of glyceollins in soybean (Glycine
max L.) and phaseollin in kidney bean (Phaseolus vulgaris L.) are localized in the membrane of
plastids. Solubilisation of these PTs required detergents, which negatively affected enzyme
activity and stability [10]. Furthermore, characterization of Sophora flavescens PT revealed
donor and acceptor specificity to be confined to the known genuine substrates (i.e. in vivo sub-
strates) [11]. An overview of recently characterized plant PTs can be found elsewhere [12]. Contrary to plant PTs, microbial PTs appear to be attractive biotechnological tools as most
of them are soluble, i.e. not membrane-bound [4] and can potentially be obtained in signifi-
cant amounts for the in vitro production of novel and bioactive prenylated compounds [13]. In the last decade, a new superfamily of soluble aromatic PTs isolated from microorganisms
was discovered. This superfamily, named ABBA, has been considered for enhancement of
molecular diversity and bioactivity of natural compounds due to their promiscuity for differ-
ent non-genuine acceptor substrates [14]. The ABBA superfamily is a group of enzymes with a
unique type of PT barrel fold comprising a series of 5 repetitive ααββ elements [13, 15]. More
specifically, this PT-barrel is formed of 10 antiparallel β strands forming a spacious central sol-
vent-filled cavity, where acceptor and donor substrates bind, surrounded by a ring of 10 α-
helices [16]. Editor: Emily J. Parker, University of Canterbury,
NEW ZEALAND Copyright: © 2017 Araya-Cloutier et al. This is an
open access article distributed under the terms of
the Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Finally, the insights obtained on acceptor substrate specificity and regioselectivity for
SrCloQ were extended to other prenyltransferases from the CloQ/NhpB family. Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files. Funding: The authors received no specific funding
for this work. Competing interests: The authors have declared
that no competing interests exist. 1 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0174665
March 29, 2017 Non-genuine phenolic acceptor substrate specificity of prenyltransferase CloQ PLOS ONE | https://doi.org/10.1371/journal.pone.0174665
March 29, 2017 PLOS ONE | https://doi.org/10.1371/journal.pone.0174665
March 29, 2017 Introduction Structural analysis of these PTs has revealed that there is a tendency for polar resi-
dues to cluster into the top half of the cavity where the pyrophosphate isoprenoid donor sub-
strate binds. Non-polar residues cluster in the lower half of the cavity where the acceptor
substrate binds [17]. Assays with microbial PTs have revealed broad aromatic acceptor sub-
strate specificity, while often preserving donor substrate selectivity [18, 19]. Phylogenetic analysis of the ABBA PT superfamily revealed two distinct homologous fami-
lies: one comprises the indole PTs, i.e. the DMATS/CymD family; the other one comprises the
phenol/phenazines PTs, i.e. the CloQ/NphB family [14, 20]. Within the CloQ/NphB family
there are PTs involved in meroterpenoid and prenylated phenazine biosynthesis (e.g. NphB,
Fnq26, SCO7190, PpzP and Fur7) and PTs involved in novobiocin and clorobiocin biosynthe-
sis (i.e. NovQ and CloQ) [4, 13]. Streptomyces roseochromogenes CloQ (SrCloQ) was one of the first members of the ABBA
superfamily discovered [15, 21]. SrCloQ is an aromatic PT catalysing the C-prenylation (C5
isoprenoid unit) of 4-hydroxyphenylpyruvate (4-HPP), as part of the biosynthesis of the anti-
biotic clorobiocin [21]. More recent crystallization and simulation studies report key residues
involved in substrate binding and on the mechanism of action of this enzyme (Fig 1). SrCloQ
is thought to perform a Friedel-Crafts type of alkylation of the acceptor 4-HPP via the forma-
tion of a carbocation on the prenyl donor. The electropositive nature of the upper cavity of the
PT barrel helps to lower the energy barrier to catalysis by facilitating the cleavage of the C–O
bond of the isoprenoid donor substrate resulting in the formation of a prenyl cation [17]. The PLOS ONE | https://doi.org/10.1371/journal.pone.0174665
March 29, 2017 2 / 19 Non-genuine phenolic acceptor substrate specificity of prenyltransferase CloQ Fig 1. Schematic representation of the proposed mechanism of prenylation by SrCloQ, adapted from previous work [17]. Important residues
interacting with the donor and acceptor substrate are shown in grey colour. The donor substrate DMAPP is shown in red, whereas the genuine substrate is
shown in blue. Fig 1. Schematic representation of the proposed mechanism of prenylation by SrCloQ, adapted from previous work [17]. Important residues
interacting with the donor and acceptor substrate are shown in grey colour. The donor substrate DMAPP is shown in red, whereas the genuine substrate is
shown in blue Fig 1. Expression and purification of SrCloQ Production of the His-tagged SrCloQ was based on previous studies [16, 21]. Calcium chloride
transformation was performed with cells of Escherichia coli Rosetta (DE3) plysS (Promega,
Madison, WI, USA). Cells harbouring Srcloq were cultured in TB medium supplemented with
chloramphenicol (34 μg mL-1) and kanamycin (50 μg mL-1) for selection. Induction with iso-
propyl thiogalactoside (IPTG, Promega) was performed for 19 h at 20˚C, after an OD600nm of
0.6 was reached. After harvesting the cells, cells were resuspended in lysis buffer (2.5 mL lysis
buffer per g cells; 50 mM TrisHCl pH 8.0, 500 mM NaCl, 10% (v/v) glycerol, 10 mM β-mer-
captoethanol, 1% (v/v) Tween 20, 20 mM imidazole, 0.5 mM phenylmethylsulfonyl fluoride,
0.5 mg mL-1 lysozyme). Lysis was performed by sonication (Sonifier S-250D, Branson, Dan-
bury, CT, USA) in 9 sets of 30 s at an amplitude of 30% (pulse was alternatively turned on and
off for periods of 10 s). The His-tagged SrCloQ was purified from the cell lysate by affinity
chromatography with an A¨KTA explorer system (GE Healthcare, Little Chalfont, UK), with a
HisTrap HP 5 mL column (GE Healthcare) and buffers A (50 mM Tris-HCl pH 7.5, 20 mM
imidazole) and B (50 mM Tris-HCl pH 7.5, 250 mM imidazole), at 5ml min-1. After equilibra-
tion of the column with buffer A, cell lysate was loaded and SrCloQ was eluted with buffer B. The fractions containing the His-tagged protein were pooled, concentrated and desalted using
Amicon Ultra-15 10K centrifugal filter devices (Merck Millipore, Billerica, MA, USA). Materials and methods
Chemicals The construction of the plasmid containing SrCloQ has been described elsewhere [21]. Daid-
zein, genistein, 4-hydroxyphenylpyruvate (4-HPP) dimethylallyl pyrophosphate (DMAPP,
donor substrate), growth media (lysogeny broth (LB) and terrific broth (TB)) and antibiotics
(kanamycin and chloramphenicol) were obtained from Sigma Aldrich (St. Louis, MO, USA). 3’-Hydroxy-daidzein was obtained from Alfa Aesar (Ward Hill, MA, USA) and all other pure
(iso)flavonoids and resveratrol were obtained from ICC Chemical Corporation (New York,
NY, USA). Acetonitrile (ACN; ULC/MS grade), water acidified with acetic acid (HOAc, 0.1%
v/v) (ULC/MS grade), and methanol (MeOH) (ULC/MS grade) were purchased from Biosolve
(Valkenswaard, The Netherlands). Water for purposes other than UHPLC was prepared using
a Milli-Q water purification system (Millipore, Molsheim, France). Glycerol was purchased
from VWR International BV (Radnor, PA, USA). Other chemicals were purchased from
Merck (Darmstadt, Germany) or Sigma Aldrich. Introduction Schematic representation of the proposed mechanism of prenylation by SrCloQ, adapted from previous work [17]. Important residues
interacting with the donor and acceptor substrate are shown in grey colour. The donor substrate DMAPP is shown in red, whereas the genuine substrate is
shown in blue. htt
//d i
/10 1371/j
l
0174665 001 https://doi.org/10.1371/journal.pone.0174665.g001 https://doi.org/10.1371/journal.pone.0174665.g001 prenyl cation, stabilized by charge delocalization, performs an electrophilic attack on the C3 of
the aromatic ring of the acceptor substrate, resulting in an intermediary positive σ complex. The residue Glu281 is ideally placed in the active site to make a hydrogen bond with the phe-
nolic hydroxyl group of 4-HPP and to neutralize the σ complex by proton abstraction. The res-
idues Arg160 and Arg176 form salt bridges with the carboxyl group of 4-HPP, which is
thought to be important for positioning the substrate [22]. The residues Glu281 and Arg160
proved to be essential for good catalytic activity, while other residues (e.g. Cys215, Cys297) sta-
bilise binding of the aromatic substrates in the active site.[17, 22] To date, there have been no studies on the acceptor substrate specificity of SrCloQ regard-
ing phenolic compounds, such as flavonoids, isoflavonoids and stilbenoids. Other studies
with closely related PTs (e.g. Streptomyces spheroids NovQ) have reported the substrate speci-
ficity with phenolic compounds as acceptor substrates [18, 19, 23], but have not studied in
detail the structural basis for non-genuine substrate specificity. In order to provide insight
on the structural basis for prenylation of phenolic compounds, (iso)flavonoid and stilbenoid
substrate specificities of SrCloQ were determined by in vitro enzymatic assays and analysis of
reaction products by means of RP-UHPLC-UV-MSn. Furthermore, we analysed in silico the
interactions between the phenolic acceptors and SrCloQ’s active site, and propose the inter-
actions between the phenolic acceptor substrates and the active site residues, governing the
substrate specificity experimentally observed. In addition, we compared the phenolic sub-
strate specificity and regioselectivity of SrCloQ with that of other members of the CloQ/
NphB family. We hypothesized that (iso)flavonoids will be prenylated by SrCloQ at the B- 3 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0174665
March 29, 2017 Non-genuine phenolic acceptor substrate specificity of prenyltransferase CloQ ring due to the similarities of this ring with the phenolic ring of SrCloQ’s genuine substrate
4-HPP. Analysis of reaction products Reaction products were analysed by Ultra High Performance Liquid Chromatography and
Mass Spectrometry (UHPLC-UV-MS). An Accela Velos UHPLC system (Thermo Scientific,
San Jose, CA, USA) was equipped with a pump, autosampler and photodiode array (PDA)
detector. Samples (1 μL) were loaded onto an Acquity UPLC BEH Shield RP18 column (2.1 i. d. mm x 150 mm, 1.7 μm particle size; Waters, Milford, MA, USA) with an Acquity UPLC
BEH Shield RP18 VanGuard pre-column (2.1 i.d. mm x 5 mm, 1.7 μm particle size; Waters). Water containing HOAc (0.1% v/v) and ACN (1% v/v), eluent A, and ACN containing
HOAc (0.1% v/v), eluent B, were used as solvents at a flow rate of 300 μL min-1. The following
elution profile was used: 1 min isocratic at 9% v/v B; 1.5 min linear gradient from 9–25% B; 7
min linear gradient from 25–50% B; 3 min isocratic on 50% B; 10 min linear gradient from
50–100% B; 2 min isocratic on 100% B, 1 min linear gradient from 100–9% B. Column temper-
ature was set at 40˚C and PDA detector was set to measure from 200–600 nm. Reaction products were analysed by Ultra High Performance Liquid Chromatography and
Mass Spectrometry (UHPLC-UV-MS). An Accela Velos UHPLC system (Thermo Scientific,
San Jose, CA, USA) was equipped with a pump, autosampler and photodiode array (PDA)
detector. Samples (1 μL) were loaded onto an Acquity UPLC BEH Shield RP18 column (2.1 i. d. mm x 150 mm, 1.7 μm particle size; Waters, Milford, MA, USA) with an Acquity UPLC
BEH Shield RP18 VanGuard pre-column (2.1 i.d. mm x 5 mm, 1.7 μm particle size; Waters). Water containing HOAc (0.1% v/v) and ACN (1% v/v), eluent A, and ACN containing
HOAc (0.1% v/v), eluent B, were used as solvents at a flow rate of 300 μL min-1. The following
elution profile was used: 1 min isocratic at 9% v/v B; 1.5 min linear gradient from 9–25% B; 7
min linear gradient from 25–50% B; 3 min isocratic on 50% B; 10 min linear gradient from
50–100% B; 2 min isocratic on 100% B, 1 min linear gradient from 100–9% B. Column temper-
ature was set at 40˚C and PDA detector was set to measure from 200–600 nm. Mass spectrometric (MS) analysis was performed on a LTQ Velos (Thermo Scientific),
which was equipped with a heated ESI-MS probe coupled to the RP-UHPLC. Protein content and composition Protein content was determined according to Bradford. A calibration curve was made with
bovine serum albumin (BSA) in concentrations of 0.25–1.0 mg mL-1. Enzyme purity was con-
firmed by SDS-PAGE under reducing conditions on a Mini-protean II system (Bio-Rad Labo-
ratories, Hercules, CA, USA), according to the manufacturer’s instructions. Commercially
prepared mini-protean TGX gels (Bio-Rad) were used with Coomassie InstantBlue (Expedion,
Cambridge, UK), with the marker Precision Plus Protein™dual colour standards (Bio-Rad). Samples (8 μL) were loaded onto a gel, and the separation was done by applying 200 V for 45
min. 4 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0174665
March 29, 2017 Non-genuine phenolic acceptor substrate specificity of prenyltransferase CloQ Assay for PT activity The reaction mixture contained: aromatic substrate (0.5 mM, 4-HPP, flavonoids, isoflavonoids
or resveratrol), DMAPP (0.5 mM), NaCl (500 mM), glycerol (10% v/v), TrisHCl buffer (pH
7.5, 100 mM), Mg2+ (7 mM in the form of MgCl2, for enhancement of activity) and purified
SrCloQ (30 μM). In analogy with previous studies on the in vitro prenylation of flavonoids
with Streptomyces sp. strain CL190 (SclNphB, 22% identity with SrCloQ) [15, 18], incubation
time of SrCloQ with the non-genuine substrates was prolonged in comparison with that of the
genuine substrate 4-HPP (24–48 h), due to the anticipated lower efficiency in conversion of
the (iso)flavonoids. The mixtures were incubated for a maximum of 48 h at 30˚C. The reaction
was ended by adding ethyl acetate (400 μL) containing formic acid (0.5% v/v). The solution
was vortexed and centrifuged (room temperature, 5 min, 10,000 g), after which the organic
layer was evaporated and the residue was re-suspended in methanol (100 μL) for analysis on
RP-UHPLC-UV-MS. Experiments were performed in duplicate. PLOS ONE | https://doi.org/10.1371/journal.pone.0174665
March 29, 2017 Analysis of reaction products Full scan MS
was performed in both negative ionisation (NI) and positive ionisation (PI) mode, in which
data were acquired in a m/z range of 90–1500 Da. For tentative annotation, data-dependent
MSn analysis on the most intense (product) ion was performed with normalised collision
energy of 35%. Prenylated products were monitored by single ion monitoring (SIM) scanning
mode followed by single reaction monitoring (SRM) on the most abundant fragment ions. The system was tuned with genistein in PI and NI mode via automatic tuning using Tune Plus
(Xcalibur v.2.2, Thermo Scientific). Nitrogen was used as sheath and auxiliary gas. The ITT
temperature was 400˚C and the source voltage was 3.50 kV (NI) or 4.50 kV (PI). The tentative annotation of prenylated reaction products was performed by means of Xcali-
bur (version 2.2., Thermo Scientific). The position of the prenyl group within the phenolic
skeleton (i.e. A- or B-ring) was elucidated by analysis of the retro-Diels-Alders (RDA) frag-
ments in PI [24]: when the C-ring of (iso)flavonoids was cleaved in MS3, one of the remaining
fragments still contained one carbon reminiscent of the prenyl chain (split in MS2), which can
be used to diagnose the ring at which the prenyl was attached [25]. Quantification of phenolic compounds was performed using the following equation Eq (1)
[26], derived from the Lambert-Beer’s law: C ¼
area Q
ε l Vinj kcell
ð1Þ ð1Þ 5 / 19 Non-genuine phenolic acceptor substrate specificity of prenyltransferase CloQ in which C is concentration (M), area is the integrated area of the UV peak at the specific wave-
length (AUs), Q is the flow rate (5 μL s-1), ε is the molar extinction coefficient (AU M-1cm-1),
l is the path length of the UV cell provided by the manufacturer (5 cm), Vinj is the injected vol-
ume of sample (1 μL), and kcell is a constant related to the cell geometry of the UV detector
[26]. This equation relates the duration of absorbance given by the UHPLC system (AUs) to
an actual absorbance value (AU) for the Lambert-Beer’s equation. The kcell represents the cor-
rection factor for the absorption of light by the coating material of the flow cell. The kcell
(0.82 ± 0.09) was determined with standard solutions of daidzein (248 nm), genistein (263 nm)
and resveratrol (310 nm) (with five concentrations each, in the range of 0.001–0.1 mg mL-1). In silico modelling Molecular Operating Environment (MOE), 2013.08 (Chemical Computing Group, Montreal,
QC, Canada) was used to analyse the PT structure and to perform docking studies. The SrCloQ
model (Protein Database entry 2XLQ) with the genuine substrate (4-HPP) bound to the active
site was used for the docking studies [17]. The location of the DMAPP substrate inside the
SrCloQ active site was modelled based on the position of the geranyl pyrophosphate (GPP)
inside SclNphB (PDB 1ZB6) [27]. The placement of DMAPP was achieved by first creating a
3D alignment of the 2XLQ and 1ZB6 structures and subsequently taking the GPP position and
transferring it to the 2XLQ structure, followed by molecular editing to convert GPP into
DMAPP. Finally, a local geometry optimization was performed, at which only DMAPP was
kept flexible. Further refinement of the DMAPP and 4-HPP position was performed using
MMFF94x energy minimization in MOE. The LigX module in MOE was used as a guide to
confirm that the placement was in line with that of the GPP inside 1ZB6. In accordance with
previous literature [17, 22], our modelled negatively charged donor substrate DMAPP
(Figure A in S1 File) made interactions with the positively charged residues Lys54, Arg66, Lys
279, and Lys120, and with the aromatic residues Tyr233 and Tyr174. The genuine substrate
4-HPP made the essential hydrogen bond with the residue Glu281, as well as with Arg160 and
Arg176. The 3D structures of the phenolic compounds were built with MOE and MMFF94x energy
minimization (gradient 0.01) was performed for all molecules. Induced fit was used as docking
mode and the predicted pose was selected based on the dock score (S) as implemented in
MOE. Analysis of reaction products The ε of the prenylated reaction products was assumed to be the same as that of the non-pre-
nylated substrate, as shown in Table A in S1 File. The percentage of conversion of the different
aromatic substrates was calculated as the μmoles of prenylated products formed from the initial
aromatic substrate concentration (500 μM), multiplied by 100. Sequence and structure comparison Alignment and sequence identity analysis of PTs from the CloQ/NphB PT family was per-
formed with UniProt (http://www.uniprot.org/align/) and visualized with ESPript 3.0 [28]. Structure superposition of SrCloQ and SclNphB was performed with MOE. PLOS ONE | https://doi.org/10.1371/journal.pone.0174665
March 29, 2017 Enzyme activity of SrCloQ SrCloQ was expressed as His-tagged protein in E. coli and purified by Ni2+ affinity chromatog-
raphy (Figure B in S1 File). The purified enzyme showed the expected molecular mass (35
kDa) [21] and an estimated purity of >90% according to the SDS-PAGE analysis (Figure C in 6 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0174665
March 29, 2017 Non-genuine phenolic acceptor substrate specificity of prenyltransferase CloQ Fig 2. Aromatic acceptor substrates and their percentage of conversion (molar %) by SrCloQ. The
atoms shared with the genuine substrate are highlighted in blue colour. Phenolic substrates with green label
represent the best acceptor substrates, whereas those with red labels were not utilized by the enzyme. https://doi org/10 1371/journal pone 0174665 g002 Fig 2. Aromatic acceptor substrates and their percentage of conversion (molar %) by SrCloQ. The
atoms shared with the genuine substrate are highlighted in blue colour. Phenolic substrates with green label
represent the best acceptor substrates, whereas those with red labels were not utilized by the enzyme. https://doi.org/10.1371/journal.pone.0174665.g002 https://doi.org/10.1371/journal.pone.0174665.g002 S1 File). Activity of SrCloQ was confirmed in control experiments with its genuine substrate
(4-HPP); incubations without SrCloQ did not yield prenylated products (Figure D in S1 File). (4-HPP); incubations without SrCloQ did not yield prenylated products (Figure D in S1 File). Fig 2 shows the structure of all aromatic acceptor substrates tested, i.e. flavonoids, isoflavo-
noids and the stilbenoid resveratrol, as well as the percentage of molar conversion by SrCloQ. Compounds had different types of substituents, such as hydroxyl, methoxyl and carbonyl
groups, at different positions of the skeleton. For optimal visualization, the structures of com-
pounds are oriented with the B-ring of the phenolics in the same direction as the phenol ring
of the genuine substrate 4-HPP. SrCloQ showed promiscuity in that it was able to use most
phenolics provided as acceptor substrates, i.e. we found reaction products for 10 out of 12 phe-
nolic compounds tested. As expected, conversion of the genuine substrate 4-HPP was more
efficient (>80% conversion) in comparison to those of the other phenolic test substrates
(< 10% conversion) under the conditions used. Of all phenolics tested, the isoflavan equol and
the flavone 4’,7-dihydroxyflavone were the best substrates for prenylation. Daidzein and genis-
tein were converted only in very small amounts, whereas isoflavone (no hydroxyl groups pres-
ent) and glycitein (methoxyl group at position C6) were not converted at all. Non-genuine phenolic acceptor substrate specificity of prenyltransferase CloQ Table 1. SrCloQ reaction products tentatively annotated by RP-UHPLC-UV-MSn. Substrate
Conversion
(% ± std)
No. RT
(min)
λmax
(nm)
[M-H]-
MS2
(rel. abundance)
MS3
(rel. abundance)
[M
+H]+
MS2
(rel. abundance)
MS3
(rel. abundance)
Tentative
annotation
4-HPP
84 ± 15
1
7.8
280
189
134 (100), 174 (10)
106 (100)
n.d. a
C-prenyl-
4-HBAL b
2
8.9
n.d. 189
134 (100), 174 (10)
105 (100), 106 (30)
n.d. C-prenyl-
4-HBAL
3
9.1
n.d. 189
134 (100), 174 (10)
106 (100)
n.d. C-prenyl-
4-HBAL
4
9.9
278
219
175 (100), 201 (10)
132 (100),107 (90), 119
(70), 106 (40), 157 (30)
n.d. C-prenyl-
4-HPA b
5
10.1
278
219
175 (100)
107 (100), 132 (80), 119
(60), 106 (50), 157 (40)
n.d. C-prenyl-
4-HPA
6
11.4
280
189
134 (100), 174 (10)
106 (100)
n.d. C-prenyl-
4-HBAL
7
12.6
285
189
134 (100), 174 (10)
106 (100)
n.d. C-prenyl-
4-HBAL
Eq
7.7 ± 1.4
8
16.9
n.d. 309
121 (100), 187 (70), 135
(30)
92 (100), 77 (25), 65 (15)
311
123 (100), 175 (90), 255
(50), 189 (40), 149 (15),
201 (10)
95 (100), 67 (35), 77 (10)
Bring-C-prenyl-
Eq
9
17.1
285
309
189 (100), 203 (20), 119
(10)
134 (100)
311
191 (100), 205 (50), 255
(20), 107 (10)
173 (100), 149 (50), 145 (10),
137 (10), 135 (10)
Aring-C-prenyl-
Eq e
7,4’-DHF
4.2 ± 1.2
10
14.5
334
321
266 (100), 265 (10)
237 (100), 223 (90), 222
(70), 238 (70), 265 (30),
135 (20)
323
267 (100), 268 (15)
239 (100), 213 (80), 240(20),
228(20)
Bring-C-prenyl-
7,4’-DHF
Lu
3.2 ± 0.6
11
13.4
n.d. 353
151 (100), 284 (95), 283
(90), 201 (70), 335 (30),
324 (20)
n.d. 355
n.d. n.d. C-prenyl-Lu
12
14.4
n.d. 353
151(100), 298 (50), 201
(30), 231 (20), 335 (15)
107 (100), 83 (10)
355
n.d. n.d. Bring-C-prenyl-
Lu
13
16.5
266,
348
353
284 (100)
256 (100)
355
287(100)
153 (100), 287 (90), 259 (50),
245 (30), 241 (30), 161 (20)
O-prenyl-Lu
4’-H-7-MF
1.7 ± 0.3
14
16.6
334
357,
335 c
342 (100), 343 (20), 207
(10)
342 (100), 341 (90), 343
(40), 325 (30), 314 (20),
205 (20)
359,
337 c
281 (100), 344 (30), 253
(30), 282 (20)
253 (100), 227 (25), 254 (20)
Bring-C-prenyl-
4’-H-7-MF
15
17.2
335
335
280 (100), 320 (80), 277
(60), 265 (15)
n.d. Structure elucidation of reaction products Table 1 shows the list of reaction products tentatively annotated by means of UHPL-
C-UV-MSn. Prenylation of substrates was confirmed by the neutral loss of 56 Da [C4H8] or 68 7 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0174665
March 29, 2017 337
281 (100), 282 (15)
253 (100), 227 (90), 254 (20),
228 (15)
Bring-C-prenyl-
4’-H-7-MF
16
18.2
327
357,
335 c
n.d. n.d. 359,
337 c
268 (100), 331 (30), 269
(20)
240 (100), 241 (20)
4’-O-prenyl-
7-MF
3-HDa
1.7 ± 0.4
17
10.9
n.d
337
268 (100), 201 (25), 309
(10), 293 (10), 135 (10)
n.d. 339
n.d. Bring-C-prenyl-
3’HDa
18
12.8
n.d
337
268 (100), 281 (40), 309
(10)
n.d. 339
n.d. prenyl-3’HDa
19
13.2
260
337
268 (100)
n.d. 339
271 (100), 283 (10)
137 (100), 243 (90), 253 (85),
215 (80), 225 (80), 161 (20), 181
(15), 201 (10)
O-prenyl-
3’HDa
Re
1.6 ± 0.3
20
13.5
n.d. 295
240 (100), 251 (80), 235
(50), 253 (35), 225 (30)
n.d. n.d. Aring-C-prenyl-
Re
21
14.2
323
295
240 (100), 251 (60), 253
(30), 225 (30)
195 (100), 212 (75), 170
(70), 225 (50), 197 (50)
n.d. Bring-C-prenyl-
Re
22
16.6
320
295
226 (100)
n.d. n.d. O-prenyl-Re
(Continued) 8 / 19 Non-genuine phenolic acceptor substrate specificity of prenyltransferase CloQ (
)
Substrate
Conversion
(% ± std)
No. RT
(min)
λmax
(nm)
[M-H]-
MS2
(rel. abundance)
MS3
(rel. abundance)
[M
+H]+
MS2
(rel. abundance)
MS3
(rel. abundance)
Tentative
annotation
Co
0.8 ± 0.3
23
18.5
349
335
279 (100), 280 (25), 292
(20)
251 (100), 279 (40), 223
(20) 252 (20), 280 (10)
337
269 (100), 270 (15), 281
(10)
241 (100), 225 (30), 197 (25),
242 (20)
C-prenyl-Co
24
19.6
n.d. 335
266 (100), 279 (10)
n.d. 337
n.d. O-prenyl-Co
4’-H-6-MF
0.8 ± 0.2
25
17.2
330
357,
335 c
342 (100), 343 (20)
298 (100), 314 (30), 251
(25)
359,
337 c
281 (100), 344 (40), 282
(20), 316 (20), 253 (15)
253 (100), 254 (20), 225 (10),
242 (10)
Bring-C-prenyl-
4’-H-6-MF
26
17.4
n.d. 357,
335 c
n.d. 359,
337 c
n.d. prenyl-4’-H-
6-MF
27
18.6
n.d. 357,
335 c
342 (100), 343 (20)
359,
337 c
n.d. prenyl-4’-H-
6-MF
Da
0.6 ± 0.2
28
15.0
n.d. 321
265 (100), 266 (60),278
(15), 252 (10)
n.d. 323
267 (100), 268 (10), 255
(10)
239 (100), 240 (15), 211 (15),
137 (10)
Bring-C-prenyl-
Da
Ge
0.4 ± 0.1
29
17.7
263
337
281 (100), 282 (30), 293
(15)
n.d. n.d. Bring-C-prenyl-
Ge
30
17.9
266
337
282 (100)
n.d. n.d. Aring-C-prenyl-
Ge
31
19.0
n.d. 337
268 (100), 255 (10)
n.d. n.d. O-prenyl-Ge
Gl
n.p. d
Is
n.p. a Not determined (n.d.). b Under alkaline conditions 4-HPP decomposes to 4-Hydroxybenzaldehyde (4-HBAL) or 4-hydroxyphenylacetic acid (4-HPA).[21]
c Parent ion formed a sodium adduct. The italic m/z represents the [M-H]- or [M+H]+ ion. d No products formed (n.p.). e Based on previous studies on the MS fragmentation of standard isoflavones[25] we proposed this product to be C8-prenyl-equol. h
//d i
/10 1371/j
l
0174665 001 Substrate
Conversion
(% ± std)
No. RT
(min)
λmax
(nm)
[M-H]-
MS2
(rel. abundance)
MS3
(rel. abundance)
[M
+H]+
MS2
(rel. abundance)
MS3
(rel. abundance)
Tentative
annotation
Co
0.8 ± 0.3
23
18.5
349
335
279 (100), 280 (25), 292
(20)
251 (100), 279 (40), 223
(20) 252 (20), 280 (10)
337
269 (100), 270 (15), 281
(10)
241 (100), 225 (30), 197 (25),
242 (20)
C-prenyl-Co
24
19.6
n.d. 335
266 (100), 279 (10)
n.d. 337
n.d. O-prenyl-Co
4’-H-6-MF
0.8 ± 0.2
25
17.2
330
357,
335 c
342 (100), 343 (20)
298 (100), 314 (30), 251
(25)
359,
337 c
281 (100), 344 (40), 282
(20), 316 (20), 253 (15)
253 (100), 254 (20), 225 (10),
242 (10)
Bring-C-prenyl-
4’-H-6-MF
26
17.4
n.d. 357,
335 c
n.d. 359,
337 c
n.d. prenyl-4’-H-
6-MF
27
18.6
n.d. 357,
335 c
342 (100), 343 (20)
359,
337 c
n.d. prenyl-4’-H-
6-MF
Da
0.6 ± 0.2
28
15.0
n.d. 321
265 (100), 266 (60),278
(15), 252 (10)
n.d. 323
267 (100), 268 (10), 255
(10)
239 (100), 240 (15), 211 (15),
137 (10)
Bring-C-prenyl-
Da
Ge
0.4 ± 0.1
29
17.7
263
337
281 (100), 282 (30), 293
(15)
n.d. n.d. Bring-C-prenyl-
Ge
30
17.9
266
337
282 (100)
n.d. n.d. Aring-C-prenyl-
Ge
31
19.0
n.d. 337
268 (100), 255 (10)
n.d. n.d. O-prenyl-Ge
Gl
n.p. d
Is
n.p. a Not determined (n.d.). b Under alkaline conditions 4-HPP decomposes to 4-Hydroxybenzaldehyde (4-HBAL) or 4-hydroxyphenylacetic acid (4-HPA).[21]
c Parent ion formed a sodium adduct. The italic m/z represents the [M-H]- or [M+H]+ ion. d No products formed (n.p.). e Based on previous studies on the MS fragmentation of standard isoflavones[25] we proposed this product to be C8-prenyl-equol. https://doi.org/10.1371/journal.pone.0174665.t001 PLOS ONE | https://doi.org/10.1371/journal.pone.0174665
March 29, 2017 9 / 19 Non-genuine phenolic acceptor substrate specificity of prenyltransferase CloQ Fig 3. SrCloQ prenylated equol products. RP-UHPLC-UV (A) and MS in NI (B) profiles of equol and prenylated equol products (products 8 and 9 in
Table 1) after incubation of equol with DMAPP and SrCloQ. MS2 spectra of C-prenylated equol (m/z 309) at retention times 16.9 (8, panel C) and 17.1 min
(9, panel D). The proposed RDA fragmentation pathways are shown as inset. https://doi org/10 1371/journal pone 0174665 g003 Fig 3. SrCloQ prenylated equol products. RP-UHPLC-UV (A) and MS in NI (B) profiles of equol and prenylated equol products (products 8 and 9 in
Table 1) after incubation of equol with DMAPP and SrCloQ. MS2 spectra of C-prenylated equol (m/z 309) at retention times 16.9 (8, panel C) and 17.1 min
(9, panel D). The proposed RDA fragmentation pathways are shown as inset. https://doi.org/10.1371/journal.pone.0174665.g003 Fig 3. SrCloQ prenylated equol products. RP-UHPLC-UV (A) and MS in NI (B) profiles of equol and prenylated equol products (products 8 and 9 in
Table 1) after incubation of equol with DMAPP and SrCloQ. MS2 spectra of C-prenylated equol (m/z 309) at retention times 16.9 (8, panel C) and 17.1 min
(9, panel D). The proposed RDA fragmentation pathways are shown as inset. https://doi.org/10.1371/journal.pone.0174665.g003 Da [C5H9] in NI and/or PI mode [29]. For more in-depth structural elucidation, tandem MS,
single-ion-monitoring (SIM) and single-reaction-monitoring (SRM) scan modes were used to
tentatively annotate the reaction products with respect to A- or B-ring prenylation and C- or
O-prenylation. Da [C5H9] in NI and/or PI mode [29]. For more in-depth structural elucidation, tandem MS,
single-ion-monitoring (SIM) and single-reaction-monitoring (SRM) scan modes were used to
tentatively annotate the reaction products with respect to A- or B-ring prenylation and C- or
O-prenylation. p
y
A- or B-ring prenylation. Analysis of the retro-Diels-Alder (RDA) fragment ions from
prenylated isomers in both NI and PI mode was used to determine the position of the prenyl
substituents (A- or B-ring). The formation of the RDA fragments upon cleavage of the C-rings
of (iso)flavonoids leave (part of) the prenyl group attached to the phenolic ring, which results
in diagnostic fragments [25]. The most common bonds in (iso)flavonoids split, resulting in A-
ring and B-ring containing ions, are the 1/3, 2/3, 0/2, 0/3, 0/4 or 2/4 bonds of the C-ring [24]. Fig 3A and 3B show the UV and MS in NI mode chromatograms of the reaction products
of equol incubated with SrCloQ. The two main prenylated products formed eluted at 16.9
min (peak 8) and 17.1 min (peak 9). Fig 3C and 3D show the MS2 spectra of these prenylated
equol isomers. Peak 8 showed high abundances of ions with m/z 121, 187 and 135. These val-
ues matched the RDA fragments of B-ring prenylated equol, i.e. 1,3A-, 1,3B- and 2,3A-, respec-
tively, as shown by the fragmentation pattern (Fig 3C). Peak 9 had different main m/z values
(189, 203 and 119). These ions were formed by the same fragmentation pathways as A- or B-ring prenylation. Analysis of the retro-Diels-Alder (RDA) fragment ions from
prenylated isomers in both NI and PI mode was used to determine the position of the prenyl
substituents (A- or B-ring). The formation of the RDA fragments upon cleavage of the C-rings
of (iso)flavonoids leave (part of) the prenyl group attached to the phenolic ring, which results
in diagnostic fragments [25]. PLOS ONE | https://doi.org/10.1371/journal.pone.0174665
March 29, 2017 The most common bonds in (iso)flavonoids split, resulting in A-
ring and B-ring containing ions, are the 1/3, 2/3, 0/2, 0/3, 0/4 or 2/4 bonds of the C-ring [24]. Fig 3A and 3B show the UV and MS in NI mode chromatograms of the reaction products
of equol incubated with SrCloQ. The two main prenylated products formed eluted at 16.9
min (peak 8) and 17.1 min (peak 9). Fig 3C and 3D show the MS2 spectra of these prenylated
l i
P
k 8 h
d hi h b
d
f i
ith
/ 121 187
d 135 Th
l A- or B-ring prenylation. Analysis of the retro-Diels-Alder (RDA) fragment ions from
prenylated isomers in both NI and PI mode was used to determine the position of the prenyl
substituents (A- or B-ring). The formation of the RDA fragments upon cleavage of the C-rings
of (iso)flavonoids leave (part of) the prenyl group attached to the phenolic ring, which results
in diagnostic fragments [25]. The most common bonds in (iso)flavonoids split, resulting in A-
ring and B-ring containing ions, are the 1/3, 2/3, 0/2, 0/3, 0/4 or 2/4 bonds of the C-ring [24]. Fig 3A and 3B show the UV and MS in NI mode chromatograms of the reaction products
of equol incubated with SrCloQ. The two main prenylated products formed eluted at 16.9
min (peak 8) and 17.1 min (peak 9). Fig 3C and 3D show the MS2 spectra of these prenylated
equol isomers. Peak 8 showed high abundances of ions with m/z 121, 187 and 135. These val-
ues matched the RDA fragments of B-ring prenylated equol, i.e. 1,3A-, 1,3B- and 2,3A-, respec-
tively, as shown by the fragmentation pattern (Fig 3C). Peak 9 had different main m/z values
(189, 203 and 119). These ions were formed by the same fragmentation pathways as PLOS ONE | https://doi.org/10.1371/journal.pone.0174665
March 29, 2017 10 / 19 Non-genuine phenolic acceptor substrate specificity of prenyltransferase CloQ Fig 4. SrCloQ prenylated 4’-hydroxy-7-methoxyflavone products. RP-UHPLC-UV (A) and MS in PI (B) profiles of 4’-hydroxy-7-methoxyflavone and
prenylated products (14, 15 and 16 in Table 1) after incubation of 4’-hydroxy-7-methoxyflavone with DMAPP and SrCloQ. MS2 spectra of C-prenylated
isomer (m/z 359, Na adduct) at retention time 17.1 min (15, panel C) and O-prenylated isomer at 18.1 min (16, panel D). The proposed RDA fragmentation
pathway is shown as inset. PLOS ONE | https://doi.org/10.1371/journal.pone.0174665
March 29, 2017 Non-genuine phenolic acceptor substrate specificity of prenyltransferase CloQ Fig 5. Molar composition of the prenylated products obtained with SrCloQ. Error bars represent the standard deviation; n.a., not annotated. https://doi.org/10.1371/journal.pone.0174665.g005 Fig 5. Molar composition of the prenylated products obtained with SrCloQ. Error bars represent the standard deviation; n.a., not annotated. https://doi.org/10.1371/journal.pone.0174665.g005 or B-ring position of the prenyl group was not possible, because the prenyl detached
completely in MS2, leaving no footprints to annotate the ring position. htt
//d i
/10 1371/j
l
0174665 004 Fig 4. SrCloQ prenylated 4’-hydroxy-7-methoxyflavone products. RP-UHPLC-UV (A) and MS in PI (B) profiles of 4’-hydroxy-7-methoxyflavone and
prenylated products (14, 15 and 16 in Table 1) after incubation of 4’-hydroxy-7-methoxyflavone with DMAPP and SrCloQ. MS2 spectra of C-prenylated
isomer (m/z 359, Na adduct) at retention time 17.1 min (15, panel C) and O-prenylated isomer at 18.1 min (16, panel D). The proposed RDA fragmentation
pathway is shown as inset. https://doi.org/10.1371/journal.pone.0174665.g004 https://doi.org/10.1371/journal.pone.0174665.g004 described before, but now the RDA fragments corresponded with A-ring prenylated equol
(Fig 3D). C- or O-prenylation. Tandem MS analysis was used to distinguish C- and O-prenylation
of the aromatic substrates. Fig 4A and 4B show the UV and MS in PI mode chromatograms of
the prenylated products of 4’-hydroxy-7-methoxyflavone produced by SrCloQ. Three main
prenylated isomers were found. Two distinct fragmentation patterns could be distinguished
for these isomers. Peak 14 and 15 showed [M+1–56]+ as main fragment (Fig 4C for peak 15;
see Table 1 for peak 14 fragmentation). Peak 16 showed [M+1–68]+ as the main fragment (Fig
4D). The prenyl group generates the fragment [M+H-56]+ when it is attached to a carbon of
an aromatic ring [29], as in the MS2 of peak 14 and 15. When it is attached to an oxygen of an
aromatic ring, the prenyl group will split off intact ([M+H-68]+) [16], leaving the original aro-
matic substrate as main daughter ion, as in the MS2 spectrum of peak 16. Moreover, the higher
retention time of peak 16 in comparison with peak 14 and 15 on the reversed phase column,
reflects that peak 16 is less polar than the other two, supporting our tentative annotation of O-
prenylation of this reaction product. In cases where O-prenylation could occur at both the A- and the B-ring (contrary to 4’-
hydroxy-7-methoxyflavone, which has only one OH group available), the annotation of the A- PLOS ONE | https://doi.org/10.1371/journal.pone.0174665
March 29, 2017 11 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0174665
March 29, 2017 Mechanism behind non-genuine acceptor substrate specificity of SrCloQ To verify the interactions of the acceptor substrates tested with the protein, we docked all the
molecules, including the genuine substrate 4-HPP, into the active site. Our docked 4-HPP
made the essential interactions [22] with the residues Glu281 and Arg160, as well as with
Arg176 and Cys297 (S1 Fig). Using this model, we docked the different phenolic substrates tested into the active site cav-
ity of SrCloQ. In principle, the active site had room to accommodate all acceptor substrates
tested, as no clashes were observed, not even for unreacted compounds such as glycitein. This
observation is in accordance with simulation analysis done previously [22], which showed that
the active site is large enough to accommodate flavonoids. The best aromatic substrate tested was equol, which was prenylated at the A- or the B-ring. Upon docking of equol into the active site of SrCloQ (Fig 6A), both the A- and B-ring can ori-
ent towards the donor substrate and make the hydrogen bond between the phenolic hydroxyl
group and Glu281. Additionally, the aromatic ring can make H-π interactions with Tyr233
and Trp122, whereas the tetrahydropyran (C-ring) can interact with Arg160. These interac-
tions are likely to help stabilizing the binding of equol inside the active site. Furthermore, this
isoflavan is one of the smallest acceptor substrates tested, and it is more amenable to torsion
than other isoflavonoids due to the lack of the C2–C3 double bond. Finally, it has less space
limitations inside the active site due to the absence of the C4 carbonyl group. This might
explain the promiscuity of SrCloQ to prenylate both the A- and the B-ring of equol. In addition, we studied the interactions of the second best acceptor substrate (i.e. 7,4’-dihy-
droxyflavone) with SrCloQ and compared them with those of its isoflavone isomer and one of
the worst acceptor substrates (i.e. daidzein) (Fig 6B). We found that 7,4’-dihydroxyflavone can
form the crucial hydrogen bond between the phenolic hydroxyl group at the B-ring and the
residue Glu281. Furthermore, the C4 carbonyl group interacts with the anchoring residue
Arg160. Also, the aromatic residue Tyr233 is able to make a hydrogen bond with the C7
hydroxyl group in the A-ring and π stacking interactions with the B-ring of the flavone. Tyr233 has been reported to stabilize the DMAPP substrate by hydrogen bonding and to make
van der Waals interactions with 4-HPP [17, 22]. SrCloQ favours B-ring C-prenylation of (iso)flavonoids and stilbenoids For all phenolic substrates tested with SrCloQ, the prenylated products obtained were anno-
tated using the above rationale. Fig 5 shows the composition of the mixture of prenylated iso-
mers formed by SrCloQ. The enzyme favoured B-ring C-prenylation of substrates in
aromatic rings. Considering the regioselectivity of SsNovQ (84% sequence identity with
SrCloQ) towards daidzein and genistein [23], we postulate that the C3’ (B-ring) is the pre-
ferred position for prenylation by SrCloQ. The only substrates that were A-ring prenylated
were equol and genistein, albeit the latter in minute amounts. It was not possible to annotate
the ring position of the prenyl group in coumestrol, as coumestrol does not undergo typical
RDA fragmentation [30, 31]. Consequently no apparent A- or B-ring fragment ions were
formed in MSn. Additionally, A-ring and B-ring prenylated coumestrol have been shown to
yield the same fragmentation pattern in MS [32, 33]. Resveratrol does not show RDA frag-
mentation due to the lack of the C-ring. Thus, reaction products of resveratrol were tenta-
tively annotated based on the fragmentation behaviour of (prenylated) resveratrol previously
reported [34, 35]. SrCloQ was able to C- or O-prenylate an acceptor molecule, such as luteolin, 4’-hydroxy-
7-methoxyflavone, 3’-hydroxydaidzein, and genistein. Luteolin and 3’-hydroxydaidzein were
primarily O-prenylated ( 80% of the reaction products). These two substrates were the only
ones with an extra phenolic hydroxyl group attached to the B-ring (meta-hydroxyl group). The
presence of hydroxyl groups proved to be essential for catalysis, as isoflavone (no hydroxyl
groups) yielded no reaction products with SrCloQ. Furthermore, the number of hydroxyl
groups on (iso)flavonoids proved to have an effect on the enzyme activity. SrCloQ yielded
slightly higher quantity of reaction products with 7,4’-dihydroxyflavone (2 hydroxyl groups) PLOS ONE | https://doi.org/10.1371/journal.pone.0174665
March 29, 2017 12 / 19 Non-genuine phenolic acceptor substrate specificity of prenyltransferase CloQ compared to luteolin (4 hydroxyl groups). Methoxylated substrates resulted in lower conver-
sion yields than their non-methoxylated derivatives, as for 4’-hydroxy-7-methoxyflavone com-
pared to 4’,7-dihydroxyflavone. PLOS ONE | https://doi.org/10.1371/journal.pone.0174665
March 29, 2017 Mechanism behind non-genuine acceptor substrate specificity of SrCloQ Residues that interact with the acceptor substrates are shown in orange: Glu281 anchors the substrate by H-
bonding with the phenolic hydroxyl group; Arg160 stabilizes binding by H-bonding with the carbonyl or ether
group of the C-ring; Tyr233 aromatic ring can make H-π bonds or π stacking interactions with the aromatic
rings of the phenolic substrates, while its carbonyl group can make H-bonding with phenolic hydroxyl groups;
Cys297 thiol group can interact with the carbonyl group of the phenolic substrate. https://doi org/10 1371/journal pone 0174665 g006 https://doi.org/10.1371/journal.pone.0174665.g006 https://doi.org/10.1371/journal.pone.0174665.g006 Mechanism behind non-genuine acceptor substrate specificity of SrCloQ Protein surface is
potential using MOE software default’s setting: yellow being lipophilic and
nor substrate DMAPP is shown in gray with the phosphate group in pink. eptor substrates are shown in orange: Glu281 anchors the substrate by H-
group; Arg160 stabilizes binding by H-bonding with the carbonyl or ether
tic ring can make H-π bonds or π stacking interactions with the aromatic
hile its carbonyl group can make H-bonding with phenolic hydroxyl groups;
h the carbonyl group of the phenolic substrate Fig 6. Phenolic aromatic substrates docked in the active site of SrCloQ (PDB 2XLQ). (A) Comparison of
equol with the A-ring (light blue) and B-ring (yellow) oriented towards the prenyl donor. (B) 7,4’-
(
)
(
) Fig 6. Phenolic aromatic substrates docked in the active site of SrCloQ (PDB 2XLQ). (A) Comparison of
equol with the A-ring (light blue) and B-ring (yellow) oriented towards the prenyl donor. (B) 7,4’-
Dihydroxyflavone (green) and daidzein (dark red) with their B-ring towards the prenyl donor. Protein surface is
coloured according to the lipophilic potential using MOE software default’s setting: yellow being lipophilic and
blue hydrophilic (cut-off of 2.5). Donor substrate DMAPP is shown in gray with the phosphate group in pink. Residues that interact with the acceptor substrates are shown in orange: Glu281 anchors the substrate by H-
bonding with the phenolic hydroxyl group; Arg160 stabilizes binding by H-bonding with the carbonyl or ether
group of the C-ring; Tyr233 aromatic ring can make H-π bonds or π stacking interactions with the aromatic
rings of the phenolic substrates, while its carbonyl group can make H-bonding with phenolic hydroxyl groups;
Cys297 thiol group can interact with the carbonyl group of the phenolic substrate. Fig 6. Phenolic aromatic substrates docked in the active site of SrCloQ (PDB 2XLQ). (A) Comparison of
equol with the A-ring (light blue) and B-ring (yellow) oriented towards the prenyl donor. (B) 7,4’-
Dihydroxyflavone (green) and daidzein (dark red) with their B-ring towards the prenyl donor. Protein surface is
coloured according to the lipophilic potential using MOE software default’s setting: yellow being lipophilic and
blue hydrophilic (cut-off of 2.5). Donor substrate DMAPP is shown in gray with the phosphate group in pink. Mechanism behind non-genuine acceptor substrate specificity of SrCloQ Daidzein, which is the isoflavone isomer of 7,4’-dihydroxyflavone, also showed the essential
interaction between the C4’ hydroxyl group and Glu281. Furthermore, the carbonyl group of
daidzein makes the interactions with Arg160 and with Cys297. In contrast to the flavone, due
to the different orientation of the A- and B-rings, daidzein does not make any interactions
with Tyr233. Overall, our results show that Arg160 plays an important role in stabilizing (iso)flavonoids
in the active site of SrCloQ via hydrogen bonds with the carbonyl or ether group in the C-ring. Moreover, our docking studies revealed that interactions with Tyr233 might contribute to the
flavone over isoflavone preference observed in this study with SrCloQ, and also in a previous
study with SsNovQ [23]. The C7 hydroxyl group at the A-ring of isoflavones is far away from
this residue and the B-ring is oriented in a different direction. Consequently, daidzein is
bl
k
h
l
i h T
233
d
h fl
M
i
di unable to make the relevant contacts with Tyr233, as opposed to the flavone. Mutation studies
are required to confirm this role of Tyr233 in flavonoid over isoflavone preference by the PTs
SrCloQ and SsNovQ. 13 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0174665
March 29, 2017 Non-genuine phenolic acceptor substrate specificity of prenyltransferase CloQ Fig 6. Phenolic aromatic substrates docked in the active site of SrCloQ (PDB 2XLQ). (A) Comparison of
equol with the A-ring (light blue) and B-ring (yellow) oriented towards the prenyl donor. (B) 7,4’-
Dihydroxyflavone (green) and daidzein (dark red) with their B-ring towards the prenyl donor. Protein surface is
coloured according to the lipophilic potential using MOE software default’s setting: yellow being lipophilic and
blue hydrophilic (cut-off of 2.5). Donor substrate DMAPP is shown in gray with the phosphate group in pink. Residues that interact with the acceptor substrates are shown in orange: Glu281 anchors the substrate by H-
bonding with the phenolic hydroxyl group; Arg160 stabilizes binding by H-bonding with the carbonyl or ether
group of the C-ring; Tyr233 aromatic ring can make H-π bonds or π stacking interactions with the aromatic
rings of the phenolic substrates, while its carbonyl group can make H-bonding with phenolic hydroxyl groups; ates docked in the active site of SrCloQ (PDB 2XLQ). (A) Comparison of
d B-ring (yellow) oriented towards the prenyl donor. (B) 7,4’-
zein (dark red) with their B-ring towards the prenyl donor. Non-genuine phenolic acceptor substrate specificity of prenyltransferase CloQ Table 2. Summary of acceptor substrate preferences and regioselectivity of SrCloQ and other closely related ABBA prenyltransferases. SrCloQ
SsNovQ[23]
Sco7190[18, 19]
SclNphB[18]
Sequence identity with SrCloQ (%)
-
85
25
21
Genuine acceptor substrate
4-HPP a
4-HPP
1,6-DHN b
1,6-DHN
Genuine donor substrate
DMAPP c
DMAPP
DMAPP
GPP d
Non-genuine acceptor substrate preferences
Flavonoids
+
+
+/-
+
Isoflavonoids
+/-
+/-
-
+/-
Substrate with no OH groups
-
n.t. n.t. n.t. Substrate with OCH3 groups
-
n.t. n.t. -
Regioselectivity
A-ring prenylation
+/-
-
+
+
B-ring prenylation
+
+
-
-
C-prenylation
++
++
+
+
O-prenylation
+
+
-
++
Double prenylation
-
+e
-
+ ubstrate preferences and regioselectivity of SrCloQ and other closely related ABBA prenyltransferases. mary of acceptor substrate preferences and regioselectivity of SrCloQ and other closely related ABBA preny Table 2. Summary of acceptor substrate preferences and regioselectivity of SrCloQ and other closely relate a 4-hydroxyphenylpyruvate. b 1,6-dihydroxynaphtalene. c dimethylallylpyrophosphate. d geranylpyrophosphate. e Only with a phenolic acid (i.e. 3,4-O-diprenyl-caffeic acid), but not with any of the (iso)flavonoids or stilbenoids tested in their study.[23] a 4-hydroxyphenylpyruvate. b 1,6-dihydroxynaphtalene. c dimethylallylpyrophosphate. d geranylpyrophosphate. e Only with a phenolic acid (i.e. 3,4-O-diprenyl-caffeic acid), but not with any of the (iso)flavonoids or stilbenoids tested in their study.[23]
https://doi org/10 1371/journal pone 0174665 t002 1,6 dihydroxynaphtalene. c dimethylallylpyrophosphate. d geranylpyrophosphate. e Only with a phenolic acid (i.e. 3,4-O-diprenyl-caffeic acid), but not with any of the (iso)flavonoids or stilbenoids tested in their study.[23] geranylpyrophosphate. e Only with a phenolic acid (i.e. 3,4-O-diprenyl-caffeic acid), but not with any of the (iso)flavonoids or stilbenoids tested in their study.[23] for the selection of appropriate acceptor substrates for the in vitro production of novel bioac-
tive prenylated compounds. Acceptor substrate specificity. For all four bacterial PTases, flavonoids were preferred
over isoflavonoids. According to our docking results, interactions of Tyr233 with flavones con-
tributes to this preference observed in SrCloQ. This Tyr233 is conserved in SsNovQ. The other
two enzymes contain alanine in that position instead (Figure E in S1 File). By superposing the
active sites of SrCloQ and SclNphB (data not shown) it was apparent that SclNphB contains a
tyrosine residue (Tyr216, also conserved in Sco7190), with its side chain at an equivalent posi-
tion as Tyr233 of SrCloQ. Comparison of non-genuine substrate preferences of different ABBA
prenyltransferases Using the information obtained from the docking studies with SrCloQ and sequence compari-
son (Figure E in S1 File), we can extend our knowledge of the non-genuine aromatic substrate
preferences of other closely related ABBA PTs [4, 15]. Table 2 shows an overview of the
sequence identity and main acceptor substrate preferences of SrCloQ compared with other
members of the CloQ/NphB PT family reported in literature. This information can be useful PLOS ONE | https://doi.org/10.1371/journal.pone.0174665
March 29, 2017 14 / 19 https://doi.org/10.1371/journal.pone.0174665.t002 PLOS ONE | https://doi.org/10.1371/journal.pone.0174665
March 29, 2017 Non-genuine phenolic acceptor substrate specificity of prenyltransferase CloQ The addition of methoxyl groups to the A-ring, as in glycitein, decreased or cancelled the
activity of SrCloQ. The same result was observed for SclNphB with the methoxylated substrate
pterostilbene [18]. The increase in molecular size by the addition of this bulky substituent pos-
sibly hampers the entrance to the active site of the PTs. The effect of hydroxylation on the
activity of SsNovQ, Sco7190 and SclNphB and the effect of methoxylation of phenolics on the
activity of SsNovQ and Sco7190 has to our knowledge not been tested, but it is tempting to pro-
pose a similar trend on activity as observed with SrCloQ. A- or B-ring regioselectivity. SrCloQ and SsNovQ favour B-ring prenylation, whereas
Sco7190 and SclNphB prefer A-ring prenylation. This preference for either the B- or A-ring
prenylation might be explained by the structure of their reported genuine substrates: SrCloQ
and SsNovQ use 4-HPP as the genuine substrate, which contains one phenyl ring thereby
resembling the B-ring of (iso)flavonoids. In contrast, Sco7190 and SclNphB use 1,6-dihydroxy-
naphtalene, which contains two connected rings, resembling the A- and C-ring of (iso)flavo-
noids. In addition, the residue Arg160, which makes interactions with the carbonyl group of
4-HPP and of the C-ring of (iso)flavonoids (Fig 6), is conserved among the B-ring prenylating
enzymes (Figure E in S1 File). This interaction is likely to assist in orienting the B-ring of the
acceptor substrate towards the donor substrate, facilitating B-ring prenylation. C- or O-prenylation. SrCloQ and SsNovQ were able to either C- or O-prenylate the same
(iso)flavonoid substrate, although C-prenylation was predominant. Sco7190 did not O-preny-
late any of the (iso)flavonoids substrates tested, whereas SclNphB predominantly showed pref-
erence for O-prenylation of (iso)flavonoids. There are no studies explaining what exactly
determines C- versus O- prenylation preference of these bacterial PTs. With regard to SrCloQ,
O-prenylation was preferred when the aromatic ring contained two neighbouring hydroxyl
groups, as with luteolin and 3’-hydroxydaidzein. Previous biochemical studies with SrCloQ
and Sco7190 showed only C-prenylation of aromatic substrates [17, 18, 21], however, none of
the acceptors tested had the neighbouring hydroxyl group. Based on our docking studies, we
observed that the C3’ hydroxyl group can be close enough to the donor substrate in the catalytic
centre and prone to electrophilic attack by the allyl cation. In contrast to SrCloQ, SsNovQ was
able to B-ring O-prenylate many (iso)flavonoid substrates without the C3’ hydroxyl group. Fur-
ther (crystallization) studies with SsNovQ may provide insight to understand this difference. With regard to the number of prenyl groups attached to the phenolic substrates used in this
study, SrCloQ produced only mono prenylated products, similarly to SsNovQ and Sco7190. In
contrast, SclNphB has been reported to produce double prenylated stilbenoids, specifically
2,4-digeranyl-resveratrol [18]. This difference might be explained by the facts that the bottom
part of the PT barrel of SrCloQ is less accessible and significantly narrower than that of
SclNphB [17]. PLOS ONE | https://doi.org/10.1371/journal.pone.0174665
March 29, 2017 The side chains of these tyrosine residues were both reported to make
equivalent interactions with the donor (i.e. stabilization of the carbocation) and genuine accep-
tor substrates (π or van der Waal contacts with the aromatic ring of the acceptor substrates) [17,
22, 36]. Due to its position and orientation, it seems unlikely that Tyr216 can interact with the
non-genuine substrates in a similar way as Tyr233. Therefore, residues other than Tyr216 seem
responsible for the non-genuine acceptor substrate preference observed with SclNphB. g
The presence of the para-hydroxyl group in 4-HPP is essential, as the substrate is anchored
with this hydroxyl by a hydrogen bond to Glu281. The potential non-genuine acceptor sub-
strate isoflavone (Fig 2) does not contain hydroxyl groups. This explains the lack of reaction
products of SrCloQ with this molecule as acceptor substrate. Glu281facilitates the formation of
the prenylated σ-complex and it neutralizes it by proton abstraction (Fig 1) [17]. Glu281 is
conserved in SsNovQ and Sco7190, but not in SclNphB. Previous studies on the mechanism of
SclNphB revealed that the genuine acceptor substrate 1,6-dihydroxynaphthalene makes con-
tacts with Ser51 and the non-genuine acceptor substrate flaviolin with Gln295 [19]. These resi-
dues are likely to help position the acceptor substrates in SclNphB, as Glu281 in the other PTs. In SclNphB, a water molecule, instead of any particular residue, is the most likely to facilitate
the proton extraction step, according to simulation studies [36]. PLOS ONE | https://doi.org/10.1371/journal.pone.0174665
March 29, 2017 15 / 19 Acknowledgments The authors would like to thank Prof. Dr. Lutz Heide from the Pharmaceutical Institute, Eber-
hard-Karls-University Tu¨bingen, Germany for kindly providing the cloq plasmid and for his
input during the writing process of this manuscript. Writing – review & editing: BM GS FL JPV HG. Writing – review & editing: BM GS FL JPV HG. Conclusions In this study we demonstrated that: (i) SrCloQ can prenylate aromatic substrates belonging to
the (iso)flavonoid and stilbenoid classes; (ii) SrCloQ is able to either C- or O-prenylate the
same acceptor substrate; (iii) SrCloQ showed a preference for C-prenylation at the B-ring of
(iso)flavonoids, as hypothesized in the introduction; (iv) the addition of a meta hydroxyl
group at the B-ring changes the preference to O-prenylation. The genuine substrate 4-HPP
showed the highest conversion yield, followed by equol and 7,4’-dihydroxyflavone. We pro-
pose, using in silico modelling, the mechanisms by which the acceptor substrate specificity and
regioselectivity observed with SrCloQ, but also of related PTs, can be explained. This informa-
tion can help to choose the appropriate acceptor substrate for a specific PT when tailoring
novel prenylated phenolic compounds. PLOS ONE | https://doi.org/10.1371/journal.pone.0174665
March 29, 2017 16 / 19 Non-genuine phenolic acceptor substrate specificity of prenyltransferase CloQ Conceptualization: CAC BM JPV HG. Formal analysis: CAC BM. Funding acquisition: HG. Investigation: BM FL. Methodology: CAC GS. Validation: CAC. Validation: CAC. Visualization: CAC GS. Writing – original draft: CAC BM JPV HG. Writing – original draft: CAC BM JPV HG. Project administration: CAC JPV HG. Project administration: CAC JPV HG. Resources: HG. Resources: HG. Supervision: CAC JPV HG. References 1. Palsuledesai CC, Distefano MD. Protein prenylation: enzymes, therapeutics, and biotechnology appli-
cations. ACS Chem Biol. 2015; 10(1):51–62. https://doi.org/10.1021/cb500791f PMID: 25402849 2. Simons R, Gruppen H, Bovee TFH, Verbruggen MA, Vincken J-P. Prenylated isoflavonoids from plants
as selective estrogen receptor modulators (phytoSERMs). Food Funct. 2012; 3(8):810–27. https://doi. org/10.1039/c2fo10290k PMID: 22684228 3. van de Schans MGM, Vincken J-P, de Waard P, Hamers ARM, Bovee TFH, Gruppen H. Glyceollins
and dehydroglyceollins isolated from soybean act as SERMs and ER subtype-selective phytoestro-
gens. J Steroid Biochem Mol Biol. 2016; 156:53–63. https://doi.org/10.1016/j.jsbmb.2015.11.020
PMID: 26655113 4. Winkelblech J, Fan A, Li S-M. Prenyltransferases as key enzymes in primary and secondary metabo-
lism. Appl Microbiol Biotechnol. 2015; 99(18):7379–97. https://doi.org/10.1007/s00253-015-6811-y
PMID: 26216239 5. Liang PH, Ko TP, Wang AHJ. Structure, mechanism and function of prenyltransferases. Eur J Biochem. 2002; 269(14):3339–54. PMID: 12135472 6. Heide L. Prenyl transfer to aromatic substrates: genetics and enzymology. Curr Opin Chem Biol. 2009;
13(2):171–9. https://doi.org/10.1016/j.cbpa.2009.02.020 PMID: 19299193 7. Yazaki K, Sasaki K, Tsurumaru Y. Prenylation of aromatic compounds, a key diversification of plant sec-
ondary metabolites. Phytochemistry. 2009; 70(15–16):1739–45. https://doi.org/10.1016/j.phytochem. 2009.08.023 PMID: 19819506 8. Veitch NC. Isoflavonoids of the Leguminosae. Nat Prod Rep. 2013; 30(7):988–1027. https://doi.org/10. 1039/c3np70024k PMID: 23736284 17 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0174665
March 29, 2017 Non-genuine phenolic acceptor substrate specificity of prenyltransferase CloQ 9. Khalivulla SI, Reddy BAK, Gunasekar D, Blond A, Bodo B, Murthy MM, et al. A new di-O-prenylated iso-
flavone from Tephrosia tinctoria. J Asian Nat Prod Res. 2008; 10(10):953–5. 10. Welle R, Grisebach H. Properties and solubilization of the prenyltransferase of isoflavonoid phytoalexin
biosynthesis in soybean. Phytochemistry. 1991; 30(2):479–84. 11. Sasaki K, Tsurumaru Y, Yamamoto H, Yazaki K. Molecular characterization of a membrane-bound pre-
nyltransferase specific for isoflavone from Sophora flavescens. J Biol Chem. 2011; 286(27):24125–34. https://doi.org/10.1074/jbc.M111.244426 PMID: 21576242 12. Yang X, Jiang Y, Yang J, He J, Sun J, Chen F, et al. Prenylated flavonoids, promising nutraceuticals
with impressive biological activities. Trends Food Sci Technol. 2015; 44(1):93–104. 13. Saleh O, Haagen Y, Seeger K, Heide L. Prenyl transfer to aromatic substrates in the biosynthesis of
aminocoumarins, meroterpenoids and phenazines: The ABBA prenyltransferase family. Phytochemis-
try. 2009; 70(15–16):1728–38. https://doi.org/10.1016/j.phytochem.2009.05.009 PMID: 19559450 14. Bonitz T, Alva V, Saleh O, Lupas AN, Heide L. Evolutionary relationships of microbial aromatic prenyl-
transferases. PLoS One. 2011; 6(11):1–8. 15. Tello M, Kuzuyama T, Heide L, Noel JP, Richard SB. The ABBA family of aromatic prenyltransferases:
broadening natural product diversity. References Cell Mol Life Sci. 2008; 65(10):1459–63. https://doi.org/10.1007/
s00018-008-7579-3 PMID: 18322648 16. Haagen Y, Unso¨ld I, Westrich L, Gust B, Richard SB, Noel JP, et al. A soluble, magnesium-independent
prenyltransferase catalyzes reverse and regular C-prenylations and O-prenylations of aromatic sub-
strates. FEBS Lett. 2007; 581(16):2889–93. https://doi.org/10.1016/j.febslet.2007.05.031 PMID:
17543953 17. Metzger U, Keller S, Stevenson CEM, Heide L, Lawson DM. Structure and mechanism of the magne-
sium-independent aromatic prenyltransferase CloQ from the clorobiocin biosynthetic pathway. J Mol
Biol. 2010; 404(4):611–26. https://doi.org/10.1016/j.jmb.2010.09.067 PMID: 20946900 18. Kumano T, Richard SB, Noel JP, Nishiyama M, Kuzuyama T. Chemoenzymatic syntheses of preny-
lated aromatic small molecules using Streptomyces prenyltransferases with relaxed substrate specifici-
ties. Bioorg Med Chem. 2008; 16(17):8117–26. https://doi.org/10.1016/j.bmc.2008.07.052 PMID:
18682327 19. Kuzuyama T, Noel JP, Richard SB. Structural basis for the promiscuous biosynthetic prenylation of aro-
matic natural products. Nature. 2005; 435(7044):983–7. https://doi.org/10.1038/nature03668 PMID:
15959519 20. Leipoldt F, Zeyhle P, Kulik A, Kalinowski J, Heide L, Kaysser L. Diversity of ABBA prenyltransferases in
marine Streptomyces sp. CNQ-509: Promiscuous enzymes for the biosynthesis of mixed terpenoid
compounds. PLoS One. 2015; 10(12):1–15. 21. Pojer F, Wemakor E, Kammerer B, Chen H, Walsh CT, Li S-M, et al. CloQ, a prenyltransferase involved
in clorobiocin biosynthesis. Proc Natl Acad Sci USA. 2003; 100(5):2316–21. https://doi.org/10.1073/
pnas.0337708100 PMID: 12618544 22. Bayse CA, Merz KM. Mechanistic insights into Mg2+-independent prenylation by CloQ from classical
molecular mechanics and hybrid quantum mechanics/molecular mechanics molecular dynamics simu-
lations. Biochemistry. 2014; 53(30):5034–41. https://doi.org/10.1021/bi500531p PMID: 25020142 23. Ozaki T, Mishima S, Nishiyama M, Kuzuyama T. NovQ is a prenyltransferase capable of catalyzing the
addition of a dimethylallyl group to both phenylpropanoids and flavonoids. J Antibiot. 2009; 62(7):385–
92. https://doi.org/10.1038/ja.2009.48 PMID: 19557032 24. Cuyckens F, Claeys M. Mass spectrometry in the structural analysis of flavonoids. J Mass Spectrom. 2004; 39(1):1–15. https://doi.org/10.1002/jms.585 PMID: 14760608 25. Aisyah S, Vincken J-P, Andini S, Mardiah Z, Gruppen H. Compositional changes in (iso)flavonoids and
estrogenic activity of three edible Lupinus species by germination and Rhizopus-elicitation. Phytochem-
istry. 2016; 122:65–75. https://doi.org/10.1016/j.phytochem.2015.12.015 PMID: 26749476 26. Kosters HA, Wierenga PA, de Vries R, Gruppen H. Characteristics and effects of specific peptides on
heat-induced aggregation of β-lactoglobulin. Biomacromolecules. 2011; 12(6):2159–70. https://doi.org/
10.1021/bm2002285 PMID: 21517078 27. Metzger U, Schall C, Zocher G, Unso¨ld I, Stec E, Li S-M, et al. The structure of dimethylallyl tryptophan
synthase reveals a common architecture of aromatic prenyltransferases in fungi and bacteria. Proc Natl
Acad Sci USA. 2009; 106(34):14309–14. PLOS ONE | https://doi.org/10.1371/journal.pone.0174665
March 29, 2017 References https://doi.org/10.1073/pnas.0904897106 PMID: 19706516 28. Robert X G P. Deciphering key features in protein structures with the new ENDscript server. Nucleic
Acid Research. 2014; 42(W1):W320–W1. 29. Simons R, Vincken J-P, Bakx EJ, Verbruggen MA, Gruppen H. A rapid screening method for prenylated
flavonoids with ultra-high-performance liquid chromatography/electrospray ionisation mass 18 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0174665
March 29, 2017 Non-genuine phenolic acceptor substrate specificity of prenyltransferase CloQ spectrometry in licorice root extracts. Rapid Commun Mass Spectrom. 2009; 23(19):3083–93. https://
doi.org/10.1002/rcm.4215 PMID: 19711301 spectrometry in licorice root extracts. Rapid Commun Mass Spectrom. 2009; 23(19):3083–93. https://
doi.org/10.1002/rcm.4215 PMID: 19711301 30. 30. van de Schans MGM, Vincken J-P, Bovee TFH, David Cervantes A, Logtenberg MJ, Gruppen H. Struc-
tural changes of 6a-hydroxy-pterocarpans upon heating modulate their estrogenicity. J Agric Food
Chem. 2014; 62(43):10475–84. https://doi.org/10.1021/jf503127c PMID: 25296697 31. Yang M, Wang W, Sun J, Zhao Y, Liu Y, Liang H, et al. Characterization of phenolic compounds in the
crude extract of Hedysarum multijugum by high-performance liquid chromatography with electrospray
ionization tandem mass spectrometry. Rapid Commun Mass Spectrom. 2007; 21(23):3833–41. PMID:
17979101 32. Morandi D, Le Quere JL. Influence of nitrogen on accumulation of isosojagol (a newly detected coume-
stan in soybean) and associated isoflavonoids in roots and nodules of mycorrhizal and non-mycorrhizal
soybean. New Phytol. 1991; 117(1):75–9. 33. O’Neill MJ. Aureol and phaseol, two new coumestans from Phaseolus aureus Roxb. Z Naturforsch C. 1983; 38(9–10):698–700. 34. Stella L, De Rosso M, Panighel A, Vedova AD, Flamini R, Traldi P. Collisionally induced fragmentation
of [M–H]−species of resveratrol and piceatannol investigated by deuterium labelling and accurate mass
measurements. Rapid Commun Mass Spectrom. 2008; 22(23):3867–72. https://doi.org/10.1002/rcm. 3811 PMID: 18980255 35. Sobolev VS, Potter TL, Horn BW. Prenylated stilbenes from peanut root mucilage. Phytochem Anal. 2006; 17(5):312–22. PMID: 17019932 36. Yang Y, Miao YP, Wang B, Cui GL, Merz KM. Catalytic Mechanism of Aromatic Prenylation by NphB. Biochemistry. 2012; 51(12):2606–18. https://doi.org/10.1021/bi201800m PMID: 22385275 19 / 19
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